WO2023147092A9 - Coronavirus vaccine - Google Patents

Coronavirus vaccine Download PDF

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WO2023147092A9
WO2023147092A9 PCT/US2023/011791 US2023011791W WO2023147092A9 WO 2023147092 A9 WO2023147092 A9 WO 2023147092A9 US 2023011791 W US2023011791 W US 2023011791W WO 2023147092 A9 WO2023147092 A9 WO 2023147092A9
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cov
sars
polypeptide
seq
fragment
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PCT/US2023/011791
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French (fr)
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WO2023147092A3 (en
WO2023147092A2 (en
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Ye Che
Kena Anne SWANSON
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BioNTech SE
Pfizer Inc.
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Publication of WO2023147092A2 publication Critical patent/WO2023147092A2/en
Publication of WO2023147092A3 publication Critical patent/WO2023147092A3/en
Publication of WO2023147092A9 publication Critical patent/WO2023147092A9/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • CORONAVIRUS VACCINE This disclosure relates to the field of RNA to prevent or treat coronavirus infection.
  • the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen ⁇ specific immune responses such as antibody and/or T cell responses. These methods and agents are, in particular, useful for the prevention or treatment of coronavirus infection.
  • Administration of RNA disclosed herein to a subject can protect the subject against coronavirus infection.
  • the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS ⁇ CoV ⁇ 2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS ⁇ CoV ⁇ 2, in the subject, i.e., vaccine RNA encoding vaccine antigen.
  • Administering to the subject RNA encoding vaccine antigen may provide (following expression of the RNA by appropriate target cells) vaccine antigen for inducing an immune response against vaccine antigen (and disease ⁇ associated antigen) in the subject.
  • Coronaviruses are positive ⁇ sense, single ⁇ stranded RNA ((+)ssRNA) enveloped viruses that encode for a total of four structural proteins, spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N).
  • the spike protein (S protein) is responsible for receptor ⁇ recognition, attachment to the cell, infection via the endosomal pathway, and the genomic release driven by fusion of viral and endosomal membranes. Though sequences between the different family members vary, there are conserved regions and motifs within the S protein making it possible to divide the S protein into two subdomains: S1 and S2.
  • SARS ⁇ CoV ⁇ 2 severe acute respiratory syndrome coronavirus 2
  • MN908947.3 The genetic sequence of SARS ⁇ CoV ⁇ 2 became available to the WHO and public (MN908947.3) and the virus was categorized into the betacoronavirus subfamily.
  • SARS ⁇ CoV ⁇ 2 infections and the resulting disease COVID ⁇ 19 have spread globally, affecting a growing number of countries.
  • the WHO characterized the COVID ⁇ 19 outbreak as a pandemic.
  • the ongoing pandemic remains a significant challenge to public health and economic stability worldwide. Every individual is at risk of infection as there is no pre ⁇ existing immunity to SARS ⁇ CoV ⁇ 2.
  • Common symptoms in hospitalized patients include fever, dry cough, shortness of breath, fatigue, myalgias, nausea/vomiting or diarrhoea, headache, weakness, and rhinorrhoea.
  • Anosmia loss of smell
  • ageusia loss of taste
  • CFR case fatality rates
  • Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers are overrepresented among COVID ⁇ 19 patients due to occupational exposure to infected patients.
  • a molecular test is used to detect SARS ⁇ CoV ⁇ 2 and confirm infection.
  • the reverse transcription polymerase chain reaction (RT ⁇ PCR) test methods targeting SARS ⁇ CoV ⁇ 2 viral RNA are the gold standard in vitro methods for diagnosing suspected cases of COVID ⁇ 19.
  • Samples to be tested are collected from the nose and/or throat with a swab.
  • the present disclosure provides insights into immune responses elicited by exposure to (e.g., by vaccination and/or infection) different SARS ⁇ CoV ⁇ 2 variants or immunogenic polypeptides (e.g., S protein), or immunogenic fragments thereof.
  • administering RNA encoding an S protein of a BA.2 and/or BA.4/5 Omicron SARS ⁇ CoV ⁇ 2 variant, or an immunogenic fragment thereof can result in an improved immune response, which includes, e.g., improved neutralization of Omicron BA.4 and/or Omicron BA.5 SARS ⁇ CoV ⁇ 2 variants and/or broader cross ⁇ neutralization of variants (e.g., Omicron variants) of concern (e.g., increased neutralization titers against a larger number of variants (e.g., Omicron variants) of concern).
  • the present disclosure provides an insight that a bivalent coronavirus vaccine (e.g., a bivalent BA.4/5 vaccine comprising a first RNA encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain or an immunogenic fragment thereof, and a second RNA encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4/5 Omicron variant or an immunogenic fragment thereof) can provide broader cross ⁇ neutralization against SARS ⁇ CoV ⁇ 2 Wuhan strain and certain variants thereof (e.g., in some embodiments variants that are prevalent and/or rapidly spreading in a relevant jurisdiction, e.g., certain Omicron variants) in certain subjects as compared to a monovalent coronavirus vaccine (e.g., a vaccine comprising RNA encoding a SARS ⁇ CoV ⁇ 2 S protein of a coronavirus strain or variant thereof).
  • a bivalent coronavirus vaccine e.g., a bivalent BA
  • such broader cross ⁇ neutralization can be observed in vaccine ⁇ na ⁇ ve subjects. In some embodiments, such broader cross ⁇ neutralization can be observed in subjects without a coronavirus infection (e.g., a SARS ⁇ CoV ⁇ 2 infection). In some embodiments, such broader cross ⁇ neutralization can be observed in subjects who previously received a SARS ⁇ CoV ⁇ 2 vaccine (e.g., in some embodiments an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein, e.g., in some embodiments of a Wuhan strain). In some embodiments, such broader cross ⁇ neutralization can be observed in in young pediatric subjects (e.g., subjects aged 6 months to less than 2 years, and/or 2 years to less than 5 years).
  • young pediatric subjects e.g., subjects aged 6 months to less than 2 years, and/or 2 years to less than 5 years.
  • the present disclosure provides an insight that exposure to at least two certain SARS ⁇ CoV ⁇ 2 variants or immunogenic polypeptides (e.g., S protein), or immunogenic fragments thereof can result in an synergistic improvement in immune response (e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape) as compared to exposure to one SARS ⁇ CoV ⁇ 2 strain and/or other combinations of SARS ⁇ CoV ⁇ 2 variants.
  • immunogenic polypeptides e.g., S protein
  • an immune response that is less susceptible to immune escape compared to exposure to one SARS ⁇ CoV ⁇ 2 strain and/or other combinations of SARS ⁇ CoV ⁇ 2 variants.
  • the present disclosure provides an insight that exposure to a S protein from a Wuhan strain or an immunogenic fragment thereof (e.g., by vaccination and/or infection), and exposure to a S protein of an Omicron BA.1 variant or an immunogenic fragment thereof (e.g., by vaccination and/or infection) can result in an synergistic improvement in immune response (e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape) as compared to exposure to one SARS ⁇ CoV ⁇ 2 strain and/or other combinations of SARS ⁇ CoV ⁇ 2 variants).
  • an immune response e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape
  • the present disclosure provides an insight that exposure to a S protein from a Wuhan strain or an immunogenic fragment thereof (e.g., by vaccination and/or infection), and exposure to a S protein of an Omicron BA.4 or BA.5 variant or an immunogenic fragment thereof (e.g., by vaccination and/or infection) can result in an synergistic improvement in immune response (e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape) as compared to exposure to one SARS ⁇ CoV ⁇ 2 strain and/or other combinations of SARS ⁇ CoV ⁇ 2 variants).
  • an immune response e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape
  • the present disclosure provides an insight that (i) exposure to a S protein from a strain/variant selected from the group consisting of Wuhan strain, an alpha variant, beta variant, delta variant, Omicron BA.1, and sublineages derived from any of the aforementioned strains/variants, or immunogenic fragments thereof (e.g., by vaccination and/or infection), combined with (ii)exposure to a S protein from a strain/variant selected from the group consisting of Omicron BA.2, Omicron BA.4, Omicron BA.5, and sublineages derived from any of the aforementioned strains/variants, or immunogenic fragments thereof (e.g., by vaccination and/or infection) can result in an synergistic improvement in immune response (e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape) as compared to exposure to one SARS ⁇ CoV ⁇ 2 strain and/or other combinations of SARS ⁇ CoV ⁇ 2 variants
  • the present disclosure also provides significant insights into how an immune response develops in subjects following exposures to (e.g., vaccinations and/or infections) multiple, different SARS ⁇ CoV ⁇ 2 strains.
  • an immune response develops in subjects following exposures to (e.g., vaccinations and/or infections) multiple, different SARS ⁇ CoV ⁇ 2 strains.
  • disclosed herein is a finding that different combinations of SARS ⁇ CoV ⁇ 2 variants elicit different immune responses.
  • the present disclosure provides an insight that exposure to certain combinations of SARS ⁇ CoV ⁇ 2 variants can elicit an improved immune response (e.g., higher neutralization titers, broader cross ⁇ neutralization, and/or an immune response that is less susceptible to immune escape).
  • an improved immune response can be produced when subjects are delivered two or more antigens (e.g., as polypeptides or RNAs encoding such polypeptides), each having few shared epitopes.
  • an improved immune response can be produced when subjects are delivered a combination of SARS ⁇ CoV ⁇ 2 S proteins (e.g., as polypeptides or RNAs encoding such polypeptides) sharing no more than 50% (e.g., no more than 40%, no more than 30%, no more 20% or more) of epitopes (including, e.g., amino acid mutations) that can be bound by neutralization antibodies.
  • an improved immune response can be produced by delivering, as polypeptides or RNAs encoding such polypeptides, (a) a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, an Alpha variant, Beta variant, or a Delta variant of SARS ⁇ CoV ⁇ 2 or an immunogenic fragment thereof, and (b) an S protein from a SARS ⁇ CoV ⁇ 2 Omicron variant or an immunogenic fragment thereof.
  • an improved immune response can be produced by delivering, as polypeptides or RNAs encoding such polypeptides, (a) a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant of SARS ⁇ CoV ⁇ 2 or an immunogenic fragment thereof, and (b) an S protein of a SARS ⁇ CoV ⁇ 2 Omicron variant that is not a BA.1 Omicron variant or an immunogenic fragment thereof.
  • an improved immune response can be produced by delivering, as polypeptides or RNAs encoding such polypeptides, (a) an S protein from a Wuhan strain, an Alpha variant, a Beta Variant, a Delta SARS ⁇ CoV ⁇ 2 variant, or a BA.1 Omicron variant or an immunogenic fragment thereof and (b) an S protein of a SARS ⁇ CoV ⁇ 2 Omicron variant that is not a BA.1 Omicron variant or an immunogenic fragment thereof.
  • an improved immune response can be produced by delivering, as polypeptides or RNAs encoding such polypeptides, (a) a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant, or an immunogenic fragment thereof and (b) an S protein of a BA.2 or a BA.4 or BA.5 SARS ⁇ CoV ⁇ 2 Omicron variant or an immunogenic fragment thereof.
  • the present disclosure also provides an insight that administration of multiple doses (e.g., at least 2, at least 3, at least 4, or more doses) of a coronavirus vaccine described herein (e.g., a bivalent vaccine described herein such as a bivalent BA.4/5 vaccine) may provide certain beneficial effect(s) on affinity of antibodies against one or more SARS ⁇ CoV ⁇ 2 strain or variants thereof.
  • a coronavirus vaccine described herein e.g., a bivalent vaccine described herein such as a bivalent BA.4/5 vaccine
  • such beneficial effect(s) on affinity of antibodies may be observed with respect to antibodies against certain Omicron variants.
  • such beneficial effect(s) on affinity of antibodies may be observed with respect to antibodies against certain Omicron variants that share at least one or more common epitopes, for example, with a Wuhan strain.
  • compositions that can produce an improved immune response (e.g., an immune response having broader cross ⁇ neutralization activity, stronger neutralization, and/or which is less susceptible to immune escape).
  • a composition described herein comprises two or more antigens or nucleic acids (e.g., RNA) that encodes such antigens that have few shared epitopes.
  • a composition described herein delivers, as polypeptides or nucleic acids encoding such polypeptides, a combination of SARS ⁇ CoV ⁇ 2 S proteins or immunogenic fragments thereof sharing no more than 50% (e.g., no more than 40%, no more than 30%, no more than 20% or more) of epitopes (including, e.g., amino acid mutations) that can be bound by neutralization antibodies.
  • a composition described herein comprises (a) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant or an immunogenic fragment thereof and (b) RNA encoding an S protein from an Omicron variant of SARS ⁇ CoV ⁇ 2 (e.g., in some embodiments an S protein from a BA.1, BA.2, or BA.4/5 Omicron variant) or an immunogenic fragment thereof.
  • a composition described herein comprises (a) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant or an immunogenic fragment thereof and (b) RNA encoding an S protein of an Omicron variant of SARS ⁇ CoV ⁇ 2 that is not a BA.1 Omicron variant or an immunogenic fragment thereof.
  • a composition described herein comprises (a) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant or a BA.1 Omicron variant or an immunogenic fragment thereof and (b) RNA encoding an S protein of a Omicron variant that is not a BA.1 Omicron variant or an immunogenic fragment thereof.
  • a composition described herein comprises (a) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain, an Alpha variant, a Beta variant or a Delta variant of SARS ⁇ CoV ⁇ 2 and (b) RNA encoding an S protein from a BA.2 or a BA.4 or BA.5 Omicron variant of SARS ⁇ CoV ⁇ 2 or an immunogenic fragment thereof.
  • a composition described herein comprises RNA encoding an S protein from a BA.2 Omicron variant of SARS ⁇ CoV ⁇ 2 or an immunogenic fragment thereof.
  • a composition comprises RNA encoding an S protein from a BA.4 or BA.5 Omicron variant of SARS ⁇ CoV ⁇ 2 or an immunogenic fragment thereof.
  • SARS ⁇ CoV ⁇ 2 is an RNA virus with four structural proteins. One of them, the spike protein is a surface protein which binds the angiotensin ⁇ converting enzyme 2 (ACE ⁇ 2) present on host cells. Therefore, the spike protein is considered a relevant antigen for vaccine development.
  • BNT162b2 (SEQ ID NO: 20) is an mRNA vaccine for prevention of COVID ⁇ 19 and demonstrated an efficacy of 95% or more at preventing COVID ⁇ 19.
  • the vaccine is made of a 5’capped mRNA encoding for the full ⁇ length SARS ⁇ CoV ⁇ 2 spike glycoprotein (S) encapsulated in lipid nanoparticles (LNPs).
  • S SARS ⁇ CoV ⁇ 2 spike glycoprotein
  • LNPs lipid nanoparticles
  • ALC ⁇ 0315 (4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate)
  • ALC ⁇ 0159 (2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide), 1,2 ⁇ Distearoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine (DSPC), cholesterol, potassium chloride, potassium dihydrogen phosphate, sodium chloride, disodium phosphate dihydrate, sucrose and water for injection.
  • a different buffer may be used in lieu of PBS.
  • the buffer is formulated in a Tris ⁇ buffered solution.
  • the formulation comprises ALC ⁇ 0315 (4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate), ALC ⁇ 0159 (2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide), DSPC (1,2 ⁇ distearoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride and water.
  • the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg/ml.
  • RNA in the pharmaceutical RNA preparation is diluted prior to administration (e.g., diluted to a concentration of about 0.05 mg/ml). In some embodiments, the administration volumes are between about 200 ⁇ l and about 300 ⁇ l. In some embodiments, the RNA in pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, and about 10% sucrose.
  • the concentration of RNA in a pharmaceutical RNA preparation is about 0.1 mg/ml, and is formulated in about 10 mM Tris buffer, about 10% sucrose and a dose of about 10 ⁇ g of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 ⁇ l of diluted pharmaceutical RNA preparation.
  • the concentration of RNA in a pharmaceutical RNA preparation is about 0.1 mg/ml, and is formulated in about 10 mM Tris buffer, about 10% sucrose and a dose of the RNA of about 10 ⁇ g is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 ⁇ l of diluted pharmaceutical RNA preparation.
  • an active substance consists of a single ⁇ stranded, 5' ⁇ capped codon ⁇ optimized mRNA that is translated into the spike antigen of SARS ⁇ CoV ⁇ 2.
  • an encoded spike antigen protein sequence contains two proline mutations, which stabilize an antigenically optimal pre ⁇ fusion confirmation (P2 S).
  • an RNA does not contain any uridines; e.g., instead of uridine the modified N1 ⁇ methylpseudouridine can be used in RNA synthesis.
  • mRNA disclosed herein can be translated into the SARS ⁇ CoV ⁇ 2 S protein in a host cell. The S protein can then be expressed on the cell surface where it can induce an adaptive immune response. The S protein can be identified as a target for neutralising antibodies against the virus and is considered a relevant vaccine component.
  • BNT162b2 can be administered intramuscularly (IM) in two 30 ⁇ g doses of the diluted vaccine solution given about 21 days apart (e.g., to adult vaccine na ⁇ ve subjects (i.e., subjects 12 years and older who have not previously been administered a SARS ⁇ CoV ⁇ 2 vaccine)).
  • IM intramuscularly
  • the recent emergence of novel circulating variants of SARS ⁇ CoV ⁇ 2 has raised significant concerns about geographic and temporal efficacy of vaccine interventions.
  • One of the earliest variants that emerged and rapidly became globally dominant was D614G.
  • the alpha variant also known as B.1.1.7, VOC202012/01, 501Y.V1 or GRY
  • the alpha variant has a large number of mutations, including several mutations in the S gene. It has been shown to be inherently more transmissible, with a growth rate that has been estimated to be 40 ⁇ 70% higher than other SARS ⁇ CoV ⁇ 2 lineages in multiple countries (Volz et al., 2021, Nature, https://doi.org/10.1038/s41586 ⁇ 021 ⁇ 03470 ⁇ x; Washington et al., 2021, Cell https://doi.org/10.1016/j.cell.2021.03.052).
  • the beta variant also known as B.1.351 or GH/501Y.V2 was first detected in South Africa. The beta variant carries several mutations in the S gene.
  • N501Y shared with alpha
  • E484K shared with K417N
  • the gamma variant also known as P.1 or GR/501Y.V3
  • the gamma variant carries several mutations that affect the spike protein, including two shared with beta (N501Y and E484K), as well as a different mutation at position 417 (K417T).
  • the delta variant also known as B.1.617.2 or G/478K.V1 was first documented in India.
  • the delta variant has several point mutations that affect the spike protein, including P681R (a mutation position shared with alpha and adjacent to the furin cleavage site), and L452R, which is in the RBD and has been linked with increased binding to ACE2 and neutralizing antibody resistance. There is also a deletion in the spike protein at position 156/157. These four VOCs have circulated globally and became dominant variants in the geographic regions where they were first identified. On 24 November 2021, the Omicron (B.1.1.529) variant was first reported to WHO from South Africa.
  • SARS ⁇ CoV ⁇ 2 Omicron and its sublineages have had a major impact on the 20 epidemiological landscape of the COVID ⁇ 19 pandemic since initial emergence in November 2021 (WHO Technical Advisory Group on SARS ⁇ CoV ⁇ 2 Virus Evolution (TAG ⁇ VE): Classification of Omicron (B.1.1.259): SARS ⁇ CoV ⁇ 2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to Omicron SARS ⁇ CoV ⁇ 2 variant: Technical brief and priority actions for Member States (2022)).
  • Omicron BA.2.12.1 subsequently displaced BA.2 to become dominant in the United States, whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia/ Pacific (H. Gruell et al., “SARS ⁇ CoV ⁇ 2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID ⁇ 19 country overview ⁇ Country overview report: Week 31 2022 (2022); J. Hadfield et al., “Nextstrain: Real ⁇ time tracking of pathogen evolution,” Bioinformatics 34, 4121–4123 (2016)).
  • Omicron XBB.1.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention.
  • Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, among which some are shared between all Omicron VOCs while others are specific to one or more Omicron sublineages.
  • BA.2.12.1 exhibits high similarity with BA.2 but not BA.1, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more so from BA.1, in line with their genealogy (A. Z.
  • BA.2 ⁇ descendant VOCs including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5 (BA.4 and BA.5 encode for the 30 same S sequence).
  • Most of these shared and VOC ⁇ specific alterations were shown to play an important role in immune escape from monoclonal antibodies and polyclonal sera raised against the wild ⁇ type S glycoprotein.
  • the BA.4/BA.5 ⁇ specific alterations are strongly implicated in immune escape of these VOCs (P. Wang et al., “Antibody resistance of SARS ⁇ CoV ⁇ 2 variants B.1.351 and B.1.1.7. Nature 593, 130–135 (2021); Q.
  • RNA i.e., vaccine RNA
  • an amino acid sequence i.e., a vaccine antigen
  • SARS ⁇ CoV ⁇ 2 S protein an immunogenic variant thereof
  • an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof i.e., an antigenic peptide or protein.
  • the vaccine antigen comprises an epitope of SARS ⁇ CoV ⁇ 2 S protein for inducing an immune response against coronavirus S protein, in particular SARS ⁇ CoV ⁇ 2 S protein, in the subject.
  • RNA encoding vaccine antigen is administered to provide (following expression of the polynucleotide by appropriate target cells) antigen for induction, i.e., stimulation, priming and/or expansion, of an immune response, e.g., antibodies and/or immune effector cells, which is targeted to target antigen (coronavirus S protein, in particular SARS ⁇ CoV ⁇ 2 S protein) or a procession product thereof.
  • the immune response which is to be induced according to the present disclosure is a B cell ⁇ mediated immune response, i.e., an antibody ⁇ mediated immune response. Additionally or alternatively, in one embodiment, the immune response which is to be induced according to the present disclosure is a T cell ⁇ mediated immune response. In one embodiment, the immune response is an anti ⁇ coronavirus, in particular anti ⁇ SARS ⁇ CoV ⁇ 2 immune response. Vaccines described herein comprise as an active principle single ⁇ stranded RNA that may be translated into protein upon entering cells of a recipient.
  • the RNA may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (e.g., 5' cap, 5' UTR, 3' UTR, poly(A) ⁇ tail, or combinations thereof). In one embodiment, the RNA contains all of these elements. In one embodiment, a cap1 structure may be utilized as specific capping structure at the 5’ ⁇ end of the RNA drug substance.
  • beta ⁇ S ⁇ ARCA(D1) (m 2 7,2' ⁇ O GppSpG) or m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG may be utilized as specific capping structure at the 5' ⁇ end of the RNA drug substances.
  • 5' ⁇ UTR sequence the 5' ⁇ UTR sequence of the human alpha ⁇ globin mRNA, optionally with an optimized ⁇ Kozak sequence ⁇ to increase transla ⁇ onal efficiency (e.g., SEQ ID NO: 12) may be used.
  • 3' ⁇ UTR sequence a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) (e.g., SEQ ID NO: 13) placed between the coding sequence and the poly(A) ⁇ tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used.
  • F amino terminal enhancer of split
  • I 12S ribosomal RNA
  • the 3‘ ⁇ UTR may be two re ⁇ iterated 3' ⁇ UTRs of the human beta ⁇ globin mRNA.
  • an RNA comprises a poly(A) ⁇ tail comprising a length of at least 90 adenosine nucleotides (including, e.g., at least about 100 adenosine nucleotides, at least about 110 adenosine nucleotides, at least about 120 adenosine nucleotides, at least about 130 adenosine nucleotides, or longer).
  • a poly(A) ⁇ tail may comprise a length of about 90 to about 150 adenosine nucleotides (e.g., about 100 to about 150 adenosine nucleotides).
  • a poly(A) ⁇ tail may comprise an interrupted poly(A) ⁇ tail.
  • a poly(A) ⁇ tail measuring about 90 to about 120 nucleotides in length (e.g., about 110 nucleotides in length), consisting of a stretch of about 30 adenosine residues (e.g., about 28, about 29, about 30, about 31, or about 32 adenosine residues), followed by a linker sequence of about 10 nucleotides (of random nucleotides, e.g., about 9, about 10, or about 11 random nucleotides) and another about 70 adenosine nucleotides (e.g., about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, or about 75 adenosine nucleotides) may be used (e.g., a poly(A) tail comprising SEQ ID NO: 14).
  • a nucleotide sequence encoding a secretory signal peptide may be fused to the antigen ⁇ encoding regions preferably in a way that the sec is translated as an N terminal tag.
  • sec corresponds to the secretory signal peptide of a SARS ⁇ CoV ⁇ 2 S protein.
  • sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins may be used as GS/Linkers to join a secretory signal and an antigenic polypeptide.
  • Vaccine RNA described herein may be complexed with proteins and/or lipids, preferably lipids, to generate RNA ⁇ particles for administration. If a combination of different RNAs is used, the RNAs may be complexed together or complexed separately with proteins and/or lipids to generate RNA ⁇ particles for administration.
  • the disclosure features a composition or medical preparation comprising an RNA encoding a SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein the SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises: (a) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P, A899P, and A942P; (2) K986P, V987P, F817P, A892P, A899P, and A942P; (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (4) K986P, V
  • the RNA comprises a modified nucleoside in place of uridine. In some embodiments, the RNA comprises modified uridines in place of all uridines. In some embodiments, the RNA comprises N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ) in place of all uridines. In some embodiments, the RNA comprises a 5’ cap. In some embodiments, the 5’ cap is or comprises m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG. In some embodiments, the RNA comprises a 5’ ⁇ UTR that is or comprises a modified human alpha ⁇ globin 5’ ⁇ UTR.
  • the RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 12.
  • the RNA comprises a 3’ ⁇ UTR that is or comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.
  • AES amino terminal enhancer of split
  • the RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 13.
  • the RNA comprises a poly ⁇ A sequence.
  • the poly ⁇ A sequence comprises at least 100 nucleotides.
  • the poly ⁇ A sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
  • the poly ⁇ A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 14.
  • the RNA is formulated or is to be formulated for intramuscular administration.
  • the RNA is formulated or is to be formulated as particles.
  • the particles are lipid nanoparticles (LNPs) or lipoplex (LPX) particles.
  • the LNPs comprise ((4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate), 2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide, 1,2 ⁇ Distearoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine, and cholesterol.
  • the lipoplex particles are obtainable by mixing the RNA with liposomes.
  • the RNA is mRNA or saRNA.
  • the composition or medical preparation is a pharmaceutical composition.
  • the composition or medical preparation is a vaccine.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the present disclosure provides a composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the
  • the present disclosure features a composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A8
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812
  • the disclosure provides a composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F8
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F8
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F8
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 9
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70: D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70: K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • composition or medical preparation comprising a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein: (a) the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70: D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
  • the present disclosure provides an insight that incorporation of a D985P mutation rather than a K986P mutation can improve protein express and/or immunogencity (e.g., improve neutralization response).
  • incorporation of D985P rather than K986P can provide such improvements when combined with one or more other proline mutations (e.g., one or more proline mutations disclosed herein).
  • incorporation of D985P rather than K986P can provide such improvements when combined with V987P (e.g., one or more proline mutations disclosed herein).
  • incorporation of D985P rather than K986P can provide such improvements when combined with one or more (e.g., all) of F817P, A892P, A899P, A942P, and V987P.
  • the present disclosure provides an insight that RNA encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more proline mutations (e.g., one or more of the proline mutations and/or combination of proline mutations disclosed herein) and a mutated furin cleavage site can provide an improved immune response (e.g., an improved immune repsonse as compared to a similar or same construct comprising an intact furin cleavage site).
  • the first RNA and the second RNA each comprise a modified nucleoside in place of uridine. In some embodiments, the first RNA and the second RNA each comprise modified uridines in place of all uridines. In some embodiments, the first RNA and the second RNA each comprise N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ) in place of all uridines. In some embodiments, the first RNA and the second RNA each comprise a 5’ cap. In some embodiments, the 5’ cap comprises m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG.
  • the first RNA and the second RNA each comprise a 5’ ⁇ UTR that is or comprises a modified human alpha ⁇ globin 5’ ⁇ UTR.
  • the first RNA and the second RNA each comprise a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 12.
  • the first RNA and the second RNA each comprise a 3’ ⁇ UTR that is or comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.
  • the first RNA and the second RNA each comprise a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 13.
  • the first RNA and the second RNA each comprise a poly ⁇ A sequence.
  • the first RNA and the second RNA each comprise a poly ⁇ A sequence that comprises at least 100 nucleotides. In some embodiments, the first RNA and the second RNA each comprise a poly ⁇ A sequence that comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
  • the first RNA and the second RNA each comprise a poly ⁇ A sequence that comprises or consists of the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 14.
  • the first RNA and the second RNA are each formulated or to be formulated for intramuscular administration.
  • the first RNA and the second RNA are each formulated or to be formulated as particles.
  • the first RNA and the second RNA are each to be formulated as lipid nanoparticles (LNPs) or lipoplex (LPX) particles.
  • the LNPs comprise ((4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate), 2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide, 1,2 ⁇ Distearoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine, and cholesterol.
  • the first RNA and the second RNA are formulated in separate LNPs.
  • the first RNA and the second RNA are formulated in the same LNP.
  • the lipoplex particles are obtainable by mixing the RNA with liposomes.
  • the first RNA and the second RNA are each mRNA. In some embodiments, the first RNA and the second RNA are each saRNA. In some embodiments, the composition or medical preparation is a pharmaceutical composition. In some embodiments, the composition or medical preparation is a vaccine. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. In another aspect, the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject a composition or medical preparation described herein thereby inducing an immune response in the subject.
  • the SARS ⁇ CoV ⁇ 2 S polypeptide comprises an amino acid sequence that does not comprise a D985P substitution relative to SEQ ID NO:1; does not comprise a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or does not comprise a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.
  • the method further comprises administering a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein the second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment is a SARS ⁇ CoV ⁇ 2 S polypeptide of an Omicron variant that is not a BA.1 Omicron variant.
  • the method further comprises administering a second, different RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, wherein the second SARC ⁇ CoV ⁇ 2 S polypeptide or fragment is selected from an SARS ⁇ CoV ⁇ 2 S polypeptide or fragment described herein.
  • Another aspect of the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject the composition or medical preparation described herein, thereby inducing an immune response in the subject.
  • the SARS ⁇ CoV ⁇ 2 S polypeptide comprises an amino acid sequence that does not comprise a D985P substitution relative to SEQ ID NO:1; does not comprise a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or does not comprise a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.
  • the method further comprises administering a second composition or medical preparation, wherein the second composition or medical preparation comprises an RNA encoding an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment of an Omicron variant that is not a BA.1 Omicron variant.
  • the method further comprises administering a second composition or medical preparation, wherein the second composition or medical preparation comprises a third RNA encoding a third SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a fourth RNA encoding a fourth SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof.
  • the third RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, and wherein the third RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA and/or that is different from the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the second RNA.
  • the fourth RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, and wherein the fourth RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA and/or that is different from the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the second RNA.
  • the third RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, and wherein the third RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA and that is different from the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the second RNA.
  • the fourth RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, and wherein the fourth RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA and that is different from the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the second RNA.
  • the third RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, wherein the third RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA and that is different from the second SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the second RNA, wherein the fourth RNA encodes an SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof that is a first or a second SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof recited in any one of claims 24 ⁇ 102, wherein the fourth RNA encodes a SARS ⁇ CoV ⁇ 2 S polypeptide or fragment that is different from the first SARS ⁇ CoV ⁇ 2 S polypeptide or fragment encoded by the first RNA
  • each of the first, second, third, and fourth RNAs encodes a different SARS ⁇ CoV ⁇ 2 S polypeptide or immunogenic fragment thereof.
  • a monovalent vaccine as described herein can be administered with a bivalent vaccine as described herein.
  • a method of inducing an immune response comprises administering to a subject (i) a composition or medical preparation described herein that comprises an RNA encoding a SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof described herein and (ii) a composition or medical preparation comprising at least a first RNA encoding a first SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof, and a second RNA encoding a second SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof as described herein.
  • the monovalent vaccine and the bivalent vaccine can be administered at least 3 weeks apart, including, e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, or longer.
  • the monovalent vaccine and the bivalent vaccine can be administered at least 3 months apart, including, e.g., at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, or longer.
  • the monovalent vaccine and the bivalent vaccine can be administered on different arms in a single session.
  • the monovalent vaccine and the bivalent vaccine can be administered as a trivalent vaccine in a single injection (e.g., mixing the monovalent and bivalent vaccines together prior to administration).
  • FIG. 1 Schematic overview of the S protein organization of the SARS ⁇ CoV ⁇ 2 S protein.
  • the sequence within the S1 subunit consists of the signal sequence (SS) and the receptor binding domain (RBD) which is the key subunit within the S protein which is relevant for binding to the human cellular receptor ACE2.
  • the S2 subunit contains the S2 protease cleavage site (S2’) followed by a fusion peptide (FP) for membrane fusion, heptad repeats (HR1 and HR2) with a central helix (CH) domain, the transmembrane domain (TM) and a cytoplasmic tail (CT).
  • S2 S2 protease cleavage site
  • FP fusion peptide
  • HR1 and HR2 heptad repeats
  • CH central helix
  • TM transmembrane domain
  • CT cytoplasmic tail
  • RNA vaccines Based on the full and wildtype S protein, we have designed different constructs encoding the (1) full protein with mutations in close distance to the first heptad repeat (HRP1) that include stabilizing mutations preserving neutralisation sensitive sites, the (2) S1 domain or the (3) RB domain (RBD) only. Furthermore, to stabilize the protein fragments a fibritin domain (F) was fused to the C ⁇ terminus. All constructs start with the signal peptide (SP) to ensure Golgi transport to the cell membrane.
  • SP signal peptide
  • RNA vaccines with 5' ⁇ cap, 5' ⁇ and 3' ⁇ untranslated regions, coding sequences with intrinsic secretory signal peptide as well as GS ⁇ linker, and poly(A) ⁇ tail. Please note that the individual elements are not drawn exactly true to scale compared to their respective sequence lengths.
  • UTR Untranslated region
  • sec Secretory signal peptide
  • RBD Receptor Binding Domain
  • GS Glycine ⁇ serine linker.
  • Figure 4 General structure of Certain RNA vaccines.
  • RNA drug substances with 5' ⁇ cap, 5' ⁇ and 3' ⁇ untranslated regions, coding sequences with intrinsic secretory signal peptide as well as GS ⁇ linker, and poly(A) ⁇ tail. Please note that the individual elements are not drawn exactly true to scale compared to their respective sequence lengths.
  • GS Glycine ⁇ serine linker
  • UTR Untranslated region
  • Sec Secretory signal peptide
  • RBD Receptor Binding Domain.
  • Figure 5 General structure of Certain RNA vaccines.
  • RNA vaccines with 5' ⁇ cap, 5' ⁇ and 3' ⁇ untranslated regions, coding sequences of the Venezuelan equine encephalitis virus (VEEV) RNA ⁇ dependent RNA polymerase replicase and the SARS ⁇ CoV ⁇ 2 antigen with intrinsic secretory signal peptide as well as GS ⁇ linker, and poly(A) ⁇ tail.
  • VEEV Venezuelan equine encephalitis virus
  • GS ⁇ linker Glycine ⁇ serine linker.
  • Anti ⁇ S protein IgG response 6, 14 and 21 d after immunization with LNP ⁇ C12 formulated modRNA coding for transmembrane ⁇ anchored RBD ⁇ based vaccine constructs BALB/c mice were immunized IM once with 4 ⁇ g of LNP ⁇ C12 ⁇ formulated transmembrane ⁇ anchored RBD ⁇ based vaccine constructs (surrogate to BNT162b3c/BNT162b3d).
  • animals were bled and the serum samples were analyzed for total amount of anti ⁇ S1 (left) and anti ⁇ RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA.
  • the values are derived from separate neutralization GMTs from the pseudovirus testing.
  • Also shown is a schematic depicting a process for developing new SARS ⁇ CoV ⁇ 2 variant specific vaccines. Fig. 12.
  • Fig. 14. Schematics of an exemplary vaccination regimen.
  • Serum neutralizing capacity was assessed using a pseudovirus and live virus neutralization test; SARS ⁇ CoV ⁇ 2 spike ⁇ specific B MEM cells were assessed via a flow cytometry ⁇ based B cell phenotyping assay using bulk PBMCs. N/A, not applicable. Fig. 16. Omicron BA.1 breakthrough infection in BNT162b2 double ⁇ and triple ⁇ vaccinated individuals induces broad neutralization of Omicron BA.1, BA.2 and other VOCs.
  • Serum was drawn from double ⁇ vaccinated individuals (BNT162b2 2 ) at 22 days after the second dose (open circles), from triple ⁇ vaccinated individuals (BNT162b2 3 ) at 28 days after the third dose (closed circles), from double ⁇ vaccinated individuals with an Omicron BA.1 breakthrough infection (BNT162b2 2 + Omi) at 46 days post ⁇ infection (open triangles), and from triple ⁇ vaccinated individuals and Omicron BA.1 breakthrough infection (BNT162b2 3 + Omi) at 44 days post ⁇ infection (closed triangles).
  • Serum was tested in duplicate; (A) shows 50% pseudovirus neutralization (pVN 50 ) geometric mean titers (GMTs), (B) shows 50% virus neutralization (VN 50 ) GMTs, and (C) shows the geometric mean ratio of SARS ⁇ CoV ⁇ 2 variant of concern (VOC) and Wuhan VN50 GMTs.
  • pVN 50 pseudovirus neutralization
  • VN 50 virus neutralization
  • VN 50 viral genome
  • C shows the geometric mean ratio of SARS ⁇ CoV ⁇ 2 variant of concern
  • VOC geometric mean ratio of SARS ⁇ CoV ⁇ 2 variant of concern
  • VOC geometric mean ratio of SARS ⁇ CoV ⁇ 2 variant of concern
  • VOC geometric mean ratio of SARS ⁇ CoV ⁇ 2 variant of concern
  • Wuhan VN50 GMTs For titer values below the limit of detection (LOD), LOD/2 values were plotted. Values above violin plots represent group GMTs. The non ⁇ parametric Friedman test with Dunn’s multiple comparisons correction was used to compare Wuhan neutralizing group GMTs
  • FIG.B shows representative flow plots of Omicron BA.1 and Wuhan S protein ⁇ and RBD ⁇ binding for each of the four groups of individuals investigated. Frequencies of B MEM binding Omicron BA.1, Wuhan, or both (shared) shown for full ⁇ length S protein in (C) and RBD shown in (D) for Omicron BA.1 ⁇ experienced and na ⁇ ve BNT162b2 double and triple vaccinees.
  • E Venn diagrams visualizing the combinatorial (Boolean) gating strategy to identify cross ⁇ reactive B MEM recognizing all four variants simultaneously (All 4 +ve) and B MEM recognizing only Omicron BA.1 (only Omi) or only Wuhan (only Wuhan) S proteins.
  • Serum was drawn from 10 individuals vaccinated with other approved COVID ⁇ 19 vaccines or mixed regimens at a median of 43 days after infection (grey diamonds). Serum was tested in duplicate; individual 50% pseudovirus neutralization (pVN 50 ) geometric mean titers (GMTs) against SARS ⁇ CoV ⁇ 2 Wuhan, Alpha, Beta, Delta and Omicron BA.1 and BA.2 variants, plus SARS ⁇ CoV ⁇ 1 were plotted.
  • pVN 50 pseudovirus neutralization
  • GTTs geometric mean titers
  • LOD/2 values were plotted. Values above violin plots represent group GMTs. The non ⁇ parametric Friedman test with Dunn’s multiple comparisons correction was used to compare Wuhan neutralizing group GMTs with titers against the indicated variants and SARS ⁇ CoV ⁇ 1. Multiplicity ⁇ adjusted p values are shown. Approved vaccines included AZD1222, BNT162b2 (in some embodiments as part of a 4 ⁇ dose series), Ad26.COV2.S, mRNA ⁇ 1273 (administered as a two ⁇ dose or three ⁇ dose series), and combinations thereof. Fig. 20.
  • b2 refers to sera from subjects administered Wuhan ⁇ specific RNA vaccine as the 4 th (booster) dose of BNT162b2.
  • OMI refers to sera from subjects administered an Omicron BA.1 ⁇ specific 4 th (booster) dose.
  • FFRNT refers to fluorescent focus reduction neutralization test. Neutralization data was obtained using an FFRNT assay, with a viral particle containing a SARS ⁇ CoV ⁇ 2 S protein from the variant indicated in the figures.
  • C Comparison of titers of neutralizing antibodies against a SARS ⁇ CoV ⁇ 2 pseudovirus comprising a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain. Sera from subjects previously or currently infected with SARS ⁇ CoV ⁇ 2 excluded.
  • D Comparison of titers of neutralizing antibodies against a SARS ⁇ CoV ⁇ 2 pseudovirus comprising a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, in sera from a population comprising individuals previously or currently infected with SARS ⁇ CoV ⁇ 2 (as determined by an antigen assay or a PCR assay, respectively.
  • mice were immunized twice with LNP ⁇ formulated vaccine comprising (i) BNT162b2 (encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain), (ii) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (Omi), (iii) RNA encoding an S protein having mutations characteristic of a delta variant, (iv) a combination of BNT162b2 and an RNA encoding an protein having mutations characteristic of an Omicron BA.1 variant (B2+Omi), or (v) RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a delta variant and RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (Delta + Omi).
  • BNT162b2 encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain
  • SARS ⁇ CoV ⁇ 2 ⁇ S pseudovirus comprising a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain, or a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a beta, delta, or Omicron BA.1 variant.
  • Graphs depict pVN 50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Mean + SEM is shown by horizontal bars with whiskers for each group. LLOD, lower limit of detection. ULOD, upper limit of detection. Fig. 22.
  • RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a Beta variant increases neutralization antibody titers against SARS ⁇ CoV ⁇ 2 when administered to patients previously administered two doses of a vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain.
  • Subjects previously administered two doses of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain were administered a third and a fourth dose of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a Beta variant.
  • Neutralization antibody titers were measured before administration of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain (D1 ⁇ PreVax), one month after administration of a second dose of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain (M1PD2), one ⁇ month after administration of a third dose of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a SARS ⁇ CoV ⁇ 2 Beta variant, and one month after administration of a fourth dose of an RNA vaccine encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a SARS ⁇ CoV ⁇ 2 Beta variant.
  • GMFR refers to the geometric mean fold rise, and is a measure of the increase in neutralization antibody titers since the previous vaccine dose (e.g., the GMFR for Post ⁇ Dose2 (PD2) is a measure of the increase in neutralization antibody titers relative to before administration of any vaccine (pre ⁇ vax)).
  • A Neutralization antibody titers measured in a viral neutralization assay that uses a viral particle comprising a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain.
  • B Neutralization antibody titers measured in a viral neutralization assay that uses a viral particle comprising a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of a Beta variant.
  • Fig. 23 50% neutralization titers of sera collected 7 days after a fourth dose of BNT162b2, an Omicron BA.1 ⁇ specific booster, or a bivalent vaccine.
  • Subjects who were previously administered two doses of BNT162b2 (30 ug), and a third (booster) dose of BNT162b2 (30 ug) received (i) a 30 ug dose of BNT162b2 (encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain), (ii) a 60 ug dose of BNT162b2, (iii) a 30 ug dose of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (e.g., as described herein (referred to herein as “Omicron ⁇ specific RNA vaccine“)), (iii) a 60 ug dose of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicro
  • GMR Geometric mean ratio
  • FFRNT fluorescent focus reduction neutralization test. Neutralization data was obtained using an FFRNT assay, with a viral particle containing a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of the variant indicated in the figures. LLOQ refers to Lower Limit of Quantification and ULOQ refers to Upper Limit of Quantification.
  • A Comparison of titers of neutralizing antibodies against a SARS ⁇ CoV ⁇ 2 pseudovirus comprising a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristics of an Omicron BA.1 variant.
  • G Geometric mean rise (GMR) of neutralization antibodies observed in subjects administered 60 ug of BNT162b2, 30 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (OMI 30 ug), 60 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (OMI 60 ug), 30 ug of a bivalent vaccine comprising 15 ug of BNT162b2 and 15 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (Bivalent 30 ug), or 60 ug of a bivalent vaccine comprising 30 ug of BNT162b2 and 30 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (Bivalent 60 ug), as compared to subjects administered
  • Results are shown both for a population pool that excludes subjects previously or currently infected with SARS ⁇ CoV ⁇ 2 and a population pool that includes these subjects.
  • Fig. 24 Reactogenicity of certain exemplary RNA (formulated in LNP) at a given dose: subjects administered a 60 ug dose of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein are more likely to exhibit a higher injection site pain and exhibit similar systemic reactions as subjects administered a 30 ug dose of RNA.
  • Subjects were administered 30 ug or 60 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain (BNT162b2, corresponding to groups G1 and G2, respectively), 30 ug or 60 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (BNT162b2 OMI, corresponding to groups G3 and G4, respectively), 30 ug of a bivalent vaccine comprising 15 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and 15 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein having mutations characteristic of an Omicron BA.1 variant (BNT162B2 (15 ug) + BNT162b2 OMI (15 ug), corresponding to group G5), or 60 ug of a bivalent vaccine comprising 30 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S
  • A Local reactions, including redness, swelling, and pain at the injection site, observed within 7 days of injection. Injection site pain was found to be increased in subjects administered 60 ug of RNA encoding a SARS ⁇ CoV ⁇ 2 S protein comprising mutations characteristic an Omicron BA.1 variant or a bivalent vaccine, as compared to other doses tested.
  • B Systemic reactions, including fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, and use of medication, observed within 7 days of injection. Systemic reactions through 7 days were observed to be broadly similar across different groups. Fatigue was found to trend higher after administration of 60 ug doses, as compared to 30 ug doses. Fig. 25.
  • This figure is an extension of Fig. 16, including data neutralizing activity against Omicron BA.4 and BA.5.
  • serum was tested in duplicate; 50% pseudovirus neutralization (pVN 50 ) geometric mean titers (GMTs) (in A and B), and the geometric mean ratio of SARS ⁇ CoV ⁇ 2 variants of concern (VOCs) and SARS ⁇ CoV ⁇ 1 pVN50 GMTs normalized against Wuhan pVN50 GMTs (in C) were plotted.
  • pVN 50 pseudovirus neutralization
  • GTTs geometric mean titers
  • VOCs geometric mean ratio of SARS ⁇ CoV ⁇ 2 variants of concern
  • SARS ⁇ CoV ⁇ 1 pVN50 GMTs normalized against Wuhan pVN50 GMTs
  • Serum neutralizing capacity was assessed using a pseudovirus neutralization test.
  • Fig. 29 50% pseudovirus neutralization (pVN50) geometric mean titers (GMTs) from the BNT162b2 3 and All Vax + Omi BA.1 breakthrough infection cohorts. Serum was drawn from Omicron ⁇ na ⁇ ve BNT162b2 triple ⁇ vaccinated individuals (BNT162b2 3 , circles) at 28 days after the third dose, and from vaccinated individuals with subsequent Omicron BA.1 breakthrough infection (all Vax + Omi BA.1, triangles) at a median 43 days post ⁇ infection.
  • pVN50 pseudovirus neutralization
  • GTTs geometric mean titers
  • Serum was drawn from BNT162b2 triple ⁇ vaccinated individuals with subsequent Omicron BA.1 breakthrough infection at a median 44 days post ⁇ infection (BNT162b2 3 + Omi BA.1, triangles), and from BNT162b2 triple ⁇ vaccinated individuals with subsequent Omicron BA.2 breakthrough infection at 38 days post ⁇ infection (BNT162b2 3 + Omi BA.2, squares).
  • 50% pseudovirus neutralization (pVN 50 ) geometric mean titers (GMTs) (in A, B), and the geometric mean ratio of SARS ⁇ CoV ⁇ 2 variants of concern (VOCs) and SARS ⁇ CoV ⁇ 1 pVN 50 GMTs normalized against Wuhan pVN50 GMTs (in C) were plotted.
  • Amino acid positions, amino acid descriptions (one letter code) and kind of mutations (substitutions, deletions, insertions) are indicated.
  • NTD N ⁇ terminal domain
  • RBD Receptor ⁇ binding domain, ⁇ , deletion; ins, insertion
  • * Cytoplasmic domain truncated for the C ⁇ terminal 19 amino acids.
  • Fig. 32 Alterations on the spike glycoprotein amino acid sequence of SARS ⁇ CoV ⁇ 2 Omicron sub ⁇ lineages. Amino acid positions, amino acid descriptions (one letter code) and kind of mutations substitutions, deletions, insertions) are indicated.
  • Fig. 33 Immunization protocol for studies with VOC boosters.
  • BALB/c mice were immunized according to the indicated schedule with two doses (1 ug each) of the original BNT162b2 vaccine, followed by at least one dose (1 ug total) of a monovalent, bivalent, or trivalent booster dose of either: (a) the original BNT162b2 (“BNT162b2”); (b) BNT162b2 OMI BA.1 (“OMI BA.1”); (c) BNT162b2 OMI BA.4/5 (“OMI BA.4/5”); or a combination thereof.
  • Fig. 34 Baseline grouped neutralizing GMTs. Sera drawn from mice immunized as depicted in Fig. 33 (day 104, pre ⁇ boost) were assessed for geometric mean titers of neutralizing antibodies against various strains. Data are presented grouped by cohort. Fig. 35. Baseline staggered neutralizing GMTs. Sera drawn from mice immunized as depicted in Fig. 33 (day 104, pre ⁇ boost) were assessed for geometric mean titers of neutralizing antibodies against various strains. Data are presented in staggered format (i.e., by strain against which neutralization was assessed). Fig. 36. Baseline cross ⁇ neutralization. Sera drawn from mice immunized as depicted in Fig.
  • Fig. 33 (day 104, pre ⁇ boost) were assessed for geometric mean titers of neutralizing antibodies against various strains. Cross ⁇ neutralization results are presented as calculated variant/Wuhan reference GMT Ratios.
  • Fig. 37 Post ⁇ boost geometric mean fold increase in GMTs. Sera drawn from mice immunized as depicted in Fig. 33 (day 111, 7 ⁇ days post ⁇ boost) were assessed for geometric mean fold increase in GMT of neutralizing antibodies against various strains.
  • Fig. 38 Post ⁇ boost grouped neutralizing GMTs. Sera drawn from mice immunized as depicted in Fig. 33 (day 111, 7 ⁇ days post ⁇ boost) were assessed for geometric mean fold increase in GMT of neutralizing antibodies against various strains.
  • Fig. 39 Post ⁇ boost cross ⁇ neutralization. Sera drawn from mice immunized as depicted in Fig. 33 (day 111, 7 ⁇ days post ⁇ boost) were assessed for geometric mean fold increase in GMT of neutralizing antibodies against various strains. Cross ⁇ neutralization results are presented as calculated variant/Wuhan reference GMT Ratios.
  • Fig. 40 Exemplary spike protein amino acid mutations. Amino acid residues that are modified are shown, and used to produce RNA vaccines encoding variant coronavirus spike proteins. In some instances, such amino acid modifications can be combined with other amino acid residue modifications, such as as shown in Fig. 41 under columns “Mutations” and “Mutation Types”.
  • the amino acid positions are numbered relative to the S protein sequence from a Wuhan sequence (SEQ ID NO: 1).
  • various combinations of amino acid mutations as described herein can be applied to different coronvavirus S protein or immunogenic fragments thereof.
  • Fig. 41 Exemplary Spike Protein Variants. Exemplary combinations of spike protein mutations are shown, including the amino acid residues that are modified, type of mutation, and furin mutations (from 682/683/684/685 RRAR to GSAS).
  • RNA constructs encoding exemplary combinations of spike protein mutations were evaluated for S protein expression, CR3022 epitope response, and ACE2 response.
  • the amino acid positions are numbered relative to the S protein sequence from a Wuhan sequence (SEQ ID NO: 1).
  • RNAs encoding exemplary spike protein variants can be applied to different coronvavirus S protein or immunogenic fragments thereof.
  • Fig. 42 Effect of RNA encoding exemplary spike protein variants on neutralization against various coronavirvus strains and/or variants.
  • RNAs encoding exemplary spike protein variants e.g., containing a P6’ backbone as shown in Fig. 40, D614G, and furin site mutations (from 682/683/684/685 RRAR to GSAS) stimulated higher neutralization titers across various VOCs.
  • Fig. 43 Effect of RNA encoding exemplary spike protein variants on neutralization against various coronavirvus strains and/or variants.
  • RNAs encoding exemplary spike protein variants e.g., containing a P6’ backbone as shown in Fig. 40, D614G, and furin site mutations (from 682/683/684/685 RRAR to GSAS) stimulate
  • BNT162b5 ⁇ format Bivalent (Wuhan + BA.4/5) is more immunogenic than BNT162b2 ⁇ format Bivalent (Wuhan + BA.4/5).
  • Mice were administered two doses of BNT162b2 21 days apart, followed by a third dose comprising (i) BNT162b2, (ii) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant, where the Spike protein encoded by each of the first and the second RNA also comprise K986P and V987P mutations (“BNT162b2 Bivalent (BA.4/5)”), or (iii) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5
  • BNT162b2 encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain, and comprising K986P and V987P mutations
  • a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.1 Omicron variant, where the Spike protein encoded by each of the first and the second RNA also comprise K986P and V987P mutations (“BNT162b2 Bivalent Omi BA.1”), or
  • a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.2 Omicron variant, where the S protein encoded by each of the first and the second RNA also comprise P6’ mutations (D985P, V98
  • Sera were collected one month after administering an RNA vaccine, and neutralization titers were collected for Wuhan (“WT”), Omicron BA.1 (“BA.1”), or Omicron BA.2 (“BA.2”) SARS ⁇ COV ⁇ 2 variants. Titers are shown for (A) all subjects, (B) subjects who showed evidence of prior SARS ⁇ CoV ⁇ 2 infection at the time a SARS ⁇ CoV ⁇ 2 vaccine was administered, and (C) subjects who showed no evidence of prior SARS ⁇ CoV ⁇ 2 infection at the time of administering a SARS ⁇ CoV ⁇ 2 vaccine. Titer values are shown above each bar. Titers were collected using a Fluroscent Focus Reduction Neutralization Titer (FFRNT) assay.
  • FFRNT Fluroscent Focus Reduction Neutralization Titer
  • mice administered two doses of BNT162b2 (encoding a SARS ⁇ CoV ⁇ 2 S protein of a Wuhan strain, and comprising K986P and V987P mutations) were administered (i) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant, where the Spike protein encoded by each of the first and the second RNA also comprise K986P and V987P mutations (“BNT162b2 Bivalent BA.4/5”), (ii) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded by each of the first and the second RNA also comprise P6’ mutations (D985P, V987
  • Fig. 46 In vitro characterization of Exemplary SARS ⁇ CoV ⁇ 2 Variants. Shown is protein expression, ACE2 binding, and CR3022 binding (a neutralizing antibody) for certain exemplary SARS ⁇ CoV ⁇ 2 variants comprising mutations described herein (mutations present in each SARS ⁇ CoV ⁇ 2 variant listed in Table 34). Fig. 47. In vitro characterization of Exemplary SARS ⁇ CoV ⁇ 2 Variants.
  • Fig. 45 Bivalent BNT162b6, and BNT162b7 provide an improved immune response when administered as a booster to vaccine ⁇ naive mice.
  • mice were administered (i) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant, where the Spike protein encoded by each of the first and the second RNA also comprise K986P and V987P mutations (“BNT162b2 Bivalent BA.4/5”), (ii) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS ⁇ CoV ⁇ 2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded by each of the first and the second RNA also comprise P6’ mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and mutations at the furin cleavage site (682/683/684/6
  • peptide comprises oligo ⁇ and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds.
  • a “therapeutic protein” has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount.
  • a therapeutic protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder.
  • a therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition.
  • therapeutic protein includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.
  • “Fragment”, with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N ⁇ terminus and/or C ⁇ terminus. A fragment shortened at the C ⁇ terminus (N ⁇ terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3' ⁇ end of the open reading frame.
  • a fragment shortened at the N ⁇ terminus is obtainable e.g. by translation of a truncated open reading frame that lacks the 5' ⁇ end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation.
  • a fragment of an amino acid sequence comprises e.g. at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.
  • a fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
  • variant herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification.
  • the parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence.
  • the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.
  • wild type or WT or “native” herein is meant an amino acid sequence that is found in nature, including allelic variations.
  • a wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.
  • variants of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • variant includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring.
  • variant includes, in particular, fragments of an amino acid sequence.
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence.
  • amino acid sequence variants having an insertion one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.
  • Amino acid addition variants comprise amino ⁇ and/or carboxy ⁇ terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein.
  • Amino acid deletion variants that comprise the deletion at the N ⁇ terminal and/or C ⁇ terminal end of the protein are also called N ⁇ terminal and/or C ⁇ terminal truncation variants.
  • Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties.
  • amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non ⁇ polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence.
  • the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids.
  • the degree of similarity or identity is given for the entire length of the reference amino acid sequence.
  • the alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity indicates the percentage of amino acids that are identical between the sequences.
  • Sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • the terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared.
  • Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences.
  • the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci.
  • NCBI National Center for Biotechnology Information
  • the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, ⁇ 2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used.
  • the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides.
  • the degree of similarity or identity is given for the entire length of the reference sequence.
  • Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.
  • the amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
  • a fragment or variant of an amino acid sequence is preferably a "functional fragment” or “functional variant".
  • the term "functional fragment” or “functional variant” of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent.
  • one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived.
  • the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence.
  • the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence.
  • immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
  • An amino acid sequence (peptide, protein or polypeptide) "derived from” a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence.
  • the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • an "instructional material” or “instructions” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure.
  • the instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions of the present disclosure or be shipped together with a container which contains the compositions.
  • the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated”, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated”.
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non ⁇ native environment such as, for example, a host cell.
  • the term "recombinant" in the context of the present disclosure means "made through genetic engineering".
  • a "recombinant object” such as a recombinant nucleic acid in the context of the present disclosure is not occurring naturally.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • Physiological pH refers to a pH of about 7.5.
  • the term “genetic modification” or simply “modification” includes the transfection of cells with nucleic acid.
  • transfection relates to the introduction of nucleic acids, in particular RNA, into a cell.
  • the term "transfection” also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient.
  • a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or an organism of a patient.
  • transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed.
  • RNA can be transfected into cells to transiently express its coded protein.
  • nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus ⁇ based systems or transposon ⁇ based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.
  • Coronavirus Coronaviruses are enveloped, positive ⁇ sense, single ⁇ stranded RNA ((+) ssRNA) viruses. They have the largest genomes (26–32 kb) among known RNA viruses and are phylogenetically divided into four genera ( ⁇ , ⁇ , ⁇ , and ⁇ ), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory diseases, although severity can be greater in infants, the elderly, and the immunocompromised.
  • SARS ⁇ CoV ⁇ 2 severe acute respiratory syndrome coronavirus ⁇ 2
  • SARS ⁇ CoV ⁇ 2 SARS ⁇ CoV ⁇ 2
  • SARS ⁇ CoV ⁇ 2 MN908947.3 belongs to betacoronavirus lineage B. It has at least 70% sequence similarity to SARS ⁇ CoV.
  • coronaviruses have four structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N), and spike (S).
  • E and M proteins have important functions in the viral assembly, and the N protein is necessary for viral RNA synthesis.
  • the critical glycoprotein S is responsible for virus binding and entry into target cells.
  • the S protein is synthesized as a single ⁇ chain inactive precursor that is cleaved by furin ⁇ like host proteases in the producing cell into two noncovalently associated subunits, S1 and S2.
  • the S1 subunit contains the receptor ⁇ binding domain (RBD), which recognizes the host ⁇ cell receptor.
  • the S2 subunit contains the fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate fusion of the viral and host ⁇ cell membranes by undergoing a large conformational rearrangement.
  • the S1 and S2 subunits trimerize to form a large prefusion spike.
  • the S precursor protein of SARS ⁇ CoV ⁇ 2 can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits.
  • the S1 subunit comprises the receptor ⁇ binding domain (RBD), which mediates virus entry into sensitive cells through the host angiotensin ⁇ converting enzyme 2 (ACE2) receptor.
  • RBD receptor ⁇ binding domain
  • RNA encoding an amino acid sequence comprising SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof.
  • the RNA encodes a peptide or protein comprising at least an epitope SARS ⁇ CoV ⁇ 2 S protein or an immunogenic variant thereof for inducing an immune response against coronavirus S protein, in particular SARS ⁇ CoV ⁇ 2 S protein in a subject.
  • amino acid sequence comprising SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof is also designated herein as "vaccine antigen”, “peptide and protein antigen", "antigen molecule” or simply "antigen”.
  • the SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof is also designated herein as "antigenic peptide or protein" or "antigenic sequence”.
  • SARS ⁇ CoV ⁇ 2 coronavirus full length spike (S) protein from the first detected SARS ⁇ CoV ⁇ 2 strain (referred to as the Wuhan strain herein) consists of 1273 amino acids and has the amino acid sequence according to SEQ ID NO: 1:
  • the above sequence is considered the wildtype or Wuhan SARS ⁇ CoV ⁇ 2 S protein amino acid sequence.
  • position numberings in a SARS ⁇ CoV ⁇ 2 S protein given herein are in relation to the amino acid sequence according to SEQ ID NO: 1.
  • One of skill in the art reading the present disclosure can determine the locations of the corresponding positions in SARS ⁇ CoV ⁇ 2 S protein variants.
  • Table 1 includes additional exemplary S proteins from various coronavirus variants, including the alpha, beta, gamma, delta, and omicron variants (including omicron BA.1, BA.2 and BA.4/5).
  • Omicron variant refers to any Omicron variant, including e.g., Omicron variants described herein and descendents thereof. Amino acid sequences were obtained from the UniProt database, accessible via the World Wide Web at uniprot.org, or the GenBank database, accessible via the World Wide Web at ncbi.nlm.nih.gov, and the UniProt or GenBank database accession numbers of each spike protein sequence are included in the Table 1.
  • amino acid sequences correspond to the amino acid sequences of native coronavirus spike proteins.
  • the amino acid sequences of native coronavirus spike proteins encoded by RNA constructs described herein may be modified, as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.
  • the amino acid sequences of native coronavirus spike proteins encoded by RNA constructs described herein are substituted, as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.
  • the amino acid sequences of spike proteins e.g., including the alpha, beta, gamma, delta, and omicron variants (including omicron BA.1, BA.2, BA.4/5) of these SARS ⁇ CoV ⁇ 2 variants encoded by RNA constructs described herein may be modified at the corresponding position, as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of the native variant coronavirus spike proteins or fragments thereof.
  • spike proteins e.g., including the alpha, beta, gamma, delta, and omicron variants (including omicron BA.1, BA.2, BA.4/5) of these SARS ⁇ CoV ⁇ 2 variants encoded by RNA constructs described herein may be modified at the corresponding position, as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of the native variant coronavirus spike proteins or fragments thereof.
  • amino acid sequences of spike proteins of these SARS ⁇ CoV ⁇ 2 variants encoded by RNA constructs described herein are substituted, as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of variant coronavirus spike proteins or fragments thereof. Additional variants not specifically set forth below are also contemplated.
  • any variant coronavirus spike protein having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the native coronavirus spike protein sequence encoded by RNA constructs described herein may be modified at the corresponding position, (e.g., substituted), as described herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are modifications of native coronavirus spike proteins or fragments thereof.
  • Table 1
  • a coronavirus spike protein sequence comprises SEQ ID NO: 105, shown below, which is the SARS ⁇ CoV ⁇ 2 (Omicron BA.4/5) sequence represented by SEQ ID NO: 104 (see Table 1), but differs by one amino acid at position 403 and comprises a R403S mutation.
  • Coronavirus Spike Protein Modifications In specific embodiments, full length spike (S) protein (e.g., the full length S protein of SEQ ID NO: 1) is modified in such a way that the prototypical prefusion conformation is stabilized.
  • a SARS ⁇ CoV ⁇ 2 S protein may be stabilized by introducing one or more glycine mutations (e.g., one or more glycine mutations in the crown of the helix turn region in the S protein, in the 12 amino acids between the heptad region 1 (HR1) and central helix (CH) or heptad regoin 2 (HR2) regions of the S2 subunit, and/or at one or more of L984, D985, K986, and V987 of (positions relative to SEQ ID NO: 1)).
  • one or more glycine mutations e.g., one or more glycine mutations in the crown of the helix turn region in the S protein, in the 12 amino acids between the heptad region 1 (HR1) and central helix (CH) or heptad regoin 2 (HR2) regions of the S2 subunit, and/or at one or more of L984, D985, K986, and V987 of (positions relative to SEQ ID
  • a Spike protein comprises glycine mutations at each of L984, D985, K986, and V987 (i.e., at positions corresponding to these residues in SEQ ID NO: 1).
  • a SARS ⁇ CoV ⁇ 2 S protein may be stabilized by introducing one or more proline mutations.
  • a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at residues 986 and/or 987 of SEQ ID NO: 1.
  • a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at one or more of residues 817, 892, 899, and 942 of SEQ ID NO: 1.
  • a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at each of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO: 1. In some embodiments, a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at residues 985 and/or 987 of SEQ ID NO: 1. In some embodiments, a SARS ⁇ CoV ⁇ 2 S protein comprises a proline substitution at each of residues 817, 892, 899, 942, 985, and 987 of SEQ ID NO: 1.
  • stabilization of the prefusion conformation may be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 in the full length spike protein.
  • spike (S) protein stabilized protein variants are obtained in a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline.
  • a SARS ⁇ CoV ⁇ 2 S protein variant wherein the prototypical prefusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 7:
  • a Spike protein can be modified in such a way as to block a pre ⁇ fusion to post ⁇ fusion conformational change (referred to herein as a “pre ⁇ post fusion block”).
  • a pre ⁇ post fusion block can be introduced by introducing two cysteine mutations at residues close to one another in the folded protein (e.g., at locations close to one another in a pre ⁇ fusion conformation of the Spike protein).
  • pre ⁇ post fusion block mutations include L984C ⁇ A989C and I980C ⁇ Q992C.
  • a Spike protein can be modified so as to decrease “shedding” (i.e., decrease separation of S1 and S2 subunits).
  • a Spike protein can be modified to decrease shedding by introducing mutations at the furin cleavage site, such that a furin protease can no longer bind and/or cleave the S protein (e.g., one or more mutations at residues 682 ⁇ 685 of SEQ ID NO: 1).
  • an S protein can be modified to reduce shedding by introducing mutations at each of residues 682, 683, and 685 (e.g., introducing mutations (i) R682G, R683S, and R685S, or (ii) R682Q, R683Q, and R685Q).
  • an S protein can be modified so as to reduce shedding by introducing cysteine mutations that can form a disulfide bond (e.g., by introducing cysteine mutations at positions that are close to one another in a folded conformation of an S protein, e.g., at residues A570 and N960).
  • one or more modifications may be introduced into a Spike protein so as to stabilize an “up” confirmation (referred to herein as “RBD Up” mutations).
  • RBD Up up confirmation of the SARS ⁇ CoV ⁇ 2 Spike protein is thought to increase exposure of neutralization sensitive residues.
  • mutations that stabilize the up conformation can produce a vaccine that is more immunogenic.
  • Table 2 lists various combinations of amino acid modifications that can be introduced into coronavirus spike protein sequences disclosed above and thus polynucleotides (e.g., RNAs) encoding immunogenic polypeptides comprising coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof can be produced.
  • a “+” symbol indicates the inclusion of the specified modification in a particular S protein sequence from a coronvirus strain or variant (e.g., SARS ⁇ CoV ⁇ 2 strains and/or variants as described in Table 1).
  • a spike protein sequence may contain any combination of the modifications in the following Table 2.
  • the amino acid positions indicated in Table 2 are numbered relative to SEQ ID.
  • SEQ ID NO: 1 (Wuhan), SEQ ID NO: 69 (Omicron BA.1), SEQ ID NO: 70 (Omicron BA.2), and SEQ ID NO: 104 (Omicron BA.4/5).
  • the amino acid positions corresponding to spike protein sequences from other coronavirus variants can determined through an alignment with SEQ ID NO: 1 (see e.g., Table 5).
  • Table 2 The amino acid positions corresponding to spike protein sequences from other coronavirus variants (e.g., alpha, beta, or delta variant) can determined through an alignment with SEQ ID NO: 1 (see e.g., Table 5).
  • Table 3 lists various combinations of amino acid modifications that can be introduced into coronavirus spike protein sequences disclosed above and thus polynucleotides (e.g., RNAs) encoding immunogenic polypeptides comprising coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof can be produced.
  • Table 3 like Table 2, lists the position of amino acid modifications (with respect to the Wuhan spike protein sequence according to SEQ ID NO: 1), and Table 3 also include the specific amino acid residue that is substituted for the native amino acid residue.
  • a “+” symbol indicates the inclusion of the specified modification in a particular S protein sequence from a coronavirus strain or variant (e.g., SARS ⁇ CoV ⁇ 2 strains and/or variants as described in Table 1).
  • a coronavirus spike protein variant encoded by an RNA vaccine may contain any combination of the modifications in Table 2 above, and for example, may include any of the specific substitutions shown in Table 3.
  • the amino acid positions indicated in Table 2 are numbered relative to SEQ ID. NO: 1 (Wuhan), SEQ ID NO: 69 (Omicron BA.1), SEQ ID NO: 70 (Omicron BA.2), and SEQ ID NO: 104 (Omicron BA.4/5).
  • the amino acid positions corresponding to spike protein sequences from other coronavirus variants can determined through an alignment with SEQ ID NO: 1 (see e.g., Table 5).
  • Table 3 The following Table 4 lists various combinations of amino acid modifications that can be introduced into coronavirus spike protein sequences disclosed herein (see e.g., Table 1) and thus polynucleotides (e.g., RNAs) encoding immunogenic polypeptides comprising coronavirus spike proteins that are variants of native coronavirus spike proteins or fragments thereof can be produced.
  • a “+” symbol indicates the inclusion of the specified modification in a particular S protein sequence from a coronavirus strain or variant (e.g., SARS ⁇ CoV ⁇ 2 strains and/or variants as described in Table 1).
  • a spike protein seqeunce may contain any combination of the modifications in the following Table 4.
  • the amino acid positions indicated in Table 4 are numbered relative to SEQ ID.
  • the amino acid corresponding to the amino acid at position 326 in SEQ ID. NO: 69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 326 in SEQ ID NO:69 can be substituted with a serine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a serine residue at 326 may be referred to herein as 326S.
  • SARS ⁇ CoV ⁇ 2 Omicron UFO69279.1 (BA.1, previously B.1.1.529) can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 364 in SEQ ID NO:69 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a phenylalanine residue at 364 may be referred to herein as 364F.
  • the amino acid corresponding to the amino acid at position 567 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 567 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 567 may be referred to herein as 567C.
  • the amino acid corresponding to the amino acid at position 611 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 611 in SEQ ID NO:69 can be substituted with a glycine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a glycine residue at 611 may be referred to herein as 611G.
  • the amino acid corresponding to the amino acid at position 814 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 814 in SEQ ID NO:69 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a phenylalanine residue at 814 may be referred to herein as 814P.
  • the amino acid corresponding to the amino acid at position 840 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 840 in SEQ ID NO:69 can be substituted with an asparagine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a asparagine residue at 840 may be referred to herein as 840N.
  • the amino acid corresponding to the amino acid at position 851 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 851 in SEQ ID NO:69 can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a phenylalanine residue at 851 may be referred to herein as 851F.
  • the amino acid corresponding to the amino acid at position 889 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 889 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 889 may be referred to herein as 889P.
  • the amino acid corresponding to the amino acid at position 896 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 896 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 896 may be referred to herein as 896P.
  • the amino acid corresponding to the amino acid at position 939 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 939 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 939 may be referred to herein as 939P.
  • the amino acid corresponding to the amino acid at position 957 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 957 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 957 may be referred to herein as 957C.
  • the amino acid corresponding to the amino acid at position 977 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 977 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 977 may be referred to herein as 977C.
  • the amino acid corresponding to the amino acid at position 981 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 981 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 981 may be referred to herein as 981C.
  • the amino acid corresponding to the amino acid at position 982 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 982 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 982 may be referred to herein as 982P.
  • the amino acid corresponding to the amino acid at position 983 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 983 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 983 may be referred to herein as 983P.
  • the amino acid corresponding to the amino acid at position 984 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 984 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a proline residue at 983 may be referred to herein as 984P.
  • the amino acid corresponding to the amino acid at position 986 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 986 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 986 may be referred to herein as 986C.
  • the amino acid corresponding to the amino acid at position 989 in SEQ ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • the amino acid corresponding to the amino acid at position 989 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant coronavirus spike protein encoded by RNA as described herein.
  • a substitution with a cysteine residue at 989 may be referred to herein as 989C.
  • a variant spike protein encoded by RNA described herein has, at least, or has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, and/or 18 of the following modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982, 983, 984, 986, 989 as set forth in SARS ⁇ CoV ⁇ 2 Omicron (BA.1, previously B.1.1.529) spike protein, UniProt Accession Number UFO69279.1, or the corresponding amino acid in the spike protein of another coronavirus, wherein in some embodiments the modification at the position or corresponding position 326 is a serine, 364 is a phenylalanine, 567 is a cysteine, 611 is a glycine, 814 is a proline, 840 is a asparagine, 851 is a phenylalanine, 889 is a proline,
  • RNA e.g., as described herein
  • an immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof.
  • these modifications may (a) increase adoption by RBDs of the variant coronavirus spike proteins of the RBD ⁇ up conformation to expose more neutralization ⁇ sensitive epitopes on the spike protein, (b) decrease adoption by RBDs of the variant coronavirus spike proteins of the RBD ⁇ down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of a S1 subunit of the variant coronavirus spike protein, and/or (f) improve localization of the variant coronavirus spike protein to a host cell membrane.
  • Mutations described herein and e.g., in Tables 2A, 2B, and 2C may be introduced into S protein sequence of other coronavirus strains or variant sequences, or immunogenic fragments thereof, and the corresponding position may be determined through a sequence alignment with SEQ ID NO: 69 (see e.g., Table 5).
  • a variant spike protein encoded by RNA described herein has at least, or has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, and/or 18 of the following modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982, 983, 984, 986, 989 as set forth in SARS ⁇ CoV ⁇ 2 Omicron (BA.1, previously B.1.1.529) spike protein, UniProt Accession Number UFO69279.1, or the corresponding amino acid in the spike protein of another coronavirus, wherein in some embodiments the modification at the position or corresponding position 326 is to any amino acid except phenylalanine, 364 is any amino acid except valine, 567 is any amino acid except alanine, 611 is any amino acid except glycine, 814 is any amino acid except phenylalanine, 840 is any amino acid except aspartic acid, 851
  • the modifications described herein may be applied alone or in combination with any one or more additional modifications described herein to produce an RNA encoding isolated immunogenic polypeptide comprising a variant coronavirus spike protein that is a variant of a native coronavirus spike protein or fragment thereof.
  • these modifications may (a) increase adoption by RBDs of the variant coronavirus spike proteins of the RBD ⁇ up conformation to expose more neutralization ⁇ sensitive epitopes on the spike protein, (b) decrease adoption by RBDs of the variant coronavirus spike proteins of the RBD ⁇ down conformation, (c) increase expression of the variant coronavirus spike protein compared to the native coronavirus spike protein, (d) increase adoption of a prefusion conformation, (e) decrease shedding of a S1 subunit of the variant coronavirus spike protein, and/or (f) improve localization of the variant coronavirus spike protein to a host cell membrane.
  • amino acids in each human coronavirus spike protein sequence and the corresponding position of that amino acid with respect to SEQ ID NO:1 can be determined based an alignment of the protein sequences.
  • Table 5 is an alignment of human coronavirus spike protein sequences (e.g., the spike protein sequences of Table 1). The highlighted positions in the below alignment correspond to the location of the amino acids to be modified identified in the Table 2 above.
  • cortavivirus Variants Those skilled in the art are aware of various spike variants, and/or resources that document them. For example, the following strains, their SARS ⁇ CoV ⁇ 2 S protein amino acid sequences and, in particular, modifications thereof compared to wildtype SARS ⁇ CoV ⁇ 2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.
  • B.1.1.7 "Variant of Concern 202012/01" (VOC ⁇ 202012/01)
  • B.1.1.7 is a variant of SARS ⁇ CoV ⁇ 2 which was first detected in October 2020 during the COVID ⁇ 19 pandemic in the United Kingdom from a sample taken the previous month, and it quickly began to spread by mid ⁇ December.
  • the B.1.1.7 variant is defined by 23 mutations: 13 non ⁇ synonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17 mutations that change proteins and six that do not).
  • the spike protein changes in B.1.1.7 include deletion 69 ⁇ 70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.
  • B.1.351 (501.V2) B.1.351 lineage and colloquially known as South African COVID ⁇ 19 variant, is a variant of SARS ⁇ CoV ⁇ 2. Preliminary results indicate that this variant may have an increased transmissibility.
  • the B.1.351 variant is defined by multiple spike protein changes including: L18F, D80A, D215G, deletion 242 ⁇ 244, R246I, K417N, E484K, N501Y, D614G and A701V. There are three mutations of particular interest in the spike region of the B.1.351 genome: K417N, E484K, N501Y.
  • B.1.1.298 (Cluster 5) B.1.1.298 was discovered in North Jutland, Denmark, and is believed to have been spread from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been confirmed. The specific mutations include deletion 69–70, Y453F, D614G, I692V, M1229I, and optionally S1147L. P.1 (B.1.1.248) Lineage B.1.1.248, known as the Brazil(ian) variant, is one of the variants of SARS ⁇ CoV ⁇ 2 which has been named P.1 lineage.
  • P.1 has a number of S ⁇ protein modifications [L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F] and is similar in certain key RBD positions (K417, E484, N501) to variant B.1.351 from South Africa.
  • B.1.427/B.1.429 has listed B.1.427/B.1.429 as "variant of concern”.
  • B.1.525 B.1.525 carries the same E484K modification as found in the P.1, and B.1.351 variants, and also carries the same ⁇ H69/ ⁇ V70 deletion as found in B.1.1.7, and B.1.1.298. It also carries the modifications D614G, Q677H and F888L.
  • B.1.526 B.1.526 was detected as an emerging lineage of viral isolates in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.
  • the following table shows an overview of circulating SARS ⁇ CoV ⁇ 2 strains which are VOI/VOC.
  • Omicron 7 B.1.1.529 B.1.529
  • BA.4 and BA.5 variants have the same S protein amino acid sequence, in which case the term “BA.4/5” may be used to refer to an amino acid sequence of an S protein that can be found in either of BA.4 or BA.5.
  • BA.4.6 and BF.7 variants have the same protein amino acid sequence, in which case the term “BA.4.6/BF.7” can be used to refer to an amino acid sequence of an S protein present in either of BA.4.6 or BF.7.
  • Table 3A Omicron Variants of Concern and Characteristic mutations
  • BA.5 comprising one of more of the following mutations in the S protein (positions shown relative to SEQ ID NO: 1): E340X (e.g., E340K), R346X (e.g., R346T, R346I, or R346S), K444X (e.g., K444N or K444T), V445X, 5 N450D, and S:N460X (e.g., N460K).
  • E340X e.g., E340K
  • R346X e.g., R346T, R346I, or R346S
  • K444X e.g., K444N or K444T
  • V445X 5 N450D
  • S:N460X e.g., N460K
  • RNA described herein comprises a nucleotide sequence encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 5 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) characteristic of an Omicron variant (e.g., one or more mutations of an Omicron variant listed in Table 3A) and one or more mutations that stabilize the S protein in a pre ⁇ fusion confirmation.
  • mutations including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 5 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more
  • characteristic of an Omicron variant e.g., one or more mutations of an Omicron variant listed in Table 3A
  • an RNA comprises a nucleotide sequence encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) listed in Table 3A.
  • one or more mutations may come from two or more variants as listed in Table 3A.
  • an RNA comprises a nucleotide sequence encoding a SARS ⁇ CoV ⁇ 2 S protein comprising each of the mutations identified in Table 3A as being characteristic of a certain Omicron variant (e.g., in some embodiments, an RNA comprises a nucleotide sequence encoding a SARS ⁇ CoV ⁇ 2 S protein comprising each of the mutations listed in Table 3A as being characteristic of an Omicron BA.1, BA.2, BA.2.12.1, BA.4/5, BA.2.75, BA.2.75.1, BA.4.6, BQ.1.1, XBB, XBB.1, XBB.2, or XBB.1.3 variant).
  • an RNA disclosed herein comprises a nucleotide sequence that encodes an immunogenic fragment of the SARS ⁇ Cov ⁇ 2 S protein (e.g., the RBD) and which comprises one or more mutations that are characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., an Omicron variant described herein).
  • an RNA comprises a nucleotide sequence encoding the RBD of an S protein of a SARS ⁇ CoV ⁇ 2 variant (e.g., a region of the S protein corresponding to amino acids 327 to 528 of SEQ ID NO: 1, and comprising one or more mutations characteristic of a variant of concern that lie within this region).
  • an RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising a subset of the mutations listed in Table 3A. In some embodiments, an RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising the mutations listed in Table 3A that are most prevalent in a certain variant (e.g., mutations that have been detected in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of sequences collected to date for a given variant sequenced).
  • a certain variant e.g., mutations that have been detected in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of sequences collected to date for a given variant sequenced.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4/5 variant.
  • the one or more mutations characteristic of a BA.4/5 variant include T19I, ⁇ 24 ⁇ 26, A27S, ⁇ O24 ⁇ 26, A27S, ⁇ 69/70, G142D, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4/5 variant and excludes R408S. In some embodiments, RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4/5 variant and excludes R408S.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of a BA.2.75 variant.
  • the one or more mutations characteristic of a BA.2.75 variant include T19I, ⁇ 24 ⁇ 26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4/5 variant and excludes R408S. In some embodiments, RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4/BA.5 variant, and which excludes R408S and N354D.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of a BA.2.75 variant.
  • the one or more mutations characteristic of a BA.2.75 variant include T19I, ⁇ 24 ⁇ 26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 25 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes N354D. In some embodiments, RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes D796Y.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes D796Y and N354D. In some embodiments, RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a BA.2.75.2 variant.
  • the one or more mutations characteristic of a BA.2.75.2 variant include T19I, ⁇ 24 ⁇ 26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and D1199N.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 30 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75.2 variant, and which excludes R346T. In some embodiments, RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a BA.4.6 or BF.7 variant.
  • the one or more mutations characteristic of a BA.4.6 or BF.7 variant include T19I, ⁇ 24 ⁇ 26, A27S, ⁇ 69/70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7 variant, and which exclude R408S.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7 variant, and which exclude N658S.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 25 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7 variant, and which exclude N658S and R408S.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron XBB variant.
  • the one or more mutations characteristic of an Omicron XBB variant include T19I, ⁇ 24 ⁇ 26, A27S, V83A, G142D, ⁇ 144, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron XBB.1 variant.
  • the one or more mutations characteristic of an Omicron XBB.1 variant include G252V.
  • the one or more mutations characteristic of an Omicron XBB.1 variant include T19I, ⁇ 24 ⁇ 26, A27S, V83A, G142D, ⁇ 144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of an Omicron XBB.1 variant and which exclude Q493R.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of an Omicron XBB variant and which exclude Q493R and G252V.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron XBB.2 variant.
  • the one or more mutations characteristic of an Omicron XBB.2 variant include D253G.
  • the one or more mutations characteristic of an Omicron XBB.2 variant include T19I, ⁇ 24 ⁇ 26, A27S, V83A, G142D, ⁇ 144, H146Q, Q183E, V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron XBB.1.3 variant.
  • the one or more mutations characteristic of an Omicron XBB.1.3 variant include G252V and A484T.
  • the one or more mutations characteristic of an Omicron XBB.1.3 variant include T19I, ⁇ 24 ⁇ 26, A27S, V83A, G142D, ⁇ 144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, A484T, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BQ.1.1 variant.
  • the one or more mutations characteristic of a BQ.1.1 variant include T19I, ⁇ 24 ⁇ 26, A27S, ⁇ 69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N463K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.
  • RNA described herein encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BQ.1.1 variant.
  • a vaccine antigen described herein comprises, consists essentially of or consists of a spike protein (S) of SARS ⁇ CoV ⁇ 2, a variant thereof, or a fragment thereof and comprises one or more of mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., one or more of mutations associated with an Omicron variant that are listed in Table 3A).
  • a vaccine antigen comprises (a) the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, and (b) one of more mutations associated with a SARS ⁇ CoV ⁇ 2 variant of concern (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7 and comprises one or more associated with a SARS ⁇ CoV ⁇ 2 variant of concern (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises (a) the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, and (b) one of more of the mutations listed in Table 3A.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80 and comprises one or more mutations associated with a SARS ⁇ CoV ⁇ 2 variant of concern (e.g., one or more mutations listed in Table 3A).
  • RNA encoding a vaccine antigen comprises (i) the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9 and/or (ii) a nucleotide sequence encoding an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence comprising the
  • RNA encoding a vaccine antigen (a) (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, and (b) comprises one or more mutations characteristics of a SARS ⁇ CoV ⁇ 2 variant of concern (e.g., one or more mutations listed in Table 3A).
  • RNA encoding a vaccine antigen comprises (a) (i) the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81, a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81 and/or (ii) a nucleotide sequence encoding encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, an amino acid sequence having
  • RNA encoding a vaccine antigen (a) (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81; and/or (ii) comprises a nucleotide sequence that encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80 or 7, and (b) comprises one or more mutations characteristics of a SARS ⁇ CoV ⁇ 2 variant of concern (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises, consists essentially of or consists of SARS ⁇ CoV ⁇ 2 spike S1 fragment (S1) (the S1 subunit of a spike protein (S) of SARS ⁇ CoV ⁇ 2), a variant thereof, or a fragment thereof, and comprises one or more mutations of a SARS ⁇ CoV ⁇ 2 variant described herein.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1 and comprises one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1 and comprises one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 80, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 80 and comprises one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., one or more mutations listed in Table 3A).
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 80 and comprises one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant (e.g., one or more mutations listed in Table 3A).
  • Vaccine Antigens and Combinations Thereof the vaccine antigen described herein comprises, consists essentially of or consists of a spike protein (S) of SARS ⁇ CoV ⁇ 2, a variant thereof, or a fragment thereof.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.
  • the vaccine antigen comprises, consists essentially of or consists of SARS ⁇ CoV ⁇ 2 spike S1 fragment (S1) (the S1 subunit of a spike protein (S) of SARS ⁇ CoV ⁇ 2), a variant thereof, or a fragment thereof.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.
  • the vaccine antigen comprises, consists essentially of or consists of the receptor binding domain (RBD) of the S1 subunit of a spike protein (S) of SARS ⁇ CoV ⁇ 2, a variant thereof, or a fragment thereof.
  • RBD receptor binding domain
  • S spike protein
  • S spike protein
  • the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, a variant thereof, or a fragment thereof is also referred to herein as "RBD" or "RBD domain”.
  • a vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.
  • a signal peptide is fused, either directly or through a linker, to a SARS ⁇ CoV ⁇ 2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein.
  • a signal peptide is fused to the above described amino acid sequences derived from SARS ⁇ CoV ⁇ 2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above.
  • Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N ⁇ terminus of the antigenic peptide or protein, without being limited thereto.
  • Signal peptides as defined herein preferably allow the transport of the antigenic peptide or protein as encoded by the RNA into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal ⁇ lysosomal compartment.
  • the signal peptide sequence as defined herein includes, without being limited thereto, the signal peptide sequence of SARS ⁇ CoV ⁇ 2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or a functional variant thereof.
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.
  • RNA encoding a signal sequence comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,
  • RNA encoding a signal sequence comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.
  • RNA encoding a signal sequence comprises the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 9
  • RNA encoding a signal sequence comprises the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.
  • an RNA comprises a sequence encoding a signal peptide.
  • a signal peptide sequence as defined herein includes, without being limited thereto, the signal peptide sequence of an immunoglobulin, e.g., the signal peptide sequence of an immunoglobulin heavy chain variable region, wherein the immunoglobulin may be human immunoglobulin.
  • the signal peptide sequence as defined herein can include a sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31 or a functional variant thereof.
  • a signal peptide sequence is functional in mammalian cells.
  • a utilized signal sequence is “intrinsic” in that it is, in nature, associated with (e.g., linked to) the encoded polypeptide.
  • a utilized signal sequence is heterologous to an encoded polypeptide, e.g., is not naturally part of a polypeptide (e.g., protein) whose sequences are included in the encoded polypeptide.
  • signal peptides are sequences, which are typically characterized by a length of about 15 to 30 amino acids. In many embodiments, signal peptides are positioned at the N ⁇ terminus of an encoded polypeptide as described herein, without being limited thereto. In some embodiments, signal peptides preferably allow the transport of the polypeptide encoded by RNAs of the present disclosure with which they are associated into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal ⁇ lysosomal compartment.
  • RNAs of the present disclosure preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal ⁇ lysosomal compartment.
  • a signal sequence is selected from an S1S2 signal peptide (aa 1 ⁇ 16 or aa 1 ⁇ 19), an immunoglobulin secretory signal peptide (aa 1 ⁇ 22), an HSV ⁇ 1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV ⁇ 2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA), a human SPARC signal peptide, a human insulin isoform 1 signal peptide, a human albumin signal peptide, etc.
  • an RNA sequence encodes an epitope that may comprise or otherwise be linked to a signal sequence (e.g., secretory sequence), such as those listed in Table A, or at least a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto.
  • a signal sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having at least 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.
  • a sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having 1, 2, 3, 4, or at most 5 amino acid differences relative thereto, is utilized.
  • a signal sequence is selected from those included in the Table A below and/or those encoded by the sequences in Table B below. Table A: Exemplary signal sequences
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.
  • a signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.
  • RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, an amino
  • RNA encoding a signal sequence comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.
  • signal peptides are preferably used in order to promote secretion of the encoded antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to an encoded antigenic peptide or protein as defined herein.
  • the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, said signal peptide preferably being fused to the antigenic peptide or protein, more preferably to the N ⁇ terminus of the antigenic peptide or protein as described herein.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4, or a fragment of the nucleotide sequence of SEQ ID NO: 4, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence of amino
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.
  • Multimerization Domains In some embodiments, an RNA utilized as described herein comprises a sequence that encodes a multimerization element (e.g., a heterologous multimerization element). In some embodiments, a heterologous multimerization element comprises a dimerization, trimerization or tetramerization element.
  • a multimerization element is one described in WO2017/081082 (e.g., SEQ ID NOs: 1116 ⁇ 1167, or fragments or variants thereof).
  • Exemplary trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4 ⁇ pll, and p53.
  • a provided encoded polypeptide(s) is able to form a trimeric complex.
  • a utilized encoded polypeptide(s) may comprise a domain allowing formation of a multimeric complex, such as for example a trimeric complex of an amino acid sequence comprising an encoded polypeptide(s) as described herein.
  • a domain allowing formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein.
  • an encoded polypeptide(s) can be modified by addition of a T4 ⁇ fibritin ⁇ derived “foldon” trimerization domain, for example, to increase its immunogenicity.
  • a trimerization domain is fused, either directly or through a linker, e.g., a glycine/serine linker, to a SARS ⁇ CoV ⁇ 2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein.
  • a trimerization domain is fused to the above described amino acid sequences derived from SARS ⁇ CoV ⁇ 2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above (which may optionally be fused to a signal peptide as described above).
  • trimerization domains are preferably located at the C ⁇ terminus of the antigenic peptide or protein, without being limited thereto.
  • Trimerization domains as defined herein preferably allow the trimerization of the antigenic peptide or protein as encoded by the RNA.
  • trimerization domains as defined herein include, without being limited thereto, foldon, the natural trimerization domain of T4 fibritin.
  • the C ⁇ terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain.
  • the trimerization domain as defined herein includes, without being limited thereto, a sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or a functional variant thereof. In one embodiment, the trimerization domain as defined herein includes, without being limited thereto, a sequence comprising the amino acid sequence of SEQ ID NO: 10 or a functional variant thereof.
  • a trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.
  • a trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.
  • RNA encoding a trimerization domain comprises the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, or a fragment of the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%
  • RNA encoding a trimerization domain comprises the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.
  • a trimerization domain comprises the amino acid sequence SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10.
  • a trimerization domain comprises the amino acid sequence of SEQ ID NO: 10.
  • RNA encoding a trimerization domain comprises the nucleotide sequence of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11, or a fragment of the nucleotide sequence of SEQ ID NO: 11, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%
  • RNA encoding a trimerization domain comprises the nucleotide sequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10.
  • trimerization domains are preferably used in order to promote trimerization of the encoded antigenic peptide or protein. More preferably, a trimerization domain as defined herein is fused to an antigenic peptide or protein as defined herein.
  • the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerization domain as defined herein, said trimerization domain preferably being fused to the antigenic peptide or protein, more preferably to the C ⁇ terminus of the antigenic peptide or protein.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 17, 21, or 26, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment of the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 18, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence of amino
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.
  • Transmembrane Domain In some embodiments, an RNA described herein comprises a sequence that encodes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.
  • a transmembrane domain can be N ⁇ terminal, C ⁇ terminal, or internal to an encoded polypeptide.
  • a coding sequence of a transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences of sequences (e.g., sequences encoding polypeptide(s)) with which it is to be linked.
  • a transmembrane domain comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV ⁇ 1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.
  • HA Hemagglutinin
  • EIAV equine infectious anaemia virus
  • MMV murine leukaemia virus
  • VSV vesicular stomatitis virus
  • Rabies virus or a seven transmembrane domain receptor.
  • a transmembrane domain is fused, either directly or through a linker, e.g., a glycine/serine linker, to a SARS ⁇ CoV ⁇ 2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a transmembrane domain is fused to a SARS ⁇ CoV ⁇ 2 S polypeptide or an immunogenic fragment thereof (antigenic peptides or proteins), which may optionally be fused to a signal peptide and/or trimerization domain as described above.
  • a linker e.g., a glycine/serine linker
  • transmembrane domains are preferably located at the C ⁇ terminus of the antigenic peptide or protein, without being limited thereto.
  • such transmembrane domains are located at the C ⁇ terminus of the trimerization domain, if present, without being limited thereto.
  • a trimerization domain is present between the SARS ⁇ CoV ⁇ 2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein, and the transmembrane domain.
  • Transmembrane domains as defined herein preferably allow the anchoring into a cellular membrane of the antigenic peptide or protein as encoded by the RNA.
  • the transmembrane domain sequence as defined herein includes, without being limited thereto, the transmembrane domain sequence of SARS ⁇ CoV ⁇ 2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or a functional variant thereof.
  • a transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.
  • a transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.
  • RNA encoding a transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an
  • RNA encoding a transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.
  • a vaccine antigen comprises the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, or a fragment of the nucleotide sequence of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32, or a fragment of the nucleotide sequence of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 9
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 27, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27, or a fragment of the nucleotide sequence of SEQ ID NO: 27, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 28
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 49.
  • the amino acid sequence of SEQ ID NO: 49 corresponds to the amino acid sequence of the full ⁇ length S protein from Omicron BA.1, which includes proline residues at positions 986 and 987 of SEQ ID NO: 49.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 50, or a fragment of the nucleotide sequence of SEQ ID NO: 50, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49.
  • the nucleotide sequence of SEQ ID NO: 50 is a nucleotide sequence designed to encode the amino acid sequence of the full ⁇ length S protein from Omicron BA.1 with proline residues at positions 986 and 987 of SEQ ID NO: 49.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 51, or a fragment of the nucleotide sequence of SEQ ID NO: 51, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49.
  • the nucleotide sequence of SEQ ID NO: 51 corresponds to an RNA construct (e.g., comprising a 5’ UTR, a S ⁇ protein ⁇ encoding sequence, a 3’ UTR, and a poly ⁇ A tail), which encodes the amino acid sequence of the full ⁇ length S protein from Omicron BA.1 with proline residues at positions 986 and 987 of SEQ ID NO: 49.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 55, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 55, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 55.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 56, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 56, or a fragment of the nucleotide sequence of SEQ ID NO: 56, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 56; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 55, or the amino acid sequence having at least 99.5%, 99%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 56; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 57, or a fragment of the nucleotide sequence of SEQ ID NO: 57, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, an amino acid sequence having at least 99.5%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 58, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 58, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 58.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 59, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 59, or a fragment of the nucleotide sequence of SEQ ID NO: 59, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 59; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58, or an immuno
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 59; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 60, or a fragment of the nucleotide sequence of SEQ ID NO: 60, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58, an amino acid sequence having at least 99.5%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 61, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 61, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 61.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 62, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 62, or a fragment of the nucleotide sequence of SEQ ID NO: 62, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 62; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61, or an immuno
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 62; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 63, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 63, or a fragment of the nucleotide sequence of SEQ ID NO: 63, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 63; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, an amino acid sequence having at least 99
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 63; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 52, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52.
  • a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 52.
  • RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 53, or a fragment of the nucleotide sequence of SEQ ID NO: 53, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 54, or a fragment of the nucleotide sequence of SEQ ID NO: 54, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 83, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 83, or a fragment of the nucleotide sequence of SEQ ID NO: 83, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 83; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80, an amino acid sequence having at least 99.5%,
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 83; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 103, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 103, or a fragment of the nucleotide sequence of SEQ ID NO: 103, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 103; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 100, an amino acid sequence having at least 99.5%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 103; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 100.
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 98, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 98, or a fragment of the nucleotide sequence of SEQ ID NO: 98, or the nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 98; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 95, an amino acid sequence having at least 99.5%
  • RNA encoding a vaccine antigen comprises the nucleotide sequence of SEQ ID NO: 98; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 95.
  • the vaccine antigens described above comprise a contiguous sequence of SARS ⁇ CoV ⁇ 2 coronavirus spike (S) protein that consists of or essentially consists of the above described amino acid sequences derived from SARS ⁇ CoV ⁇ 2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above.
  • the vaccine antigens described above comprise a contiguous sequence of SARS ⁇ CoV ⁇ 2 coronavirus spike (S) protein of no more than 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2), or BNT162b3 (e.g., BNT162b3c).
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) described herein as RBP020.2.
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) described herein as BNT162b3 (e.g., BNT162b3c).
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5.
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 50, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 51, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 57, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 60, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 63, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 63, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 63; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 53, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 54, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52.
  • modRNA nucleoside modified messenger RNA
  • RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.
  • the term "vaccine” refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.
  • the RNA encoding the antigen molecule is expressed in cells of the subject to provide the antigen molecule. In one embodiment, expression of the antigen molecule is at the cell surface or into the extracellular space. In one embodiment, the antigen molecule is presented in the context of MHC.
  • the RNA encoding the antigen molecule is transiently expressed in cells of the subject. In one embodiment, after administration of the RNA encoding the antigen molecule, in particular after intramuscular administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in muscle occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in spleen occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in antigen presenting cells, preferably professional antigen presenting cells occurs.
  • the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells.
  • the RNA encoding the antigen molecule after administration of the RNA encoding the antigen molecule, no or essentially no expression of the RNA encoding the antigen molecule in lung and/or liver occurs.
  • expression of the RNA encoding the antigen molecule in spleen is at least 5 ⁇ fold the amount of expression in lung.
  • the methods and agents e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to lymph nodes and/or spleen.
  • RNA encoding a vaccine antigen is detectable in lymph nodes and/or spleen 6 hours or later following administration and preferably up to 6 days or longer.
  • the methods and agents e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cell follicles, subcapsular sinus, and/or T cell zone, in particular B cell follicles and/or subcapsular sinus of lymph nodes.
  • the methods and agents e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cells (CD19+), subcapsular sinus macrophages (CD169+) and/or dendritic cells (CD11c+) in the T cell zone and intermediary sinus of lymph nodes, in particular to B cells (CD19+) and/or subcapsular sinus macrophages (CD169+) of lymph nodes.
  • B cells CD19+
  • subcapsular sinus macrophages CD169+
  • CD11c+ dendritic cells
  • the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to white pulp of spleen.
  • the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cells, DCs (CD11c+), in particular those surrounding the B cells, and/or macrophages of spleen, in particular to B cells and/or DCs (CD11c+).
  • the vaccine antigen is expressed in lymph node and/or spleen, in particular in the cells of lymph node and/or spleen described above.
  • the peptide and protein antigens suitable for use according to the disclosure typically include a peptide or protein comprising an epitope of SARS ⁇ CoV ⁇ 2 S protein or a functional variant thereof for inducing an immune response.
  • the peptide or protein or epitope may be derived from a target antigen, i.e. the antigen against which an immune response is to be elicited.
  • the peptide or protein antigen or the epitope contained within the peptide or protein antigen may be a target antigen or a fragment or variant of a target antigen.
  • the target antigen may be a coronavirus S protein, in particular SARS ⁇ CoV ⁇ 2 S protein.
  • the antigen molecule or a procession product thereof, e.g., a fragment thereof, may bind to an antigen receptor such as a BCR or TCR carried by immune effector cells, or to antibodies.
  • a peptide and protein antigen which is provided to a subject according to the present disclosure by administering RNA encoding the peptide and protein antigen, i.e., a vaccine antigen preferably results in the induction of an immune response, e.g., a humoral and/or cellular immune response in the subject being provided the peptide or protein antigen.
  • a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragment or variant is immunologically equivalent to the target antigen.
  • fragment of an antigen or "variant of an antigen” means an agent which results in the induction of an immune response which immune response targets the antigen, i.e. a target antigen.
  • the vaccine antigen may correspond to or may comprise the target antigen, may correspond to or may comprise a fragment of the target antigen or may correspond to or may comprise an antigen which is homologous to the target antigen or a fragment thereof.
  • a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence being homologous to an immunogenic fragment of a target antigen.
  • An "immunogenic fragment of an antigen" according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against the target antigen.
  • the vaccine antigen may be a recombinant antigen.
  • immunologically equivalent means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect.
  • immunologically equivalent is preferably used with respect to the immunological effects or properties of antigens or antigen variants used for immunization.
  • an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.
  • Activation refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions.
  • activated immune effector cells refers to, among other things, immune effector cells that are undergoing cell division.
  • primary refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.
  • clonal expansion or “expansion” refers to a process wherein a specific entity is multiplied.
  • the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified.
  • clonal expansion leads to differentiation of the immune effector cells.
  • the term "antigen” relates to an agent comprising an epitope against which an immune response can be generated.
  • the term "antigen” includes, in particular, proteins and peptides.
  • an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages.
  • an antigen or a procession product thereof such as a T ⁇ cell epitope is in one embodiment bound by a T ⁇ or B ⁇ cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a procession product thereof may react specifically with antibodies or T lymphocytes (T cells).
  • an antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS ⁇ CoV ⁇ 2 S protein, and an epitope is derived from such antigen.
  • viral antigen refers to any viral component having antigenic properties, i.e. being able to provoke an immune response in an individual.
  • the viral antigen may be coronavirus S protein, e.g., SARS ⁇ CoV ⁇ 2 S protein.
  • the term “expressed on the cell surface” or “associated with the cell surface” means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell.
  • a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids.
  • the association may be direct or indirect.
  • the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell.
  • a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.
  • Cell surface or “surface of a cell” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules.
  • an antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen ⁇ specific antibodies added to the cells.
  • extracellular portion or “exodomain” in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.
  • the term refers to one or more extracellular loops or domains or a fragment thereof.
  • epipe refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system.
  • the epitope may be recognized by T cells, B cells or antibodies.
  • An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length.
  • epitope includes T cell epitopes.
  • T cell epitope refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules.
  • MHC major histocompatibility complex
  • MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells.
  • the proteins encoded by the MHC are expressed on the surface of cells, and display both self ⁇ antigens (peptide fragments from the cell itself) and non ⁇ self ⁇ antigens (e.g., fragments of invading microorganisms) to a T cell.
  • the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective.
  • the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.
  • the peptide and protein antigen can be 2 ⁇ 100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids.
  • the peptide can be greater than 100 amino acids.
  • the peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein.
  • vaccine antigen is recognized by an immune effector cell.
  • the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co ⁇ stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen.
  • the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell.
  • an antigen is presented by a diseased cell such as a virus ⁇ infected cell.
  • an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells.
  • binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g. perforins and granzymes.
  • an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen.
  • an antibody or B cell receptor binds to native epitopes of an antigen.
  • Bivalent Vaccine Combinations Multiple various spike protein (S) of SARS ⁇ CoV ⁇ 2 variants as described herein may be delivered in combination, for example by a bivalent RNA vaccine comprising at least one RNA encoding two or more spike proteins (S) or any variants thereof (e.g., as described herein). Exemplary combinations of spike proteins are described herein and shown, e.g., in Tables below. Bivalent vaccines may include any of these described combinations in either spike protein encoded by the RNA vaccine.
  • an RNA vaccine comprises at least one RNA encoding one or more coronavirus spike proteins (e.g., a spike protein variant described in Table 7).
  • an RNA vaccine comprises at least two RNA each encoding a distinct coronavirus spike protein (e.g., a spike protein variant described in Table 7).
  • Coronavius spike protein antigens may be administered as single ⁇ stranded, 5' capped mRNA that is translated into the respective protein upon entering cells of a subject being administered the RNA.
  • the RNA contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence).
  • beta ⁇ S ⁇ ARCA(D1) is utilized as specific capping structure at the 5' ⁇ end of the RNA.
  • m27,3’ ⁇ OGppp(m12’ ⁇ O)ApG is utilized as specific capping structure at the 5' ⁇ end of the RNA.
  • the 5' ⁇ UTR sequence is derived from the human alpha ⁇ globin mRNA and optionally has an optimized ⁇ Kozak sequence ⁇ to increase translational efficiency.
  • a combination of two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • two re ⁇ iterated 3' ⁇ UTRs derived from the human beta ⁇ globin mRNA are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • a poly(A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is used. This poly(A) sequence was designed to enhance RNA stability and translational efficiency.
  • RNA vaccines encoding any of the coronavirus spike protein variants described herein (and e.g., in Table 7) may include any of the other nucleic acid modification and RNA construct components described herein.
  • RNA moelcules may be formulated in the lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., two populations of RNAs are mixed prior to LNP formulation; or each RNA is formulated in a separate LNP composition, followed by mixing together).
  • LNPs lipid nanoparticles
  • Combinations of exemplary spike protein variants described herein may be utilized in a bivalent RNA vaccine.
  • Exemplary combinations of spike proteins that can be utilized in a bivalent RNA vaccine are shown in Table 8 below.
  • Nucleic acids The term "polynucleotide” or “nucleic acid”, as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • a nucleic acid may be single ⁇ stranded or double ⁇ stranded.
  • RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.
  • IVT RNA in vitro transcribed RNA
  • a polynucleotide is preferably isolated.
  • Nucleic acids may be comprised in a vector.
  • vector includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors.
  • Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
  • Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
  • the RNA encoding the vaccine antigen is expressed in cells such as antigen presenting cells of the subject treated to provide the vaccine antigen.
  • the nucleic acids described herein may be recombinant and/or isolated molecules.
  • RNA relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2' ⁇ position of a ⁇ D ⁇ ribofuranosyl group.
  • RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non ⁇ nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non ⁇ standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • the RNA is messenger RNA (mRNA) that relates to a RNA transcript which encodes a peptide or protein.
  • mRNA messenger RNA
  • mRNA generally contains a 5' untranslated region (5' ⁇ UTR), a peptide coding region and a 3' untranslated region (3' ⁇ UTR).
  • the RNA is produced by in vitro transcription or chemical synthesis.
  • the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.
  • RNA is in vitro transcribed RNA (IVT ⁇ RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the RNA is "replicon RNA” or simply a "replicon”, in particular "self ⁇ replicating RNA” or “self ⁇ amplifying RNA”.
  • the replicon or self ⁇ replicating RNA is derived from or comprises elements derived from a ssRNA virus, in particular a positive ⁇ stranded ssRNA virus such as an alphavirus.
  • Alphaviruses are typical representatives of positive ⁇ stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837–856).
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’ ⁇ cap, and a 3’ poly(A) tail.
  • the genome of alphaviruses encodes non ⁇ structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non ⁇ structural proteins (nsP1–nsP4) are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome.
  • the first ORF is larger than the second ORF, the ratio being roughly 2:1.
  • RNA RNA molecule that resembles eukaryotic messenger RNA
  • mRNA messenger RNA
  • (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non ⁇ structural poly ⁇ protein (nsP1234).
  • Alphavirus ⁇ derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • Alphavirus ⁇ based trans ⁇ replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans ⁇ replication system).
  • Trans ⁇ replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
  • the RNA described herein may have modified nucleosides.
  • the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
  • uracil describes one of the nucleobases that can occur in the nucleic acid of RNA.
  • uridine describes one of the nucleosides that can occur in RNA.
  • the structure of uridine is:
  • UTP uridine 5’ ⁇ triphosphate
  • Pseudo ⁇ UTP pseudouridine 5’ ⁇ triphosphate
  • Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon ⁇ carbon bond instead of a nitrogen ⁇ carbon glycosidic bond.
  • Another exemplary modified nucleoside is N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ), which has the structure: N1 ⁇ methyl ⁇ pseudo ⁇ UTP has the following structure:
  • RNA comprises a modified nucleoside in place of at least one uridine.
  • RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is independently selected from pseudouridine ( ⁇ ), N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ), and 5 ⁇ methyl ⁇ uridine (m5U).
  • the modified nucleoside comprises pseudouridine ( ⁇ ).
  • the modified nucleoside comprises N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ). In some embodiments, the modified nucleoside comprises 5 ⁇ methyl ⁇ uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine ( ⁇ ), N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ), and 5 ⁇ methyl ⁇ uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine ( ⁇ ) and N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ). In some embodiments, the modified nucleosides comprise pseudouridine ( ⁇ ) and 5 ⁇ methyl ⁇ uridine (m5U).
  • the modified nucleosides comprise N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ) and 5 ⁇ methyl ⁇ uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine ( ⁇ ), N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ), and 5 ⁇ methyl ⁇ uridine (m5U).
  • the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3 ⁇ methyl ⁇ uridine (m 3 U), 5 ⁇ methoxy ⁇ uridine (mo 5 U), 5 ⁇ aza ⁇ uridine, 6 ⁇ aza ⁇ uridine, 2 ⁇ thio ⁇ 5 ⁇ aza ⁇ uridine, 2 ⁇ thio ⁇ uridine (s 2 U), 4 ⁇ thio ⁇ uridine (s 4 U), 4 ⁇ thio ⁇ pseudouridine, 2 ⁇ thio ⁇ pseudouridine, 5 ⁇ hydroxy ⁇ uridine (ho 5 U), 5 ⁇ aminoallyl ⁇ uridine, 5 ⁇ halo ⁇ uridine (e.g., 5 ⁇ iodo ⁇ uridine or 5 ⁇ bromo ⁇ uridine), uridine 5 ⁇ oxyacetic acid (cmo 5 U), uridine 5 ⁇ oxyacetic acid methyl ester (mcmo 5 U), 5 ⁇ carboxymethyl ⁇ uridine (cm 5 U), 1 ⁇ carboxymethyl ⁇ pseudouridine
  • the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine.
  • modified cytidine e.g., in the RNA 5 ⁇ methylcytidine is substituted partially or completely, preferably completely, for cytidine.
  • the RNA comprises 5 ⁇ methylcytidine and one or more selected from pseudouridine ( ⁇ ), N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ), and 5 ⁇ methyl ⁇ uridine (m5U).
  • the RNA comprises 5 ⁇ methylcytidine and N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ).
  • the RNA comprises 5 ⁇ methylcytidine in place of each cytidine and N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ) in place of each uridine.
  • the RNA according to the present disclosure comprises a 5’ ⁇ cap.
  • the RNA of the present disclosure does not have uncapped 5' ⁇ triphosphates.
  • the RNA may be modified by a 5' ⁇ cap analog.
  • RNA e.g., mRNA
  • 5' ⁇ cap refers to a structure found on the 5' ⁇ end of an RNA (e.g., mRNA) molecule and generally consists of a guanosine nucleotide connected to the RNA (e.g., mRNA) via a 5' ⁇ to 5' ⁇ triphosphate linkage. In one embodiment, this guanosine is methylated at the 7 ⁇ position.
  • Providing an RNA with a 5' ⁇ cap or 5' ⁇ cap analog may be achieved by in vitro transcription, in which the 5' ⁇ cap is co ⁇ transcriptionally expressed into the RNA strand, or may be attached to RNA post ⁇ transcriptionally using capping enzymes.
  • RNA comprises a cap0, cap1, or cap2, preferably cap1 or cap2, more preferably cap1.
  • cap0 comprises the structure "m 7 GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'.
  • cap1 comprises the structure "m 7 GpppNm”, wherein Nm is any nucleoside bearing an OCH 3 moiety at position 2'.
  • cap2 comprises the structure "m 7 GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH 3 moiety at position 2'.
  • the building block cap for RNA is m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG (also sometimes referred to as m 2 7,3 ⁇ O G(5’)ppp(5’)m 2’ ⁇ O ApG), which has the following structure:
  • Cap1 RNA which comprises RNA and m 2 7,3 ⁇ O G(5’)ppp(5’)m 2’ ⁇ O ApG:
  • another exemplary Cap1 RNA (no cap analog): .
  • the RNA is modified with "Cap0" structures using, in one embodiment, the cap analog anti ⁇ reverse cap (ARCA Cap (m 2 7,3 ⁇ O G(5’)ppp(5’)G)) with the structure: .
  • Cap0 RNA comprising RNA and m 2 7,3 ⁇ O G(5’)ppp(5’)G:
  • the "Cap0" structures are generated using the cap analog Beta ⁇ S ⁇ ARCA (m 2 7,2 ⁇ O G(5’)ppSp(5’)G) with the structure:
  • Beta ⁇ S ⁇ ARCA m 2 7,2 ⁇ O G(5’)ppSp(5’)G
  • RNA RNA:
  • the "D1" diastereomer of beta ⁇ S ⁇ ARCA or "beta ⁇ S ⁇ ARCA(D1)” is the diastereomer of beta ⁇ S ⁇ ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta ⁇ S ⁇ ARCA (beta ⁇ S ⁇ ARCA(D2)) and thus exhibits a shorter retention time (cf., WO 2011/015347, herein incorporated by reference).
  • a particularly preferred cap is beta ⁇ S ⁇ ARCA(D1) (m 2 7,2' ⁇ O GppSpG) or m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG.
  • RNA according to the present disclosure comprises a 5' ⁇ UTR and/or a 3' ⁇ UTR.
  • the term "untranslated region" or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5' ⁇ UTR) and/or 3' (downstream) of an open reading frame (3' ⁇ UTR).
  • a 5' ⁇ UTR if present, is located at the 5' end, upstream of the start codon of a protein ⁇ encoding region.
  • a 5' ⁇ UTR is downstream of the 5' ⁇ cap (if present), e.g. directly adjacent to the 5' ⁇ cap.
  • a 3' ⁇ UTR if present, is located at the 3' end, downstream of the termination codon of a protein ⁇ encoding region, but the term "3' ⁇ UTR" does preferably not include the poly(A) sequence.
  • the 3' ⁇ UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.
  • RNA comprises a 5’ ⁇ UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.
  • RNA comprises a 3’ ⁇ UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.
  • a particularly preferred 5’ ⁇ UTR comprises the nucleotide sequence of SEQ ID NO: 12.
  • a particularly preferred 3’ ⁇ UTR comprises the nucleotide sequence of SEQ ID NO: 13.
  • the RNA according to the present disclosure comprises a 3' ⁇ poly(A) sequence.
  • poly(A) sequence or “poly ⁇ A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' ⁇ end of an RNA molecule.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3’ ⁇ UTR in the RNAs described herein.
  • An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3' ⁇ end of the RNA by a template ⁇ independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template ⁇ dependent RNA polymerase. It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009 ⁇ 4017).
  • the poly(A) sequence may be of any length.
  • a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly(A) sequence typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in certain enbodiments of the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly(A) sequence contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U).
  • Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its 3' ⁇ end, i.e., the poly(A) sequence is not masked or followed at its 3' ⁇ end by a nucleotide other than A.
  • the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides.
  • the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.
  • a poly(A) sequence included in an RNA described herein is a interrupted poly(A) sequence, e.g., as described in WO2016/005324, the entire content of which is incorporated herein by reference for purposes described herein.
  • a poly(A) sequence comprises a stretch of at least 20 adenosine residues (including, e.g., at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues), followed by a linker sequence (e.g., in some embodiments comprising non ⁇ A nucleotides) and another stretch of at least 20 adenosine residues (including, e.g., at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues).
  • a linker sequence e.g., in some embodiments comprising non ⁇ A nucleotides
  • such a linker sequence may be 3 ⁇ 50 nucleotides in length, or 5 ⁇ 25 nucleotides in length, or 10 ⁇ 15 nucleotides in length.
  • a poly(A) sequence comprises a stretch of about 30 adenosine residues, followed by a linker sequence having a length of about 5 ⁇ 15 nucleoties (e.g., in some embodiments comprising non ⁇ A nucleotides) and another stretch of about 70 adenosine residues.
  • RNA comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 14.
  • a particularly preferred poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 14.
  • vaccine antigen is preferably administered as single ⁇ stranded, 5' ⁇ capped mRNA that is translated into the respective protein upon entering cells of a subject being administered the RNA.
  • the RNA contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' ⁇ cap, 5' ⁇ UTR, 3' ⁇ UTR, poly(A) sequence).
  • beta ⁇ S ⁇ ARCA(D1) is utilized as specific capping structure at the 5' ⁇ end of the RNA.
  • m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG is utilized as specific capping structure at the 5' ⁇ end of the RNA.
  • the 5' ⁇ UTR sequence is derived from the human alpha ⁇ globin mRNA and optionally has an optimized ⁇ Kozak sequence ⁇ to increase transla ⁇ onal efficiency.
  • FI element a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • F amino terminal enhancer of split
  • I mitochondrial encoded 12S ribosomal RNA
  • two re ⁇ iterated 3' ⁇ UTRs derived from the human beta ⁇ globin mRNA are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • a poly(A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is used.
  • This poly(A) sequence was designed to enhance RNA stability and translational efficiency.
  • RNA encoding a vaccine antigen is expressed in cells of the subject treated to provide the vaccine antigen.
  • the RNA is transiently expressed in cells of the subject.
  • the RNA is in vitro transcribed RNA.
  • expression of the vaccine antigen is at the cell surface.
  • the vaccine antigen is expressed and presented in the context of MHC. In one embodiment of all aspects of the present disclosure, expression of the vaccine antigen is into the extracellular space, i.e., the vaccine antigen is secreted.
  • the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.
  • the term “transcription” comprises "in vitro transcription", wherein the term “in vitro transcription” relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell ⁇ free system, preferably using appropriate cell extracts.
  • cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present disclosure encompassed by the term "vector".
  • the RNA used in certain embodiments of the present disclosure preferably is in vitro transcribed RNA (IVT ⁇ RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription according to the present disclosure is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the term "expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.
  • at least a portion of the RNA is delivered to a target cell.
  • at least a portion of the RNA is delivered to the cytosol of the target cell.
  • the RNA is translated by the target cell to produce the peptide or protein it encodes.
  • the target cell is a spleen cell.
  • the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen.
  • the target cell is a dendritic cell or macrophage.
  • RNA particles such as RNA lipid particles described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject comprising the administration of the RNA particles described herein to the subject. In one embodiment, the RNA is delivered to the cytosol of the target cell.
  • the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • RNA encoding vaccine antigen to be administered according to the present disclosure is non ⁇ immunogenic.
  • RNA encoding immunostimulant may be administered according to the present disclosure to provide an adjuvant effect.
  • the RNA encoding immunostimulant may be standard RNA or non ⁇ immunogenic RNA.
  • non ⁇ immunogenic RNA refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non ⁇ immunogenic RNA non ⁇ immunogenic, i.e., than would have been induced by standard RNA (stdRNA).
  • stdRNA standard RNA
  • non ⁇ immunogenic RNA which is also termed modified RNA (modRNA) herein, is rendered non ⁇ immunogenic by incorporating modified nucleosides suppressing RNA ⁇ mediated activation of innate immune receptors into the RNA and removing double ⁇ stranded RNA (dsRNA).
  • modified RNA any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA.
  • modified nucleosides that suppress RNA ⁇ mediated activation of innate immune receptors.
  • the modified nucleosides comprises a replacement of one or more uridines with a nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a modified uracil.
  • the nucleoside comprising a modified nucleobase is selected from the group consisting of 3 ⁇ methyl ⁇ uridine (m 3 U), 5 ⁇ methoxy ⁇ uridine (mo 5 U), 5 ⁇ aza ⁇ uridine, 6 ⁇ aza ⁇ uridine, 2 ⁇ thio ⁇ 5 ⁇ aza ⁇ uridine, 2 ⁇ thio ⁇ uridine (s 2 U), 4 ⁇ thio ⁇ uridine (s 4 U), 4 ⁇ thio ⁇ pseudouridine, 2 ⁇ thio ⁇ pseudouridine, 5 ⁇ hydroxy ⁇ uridine (ho 5 U), 5 ⁇ aminoallyl ⁇ uridine, 5 ⁇ halo ⁇ uridine (e.g., 5 ⁇ iodo ⁇ uridine or 5 ⁇ bromo ⁇ uridine), uridine 5 ⁇ oxyacetic acid (cmo 5 U),
  • the nucleoside comprising a modified nucleobase is pseudouridine ( ⁇ ), N1 ⁇ methyl ⁇ pseudouridine (m1 ⁇ ) or 5 ⁇ methyl ⁇ uridine (m5U), in particular N1 ⁇ methyl ⁇ pseudouridine.
  • the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.
  • RNA e.g., mRNA
  • IVT in vitro transcription
  • dsRNA double ⁇ stranded RNA
  • dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition.
  • dsRNA can be removed from RNA such as IVT RNA, for example, by ion ⁇ pair reversed phase HPLC using a non ⁇ porous or porous C ⁇ 18 polystyrene ⁇ divinylbenzene (PS ⁇ DVB) matrix.
  • PS ⁇ DVB non ⁇ porous or porous C ⁇ 18 polystyrene ⁇ divinylbenzene
  • dsRNA can be separated from ssRNA by using a cellulose material.
  • an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.
  • remove or “removal” refers to the characteristic of a population of first substances, such as non ⁇ immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance.
  • a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non ⁇ separated mixture of first and second substances.
  • the removal of dsRNA from non ⁇ immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non ⁇ immunogenic RNA composition is dsRNA.
  • the non ⁇ immunogenic RNA is free or essentially free of dsRNA.
  • the non ⁇ immunogenic RNA composition comprises a purified preparation of single ⁇ stranded nucleoside modified RNA.
  • the purified preparation of single ⁇ stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA).
  • the purified preparation is at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).
  • the non ⁇ immunogenic RNA is translated in a cell more efficiently than standard RNA with the same sequence.
  • translation is enhanced by a factor of 2 ⁇ fold relative to its unmodified counterpart. In one embodiment, translation is enhanced by a 3 ⁇ fold factor. In one embodiment, translation is enhanced by a 4 ⁇ fold factor. In one embodiment, translation is enhanced by a 5 ⁇ fold factor. In one embodiment, translation is enhanced by a 6 ⁇ fold factor. In one embodiment, translation is enhanced by a 7 ⁇ fold factor. In one embodiment, translation is enhanced by an 8 ⁇ fold factor. In one embodiment, translation is enhanced by a 9 ⁇ fold factor. In one embodiment, translation is enhanced by a 10 ⁇ fold factor. In one embodiment, translation is enhanced by a 15 ⁇ fold factor. In one embodiment, translation is enhanced by a 20 ⁇ fold factor. In one embodiment, translation is enhanced by a 50 ⁇ fold factor.
  • translation is enhanced by a 100 ⁇ fold factor. In one embodiment, translation is enhanced by a 200 ⁇ fold factor. In one embodiment, translation is enhanced by a 500 ⁇ fold factor. In one embodiment, translation is enhanced by a 1000 ⁇ fold factor. In one embodiment, translation is enhanced by a 2000 ⁇ fold factor. In one embodiment, the factor is 10 ⁇ 1000 ⁇ fold. In one embodiment, the factor is 10 ⁇ 100 ⁇ fold. In one embodiment, the factor is 10 ⁇ 200 ⁇ fold. In one embodiment, the factor is 10 ⁇ 300 ⁇ fold. In one embodiment, the factor is 10 ⁇ 500 ⁇ fold. In one embodiment, the factor is 20 ⁇ 1000 ⁇ fold. In one embodiment, the factor is 30 ⁇ 1000 ⁇ fold. In one embodiment, the factor is 50 ⁇ 1000 ⁇ fold. In one embodiment, the factor is 100 ⁇ 1000 ⁇ fold.
  • the factor is 200 ⁇ 1000 ⁇ fold. In one embodiment, translation is enhanced by any other significant amount or range of amounts.
  • the non ⁇ immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In one embodiment, the non ⁇ immunogenic RNA exhibits an innate immune response that is 2 ⁇ fold less than its unmodified counterpart. In one embodiment, innate immunogenicity is reduced by a 3 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 4 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 5 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 6 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 7 ⁇ fold factor.
  • innate immunogenicity is reduced by a 8 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 9 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 10 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 15 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 20 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 50 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 100 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 200 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 500 ⁇ fold factor. In one embodiment, innate immunogenicity is reduced by a 1000 ⁇ fold factor.
  • innate immunogenicity is reduced by a 2000 ⁇ fold factor.
  • the term “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity.
  • the term refers to a decrease such that an effective amount of the non ⁇ immunogenic RNA can be administered without triggering a detectable innate immune response.
  • the term refers to a decrease such that the non ⁇ immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non ⁇ immunogenic RNA.
  • the decrease is such that the non ⁇ immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non ⁇ immunogenic RNA.
  • Immunogenicity is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal.
  • the innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term "expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.
  • the terms “linked,” “fused”, or “fusion” are used interchangeably. These terms refer to the joining together of two or more elements or components or domains. Codon ⁇ optimization / Increase in G/C content
  • the amino acid sequence comprising a SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof described herein is encoded by a coding sequence which is codon ⁇ optimized and/or the G/C content of which is increased compared to wild type coding sequence.
  • This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon ⁇ optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence.
  • the codon ⁇ optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
  • codon ⁇ optimized refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule.
  • coding regions are preferably codon ⁇ optimized for optimal expression in a subject to be treated using the RNA molecules described herein. Codon ⁇ optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".
  • the guanosine/cytosine (G/C) content of the coding region of the RNA described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA.
  • This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA (e.g., mRNA). Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content.
  • codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.
  • the G/C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.
  • G/C content of a coding region is increased by about 10% to about 60% (e.g., by about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, or by about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%) compared to the G/C content of the coding region of the wild type RNA.
  • RNA disclosed herein comprises a sequence disclosed herein (e.g., SEQ ID NO: 9), that has been modified to encode one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 varaint (e.g., a BA.2 or a BA.4/5 Omicron variant).
  • RNA can be modified to encode one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant by making as few nucleotide changes as possible.
  • RNA can be modified to encode one or more mutations that are characteristic of a SARS ⁇ CoV ⁇ 2 variant by introducing mutations that result in high codon ⁇ optimization and/or increased G/C content.
  • one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant are introduced onto a full ⁇ length S protein (e.g., an S protein comprising SEQ ID NO: 1).
  • one or more mutations characteristic of a SARS ⁇ CoV ⁇ 2 variant are introduced onto a full ⁇ length S protein having one or more proline mutations that increase stability of a prefusion confirmation.
  • proline substitutions are made at positions corresponding to positions 986 and 987 of SEQ ID NO: 1.
  • proline substitutions are made at positions corresponding to positions 985 and 987 of SEQ ID NO: 1.
  • at least 4 proline substitutions are made.
  • At least four of such proline mutations include mutations at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1.
  • such a SARS ⁇ CoV ⁇ 2 protein comprising proline substitutions at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1 may further comprise proline substitutions at positions corresponding to residues 986 and 987 of SEQ ID NO: 1.
  • such a SARS ⁇ CoV ⁇ 2 protein comprising proline substitutions at positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1 may further comprise proline substitutions at positions corresponding to residues 985 and 987 of SEQ ID NO: 1.
  • RNA e.g., mRNA
  • the present disclosure provides an RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide that comprises at least a portion of a SARS ⁇ CoV ⁇ 2 S protein.
  • the RNA is suitable for intracellular expression of the polypeptide.
  • such an encoded polypeptide comprises a sequence corresponding to the complete S protein.
  • such an encoded polypeptide does not comprise a sequence corresponding to the complete S protein.
  • the encoded polypeptide comprises a sequence that corresponds to the receptor binding domain (RBD).
  • the encoded polypeptide comprises a sequence that corresponds to the RBD, and further comprises a trimerization domain (e.g., a trimerization domain as disclosed herein, such as a fibritin domain).
  • an RBD comprises a signaling domain (e.g., a signaling domain as disclosed herein).
  • an RBD comprises a transmembrane domain (e.g., a transmembrane domain as disclosed herein).
  • an RBD comprises a signaling domain and a trimerization domain.
  • an RBD comprises a signaling domain, a trimerization domain, and transmembrane domain.
  • the encoded polypeptide comprises a sequence that corresponds to two receptor binding domains.
  • the encoded polypeptide comprises a sequence that corresponds to two receptor binding domains in tandem in an amino acid chain, e.g., as disclosed in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against COVID ⁇ 19, MERS, and SARS," Cell 182.3 (2020): 722 ⁇ 733, the contents of which are incorporated by reference herein in their entirety.
  • a SARS ⁇ CoV ⁇ 2 S protein, or an immunogenic fragment thereof comprises one or more mutations to alter, add, or remove a glycosylation site, e.g., as described in WO2022221835A2, US20220323574A1, WO2022266012A1, or WO2022195351A1.
  • compositions or medical preparations described herein comprise RNA encoding an amino acid sequence comprising SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof.
  • methods described herein comprise administration of such RNA.
  • the active platform for use herein is based on an antigen ⁇ coding RNA vaccine to induce robust neutralising antibodies and accompanying/concomitant T cell response to achieve protective immunization with preferably minimal vaccine doses.
  • the RNA administered is preferably in ⁇ vitro transcribed RNA.
  • Three different RNA platforms are particularly preferred, namely non ⁇ modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) and self ⁇ amplifying RNA (saRNA).
  • uRNA non ⁇ modified uridine containing mRNA
  • modRNA nucleoside modified mRNA
  • saRNA self ⁇ amplifying RNA
  • the RNA is in vitro transcribed RNA.
  • uRNA is mRNA.
  • modRNA is mRNA.
  • S1S2 protein/S1S2 RBD Sequences encoding the respective antigen of SARS ⁇ CoV ⁇ 2.
  • nsP1, nsP2, nsP3, and nsP4 Wildtype sequences encoding the Venezuelan equine encephalitis virus (VEEV) RNA ⁇ dependent RNA polymerase replicase and a subgenomic promotor plus conserved sequence elements supporting replication and translation.
  • VEEV Venezuelan equine encephalitis virus
  • virUTR Viral untranslated region encoding parts of the subgenomic promotor as well as replication and translation supporting sequence elements.
  • hAg ⁇ Kozak 5' ⁇ UTR sequence of the human alpha ⁇ globin mRNA with an optimized ⁇ Kozak sequence ⁇ to increase transla ⁇ onal efficiency.
  • Sec corresponds to a secretory signal peptide (sec), which guides translocation of the nascent polypeptide chain into the endoplasmatic reticulum.
  • a secretory signal peptide includes the intrinsic S1S2 secretory signal peptide of a SARS ⁇ CoV ⁇ 2 S protein.
  • such a secretory signal peptide is a secretory signal peptide from a non ⁇ S1S2 protein.
  • an immunoglobulin secretory signal peptide (aa 1 ⁇ 22), an HSV ⁇ 1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV ⁇ 2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA); a human SPARC signal peptide, a human insulin isoform 1 signal peptide, a human albumin signal peptide, or any other signal peptide described herein.
  • Glycine ⁇ serine linker (GS): Sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.
  • Fibritin Partial sequence of T4 fibritin (foldon), used as artificial trimerization domain.
  • TM TM sequence corresponds to the transmembrane part of a protein.
  • a transmembrane domain can be N ⁇ terminal, C ⁇ terminal, or internal to an encoded polypeptide.
  • a coding sequence of a transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences of sequences (e.g., sequences encoding polypeptide(s)) with which it is to be linked.
  • a transmembrane domain comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV ⁇ 1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.
  • HA Hemagglutinin
  • EIAV equine infectious anaemia virus
  • MMV murine leukaemia virus
  • VSV vesicular stomatitis virus
  • Rabies virus or a seven transmembrane domain receptor.
  • the transmembrane part of a protein is from the S1S2 protein.
  • FI element The 3' ⁇ UTR is a combination of two sequence elements derived from the “amino terminal enhancer of split” (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.
  • A30L70 A poly(A) ⁇ tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.
  • vaccine RNA described herein may comprise, from 5' to 3', one of the following structures: Cap ⁇ 5' ⁇ UTR ⁇ Vaccine Antigen ⁇ Encoding Sequence ⁇ 3' ⁇ UTR ⁇ Poly(A) or Cap ⁇ hAg ⁇ Kozak ⁇ Vaccine Antigen ⁇ Encoding Sequence ⁇ FI ⁇ A30L70.
  • a vaccine antigen described herein may comprise a full ⁇ length S protein or an immunogenic fragment thereof (e.g., RBD).
  • a vaccine antigen comprises a full ⁇ length S protein
  • its secretory signal peptide and/or transmembrane domain may be replaced by a heterologous secretory signal peptide (e.g., as described herein) and/or a heterologous transmembrane domain (e.g., as described herein).
  • a vaccine antigen described herein may comprise, from N ⁇ terminus to C ⁇ terminus, one of the following structures: Signal Sequence ⁇ RBD ⁇ Trimerization Domain or Signal Sequence ⁇ RBD ⁇ Trimerization Domain ⁇ Transmembrane Domain.
  • RBD and Trimerization Domain may be separated by a linker, in particular a GS linker such as a linker having the amino acid sequence GSPGSGSGS.
  • Trimerization Domain and Transmembrane Domain may be separated by a linker, in particular a GS linker such as a linker having the amino acid sequence GSGSGS.
  • Signal Sequence may be a signal sequence as described herein.
  • RBD may be a RBD domain as described herein.
  • Trimerization Domain may be a trimerization domain as described herein.
  • Transmembrane Domain may be a transmembrane domain as described herein.
  • Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence
  • RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence
  • Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence
  • Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 9
  • Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31
  • RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1
  • Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10
  • Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.
  • an RNA polynucleotide comprising a sequence encoding a vaccine antigen (e.g., a SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof) or comprising an open reading frame encoding a vaccine antigen (e.g., a SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof) such as the nucleotide sequence of SEQ ID NO: 50 or the nucleotide sequence of SEQ ID NO: 53, a variant or fragment thereof, further comprises a 5’ cap, e.g., a 5’ cap comprising a Cap1 structure, a 5’ UTR sequence, e.g., a 5’ UTR sequence comprising the nucleotide sequence of SEQ ID NO: 12, a 3’ UTR sequence, e.g.,
  • the RNA polynucleotide is formulated in a composition comprising ((4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate), cholesterol, distearoylphosphatidylcholine, and (2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide).
  • the RNA described herein or RNA encoding the vaccine antigen described herein may be non ⁇ modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) or self ⁇ amplifying RNA (saRNA).
  • RNA described herein or RNA encoding the vaccine antigen described herein is nucleoside modified mRNA (modRNA).
  • modRNA nucleoside modified mRNA
  • RNA disclosed herein encodes an S protein comprising one or more mutations that are characteristic of a SARS ⁇ CoV ⁇ 2 variant.
  • RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Alpha variant.
  • RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a Beta variant.
  • RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a Delta variant.
  • RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron variant (e.g., an S protein comprising one or more mutations characteristic of a BA.1, BA.2, or BA.4/5 Omicron variant). In some embodiments, RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an BA.1 Omicron variant. In some embodiments, RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an BA.2 Omicron variant. In some embodiments, RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an BA.2.12.1 Omicron variant.
  • RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a BA.3 Omicron variant. In some embodiments, RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a BA.4 Omicron variant. In some embodiments, RNA encodes a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of a BA.5 Omicron variant.
  • Non ⁇ modified uridine messenger RNA (uRNA) The active principle of the non ⁇ modified messenger RNA (uRNA) drug substance is a single ⁇ stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding the coronavirus vaccine antigen (i.e.
  • each uRNA preferably contains common structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5′ ⁇ cap, 5′ ⁇ UTR, 3′ ⁇ UTR, poly(A) ⁇ tail).
  • the preferred 5’ cap structure is beta ⁇ S ⁇ ARCA(D1) (m 2 7,2' ⁇ O GppSpG).
  • the preferred 5′ ⁇ UTR and 3′ ⁇ UTR comprise the nucleotide sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQ ID NO: 13, respectively.
  • the preferred poly(A) ⁇ tail comprises the sequence of SEQ ID NO: 14.
  • RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ hAg ⁇ Kozak ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant)
  • RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ hAg ⁇ Kozak ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant) BNT162a1;
  • RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ hAg ⁇ Kozak ⁇ RBD ⁇ GS ⁇ Fibritin ⁇ FI ⁇ A30L70 Encoded antigen Viral
  • Nucleoside modified messenger RNA The active principle of the nucleoside modified messenger RNA (modRNA) drug substance is as well a single ⁇ stranded mRNA that is translated upon entering a cell.
  • each modRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5′ ⁇ cap, 5′ ⁇ UTR, 3′ ⁇ UTR, poly(A) ⁇ tail). Compared to the uRNA, modRNA contains 1 ⁇ methyl ⁇ pseudouridine instead of uridine.
  • the preferred 5’ cap structure is m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG.
  • the preferred 5′ ⁇ UTR and 3′ ⁇ UTR comprise the nucleotide sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQ ID NO: 13, respectively.
  • the preferred poly(A) ⁇ tail comprises the sequence of SEQ ID NO: 14.
  • BNT162b3c (SEQ ID NO: 29; SEQ ID NO: 30) Structure m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG ⁇ hAg ⁇ Kozak ⁇ RBD ⁇ GS ⁇ Fibritin ⁇ GS ⁇ TM ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused to Transmembrane Domain (TM) of S1S2 protein); intrinsic S1S2 protein secretory signal peptide (aa 1 ⁇ 19) at the N ⁇ terminus of the antigen sequence BNT162b3d (SEQ ID NO: 31; SEQ ID NO: 32) Structure m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG ⁇ hAg ⁇ Kozak ⁇ RBD ⁇ GS ⁇ Fibritin ⁇ GS ⁇ TM ⁇ FI ⁇ A30L70
  • RBP020.11 Structure m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG) ⁇ hAg ⁇ Kozak ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant), comprising mutations characteristic of the Beta variant of SARS ⁇ CoV ⁇ 2 BNT162b2 – Alpha variant; RBP020.14 (SEQ ID NO: 60; SEQ ID NO: 58) Structure m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG) ⁇ hAg ⁇ Kozak ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant), comprising mutations
  • RNA constructs encoding SARS ⁇ CoV ⁇ 2 spike sequence variants are shown in Tables 8 ⁇ 18.
  • the spike protein sequence and encoding DNA and RNA sequence are provided.
  • exemplary full length RNA vaccine and corresponding DNA sequences are provided.
  • U may represent a naturally ⁇ occurring uridine or a modified uridine, e.g., pseudouridine.
  • a poly ⁇ A tail is included in the sequence.
  • RNA and DNA sequences described herein may include a polyA tail that is shorter or longer than what is shown, e.g., by at least 1, at least 2, at least 3, at least 4 nucletodides and up to at least 10 “A” nucleotides.
  • an RNA construct encoding a spike protein from a coronavirus variant as described in Tables 7 ⁇ 18a has a structure as shown below: m 2 7,3’ ⁇ O Gppp(m 1 2’ ⁇ O )ApG) ⁇ hAg ⁇ Kozak ⁇ Antigen ⁇ FI ⁇ A30L70, wherein the encoded “Antigen” is the viral spike protein (S1S2 protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein) as indicated in Tables 7 ⁇ 18a.
  • I S S V L N D I mut4 I R A A E I mut4 y Sequences of RBP020.14 are also shown in Table 11.
  • Table 25 Description of sequences of RBP020.23 (Omicron BA.4/BA.5-specific RNA vaccine) as described in Table 24 above
  • saRNA Self ⁇ amplifying RNA
  • the active principle of the self ⁇ amplifying mRNA (saRNA) drug substance is a single ⁇ stranded RNA, which self ⁇ amplifies upon entering a cell, and the coronavirus vaccine antigen is translated thereafter.
  • the coding region of saRNA contains two open reading frames (ORFs).
  • the 5’ ⁇ ORF encodes the RNA ⁇ dependent RNA polymerase such as Venezuelan equine encephalitis virus (VEEV) RNA ⁇ dependent RNA polymerase (replicase).
  • VEEV Venezuelan equine encephalitis virus
  • replicase RNA ⁇ dependent RNA polymerase
  • the replicase ORF is followed 3’ by a subgenomic promoter and a second ORF encoding the antigen.
  • saRNA UTRs contain 5’ and 3’ conserved sequence elements (CSEs) required for self ⁇ amplification.
  • the saRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5′ ⁇ cap, 5′ ⁇ UTR, 3′ ⁇ UTR, poly(A) ⁇ tail).
  • the saRNA preferably contains uridine.
  • the saRNA comprises one or more nucleoside modifications as described herein.
  • the preferred 5’ cap structure is beta ⁇ S ⁇ ARCA(D1) (m 2 7,2' ⁇ O GppSpG).
  • an saRNA described herein encodes a single antigen (e.g., one SARS ⁇ CoV ⁇ 2 S polypeptide).
  • an saRNA utilized in accordance with the present disclosure encodes two or more antigens (e.g., two or more SARS ⁇ CoV ⁇ 2 S polypeptides,).
  • an saRNA encodes two S polypeptides, each from a different SARS ⁇ CoV ⁇ 2 variant.
  • an saRNA platform can provide certain advantages as compared to other RNA platforms. For example, in some embodiments, saRNA can provide increased duration of expression of an antigen, lower dose levels, improved tolerability, and/or increased antigen capacity, while maintaining a robust antibody and T cell response. Cytoplasmic delivery of saRNA initiates an alphavirus ⁇ like life cycle.
  • the saRNA does not encode for alphaviral structural proteins that are required for genome packaging or cell entry, therefore generation of replication competent viral particles is very unlikely to not possible. Replication does not involve any intermediate steps that generate DNA. The use/uptake of saRNA therefore poses no risk of genomic integration or other permanent genetic modification within the target cell. Furthermore, the saRNA itself prevents its persistent replication by effectively activating innate immune response via recognition of dsRNA intermediates.
  • RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ replicase ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant)
  • RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ replicase ⁇ S1S2 ⁇ PP ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S protein) of the SARS ⁇ CoV ⁇ 2 (S1S2 full ⁇ length protein, sequence variant) BNT162c1;
  • RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5) Structure beta ⁇ S ⁇ ARCA(D1) ⁇ replicase ⁇ RBD ⁇ GS ⁇ Fibritin ⁇ FI ⁇ A30L70 Encoded antigen Viral spike protein (S protein) of the SARS ⁇ CoV ⁇ 2 (par
  • vaccine RNA described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, 19, 20, 21, 24, 25, 26, 27, 30, and 32.
  • a particularly preferred vaccine RNA described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 17, 19, 21, 25, 26, 30, and 32 such as selected from the group consisting of SEQ ID NO: 17, 19, 21, 26, 30, and 32.
  • RNA described herein is formulated in lipid nanoparticles, lipoplex, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles.
  • RNA described herein is preferably formulated in lipid nanoparticles (LNP).
  • the LNP comprise a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA.
  • the cationic lipid is ALC ⁇ 0315
  • the neutral lipid is DSPC
  • the steroid is cholesterol
  • the polymer conjugated lipid is ALC ⁇ 0159.
  • the preferred mode of administration is intramuscular administration, more preferably in aqueous cryoprotectant buffer for intramuscular administration.
  • the drug product is a preferably a preservative ⁇ free, sterile dispersion of RNA formulated in lipid nanoparticles (LNP) in aqueous cryoprotectant buffer for intramuscular administration.
  • particles disclosed herein are formulated in a solution comprising 10 mM Tris and 10% sucrose, and optionally having a pH of about 7.4. In some embodiments, particles disclosed herein are formulated in a solution comprising about 103 mg/ml sucrose, about 0.20 mg/ml tromethamine (Tris base), and about 1.32 mg/ml Tris.
  • a composition comprises: (a) about 0.1 mg/mL RNA comprising an open reading frame encoding a polypeptide that comprises a SARS ⁇ CoV ⁇ 2 protein or an immunogenic fragment or variant thereof, (b) about 1.43 mg/ml ALC ⁇ 0315, (c) about 0.18 mg/ml ALC ⁇ 0159 (d) about 0.31 mg/ml DSPC, (e) about 0.62 mg/ml cholesterol, (f) about 103 mg/ml sucrose, (g) about 0.20 mg/ml tromethamine (Tris base), (h) about 1.32 mg/ml Tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl), and (i) q.s. water.
  • the ratio of RNA (e.g., mRNA) to total lipid (N/P) is between 6.0 and 6.5 such as about 6.0 or about 6.3.
  • Nucleic acid containing particles Nucleic acids described herein such as RNA encoding a vaccine antigen may be administered formulated as particles.
  • the term "particle” relates to a structured entity formed by molecules or molecule complexes.
  • the term "particle” relates to a micro ⁇ or nano ⁇ sized structure, such as a micro ⁇ or nano ⁇ sized compact structure dispersed in a medium.
  • a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof.
  • nucleic acid particle is a nanoparticle.
  • nanoparticle refers to a particle having an average diameter suitable for parenteral administration.
  • a “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like).
  • a nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid ⁇ like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid.
  • exemplary nucleic acid particles include lipid nanoparticles, polyplexes (PLX), lapidated polyplexes (LPLX), (LNP) ⁇ based and lipoplex (LPX) ⁇ based formulations, liposomes, or polysaccharide nanoparticles.
  • RNA encoding an amino acid sequence comprising a SARS ⁇ CoV ⁇ 2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS ⁇ CoV ⁇ 2 S protein or the immunogenic variant thereof is formulated as LNPs.
  • LNPs comprise one or more cationically ionizable lipids; one or more neutral lipids (e.g., in some embodiments a sterol such as, e.g., cholesterol; and/or phospholipids), and one or more polymer ⁇ conjugated lipids.
  • the formulation comprises ALC ⁇ 0315 (4 ⁇ hydroxybutyl)azanediyl)bis(hexane ⁇ 6,1 ⁇ diyl)bis(2 ⁇ hexyldecanoate), ALC ⁇ 0159 (2 ⁇ [(polyethylene glycol) ⁇ 2000] ⁇ N,N ⁇ ditetradecylacetamide), DSPC (1,2 ⁇ distearoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride and water.
  • RNA particles described herein include nanoparticles.
  • exemplary nanoparticles include lipid nanoparticles, lipoplex, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles.
  • Polyplexes (PLX), polysaccharide nanoparticles, and liposomes are all delivery technologies that are well known to a person of skill in the art. See, e.g., Lächelt, Ulrich, and Ernst Wagner.
  • the concentration of RNA in a pharmaceutical RNA preparation is about 0.1 mg/ml. In some embodiments, the concentration of RNA in a pharmaceutical RNA preparation is about 30 ⁇ g/ml to about 100 ⁇ g/ml. In some embodiments, the concentration of RNA in a pharmaceutical RNA preparation is about 50 ⁇ g/ml to about 100 ⁇ g/ml. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid ⁇ like material and/or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.
  • particles described herein further comprise at least one lipid or lipid ⁇ like material other than a cationic or cationically ionizable lipid or lipid ⁇ like material, at least one polymer other than a cationic polymer, or a mixture thereof
  • nucleic acid particles comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.
  • Nucleic acid particles described herein may have an average diameter that in one embodiment ranges from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.
  • Nucleic acid particles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less.
  • the nucleic acid particles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.
  • the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged.
  • the N/P ratio where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.
  • Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid ⁇ like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles.
  • the term "colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers.
  • the mixture may be termed a colloid or a colloidal suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not the entire suspension.
  • colloids comprising at least one cationic or cationically ionizable lipid or lipid ⁇ like material and/or at least one cationic polymer methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted.
  • the most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).
  • film hydration method lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask.
  • the obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included.
  • Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water ⁇ in ⁇ oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.
  • ethanol injection technique refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle.
  • the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion.
  • colloidal liposome dispersion is, in one embodiment, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring.
  • the RNA lipoplex particles described herein are obtainable without a step of extrusion.
  • the term "extruding" or "extrusion” refers to the creation of particles having a fixed, cross ⁇ sectional profile.
  • LNPs typically comprise four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid such as polyethylene glycol (PEG) ⁇ lipids. Each component is responsible for payload protection, and enables effective intracellular delivery. LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid in an aqueous buffer.
  • average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so ⁇ called cumulant algorithm, which provides as results the so ⁇ called Z average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814 ⁇ 4820, ISO 13321).
  • PI polydispersity index
  • average diameter "diameter” or size” for particles is used synonymously with this value of the Z average .
  • the "polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so ⁇ called cumulant analysis as mentioned in the definition of the "average diameter”.
  • nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60).
  • nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.
  • the present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid ⁇ like material, and/or at least one cationic polymer which associate with nucleic acid to form nucleic acid particles and compositions comprising such particles.
  • the nucleic acid particles may comprise nucleic acid which is complexed in different forms by non ⁇ covalent interactions to the particle.
  • the particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells.
  • Suitable cationic or cationically ionizable lipids or lipid ⁇ like materials and cationic polymers are those that form nucleic acid particles and are included by the term "particle forming components" or “particle forming agents".
  • the term “particle forming components” or “particle forming agents” relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles.
  • a nucleic acid containing particle e.g., a lipid nanoparticle (LNP)
  • LNP lipid nanoparticle
  • a nucleic acid containing particle comprises two or more RNA molecules, each encoding a different immunogenic polypeptide or immunogenic fragment thereof.
  • two or more RNA molecules present in a nucleic acid containing particle comprise: a first RNA molecule encodes an immunogenic polypeptide or immunogenic fragment thereof from a coronavirus and a second RNA molecule encodes an immunogenic polypeptide or immunogenic fragment thereof from an infectious disease pathogen (e.g., virus, bacteria, parasite, etc.).
  • infectious disease pathogen e.g., virus, bacteria, parasite, etc.
  • two or more RNA molecules present in a nucleic acid containing particle comprise: a first RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a coronavirus (e.g., in some embodiments SARS ⁇ CoV ⁇ 2 Wuhan strain or a variant thereof, e.g., a SARS ⁇ CoV ⁇ 2 having one or more mutations characteristic of an Omicron variant) and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from an influenza virus.
  • a coronavirus e.g., in some embodiments SARS ⁇ CoV ⁇ 2 Wuhan strain or a variant thereof, e.g., a SARS ⁇ CoV ⁇ 2 having one or more mutations characteristic of an Omicron variant
  • a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from an influenza virus.
  • a nucleic acid containing particle comprises: a first RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a first coronavirus (e.g., as described herein) and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from a second coronavirus (e.g., as described herein).
  • a first coronavirus is different from a second coronavirus.
  • a first and/or second coronavirus is independently from a SARS ⁇ CoV ⁇ 2 Wuhan strain or a variant thereof, e.g., a SARS ⁇ CoV ⁇ 2 having one or more mutations characteristic of an Omicron variant.
  • two or more RNA molecules present in a nucleic acid containing particle each encode an immunogenic polypeptide or an immunogenic fragment thereof from the same and/or different strains and/or variants of coronavirus (e.g., in some embodiments SARS ⁇ CoV ⁇ 2 strains or variants).
  • two or more RNA molecules present in a nucleic acid containing particle each encode a different immunogenic polypeptide or immunogenic fragment thereof from a coronavirus membrane protein, a coronavirus nucleocapsid protein, a coronavirus spike protein, a coronavirus non ⁇ structural protein and/or a coronavirus accessory protein.
  • such immunogenic polypeptides or immunogenic fragments thereof may be from the same or a different coronavirus (e.g., in some embodiments a SARS ⁇ CoV ⁇ 2 Wuhan strain or variants thereof, for example, in some embodiments a variant having one or more mutations characteristic of a prevalent variant such as an Omicron variant).
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein or an immunogenic fragment thereof from a first strain or variant, and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein or an immunogenic fragment thereof from a second strain or variant, wherein the second strain or variant is different from the first strain or variant.
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 Omicron variant).
  • an Omicron variant e.g., a BA.1, BA.2, BA.3, BA.4, or BA.5 Omicron variant
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of an Omicron BA.1 variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of an Omicron BA.2 variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of an Omicron BA.3 variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of an Omicron BA.4 or BA.5 variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a first Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a second Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.1 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.2 Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.1 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.3 Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.1 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4 or BA.5 Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.2 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.3 Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.2 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4 or BA.5 Omicron variant.
  • a nucleic acid containing particle comprises a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a BA.3 Omicron variant and a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations that are characteristic of a BA.4 or BA.5 Omicron variant.
  • a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7.
  • a first RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein from a Wuhan strain comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 9.
  • a first RNA molecule encoding a SARS ⁇ COV ⁇ 2 S protein from a Wuhan strain comprises a nucleotide sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) SEQ ID NO: 20.
  • a first RNA molecule encoding a SARS ⁇ COV ⁇ 2 S protein from a Wuhan strain comprises a nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) SEQ ID NO: 7.
  • a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein having one or more mutations that are characteristic of an Omicron variant comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49.
  • a second RNA molecule encoding a SARS ⁇ CoV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron variant comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO: 50.
  • a second RNA molecule encoding a SARS ⁇ COV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron variant comprises a nucleotide sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) SEQ ID NO: 51.
  • a second RNA molecule encoding a SARS ⁇ COV ⁇ 2 S protein comprising one or more mutations characteristic of an Omicron variant comprises a nucleotide sequence that encodes an amino acid sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to) SEQ ID NO: 49.
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 7); and a second RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO:
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 9); and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 50.
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 20; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 51.
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 7); and a second RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 55, 58, or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 9; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 56, 59, or 62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 20; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 57, 60, or 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 58; and a second RNA molecule comprising a nucleotide sequence that encodes an amino acid sequence of SEQ ID NO: 49, 55, or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 59 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 59; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 50, 56, or 62, or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher)
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 60 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 60; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 51, 57, or 63, or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 49; and a second RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 55 or 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher)
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 50; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 56 or 62 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO:
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 51; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 57 or 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 51;
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 55; and a second RNA molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 56; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO:
  • a nucleic acid containing particle (e.g., in some embodiments an LNP as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ ID NO: 57 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 57; and a second RNA molecule comprising a nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO
  • a particle e.g., in some embodiments an LNP
  • nucleic acids e.g., RNAs
  • a particle ⁇ forming components e.g., lipids
  • nucleic acids e.g., RNAs
  • nucleic acids e.g., RNAs
  • nucleic acids e.g., RNAs
  • nucleic acids e.g., RNAs
  • nucleic acids encoding different polypeptides
  • can be mixed e.g., in some embodiments in substantially equal proportions, e.g., in some embodiments at a 1:1 ratio when two RNA molecules are present
  • particle ⁇ forming components e.g., lipids
  • two or more RNA molecules each encoding a different polypeptide can be mixed with particle ⁇ forming agents to form nucleic acid containing particles as described above.
  • two or more RNA molecules each encoding a different polypeptide can be formulated into separate particle compositions, which are then mixed together.
  • individual populations of nucleic acid containing particles each population comprising an RNA molecule encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., as described herein), can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration (e.g., by an administering health ⁇ care professional)).
  • a composition comprises two or more populations of particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one RNA molecule encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., a SARS ⁇ CoV ⁇ 2 S protein, or fragments thereof, from a different variant).
  • each population may be provided in a composition at a desirable proportion (e.g., in some embodiments, each population may be provided in a composition in an amount that provides the same amount of RNA molecules).
  • Cationic polymer Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle ⁇ based delivery.
  • cationic polymers are used to electrostatically condense the negatively charged nucleic acid into nanoparticles.
  • These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture.
  • Polymers such as poly ⁇ L ⁇ lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein.
  • some investigators have synthesized polymers specifically for nucleic acid delivery.
  • the polymer is biologically derived, i.e., a biopolymer such as a protein.
  • additional moieties can also be present in the polymer, for example targeting moieties such as those described herein.
  • the polymer is said to be a "copolymer.” It is to be understood that the polymer being employed herein can be a copolymer.
  • the repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc.
  • Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
  • the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body. In certain embodiments, polymer may be protamine or polyalkyleneimine, in particular protamine.
  • protamine refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish).
  • protamine refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long ⁇ acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
  • the term "protamine” as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.
  • the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine.
  • a preferred polyalkyleneimine is polyethyleneimine (PEI).
  • the average molecular weight of PEI is preferably 0.75 ⁇ 10 2 to 10 7 Da, preferably 1000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.
  • Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).
  • Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid.
  • cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g.
  • lipid and lipid ⁇ like material are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles.
  • Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self ⁇ assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long ⁇ chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • amphiphilic refers to a molecule having both a polar portion and a non ⁇ polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non ⁇ polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge.
  • the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non ⁇ natural lipids and lipid ⁇ like compounds.
  • the term "lipid ⁇ like material", “lipid ⁇ like compound” or “lipid ⁇ like molecule” relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • lipid is to be construed to cover both lipids and lipid ⁇ like materials unless otherwise indicated herein or clearly contradicted by context.
  • amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • the amphiphilic compound is a lipid.
  • lipid refers to a group of organic compounds that are characterized by being insoluble in water, but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri ⁇ , di ⁇ , monoglycerides, and phospholipids), as well as sterol ⁇ containing metabolites such as cholesterol. Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis ⁇ double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Glycerolipids are composed of mono ⁇ , di ⁇ , and tri ⁇ substituted glycerols, the best ⁇ known being the fatty acid triesters of glycerol, called triglycerides.
  • triacylglycerol is sometimes used synonymously with "triglyceride”.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids.
  • Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • the glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid ⁇ derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • Examples of glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone.
  • the major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides N ⁇ acyl ⁇ sphingoid bases
  • Ceramides are a major subclass of sphingoid base derivatives with an amide ⁇ linked fatty acid.
  • the fatty acids are typically saturated or mono ⁇ unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose ⁇ containing headgroups.
  • glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterol lipids such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins. Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • saccharolipids a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram ⁇ negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty ⁇ acyl chains. The minimal lipopolysaccharide required for growth in E.
  • coli is Kdo2 ⁇ Lipid A, a hexa ⁇ acylated disaccharide of glucosamine that is glycosylated with two 3 ⁇ deoxy ⁇ D ⁇ manno ⁇ octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity.
  • lipids and lipid ⁇ like materials may be cationic, anionic or neutral.
  • Neutral lipids or lipid ⁇ like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • Cationic or cationically ionizable lipids or lipid ⁇ like materials The nucleic acid particles described herein may comprise at least one cationic or cationically ionizable lipid or lipid ⁇ like material as particle forming agent.
  • Cationic or cationically ionizable lipids or lipid ⁇ like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid ⁇ like materials which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids or lipid ⁇ like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • a "cationic lipid” or “cationic lipid ⁇ like material” refers to a lipid or lipid ⁇ like material having a net positive charge. Cationic lipids or lipid ⁇ like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid or lipid ⁇ like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH.
  • This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • such "cationically ionizable" lipids or lipid ⁇ like materials are comprised by the term "cationic lipid or lipid ⁇ like material" unless contradicted by the circumstances.
  • the cationic or cationically ionizable lipid or lipid ⁇ like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.
  • cationic lipids include, but are not limited to 1,2 ⁇ dioleoyl ⁇ 3 ⁇ trimethylammonium propane (DOTAP); N,N ⁇ dimethyl ⁇ 2,3 ⁇ dioleyloxypropylamine (DODMA), 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane (DOTMA), 3 ⁇ (N—(N′,N′ ⁇ dimethylaminoethane) ⁇ carbamoyl)cholesterol (DC ⁇ Chol), dimethyldioctadecylammonium (DDAB); 1,2 ⁇ dioleoyl ⁇ 3 ⁇ dimethylammonium ⁇ propane (DODAP); 1,2 ⁇ diacyloxy ⁇ 3 ⁇ dimethylammonium propanes; 1,2 ⁇ dio
  • the cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle.
  • Additional lipids or lipid ⁇ like materials Particles described herein may also comprise lipids or lipid ⁇ like materials other than cationic or cationically ionizable lipids or lipid ⁇ like materials, i.e., non ⁇ cationic lipids or lipid ⁇ like materials (including non ⁇ cationically ionizable lipids or lipid ⁇ like materials).
  • anionic and neutral lipids or lipid ⁇ like materials are referred to herein as non ⁇ cationic lipids or lipid ⁇ like materials.
  • Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid ⁇ like material may enhance particle stability and efficacy of nucleic acid delivery.
  • An additional lipid or lipid ⁇ like material may be incorporated which may or may not affect the overall charge of the nucleic acid particles.
  • the additional lipid or lipid ⁇ like material is a non ⁇ cationic lipid or lipid ⁇ like material.
  • the non ⁇ cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • an "anionic lipid” refers to any lipid that is negatively charged at a selected pH.
  • a "neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl ⁇ 2' ⁇ hydroxyethyl ether, cholesteryl ⁇ 4' ⁇ hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl ⁇ phosphatidylcholine (POPC), 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine (18:0 Diether PC), 1 ⁇ ole
  • the additional lipid is DSPC or DSPC and cholesterol.
  • the nucleic acid particles include both a cationic lipid and an additional lipid.
  • particles described herein include a polymer conjugated lipid such as a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.
  • the non ⁇ cationic lipid, in particular neutral lipid, may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in the particle.
  • RNA lipoplex particles In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.
  • the term "RNA lipoplex particle” relates to a particle that contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE.
  • a RNA lipoplex particle is a nanoparticle.
  • the RNA lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.
  • RNA lipoplex particles described herein have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.
  • the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.
  • the RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration.
  • the RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase.
  • the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM.
  • Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane (DOTMA) and/or 1,2 ⁇ dioleoyl ⁇ 3 ⁇ trimethylammonium ⁇ propane (DOTAP).
  • DOTMA 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane
  • DOTAP 1,2 ⁇ dioleoyl ⁇ 3 ⁇ trimethylammonium ⁇ propane
  • the at least one additional lipid comprises 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2 ⁇ dioleoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphocholine (DOPC).
  • the at least one cationic lipid comprises 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine (DOPE).
  • DOPE 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine
  • DOPE 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine
  • the liposomes and RNA lipoplex particles comprise 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane (DOTMA) and 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine (DOPE).
  • DOTMA 1,2 ⁇ di ⁇ O ⁇ octadecenyl ⁇ 3 ⁇ trimethylammonium propane
  • DOPE 1,2 ⁇ di ⁇ (9Z ⁇ octadecenoyl) ⁇ sn ⁇ glycero ⁇ 3 ⁇ phosphoethanolamine
  • Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen ⁇ presenting cells, in particular dendritic cells.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen.
  • no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs.
  • RNA accumulation and/or RNA expression in antigen presenting cells such as professional antigen presenting cells in the spleen occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells.
  • the antigen presenting cells are dendritic cells and/or macrophages.
  • Lipid nanoparticles LNPs
  • nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs).
  • LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
  • the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.
  • the LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid.
  • the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.
  • the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.
  • the steroid is present in a concentration ranging from 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43 mol percent.
  • the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.
  • the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugated lipid.
  • the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.
  • the mol percent is determined based on total mol of lipid present in the lipid nanoparticle.
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In one embodiment, the neutral lipid is DSPC. In one embodiment, the steroid is cholesterol. In one embodiment, the polymer conjugated lipid is a pegylated lipid.
  • the pegylated lipid has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In one embodiment, R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In one embodiment, w has a mean value ranging from 40 to 55. In one embodiment, the average w is about 45.
  • R 12 and R 13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.
  • the pegylated lipid is DMG ⁇ PEG 2000, e.g., having the following structure:
  • the lipid has one of the following structures (IIIA) or (IIIB): (IIIA) (IIIB) wherein: A is a 3 to 8 ⁇ membered cycloalkyl or cycloalkylene ring; R 6 is, at each occurrence, independently H, OH or C 1 ⁇ C 24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or (IIID): (IIIC) (IIID) wherein y and z are each independently integers ranging from 1 to 12.
  • the lipid has one of the following structures (IIIE) or (IIIF): (IIIE) (IIIF)
  • the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): (IIIG) (IIIH) .
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4.
  • n is 5.
  • n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H.
  • R 6 is C 1 ⁇ C 24 alkyl.
  • R 6 is OH.
  • G 3 is unsubstituted.
  • G3 is substituted.
  • G 3 is linear C 1 ⁇ C 24 alkylene or linear C 1 ⁇ C 24 alkenylene.
  • R 1 or R 2 is C 6 ⁇ C 24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: , wherein: R 7a and R 7b are, at each occurrence, independently H or C 1 ⁇ C 12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • at least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C 1 ⁇ C 8 alkyl.
  • C 1 ⁇ C 8 alkyl is methyl, ethyl, n ⁇ propyl, iso ⁇ propyl, n ⁇ butyl, iso ⁇ butyl, tert ⁇ butyl, n ⁇ hexyl or n ⁇ octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in the table below. Table 26: Representative Compounds of Formula (III).
  • the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid and a pegylated lipid.
  • the lipid of Formula (III) is compound III ⁇ 3.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is ALC ⁇ 0159.
  • the cationic lipid is present in the LNP in an amount from about 40 to about 50 mole percent.
  • the neutral lipid is present in the LNP in an amount from about 5 to about 15 mole percent.
  • the steroid is present in the LNP in an amount from about 35 to about 45 mole percent.
  • the pegylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent.
  • the LNP comprises compound III ⁇ 3 in an amount from about 40 to about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol in an amount from about 35 to about 45 mole percent, and ALC ⁇ 0159 in an amount from about 1 to about 10 mole percent. In some embodiments, the LNP comprises compound III ⁇ 3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC ⁇ 0159 in an amount of about 1.8 mole percent. In various different embodiments, the cationic lipid has one of the structures set forth in the table below. Table 27: Representative cationic lipids.
  • the LNP comprises a cationic lipid shown in the above table, e.g., a cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid and a pegylated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is DMG ⁇ PEG 2000.
  • the LNP comprises a cationic lipid that is an ionizable lipid ⁇ like material (lipidoid).
  • the cationic lipid has the following structure: The N/P value is preferably at least about 4.
  • the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.
  • LNP described herein may have an average diameter that in one embodiment ranges from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.
  • RNA Targeting Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein (e.g., RNA encoding vaccine antigens and/or immunostimulants). In one embodiment, the disclosure involves targeting lung. Targeting lung is in particular preferred if the RNA administered is RNA encoding vaccine antigen.
  • RNA may be delivered to lung, for example, by administering the RNA which may be formulated as particles as described herein, e.g., lipid particles, by inhalation.
  • the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular preferred if the RNA administered is RNA encoding vaccine antigen.
  • the target cell is a spleen cell.
  • the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen.
  • the target cell is a dendritic cell in the spleen.
  • the "lymphatic system” is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph.
  • the lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph.
  • the primary or central lymphoid organs generate lymphocytes from immature progenitor cells.
  • the thymus and the bone marrow constitute the primary lymphoid organs.
  • Secondary or peripheral lymphoid organs which include lymph nodes and the spleen, maintain mature na ⁇ ve lymphocytes and initiate an adaptive immune response.
  • RNA may be delivered to spleen by so ⁇ called lipoplex formulations, in which the RNA is bound to liposomes comprising a cationic lipid and optionally an additional or helper lipid to form injectable nanoparticle formulations.
  • the liposomes may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase.
  • RNA lipoplex particles may be prepared by mixing the liposomes with RNA. Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference.
  • RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen ⁇ presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells.
  • the antigen presenting cells are dendritic cells and/or macrophages.
  • the electric charge of the RNA lipoplex particles of the present disclosure is the sum of the electric charges present in the at least one cationic lipid and the electric charges present in the RNA.
  • the charge ratio is the ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA.
  • the spleen targeting RNA lipoplex particles described herein at physiological pH preferably have a net negative charge such as a charge ratio of positive charges to negative charges from about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2.
  • the charge ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.
  • Immunostimulants may be provided to a subject by administering to the subject RNA encoding an immunostimulant in a formulation for preferential delivery of RNA to liver or liver tissue. The delivery of RNA to such target organ or tissue is preferred, in particular, if it is desired to express large amounts of the immunostimulant and/or if systemic presence of the immunostimulant, in particular in significant amounts, is desired or required.
  • RNA delivery systems have an inherent preference to the liver. This pertains to lipid ⁇ based particles, cationic and neutral nanoparticles, in particular lipid nanoparticles such as liposomes, nanomicelles and lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates).
  • a drug delivery system may be used to transport the RNA into the liver by preventing its degradation.
  • polyplex nanomicelles consisting of a poly(ethylene glycol) (PEG) ⁇ coated surface and an RNA (e.g., mRNA) ⁇ containing core is a useful system because the nanomicelles provide excellent in vivo stability of the RNA, under physiological conditions. Furthermore, the stealth property provided by the polyplex nanomicelle surface, composed of dense PEG palisades, effectively evades host immune defenses.
  • suitable immunostimulants for targeting liver are cytokines involved in T cell proliferation and/or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended ⁇ PK cytokines.
  • RNA encoding an immunostimulant may be administered in a formulation for preferential delivery of RNA to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen.
  • the delivery of an immunostimulant to such target tissue is preferred, in particular, if presence of the immunostimulant in this organ or tissue is desired (e.g., for inducing an immune response, in particular in case immunostimulants such as cytokines are required during T ⁇ cell priming or for activation of resident immune cells), while it is not desired that the immunostimulant is present systemically, in particular in significant amounts (e.g., because the immunostimulant has systemic toxicity).
  • suitable immunostimulants are cytokines involved in T cell priming.
  • Suitable cytokines include IL12, IL15, IFN ⁇ , or IFN ⁇ , fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended ⁇ PK cytokines.
  • Immunostimulants In one embodiment, the RNA encoding vaccine antigen may be non ⁇ immunogenic. In this and other embodiments, the RNA encoding vaccine antigen may be co ⁇ administered with an immunostimulant or RNA encoding an immunostimulant. The methods and agents described herein are particularly effective if the immunostimulant is attached to a pharmacokinetic modifying group (hereafter referred to as "extended ⁇ pharmacokinetic (PK)" immunostimulant).
  • PK pharmacokinetic modifying group
  • RNA encoding an immunostimulant is administered in the form of RNA encoding an immunostimulant.
  • said RNA is targeted to the liver for systemic availability. Liver cells can be efficiently transfected and are able to produce large amounts of protein.
  • An “immunostimulant” is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells.
  • the immunostimulant may be pro ⁇ inflammatory.
  • the immunostimulant is a cytokine or a variant thereof.
  • cytokines examples include interferons, such as interferon ⁇ alpha (IFN ⁇ ) or interferon ⁇ gamma (IFN ⁇ ), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M ⁇ CSF and GM ⁇ CSF, and tumor necrosis factor.
  • the immunostimulant includes an adjuvant ⁇ type immunostimulatory agent such as APC Toll ⁇ like Receptor agonists or costimulatory/cell adhesion membrane proteins.
  • Toll ⁇ like Receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM ⁇ 1.
  • Cytokines are a category of small proteins ( ⁇ 5–20 kDa) that are important in cell signaling. Their release has an effect on the behavior of cells around them. Cytokines are involved in autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells.
  • a given cytokine may be produced by more than one type of cell.
  • Cytokines act through receptors, and are especially important in the immune system; cytokines modulate the balance between humoral and cell ⁇ based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.
  • a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof.
  • a cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal.
  • One particularly preferred cytokine is interferon ⁇ .
  • Interferons Interferons are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus ⁇ infected cell will release interferons causing nearby cells to heighten their anti ⁇ viral defenses. Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon. All type I interferons bind to a specific cell surface receptor complex known as the IFN ⁇ / ⁇ receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.
  • IFNAR IFN ⁇ / ⁇ receptor
  • type I interferons present in humans are IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ and IFN ⁇ .
  • type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.
  • the IFN ⁇ proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection.
  • the genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.
  • the IFN ⁇ proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN ⁇ have been described, IFN ⁇ 1 and IFN ⁇ 3. The natural and recombinant forms of IFN ⁇ 1 have antiviral, antibacterial, and anticancer properties.
  • Type II interferon (IFN ⁇ in humans) is also known as immune interferon and is activated by IL12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells. Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2 ⁇ 4) and IFNLR1 (also called CRF2 ⁇ 12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus or fungal infections. In general, type I and II interferons are responsible for regulating and activating the immune response. According to the disclosure, a type I interferon is preferably IFN ⁇ or IFN ⁇ , more preferably IFN ⁇ .
  • an interferon may be a naturally occurring interferon or a functional fragment or variant thereof.
  • An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.
  • Interleukins Interleukins are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15–25% identity). The human genome encodes more than 50 interleukins and related proteins.
  • an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof.
  • An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.
  • Extended ⁇ PK group Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant).
  • the immunostimulant may be fused to an extended ⁇ PK group, which increases circulation half ⁇ life.
  • extended ⁇ PK groups are described infra. It should be understood that other PK groups that increase the circulation half ⁇ life of immunostimulants such as cytokines, or variants thereof, are also applicable to the present disclosure.
  • the extended ⁇ PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).
  • PK is an acronym for "pharmacokinetic” and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject.
  • an "extended ⁇ PK group” refers to a protein, peptide, or moiety that increases the circulation half ⁇ life of a biologically active molecule when fused to or administered together with the biologically active molecule.
  • an extended ⁇ PK group examples include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549).
  • HSA human serum albumin
  • Other exemplary extended ⁇ PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903 ⁇ 15 which is herein incorporated by reference in its entirety.
  • an "extended ⁇ PK" immunostimulant refers to an immunostimulant moiety in combination with an extended ⁇ PK group.
  • the extended ⁇ PK immunostimulant is a fusion protein in which an immunostimulant moiety is linked or fused to an extended ⁇ PK group.
  • the serum half ⁇ life of an extended ⁇ PK immunostimulant is increased relative to the immunostimulant alone (i.e., the immunostimulant not fused to an extended ⁇ PK group).
  • the serum half ⁇ life of the extended ⁇ PK immunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer relative to the serum half ⁇ life of the immunostimulant alone.
  • the serum half ⁇ life of the extended ⁇ PK immunostimulant is at least 1.5 ⁇ fold, 2 ⁇ fold, 2.5 ⁇ fold, 3 ⁇ fold, 3.5 fold, 4 ⁇ fold, 4.5 ⁇ fold, 5 ⁇ fold, 6 ⁇ fold, 7 ⁇ fold, 8 ⁇ fold, 10 ⁇ fold, 12 ⁇ fold, 13 ⁇ fold, 15 ⁇ fold, 17 ⁇ fold, 20 ⁇ fold, 22 ⁇ fold, 25 ⁇ fold, 27 ⁇ fold, 30 ⁇ fold, 35 ⁇ fold, 40 ⁇ fold, or 50 ⁇ fold greater than the serum half ⁇ life of the immunostimulant alone.
  • the serum half ⁇ life of the extended ⁇ PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.
  • half ⁇ life refers to the time taken for the serum or plasma concentration of a compound such as a peptide or protein to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms.
  • An extended ⁇ PK immunostimulant suitable for use herein is stabilized in vivo and its half ⁇ life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and/or clearance or sequestration.
  • the half ⁇ life can be determined in any manner known per se, such as by pharmacokinetic analysis.
  • Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996).
  • the extended ⁇ PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "albumin").
  • Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.
  • albumin fusion protein refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular an immunostimulant.
  • the albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in ⁇ frame with a polynucleotide encoding an albumin.
  • the therapeutic protein and albumin, once part of the albumin fusion protein may each be referred to as a “portion”, “region” or “moiety” of the albumin fusion protein (e.g., a “therapeutic protein portion” or an “albumin protein portion”).
  • an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin).
  • an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, e.g. a liver cell, and secreted into the circulation.
  • Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N ⁇ and O ⁇ linked glycosylation); specific proteolytic cleavages; and/or assembly into multimeric proteins.
  • An albumin fusion protein is preferably encoded by RNA in a non ⁇ processed form which in particular has a signal peptide at its N ⁇ terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off.
  • albumin fusion protein refers to an albumin fusion protein product which has undergone N ⁇ terminal signal peptide cleavage, herein also referred to as a “mature albumin fusion protein”.
  • albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin.
  • Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half ⁇ life of the therapeutic protein in the bloodstream.
  • albumin refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin.
  • albumin refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules.
  • the albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig.
  • Non ⁇ mammalian albumins include, but are not limited to, hen and salmon.
  • the albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.
  • the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.
  • HSA human serum albumin
  • HA human albumin
  • albumin and “serum albumin” are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).
  • a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non ⁇ fusion state.
  • the albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability.
  • Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin.
  • an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.
  • albumin may be naturally occurring albumin or a fragment or variant thereof.
  • Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.
  • the albumin fusion protein comprises albumin as the N ⁇ terminal portion, and a therapeutic protein as the C ⁇ terminal portion.
  • an albumin fusion protein comprising albumin as the C ⁇ terminal portion, and a therapeutic protein as the N ⁇ terminal portion may also be used.
  • the albumin fusion protein has a therapeutic protein fused to both the N ⁇ terminus and the C ⁇ terminus of albumin.
  • the therapeutic proteins fused at the N ⁇ and C ⁇ termini are the same therapeutic proteins.
  • the therapeutic proteins fused at the N ⁇ and C ⁇ termini are different therapeutic proteins.
  • the different therapeutic proteins are both cytokines.
  • the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s).
  • a linker peptide between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids such that it is flexible or more rigid.
  • the linker sequence may be cleavable by a protease or chemically.
  • the term "Fc region” refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains.
  • the term "Fc domain” refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain.
  • an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C ⁇ terminus of the antibody.
  • a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.
  • an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof.
  • an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain).
  • an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof).
  • an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
  • An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy ⁇ chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain.
  • the Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
  • the Fc domain encompasses native Fc and Fc variant molecules.
  • any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule.
  • the Fc domain has reduced effector function (e.g., Fc ⁇ R binding).
  • the Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules.
  • an Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.
  • an Fc domain can comprise a chimeric hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule.
  • an Fc domain can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.
  • an extended ⁇ PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term "Fc domain").
  • the Fc domain does not contain a variable region that binds to antigen.
  • Fc domains suitable for use in the present disclosure may be obtained from a number of different sources.
  • an Fc domain is derived from a human immunoglobulin.
  • the Fc domain is from a human IgG1 constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non ⁇ human primate (e.g. chimpanzee, macaque) species.
  • a rodent e.g. a mouse, rat, rabbit, guinea pig
  • non ⁇ human primate e.g. chimpanzee, macaque
  • the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.
  • immunoglobulin class including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.
  • Fc domain gene sequences e.g., mouse and human constant region gene sequences
  • Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and/or with a particular modification to reduce immunogenicity.
  • Many sequences of antibodies and antibody ⁇ encoding genes have been published and suitable Fc domain sequences (e.g.
  • the extended ⁇ PK group is a serum albumin binding protein such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, and WO2009/133208, which are herein incorporated by reference in their entirety.
  • the extended ⁇ PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are herein incorporated by reference in their entirety.
  • the extended ⁇ PK group is a serum immunoglobulin binding protein such as those disclosed in US2007/0178082, US2014/0220017, and US2017/0145062, which are herein incorporated by reference in their entirety.
  • the extended ⁇ PK group is a fibronectin (Fn) ⁇ based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012/0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin ⁇ based scaffold domain proteins are also disclosed in US2012/0094909.
  • Fn3 ⁇ based extended ⁇ PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.
  • the extended ⁇ PK immunostimulant can employ one or more peptide linkers.
  • peptide linker refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended ⁇ PK moiety and an immunostimulant moiety) in a linear amino acid sequence of a polypeptide chain.
  • peptide linkers may be used to connect an immunostimulant moiety to a HSA domain.
  • Linkers suitable for fusing the extended ⁇ PK group to e.g. an immunostimulant are well known in the art.
  • linkers include glycine ⁇ serine ⁇ polypeptide linkers, glycine ⁇ proline ⁇ polypeptide linkers, and proline ⁇ alanine polypeptide linkers.
  • the linker is a glycine ⁇ serine ⁇ polypeptide linker, i.e., a peptide that consists of glycine and serine residues.
  • an immunostimulant polypeptide described herein can contain sequences encoding a "marker" or "reporter".
  • marker or reporter genes include ⁇ lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin ⁇ B ⁇ hosphotransferase (HPH), thymidine kinase (TK), ⁇ galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).
  • CAT chloramphenicol acetyltransferase
  • ADA adenosine deaminase
  • DHFR dihydrofolate reductase
  • HPH hygromycin ⁇ B ⁇ hosphotransferase
  • TK thymidine kinase
  • XGPRT xanthine guanine phosphoribosyltransferase
  • the pharmaceutical composition described herein is an immunogenic composition for inducing an immune response against coronavirus in a subject.
  • the immunogenic composition is a vaccine.
  • the components described herein such as RNA encoding a vaccine antigen may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers etc.
  • the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a coronavirus infection.
  • composition relates to a formulation comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject.
  • a pharmaceutical composition is also known in the art as a pharmaceutical formulation.
  • the pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants.
  • adjuvant relates to a compound which prolongs, enhances or accelerates an immune response.
  • Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune ⁇ stimulating complexes.
  • oil emulsions e.g., Freund's adjuvants
  • mineral compounds such as alum
  • bacterial products such as Bordetella pertussis toxin
  • immune ⁇ stimulating complexes include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines.
  • the cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFN ⁇ , IFN ⁇ , GM ⁇ CSF, LT ⁇ a.
  • Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51.
  • Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.
  • the pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".
  • pharmaceutically acceptable refers to the non ⁇ toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
  • the term "pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease.
  • the desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition.
  • compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • the pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents.
  • the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • excipient refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
  • diluting and/or thinning agent relates a diluting and/or thinning agent.
  • the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.
  • carrier refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition.
  • a carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject.
  • Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy ⁇ propylene copolymers.
  • the pharmaceutical composition of the present disclosure includes isotonic saline.
  • Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
  • compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly.
  • the pharmaceutical composition is formulated for local administration or systemic administration.
  • Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration.
  • parenteral administration refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection.
  • the pharmaceutical composition is formulated for intramuscular administration.
  • the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration.
  • co ⁇ administering means a process whereby different compounds or compositions (e.g., RNA encoding an antigen and RNA encoding an immunostimulant) are administered to the same patient.
  • the different compounds or compositions may be administered simultaneously, at essentially the same time, or sequentially.
  • the pharmaceutical compositions and products described herein may be provided as a frozen concentrate for solution for injection, e.g., at a concentration of 0.50 mg/mL.
  • a drug product is thawed and diluted with isotonic sodium chloride solution (e.g., 0.9% NaCl, saline), e.g., by a one ⁇ step dilution process.
  • bacteriostatic sodium chloride solution e.g., 0.9% NaCl, saline
  • a diluted drug product is an off ⁇ white suspension.
  • concentration of the final solution for injection varies depending on the respective dose level to be administered.
  • administration is performed within 6 h after begin of preparation due to the risk of microbial contamination and considering the multiple ⁇ dose approach of the preparation process.
  • two conditions are allowed: room temperature for preparation, handling and transfer as well as 2 to 8°C for storage.
  • compositions described herein may be shipped and/or stored under temperature ⁇ controlled conditions, e.g., temperature conditions of about 4 ⁇ 5 o C or below, about ⁇ 20 o C or below, ⁇ 70°C ⁇ 10°C (e.g., ⁇ 80°C to ⁇ 60°C), e.g., utilizing a cooling system (e.g., that may be or include dry ice) to maintain the desired temperature.
  • compositions described herein are shipped in temperature ⁇ controlled thermal shippers. Such shippers may contain a GPS ⁇ enabled thermal sensor to track the location and temperature of each shipment. The compositions can be stored by refilling with, e.g., dry ice.
  • the present disclosure provides methods and agents for inducing an adaptive immune response against coronavirus in a subject comprising administering an effective amount of a composition comprising RNA encoding a coronavirus vaccine antigen described herein.
  • the methods and agents described herein provide immunity in a subject to coronavirus, coronavirus infection, or to a disease or disorder associated with coronavirus.
  • the present disclsoure thus provides methods and agents for treating or preventing the infection, disease, or disorder associated with coronavirus.
  • the methods and agents described herein are administered to a subject having an infection, disease, or disorder associated with coronavirus.
  • the methods and agents described herein are administered to a subject at risk for developing the infection, disease, or disorder associated with coronavirus.
  • the methods and agents described herein may be administered to a subject who is at risk for being in contact with coronavirus.
  • the methods and agents described herein are administered to a subject who lives in, traveled to, or is expected to travel to a geographic region in which coronavirus is prevalent.
  • the methods and agents described herein are administered to a subject who is in contact with or expected to be in contact with another person who lives in, traveled to, or is expected to travel to a geographic region in which coronavirus is prevalent.
  • the methods and agents described herein are administered to a subject who has knowingly been exposed to coronavirus through their occupation, or other contact.
  • a coronavirus is SARS ⁇ CoV ⁇ 2.
  • methods and agents described herein are administered to a subject with evidence of prior exposure to and/or infection with SARS ⁇ CoV ⁇ 2 and/or an antigen or epitope thereof or cross ⁇ reactive therewith.
  • methods and agents described herein are administered to a subject in whom antibodies, B cells, and/or T cells reactive with one or more epitopes of a SARS ⁇ CoV ⁇ 2 spike protein are detectable and/or have been detected.
  • the composition must induce an immune response against the coronavirus antigen in a cell, tissue or subject (e.g., a human).
  • the composition induces an immune response against the coronavirus antigen in a cell, tissue or subject (e.g., a human).
  • the vaccine induces a protective immune response in a mammal.
  • the therapeutic compounds or compositions of the present disclosure may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods.
  • prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression.
  • the term "prevent” encompasses any activity, which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease ⁇ related complications.
  • a regimen described herein includes at least one dose.
  • a regimen includes a first dose and at least one subsequent dose.
  • the first dose is the same amount as at least one subsequent dose.
  • the first dose is the same amount as all subsequent doses.
  • the first dose is a different amount as at least one subsequent dose.
  • the first dose is a different amount than all subsequent doses.
  • a regimen comprises two doses. In some embodiments, a provided regimen consists of two doses. In some embodiments, a regimen comprises three doses. In one embodiment, the present disclosure envisions administration of a single dose. In one embodiment, the present disclosure envisions administration of a priming dose followed by one or more booster doses. The booster dose or the first booster dose may be administered 7 to 28 days or 14 to 24 days following administration of the priming dose. In some embodiments, a first booster dose may be administered 1 week to 3 months (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks) following administration of a priming dose.
  • 1 week to 3 months e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks
  • a subsequent booster dose may be adminsitered at least 1 week or longer, including, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer, following a preceding booster dose.
  • subsequent booster doses may be administered about 5 ⁇ 9 weeks or 6 ⁇ 8 weeks apart.
  • At least one subsequent booster dose may be administered at least 3 months or longer, including, e.g., at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, or longer, following a preceding dose.
  • a subsequent dose given to an individual e.g., as part of a primary regimen or booster regimen
  • a subsequent dose given to an individual e.g., as part of a primary regimen or booster regimen
  • a subsequent dose can be higher or lower than the prior dose, for example, based on consideration of various factors, including, e.g., immunogenicity and/or reactogenicity induced by the prior dose, prevalence of the disease, etc.
  • a subsequent dose can be higher than a prior dose by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or higher.
  • a subsequent dose can be higher than a prior dose by at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.5 fold, at least 3 ⁇ fold, or higher.
  • a subsequent dose can be higher than a prior dose by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or higher. In some embodiments, a subsequent dose can be lower than a prior dose by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or lower.In some embodiments, an amount the RNA described herein from 0.1 ⁇ g to 300 ⁇ g, 0.5 ⁇ g to 200 ⁇ g, or 1 ⁇ g to 100 ⁇ g, such as about 1 ⁇ g, about 2 ⁇ g, about 3 ⁇ g, about 4 ⁇ g, about 5 ⁇ g, about 6 ⁇ g, about 7 ⁇ g, about 8 ⁇ g, about 9 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, about 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 35 ⁇ g, about 40 ⁇ g, about 45 ⁇ g, about 50 ⁇ g,
  • an amount of the RNA described herein of 60 ⁇ g or lower, 55 ⁇ g or lower, 50 ⁇ g or lower, 45 ⁇ g or lower, 40 ⁇ g or lower, 35 ⁇ g or lower, 30 ⁇ g or lower, 25 ⁇ g or lower, 20 ⁇ g or lower, 15 ⁇ g or lower, 10 ⁇ g or lower, 5 ⁇ g or lower, 3 ⁇ g or lower, 2.5 ⁇ g or lower, or 1 ⁇ g or lower may be administered per dose (e.g., in a given dose).
  • an amount of the RNA described herein of at least 0.25 ⁇ g, at least 0.5 ⁇ g, at least 1 ⁇ g, at least 2 ⁇ g, at least 3 ⁇ g, at least 4 ⁇ g, at least 5 ⁇ g, at least 10 ⁇ g, at least 15 ⁇ g, at least 20 ⁇ g, at least 25 ⁇ g, at least 30 ⁇ g, at least 40 ⁇ g, at least 50 ⁇ g, or at least 60 ⁇ g may be administered per dose (e.g., in a given dose). In some embodiments, an amount of the RNA described herein of at least 3 ug may be administered in at least one of given doses.
  • an amount of the RNA described herein of at least 10 ug may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of at least 15 ug may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of at least 20 ug may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of at least 25 ug may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of at least 30 ug may be administered in at least one of given doses.
  • an amount of the RNA described herein of at least 50 ug may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of at least 60 ug may be administered in at least one of given doses. In some embodiments, combinations of aforementioned amounts may be administered in a regimen comprising two or more doses (e.g., a prior dose and a subsequent dose can be of different amounts as described herein). In some embodiments, combinations of aforementioned amounts may be administered in a primary regimen and a booster regimen (e.g., different doses can be given in a primary regimen and a booster regimen).
  • an amount of the RNA described herein of 0.25 ⁇ g to 60 ⁇ g, 0.5 ⁇ g to 55 ⁇ g, 1 ⁇ g to 50 ⁇ g, 5 ⁇ g to 40 ⁇ g, or 10 ⁇ g to 30 ⁇ g may be administered per dose.
  • an amount of the RNA described herein of 3 ⁇ g to 30 ⁇ g may be administered in at least one of given doses.
  • an amount of the RNA described herein of 3 ⁇ g to 20 ⁇ g may be administered in at least one of given doses.
  • an amount of the RNA described herein of 3 ⁇ g to 15 ⁇ g may be administered in at least one of given doses.
  • an amount of the RNA described herein of 3 ⁇ g to 10 ⁇ g may be administered in at least one of given doses. In some embodiments, an amount of the RNA described herein of 10 ⁇ g to 30 ⁇ g may be administered in at least one of given doses.
  • a regimen administered to a subject may comprise a plurality of doses (e.g., at least two doses, at least three doses, or more). In some embodiments, a regimen administered to a subject may comprise a first dose and a second dose, which are given at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, or more.
  • such doses may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart.
  • doses may be administered days apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart.
  • doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about 6 weeks apart, or about 1 to more than 6 weeks apart.
  • doses may be separated by a period of about 7 to about 60 days, such as for example about 14 to about 48 days, etc.
  • a minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more.
  • a maximum number of days between doses may be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or fewer.
  • doses may be about 21 to about 28 days apart.
  • doses may be about 19 to about 42 days apart.
  • doses may be about 7 to about 28 days apart.
  • doses may be about 14 to about 24 days.
  • doses may be about 21 to about 42 days.
  • a vaccination regimen comprises a first dose and a second dose. In some embodiments, a first dose and a second dose are administered by at least 21 days apart. In some embodiments, a first dose and a second dose are administered by at least 28 days apart. In some embodiments, a vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is the same as the amount of RNA administered in the second dose. In some embodiments, a vaccination regimen comprises a first dose and a second dose wherein the amount of RNA administered in the first dose differs from that administered in the second dose.
  • a vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is less than that administered in the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% ⁇ 90% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% ⁇ 50% of the second dose. In some embodiments, the amount of RNA administered in the first dose is 10% ⁇ 20% of the second dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, including, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart or longer. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart.
  • a first dose comprises less than about 30 ug of RNA and a second dose comprises at least about 30 ug of RNA.
  • a first dose comprises about 1 to less than about 30 ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or less than about 30 ug of RNA) and a second dose comprises about 30 to about 100 ug of RNA (e.g., about 30, about 40, about 50, or about 60 ug of RNA).
  • a first dose comprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA, or about 1 to about 5 ug of RNA and a second dose comprises about 30 to about 60 ug of RNA.
  • a first dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA) and a second dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA).
  • a first dose comprises about 1 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments, a first dose comprises about 5 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments, a first dose comprises about 10 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments, a first dose comprises about 15 ug of RNA and a second dose comprises about 30 ug of RNA.
  • a first dose comprises about 1 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 5 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 6 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 10 ug of RNA and a second dose comprises about 60 ug of RNA.
  • a first dose comprises about 15 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 20 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 25 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments, a first dose comprises less than about 10 ug of RNA and a second dose comprises at least about 10 ug of RNA.
  • a first dose comprises about 0.1 to less than about 10 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 ug of RNA) and a second dose comprises about 10 to about 30 ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 ug of RNA).
  • a first dose comprises about 0.1 to about 10 ug of RNA, about 1 to about 5 ug of RNA, or about 0.1 to about 3 ug of RNA and a second dose comprises about 10 to about 30 ug of RNA.
  • a first dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5ug of RNA) and a second dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20ug of RNA).
  • a first dose comprises about 0.1 ug of RNA and a second dose comprises about 10 ug of RNA.
  • a first dose comprises about 0.3 ug of RNA and a second dose comprises about 10 ug of RNA.
  • a first dose comprises about 1 ug of RNA and a second dose comprises about 10 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 10 ug of RNA. In some embodiments, a first dose comprises less than about 3 ug of RNA and a second dose comprises at least about 3 ug of RNA.
  • a first dose comprises about 0.1 to less than about 3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5 ug of RNA) and a second dose comprises about 3 to about 10 ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10 ug of RNA).
  • a first dose comprises about 0.1 to about 3 ug of RNA, about 0.1 to about 1 ug of RNA, or about 0.1 to about 0.5 ug of RNA and a second dose comprises about 3 to about 10 ug of RNA.
  • a first dose comprises about 0.1 to about 1.0 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA) and a second dose comprises about 1 to about 3 ug of RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA).
  • a first dose comprises about 0.1 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 0.3 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 0.5 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 1 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is greater than that administered in the second dose.
  • the amount of RNA administered in the second dose is 10% ⁇ 90% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% ⁇ 50% of the first dose. In some embodiments, the amount of RNA administered in the second dose is 10% ⁇ 20% of the first dose. In some embodiments, the first dose and the second dose are administered at least 2 weeks apart, including, at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart or longer. In some embodiments, the first dose and the second dose are administered at least 3 weeks apart In some embodiments, a first dose comprises at least about 30 ug of RNA and a second dose comprises less than about 30 ug of RNA.
  • a first dose comprises about 30 to about 100 ug of RNA (e.g., about 30, about 40, about 50, or about 60 ug of RNA) and a second dose comprises about 1 to about 30 ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30 ug of RNA).
  • a second dose comprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA, or about 1 to 5 ug of RNA.
  • a first dose comprises about 30 to about 60 ug of RNA and a second dose comprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA, or about 0.1 to about 3 ug of RNA.
  • a first dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA) and a second dose comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA).
  • a first dose comprises about 30 ug of RNA and a second dose comprises about 1 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about 5 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about 10 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about 15 ug of RNA.
  • a first dose comprises about 60 ug of RNA and a second dose comprises about 1 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 5 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 6 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 10 ug of RNA.
  • a first dose comprises about 60 ug of RNA and a second dose comprises about 15 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 20 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 25 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments, a first dose comprises at least about 10 ug of RNA and a second dose comprises less than about 10 ug of RNA.
  • a first dose comprises about 10 to about 30 ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 ug of RNA) and a second dose comprises about 0.1 to less than about 10 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about 10 ug of RNA).
  • a first dose comprises about 10 to about 30 ug of RNA, or about 0.1 to about 3 ug of RNA and a second dose comprises about 1 to about 10 ug of RNA, or about 1 to about 5 ug of RNA.
  • a first dose comprises about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20 ug of RNA) and a second dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5 ug of RNA).
  • a first dose comprises about 10 ug of RNA and a second dose comprises about 0.1 ug of RNA.
  • a first dose comprises about 10 ug of RNA and a second dose comprises about 0.3 ug of RNA.
  • a first dose comprises about 10 ug of RNA and a second dose comprises about 1 ug of RNA. In some embodiments, a first dose comprises about 10 ug of RNA and a second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises at least about 3 ug of RNA and a second dose comprises less than about 3 ug of RNA.
  • a first dose comprises about 3 to about 10 ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10 ug of RNA) and a second dose comprises 0.1 to less than about 3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5 about 2.0, or about 2.5 ug of RNA).
  • a first dose comprises about 3 to about 10 ug of RNA and a second dose comprises about 0.1 to about 3 ug of RNA, about 0.1 to about 1 ug of RNA, or about 0.1 to about 0.5 ug of RNA.
  • a first dose comprises about 1 to about 3 ug of RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA) and a second dose comprises about 0.1 to 0.3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA).
  • a first dose comprises about 3 ug of RNA and a second dose comprises about 0.1 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 0.3 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 0.6 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about 1 ug of RNA. In some embodiments, a vaccination regimen comprises at least two doses, including, e.g., at least three doses, at least four doses or more. In some embodiments, a vaccination regimen comprises three doses.
  • the time interval between the first dose and the second dose can be the same as the time interval between the second dose and the third dose. In some embodiments, the time interval between the first dose and the second dose can be longer than the time interval between the second dose and the third dose, e.g., by days or weeks (including, e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer).
  • the time interval between the first dose and the second dose can be shorter than the time interval between the second dose and the third dose, e.g., by days or weeks (including, e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer).
  • the time interval between the first dose and the second dose can be shorter than the time interval between the second dose and the third dose, e.g., by at least 1 month (including, e.g., at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or longer).
  • a last dose of a primary regimen and a first dose of a booster regimen are given at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart.
  • a primary regimen may comprises two doses.
  • a primary regimen may comprises three doses.
  • a first dose and a second dose (and/or other subsequent dose) may be administered by intramuscular injection.
  • a first dose and a second dose (and/or other subsequent dose) may be administered in the deltoid muscle.
  • a first dose and a second dose (and/or other subsequent dose) may be administered in the same arm.
  • an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 mL each) 21 days apart.
  • an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) as a series of two doses (e.g., 0.2 mL each) 21 days apart.
  • an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) as a series of three doses (e.g., 0.3 mL or lower including, e.g., 0.2 mL), wherein doses are given at least 3 weeks apart.
  • the first and second doses may be administered 3 weeks apart, while the second and third doses may be administered at a longer time interval than that between the first and the second doses, e.g., at least 4 weeks apart or longer (including, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or longer).
  • each dose is about 60 ug.
  • each dose is about 50 ug. In some embodiments, each dose is about 30 ug. In some embodiments, each dose is about 25 ug. In some embodiments, each dose is about 20 ug. In some embodiments, each dose is about 15 ug. In some embodiments, each dose is about 10 ug. In some embodiments, each dose is about 3 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 60 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 50 ug.
  • a vaccination regimen e.g., a primary vaccination regimen and/or a booster vaccination regimen
  • At least one dose given in a vaccination regimen is about 30 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 25 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 20 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 15 ug.
  • At least one dose given in a vaccination regimen is about 10 ug. In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3 ug. In one embodiment, an amount of the RNA described herein of about 60 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 50 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 30 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 25 ⁇ g is administered per dose.
  • an amount of the RNA described herein of about 20 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 15 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 10 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 5 ⁇ g is administered per dose. In one embodiment, an amount of the RNA described herein of about 3 ⁇ g is administered per dose. In one embodiment, at least two of such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose.
  • the efficacy of the RNA vaccine described herein is at least 70%, at least 80%, at least 90, or at least 95% beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose if a second dose is administered 21 days following administration of the first dose).
  • such efficacy is observed in populations of age of at least 50, at least 55, at least 60, at least 65, at least 70, or older.
  • the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein a second dose may be administered about 21 days following administration of the first dose, and administered, for example, in an amount of about 30 ⁇ g per dose) beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose if a second dose is administered 21 days following administration of the first dose) in populations of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70, is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.
  • Such efficacy may be observed over time periods of up to 1 month, 2 months, 3 months, 6 months or even longer.
  • vaccine efficacy is defined as the percent reduction in the number of subjects with evidence of infection (vaccinated subjects vs. non ⁇ vaccinated subjects).
  • efficacy is assessed through surveillance for potential cases of COVID ⁇ 19. If, at any time, a patient develops acute respiratory illness, for the purposes herein, the patient can be considered to potentially have COVID ⁇ 19 illness.
  • the assessments can include a nasal (midturbinate) swab, which may be tested using a reverse transcription ⁇ polymerase chain reaction (RT ⁇ PCR) test to detect SARS ⁇ CoV ⁇ 2.
  • RT ⁇ PCR reverse transcription ⁇ polymerase chain reaction
  • clinical information and results from local standard ⁇ of ⁇ care tests can be assessed.
  • efficacy assessments may utilize a definition of SARS ⁇ CoV ⁇ 2 ⁇ related cases wherein: • Confirmed COVID ⁇ 19: presence of at least 1 of the following symptoms and SARS ⁇ CoV ⁇ 2 NAAT (nucleic acid amplification ⁇ based test) positive during, or within 4 days before or after, the symptomatic period: fever; new or increased cough; new or increased shortness of breath; chills; new or increased muscle pain; new loss of taste or smell; sore throat; diarrhea; vomiting.
  • efficacy assessments may utilize a definition of SARS ⁇ CoV ⁇ 2 ⁇ related cases wherein one or more of the following additional symptoms defined by the CDC can be considered: fatigue; headache; nasal congestion or runny nose; nausea.
  • efficacy assessments may utilize a definition of SARS ⁇ CoV ⁇ 2 ⁇ related severe cases • Confirmed severe COVID ⁇ 19: confirmed COVID ⁇ 19 and presence of at least 1 of the following: clinical signs at rest indicative of severe systemic illness (e.g., RR ⁇ 30 breaths per minute, HR ⁇ 125 beats per minute, SpO 2 ⁇ 93% on room air at sea level, or PaO 2 /FiO 2 ⁇ 300mm Hg); respiratory failure (which can be defined as needing high ⁇ flow oxygen, noninvasive ventilation, mechanical ventilation, or ECMO); evidence of shock (e.g., SBP ⁇ 90 mm Hg, DBP ⁇ 60 mm Hg, or requiring vasopressors); significant acute renal, hepatic, or neurologic dysfunction; admission to an ICU; death.
  • clinical signs at rest indicative of severe systemic illness e.g., RR ⁇ 30 breaths per minute, HR ⁇ 125 beats per minute, SpO 2 ⁇ 93% on room air at sea level, or
  • a serological definition can be used for patients without clinical presentation of COVID ⁇ 19: e.g., confirmed seroconversion to SARS ⁇ CoV ⁇ 2 without confirmed COVID ⁇ 19: e.g., positive N ⁇ binding antibody result in a patient with a prior negative N ⁇ binding antibody result.
  • any or all of the following assays can be performed on serum samples: SARS ⁇ CoV ⁇ 2 neutralization assay; S1 ⁇ binding IgG level assay; RBD ⁇ binding IgG level assay; N ⁇ binding antibody assay.
  • methods and agents described herein are administered to a paediatric population.
  • the paediatric population comprises or consists of subjects under 18 years, e.g., 5 to less than 18 years of age, 12 to less than 18 years of age, 16 to less than 18 years of age, 12 to less than 16 years of age, or 5 to less than 12 years of age.
  • the paediatric population comprises or consists of subjects under 5 years, e.g., 2 to less than 5 years of age, 12 to less than 24 months of age, 7 to less than 12 months of age, or less than 6 months of age.
  • an RNA (e.g., mRNA) composition described herein is administered to subjects of less than 2 years old, for example, 6 months to less than 2 years old.
  • an RNA (e.g., mRNA) composition described herein is administered to subjects of less than 6 months old, for example, 1 month to less than 4 months old.
  • a dosing regimen e.g., doses and/or dosing schedule
  • a paediatric population may vary for different age groups.
  • a subject 6 months through 4 years of age may be administered according to a primary regimen comprising at least three doses, in which the initial two doses are adminsitered at least 3 weeks (including, e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart followed by a third dose administered at least 8 weeks (including, e.g., at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer) after the second dose.
  • at least one dose administered is 3 ug RNA described herein.
  • a subject 5 years of age and older may be administered according to a primary regimen comprising at least two doses, in which the two doses are administered at least 3 weeks (including, e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart.
  • at least one dose administered is 10 ug RNA described herein.
  • a subject 5 years of age and older who are immunocompromised may be administered according to a primary regimen comprising at least three doses, in which the initial two doses are administered at least 3 weeks (including, e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart, followed by a third dose administered at least 4 weeks (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer) after the second dose.
  • a primary regimen comprising at least three doses, in which the initial two doses are administered at least 3 weeks (including, e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer) apart, followed by a third dose administered at least 4 weeks (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at
  • an RNA (e.g., mRNA) composition described herein is administered to subjects of age 12 or older and each dose is about 30 ug.
  • an RNA (e.g., mRNA) composition described herein is administered to subjects of age 12 or older (including, e.g., age 18 or older) and each dose is higher than 30 ug, including, e.g., 35 ug, 40 ug, 45 ug, 50 ug, 55 ug, 60 ug, 65 ug , 70 ug, or higher.
  • an RNA (e.g., mRNA) composition described herein is administered to subjects of age 12 or older and each dose is about 60 ug.

Abstract

This disclosure relates to the field of RNA to prevent or treat coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses such as antibody and/or T cell responses. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccine antigen.

Description

CORONAVIRUS VACCINE    This  disclosure  relates  to  the  field  of  RNA  to  prevent  or  treat  coronavirus  infection.  In  particular,  the  present  disclosure  relates  to methods  and  agents  for  vaccination  against  coronavirus infection and  inducing effective coronavirus antigen‐specific immune responses  such as antibody and/or T cell responses. These methods and agents are, in particular, useful  for  the prevention or  treatment of  coronavirus  infection. Administration of RNA disclosed  herein to a subject can protect the subject against coronavirus infection. Specifically, in one  embodiment, the present disclosure relates to methods comprising administering to a subject  RNA encoding  a peptide or protein  comprising  an epitope of  SARS‐CoV‐2  spike protein  (S  protein) for inducing an immune response against coronavirus S protein, in particular S protein  of SARS‐CoV‐2, in the subject, i.e., vaccine RNA encoding vaccine antigen. Administering to the  subject  RNA  encoding  vaccine  antigen may  provide  (following  expression  of  the  RNA  by  appropriate  target cells) vaccine antigen  for  inducing an  immune  response against vaccine  antigen (and disease‐associated antigen) in the subject.    Coronaviruses  are  positive‐sense,  single‐stranded  RNA  ((+)ssRNA)  enveloped  viruses  that  encode  for  a  total  of  four  structural  proteins,  spike  protein  (S),  envelope  protein  (E),  membrane  protein  (M)  and  nucleocapsid  protein  (N).  The  spike  protein  (S  protein)  is  responsible  for  receptor‐recognition,  attachment  to  the  cell,  infection  via  the  endosomal  pathway,  and  the  genomic  release  driven  by  fusion  of  viral  and  endosomal membranes.  Though sequences between the different family members vary, there are conserved regions  and motifs within the S protein making it possible to divide the S protein into two subdomains:  S1 and S2. While the S2, with its transmembrane domain, is responsible for membrane fusion,  the S1 domain recognizes the virus‐specific receptor and binds to the target host cell. Within  several coronavirus isolates, the receptor binding domain (RBD) was identified and a general  structure of the S protein defined (Figure 1).  In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China and  it became clear that a novel coronavirus (severe acute respiratory syndrome coronavirus 2;  SARS‐CoV‐2) was the underlying cause. The genetic sequence of SARS‐CoV‐2 became available  to the WHO and public (MN908947.3) and the virus was categorized into the betacoronavirus  subfamily. By sequence analysis, the phylogenetic tree revealed a closer relationship to severe  acute  respiratory  syndrome  (SARS)  virus  isolates  than  to  another  coronavirus  infecting  humans, namely the Middle East respiratory syndrome (MERS) virus.  SARS‐CoV‐2  infections and the resulting disease COVID‐19 have spread globally, affecting a  growing  number  of  countries.  On  11 March  2020  the WHO  characterized  the  COVID‐19  outbreak  as  a  pandemic.  As  of  01 December  2020,  there  have  been  >63 million  globally  confirmed COVID‐19 cases and >1.4 million deaths, with 191 countries/regions affected. The  ongoing pandemic  remains  a  significant  challenge  to public  health  and  economic  stability  worldwide.   Every  individual  is at  risk of  infection as  there  is no pre‐existing  immunity  to  SARS‐CoV‐2.  Following  infection  some  but  not  all  individuals  develop  protective  immunity  in  terms  of  neutralising  antibody  responses  and  cell  mediated  immunity.  However,  it  is  currently  unknown to what extent and  for how  long this protection  lasts. According to WHO 80% of  infected individuals recover without need for hospital care, while 15% develop more severe  disease and 5% need  intensive care.  Increasing age and underlying medical conditions are  considered risk factors for developing severe disease.   The  presentation  of  COVID‐19  is  generally with  cough  and  fever, with  chest  radiography  showing  ground‐glass  opacities  or  patchy  shadowing.  However,  many  patients  present  without fever or radiographic changes, and infections may be asymptomatic which is relevant  to  controlling  transmission.  For  symptomatic  subjects, progression of disease may  lead  to  acute respiratory distress syndrome requiring ventilation and subsequent multi‐organ failure  and  death.  Common  symptoms  in  hospitalized  patients  (in  order  of  highest  to  lowest  frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea/vomiting  or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss  of taste) may be the sole presenting symptom in approximately 3% of individuals who have  COVID‐19.  All ages may present with  the disease, but notably case  fatality rates  (CFR) are elevated  in  persons >60  years of age. Comorbidities are also associated with  increased CFR,  including  cardiovascular disease, diabetes, hypertension, and chronic  respiratory disease. Healthcare  workers  are  overrepresented  among  COVID‐19  patients  due  to  occupational  exposure  to  infected patients.  In most situations, a molecular test is used to detect SARS‐CoV‐2 and confirm infection. The  reverse transcription polymerase chain reaction (RT‐PCR) test methods targeting SARS‐CoV‐2  viral RNA are the gold standard in vitro methods for diagnosing suspected cases of COVID‐19.  Samples to be tested are collected from the nose and/or throat with a swab.   Among other things, the present disclosure provides insights into immune responses elicited  by  exposure  to  (e.g.,  by  vaccination  and/or  infection)  different  SARS‐CoV‐2  variants  or  immunogenic polypeptides (e.g., S protein), or immunogenic fragments thereof.  For example,  in  some embodiments, administering RNA encoding an  S protein of a BA.2 and/or BA.4/5  Omicron SARS‐CoV‐2 variant, or an immunogenic fragment thereof, can result in an improved  immune  response, which  includes,  e.g.,  improved  neutralization  of  Omicron  BA.4  and/or  Omicron  BA.5  SARS‐CoV‐2  variants  and/or  broader  cross‐neutralization  of  variants  (e.g.,  Omicron variants) of concern (e.g., increased neutralization titers against a larger number of  variants (e.g., Omicron variants) of concern).  In some embodiments, the present disclosure  provides  an  insight  that  a  bivalent  coronavirus  vaccine  (e.g.,  a  bivalent  BA.4/5  vaccine  comprising a first RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain or an immunogenic  fragment thereof, and a second RNA encoding a SARS‐CoV‐2 S protein comprising one or more  mutations that are characteristic of a BA.4/5 Omicron variant or an  immunogenic fragment  thereof)  can  provide  broader  cross‐neutralization  against  SARS‐CoV‐2 Wuhan  strain  and  certain variants thereof (e.g., in some embodiments variants that are prevalent and/or rapidly  spreading  in  a  relevant  jurisdiction,  e.g.,  certain  Omicron  variants)  in  certain  subjects  as  compared to a monovalent coronavirus vaccine (e.g., a vaccine comprising RNA encoding a  SARS‐CoV‐2 S protein of a coronavirus strain or variant thereof). In some embodiments, such  broader  cross‐neutralization  can  be  observed  in  vaccine‐naïve  subjects.  In  some  embodiments,  such  broader  cross‐neutralization  can  be  observed  in  subjects  without  a  coronavirus  infection  (e.g.,  a  SARS‐CoV‐2  infection).  In  some  embodiments,  such  broader  cross‐neutralization  can  be  observed  in  subjects  who  previously  received  a  SARS‐CoV‐2  vaccine (e.g., in some embodiments an RNA vaccine encoding a SARS‐CoV‐2 S protein, e.g., in  some  embodiments  of  a  Wuhan  strain).  In  some  embodiments,  such  broader  cross‐ neutralization can be observed in in young pediatric subjects (e.g., subjects aged 6 months to  less than 2 years, and/or 2 years to less than 5 years).  In some embodiments, the present disclosure provides an insight that exposure to at least two  certain SARS‐CoV‐2 variants or immunogenic polypeptides (e.g., S protein), or immunogenic  fragments thereof can result in an synergistic improvement in immune response (e.g., higher  neutralization  titers, broader  cross‐neutralization, and/or an  immune  response  that  is  less  susceptible  to  immune escape) as compared  to exposure  to one SARS‐CoV‐2 strain and/or  other combinations of SARS‐CoV‐2 variants.    In some embodiments, the present disclosure  provides an  insight  that exposure  to a S protein  from a Wuhan  strain or an  immunogenic  fragment  thereof  (e.g., by vaccination and/or  infection), and exposure  to a S protein of an  Omicron  BA.1  variant  or  an  immunogenic  fragment  thereof  (e.g.,  by  vaccination  and/or  infection)    can  result  in  an  synergistic  improvement  in  immune  response  (e.g.,  higher  neutralization  titers, broader  cross‐neutralization, and/or an  immune  response  that  is  less  susceptible  to  immune escape) as compared  to exposure  to one SARS‐CoV‐2 strain and/or  other combinations of SARS‐CoV‐2 variants).  In some embodiments,  the present disclosure  provides an  insight  that exposure  to a S protein  from a Wuhan  strain or an  immunogenic  fragment  thereof  (e.g., by vaccination and/or  infection), and exposure  to a S protein of an  Omicron  BA.4  or  BA.5  variant  or  an  immunogenic  fragment  thereof  (e.g.,  by  vaccination  and/or infection)  can result in an synergistic improvement in immune response (e.g., higher  neutralization  titers, broader  cross‐neutralization, and/or an  immune  response  that  is  less  susceptible  to  immune escape) as compared  to exposure  to one SARS‐CoV‐2 strain and/or  other combinations of SARS‐CoV‐2 variants).  In some embodiments,  the present disclosure  provides an  insight  that  (i) exposure  to a S protein  from a strain/variant selected  from  the  group consisting of Wuhan strain, an alpha variant, beta variant, delta variant, Omicron BA.1,  and sublineages derived  from any of the aforementioned strains/variants, or  immunogenic  fragments thereof (e.g., by vaccination and/or  infection), combined with (ii)exposure to a S  protein from a strain/variant selected from the group consisting of Omicron BA.2, Omicron  BA.4,    Omicron  BA.5,  and  sublineages  derived  from  any  of  the  aforementioned  strains/variants, or immunogenic fragments thereof (e.g., by vaccination and/or infection)  can  result  in an synergistic  improvement  in  immune response  (e.g., higher neutralization titers,  broader cross‐neutralization, and/or an immune response that is less susceptible to immune  escape) as  compared  to exposure  to one SARS‐CoV‐2  strain and/or other  combinations of  SARS‐CoV‐2 variants).      The  present  disclosure  also  provides  significant  insights  into  how  an  immune  response  develops  in  subjects  following exposures  to  (e.g.,  vaccinations and/or  infections) multiple,  different SARS‐CoV‐2 strains.  Among other things, disclosed herein is a finding that different  combinations  of  SARS‐CoV‐2  variants  elicit  different  immune  responses.    Specifically,  the  present disclosure provides an insight that exposure to certain combinations of SARS‐CoV‐2  variants can elicit an  improved  immune response (e.g., higher neutralization titers, broader  cross‐neutralization, and/or an immune response that is less susceptible to immune escape).   In some embodiments, an  improved  immune response can be produced when subjects are  delivered two or more antigens (e.g., as polypeptides or RNAs encoding such polypeptides),  each having few shared epitopes.  In some embodiments, an improved immune response can  be produced when subjects are delivered a combination of SARS‐CoV‐2 S proteins  (e.g., as  polypeptides or RNAs encoding such polypeptides) sharing no more than 50% (e.g., no more  than 40%, no more than 30%, no more 20% or more) of epitopes (including, e.g., amino acid  mutations)  that  can  be  bound  by  neutralization  antibodies.    In  some  embodiments,  an  improved immune response can be produced by delivering, as polypeptides or RNAs encoding  such polypeptides,  (a) a SARS‐CoV‐2 S protein  from a Wuhan strain, an Alpha variant, Beta  variant, or a Delta variant of SARS‐CoV‐2 or an immunogenic fragment thereof, and (b) an S  protein from a SARS‐CoV‐2 Omicron variant or an immunogenic fragment thereof.   In some  embodiments, an improved immune response can be produced by delivering, as polypeptides  or RNAs encoding such polypeptides,    (a) a SARS‐CoV‐2 S protein  from a Wuhan strain, an  Alpha variant, a Beta variant, or a Delta variant of SARS‐CoV‐2 or an immunogenic fragment  thereof, and  (b) an S protein of a SARS‐CoV‐2 Omicron variant  that  is not a BA.1 Omicron  variant or an immunogenic fragment thereof.  In some embodiments, an improved immune  response can be produced by delivering, as polypeptides or RNAs encoding such polypeptides,  (a) an S protein from a Wuhan strain, an Alpha variant, a Beta Variant, a Delta SARS‐CoV‐2  variant, or a BA.1 Omicron variant or an immunogenic fragment thereof and (b) an S protein  of  a  SARS‐CoV‐2 Omicron  variant  that  is  not  a  BA.1 Omicron  variant  or  an  immunogenic  fragment thereof.  In some embodiments, an improved immune response can be produced by  delivering, as polypeptides or RNAs encoding such polypeptides, (a) a SARS‐CoV‐2 S protein  from a Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant, or an immunogenic  fragment thereof and (b) an S protein of a BA.2 or a BA.4 or BA.5 SARS‐CoV‐2 Omicron variant  or an immunogenic fragment thereof.    In some embodiments, the present disclosure also provides an insight that administration of  multiple doses (e.g., at least 2, at least 3, at least 4, or more doses) of a coronavirus vaccine  described herein (e.g., a bivalent vaccine described herein such as a bivalent BA.4/5 vaccine)  may provide certain beneficial effect(s) on affinity of antibodies against one or more SARS‐‐ CoV‐2 strain or variants thereof. In some embodiments, such beneficial effect(s) on affinity of  antibodies may be observed with respect to antibodies against certain Omicron variants. By  way of example only, in some embodiments, such beneficial effect(s) on affinity of antibodies  may be observed with respect to antibodies against certain Omicron variants that share at  least one or more common epitopes, for example, with a Wuhan strain.  Also disclosed herein are compositions that can produce an improved immune response (e.g.,  an  immune  response  having  broader  cross‐neutralization  activity,  stronger  neutralization,  and/or which  is  less susceptible to  immune escape).    In some embodiments, a composition  described herein comprises two or more antigens or nucleic acids (e.g., RNA) that encodes  such antigens that have few shared epitopes.  In some embodiments, a composition described  herein delivers, as polypeptides or nucleic acids encoding such polypeptides, a combination of  SARS‐CoV‐2 S proteins or immunogenic fragments thereof sharing no more than 50% (e.g., no  more than 40%, no more than 30%, no more than 20% or more) of epitopes (including, e.g.,  amino acid mutations) that can be bound by neutralization antibodies.  In some embodiments,  a composition described herein comprises (a) RNA encoding a SARS‐CoV‐2 S protein from a  Wuhan strain, an Alpha variant, a Beta variant, or a Delta variant or an immunogenic fragment  thereof and (b) RNA encoding an S protein from an Omicron variant of SARS‐CoV‐2 (e.g.,  in  some  embodiments  an  S  protein  from  a  BA.1,  BA.2,  or  BA.4/5  Omicron  variant)  or  an  immunogenic  fragment  thereof.    In  some  embodiments,  a  composition  described  herein  comprises (a) RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, an Alpha variant, a  Beta variant, or a Delta variant or an immunogenic fragment thereof and (b) RNA encoding an  S  protein  of  an Omicron  variant  of  SARS‐CoV‐2  that  is  not  a  BA.1 Omicron  variant  or  an  immunogenic  fragment  thereof.    In  some  embodiments,  a  composition  described  herein  comprises (a) RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain, an Alpha variant, a  Beta variant, or a Delta variant or a BA.1 Omicron variant or an immunogenic fragment thereof  and (b) RNA encoding an S protein of a Omicron variant that is not a BA.1 Omicron variant or  an  immunogenic fragment thereof.   In some embodiments, a composition described herein  comprises (a) RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain, an Alpha variant, a  Beta variant or a Delta variant of SARS‐CoV‐2 and (b) RNA encoding an S protein from a BA.2  or a BA.4 or BA.5 Omicron variant of SARS‐CoV‐2 or an  immunogenic fragment thereof.    In  some embodiments, a composition described herein comprises RNA encoding an S protein  from a BA.2 Omicron variant of SARS‐CoV‐2 or an immunogenic fragment thereof.  In some  embodiments,  a  composition  comprises  RNA  encoding  an  S  protein  from  a  BA.4  or  BA.5  Omicron variant of SARS‐CoV‐2 or an immunogenic fragment thereof.    SARS‐CoV‐2 is an RNA virus with four structural proteins. One of them, the spike protein is a  surface protein which binds  the angiotensin‐converting enzyme 2  (ACE‐2) present on host  cells. Therefore, the spike protein is considered a relevant antigen for vaccine development.   BNT162b2 (SEQ ID NO: 20) is an mRNA vaccine for prevention of COVID‐19 and demonstrated  an efficacy of 95% or more at preventing COVID‐19. The vaccine is made of a 5’capped mRNA  encoding  for  the  full‐length  SARS‐CoV‐2  spike  glycoprotein  (S)  encapsulated  in  lipid  nanoparticles  (LNPs). The  finished product  is presented as a concentrate  for dispersion  for  injection  containing  BNT162b2  as  active  substance.  Other  ingredients  are:  ALC‐0315  (4‐ hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  ALC‐0159  (2‐ [(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide),  1,2‐Distearoyl‐sn‐glycero‐3‐ phosphocholine  (DSPC), cholesterol, potassium chloride, potassium dihydrogen phosphate,  sodium chloride, disodium phosphate dihydrate, sucrose and water for injection.  In some embodiments, a different buffer may be used in lieu of PBS.  In some embodiments,  the buffer is formulated in a Tris‐buffered solution.  In some embodiments, the formulation  comprises  ALC‐0315  (4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  ALC‐0159  (2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide), DSPC  (1,2‐distearoyl‐ sn‐glycero‐3‐phosphocholine),  cholesterol,  sucrose,  trometamol  (Tris),  trometamol  hydrochloride and water.   In some embodiments, the concentration of the RNA in the pharmaceutical RNA preparation  is  about  0.1  mg/ml.    In  some  embodiments  about  30  ug  of  RNA  is  administered  by  administering  about  200  uL  of  RNA  preparation.    In  some  embodiments,  the  RNA  in  the  pharmaceutical  RNA  preparation  is  diluted  prior  to  administration  (e.g.,  diluted  to  a  concentration of about 0.05 mg/ml).  In some embodiments, the administration volumes are  between about 200 µl and about 300 µl.  In some embodiments, the RNA in pharmaceutical  RNA preparation is formulated in about 10 mM Tris buffer, and about 10% sucrose.  In  some  embodiments,  the  concentration of RNA  in  a  pharmaceutical RNA preparation  is  about 0.1 mg/ml, and is formulated in about 10 mM Tris buffer, about 10% sucrose and a dose  of about 10 µg of RNA is administered by diluting the pharmaceutical RNA preparation about  1:1  and  administering  about  200 µl  of diluted  pharmaceutical RNA preparation.    In  some  embodiments,  the concentration of RNA  in a pharmaceutical RNA preparation  is about 0.1  mg/ml, and is formulated in about 10 mM Tris buffer, about 10% sucrose and a dose of the  RNA of about 10 µg  is administered by diluting the pharmaceutical RNA preparation about  1:5.75 and administering about 200 µl of diluted pharmaceutical RNA preparation.  The sequence of  the SARS‐CoV‐2 S protein of a Wuhan strain disclosed herein was chosen  based on  the sequence  for  the “SARS‐CoV‐2  isolate Wuhan‐Hu  ‐1”: GenBank: MN908947.3  (complete genome) and GenBank: QHD43416.1 (spike surface glycoprotein).   In some embodiments, an active substance consists of a single‐stranded, 5'‐capped codon‐ optimized  mRNA  that  is  translated  into  the  spike  antigen  of  SARS‐CoV‐2.  In  some  embodiments, an encoded spike antigen protein sequence contains two proline mutations,  which stabilize an antigenically optimal pre‐fusion confirmation (P2 S). In some embodiments,  an  RNA  does  not  contain  any  uridines;  e.g.,  instead  of  uridine  the  modified  N1‐ methylpseudouridine can be used in RNA synthesis. mRNA disclosed herein can be translated  into the SARS‐CoV‐2 S protein in a host cell. The S protein can then be expressed on the cell  surface where it can induce an adaptive immune response. The S protein can be identified as  a  target  for neutralising  antibodies  against  the  virus  and  is  considered  a  relevant  vaccine  component.   BNT162b2 can be administered intramuscularly (IM) in two 30 μg doses of the diluted vaccine  solution given about 21 days apart (e.g., to adult vaccine naïve subjects (i.e., subjects 12 years  and older who have not previously been administered a SARS‐CoV‐2 vaccine)).   The  recent  emergence  of  novel  circulating  variants  of  SARS‐CoV‐2  has  raised  significant  concerns about geographic and temporal efficacy of vaccine interventions. One of the earliest  variants that emerged and rapidly became globally dominant was D614G.   The  alpha  variant  (also  known  as  B.1.1.7,  VOC202012/01,  501Y.V1  or  GRY)  was  initially  detected in the United Kingdom. The alpha variant has a large number of mutations, including  several mutations in the S gene. It has been shown to be inherently more transmissible, with  a growth rate that has been estimated to be 40‐70% higher than other SARS‐CoV‐2 lineages in  multiple  countries  (Volz et al., 2021, Nature, https://doi.org/10.1038/s41586‐021‐03470‐x;  Washington et al., 2021, Cell  https://doi.org/10.1016/j.cell.2021.03.052).  The beta variant (also known as B.1.351 or GH/501Y.V2) was first detected in South Africa. The  beta variant carries several mutations in the S gene. Three of these mutations are at sites in  the RBD that are associated with immune evasion: N501Y (shared with alpha) and E484K and  K417N.   The gamma variant (also known as P.1 or GR/501Y.V3) was first detected in Brazil. The gamma  variant carries several mutations that affect the spike protein, including two shared with beta  (N501Y and E484K), as well as a different mutation at position 417 (K417T).  The delta variant (also known as B.1.617.2 or G/478K.V1) was first documented in India. The  delta variant has  several point mutations  that affect  the  spike protein,  including P681R  (a  mutation position shared with alpha and adjacent to the furin cleavage site), and L452R, which  is  in the RBD and has been linked with  increased binding to ACE2 and neutralizing antibody  resistance. There is also a deletion in the spike protein at position 156/157.  These  four VOCs have circulated globally and became dominant variants  in  the geographic  regions where they were first identified.  On 24 November 2021, the Omicron (B.1.1.529) variant was first reported to WHO from South  Africa.   SARS‐CoV‐2 Omicron and its sublineages have had a major impact on the 20 epidemiological  landscape  of  the  COVID‐19  pandemic  since  initial  emergence  in  November  2021  (WHO  Technical Advisory Group on SARS‐CoV‐2 Virus Evolution (TAG‐VE): Classification of Omicron  (B.1.1.259): SARS‐CoV‐2 Variant of Concern  (2021); WHO Headquarters  (HQ), WHO Health  Emergencies Programme, Enhancing Response to Omicron SARS‐CoV‐2 variant: Technical brief  and  priority  actions  for  Member  States  (2022)).  Significant  alterations  in  the  spike  (S)  glycoprotein of the first Omicron variant BA.1 leading to the loss of many neutralizing antibody  epitopes  (M.  Hoffmann  et  al.,  “The  Omicron  variant  is  highly  resistant  against  antibody  mediated neutralization: Implications for control of the COVID‐19 pandemic”, Cell 185, 447– 456.e11 (2022)) rendered BA.1 capable of partially escaping previously established SARS‐CoV‐ 2  wild‐type  strain  (Wuhan‐Hu‐1)‐based  immunity  (V.  Servellita,  et  al.,  “Neutralizing  30  immunity  in  vaccine  breakthrough  infections  from  the  SARS‐CoV‐2  Omicron  and  Delta  variants”, Cell  185, 1539–1548.e5  (2022);  Y. Cao  et  al.,  “Omicron  escapes  the majority of  existing  SARS‐CoV‐2  neutralizing  antibodies”,  Nature  602,  657–663  (2022)).  Hence,  breakthrough infection of vaccinated individuals with Omicron are more common than with  previous Variants of Concern (VOCs). While Omicron BA.1 was displaced by the BA.2 variant  in many countries around the globe, other variants such as BA.1.1 and BA.3 temporarily and/or  locally  gained momentum  but  did  not  become  globally  dominant  (S.  Xia  et  al.,  “Origin,  virological  features,  immune evasion and  intervention of SARS‐CoV‐2 Omicron sublineages.  Signal  Transduct.  Target.  Ther.  7,  241  (2022);  H.  Gruell  et  al.,  “SARS‐CoV‐2  Omicron  sublineages  exhibit  distinct  antibody  escape  patterns,  Cell  Host Microbe  7,  241  (2022).).  Omicron BA.2.12.1 subsequently displaced BA.2  to become dominant  in  the United States,  whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia/Pacific (H. Gruell et  al.,  “SARS‐CoV‐2 Omicron  sublineages exhibit distinct antibody escape patterns,” Cell Host  Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID‐  19  country  overview  ‐Country  overview  report: Week  31  2022  (2022);  J.  Hadfield  et  al.,  “Nextstrain: Real‐time tracking of pathogen evolution,” Bioinformatics 34, 4121–4123 (2018)).  Currently, Omicron XBB.1.5  is dominant globally,  including  in the United States (Centers for  Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health  and Human Services, CDC; 2023, January 22. https://covid.cdc.gov/coviddata‐tracker (2022)).  Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions)  in the S glycoprotein, among which some are shared between all Omicron VOCs while others  are  specific  to  one  or  more  Omicron  sublineages.  Antigenically,  BA.2.12.1  exhibits  high  similarity with  BA.2  but  not  BA.1, whereas  BA.4  and  BA.5  differ  considerably  from  their  ancestor BA.2 and even more so from BA.1, in line with their genealogy (A. Z. Mykytyn et al.,  “Antigenic cartography of SARS‐CoV‐2 reveals that Omicron BA.1 and BA.2 are antigenically  distinct,” Sci. Immunol. 7, eabq4450 (2022).). Major differences of BA.1 from the remaining  Omicron VOCs include Δ143–145, L212I, or ins214EPE in the S glycoprotein N‐terminal domain  and G446S or G496S in the receptor binding domain (RBD). Amino acid changes T376A, D405N,  and R408S in the RBD are in turn common to BA.2 and its descendants but not found in BA.1.  In  addition,  some  alterations  are  specific  for  individual  BA.2‐descendant  VOCs,  including  L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5 (BA.4 and BA.5 encode for the 30  same S sequence). Most of these shared and VOC‐specific alterations were shown to play an  important  role  in  immune  escape  from monoclonal  antibodies  and  polyclonal  sera  raised  against  the  wild‐type  S  glycoprotein.  In  particular,  the  BA.4/BA.5‐specific  alterations  are  strongly implicated in immune escape of these VOCs (P. Wang et al., “Antibody resistance of  SARS‐CoV‐2  variants  B.1.351  and  B.1.1.7.  Nature  593,  130–135  (2021);  Q.  Wang  et  al.,  “Antibody evasion by SARS‐CoV‐2 Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 5  603–608 (2022)).    Summary  The present disclosure generally embraces  the  immunotherapeutic  treatment of a  subject  comprising the administration of RNA,  i.e., vaccine RNA, encoding an amino acid sequence,  i.e., a vaccine antigen, comprising SARS‐CoV‐2 S protein, an immunogenic variant thereof, or  an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof,  i.e., an antigenic peptide or protein. Thus, the vaccine antigen comprises an epitope of SARS‐ CoV‐2 S protein for inducing an immune response against coronavirus S protein, in particular  SARS‐CoV‐2 S protein, in the subject. RNA encoding vaccine antigen is administered to provide  (following expression of the polynucleotide by appropriate target cells) antigen for induction,  i.e., stimulation, priming and/or expansion, of an  immune response, e.g., antibodies and/or  immune effector cells, which is targeted to target antigen (coronavirus S protein, in particular  SARS‐CoV‐2  S protein) or  a procession product  thereof.  In one  embodiment,  the  immune  response which  is  to  be  induced  according  to  the  present  disclosure  is  a  B  cell‐mediated  immune response, i.e., an antibody‐mediated immune response. Additionally or alternatively,  in one embodiment, the  immune response which is to be  induced according to the present  disclosure is a T cell‐mediated immune response. In one embodiment, the immune response  is an anti‐coronavirus, in particular anti‐SARS‐CoV‐2 immune response.   Vaccines described herein comprise as an active principle single‐stranded RNA that may be  translated  into protein upon entering cells of a recipient.  In addition to wildtype or codon‐ optimized  sequences  encoding  the  antigen  sequence,  the  RNA may  contain  one  or more  structural elements optimized for maximal efficacy of the RNA with respect to stability and  translational efficiency (e.g., 5' cap, 5' UTR, 3' UTR, poly(A)‐tail, or combinations thereof). In  one  embodiment,  the  RNA  contains  all  of  these  elements.  In  one  embodiment,  a  cap1  structure may be utilized as specific capping structure at the 5’‐end of the RNA drug substance.  In  one  embodiment,  beta‐S‐ARCA(D1)  (m2 7,2'‐OGppSpG)  or m2 7,3’‐OGppp(m1 2’‐O)ApG may  be  utilized as  specific  capping  structure at  the 5'‐end of  the RNA drug  substances. As 5'‐UTR  sequence,  the  5'‐UTR  sequence  of  the  human  alpha‐globin  mRNA,  optionally  with  an  optimized ʻKozak sequenceʼ to increase translaƟonal efficiency (e.g., SEQ ID NO: 12) may be  used. As 3'‐UTR sequence, a combination of two sequence elements (FI element) derived from  the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded  12S ribosomal RNA (called I) (e.g., SEQ ID NO: 13) placed between the coding sequence and  the poly(A)‐tail  to assure higher maximum protein  levels and prolonged persistence of  the  mRNA may be used. These were identified by an ex vivo selection process for sequences that  confer  RNA  stability  and  augment  total  protein  expression  (see WO  2017/060314,  herein  incorporated by reference). Alternatively, the 3‘‐UTR may be two re‐iterated 3'‐UTRs of the  human beta‐globin mRNA.   Additionally  or  alternatively,  in  some  embodiments,  an  RNA  comprises  a  poly(A)‐tail  comprising a  length of at  least 90 adenosine nucleotides (including, e.g., at  least about 100  adenosine  nucleotides,  at  least  about  110  adenosine  nucleotides,  at  least  about  120  adenosine  nucleotides,  at  least  about  130  adenosine  nucleotides,  or  longer).  In  some  embodiments,  a  poly(A)‐tail may  comprise  a  length  of  about  90  to  about  150  adenosine  nucleotides  (e.g., about 100 to about 150 adenosine nucleotides).  In some embodiments a  poly(A)‐tail  may  comprise  an  interrupted  poly(A)‐tail.  For  example,  in  some  such  embodiments,  a poly(A)‐tail measuring  about 90  to  about 120 nucleotides  in  length  (e.g.,  about 110 nucleotides in length), consisting of a stretch of about 30 adenosine residues (e.g.,  about 28, about 29, about 30, about 31, or about 32 adenosine residues), followed by a linker  sequence of about 10 nucleotides (of random nucleotides, e.g., about 9, about 10, or about  11 random nucleotides) and another about 70 adenosine nucleotides (e.g., about 65, about  66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, or about  75 adenosine nucleotides) may be used (e.g., a poly(A) tail comprising SEQ  ID NO: 14). This  poly(A)‐tail sequence was designed to enhance RNA stability and translational efficiency.  Furthermore,  in  some  embodiments,  a  nucleotide  sequence  encoding  a  secretory  signal  peptide (sec) may be fused to the antigen‐encoding regions preferably in a way that the sec is  translated as an N terminal tag. In one embodiment, sec corresponds to the secretory signal  peptide of a SARS‐CoV‐2 S protein. In some embodiments, sequences coding for short linker  peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly  used for fusion proteins may be used as GS/Linkers to join a secretory signal and an antigenic  polypeptide.  Vaccine RNA described herein may be complexed with proteins and/or lipids, preferably lipids,  to generate RNA‐particles for administration. If a combination of different RNAs is used, the  RNAs may be  complexed  together or  complexed  separately with proteins  and/or  lipids  to  generate RNA‐particles for administration.  In one aspect,  the disclosure  features a composition or medical preparation comprising an  RNA encoding a SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein the  SARS‐CoV‐2 S polypeptide or fragment comprises: (a) an amino acid sequence having at least  85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1,  and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:1: (1)  D985P, V987P, F817P, A892P, A899P, and A942P; (2) K986P, V987P, F817P, A892P, A899P, and  A942P; (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (4)  K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (5) D985P,  K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (6) D985P,  V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;  (7) K986P, V987P, F817P,  A892P, A899P, A942P, R682G, R683S, and R685S; or  (8) D985P, K986P, V987P, F817P, A892P,  A899P, A942P, R682G, R683S, and R685S; (b)   an amino acid sequence having at  least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; (c) an amino acid sequence having at least  85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70,  and comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1)  D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and  A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (d)  an amino acid sequence having  at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.    In some embodiments, the RNA comprises a modified nucleoside in place of uridine. In some  embodiments,  the  RNA  comprises  modified  uridines  in  place  of  all  uridines.  In  some  embodiments, the RNA comprises N1‐methyl‐pseudouridine (m1ψ) in place of all uridines. In  some  embodiments,  the RNA  comprises  a 5’  cap.  In  some embodiments,  the 5’  cap  is or  comprises m2 7,3’‐OGppp(m1 2’‐O)ApG.   In some embodiments, the RNA comprises a 5’‐UTR that  is or comprises a modified human  alpha‐globin  5’‐UTR.  In  some  embodiments,  the  RNA  comprises  a  5’ UTR  comprising  the  nucleotide sequence of SEQ  ID NO: 12, or a nucleotide sequence that  is at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 12.   In some embodiments, the RNA comprises a 3’‐UTR that is or comprises a first sequence from  the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the  mitochondrial encoded 12S ribosomal RNA.  In some embodiments, the RNA comprises a 3’  UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence that is at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ  ID NO: 13.  In  some embodiments,  the RNA comprises a poly‐A  sequence.  In  some embodiments,  the  poly‐A  sequence  comprises  at  least  100  nucleotides.  In  some  embodiments,  the  poly‐A  sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the  30 adenine nucleotides and 70 adenine nucleotides are separated by a  linker sequence.  In  some embodiments, the poly‐A sequence comprises or consists of the nucleotide sequence of  SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identical to the nucleotide sequence of SEQ ID NO: 14.   In  some  embodiments,  the  RNA  is  formulated  or  is  to  be  formulated  for  intramuscular  administration.  In  some  embodiments,  the  RNA  is  formulated  or  is  to  be  formulated  as  particles. In some embodiments, the particles are lipid nanoparticles (LNPs) or lipoplex (LPX)  particles.  In some embodiments,  the LNPs comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐ 6,1‐diyl)bis(2‐hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐sn‐glycero‐3‐phosphocholine,  and  cholesterol.  In  some  embodiments,  the  lipoplex particles are obtainable by mixing the RNA with liposomes.  In some embodiments, the RNA is mRNA or saRNA.  In  some  embodiments,  the  composition  or  medical  preparation  is  a  pharmaceutical  composition. In some embodiments, the composition or medical preparation is a vaccine.  In  some embodiments, wherein  the pharmaceutical composition  further comprises one or  more pharmaceutically acceptable carriers, diluents and/or excipients.  In  another  aspect,  the  present  disclosure  provides  a  composition  or medical  preparation  comprising a first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment  thereof, and a second RNA encoding a second SARS‐CoV‐2 S polypeptide or an immunogenic  fragment thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an  amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%,  or  100%  identity  to  SEQ  ID NO:1;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P,  A899P, and A942P; (2) K986P, V987P, F817P, A892P, A899P, and A942P; (3) D985P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (4) K986P, V987P, F817P,  A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (5) D985P, K986P, V987P, F817P,  A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (6) D985P, V987P, F817P, A892P,  A899P, A942P, R682G, R683S, and R685S;  (7) K986P, V987P, F817P, A892P, A899P, A942P,  R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G,  R683S, and R685S.  In  one  aspect,  the  present  disclosure  features  a  composition  or  medical  preparation  comprising a first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment  thereof, and a second RNA encoding a second SARS‐CoV‐2 S polypeptide or an immunogenic  fragment thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an  amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%,  or  100%  identity  to  SEQ  ID NO:1;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:69:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:1; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to SEQ  ID NO:70, and comprises one of  the  following sets of amino  substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P,  A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G,  R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:1; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity to SEQ ID NO:104 or 105, and comprises one of the following sets of  amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812P, A887P, A894P,  and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P,  A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P,  R677G, R678S, and R680S.  Another aspect of the disclosure provides a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to  SEQ  ID NO:1, and  comprises one of  the  following  sets of amino  substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P, A899P, and A942P; (2)  K986P, V987P, F817P, A892P, A899P, and A942P;  (3) D985P, V987P, F817P, A892P, A899P,  A942P, D614G, R682G, R683S, and R685S;  (4) K986P, V987P, F817P, A892P, A899P, A942P,  D614G, R682G, R683S, and R685S;  (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P,  D614G, R682G, R683S, and R685S; (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G,  R683S, and R685S; (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;  or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to SEQ  ID NO:69, and comprises one of  the  following sets of amino  substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P,  A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G,  R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to SEQ  ID NO:70, and comprises one of  the  following sets of amino  substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P,  A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G,  R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity to SEQ ID NO:104 or 105, and comprises one of the following sets of  amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812P, A887P, A894P,  and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P,  A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P,  R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to  SEQ  ID NO:1, and  comprises one of  the  following  sets of amino  substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P, A899P, and A942P; (2)  K986P, V987P, F817P, A892P, A899P, and A942P;  (3) D985P, V987P, F817P, A892P, A899P,  A942P, D614G, R682G, R683S, and R685S;  (4) K986P, V987P, F817P, A892P, A899P, A942P,  D614G, R682G, R683S, and R685S;  (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P,  D614G, R682G, R683S, and R685S; (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G,  R683S, and R685S; (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;  or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S.  Another aspect of the disclosure features composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to SEQ  ID NO:69, and comprises one of  the  following sets of amino  substitutions relative to SEQ ID NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P,  A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G,  R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity  to SEQ  ID NO:70, and comprises one of  the  following sets of amino  substitutions relative to SEQ ID NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P,  A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G,  R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70; and (b) the second SARS‐CoV‐2 S polypeptide or fragment comprises  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,  99%, or 100%  identity to SEQ ID NO:104 or 105, and comprises one of the following sets of  amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P, F812P, A887P, A894P,  and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P,  A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P,  R677G, R678S, and R680S.  In one aspect, the disclosure provides a composition or medical preparation comprising a first  RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and a  second  RNA  encoding  a  second  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS‐CoV‐2 S polypeptide or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following sets of amino substitutions relative to SEQ ID NO:1: (1) D985P, V987P, F817P, A892P,  A899P, and A942P; (2) K986P, V987P, F817P, A892P, A899P, and A942P; (3) D985P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (4) K986P, V987P, F817P,  A892P, A899P, A942P, D614G, R682G, R683S, and R685S;  (5) D985P, K986P, V987P, F817P,  A892P, A899P, A942P, D614G, R682G, R683S, and R685S; (6) D985P, V987P, F817P, A892P,  A899P, A942P, R682G, R683S, and R685S;  (7) K986P, V987P, F817P, A892P, A899P, A942P,  R682G, R683S, and R685S; or (8) D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G,  R683S, and R685S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS‐CoV‐2 S polypeptide or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:69:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS‐CoV‐2 S polypeptide or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:70:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:104 or SEQ ID NO:105; and (b) the second SARS‐CoV‐2 S polypeptide or  fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of  the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P,  F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3)  D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P,  V987P,  F817P,  A892P,  A899P,  and  A942P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises  one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P, V984P,  F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3)  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:69:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P; (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein:  (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:69,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:69,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:69: (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S polypeptide or  fragment  comprises  an amino  acid  sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:69, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:69:  (1) D982P, V984P,  F814P, A889P, A896P,  and A939P;  (2) K983P, V984P,  F814P,  A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5)  D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:69: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein:  (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P,  V987P,  F817P,  A892P,  A899P,  and  A942P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises  one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P,  F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3)  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein:  (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:70:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:70,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:70,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S polypeptide or  fragment  comprises  an amino  acid  sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:70, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:70:  (1) D982P, V984P,  F814P, A889P, A896P,  and A939P;  (2) K983P, V984P,  F814P,  A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5)  D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:70,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P,  V987P,  F817P,  A892P,  A899P,  and  A942P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:104 or 105, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P,  and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; (4)  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P,  V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, and A942P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of  the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P,  F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3)  D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S polypeptide or  fragment  comprises  an amino  acid  sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:104 or 105, and comprises one of the following sets of amino substitutions relative  to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P,  V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P,  R677G, R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and  R680S; or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:1, and comprises the following substitutions relative to SEQ  ID NO:1:  D985P, K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P,  V984P,  F814P,  A889P,  A896P,  and  A939P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises  one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P, V984P,  F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3)  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%,  96%,  97%,  98%,  99%,  or  100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets  of  amino  substitutions  relative  to  SEQ  ID NO:70:  (1) D982P, V984P,  F814P,  A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P, A889P,  A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:70: (1) D982P,  V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or (5) D982P, K983P, V984P, F814P,  A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:70,  and  comprises one of  the  following  sets of  amino  substitutions  relative  to  SEQ  ID  NO:70: (1) D982P, V984P, F814P, A889P, A896P, and A939P; (2) K983P, V984P, F814P, A889P,  A896P, and A939P; (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S,  and R682S; or  (5) D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P,  V984P,  F814P,  A889P,  A896P,  and  A939P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:104 or 105, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P,  and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; (4)  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P,  V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of  the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P,  F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3)  D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:69, and comprises the following substitutions relative to SEQ ID NO:69:  D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70:  D982P,  V984P,  F814P,  A889P,  A896P,  and  A939P;  and  (b)  the  second  SARS‐CoV‐2  S  polypeptide or fragment comprises an amino acid sequence having at least 85%, 90%, 91%,  92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ  ID NO:104 or 105, and  comprises one of the following sets of amino substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P, A894P,  and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; (4)  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P,  V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70:  K983P, V984P, F814P, A889P, A896P, and A939P; and (b) the second SARS‐CoV‐2 S polypeptide  or fragment comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,  95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of  the following sets of amino substitutions relative to SEQ ID NO:104 or 105: (1) D980P, V982P,  F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3)  D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or (5) D980P, K981P, V982P, F812P,  A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70:  D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein:  (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70:  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the second  SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at least 85%,  90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or  105, and comprises one of the following sets of amino substitutions relative to SEQ ID NO:104  or 105: (1) D980P, V982P, F812P, A887P, A894P, and A937P; (2) K981P, V982P, F812P, A887P,  A894P, and A937P; (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S,  and R680S; or  (5) D980P,  K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  Another aspect of the disclosure features a composition or medical preparation comprising a  first RNA encoding a first SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, and  a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an  immunogenic  fragment  thereof, wherein: (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity to SEQ ID NO:70, and comprises the following substitutions relative to SEQ ID NO:70:  D982P, K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and (b) the  second SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence having at  least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID  NO:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ  ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P,  F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G,  R678S, and R680S; (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  or (5) D980P, K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S.  The present disclosure, among other things, provides an insight that incorporation of a D985P  mutation rather than a K986P mutation can improve protein express and/or immunogencity  (e.g., improve neutralization response).  In some embodiments, incorporation of D985P rather  than K986P can provide such improvements when combined with one or more other proline  mutations  (e.g., one or more proline mutations disclosed herein).    In  some embodiments,  incorporation of D985P rather than K986P can provide such improvements when combined  with V987P (e.g., one or more proline mutations disclosed herein).    In some embodiments,  incorporation of D985P rather than K986P can provide such improvements when combined  with one or more (e.g., all) of F817P, A892P, A899P, A942P, and V987P.  In some embodiments, the present disclosure provides an insight that RNA encoding a SARS‐ CoV‐2 S protein comprising one or more proline mutations (e.g., one or more of the proline  mutations and/or combination of proline mutations disclosed herein) and a mutated  furin  cleavage site can provide an improved immune response (e.g., an improved immune repsonse  as compared to a similar or same construct comprising an intact furin cleavage site).   In some embodiments, the first RNA and the second RNA each comprise a modified nucleoside  in place of uridine. In some embodiments, the first RNA and the second RNA each comprise  modified uridines in place of all uridines. In some embodiments, the first RNA and the second  RNA each comprise N1‐methyl‐pseudouridine (m1ψ) in place of all uridines.  In some embodiments, the  first RNA and  the second RNA each comprise a 5’ cap.  In some  embodiments, the 5’ cap comprises m2 7,3’‐OGppp(m1 2’‐O)ApG.  In some embodiments, the first RNA and the second RNA each comprise a 5’‐UTR that  is or  comprises a modified human alpha‐globin 5’‐UTR.  In some embodiments, the first RNA and  the second RNA each comprise a 5’ UTR comprising the nucleotide sequence of SEQ ID NO:  12, or a nucleotide sequence  that  is at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identical to the nucleotide sequence of SEQ ID NO: 12.  In some embodiments, the first RNA and the second RNA each comprise a 3’‐UTR that  is or  comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA  and  a  second  sequence  from  the  mitochondrial  encoded  12S  ribosomal  RNA.  In  some  embodiments,  the  first RNA  and  the  second RNA  each  comprise  a  3’ UTR  comprising  the  nucleotide sequence of SEQ  ID NO: 13, or a nucleotide sequence that  is at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 13.  In some embodiments, the first RNA and the second RNA each comprise a poly‐A sequence.  In some embodiments, the first RNA and the second RNA each comprise a poly‐A sequence  that comprises at least 100 nucleotides. In some embodiments, the first RNA and the second  RNA each comprise a poly‐A sequence that comprises 30 adenine nucleotides followed by 70  adenine nucleotides, wherein  the 30  adenine nucleotides and 70  adenine nucleotides are  separated by a linker sequence. In some embodiments, the first RNA and the second RNA each  comprise a poly‐A sequence that comprises or consists of the nucleotide sequence of SEQ ID  NO: 14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identical to the nucleotide sequence of SEQ ID NO: 14.   In  some  embodiments,  the  first RNA  and  the  second  RNA  are  each  formulated  or  to  be  formulated for intramuscular administration.  In  some  embodiments,  the  first  RNA  and  the  second  RNA  are  each  formulated  or  to  be  formulated as particles. In some embodiments,the first RNA and the second RNA are each to  be  formulated  as  lipid  nanoparticles  (LNPs)  or  lipoplex  (LPX)  particles.  In  some  embodiments,the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.  In some embodiments, the first RNA and the second RNA are formulated in separate LNPs. In  some embodiments, the first RNA and the second RNA are  formulated  in the same LNP.  In  some embodiments, the lipoplex particles are obtainable by mixing the RNA with liposomes.  In  some  embodiments,  the  first  RNA  and  the  second  RNA  are  each  mRNA.    In  some  embodiments, the first RNA and the second RNA are each saRNA.  In  some  embodiments,  the  composition  or  medical  preparation  is  a  pharmaceutical  composition. In some embodiments, the composition or medical preparation is a vaccine.  In  some  embodiments,  the  pharmaceutical  composition  further  comprises  one  or  more  pharmaceutically acceptable carriers, diluents and/or excipients.  In another aspect,  the disclosure provides a method of  inducing an  immune  response  in a  subject,  the  method  comprising  administering  to  the  subject  a  composition  or  medical  preparation described herein thereby inducing an immune response in the subject.  In some embodiments, the SARS‐CoV‐2 S polypeptide comprises an amino acid sequence that  does not comprise a D985P substitution relative to SEQ ID NO:1; does not comprise a D982P  substitution  relative  to  SEQ  ID  NO:69  or  SEQ  ID  NO:70,  or  does  not  comprise  a  D980P  substitution relative to SEQ ID NO:104 or SEQ ID NO:105.  In some embodiments, the method further comprises administering a second RNA encoding  a second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein the second  SARS‐CoV‐2  S polypeptide or  immunogenic  fragment  is  a  SARS‐CoV‐2  S polypeptide of  an  Omicron variant that is not a BA.1 Omicron variant.  In some embodiments, the method further comprises administering a second, different RNA  encoding a second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein  the  second  SARC‐CoV‐2  S  polypeptide  or  fragment  is  selected  from  an  SARS‐CoV‐2  S  polypeptide or fragment described herein.  Another aspect of  the disclosure provides a method of  inducing an  immune  response  in a  subject,  the method  comprising  administering  to  the  subject  the  composition  or medical  preparation described herein, thereby inducing an immune response in the subject. In some  embodiments, the SARS‐CoV‐2 S polypeptide comprises an amino acid sequence that does not  comprise  a  D985P  substitution  relative  to  SEQ  ID  NO:1;  does  not  comprise  a  D982P  substitution  relative  to  SEQ  ID  NO:69  or  SEQ  ID  NO:70,  or  does  not  comprise  a  D980P  substitution relative to SEQ ID NO:104 or SEQ ID NO:105.  In some embodiments, the method further comprises administering a second composition or  medical preparation, wherein the second composition or medical preparation comprises an  RNA  encoding  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  of  an Omicron  variant that is not a BA.1 Omicron variant.    In some embodiments, the method further comprises administering a second composition or  medical preparation, wherein  the  second composition or medical preparation comprises a  third RNA encoding a third SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof,  and a fourth RNA encoding a fourth SARS‐CoV‐2 S polypeptide or an immunogenic fragment  thereof.  In  some  embodiments,  the  third  RNA  encodes  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof  that  is  a  first  or  a  second  SARS‐CoV‐2  S  polypeptide  or  immunogenic  fragment  thereof recited  in any one of claims 24‐102, and wherein  the third  RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that  is different from the first SARS‐ CoV‐2 S polypeptide or fragment encoded by the first RNA and/or that is different from the  second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.  In  some  embodiments,  the  fourth  RNA  encodes  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof  that  is  a  first  or  a  second  SARS‐CoV‐2  S  polypeptide  or  immunogenic fragment thereof recited in any one of claims 24‐102, and wherein the fourth  RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that  is different from the first SARS‐ CoV‐2 S polypeptide or fragment encoded by the first RNA and/or that is different from the  second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.    In  some  embodiments,  the  third  RNA  encodes  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof  that  is  a  first  or  a  second  SARS‐CoV‐2  S  polypeptide  or  immunogenic  fragment  thereof recited  in any one of claims 24‐102, and wherein  the third  RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that  is different from the first SARS‐ CoV‐2  S polypeptide or  fragment encoded by  the  first RNA  and  that  is different  from  the  second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.    In  some  embodiments,  the  fourth  RNA  encodes  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof  that  is  a  first  or  a  second  SARS‐CoV‐2  S  polypeptide  or  immunogenic fragment thereof recited in any one of claims 24‐102, and wherein the fourth  RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that  is different from the first SARS‐ CoV‐2  S polypeptide or  fragment encoded by  the  first RNA  and  that  is different  from  the  second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.    In  some  embodiments,  the  third  RNA  encodes  an  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof  that  is  a  first  or  a  second  SARS‐CoV‐2  S  polypeptide  or  immunogenic fragment thereof recited  in any one of claims 24‐102, wherein the third RNA  encodes a SARS‐CoV‐2 S polypeptide or fragment that is different from the first SARS‐CoV‐2 S  polypeptide or fragment encoded by the first RNA and that is different from the second SARS‐ CoV‐2  S  polypeptide  or  fragment  encoded  by  the  second  RNA,  wherein  the  fourth  RNA  encodes an SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof that is a first or a  second SARS‐CoV‐2 S polypeptide or  immunogenic  fragment  thereof  recited  in any one of  claims 24‐102, wherein the fourth RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that  is different from the first SARS‐CoV‐2 S polypeptide or fragment encoded by the first RNA and  that  is  different  from  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  encoded  by  the  second RNA.    In some embodiments, each of the first, second, third, and fourth RNAs encodes a different  SARS‐CoV‐2 S polypeptide or immunogenic fragment thereof.  In  another  aspect,  a monovalent  vaccine  as described  herein  can  be  administered with  a  bivalent  vaccine  as  described  herein.  For  example,  in  some  embodiments,  a method  of  inducing  an  immune  response  comprises  administering  to  a  subject  (i)  a  composition  or  medical  preparation  described  herein  that  comprises  an  RNA  encoding  a  SARS‐CoV‐2  S  polypeptide or an immunogenic fragment thereof described herein and (ii) a composition or  medical preparation comprising at least a first RNA encoding a first SARS‐CoV‐2 S polypeptide  or an  immunogenic  fragment  thereof, and a second RNA encoding a second SARS‐CoV‐2 S  polypeptide  or  an  immunogenic  fragment  thereof  as  described  herein.[DDM1]      In  some  embodiments, the monovalent vaccine and the bivalent vaccine can be administered at least  3 weeks apart, including, e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7  weeks, at  least 8 weeks, or  longer.  In some embodiments, the monovalent vaccine and the  bivalent vaccine can be administered at least 3 months apart, including, e.g., at least 4 months,  at least 5 months, at least 6 months, at least 7 months, at least 8 months, or longer. In some  embodiments,  the monovalent  vaccine  and  the  bivalent  vaccine  can  be  administered  on  different arms  in a single  session.  In some embodiments,  the monovalent vaccine and  the  bivalent vaccine can be administered as a trivalent vaccine in a single injection (e.g., mixing  the monovalent and bivalent vaccines together prior to administration).       
Brief description of the drawings    Figure 1. Schematic overview of the S protein organization of the SARS‐CoV‐2 S protein.  The  sequence within  the S1  subunit  consists of  the  signal  sequence  (SS) and  the  receptor  binding domain  (RBD) which  is  the  key  subunit within  the  S protein which  is  relevant  for  binding to the human cellular receptor ACE2. The S2 subunit contains the S2 protease cleavage  site  (S2’)  followed by a  fusion peptide  (FP)  for membrane  fusion, heptad repeats  (HR1 and  HR2) with a central helix (CH) domain, the transmembrane domain (TM) and a cytoplasmic tail  (CT).    Figure 2. Exemplary SARS‐CoV‐2 vaccine constructs.   Based on the full and wildtype S protein, we have designed different constructs encoding the  (1) full protein with mutations in close distance to the first heptad repeat (HRP1) that include  stabilizing mutations preserving neutralisation sensitive sites, the (2) S1 domain or the (3) RB  domain (RBD) only. Furthermore, to stabilize the protein fragments a fibritin domain (F) was  fused  to  the  C‐terminus.  All  constructs  start with  the  signal  peptide  (SP)  to  ensure Golgi  transport to the cell membrane.    Figure 3. General structure of Certain RNA vaccines.  Schematic illustration of the general structure of certain RNA vaccines with 5'‐cap, 5'‐ and 3'‐ untranslated regions, coding sequences with intrinsic secretory signal peptide as well as GS‐ linker, and poly(A)‐tail. Please note that the individual elements are not drawn exactly true to  scale compared to their respective sequence lengths.  UTR = Untranslated region; sec = Secretory signal peptide; RBD = Receptor Binding Domain;  GS = Glycine‐serine linker.    Figure 4. General structure of Certain RNA vaccines.  Schematic illustration of the general structure of certain RNA drug substances with 5'‐cap, 5'‐  and 3'‐untranslated regions, coding sequences with intrinsic secretory signal peptide as well  as GS‐linker, and poly(A)‐tail. Please note that the individual elements are not drawn exactly  true to scale compared to their respective sequence lengths.  GS = Glycine‐serine linker; UTR = Untranslated region; Sec = Secretory signal peptide; RBD =  Receptor Binding Domain.    Figure 5. General structure of Certain RNA vaccines.  Schematic  illustration  of  the  general  structure  of  RNA  vaccines  with  5'‐cap,  5'‐  and  3'‐ untranslated regions, coding sequences of the Venezuelan equine encephalitis virus  (VEEV)  RNA‐dependent  RNA  polymerase  replicase  and  the  SARS‐CoV‐2  antigen  with  intrinsic  secretory signal peptide as well as GS‐linker, and poly(A)‐tail. Please note that the individual  elements are not drawn exactly true to scale compared to their respective sequence lengths.  UTR = Untranslated region; Sec = Secretory signal peptide; RBD = Receptor Binding Domain;  GS = Glycine‐serine linker.    Figure 6. Schematic overview of the S protein organization of the SARS‐CoV‐2 S protein and  constructs for the development of a SARS‐CoV‐2 vaccine.  Based on the wildtype S protein, we have designed two different transmembrane‐anchored  RBD‐based vaccine constructs encoding the RBD fragment fused to the T4 fibritin trimerization  domain (F) and the autochthonus transmembrane domain (TM). Construct (1) starts with the  SARS‐CoV‐2‐S signal peptide (SP; AA 1‐19 of the S protein) whereas construct (2) starts with  the  human  Ig  heavy  chain  signal  peptide  (huSec)  to  ensure  Golgi  transport  to  the  cell  membrane.    Figure  7.  Anti‐S  protein  IgG  response  6,  14  and  21 d  after  immunization  with  LNP‐C12  formulated modRNA coding for transmembrane‐anchored RBD‐based vaccine constructs.  BALB/c mice were  immunized  IM once with 4 µg of  LNP‐C12‐formulated  transmembrane‐ anchored RBD‐based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). On day 6, 14  and 21 after immunization, animals were bled and the serum samples were analyzed for total  amount  of  anti‐S1  (left)  and  anti‐RBD  (right)  antigen  specific  immunoglobulin  G  (IgG)  measured  via  ELISA.  For  day  6  (1:50),  day  14  (1:300)  and  day  21  (1:900)  different  serum  dilution were included in the graph. One point in the graph stands for one mouse, every mouse  sample was measured in duplicates (group size n=8; mean + SEM is included for the groups).    Figure 8. Neutralization of SARS‐CoV‐2 pseudovirus 6, 14 and 21 d after immunization with  LNP‐C12  formulated  modRNA  coding  for  transmembrane‐anchored  RBD‐based  vaccine  constructs.  BALB/c mice were  immunized  IM once with 4 µg of  LNP‐C12‐formulated  transmembrane‐ anchored RBD‐based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). On day 6, 14  and  21  after  immunization,  animals were  bled  and  the  sera were  tested  for  SARS  CoV‐2  pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious  events, compared to positive controls without serum). One point in the graphs stands for one  mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean + SEM is shown  by horizontal bars with whiskers for each group. LLOQ,  lower  limit of quantification. ULOQ,  upper limit of quantification.    Fig. 9. 50% pseudovirus neutralization  titers  (pVNT50) of  sera collected 21 days after  the  second  dose  and  1 month  after  the  third  dose  of  BNT162b2  against  VSV‐SARS‐CoV‐2‐S  pseudovirus  bearing  the Wuhan Hu‐1  reference  or Omicron  BA.1  lineage  spike  protein.  N=19‐20 sera from immunized subjects collected either 21 days after the second BNT162b2  dose or 1 months after the third BNT162b2 dose were tested. For values below the limit of  detection  (LOD; 10),  LOD/2 values are plotted. Group GMTs  (values above bars) with 95%  confidence intervals are shown.    Fig.  10.  CD8+  T  cell  epitopes  in  BNT162b2  vaccine  remain  largely  unaffected   by Omicron variant mutations.  Shown is the number of previously identified MHC‐I epitopes  affected  in various variants of concern  (VOCs).   Approximately 80% of previously  identified  CD8+ epitopes are not affected by the mutations in the Omicron BA.1 variant, suggesting that  two doses of BNT162b2 may still induce protection against severe disease.    Fig. 11. Neutralization of Omicron BA.1 after two doses of BNT162b2 and variant specific  booster.    Shown  is  neutralization  of  the  Omicron  BA.1  variant  from  sera  of  patients  administered two doses of BNT162b2 and (i) a third booster dose of BNT162b2, or (ii) a third  booster dose of an RNA encoding a Spike protein with alpha or delta variant mutations, or a  third booster dose of both a Spike protein comprising alpha mutations and a Spike protein  comprising delta mutations.  The values are derived from separate neutralization GMTs from  the pseudovirus testing. Also shown  is a schematic depicting a process  for developing new  SARS‐CoV‐2 variant specific vaccines.    Fig. 12. Longitudinal analysis of neutralizing antibody responses against VSV‐SARS‐CoV‐2‐S  pseudovirus bearing the Wuhan or Omicron BA.1 variant spike protein in a subset of study  participants. Sera  from n=9 participants drawn at 21 days after dose 2, prior  to dose 3, 1  month after dose 3 and 3 months after dose 3 were tested. Each serum was tested in duplicate  and  individual geometric mean 50% pseudovirus neutralizing titers (GMTs) were calculated.  For  values  below  the  limit  of  detection  (LOD),  LOD/2  values were  assigned. Group GMTs  (values in table) and 95% confidence intervals per time point are indicated.    Fig. 13. Analysis of HLA class I T cell epitopes conservation between the Wuhan and  Omicron BA.1 variants. HLA class I restricted Spike protein epitopes with T cell reactivity  identified based on their recognition by CD8+ T cells and reported in IEDB (n=244) are  plotted by their position (top row) along the Spike protein (bottom row). Epitope indications  are positioned by the amino acid position of the center of the epitope; epitopes conserved in  both variants are marked in light gray (n=208);  while epitopes spanning an Omicron BA.1  mutation site are marked dark gray (n=36). NTD=N‐terminal domain; RBD=Receptor‐binding  domain; FCS=Furin cleavage site. The S1 and S2 regions of the Spike protein are indicated.    Fig. 14. Schematics of an exemplary vaccination regimen.    Fig. 15. Cohorts, sampling and experimental setup for characterization of immune  response in Omicron BA.1 breakthrough cases.  Blood samples were drawn from four  cohorts: Omicron‐naïve individuals double‐ or triple‐vaccinated with BNT162b2, and  individuals double‐ or triple‐vaccinated with BNT162b2 that subsequently had a  breakthrough infection with Omicron BA.1. PBMCs and sera were isolated in the Omicron‐ naïve cohorts at the time‐points indicated following their most recent vaccination; for  convalescent cohorts, the time from their most recent vaccination to Omicron BA.1  infection, and infection to PBMC and serum isolation are indicated (all values specified as  median‐range). Serum neutralizing capacity was assessed using a pseudovirus and live virus  neutralization test; SARS‐CoV‐2 spike‐specific BMEM cells were assessed via a flow cytometry‐ based B cell phenotyping assay using bulk PBMCs. N/A, not applicable.    Fig. 16. Omicron BA.1 breakthrough infection in BNT162b2 double‐ and triple‐vaccinated  individuals induces broad neutralization of Omicron BA.1, BA.2 and other VOCs.   Serum was drawn from double‐vaccinated individuals (BNT162b22) at 22 days after the  second dose (open circles), from triple‐vaccinated individuals (BNT162b23) at 28 days after  the third dose (closed circles), from double‐vaccinated individuals with an Omicron BA.1  breakthrough infection (BNT162b22 + Omi) at 46 days post‐infection (open triangles), and  from triple‐vaccinated individuals and Omicron BA.1 breakthrough infection (BNT162b23 +  Omi) at 44 days post‐infection (closed triangles). Serum was tested in duplicate; (A) shows  50% pseudovirus neutralization (pVN50) geometric mean titers (GMTs), (B) shows 50% virus  neutralization (VN50) GMTs, and (C) shows the geometric mean ratio of SARS‐CoV‐2 variant  of concern (VOC) and Wuhan VN50 GMTs. For titer values below the limit of detection (LOD),  LOD/2 values were plotted. Values above violin plots represent group GMTs. The non‐ parametric Friedman test with Dunn’s multiple comparisons correction was used to compare  Wuhan neutralizing group GMTs with titers against the indicated variants and SARS‐CoV‐1.  Multiplicity‐adjusted p values are shown. (A) pVN50 GMTs against Wuhan, VOC and SARS‐ CoV‐1 pseudovirus. (B) VN50 GMTs against live SARS‐CoV‐2 Wuhan, Beta, Delta and Omicron  BA.1. (C) Group geometric mean ratios with 95% confidence intervals for all cohorts shown  in (B).    Fig.  17.  BMEM  cells  of  individuals  double‐  and  triple‐vaccinated  with  BNT162b2  broadly  recognize VOCs and are  further boosted by Omicron BA.1 breakthrough  infection. PBMC  samples  from double‐vaccinated  individuals  (BNT162b22) at 22 days after  the second dose  (open  squares)  and 5 months  after  the  second dose  (open  circles),  from  triple‐vaccinated  individuals  (BNT162b23)  at  84  days  after  the  third  dose  (closed  circles),  from  double‐ vaccinated  individuals with Omicron BA.1 breakthrough  infection  (BNT162b22 + Omi) at 46  days post‐infection (open triangles), and from triple‐vaccinated individuals with Omicron BA.1  breakthrough infection (BNT162b23 + Omi) at 44 days post‐infection (closed triangles) were  analyzed via flow cytometry for SARS‐CoV‐2‐specific BMEM cell (BMEM – CD3‐CD19+CD20+IgD‐ CD38int/low) frequencies via B cell bait staining. (A) Schematic of one‐dimensional staining of  BMEM  cells  with  fluorochrome‐labeled  SARS‐CoV‐2  S  protein  tetramer  bait  allowing  discrimination of variant recognition. Frequencies of Wuhan or VOC full‐length S protein‐ (B)  and RBD‐  (C)  specific BMEM  cells  for  the  four groups of  individuals were analyzed. Variant‐ specific BMEM cell  frequencies were normalized to Wuhan  frequencies  for S protein  (D) and  RBD‐ (E) binding. (F) Depicts the frequency ratios of RBD protein specific BMEM cells over full‐ length S protein‐specific BMEM cells.    Fig. 18. Omicron BA.1 breakthrough  infection of BNT162b2 double‐ and  triple‐vaccinated  individuals primarily boosts BMEM  against  conserved epitopes  shared broadly between  S  proteins of Wuhan and other VOCs rather than strictly Omicron S‐specific epitopes.  PBMC  samples  from double‐vaccinated  individuals  (BNT162b22) at 22 days after  the second dose  (open  squares)  and 5 months  after  the  second dose  (open  circles),  from  triple‐vaccinated  individuals  (BNT162b23)  at  84  days  after  the  third  dose  (closed  circles),  from  double‐ vaccinated  individuals with Omicron BA.1 breakthrough  infection  (BNT162b22 + Omi) at 46  days post‐infection (open triangles), and from triple‐vaccinated individuals with Omicron BA.1  breakthrough infection (BNT162b23 + Omi) at 44 days post‐infection (closed triangles) were  analyzed  via  flow  cytometry  for  SARS‐CoV‐2‐specific  memory  B  cell  (B MEM  –  CD3‐ CD19+CD20+IgD‐CD38int/low) frequencies via B cell bait staining (schematic shown in (A)). (B)  shows representative flow plots of Omicron BA.1 and Wuhan S protein‐ and RBD‐binding for  each of the four groups of individuals investigated. Frequencies of BMEM binding Omicron BA.1,  Wuhan, or both  (shared)  shown  for  full‐length  S protein  in  (C)  and RBD  shown  in  (D)  for  Omicron  BA.1‐experienced  and  naïve  BNT162b2  double  and  triple  vaccinees.  (E)  Venn  diagrams  visualizing  the  combinatorial  (Boolean)  gating  strategy  to  identify  cross‐reactive  BMEM  recognizing  all  four  variants  simultaneously  (All  4  +ve)  and  BMEM  recognizing  only  Omicron BA.1 (only Omi) or only Wuhan (only Wuhan) S proteins. Frequencies for these three  BMEM sub‐groups were compared for full‐length S protein (F) and RBD (G) in the four different  groups  of  individuals  investigated.  RBD  variant  recognition  pattern  by  BMEM was  assessed  through Boolean flow cytometric gating strategy and frequencies recognizing combinations of  Wuhan and/or variant RBDs are displayed in (H), for all Omicron convalescent subjects (double  and triple vaccinees pooled, n=13). (I) Conserved site within the RBD domain recognized by  RBD‐specific BMEM after Omicron BA.1 break‐through infection. Mean values are indicated in  C, D, F, and G. n = number of individuals per group.    Fig. 19. Omicron  BA.1 breakthrough infection of individuals vaccinated with other approved  COVID‐19  vaccines  or  mixed  regimens  results  in  immune  sera  that  broadly  neutralize  Omicron BA.1, BA.2 and other VOCs plus SARS‐CoV‐1. Serum was drawn from 10 individuals  vaccinated with other approved COVID‐19 vaccines or mixed regimens at a median of 43 days  after  infection  (grey diamonds). Serum was tested  in duplicate;  individual 50% pseudovirus  neutralization (pVN50) geometric mean titers (GMTs) against SARS‐CoV‐2 Wuhan, Alpha, Beta,  Delta and Omicron BA.1 and BA.2 variants, plus SARS‐CoV‐1 were plotted. For  titer values  below  the  limit  of  detection  (LOD),  LOD/2  values were  plotted. Values  above  violin  plots  represent group GMTs. The non‐parametric Friedman test with Dunn’s multiple comparisons  correction was  used  to  compare Wuhan  neutralizing  group  GMTs with  titers  against  the  indicated  variants  and  SARS‐CoV‐1. Multiplicity‐adjusted  p  values  are  shown.    Approved  vaccines  included AZD1222, BNT162b2  (in  some embodiments as part of a 4‐dose  series),  Ad26.COV2.S,  mRNA‐1273  (administered  as  a  two‐dose  or  three‐dose  series),  and  combinations thereof.    Fig. 20. 50% neutralization titers of sera collected 1 month after a fourth dose of BNT162b2  or an Omicron‐specific booster.  Subjects who were previously administered  two doses of  BNT162b2, and a third (booster) dose of BNT162b2 (30 ug) received a dose (30 ug) of (i) an  RNA encoding a SARS‐CoV‐2 S protein from an Omicron BA.1 variant (e.g., as described herein  (referred to herein as “Omicron‐specific RNA vaccine“), or (ii) BNT162b2, as a fourth (boster)  dose.  Sera  from  the  subjects  were  collected  one month  after  administration  of  the  4th  (booster) dose.  Group GMTs (values above bars) with 95% confidence intervals are shown.   “b2”  refers  to  sera  from  subjects  administered  Wuhan‐specific  RNA  vaccine  as  the  4th  (booster) dose of BNT162b2.   “OMI” refers to sera from subjects administered an Omicron  BA.1‐specific  4th  (booster)  dose.    Also  shown  is  the  fold‐change  in  titer  from  before  administration of the 4th dose to after administration of the 4th dose (Pre/Post Vax Fold‐Rise),  and the ratio of geometric mean ratio (GMR) and geometric mean fold rise (GMFR) observed  in subjects administered a 4th dose of an Omicron BA.1‐specific RNA vaccine as the 4th dose,  as compared to subjects administered BNT162b2 as the 4th dose of an Omicron BA.1‐specific  RNA vaccine. “FFRNT” refers to fluorescent focus reduction neutralization test.  Neutralization  data was obtained using an FFRNT assay, with a viral particle containing a SARS‐CoV‐2 S protein  from the variant indicated in the figures.  (A) Comparison of titers of neutralizing antibodies  against  a  SARS‐CoV‐2‐S  pseudovirus  comprising  a  SARS‐CoV‐2  S  protein  having mutations  characteristics of an Omicron BA.1 variant.  Sera from subjects previously or currently infected  with SARS‐CoV‐2 excluded.  (B) Comparison of titers of neutralizing antibodies against a SARS‐ CoV‐2 pseudovirus comprising a SARS‐CoV‐2 S protein having mutations characteristics of an  Omicron BA.1 variant in sera from a population that includes subjects previously or currently  infected with SARS‐CoV‐2 (as determined by an antigen assay or a PCR assay respectively). (C)  Comparison of titers of neutralizing antibodies against a SARS‐CoV‐2 pseudovirus comprising  a  SARS‐CoV‐2  S  protein  from  a Wuhan  strain.  Sera  from  subjects  previously  or  currently  infected with  SARS‐CoV‐2  excluded.    (D)    Comparison  of  titers  of  neutralizing  antibodies  against a SARS‐CoV‐2 pseudovirus comprising a SARS‐CoV‐2 S protein from a Wuhan strain, in  sera from a population comprising individuals previously or currently infected with SARS‐CoV‐ 2 (as determined by an antigen assay or a PCR assay, respectively. (E) Comparison of titers of  neutralizing antibodies against a SARS‐CoV‐2 pseudovirus comprising a SARS‐CoV‐2 S protein  having mutations characteristics of a delta variant. Sera from subjects previously or currently  infected with  SARS‐CoV‐2 excluded.    (F)   Comparison of  titers of neutralization antibodies  against  a  SARS‐CoV‐2  pseudovirus  comprising  a  SARS‐CoV‐2  protein  having  mutations  characteristic of a delta variant,  in  sera  from a population  including  subjects previously or  currently  infected  with  SARS‐CoV‐2  (as  determined  by  an  antigen  assay  or  a  PCR  assay,  respectively).     Fig. 21. Neutralization of SARS‐CoV‐2 pseudovirus 7 days after immunization with modRNA  coding  for  variant  specific  S  proteins.   Mice were  immunized  twice with  LNP‐formulated  vaccine comprising (i) BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan strain), (ii)  RNA encoding a SARS‐CoV‐2 S protein having mutations characteristic of an Omicron BA.1  variant (Omi), (iii) RNA encoding an S protein having mutations characteristic of a delta variant,  (iv)  a  combination  of  BNT162b2  and  an  RNA  encoding  an  protein  having  mutations  characteristic  of  an Omicron  BA.1  variant  (B2+Omi),  or  (v)  RNA  encoding  a  SARS‐CoV‐2  S  protein having mutations characteristic of a delta variant and RNA encoding a SARS‐CoV‐2 S  protein having mutations characteristic of an Omicron BA.1 variant (Delta + Omi). 7 days after  the second immunization, animals were bled and sera was tested for neutralization of a SARS‐ CoV‐2‐S pseudovirus comprising a SARS‐CoV‐2 S protein from a Wuhan strain, or a SARS‐CoV‐ 2 S protein having mutations characteristic of a beta, delta, or Omicron BA.1 variant. Graphs  depict  pVN50  serum  dilutions  (50%  reduction  of  infectious  events,  compared  to  positive  controls without serum). One point in the graphs stands for one mouse. Every mouse sample  was measured in duplicate. Mean + SEM is shown by horizontal bars with whiskers for each  group. LLOD, lower limit of detection. ULOD, upper limit of detection.    Fig. 22.   RNA encoding a SARS‐CoV‐2 S protein having mutations characteristic of a Beta  variant  increases neutralization antibody titers against SARS‐CoV‐2 when administered to  patients previously administered two doses of a vaccine encoding a SARS‐CoV‐2 S protein of  a Wuhan strain.  Subjects previously administered two doses of an RNA vaccine encoding a  SARS‐CoV‐2 S protein of a Wuhan strain were administered a third and a fourth dose of an  RNA  vaccine  encoding  a  SARS‐CoV‐2  S  protein  having mutations  characteristic  of  a  Beta  variant.    Neutralization  antibody  titers  were measured  before  administration  of  an  RNA  vaccine encoding a SARS‐CoV‐2 S protein of a Wuhan strain  (D1‐PreVax), one month after  administration of a  second dose of an RNA vaccine encoding a SARS‐CoV‐2 S protein of a  Wuhan strain  (M1PD2), one‐month after administration of a  third dose of an RNA vaccine  encoding a SARS‐CoV‐2 S protein having mutations characteristic of a SARS‐CoV‐2 Beta variant,  and one month after administration of a fourth dose of an RNA vaccine encoding a SARS‐CoV‐ 2 S protein having mutations characteristic of a SARS‐CoV‐2 Beta variant.  The third and fourth  doses were administered 1 month apart  from one another.   GMFR refers to the geometric  mean  fold rise, and  is a measure of  the  increase  in neutralization antibody  titers since  the  previous vaccine dose (e.g., the GMFR for Post‐Dose2 (PD2)  is a measure of the  increase  in  neutralization antibody titers relative to before administration of any vaccine (pre‐vax)).  (A)  Neutralization antibody titers measured in a viral neutralization assay that uses a viral particle  comprising  a  SARS‐CoV‐2  S  protein  of  a Wuhan  strain.    (B) Neutralization  antibody  titers  measured in a viral neutralization assay that uses a viral particle comprising a SARS‐CoV‐2 S  protein having mutations characteristic of a Beta variant.       Fig. 23. 50% neutralization titers of sera collected 7 days after a fourth dose of BNT162b2,  an  Omicron  BA.1‐specific  booster,  or  a  bivalent  vaccine.  Subjects  who  were  previously  administered two doses of BNT162b2 (30 ug), and a third (booster) dose of BNT162b2 (30 ug)  received (i) a 30 ug dose of BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan strain),  (ii) a 60 ug dose of BNT162b2, (iii) a 30 ug dose of RNA encoding a SARS‐CoV‐2 S protein having  mutations characteristic of an Omicron BA.1 variant  (e.g., as described herein  (referred  to  herein as “Omicron‐specific RNA vaccine“)), (iii) a 60 ug dose of RNA encoding a SARS‐CoV‐2 S  protein having mutations characteristic of an Omicron BA.1 variant,  (iv) a 30 ug dose of a  bivalent vaccine, comprising 15 ug of BNT162b2 and 15 ug of RNA encoding a SARS‐CoV‐2 S  protein comprising mutations characteristic of an Omicron BA.1 variant, or (v) a 60 ug dose of  a bivalent vaccine, comprising 30 ug of BNT162b2 and 30 ug of RNA encoding a SARS‐CoV‐2 S  protein comprising mutations characteristic of an Omicron BA.1 variant. Geometric mean ratio  (GMR) of  titers  in serum  from subjects were collected 7 days after administration of a 4th  dose.  “b2” refers to sera from subjects administered a Wuhan‐specific RNA vaccine as a 4th  dose of BNT162b2.  “OMI” refers to sera from subjects administered an Omicron BA.1‐specific  4th  dose.    “Bivalent”  refers  to  sera  from  subjects  administered  a  composition  comprising  BNT162b2  and  an  RNA  encoding  a  SARS‐CoV‐2  S  protein  comprising mutations  that  are  characteristic of an Omicron BA.1 variant as a 4th dose.  Also shown is the fold‐rise in titer from  before administration of a 4th dose to 7 days after administration of a 4th dose (*Fold‐Rise).  “FFRNT”  refers  to  fluorescent  focus  reduction neutralization  test.   Neutralization data was  obtained using an FFRNT assay, with a viral particle containing a SARS‐CoV‐2 S protein having  mutations characteristic of the variant indicated in the figures.  LLOQ refers to Lower Limit of  Quantification and ULOQ refers to Upper Limit of Quantification. (A) Comparison of titers of  neutralizing antibodies against a SARS‐CoV‐2 pseudovirus comprising a SARS‐CoV‐2 S protein  having mutations characteristics of an Omicron BA.1 variant.  Sera from subjects previously or  currently  infected  with  SARS‐CoV‐2  excluded.    (B)  Comparison  of  titers  of  neutralizing  antibodies  against  a  SARS‐CoV‐2  pseudovirus  comprising  a  SARS‐CoV‐2  S  protein  having  mutations characteristics of an Omicron BA.1 variant in sera from a population that includes  subjects previously or currently infected with SARS‐CoV‐2 (e.g., as determined by an antibody  test or a PCR assay respectively). (C) Comparison of titers of neutralizing antibodies against a  SARS‐CoV‐2 pseudovirus  comprising a  SARS‐CoV‐2  S protein of a Wuhan  strain. Sera  from  subjects previously or currently infected with SARS‐CoV‐2 excluded.  (D)  Comparison of titers  of  neutralizing  antibodies  against  a  SARS‐CoV‐2  pseudovirus  comprising  a  SARS‐CoV‐2  S  protein of a Wuhan strain,  in sera from a population that  includes  individuals previously or  currently infected with SARS‐CoV‐2. (E) Comparison of titers of neutralizing antibodies against  a SARS‐CoV‐2 pseudovirus comprising a SARS‐CoV‐2 S protein having mutations characteristics  of  a  Delta  variant.  Sera  from  subjects  previously  or  currently  infected  with  SARS‐CoV‐2  excluded.    (F)    Comparison  of  titers  of  neutralization  antibodies  against  a  SARS‐CoV‐2  pseudovirus  comprising a  SARS‐CoV‐2  S protein having mutations  characteristic of a Delta  variant,  in  sera  from  a population  including  subjects previously or  currently  infected with  SARS‐CoV‐2. (G) Geometric mean rise (GMR) of neutralization antibodies observed in subjects  administered  60  ug  of BNT162b2,  30  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  mutations characteristic of an Omicron BA.1 variant  (OMI 30 ug), 60 ug of RNA encoding a  SARS‐CoV‐2 S protein having mutations characteristic of an Omicron BA.1 variant (OMI 60 ug),  30 ug of a bivalent vaccine comprising 15 ug of BNT162b2 and 15 ug of RNA encoding a SARS‐ CoV‐2 S protein having mutations characteristic of an Omicron BA.1 variant (Bivalent 30 ug),  or 60 ug of a bivalent vaccine comprising 30 ug of BNT162b2 and 30 ug of RNA encoding a  SARS‐CoV‐2 S protein having mutations characteristic of an Omicron BA.1 variant (Bivalent 60  ug), as compared to subjects administered 30 ug of BNT162b2 as a 4th dose.  Results are shown  both for a population pool that excludes subjects previously or currently infected with SARS‐ CoV‐2 and a population pool that includes these subjects.    Fig.  24.  Reactogenicity  of  certain  exemplary  RNA  (formulated  in  LNP)  at  a  given  dose:  subjects administered a 60 ug dose of RNA encoding a SARS‐CoV‐2 S protein are more likely  to  exhibit  a  higher  injection  site  pain  and  exhibit  similar  systemic  reactions  as  subjects  administered  a  30  ug dose  of RNA.    Subjects were  administered  30  ug  or  60  ug  of RNA  encoding a SARS‐CoV‐2 S protein from a Wuhan strain (BNT162b2, corresponding to groups  G1 and G2,  respectively), 30 ug or 60 ug of RNA encoding a SARS‐CoV‐2  S protein having  mutations characteristic of an Omicron BA.1 variant (BNT162b2 OMI, corresponding to groups  G3 and G4, respectively), 30 ug of a bivalent vaccine comprising 15 ug of RNA encoding a SARS‐ CoV‐2 S protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having  mutations characteristic of an Omicron BA.1 variant (BNT162B2 (15 ug) + BNT162b2 OMI (15  ug),  corresponding  to group G5), or 60 ug of  a bivalent  vaccine  comprising 30 ug of RNA  encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐ 2 S protein having mutations characteristic of an Omicron BA.1 variant (BNT162b2 (30 ug) +  BNT162b2 OMI (30 ug), corresponding to group G6).   (A) Local reactions, including redness,  swelling, and pain at the injection site, observed within 7 days of injection.  Injection site pain  was found to be  increased  in subjects administered 60 ug of RNA encoding a SARS‐CoV‐2 S  protein comprising mutations characteristic an Omicron BA.1 variant or a bivalent vaccine, as  compared to other doses tested.  (B) Systemic reactions, including fever, fatigue, headache,  chills, vomiting, diarrhea, muscle pain,  joint pain, and use of medication, observed within 7  days of  injection.   Systemic  reactions  through 7 days were observed  to be broadly  similar  across different groups.  Fatigue was found to trend higher after administration of 60 ug doses,  as compared to 30 ug doses.      Fig. 25. Omicron BA.1 breakthrough infection of BNT162b2 double‐ and triple‐vaccinated  individuals induces broad neutralization of Omicron BA.1, BA.2 and other VOCs, but to a  lesser extent against BA.4 and BA.5.  This figure is an extension of Fig. 16, including data  neutralizing activity against Omicron BA.4 and BA.5. As described in Fig. 16, serum was  tested in duplicate; 50% pseudovirus neutralization (pVN50) geometric mean titers (GMTs) (in  A and B), and the geometric mean ratio of SARS‐CoV‐2 variants of concern (VOCs) and SARS‐ CoV‐1 pVN50 GMTs normalized against Wuhan pVN50 GMTs (in C) were plotted. For titer  values below the limit of detection (LOD), LOD/2 values were plotted. Values above violin  plots represent the group GMTs. The nonparametric Friedman test with Dunn’s multiple  comparisons correction was used to compare Wuhan neutralizing group GMTs with titers  against the indicated variants and SARS‐CoV‐1. Multiplicity adjusted p values are shown. (A)  pVN50 GMTs against Wuhan, VOC and SARS‐CoV‐1 pseudovirus in patients who received two  doses or three doses of BNT162b2. (B) pVN50 GMTs against Wuhan, VOC and SARS‐CoV‐1  pseudovirus in patients who received two doses or three doses of BNT162b2 and who have  been previously infected with an Omicron BA.1 variant of SARS‐CoV‐2. (C) Group geometric  mean ratios with 95% confidence intervals for all cohorts shown in (A) and (B).    Fig. 26. Omicron BA.1 breakthrough infection of individuals vaccinated with other  approved COVID‐19 vaccines or mixed regimens results in immune sera that broadly  neutralize Omicron BA.1, BA.2 and other VOCs, but to a lesser extent against BA.4 and BA.5.   This figure is an extension of Fig. 19, including data neutralizing activity against Omicron BA.4  and BA.5. As described in Fig. 19, serum was tested in duplicate; individual 50% pseudovirus  neutralization (pVN50) geometric mean titers (GMTs) against SARS‐CoV‐2 Wuhan, Alpha, Beta,  Delta and Omicron BA.1, BA.2 and BA.4/5 variants, plus SARS‐CoV‐1 were plotted. For titer  values below the limit of detection (LOD), LOD/2 values were plotted. Values above violin plots  represent group GMTs. The nonparametric Friedman test with Dunn’s multiple comparisons  correction was  used  to  compare Wuhan  neutralizing  group  GMTs with  titers  against  the  indicated variants and SARS‐CoV‐1. Multiplicity‐adjusted p values are shown.    Fig. 27. Sequences of RBDs of SARS‐COV‐2 Wuhan strain and variants thereof.  Top sequence  corresponds to Wuhan, second sequence corresponds to the Alpha variant, third sequence  corresponds to the Delta variant, 4th sequence corresponds to Omicron BA.1 variant, and 5th  sequence corresponds to Omicron BA.4/5 variant.  Variant‐specific amino acid alterations are  indicated in bold.    Fig. 28. Cohorts and sampling for the study described in Example 14.  A schematic is shown  for testing immune responses in triple‐vaccinated patients who are (i) Omicron naïve, (ii) have  been  infected with a BA.1 Omicron variant, or (iii) have been  infected with a BA.2 Omicron  variant.   Blood  samples were drawn  from  three  cohorts: Omicron‐naïve  individuals  triple‐ vaccinated  with  BNT162b2  (BNT162b23),  and  individuals  vaccinated  with  homologous  or  heterologous  three doses  regimens  that  subsequently had either a breakthrough  infection  with Omicron at a time of BA.1 dominance (November 2021 to January 2022; all Vax + BA.1)  or at a time of BA.2 dominance (March to May 2022; all Vax + BA.2) in Germany. Sera (droplet)  were  isolated  in the Omicron‐naïve cohort at the time‐point  indicated  following their most  recent vaccination; for convalescent cohorts, the time from their most recent vaccination to  Omicron  infection,  and  infection  to  serum  isolation  are  indicated.  All  values  specified  as  median‐range. Serum neutralizing capacity was assessed using a pseudovirus neutralization  test.  Fig.  29.  50%  pseudovirus  neutralization  (pVN50)  geometric mean  titers  (GMTs)  from  the  BNT162b23 and All Vax + Omi BA.1 breakthrough infection cohorts. Serum was drawn from  Omicron‐naïve BNT162b2 triple‐vaccinated  individuals  (BNT162b23, circles) at 28 days after  the third dose, and from vaccinated individuals with subsequent Omicron BA.1 breakthrough  infection (all Vax + Omi BA.1, triangles) at a median 43 days post‐infection. 50% pseudovirus  neutralization (pVN50) geometric mean titers (GMTs) for Omicron‐naive individuals are plotted  in (A) and for BA.1 breakthrough infected individuals in (B). This data was previously published  in Quandt et al. („Omicron BA.1 breakthrough infection drives cross‐variant neutralization and  memory B cell formation against conserved epitopes.“ Science immunology, eabq2427 (2022),  doi:10.1126/sciimmunol.abq2427),  except  for  BA.2.12.1  neutralization  data.  Serum  was  tested  in duplicate. For  titer values below  the  limit of detection  (LOD), LOD/2 values were  plotted. Values above violin plots represent group GMTs. The non‐parametric Friedman test  with Dunn’s multiple comparisons correction was used to compare Wuhan neutralizing group  GMTs with titers against the indicated variants and SARS‐CoV‐1. Multiplicity‐adjusted p values  are shown.    Fig. 30. Omicron BA.2 breakthrough infection of previously vaccinated individuals refocuses  neutralization  against  Omicron  BA.2  and  the  BA.2‐derived  subvariants  BA.2.12.1  and  BA.4/BA.5.  Serum was drawn from BNT162b2 triple‐vaccinated individuals with subsequent  Omicron BA.1 breakthrough infection at a median 44 days post‐infection (BNT162b23 + Omi  BA.1, triangles), and from BNT162b2 triple‐vaccinated  individuals with subsequent Omicron  BA.2 breakthrough infection at 38 days post‐infection (BNT162b23 + Omi BA.2, squares). 50%  pseudovirus neutralization (pVN50) geometric mean titers (GMTs) (in A, B), and the geometric  mean ratio of SARS‐CoV‐2 variants of concern (VOCs) and SARS‐CoV‐1 pVN50 GMTs normalized  against Wuhan pVN50 GMTs (in C) were plotted. pVN50 GMT and geometric mean ratio data  for Omicron‐naïve BNT162b2 triple‐vaccinated individuals (BNT162b23, circles) and BNT162b2  triple‐vaccinated  individuals  with  Omicron  BA.1  breakthrough  infection  was  previously  published  in  Quandt  et  al.  („Omicron  BA.1  breakthrough  infection  drives  cross‐variant  neutralization  and  memory  B  cell  formation  against  conserved  epitopes.“  Science  immunology,  eabq2427  (2022),  doi:10.1126/sciimmunol.abq2427),  except  for  BA.2.12.1  neutralization data. Serum was tested in duplicate. For titer values below the limit of detection  (LOD), LOD/2 values were plotted. Values above violin plots represent group GMTs. The non‐ parametric Friedman test with Dunn’s multiple comparisons correction was used to compare  Wuhan neutralizing group GMTs with  titers against  the  indicated variants and SARS‐CoV‐1.  Multiplicity‐adjusted p values are shown. (A, B) pVN50 GMTs against Wuhan, VOC and SARS‐ CoV‐1 pseudovirus. (C) Group geometric mean ratios with 95% confidence intervals.   Fig.  31.  Characteristics  of  SARS‐CoV‐2  S  glycoproteins  used  in  the  VSV‐SARS‐CoV‐2  pseudovirus based neutralization assays. The sequence of the Wuhan‐Hu‐1 isolate SARS‐CoV‐ 2 S glycoprotein (GenBank: QHD43416.1) was used as reference. Amino acid positions, amino  acid descriptions (one letter code) and kind of mutations (substitutions, deletions, insertions)  are  indicated.  NTD,  N‐terminal  domain;  RBD,  Receptor‐binding  domain,  Δ,  deletion;  ins,  insertion; *, Cytoplasmic domain truncated for the C‐terminal 19 amino acids.    Fig. 32. Alterations on the spike glycoprotein amino acid sequence of SARS‐CoV‐2 Omicron  sub‐lineages. Amino acid positions, amino acid descriptions (one letter code) and kind of  mutations  substitutions, deletions, insertions) are indicated. White letters in boxes indicate  the amino acid substitution per sub‐lineage; Δ, deletion; ins, insertion; NTD, N‐terminal  domain; RBD, receptor‐binding domain.    Fig. 33.  Immunization protocol for studies with VOC boosters.  BALB/c mice were immunized  according  to  the  indicated  schedule with  two doses  (1 ug each) of  the original BNT162b2  vaccine,  followed by  at  least one dose  (1 ug  total) of a monovalent, bivalent, or  trivalent  booster  dose of  either:  (a)  the original BNT162b2  (“BNT162b2”);  (b) BNT162b2 OMI BA.1  (“OMI BA.1”); (c) BNT162b2 OMI BA.4/5 (“OMI BA.4/5”); or a combination thereof.      Fig. 34.  Baseline grouped neutralizing GMTs.  Sera drawn from mice immunized as depicted  in  Fig.  33  (day  104,  pre‐boost)  were  assessed  for  geometric mean  titers  of  neutralizing  antibodies against various strains.  Data are presented grouped by cohort.    Fig. 35.  Baseline staggered neutralizing GMTs.  Sera drawn from mice immunized as depicted  in  Fig.  33  (day  104,  pre‐boost)  were  assessed  for  geometric mean  titers  of  neutralizing  antibodies  against  various  strains.   Data  are presented  in  staggered  format  (i.e., by  strain  against which neutralization was assessed).    Fig. 36.  Baseline cross‐neutralization.  Sera drawn from mice immunized as depicted in Fig.  33 (day 104, pre‐boost) were assessed for geometric mean titers of neutralizing antibodies  against  various  strains.    Cross‐neutralization  results  are  presented  as  calculated  variant/Wuhan reference GMT Ratios.    Fig. 37.  Post‐boost geometric mean fold increase in GMTs.  Sera drawn from mice immunized  as depicted  in Fig. 33  (day 111, 7‐days post‐boost) were assessed  for geometric mean  fold  increase in GMT of neutralizing antibodies against various strains.      Fig.  38.    Post‐boost  grouped  neutralizing  GMTs.    Sera  drawn  from mice  immunized  as  depicted  in  Fig.  33  (day  111,  7‐days  post‐boost) were  assessed  for  geometric mean  fold  increase in GMT of neutralizing antibodies against various strains.  Data are presented grouped  by cohort.    Fig. 39.  Post‐boost cross‐neutralization.  Sera drawn from mice immunized as depicted in Fig.  33 (day 111, 7‐days post‐boost) were assessed for geometric mean fold  increase  in GMT of  neutralizing antibodies against various strains.  Cross‐neutralization results are presented as  calculated variant/Wuhan reference GMT Ratios.    Fig. 40. Exemplary spike protein amino acid mutations. Amino acid residues that are modified  are shown, and used to produce RNA vaccines encoding variant coronavirus spike proteins.  In  some instances, such amino acid modifications can be combined with other amino acid residue  modifications, such as as shown in Fig. 41 under columns “Mutations” and “Mutation Types”.  The amino acid positions are numbered  relative  to  the S protein  sequence  from a Wuhan  sequence  (SEQ  ID  NO:  1).  In  some  embodiments,  various  combinations  of  amino  acid  mutations  as  described  herein  can  be  applied  to  different  coronvavirus  S  protein  or  immunogenic fragments thereof.     Fig. 41. Exemplary Spike Protein Variants. Exemplary combinations of spike protein mutations  are shown, including the amino acid residues that are modified, type of mutation, and furin  mutations  (from  682/683/684/685  RRAR  to  GSAS).  RNA  constructs  encoding  exemplary  combinations of  spike protein mutations were evaluated  for S protein expression, CR3022  epitope response, and ACE2 response. The amino acid positions are numbered relative to the  S protein sequence from a Wuhan sequence (SEQ ID NO: 1). In some embodiments, various  combinations  of  amino  acid  mutations  as  described  herein  can  be  applied  to  different  coronvavirus S protein or immunogenic fragments thereof.    Fig. 42. Effect of RNA encoding exemplary spike protein variants on neutralization against   various  coronavirvus  strains  and/or  variants.    RNAs  encoding  exemplary  spike  protein  variants (e.g., containing a P6’ backbone as shown in Fig. 40, D614G, and furin site mutations  (from 682/683/684/685 RRAR to GSAS)) stimulated higher neutralization titers across various  VOCs.    Fig. 43. BNT162b5‐format Bivalent (Wuhan + BA.4/5) is more immunogenic than BNT162b2‐ format Bivalent (Wuhan + BA.4/5).  Mice were administered two doses of BNT162b2 21 days  apart, followed by a third dose comprising (i) BNT162b2, (ii) a bivalent vaccine comprising a  first RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS‐CoV‐2 Spike  protein  comprising mutations  characteristic of a BA.4/5 Omicron variant, where  the  Spike  protein encoded by each of  the  first and  the second RNA also comprise K986P and V987P  mutations  (“BNT162b2 Bivalent  (BA.4/5)”), or  (iii) a bivalent vaccine comprising a  first RNA  encoding a Wuhan  Spike protein and  a  second RNA encoding  a  SARS‐CoV‐2  Spike protein  comprising mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded  by each of the first and the second RNA also comprise P6’ mutations (D985P, V987P, F817P,  A892P, A899P, and A942P), D614G, and mutations at the furin cleavage site (682/683/684/685  RRAR to GSAS) (“BNT162b5 Bivalent (BA.4/5)”).  Sera samples were collected 1 month after  the third dose, and neutralization titers were determined for Wuhan and Omicron variants  BA.1, BA.2, BA.2.12.1, and BA.4/5 using a pseudovirus neutralization assay (50% pVNT Titers).    Fig. 44. Bivalent BNT162b5 provides an improved immune response in vaccine‐experienced  human  subjects.    Human  subjects  previously  administered  three  doses  of  BNT162b2  (encoding  a  SARS‐CoV‐2  S  protein  of  a Wuhan  strain,  and  comprising  K986P  and  V987P  mutations)  were administered (i) a bivalent vaccine comprising a first RNA encoding a Wuhan  Spike protein and a second RNA encoding a SARS‐CoV‐2 Spike protein comprising mutations  characteristic of a BA.1 Omicron variant, where the Spike protein encoded by each of the first  and  the  second RNA also comprise K986P and V987P mutations  (“BNT162b2 Bivalent Omi  BA.1”), or (ii) a bivalent vaccine comprising a first RNA encoding a Wuhan Spike protein and a  second RNA encoding a SARS‐CoV‐2 Spike protein comprising mutations characteristic of a  BA.2 Omicron variant, where the S protein encoded by each of the first and the second RNA  also comprise P6’ mutations (D985P, V987P, F817P, A892P, A899P, and A942P), D614G, and  mutations at the furin cleavage site (682/683/684/685 RRAR to GSAS) (“BNT162b5 Bivalent  (BA.2)”).    Sera  were  collected  one  month  after  administering  an  RNA  vaccine,  and  neutralization  titers were collected  for Wuhan  (“WT”), Omicron BA.1  (“BA.1”), or Omicron  BA.2  (“BA.2”) SARS‐COV‐2 variants.   Titers are  shown  for  (A) all subjects,  (B) subjects who  showed  evidence  of  prior  SARS‐CoV‐2  infection  at  the  time  a  SARS‐CoV‐2  vaccine  was  administered, and (C) subjects who showed no evidence of prior SARS‐CoV‐2 infection at the  time of administering a SARS‐CoV‐2 vaccine.  Titer values are shown above each bar.  Titers  were  collected  using  a  Fluroscent  Focus  Reduction  Neutralization  Titer  (FFRNT)  assay.   “1MPD4” refers to one‐month, post dose 4.  “WT” refers to Wuhan strain.  “LLOQ” stands for  Lower Limit of Quantitation.  Fig. 45. Bivalent BNT162b5 and BNT162b6 provide an  improved  immune  response when  administered as a booster  to vaccine‐experienced mice.   Mice administered  two doses of  BNT162b2 (encoding a SARS‐CoV‐2 S protein of a Wuhan strain, and comprising K986P and  V987P mutations)  were administered (i) a bivalent vaccine comprising a first RNA encoding a  Wuhan  Spike protein  and  a  second RNA  encoding a  SARS‐CoV‐2  Spike protein  comprising  mutations characteristic of a BA.4/5 Omicron variant, where  the Spike protein encoded by  each of the first and the second RNA also comprise K986P and V987P mutations (“BNT162b2  Bivalent  BA.4/5”),  (ii)  a  bivalent  vaccine  comprising  a  first  RNA  encoding  a Wuhan  Spike  protein  and  a  second  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  comprising  mutations  characteristic of a BA.4/5 Omicron variant, where the S protein encoded by each of the first  and the second RNA also comprise P6’ mutations (D985P, V987P, F817P, A892P, A899P, and  A942P), D614G, and mutations at the  furin cleavage site  (682/683/684/685 RRAR to GSAS)  (“BNT162b5 Bivalent (BA.4/5)”), or (iii) a bivalent vaccine comprising a first RNA encoding a  Wuhan  Spike protein  and  a  second RNA  encoding a  SARS‐CoV‐2  Spike protein  comprising  mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded by each of  the  first  and  the  second  RNA  also  comprise  P6 mutations  (K986P,  V987P,  F817P,  A892P,  A899P, and A942P), D614G, mutations at the furin cleavage site (682/683/684/685 RRAR to  GSAS),  and mutations  to  introduce  a  disulfide  bond  (T547C, N978C)  (“BNT162b6  Bivalent  (BA.4/5)”).    Sera  were  collected  one  month  after  administering  an  RNA  vaccine,  and  neutralization titers were deteremined for a collection of variants of concern (indicated in the  legend).  50% neutralization titers (“50% pVNT Titer”) are shown.  “LOD” stands for Limit of  Detection.    Fig.  46.  In  vitro  characterization  of  Exemplary  SARS‐CoV‐2  Variants.    Shown  is  protein  expression, ACE2 binding, and CR3022 binding (a neutralizing antibody) for certain exemplary  SARS‐CoV‐2 variants comprising mutations described herein (mutations present in each SARS‐ CoV‐2 variant listed in Table 34).      Fig.  47.  In  vitro  characterization  of  Exemplary  SARS‐CoV‐2  Variants.    Shown  is  protein  expression, ACE2 binding, and CR3022 binding (a neutralizing antibody) for certain exemplary  SARS‐CoV‐2 variants comprising mutations described herein (mutations present in each SARS‐ CoV‐2 variant listed in Table 34).    Fig. 45. Bivalent BNT162b6, and BNT162b7 provide an  improved  immune  response when  administered  as  a booster  to  vaccine‐naive mice.   Mice were  administered  (i)  a bivalent  vaccine comprising a first RNA encoding a Wuhan Spike protein and a second RNA encoding a  SARS‐CoV‐2 Spike protein comprising mutations characteristic of a BA.4/5 Omicron variant,  where the Spike protein encoded by each of the first and the second RNA also comprise K986P  and V987P mutations (“BNT162b2 Bivalent BA.4/5”), (ii) a bivalent vaccine comprising a first  RNA encoding a Wuhan Spike protein and a second RNA encoding a SARS‐CoV‐2 Spike protein  comprising mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded  by each of the first and the second RNA also comprise P6’ mutations (D985P, V987P, F817P,  A892P, A899P, and A942P), D614G, and mutations at the furin cleavage site (682/683/684/685  RRAR to GSAS) (“BNT162b5 Bivalent (BA.4/5)”), (iii) a bivalent vaccine comprising a first RNA  encoding a Wuhan  Spike protein and  a  second RNA encoding  a  SARS‐CoV‐2  Spike protein  comprising mutations characteristic of a BA.4/5 Omicron variant, where the S protein encoded  by each of the first and the second RNA also comprise P6 mutations (K986P, V987P, F817P,  A892P, A899P, and A942P), D614G, mutations at  the  furin cleavage site  (682/683/684/685  RRAR  to GSAS),  and mutations  to  introduce  a disulfide bond  (T547C, N978C)  (“BNT162b6  Bivalent  (BA.2)”), or  (iv) a bivalent vaccine comprising a  first RNA encoding a Wuhan Spike  protein  and  a  second  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  comprising  mutations  characteristic of a BA.4/5 Omicron variant, where the S protein encoded by each of the first  and the second RNA also comprise P6 mutations (K986P, V987P, F817P, A892P, A899P, and  A942P), D614G, mutations at the furin cleavage site (682/683/684/685 RRAR to GSAS), and  mutations to introduce a disulfide bond (T547C, S982C) (“BNT162b7 Bivalent (BA.4/5)”).  Sera  were collected one month after administering an RNA vaccine, and neutralization titers were  determined for a collection of variants of concern (indicated in the legend).  50% neutralization  titers  (“50%  pVNT  Titer”)  are  shown  in  (A)  and  (B),  and  neutralization  titers  relative  to  BNT162b2 titers are shown in (C).    Detailed description  Although the present disclosure is described in detail below, it is to be understood that this  disclosure  is not  limited  to  the particular methodologies, protocols and reagents described  herein as these may vary. It is also to be understood that the terminology used herein is for  the purpose of describing particular embodiments only, and is not intended to limit the scope  of the present disclosure which will be limited only by the appended claims. Unless defined  otherwise, all technical and scientific terms used herein have the same meanings as commonly  understood by one of ordinary skill in the art.  Preferably,  the  terms  used  herein  are  defined  as  described  in  "A multilingual  glossary  of  biotechnological  terms:  (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H.  Kölbl, Eds., Helvetica Chimica Acta, CH‐4010 Basel, Switzerland, (1995).   The practice of the present disclosure will employ, unless otherwise indicated, conventional  methods  of  chemistry,  biochemistry,  cell  biology,  immunology,  and  recombinant  DNA  techniques which are explained  in the  literature  in  the  field  (cf., e.g., Molecular Cloning: A  Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,  Cold Spring Harbor 1989).  In the following, the elements of the present disclosure will be described. These elements are  listed  with  specific  embodiments,  however,  it  should  be  understood  that  they  may  be  combined in any manner and in any number to create additional embodiments. The variously  described examples and embodiments should not be construed to limit the present disclosure  to  only  the  explicitly  described  embodiments.  This  description  should  be  understood  to  disclose and encompass embodiments which combine the explicitly described embodiments  with any number of the disclosed elements. Furthermore, any permutations and combinations  of all described elements should be considered disclosed by this description unless the context  indicates otherwise.   Several documents are cited throughout the text of this specification. Each of the documents  cited herein (including all patents, patent applications, scientific publications, manufacturer's  specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference  in their entirety. Nothing herein is to be construed as an admission that the present disclosure  was not entitled to antedate such disclosure.  Definitions  In  the  following,  definitions  will  be  provided  which  apply  to  all  aspects  of  the  present  disclosure. The following terms have the following meanings unless otherwise indicated. Any  undefined terms have their art recognized meanings.  The term "about" means approximately or nearly, and in the context of a numerical value or  range set forth herein in one embodiment means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical  value or range recited or claimed.  The terms "a" and "an" and "the" and similar reference used in the context of describing the  disclosure  (especially  in  the  context of  the  claims)  are  to be  construed  to  cover both  the  singular and the plural, unless otherwise indicated herein or clearly contradicted by context.  Recitation of ranges of values herein  is merely  intended to serve as a shorthand method of  referring  individually  to  each  separate  value  falling  within  the  range.  Unless  otherwise  indicated  herein,  each  individual  value  is  incorporated  into  the  specification  as  if  it was  individually  recited herein. All methods described herein can be performed  in any suitable  order unless otherwise indicated herein or otherwise clearly contradicted by context. The use  of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended  merely to better illustrate the disclosure and does not pose a limitation on the scope of the  claims. No  language  in the specification should be construed as  indicating any non‐claimed  element essential to the practice of the disclosure.  Unless expressly  specified otherwise,  the  term  "comprising"  is used  in  the  context of  the  present document to indicate that further members may optionally be present in addition to  the members of the list introduced by "comprising". It is, however, contemplated as a specific  embodiment of the present disclosure that the term "comprising" encompasses the possibility  of no further members being present, i.e., for the purpose of this embodiment "comprising"  is to be understood as having the meaning of "consisting of" or "consisting essentially of".  Terms such as "reduce", "decrease", "inhibit" or “impair” as used herein relate to an overall  reduction or the ability to cause an overall reduction, preferably of at least 5%, at least 10%,  at  least 20%, at  least 50%, at  least 75% or even more,  in  the  level. These  terms  include a  complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.  Terms  such  as  "increase",  "enhance"  or  “exceed”  preferably  relate  to  an  increase  or  enhancement by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least  80%, at least 100%, at least 200%, at least 500%, or even more.   According to the disclosure, the term "peptide" comprises oligo‐ and polypeptides and refers  to substances which comprise about two or more, about 3 or more, about 4 or more, about 6  or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20  or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one  another via peptide bonds. The term "protein" or "polypeptide" refers to  large peptides,  in  particular peptides having at least about 150 amino acids, but the terms "peptide", "protein"  and "polypeptide" are used herein usually as synonyms.  A "therapeutic protein" has a positive or advantageous effect on a condition or disease state  of  a  subject when  provided  to  the  subject  in  a  therapeutically  effective  amount.  In  one  embodiment,  a  therapeutic  protein  has  curative  or  palliative  properties  and  may  be  administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of  one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic  properties and may be used to delay the onset of a disease or to lessen the severity of such  disease or pathological condition. The term "therapeutic protein" includes entire proteins or  peptides, and can also refer to therapeutically active fragments thereof.  It can also  include  therapeutically  active  variants  of  a  protein.  Examples  of  therapeutically  active  proteins  include,  but  are  not  limited  to,  antigens  for  vaccination  and  immunostimulants  such  as  cytokines.  "Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part  of  an  amino  acid  sequence,  i.e.  a  sequence  which  represents  the  amino  acid  sequence  shortened at the N‐terminus and/or C‐terminus. A fragment shortened at the C‐terminus (N‐ terminal fragment)  is obtainable e.g. by translation of a truncated open reading frame that  lacks  the  3'‐end  of  the  open  reading  frame. A  fragment  shortened  at  the N‐terminus  (C‐ terminal fragment)  is obtainable e.g. by translation of a truncated open reading frame that  lacks  the 5'‐end of  the open  reading  frame, as  long  as  the  truncated open  reading  frame  comprises  a  start  codon  that  serves  to  initiate  translation.  A  fragment  of  an  amino  acid  sequence comprises e.g. at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90%  of  the  amino  acid  residues  from  an  amino  acid  sequence.  A  fragment  of  an  amino  acid  sequence preferably comprises at  least 6,  in particular at  least 8, at  least 12, at  least 15, at  least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid  sequence.  By "variant" herein  is meant an amino acid sequence that differs from a parent amino acid  sequence by virtue of at least one amino acid modification. The parent amino acid sequence  may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified  version of a wild type amino acid sequence. Preferably, the variant amino acid sequence has  at least one amino acid modification compared to the parent amino acid sequence, e.g., from  1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about  5 amino acid modifications compared to the parent.  By "wild type" or "WT" or "native" herein is meant an amino acid sequence that is found in  nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an  amino acid sequence that has not been intentionally modified.  For the purposes of the present disclosure, "variants" of an amino acid sequence  (peptide,  protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants,  amino  acid  deletion  variants  and/or  amino  acid  substitution  variants.  The  term  "variant"  includes  all mutants,  splice  variants,  posttranslationally modified  variants,  conformations,  isoforms, allelic variants, species variants, and species homologs, in particular those which are  naturally occurring. The  term  "variant"  includes,  in particular,  fragments of an amino acid  sequence.  Amino acid insertion variants comprise  insertions of single or two or more amino acids  in a  particular  amino  acid  sequence.  In  the  case  of  amino  acid  sequence  variants  having  an  insertion, one or more amino acid residues are inserted into a particular site in an amino acid  sequence, although random  insertion with appropriate screening of the resulting product  is  also possible. Amino acid addition variants comprise amino‐ and/or carboxy‐terminal fusions  of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid  deletion  variants  are  characterized by  the  removal  of one or more  amino  acids  from  the  sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions  may be in any position of the protein. Amino acid deletion variants that comprise the deletion  at the N‐terminal and/or C‐terminal end of the protein are also called N‐terminal and/or C‐ terminal truncation variants. Amino acid substitution variants are characterized by at least one  residue  in  the  sequence  being  removed  and  another  residue  being  inserted  in  its  place.  Preference is given to the modifications being in positions in the amino acid sequence which  are not conserved between homologous proteins or peptides and/or to replacing amino acids  with  other  ones  having  similar  properties.  Preferably,  amino  acid  changes  in peptide  and  protein variants are conservative amino acid changes, i.e., substitutions of similarly charged  or uncharged amino acids. A conservative amino acid change involves substitution of one of a  family of amino acids which are related in their side chains. Naturally occurring amino acids  are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine,  histidine), non‐polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,  tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine,  tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly  as aromatic amino acids. In one embodiment, conservative amino acid substitutions include  substitutions within the following groups:   glycine, alanine;   valine, isoleucine, leucine;   aspartic acid, glutamic acid;   asparagine, glutamine;   serine, threonine;   lysine, arginine; and   phenylalanine, tyrosine.   Preferably the degree of similarity, preferably identity between a given amino acid sequence  and an amino acid sequence which is a variant of said given amino acid sequence will be at  least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,  93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably  for an amino acid region which is at least about 10%, at least about 20%, at least about 30%,  at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about  80%,  at  least about 90% or  about 100% of  the entire  length of  the  reference  amino  acid  sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the  degree of similarity or identity is given preferably for at least about 20, at least about 40, at  least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at  least  about  160,  at  least  about  180,  or  about  200  amino  acids,  in  some  embodiments  continuous amino acids. In some embodiments, the degree of similarity or identity is given for  the  entire  length  of  the  reference  amino  acid  sequence.  The  alignment  for  determining  sequence similarity, preferably sequence identity can be done with art known tools, preferably  using  the  best  sequence  alignment,  for  example,  using  Align,  using  standard  settings,  preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.  "Sequence similarity" indicates the percentage of amino acids that either are identical or that  represent conservative amino acid substitutions. "Sequence identity" between two amino acid  sequences indicates the percentage of amino acids that are identical between the sequences.  "Sequence  identity"  between  two  nucleic  acid  sequences  indicates  the  percentage  of  nucleotides that are identical between the sequences.  The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to  the percentage of nucleotides or amino acids which are  identical  in an optimal alignment  between  the  sequences  to  be  compared.  Said  percentage  is  purely  statistical,  and  the  differences between the two sequences may be but are not necessarily randomly distributed  over the entire length of the sequences to be compared. Comparisons of two sequences are  usually carried out by comparing the sequences, after optimal alignment, with respect to a  segment  or  "window  of  comparison",  in  order  to  identify  local  regions  of  corresponding  sequences. The optimal alignment for a comparison may be carried out manually or with the  aid of the  local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482,  with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol.  48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc.  Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP,  BESTFIT,  FASTA,  BLAST  P,  BLAST  N  and  TFASTA  in Wisconsin  Genetics  Software  Package,  Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent  identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available  on the United States National Center for Biotechnology  Information (NCBI) website (e.g., at  blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on  the NCBI website  include:  (i) Expect Threshold set  to 10;  (ii) Word Size  set  to 28;  (iii) Max  matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, ‐2; (v) Gap Costs set  to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the  algorithm  parameters  used  for  BLASTP  algorithm  on  the NCBI website  include:  (i)  Expect  Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches  in a query range set to 0; (iv)  Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional  compositional score matrix adjustment.          Percentage identity is obtained by determining the number of identical positions at which the  sequences  to  be  compared  correspond,  dividing  this  number  by  the  number  of  positions  compared (e.g., the number of positions in the reference sequence) and multiplying this result  by 100.  In some embodiments, the degree of similarity or identity is given for a region which is at least  about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or  about 100% of  the entire  length of  the  reference  sequence. For example,  if  the  reference  nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least  about 100, at least about 120, at least about 140, at least about 160, at least about 180, or  about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments,  the degree of similarity or identity is given for the entire length of the reference sequence.  Homologous  amino  acid  sequences  exhibit  according  to  the  disclosure  at  least  40%,  in  particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at  least 95%, at least 98 or at least 99% identity of the amino acid residues.  The amino acid sequence variants described herein may readily be prepared by  the skilled  person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences  for preparing peptides or proteins having substitutions, additions, insertions or deletions,  is  described  in detail  in Sambrook et al.  (1989),  for example. Furthermore,  the peptides and  amino acid variants described herein may be readily prepared with the aid of known peptide  synthesis techniques such as, for example, by solid phase synthesis and similar methods.  In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is  preferably a "functional fragment" or "functional variant". The term "functional fragment" or  "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting  one or more  functional properties  identical or similar to those of the amino acid sequence  from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic  sequences, one particular  function  is one or more  immunogenic activities displayed by  the  amino acid  sequence  from which  the  fragment or variant  is derived. The  term  “functional  fragment” or “functional variant”, as used herein, in particular refers to a variant molecule or  sequence that comprises an amino acid sequence that is altered by one or more amino acids  compared to the amino acid sequence of the parent molecule or sequence and that  is still  capable of fulfilling one or more of the  functions of the parent molecule or sequence, e.g.,  inducing  an  immune  response.  In  one  embodiment,  the modifications  in  the  amino  acid  sequence  of  the  parent  molecule  or  sequence  do  not  significantly  affect  or  alter  the  characteristics of  the molecule or sequence.  In different embodiments, the  function of the  functional fragment or functional variant may be reduced but still significantly present, e.g.,  immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at  least  80%,  or  at  least  90%  of  the  parent  molecule  or  sequence.  However,  in  other  embodiments,  immunogenicity  of  the  functional  fragment  or  functional  variant  may  be  enhanced compared to the parent molecule or sequence.  An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino  acid sequence  (peptide, protein or polypeptide) refers  to  the origin of  the  first amino acid  sequence. Preferably, the amino acid sequence which is derived from a particular amino acid  sequence has an amino acid sequence that is identical, essentially identical or homologous to  that  particular  sequence  or  a  fragment  thereof.  Amino  acid  sequences  derived  from  a  particular amino acid sequence may be variants of  that particular sequence or a  fragment  thereof. For example, it will be understood by one of ordinary skill in the art that the antigens  suitable  for use herein may be altered such  that  they vary  in sequence  from  the naturally  occurring or native sequences  from which they were derived, while retaining the desirable  activity of the native sequences.  As used herein, an "instructional material" or "instructions" includes a publication, a recording,  a  diagram,  or  any  other medium  of  expression which  can  be  used  to  communicate  the  usefulness  of  the  compositions  and methods  of  the  present  disclosure.  The  instructional  material of the kit of the present disclosure may, for example, be affixed to a container which  contains the compositions of the present disclosure or be shipped together with a container  which  contains  the  compositions. Alternatively,  the  instructional material may be  shipped  separately  from  the  container with  the  intention  that  the  instructional material  and  the  compositions be used cooperatively by the recipient.   "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a  peptide naturally present  in a  living animal  is not  "isolated", but  the  same nucleic acid or  peptide partially or completely separated from the coexisting materials of its natural state is  "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can  exist in a non‐native environment such as, for example, a host cell.   The  term  "recombinant"  in  the  context  of  the  present  disclosure means  "made  through  genetic engineering". Preferably, a "recombinant object" such as a recombinant nucleic acid  in the context of the present disclosure is not occurring naturally.  The term "naturally occurring" as used herein refers to the fact that an object can be found in  nature. For example, a peptide or nucleic acid that is present in an organism (including viruses)  and can be isolated from a source in nature and which has not been intentionally modified by  man in the laboratory is naturally occurring.   "Physiological pH" as used herein refers to a pH of about 7.5.   The  term “genetic modification” or simply “modification”  includes  the  transfection of cells  with  nucleic  acid.  The  term  "transfection"  relates  to  the  introduction  of  nucleic  acids,  in  particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also  includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such  cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present  disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or  in  vivo, e.g.  the  cell  can  form part of an organ,  a  tissue and/or an organism of a patient.  According  to  the  present  disclosure,  transfection  can  be  transient  or  stable.  For  some  applications of transfection, it is sufficient if the transfected genetic material is only transiently  expressed. RNA can be transfected into cells to transiently express its coded protein. Since the  nucleic acid introduced in the transfection process is usually not integrated into the nuclear  genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing  episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that  the transfected nucleic acid actually remains in the genome of the cell and its daughter cells,  a stable transfection must occur. Such stable transfection can be achieved by using virus‐based  systems  or  transposon‐based  systems  for  transfection.  Generally,  nucleic  acid  encoding  antigen  is transiently transfected  into cells. RNA can be transfected  into cells to transiently  express its coded protein.  The term "seroconversion"  includes a ≥4‐fold rise  from before vaccination to 1‐month post  Dose 2.      Coronavirus  Coronaviruses are enveloped, positive‐sense, single‐stranded RNA ((+) ssRNA) viruses. They  have  the  largest genomes  (26–32  kb) among  known RNA viruses and are phylogenetically  divided into four genera (α, β, γ, and δ), with betacoronaviruses further subdivided into four  lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species,  including  humans.  Some  human  coronaviruses  generally  cause mild  respiratory  diseases,  although severity can be greater in infants, the elderly, and the immunocompromised. Middle  East respiratory syndrome coronavirus  (MERS‐CoV) and severe acute respiratory syndrome  coronavirus  (SARS‐CoV),  belonging  to  betacoronavirus  lineages  C  and  B,  respectively,  are  highly pathogenic. Both viruses emerged into the human population from animal reservoirs  within the last 15 years and caused outbreaks with high case‐fatality rates. The outbreak of  severe  acute  respiratory  syndrome  coronavirus‐2  (SARS‐CoV‐2)  that  causes  atypical  pneumonia  (coronavirus disease 2019; COVID‐19) has  raged  in China  since mid‐December  2019, and has developed to be a public health emergency of international concern. SARS‐CoV‐ 2 (MN908947.3) belongs to betacoronavirus lineage B. It has at least 70% sequence similarity  to SARS‐CoV.  In general, coronaviruses have four structural proteins, namely, envelope (E), membrane (M),  nucleocapsid (N), and spike (S). The E and M proteins have  important functions  in the viral  assembly, and the N protein is necessary for viral RNA synthesis. The critical glycoprotein S is  responsible for virus binding and entry into target cells. The S protein is synthesized as a single‐ chain inactive precursor that is cleaved by furin‐like host proteases in the producing cell into  two  noncovalently  associated  subunits,  S1  and  S2.  The  S1  subunit  contains  the  receptor‐ binding domain (RBD), which recognizes the host‐cell receptor. The S2 subunit contains the  fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required  to mediate fusion of the viral and host‐cell membranes by undergoing a large conformational  rearrangement. The S1 and S2 subunits trimerize to form a large prefusion spike.  The S precursor protein of SARS‐CoV‐2 can be proteolytically cleaved into S1 (685 aa) and S2  (588  aa)  subunits.  The  S1  subunit  comprises  the  receptor‐binding  domain  (RBD),  which  mediates virus entry  into sensitive cells  through  the host angiotensin‐converting enzyme 2  (ACE2) receptor.     Antigen  Many embodiments of the present disclosure comprises the use of RNA encoding an amino  acid  sequence  comprising  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of  the  SARS‐CoV‐2  S protein or  the  immunogenic  variant  thereof.  Thus,  the RNA encodes a peptide or protein  comprising at  least an epitope  SARS‐CoV‐2  S  protein  or  an  immunogenic  variant  thereof  for  inducing  an  immune  response  against  coronavirus S protein, in particular SARS‐CoV‐2 S protein in a subject. The amino acid sequence  comprising  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof (i.e., the antigenic  peptide  or  protein)  is  also  designated  herein  as  "vaccine  antigen",  "peptide  and  protein  antigen", "antigen molecule" or simply "antigen". The SARS‐CoV‐2 S protein, an immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant  thereof  is  also  designated  herein  as  "antigenic  peptide  or  protein"  or  "antigenic  sequence".  SARS‐CoV‐2 coronavirus full length spike (S) protein from the first detected SARS‐CoV‐2 strain  (referred to as the Wuhan strain herein) consists of 1273 amino acids and has the amino acid  sequence according to SEQ ID NO: 1:   
Figure imgf000075_0001
Figure imgf000076_0001
For purposes of  the present disclosure,  the above  sequence  is  considered  the wildtype or  Wuhan  SARS‐CoV‐2  S  protein  amino  acid  sequence.  Unless  otherwise  indicated,  position  numberings in a SARS‐CoV‐2 S protein given herein are in relation to the amino acid sequence  according to SEQ ID NO: 1.  One of skill in the art reading the present disclosure can determine  the locations of the corresponding positions in SARS‐CoV‐2 S protein variants.  The  following  Table  1  includes  additional  exemplary  S  proteins  from  various  coronavirus  variants,  including  the alpha, beta, gamma, delta, and omicron variants  (including omicron  BA.1, BA.2 and BA.4/5). Unless specified otherwise, “Omicron variant“, as used herein, refers  to any Omicron variant,  including e.g., Omicron variants described herein and descendents  thereof.    Amino acid sequences were obtained  from  the UniProt database, accessible via  the World  Wide Web at uniprot.org, or the GenBank database, accessible via the World Wide Web at  ncbi.nlm.nih.gov,  and  the UniProt  or GenBank  database  accession  numbers  of  each  spike  protein sequence are included in the Table 1.  These amino acid sequences correspond to the  amino acid sequences of native coronavirus spike proteins.  In some aspects, the amino acid  sequences of native coronavirus spike proteins encoded by RNA constructs described herein  may  be modified,  as  described  herein,  to  produce  immunogenic  polypeptides  comprising  variant coronavirus spike proteins that are modifications of native coronavirus spike proteins  or  fragments  thereof.   For example,  in  some aspects,  the amino acid  sequences of native  coronavirus spike proteins encoded by RNA constructs described herein are substituted, as  described herein, to produce immunogenic polypeptides comprising variant coronavirus spike  proteins that are modifications of native coronavirus spike proteins or fragments thereof.  Like the amino acid sequences of native coronavirus spike proteins, the amino acid sequences  of  spike  proteins  (e.g.,  including  the  alpha,  beta,  gamma,  delta,  and  omicron  variants  (including  omicron  BA.1,  BA.2,  BA.4/5)  of  these  SARS‐CoV‐2  variants  encoded  by  RNA  constructs  described  herein may  be modified  at  the  corresponding  position,  as  described  herein, to produce immunogenic polypeptides comprising variant coronavirus spike proteins  that are modifications of the native variant coronavirus spike proteins or fragments thereof.   For example, in some aspects, the amino acid sequences of spike proteins of these SARS‐CoV‐ 2 variants encoded by RNA constructs described herein are substituted, as described herein,  to produce immunogenic polypeptides comprising variant coronavirus spike proteins that are  modifications of variant coronavirus spike proteins or fragments thereof.  Additional variants  not specifically set forth below are also contemplated.  For example, any variant coronavirus  spike  protein  having  70%,  75%,  80%,  85%,  90%,  95%,  96%,  97%,  98%,  or  99%  or  higher  sequence  identity  with  the  native  coronavirus  spike  protein  sequence  encoded  by  RNA  constructs  described  herein  may  be  modified  at  the  corresponding  position,  (e.g.,  substituted), as described herein, to produce immunogenic polypeptides comprising variant  coronavirus  spike  proteins  that  are modifications  of  native  coronavirus  spike  proteins  or  fragments thereof.  Table 1 
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
In  some  embodiments,  a  coronavirus  spike  protein  sequence  comprises  SEQ  ID NO:  105,  shown below, which is the SARS‐CoV‐2 (Omicron BA.4/5) sequence represented by SEQ ID NO:  104  (see  Table  1),  but  differs  by  one  amino  acid  at  position  403  and  comprises  a  R403S  mutation. 
Figure imgf000082_0001
Coronavirus Spike Protein Modifications   In specific embodiments, full length spike (S) protein (e.g., the full length S protein of SEQ ID  NO: 1)  is modified  in such a way that the prototypical prefusion conformation  is stabilized.  Certain mutations  that  stabilize  a  prefusion  confirmation  are  known  in  the  art,  e.g.,  as  disclosed in WO 2022/266010 A1, WO 2021243122 A2 and Hsieh, Ching‐Lin, et al. ("Structure‐ based  design  of  prefusion‐stabilized  SARS‐CoV‐2  spikes,"  Science  369.6510  (2020):  1501‐ 1505), the contents of each which are incorporated by reference herein in their entirety.  In  some embodiments, a SARS‐CoV‐2 S protein may be stabilized by  introducing one or more  glycine mutations (e.g., one or more glycine mutations in the crown of the helix turn region in  the S protein, in the 12 amino acids between the heptad region 1 (HR1) and central helix (CH)  or heptad regoin 2 (HR2) regions of the S2 subunit, and/or at one or more of L984, D985, K986,  and V987 of  (positions  relative  to SEQ  ID NO: 1)).    In  some embodiments, a Spike protein  comprises  glycine  mutations  at  each  of  L984,  D985,  K986,  and  V987  (i.e.,  at  positions  corresponding  to  these  residues  in SEQ  ID NO: 1).  In  some embodiments, a SARS‐CoV‐2 S  protein  may  be  stabilized  by  introducing  one  or  more  proline  mutations.    In  some  embodiments, a SARS‐CoV‐2 S protein comprises a proline substitution at residues 986 and/or  987 of SEQ  ID NO: 1.    In  some embodiments, a SARS‐CoV‐2 S protein comprises a proline  substitution at one or more of  residues 817, 892, 899, and 942 of SEQ  ID NO: 1.    In some  embodiments, a SARS‐CoV‐2 S protein comprises a proline substitution at each of residues  817, 892, 899,  and 942 of  SEQ  ID NO: 1.    In  some embodiments, a  SARS‐CoV‐2  S protein  comprises a proline substitution at each of residues 817, 892, 899, 942, 986, and 987 of SEQ  ID NO: 1.  In some embodiments, a SARS‐CoV‐2 S protein comprises a proline substitution at residues  985 and/or 987 of SEQ ID NO: 1.  In some embodiments, a SARS‐CoV‐2 S protein comprises a  proline substitution at each of residues 817, 892, 899, 942, 985, and 987 of SEQ ID NO: 1.    In  some  embodiments,  stabilization  of  the  prefusion  conformation may  be  obtained  by  introducing two consecutive proline substitutions at AS residues 986 and 987 in the full length  spike protein. Specifically, spike (S) protein stabilized protein variants are obtained in a way  that the amino acid residue at position 986 is exchanged to proline and the amino acid residue  at position 987  is also exchanged  to proline.  In one embodiment, a SARS‐CoV‐2  S protein  variant wherein  the prototypical prefusion conformation  is  stabilized comprises  the amino  acid sequence shown in SEQ ID NO: 7:   
Figure imgf000083_0001
Figure imgf000084_0001
  In some embodiments, a Spike protein can be modified in such a way as to block a pre‐fusion  to post‐fusion conformational change  (referred  to herein as a “pre‐post  fusion block”).    In  some embodiments, a pre‐post fusion block can be  introduced by  introducing two cysteine  mutations at residues close to one another in the folded protein (e.g., at locations close to one  another in a pre‐fusion conformation of the Spike protein). Examples of pre‐post fusion block  mutations include L984C‐A989C and I980C‐Q992C.  In some embodiments, a Spike protein can be modified so as  to decrease “shedding”  (i.e.,  decrease separation of S1 and S2 subunits).    In some embodiments, a Spike protein can be  modified to decrease shedding by introducing mutations at the furin cleavage site, such that  a furin protease can no longer bind and/or cleave the S protein (e.g., one or more mutations  at residues 682‐685 of SEQ ID NO: 1).  In some embodiments, an S protein can be modified to  reduce  shedding  by  introducing mutations  at  each  of  residues  682,  683,  and  685  (e.g.,  introducing mutations (i) R682G, R683S, and R685S, or (ii) R682Q, R683Q, and R685Q).    In some embodiments, an S protein can be modified so as to reduce shedding by introducing  cysteine mutations that can form a disulfide bond (e.g., by introducing cysteine mutations at  positions  that  are  close  to one  another  in  a  folded  conformation of  an  S protein,  e.g.,  at  residues A570 and N960).  In some embodiments, one or more modifications may be introduced into a Spike protein so  as  to  stabilize an “up” confirmation  (referred  to herein as “RBD Up” mutations).   Without  wishing to be bound by theory, the up confirmation of the SARS‐CoV‐2 Spike protein is thought  to  increase exposure of neutralization sensitive residues.  Thus, mutations that stabilize the  up conformation can produce a vaccine that is more immunogenic.  The  following  Table  2  lists  various  combinations  of  amino  acid modifications  that  can  be  introduced  into  coronavirus  spike  protein  sequences  disclosed  above  and  thus  polynucleotides  (e.g.,  RNAs)  encoding  immunogenic  polypeptides  comprising  coronavirus  spike proteins that are variants of native coronavirus spike proteins or fragments thereof can  be produced.  A “+” symbol indicates the inclusion of the specified modification in a particular  S  protein  sequence  from  a  coronvirus  strain  or  variant  (e.g.,  SARS‐CoV‐2  strains  and/or  variants as described in Table 1). In some instances, a spike protein sequence may contain any  combination of the modifications in the following Table 2.  The amino acid positions indicated  in Table 2 are numbered relative to SEQ ID. NO: 1 (Wuhan), SEQ ID NO: 69 (Omicron BA.1),  SEQ  ID NO:  70  (Omicron  BA.2),  and  SEQ  ID NO:  104  (Omicron  BA.4/5).    The  amino  acid  positions  corresponding  to  spike protein  sequences  from other  coronavirus  variants  (e.g.,  alpha, beta, or delta variant) can determined through an alignment with SEQ  ID NO: 1 (see  e.g., Table 5).    Table 2                                                                                                                                            
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
  The  following  Table  3  lists  various  combinations  of  amino  acid modifications  that  can  be  introduced  into  coronavirus  spike  protein  sequences  disclosed  above  and  thus  polynucleotides  (e.g.,  RNAs)  encoding  immunogenic  polypeptides  comprising  coronavirus  spike proteins that are variants of native coronavirus spike proteins or fragments thereof can  be produced.  Table 3, like Table 2, lists the position of amino acid modifications (with respect  to the Wuhan spike protein sequence according to SEQ ID NO: 1), and Table 3 also include the  specific amino acid residue that is substituted for the native amino acid residue.  A “+” symbol  indicates the inclusion of the specified modification in a particular S protein sequence from a  coronavirus strain or variant (e.g., SARS‐CoV‐2 strains and/or variants as described in Table 1).  In some instances, a coronavirus spike protein variant encoded by an RNA vaccine may contain  any combination of the modifications in Table 2 above, and for example, may include any of  the specific substitutions shown in Table 3.  The amino acid positions indicated in Table 2 are  numbered relative to SEQ ID. NO: 1 (Wuhan), SEQ ID NO: 69 (Omicron BA.1), SEQ ID NO: 70  (Omicron  BA.2),  and  SEQ  ID  NO:  104  (Omicron  BA.4/5).    The  amino  acid  positions  corresponding to spike protein sequences from other coronavirus variants (e.g., alpha, beta,  or delta variant) can determined through an alignment with SEQ ID NO: 1 (see e.g., Table 5).      Table 3                                                                                                                                               
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
  The  following  Table  4  lists  various  combinations  of  amino  acid modifications  that  can  be  introduced into coronavirus spike protein sequences disclosed herein (see e.g., Table 1) and  thus polynucleotides (e.g., RNAs) encoding immunogenic polypeptides comprising coronavirus  spike proteins that are variants of native coronavirus spike proteins or fragments thereof can  be produced.  A “+” symbol indicates the inclusion of the specified modification in a particular  S  protein  sequence  from  a  coronavirus  strain  or  variant  (e.g.,  SARS‐CoV‐2  strains  and/or  variants as described in Table 1). In some instances, a spike protein seqeunce may contain any  combination of the modifications in the following Table 4.  The amino acid positions indicated  in Table 4 are numbered relative to SEQ ID. NO: 69 (SARS‐CoV‐2 Omicron UFO69279.1 (BA.1,  previously  B.1.1.529)  in  Table  1),  and  the  corresponding  amino  acid  positions  in  other  coronavirus  spike  proteins  can  be  determined  through  sequence  alignments  (see  e.g.,  alignment of various coronavirus spike protein sequences in Table 5).      Table 4  Non‐Inclusive Coronavirus Spike Protein Modification Combinations 
Figure imgf000097_0002
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
In some embodiments, the amino acid corresponding to the amino acid at position 326 in SEQ  ID. NO: 69 (SARS‐CoV‐2 Omicron UFO69279.1 (BA.1, previously B.1.1.529) can be substituted  to produce a variant coronavirus spike protein encoded by RNA as described herein.  In some  embodiments, the amino acid corresponding to the amino acid at position 326 in SEQ ID NO:69   can  be  substituted with  a  serine  residue  to  produce  a  variant  coronavirus  spike  protein  encoded by RNA as described herein.   A substitution with a serine  residue at 326 may be  referred to herein as 326S.     In some embodiments, the amino acid corresponding to the amino acid at position 364 in SEQ  ID. NO: 69 (SARS‐CoV‐2 Omicron UFO69279.1 (BA.1, previously B.1.1.529) can be substituted  to produce a variant coronavirus spike protein encoded by RNA as described herein.  In some  embodiments, the amino acid corresponding to the amino acid at position 364 in SEQ ID NO:69  can be substituted with a phenylalanine residue to produce a variant coronavirus spike protein  encoded by RNA as described herein.  A substitution with a phenylalanine residue at 364 may  be referred to herein as 364F.  In some embodiments, the amino acid corresponding to the amino acid at position 567 in SEQ  ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 567 in SEQ ID NO:69 can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 567 may be referred to herein as 567C.  In some embodiments, the amino acid corresponding to the amino acid at position 611 in SEQ  ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 611 in SEQ ID NO:69 can be substituted with a glycine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a glycine  residue at 611 may be referred to herein as 611G.  In some embodiments, the amino acid corresponding to the amino acid at position 814 in SEQ  ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 814 in SEQ ID NO:69 can be substituted with a phenylalanine residue to produce a  variant coronavirus spike protein encoded by RNA as described herein.  A substitution with a  phenylalanine residue at 814 may be referred to herein as 814P.  In some embodiments, the amino acid corresponding to the amino acid at position 840 in SEQ  ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 840  in SEQ ID NO:69can be substituted with an asparagine residue to produce a  variant coronavirus spike protein encoded by RNA as described herein.  A substitution with a  asparagine residue at 840 may be referred to herein as 840N.  In some embodiments, the amino acid corresponding to the amino acid at position 851 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 851 in SEQ ID NO:69can be substituted with a phenylalanine residue to produce a  variant coronavirus spike protein encoded by RNA as described herein.  A substitution with a  phenylalanine residue at 851 may be referred to herein as 851F.   In some embodiments, the amino acid corresponding to the amino acid at position 889 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 889 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 889 may be referred to herein as 889P.  In some embodiments, the amino acid corresponding to the amino acid at position 896 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 896 in SEQ ID NO:69 can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 896 may be referred to herein as 896P.  In some embodiments, the amino acid corresponding to the amino acid at position 939 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 939 in SEQ ID NO:69can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 939 may be referred to herein as 939P.  In some embodiments, the amino acid corresponding to the amino acid at position 957 in SEQ  ID NO:69 can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 957 in SEQ ID NO:69can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 957 may be referred to herein as 957C.    In some embodiments, the amino acid corresponding to the amino acid at position 977 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 977 in SEQ ID NO:69can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 977 may be referred to herein as 977C.  In some embodiments, the amino acid corresponding to the amino acid at position 981 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 981 in SEQ ID NO:69can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 981 may be referred to herein as 981C.  In some embodiments, the amino acid corresponding to the amino acid at position 982 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 982 in SEQ ID NO:69can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 982 may be referred to herein as 982P.  In some embodiments, the amino acid corresponding to the amino acid at position 983 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 983 in SEQ ID NO:69can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 983 may be referred to herein as 983P.  In some embodiments, the amino acid corresponding to the amino acid at position 984 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 984 in SEQ ID NO:69can be substituted with a proline residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a proline  residue at 983 may be referred to herein as 984P.  In some embodiments, the amino acid corresponding to the amino acid at position 986 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 986 in SEQ ID NO:69can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 986 may be referred to herein as 986C.  In some embodiments, the amino acid corresponding to the amino acid at position 989 in SEQ  ID NO:69can be substituted to produce a variant coronavirus spike protein encoded by RNA  as described herein.  In some embodiments, the amino acid corresponding to the amino acid  at position 989 in SEQ ID NO:69can be substituted with a cysteine residue to produce a variant  coronavirus spike protein encoded by RNA as described herein.  A substitution with a cysteine  residue at 989 may be referred to herein as 989C.  In some embodiments, a variant spike protein encoded by RNA described herein has, at least,  or has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, and/or 18 of the following  modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982,  983,  984,  986,  989  as  set  forth  in  SARS‐CoV‐2 Omicron  (BA.1, previously B.1.1.529)  spike  protein, UniProt Accession Number UFO69279.1, or the corresponding amino acid in the spike  protein  of  another  coronavirus,  wherein  in  some  embodiments  the  modification  at  the  position or corresponding position 326  is a serine, 364 is a phenylalanine, 567  is a cysteine,  611 is a glycine, 814 is a proline, 840 is a asparagine, 851 is a phenylalanine, 889 is a proline,  896 is a proline, 939 is a proline, 957 is a cysteine, 977 is a cysteine, 981 is a cysteine, 982 is a  proline, 983 is a proline, 984 is a proline, 986 is a cysteine, and 989 is a cysteine and wherein  in further embodiments the variant spike protein has at least 80%, 81%, 82%, 83%, 84%, 85%,  86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,  99.3%,  99.4%,  99.5%,  99.6%,  99.7%,  99.8%,  or  99.9%  sequence  identity  to  a  SARS‐CoV‐2  Omicron (BA.1, previously B.1.1.529) spike protein, UniProt Accession Number UFO69279.1.   In some instances, the modifications described herein may be applied alone or in combination  with any one or more additional modifications described herein to produce an RNA (e.g., as  described herein) encoding an  immunogenic polypeptide  comprising a  variant  coronavirus  spike protein that  is a variant of a native coronavirus spike protein or fragment thereof.   In  some embodiments,  these modifications may  (a)  increase adoption by RBDs of  the variant  coronavirus  spike  proteins  of  the  RBD‐up  conformation  to  expose  more  neutralization‐ sensitive  epitopes  on  the  spike  protein,  (b)  decrease  adoption  by  RBDs  of  the  variant  coronavirus  spike  proteins  of  the RBD‐down  conformation,  (c)  increase  expression  of  the  variant  coronavirus  spike  protein  compared  to  the  native  coronavirus  spike  protein,  (d)  increase adoption of a prefusion conformation, (e) decrease shedding of a S1 subunit of the  variant coronavirus spike protein, and/or (f)  improve  localization of the variant coronavirus  spike protein to a host cell membrane.  Mutations described herein and e.g., in Tables 2A, 2B, and 2C may be introduced into S protein  sequence  of  other  coronavirus  strains  or  variant  sequences,  or  immunogenic  fragments  thereof, and the corresponding position may be determined through a sequence alignment  with SEQ ID NO: 69 (see e.g., Table 5).  In some embodiments, a variant spike protein encoded by RNA described herein, has at least,  or has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, and/or 18 of the following  modifications at positions 326, 364, 567, 611, 814, 840, 851, 889, 896, 939, 957, 977, 981, 982,  983,  984,  986,  989  as  set  forth  in  SARS‐CoV‐2 Omicron  (BA.1, previously B.1.1.529)  spike  protein, UniProt Accession Number UFO69279.1, or the corresponding amino acid in the spike  protein  of  another  coronavirus,  wherein  in  some  embodiments  the  modification  at  the  position or corresponding position 326 is to any amino acid except phenylalanine, 364 is any  amino acid except valine, 567 is any amino acid except alanine, 611 is any amino acid except  glycine, 814  is any amino acid except phenylalanine, 840  is any amino acid except aspartic  acid, 851  is any amino acid except  lysine, 889  is any amino acid except alanine, 896  is any  amino acid except alanine, 939 is any amino acid except alanine, 957 is any amino acid except  asparagine, 977 is any amino acid except isoleucine, 981 is any amino acid except leucine, 982  is any amino acid except aspartic acid, 983 is any amino acid except lysine, 984 is any amino  acid except valine, 986  is any amino acid except alanine, and 989  is any amino acid except  glutamine, and wherein  in further embodiments the variant spike protein has at  least 80%,  81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,  98%,  99%,  99.1%,  99.2%,  99.3%,  99.4%,  99.5%,  99.6%,  99.7%,  99.8%,  or  99.9%  sequence  identity to a SARS‐CoV‐2 Omicron (BA.1, previously B.1.1.529) spike protein, UniProt Accession  Number UFO69279.1.  In some instances, the modifications described herein may be applied  alone or  in combination with any one or more additional modifications described herein to  produce an RNA encoding isolated immunogenic polypeptide comprising a variant coronavirus  spike protein that  is a variant of a native coronavirus spike protein or fragment thereof.   In  some embodiments,  these modifications may  (a)  increase adoption by RBDs of  the variant  coronavirus  spike  proteins  of  the  RBD‐up  conformation  to  expose  more  neutralization‐ sensitive  epitopes  on  the  spike  protein,  (b)  decrease  adoption  by  RBDs  of  the  variant  coronavirus  spike  proteins  of  the RBD‐down  conformation,  (c)  increase  expression  of  the  variant  coronavirus  spike  protein  compared  to  the  native  coronavirus  spike  protein,  (d)  increase adoption of a prefusion conformation, (e) decrease shedding of a S1 subunit of the  variant coronavirus spike protein, and/or (f)  improve  localization of the variant coronavirus  spike protein to a host cell membrane.  The amino acids  in each human coronavirus spike protein sequence and the corresponding  position of that amino acid with respect to SEQ ID NO:1 can be determined based an alignment  of the protein sequences.  Below in Table 5 is an alignment of human coronavirus spike protein  sequences  (e.g.,  the  spike protein  sequences of Table 1).   The highlighted positions  in  the  below alignment correspond to the  location of the amino acids to be modified  identified  in  the Table 2 above.    
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Coronavirus Variants  Those skilled in the art are aware of various spike variants, and/or resources that document  them. For example, the following strains, their SARS‐CoV‐2 S protein amino acid sequences  and,  in particular, modifications thereof compared to wildtype SARS‐CoV‐2 S protein amino  acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.  B.1.1.7 ("Variant of Concern 202012/01" (VOC‐202012/01)  B.1.1.7 is a variant of SARS‐CoV‐2 which was first detected in October 2020 during the COVID‐ 19 pandemic in the United Kingdom from a sample taken the previous month, and it quickly  began to spread by mid‐December.  It  is correlated with a significant  increase  in the rate of  COVID‐19 infection in United Kingdom; this increase is thought to be at least partly because  of change N501Y inside the spike glycoprotein's receptor‐binding domain, which is needed for  binding  to ACE2  in  human  cells.  The  B.1.1.7  variant  is  defined  by  23 mutations:  13  non‐ synonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17 mutations  that change proteins and six that do not). The spike protein changes in B.1.1.7 include deletion  69‐70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.   B.1.351 (501.V2)  B.1.351 lineage and colloquially known as South African COVID‐19 variant, is a variant of SARS‐ CoV‐2. Preliminary results  indicate that this variant may have an  increased transmissibility.  The B.1.351 variant is defined by multiple spike protein changes including: L18F, D80A, D215G,  deletion 242‐244, R246I, K417N, E484K, N501Y, D614G and A701V. There are three mutations  of particular interest in the spike region of the B.1.351 genome: K417N, E484K, N501Y.  B.1.1.298 (Cluster 5)  B.1.1.298 was discovered in North Jutland, Denmark, and is believed to have been spread from  minks to humans via mink farms. Several different mutations in the spike protein of the virus  have been confirmed. The specific mutations  include deletion 69–70, Y453F, D614G,  I692V,  M1229I, and optionally S1147L.  P.1 (B.1.1.248)  Lineage B.1.1.248, known as the Brazil(ian) variant, is one of the variants of SARS‐CoV‐2 which  has been named P.1 lineage. P.1 has a number of S‐protein modifications [L18F, T20N, P26S,  D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F] and is similar in certain  key RBD positions (K417, E484, N501) to variant B.1.351 from South Africa.  B.1.427/B.1.429 (CAL.20C)  Lineage B.1.427/B.1.429, also known as CAL.20C, is defined by the following modifications in  the S‐protein: S13I, W152C, L452R, and D614G of which the L452R modification is of particular  concern. CDC has listed B.1.427/B.1.429 as "variant of concern".  B.1.525  B.1.525 carries the same E484K modification as found  in the P.1, and B.1.351 variants, and  also carries the same ΔH69/ΔV70 deletion as found in B.1.1.7, and B.1.1.298. It also carries the  modifications D614G, Q677H and F888L.   B.1.526  B.1.526 was detected as an emerging  lineage of viral  isolates  in  the New York  region  that  shares mutations with previously reported variants. The most common sets of spike mutations  in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.    The following table shows an overview of circulating SARS‐CoV‐2 strains which are VOI/VOC.     
  7                                                          
Figure imgf000119_0001
B.1.1.529  B.1.529 (“Omicron”) was first detected in South Africa in November 2021.  Omicron has been  found  to  multiply  around  70  times  faster  than  Delta  variants,  and  quickly  became  the  dominant strain of SARS‐CoV‐2 worldwide.  Since its initial detection, a number of sublineages  have arisen.  Listed in the below Table 3A are current Omicron variants of concern, along with  certain  characteristic mutations associated with  the  S protein of each  (mutation positions  shown relative to SEQ ID NO: 1).  In some embodiments, BA.4 and BA.5 variants have the same  S protein amino acid sequence, in which case the term “BA.4/5” may be used to refer to an  amino acid sequence of an S protein that can be found in either of BA.4 or BA.5.  Similarily, in  some embodiments, BA.4.6 and BF.7 variants have the same protein amino acid sequence, in  which case the term “BA.4.6/BF.7” can be used to refer to an amino acid sequence of an S  protein present in either of BA.4.6 or BF.7.  Table 3A: Omicron Variants of Concern and Characteristic mutations 
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
  In addition to the above Omicron variants, further variants of BA.5 have been observed (such  variants  including, e.g., BF.14) comprising one of more of  the  following mutations  in  the S  protein (positions shown relative to SEQ ID NO: 1): E340X (e.g., E340K), R346X (e.g., R346T,  R346I, or R346S), K444X (e.g., K444N or K444T), V445X, 5  N450D, and S:N460X (e.g., N460K).  In some embodiments, RNA described herein comprises a nucleotide sequence encoding a  SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 5  10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) characteristic of an Omicron variant (e.g.,  one or more mutations of an Omicron variant listed in Table 3A) and one or more mutations  that  stabilize  the  S  protein  in  a  pre‐fusion  confirmation.  In  some  embodiments,  an  RNA  comprises a nucleotide sequence encoding a SARS‐CoV‐2 S protein comprising one or more  mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more)  listed in Table 3A. In some such embodiments, one or more mutations may come from two or  more variants as  listed  in Table 3A.  In some embodiments, an RNA comprises a nucleotide  sequence encoding a SARS‐CoV‐2 S protein comprising each of  the mutations  identified  in  Table 3A as being characteristic of a certain Omicron variant (e.g., in some embodiments, an  RNA comprises a nucleotide sequence encoding a SARS‐CoV‐2 S protein comprising each of  the mutations listed in Table 3A as being characteristic of an Omicron BA.1, BA.2, BA.2.12.1,  BA.4/5, BA.2.75, BA.2.75.1, BA.4.6, BQ.1.1, XBB, XBB.1, XBB.2, or XBB.1.3 variant).  In  some  embodiments,  an  RNA  disclosed  herein  comprises  a  nucleotide  sequence  that  encodes an  immunogenic  fragment of  the SARS‐Cov‐2 S protein  (e.g.,  the RBD) and which  comprises one or more mutations  that are characteristic of a SARS‐CoV‐2 variant  (e.g., an  Omicron variant described herein). For example, in some embodiments, an RNA comprises a  nucleotide sequence encoding the RBD of an S protein of a SARS‐CoV‐2 variant (e.g., a region  of the S protein corresponding to amino acids 327 to 528 of SEQ ID NO: 1, and comprising one  or more mutations characteristic of a variant of concern that lie within this region).  In some embodiments, an RNA encodes a SARS‐CoV‐2 S protein comprising a subset of the  mutations listed in Table 3A. In some embodiments, an RNA encodes a SARS‐CoV‐2 S protein  comprising the mutations listed in Table 3A that are most prevalent in a certain variant (e.g.,  mutations that have been detected in at least 30%, at least 40%, at least 50%, at least 60%, at  least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at  least 99%, or 100% of sequences collected to date for a given variant sequenced). Mutation  prevalence can be determined, e.g., based on published sequences (e.g., sequences that are  collected and made available to the public by GISAID).  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations that are characteristic of a BA.4/5 variant. In some embodiments, the one  or more mutations characteristic of a BA.4/5 variant  include T19I, Δ24‐26, A27S, ΔO24‐26,  A27S, Δ69/70, G142D, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, K417N,  N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H,  and N969K.  In  some  embodiments,  RNA  described  herein  encodes a SARS‐CoV‐2 S protein comprising one or more mutations that are characteristic of  a BA.4/5 variant and excludes R408S. In some embodiments, RNA described herein encodes a  SARS‐ CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9,  10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4/5 variant  and excludes R408S.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more)  mutations  characteristic  of  a  BA.2.75  variant.  In  some  embodiments,  the  one  or  more  mutations  characteristic  of  a  BA.2.75  variant  include  T19I,  Δ24‐26,  A27S,  G142D,  K147E,  W152R, F157L,  I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S,  K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  Q954H,  and  N969K.  In  some  embodiments,  RNA  described herein encodes a SARS‐CoV‐2 S protein comprising one or more mutations that are  characteristic of a BA.4/5 variant and excludes R408S. In some embodiments, RNA described  herein encodes a SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2,  3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic  of a BA.4/BA.5 variant, and which excludes R408S and N354D.  In some embodiments, RNA  described herein encodes a SARS‐CoV‐2 S protein comprising one or more (including, e.g., 2,  3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of  a  BA.2.75  variant.  In  some  embodiments,  the  one  or more mutations  characteristic  of  a  BA.2.75  variant  include  T19I,  Δ24‐26, A27S, G142D,  K147E, W152R,  F157L,  I210V, V213G,  G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K,  S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, Q954H,  and N969K.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein  comprising one or more mutations (including, e.g., 2, 3, 4, 5, 25 6, 7, 8, 9, 10, 11, 12, 13, 14,  15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes  N354D.  In  some  embodiments,  RNA  described  herein  encodes  a  SARS‐CoV‐2  S  protein  comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,  16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes  D796Y.  In  some  embodiments,  RNA  described  herein  encodes  a  SARS‐CoV‐2  S  protein  comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,  16, 17, 18, 19, 20, or more) that are characteristic of a BA.2.75 variant, and which excludes  D796Y and N354D.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of a BA.2.75.2 variant.  In some embodiments, the one or  more mutations  characteristic  of  a  BA.2.75.2  variant  include  T19I,  Δ24‐26,  A27S,  G142D,  K147E, W152R, F157L,  I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F,  T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R,  N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and D1199N. In  some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one or  more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 30 14, 15, 16, 17, 18, 19,  20, or more) that are characteristic of a BA.2.75.2 variant, and which excludes R346T.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of a BA.4.6 or BF.7 variant. In some embodiments, the one  or more mutations  characteristic  of  a  BA.4.6  or  BF.7  variant  include  T19I,  Δ24‐26,  A27S,  Δ69/70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, K417N, N440K,  L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,  N764K, D796Y, Q954H, and N969K. In some embodiments, RNA described herein encodes a  SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9,  10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7  variant, and which exclude R408S.  In some embodiments, RNA described herein encodes a  SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9,  10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7  variant, and which exclude N658S.  In some embodiments, RNA described herein encodes a  SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9,  25 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BA.4.6 or BF.7  variant, and which exclude N658S and R408S.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of an Omicron XBB variant. In some embodiments, the one  or more mutations characteristic of an Omicron XBB variant include T19I, Δ24‐26, A27S, V83A,  G142D,  Δ144,  H146Q, Q183E,  V213E, G339H,  R346T,  L368I,  S371F,  S373P,  S375F,  T376A,  D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S,  Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of an Omicron XBB.1 variant. In some embodiments, the  one or more mutations characteristic of an Omicron XBB.1 variant  include G252V.  In some  embodiments, the one or more mutations characteristic of an Omicron XBB.1 variant include  T19I, Δ24‐26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I,  S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N,  T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,  D796Y, Q954H, and N969K.  In some embodiments, RNA described herein encodes a SARS‐ CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,  12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of an Omicron XBB.1 variant  and which exclude Q493R. In some embodiments, RNA described herein encodes a SARS‐CoV‐ 2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,  15 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of an Omicron XBB variant  and which exclude Q493R and G252V.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of an Omicron XBB.2 variant. In some embodiments, the one  or  more  mutations  characteristic  of  an  Omicron  XBB.2  variant  include  D253G.  In  some  embodiments, the one or more mutations characteristic of an Omicron XBB.2 variant include  T19I, Δ24‐26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, D253G, G339H, R346T, L368I,  S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N,  T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H,  N764K, D796Y, Q954H, and N969K.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of an Omicron XBB.1.3 variant. In some embodiments, the  one or more mutations characteristic of an Omicron XBB.1.3 variant include G252V and A484T.  In  some  embodiments,  the  one  or more mutations  characteristic  of  an Omicron  XBB.1.3  variant  include  T19I,  Δ24‐26,  A27S,  V83A,  G142D,  Δ144,  H146Q,  Q183E,  V213E,  G252V,   G339H, R346T,  L368I,  S371F,  S373P,  S375F, T376A, D405N, R408S, K417N, N440K, V445P,  G446S, N460K, S477N, T478K, A484T, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K.  In some embodiments, RNA described herein encodes a SARS‐CoV‐2 S protein comprising one  or more mutations that are characteristic of a BQ.1.1 variant. In some embodiments, the one  or more mutations  characteristic of  a  BQ.1.1  variant  include  T19I,  Δ24‐26, A27S,  Δ69/70,  G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T,  L452R, N463K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H,  and N969K.  In  some  embodiments,  RNA  described  herein  encodes a SARS‐CoV‐2 S protein comprising one or more mutations (including, e.g., 2, 3, 4, 5,  6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) that are characteristic of a BQ.1.1  variant.  In one embodiment, a vaccine antigen described herein comprises, consists essentially of or  consists of a spike protein  (S) of SARS‐CoV‐2, a variant  thereof, or a  fragment  thereof and  comprises one or more of mutations characteristic of a SARS‐CoV‐2 variant (e.g., one or more  of mutations associated with an Omicron variant that are listed in Table 3A).  In one embodiment, a vaccine antigen comprises (a) the amino acid sequence of amino acids  17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ  ID NO: 1 or 7, an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273  of SEQ ID NO: 1 or 7, or an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to an immunogenic fragment of the amino acid sequence of amino  acids 17 to 1273 of SEQ ID NO: 1 or 7, and (b) one of more mutations associated with a SARS‐ CoV‐2 variant of concern (e.g., one or more mutations listed in Table 3A). In one embodiment,  a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO:  1 or 7 and comprises one or more associated with a SARS‐CoV‐2 variant of concern (e.g., one  or more mutations listed in Table 3A).  In one embodiment, a vaccine antigen comprises (a) the amino acid sequence of amino acids  17 to 1273 of SEQ  ID NO: 80, an amino acid sequence having at  least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ  ID NO: 80, an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273  of SEQ ID NO: 80, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80,  and (b) one of more of the mutations listed in Table 3A. In one embodiment, a vaccine antigen  comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80 and comprises  one or more mutations associated with a SARS‐CoV‐2 variant of concern (e.g., one or more  mutations listed in Table 3A).  In one embodiment, RNA encoding a vaccine antigen (a) comprises (i) the nucleotide sequence  of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49  to 3819 of SEQ ID NO: 2, 8 or 9, a fragment of the nucleotide sequence of nucleotides 49 to  3819 of SEQ ID NO: 2, 8 or 9, or a nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to a fragment of the nucleotide sequence of nucleotides 49 to  3819  of  SEQ  ID NO:  2,  8  or  9  and/or  (ii)  a  nucleotide  sequence  encoding  an  amino  acid  sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or  7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an immunogenic  fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  an immongenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO:  1 or 7, and (b) comprises a nucleotide sequence encoding a SARS‐CoV‐2 S protein comprising  one or more mutations associated with a SARS‐CoV‐2 variant of concern (e.g., one or more  mutations  listed  in Table 3A)).     In one embodiment, RNA encoding a vaccine antigen (a) (i)  comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or  (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to  1273 of SEQ ID NO: 1 or 7, and (b) comprises one or more mutations characteristics of a SARS‐ CoV‐2 variant of concern (e.g., one or more mutations listed in Table 3A).  In one embodiment, RNA encoding a vaccine antigen comprises (a) (i) the nucleotide sequence  of nucleotides 49 to 3819 of SEQ ID NO: 81, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to  3819 of SEQ ID NO: 81, a fragment of the nucleotide sequence of nucleotides 49 to 3819 of  SEQ ID NO: 81, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID  NO:  81  and/or  (ii)  a  nucleotide  sequence  encoding  encodes  an  amino  acid  sequence  comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, an amino  acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to the  amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80, an immunogenic fragment  of the amino acid sequence of amino acids 17 to 1273 of SEQ  ID NO: 80, or an amino acid  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  an  immongenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO:  80, and (b) comprises one or more mutations associated with a SARS‐CoV‐2 variant of concern  (e.g., one or more mutations listed in Table 3A)).  In one embodiment, RNA encoding a vaccine  antigen (a) (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 81;  and/or (ii) comprises a nucleotide sequence that encodes an amino acid sequence comprising  the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 80 or 7, and (b) comprises  one or more mutations characteristics of a SARS‐CoV‐2 variant of concern (e.g., one or more  mutations listed in Table 3A).  In one embodiment, a vaccine antigen comprises, consists essentially of or consists of SARS‐‐  CoV‐2 spike S1 fragment (S1) (the S1 subunit of a spike protein (S) of SARS‐CoV‐2), a variant  thereof, or a fragment thereof, and comprises one or more mutations of a SARS‐CoV‐2 variant  described herein.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17  to 683 of SEQ  ID NO: 1, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:  1, an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID  NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of  SEQ  ID NO: 1 and comprises one or more mutations characteristic of a SARS‐CoV‐2 variant  (e.g.,  one  or more mutations  listed  in  Table  3A).  In  one  embodiment,  a  vaccine  antigen  comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1 and comprises  one or more mutations characteristic of a SARS‐CoV‐2 variant (e.g., one or more mutations  listed in Table 3A).  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17  to 683 of SEQ ID NO: 80, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:  1, an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID  NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of  SEQ ID NO: 80 and comprises one or more mutations characteristic of a SARS‐CoV‐2 variant  (e.g.,  one  or more mutations  listed  in  Table  3A).  In  one  embodiment,  a  vaccine  antigen  comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 80 and comprises  one or more mutations characteristic of a SARS‐CoV‐2 variant (e.g., one or more mutations  listed in Table 3A).    Vaccine Antigens and Combinations Thereof  In one embodiment, the vaccine antigen described herein comprises, consists essentially of or  consists of a spike protein (S) of SARS‐CoV‐2, a variant thereof, or a fragment thereof.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17  to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ  ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to  1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ  ID NO: 1 or 7. In one embodiment, a vaccine antigen comprises the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at  least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49  to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to  3819 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ  ID NO:  2,  8  or  9;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having  at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of  amino acids 17 to 1273 of SEQ  ID NO: 1 or 7.  In one embodiment, RNA encoding a vaccine  antigen (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or  9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino  acids 17 to 1273 of SEQ ID NO: 1 or 7.  In one embodiment, the vaccine antigen comprises, consists essentially of or consists of SARS‐ CoV‐2 spike S1 fragment (S1) (the S1 subunit of a spike protein (S) of SARS‐CoV‐2), a variant  thereof, or a fragment thereof.   In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17  to 683 of SEQ  ID NO: 1, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO:  1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ  ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the amino acid sequence of amino acids 17 to 683 of SEQ  ID NO: 1.  In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683  of SEQ ID NO: 1.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at  least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49  to 2049 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to  2049 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ  ID NO:  2,  8  or  9;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of  amino acids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to  683 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the  nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID  NO: 1.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17  to 685 of SEQ  ID NO: 1, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO:  1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ  ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the amino acid sequence of amino acids 17 to 685 of SEQ  ID NO: 1.  In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 685  of SEQ ID NO: 1.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at  least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49  to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to  2055 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ  ID NO:  2,  8  or  9;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of  amino acids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to  685 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the  nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID  NO: 1.  In one embodiment, the vaccine antigen comprises, consists essentially of or consists of the  receptor binding domain (RBD) of the S1 subunit of a spike protein (S) of SARS‐CoV‐2, a variant  thereof, or a fragment thereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID  NO: 1, a variant thereof, or a  fragment thereof  is also referred to herein as "RBD" or "RBD  domain".  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 327  to 528 of SEQ  ID NO: 1, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID  NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of  SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 327 to 528  of SEQ ID NO: 1.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to the nucleotide sequence of nucleotides  979 to 1584  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides  979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584  of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid  sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of  amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to  528 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the  nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ  ID NO: 1.  According to certain embodiments, a signal peptide is fused, either directly or through a linker,  to a SARS‐CoV‐2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide  or protein. Accordingly, in one embodiment, a signal peptide is fused to the above described  amino acid sequences derived from SARS‐CoV‐2 S protein or immunogenic fragments thereof  (antigenic peptides or proteins) comprised by the vaccine antigens described above.  Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino  acids and are preferably located at the N‐terminus of the antigenic peptide or protein, without  being limited thereto. Signal peptides as defined herein preferably allow the transport of the  antigenic peptide or protein as encoded by  the RNA  into a defined  cellular  compartment,  preferably  the  cell  surface,  the  endoplasmic  reticulum  (ER)  or  the  endosomal‐lysosomal  compartment.  In one embodiment, the signal peptide sequence as defined herein  includes,  without  being  limited  thereto,  the  signal  peptide  sequence  of  SARS‐CoV‐2  S  protein,  in  particular a sequence comprising the amino acid sequence of amino acids 1 to 16 or 1 to 19 of  SEQ ID NO: 1 or a functional variant thereof.   In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1  to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a  functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1. In one embodiment, a signal  sequence comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.  In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence  of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ  ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or  9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino  acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID  NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID  NO: 1, or the amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the amino acid  sequence of amino acids 1  to 16 of SEQ  ID NO: 1.  In one  embodiment,  RNA  encoding  a  signal  sequence  (i)  comprises  the  nucleotide  sequence  of  nucleotides 1  to 48 of  SEQ  ID NO: 2, 8 or 9;  and/or  (ii) encodes an amino  acid  sequence  comprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.  In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1  to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a  functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or the  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1. In one embodiment, a signal  sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.  In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence  of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 57 of SEQ  ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or  9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino  acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID  NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID  NO: 1, or the amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the amino acid  sequence of amino acids 1  to 19 of SEQ  ID NO: 1.  In one  embodiment,  RNA  encoding  a  signal  sequence  (i)  comprises  the  nucleotide  sequence  of  nucleotides 1  to 57 of  SEQ  ID NO: 2, 8 or 9;  and/or  (ii) encodes an amino  acid  sequence  comprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.  In  some embodiments, an RNA comprises a sequence encoding a signal peptide.    In  some  embodiments, a signal peptide sequence as defined herein  includes, without being  limited  thereto, the signal peptide sequence of an immunoglobulin, e.g., the signal peptide sequence  of  an  immunoglobulin  heavy  chain  variable  region, wherein  the  immunoglobulin may  be  human immunoglobulin. In particular, in some embodiments, the signal peptide sequence as  defined herein can include a sequence comprising the amino acid sequence of amino acids 1  to 22 of SEQ ID NO: 31 or a functional variant thereof.  In some embodiments, a signal peptide sequence is functional in mammalian cells.  In some  embodiments, a utilized signal sequence is “intrinsic” in that it is, in nature, associated with  (e.g., linked to) the encoded polypeptide.  In some embodiments, a utilized signal sequence is  heterologous  to an encoded polypeptide, e.g.,  is not naturally part of a polypeptide  (e.g.,  protein) whose sequences are included in the encoded polypeptide.  In some embodiments,  signal peptides are sequences, which are typically characterized by a length of about 15 to 30  amino acids.  In many embodiments, signal peptides are positioned at the N‐terminus of an  encoded  polypeptide  as  described  herein,  without  being  limited  thereto.  In  some  embodiments, signal peptides preferably allow the transport of the polypeptide encoded by  RNAs  of  the  present  disclosure  with  which  they  are  associated  into  a  defined  cellular  compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal‐ lysosomal compartment.  In some embodiments, a signal sequence is selected from an S1S2  signal peptide (aa 1‐16 or aa 1‐19), an immunoglobulin secretory signal peptide (aa 1‐22), an  HSV‐1  gD  signal peptide  (MGGAAARLGAVILFVVIVGLHGVRSKY),  an HSV‐2  gD  signal  peptide  (MGRLTSGVGTAALLVVAVGLRVVCA), a human SPARC signal peptide, a human insulin isoform  1 signal peptide, a human albumin signal peptide, etc. Those skilled in the art will be aware of  other secretory signal peptides such as,  for example, as disclosed  in WO2017/0810822220  (e.g., SEQ ID NOs: 1‐1115 and 1728, or fragments variants thereof) and WO2019008001.  In  some embodiments, an RNA sequence encodes an epitope that may comprise or otherwise  be linked to a signal sequence (e.g., secretory sequence), such as those listed in Table A, or at  least  a  sequence  having  1,  2,  3,  4,  or  5  amino  acid  differences  relative  thereto.  In  some  embodiments, a signal sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having at  least 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.   In some  embodiments, a sequence such as MFVFLVLLPLVSSQCVNLT, or a sequence having 1, 2, 3, 4, or  at most 5 amino acid differences relative thereto, is utilized.  In some embodiments, a signal  sequence is selected from those included in the Table A below and/or those encoded by the  sequences in Table B below.    Table A: Exemplary signal sequences 
Figure imgf000138_0001
Figure imgf000139_0001
  Table B: Exemplary nucleotide sequences encoding signal sequences   
Figure imgf000139_0002
  In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1  to 22 of SEQ  ID NO: 31, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:  31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO:  31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  22  of  SEQ  ID  NO:  31.  In  one  embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 22 of  SEQ ID NO: 31.  In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence  of nucleotides 54 to 119 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to  119 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 119 of  SEQ  ID NO: 32, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32;  and/or  (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino  acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID  NO: 31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID  NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the amino acid sequence of amino acids 1  to 22 of SEQ  ID NO: 31.  In one  embodiment,  RNA  encoding  a  signal  sequence  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.  Such  signal  peptides  are  preferably  used  in  order  to  promote  secretion  of  the  encoded  antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to  an encoded antigenic peptide or protein as defined herein.  Accordingly,  in particularly preferred embodiments, the RNA described herein comprises at  least one coding region encoding an antigenic peptide or protein and a signal peptide, said  signal peptide preferably being fused to the antigenic peptide or protein, more preferably to  the N‐terminus of the antigenic peptide or protein as described herein.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or  7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino  acid sequence of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a fragment of the  nucleotide sequence of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2,  8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of  SEQ ID NO: 1 or 7, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80%  identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an  immunogenic  fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or the amino acid sequence having  at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of  SEQ  ID NO: 1 or 7.  In one embodiment, RNA encoding a vaccine antigen  (i) comprises  the  nucleotide sequence of SEQ  ID NO: 2, 8 or 9; and/or  (ii) encodes an amino acid sequence  comprising the amino acid sequence of SEQ ID NO: 1 or 7.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ  ID NO: 7, or an  immunogenic  fragment of the amino acid  sequence of SEQ ID NO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  7.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ  ID NO: 15, 16, 19, 20, 24, or 25, a nucleotide sequence having at  least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20,  24, or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or  the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the nucleotide sequence of SEQ  ID NO: 15, 16, 19, 20, 24, or 25; and/or  (ii) encodes an  amino acid sequence comprising  the amino acid sequence of SEQ  ID NO: 7, an amino acid  sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino  acid sequence of SEQ ID NO: 7, or an immunogenic fragment of the amino acid sequence of  SEQ ID NO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID NO:  7.  In  one  embodiment,  RNA  encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19,  20, 24, or 25; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence  of SEQ ID NO: 7.   In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:  1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  683  of  SEQ  ID  NO:  1.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 683  of SEQ ID NO: 1.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of  SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO:  2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of  amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of  SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to  683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO:  1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence  of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence  comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:  1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  685  of  SEQ  ID  NO:  1.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 685  of SEQ ID NO: 1.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of  SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO:  2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of  amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of  SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to  685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:  1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence  of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence  comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.   In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ  ID NO: 3, or an  immunogenic  fragment of the amino acid  sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  3.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 4, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the nucleotide sequence of SEQ  ID NO: 4, or a  fragment of  the nucleotide  sequence of SEQ ID NO: 4, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence  of  SEQ  ID NO:  4;  and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ  ID NO: 3, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ  ID NO: 3, or an  immunogenic  fragment of the amino acid  sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  3.  In  one  embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ  ID NO: 4; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of  SEQ ID NO: 3.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  221 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 of SEQ ID NO:  29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  221  of  SEQ  ID  NO:  29.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 221  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 716 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 716 of  SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 716 of SEQ ID  NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 221  of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  221  of  SEQ  ID  NO:  29.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  224 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 of SEQ ID NO:  31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  224  of  SEQ  ID  NO:  31.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 224  of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 725 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 725 of  SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 725 of SEQ ID  NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 224  of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  224  of  SEQ  ID  NO:  31.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.   Multimerization Domains  In some embodiments, an RNA utilized as described herein comprises a sequence that encodes  a  multimerization  element  (e.g.,  a  heterologous  multimerization  element).  In  some  embodiments,  a  heterologous  multimerization  element  comprises  a  dimerization,  trimerization or tetramerization element. In some embodiments, a multimerization element  is one described in WO2017/081082 (e.g., SEQ ID NOs: 1116‐1167, or fragments or variants  thereof).  Exemplary trimerization and tetramerization elements include, but are not limited  to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll,  GCN4‐pll, and p53. In some embodiments, a provided encoded polypeptide(s) is able to form  a trimeric complex. For example, a utilized encoded polypeptide(s) may comprise a domain  allowing  formation of a multimeric complex, such as  for example a  trimeric complex of an  amino  acid  sequence  comprising  an  encoded polypeptide(s)  as described herein.  In  some  embodiments,  a  domain  allowing  formation  of  a  multimeric  complex  comprises  a  trimerization  domain,  for  example,  a  trimerization  domain  as  described  herein.    In  some  embodiments, an encoded polypeptide(s) can be modified by addition of a T4‐fibritin‐ derived  “foldon” trimerization domain, for example, to increase its immunogenicity.  According to certain embodiments, a trimerization domain is fused, either directly or through  a linker, e.g., a glycine/serine linker, to a SARS‐CoV‐2 S protein, a variant thereof, or a fragment  thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a trimerization  domain  is  fused  to  the above described amino acid sequences derived  from SARS‐CoV‐2 S  protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the  vaccine  antigens  described  above  (which may  optionally  be  fused  to  a  signal  peptide  as  described above).  Such trimerization domains are preferably located at the C‐terminus of the antigenic peptide  or protein, without being limited thereto. Trimerization domains as defined herein preferably  allow the trimerization of the antigenic peptide or protein as encoded by the RNA. Examples  of trimerization domains as defined herein include, without being limited thereto, foldon, the  natural  trimerization domain of T4  fibritin. The C‐terminal domain of T4  fibritin  (foldon)  is  obligatory  for the  formation of  the  fibritin  trimer structure and can be used as an artificial  trimerization  domain.  In  one  embodiment,  the  trimerization  domain  as  defined  herein  includes, without being limited thereto, a sequence comprising the amino acid sequence of  amino acids 3 to 29 of SEQ ID NO: 10 or a functional variant thereof. In one embodiment, the  trimerization domain as defined herein  includes, without being  limited thereto, a sequence  comprising the amino acid sequence of SEQ ID NO: 10 or a functional variant thereof.  In one embodiment, a  trimerization domain comprises  the amino acid  sequence of amino  acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID  NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID  NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the amino acid sequence of amino acids 3  to 29 of SEQ  ID NO: 10.  In one  embodiment, a trimerization domain comprises the amino acid sequence of amino acids 3 to  29 of SEQ ID NO: 10.   In  one  embodiment,  RNA  encoding  a  trimerization  domain  (i)  comprises  the  nucleotide  sequence of nucleotides 7 to 87 of SEQ ID NO: 11, a nucleotide sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7  to 87 of SEQ ID NO: 11, or a fragment of the nucleotide sequence of nucleotides 7 to 87 of SEQ  ID NO: 11, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 3 to 29  of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80%  identity to the amino acid sequence of amino acids 3 to 29 of SEQ  ID NO: 10, or a  functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or  the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10. In one embodiment, RNA  encoding a trimerization domain (i) comprises the nucleotide sequence of nucleotides 7 to 87  of SEQ  ID NO: 11; and/or  (ii) encodes an amino acid  sequence  comprising  the amino acid  sequence of amino acids 3 to 29 of SEQ ID NO: 10.  In one embodiment, a trimerization domain comprises the amino acid sequence SEQ ID NO:  10, an amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino  acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80%  identity to the amino acid sequence of SEQ  ID NO: 10.  In one  embodiment, a trimerization domain comprises the amino acid sequence of SEQ ID NO: 10.   In  one  embodiment,  RNA  encoding  a  trimerization  domain  (i)  comprises  the  nucleotide  sequence of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11, or a fragment of the  nucleotide sequence of SEQ ID NO: 11, or the nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the nucleotide  sequence of SEQ  ID NO: 11;  and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ  ID  NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino  acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80%  identity to the amino acid sequence of SEQ  ID NO: 10.  In one  embodiment, RNA encoding a trimerization domain (i) comprises the nucleotide sequence of  SEQ  ID  NO:  11;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ ID NO: 10.  Such  trimerization  domains  are  preferably  used  in  order  to  promote  trimerization  of  the  encoded antigenic peptide or protein. More preferably, a  trimerization domain as defined  herein is fused to an antigenic peptide or protein as defined herein.  Accordingly,  in particularly preferred embodiments, the RNA described herein comprises at  least one coding region encoding an antigenic peptide or protein and a trimerization domain  as defined herein, said trimerization domain preferably being fused to the antigenic peptide  or protein, more preferably to the C‐terminus of the antigenic peptide or protein.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ  ID NO: 5, or an  immunogenic  fragment of the amino acid  sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  5.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity  to  the nucleotide sequence of SEQ  ID NO: 6, or a  fragment of  the nucleotide  sequence of SEQ ID NO: 6, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence  of  SEQ  ID NO:  6;  and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ  ID NO: 5, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ  ID NO: 5, or an  immunogenic  fragment of the amino acid  sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  5.  In  one  embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ  ID NO: 6; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of  SEQ ID NO: 5.   In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ  ID NO: 17, 21, or 26, a nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%,  90%,  85%,  or  80%  identity  to  the nucleotide  sequence of  SEQ  ID NO:  17,  21, or  26, or  a  fragment of the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or the nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an  immunogenic  fragment  of  the  amino  acid  sequence  of  SEQ  ID NO:  5,  or  the  amino  acid  sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino  acid  sequence  of  SEQ  ID NO:  5.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino  acid sequence comprising the amino acid sequence of SEQ ID NO: 5.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18,  an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the amino acid sequence of SEQ ID NO: 18, or an immunogenic fragment of the amino acid  sequence of SEQ ID NO: 18, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  18.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18.   In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO:  29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  257  of  SEQ  ID  NO:  29.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 257  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 824 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of  SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 824 of SEQ ID  NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257  of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  257  of  SEQ  ID  NO:  29.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 of SEQ ID NO:  31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  260  of  SEQ  ID  NO:  31.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 260  of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 833 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of  SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 833 of SEQ ID  NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 260  of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  260  of  SEQ  ID  NO:  31.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20  to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO:  29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 of SEQ  ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29. In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 257  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 111 to 824 of SEQ  ID NO: 30, a nucleotide sequence having at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to  824 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 824 of  SEQ  ID NO: 30, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30;  and/or  (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino  acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ  ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257  of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides  111  to  824  of  SEQ  ID  NO:  30;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23  to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO:  31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 of SEQ  ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31. In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 260  of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 120 to 833 of SEQ  ID NO: 32, a nucleotide sequence having at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to  833 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 833 of  SEQ  ID NO: 32, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32;  and/or  (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino  acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ  ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260  of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides  120  to  833  of  SEQ  ID  NO:  32;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.  Transmembrane Domain  In  some  embodiments,  an  RNA  described  herein  comprises  a  sequence  that  encodes  a  membrane association element (e.g., a heterologous membrane association element), such as  a  transmembrane  domain.    A  transmembrane  domain  can  be  N‐terminal,  C‐terminal,  or  internal  to  an  encoded  polypeptide.  A  coding  sequence  of  a  transmembrane  element  is  typically  placed  in  frame  (i.e.,  in  the  same  reading  frame),  5',  3',  or  internal  to  coding  sequences of sequences (e.g., sequences encoding polypeptide(s)) with which it is to be linked.   In some embodiments, a transmembrane domain comprises or is a transmembrane domain  of Hemagglutinin (HA) of Influenza virus, Env of HIV‐1, equine infectious anaemia virus (EIAV),  murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis  virus (VSV), Rabies virus, or a seven transmembrane domain receptor.  According  to  certain  embodiments,  a  transmembrane  domain  is  fused,  either  directly  or  through a linker, e.g., a glycine/serine linker, to a SARS‐CoV‐2 S protein, a variant thereof, or  a fragment thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a  transmembrane domain is fused to a SARS‐CoV‐2 S polypeptide or an immunogenic fragment  thereof  (antigenic peptides or proteins), which may optionally be  fused to a signal peptide  and/or trimerization domain as described above.  Secretory Signals  Such  transmembrane  domains  are  preferably  located  at  the  C‐terminus  of  the  antigenic  peptide or protein, without being limited thereto. Preferably, such transmembrane domains  are  located at the C‐terminus of the trimerization domain,  if present, without being  limited  thereto.  In one embodiment, a  trimerization domain  is present between  the SARS‐CoV‐2 S  protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein, and the  transmembrane domain.   Transmembrane  domains  as defined  herein preferably  allow  the  anchoring  into  a  cellular  membrane of the antigenic peptide or protein as encoded by the RNA.   In  one  embodiment,  the  transmembrane  domain  sequence  as  defined  herein  includes,  without being limited thereto, the transmembrane domain sequence of SARS‐CoV‐2 S protein,  in particular a sequence comprising the amino acid sequence of amino acids 1207 to 1254 of  SEQ ID NO: 1 or a functional variant thereof.   In one embodiment, a transmembrane domain sequence comprises the amino acid sequence  of amino acids 1207 to 1254 of SEQ  ID NO: 1, an amino acid sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids  1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino  acids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254  of  SEQ  ID NO:  1.  In one  embodiment,  a  transmembrane domain  sequence  comprises  the  amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.  In  one  embodiment, RNA  encoding  a  transmembrane  domain  sequence  (i)  comprises  the  nucleotide  sequence  of  nucleotides  3619  to  3762  of  SEQ  ID NO:  2,  8  or  9,  a  nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or a fragment of the  nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or the nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of  SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a  functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the amino acid sequence of amino acids 1207  to 1254 of SEQ  ID NO: 1.  In one  embodiment, RNA encoding a transmembrane domain sequence (i) comprises the nucleotide  sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino  acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO:  1.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:  29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  311  of  SEQ  ID  NO:  29.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 311  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of  SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 986 of SEQ ID  NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311  of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  311  of  SEQ  ID  NO:  29.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to  314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or  an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO:  31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  314  of  SEQ  ID  NO:  31.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 314  of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 54 to 995 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 995 of  SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 995 of SEQ ID  NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii)  encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314  of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or an  immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31,  or  the amino acid  sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the  amino  acid  sequence  of  amino  acids  1  to  314  of  SEQ  ID  NO:  31.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  (i)  comprises  the  nucleotide  sequence  of  nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20  to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:  29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ  ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29. In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 311  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 111 to 986 of SEQ  ID NO: 30, a nucleotide sequence having at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to  986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 986 of  SEQ  ID NO: 30, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30;  and/or  (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino  acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ  ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311  of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides  111  to  986  of  SEQ  ID  NO:  30;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.  In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23  to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO:  31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314 of SEQ  ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31. In one  embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 314  of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides 120 to 995 of SEQ  ID NO: 32, a nucleotide sequence having at  least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to  995 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 995 of  SEQ  ID NO: 32, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32;  and/or  (ii) encodes an amino acid sequence comprising  the amino acid sequence of amino  acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ  ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314  of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  nucleotides  120  to  995  of  SEQ  ID  NO:  32;  and/or  (ii)  encodes  an  amino  acid  sequence  comprising the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the nucleotide sequence of SEQ  ID NO: 30, or a fragment of the nucleotide  sequence of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80%  identity  to  the nucleotide  sequence of SEQ  ID NO: 30; and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ ID NO: 29, or an  immunogenic fragment of the amino acid  sequence of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  29.  In  one  embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ  ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence  of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the nucleotide sequence of SEQ  ID NO: 32, or a fragment of the nucleotide  sequence of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80%  identity  to  the nucleotide  sequence of SEQ  ID NO: 32; and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ ID NO: 31, or an  immunogenic fragment of the amino acid  sequence of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  31.  In  one  embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ  ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence  of SEQ ID NO: 31.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28,  an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to the amino acid sequence of SEQ ID NO: 28, or an immunogenic fragment of the amino acid  sequence of SEQ ID NO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  28.  In  one  embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 27, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the nucleotide sequence of SEQ  ID NO: 27, or a fragment of the nucleotide  sequence of SEQ ID NO: 27, or the nucleotide sequence having at least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80%  identity  to  the nucleotide  sequence of SEQ  ID NO: 27; and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28, an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of SEQ ID NO: 28, or an  immunogenic fragment of the amino acid  sequence of SEQ ID NO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  28.  In  one  embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ  ID NO: 27; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence  of SEQ ID NO: 28.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 49,  an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the  amino  acid  sequence  of  SEQ  ID NO:  49,  or  an  immunogenic  fragment  of  the  amino  acid  sequence of SEQ ID NO: 49, or the amino acid sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49. In one embodiment,  a  vaccine  antigen  comprises  the  amino  acid  sequence  of  SEQ  ID NO:  49.  The  amino  acid  sequence of SEQ ID NO: 49 corresponds to the amino acid sequence of the full‐length S protein  from Omicron BA.1, which includes proline residues at positions 986 and 987 of SEQ ID NO:  49.   In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  50,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 50, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 49, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 49. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 50; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49. The nucleotide  sequence of  SEQ  ID NO: 50  is  a nucleotide  sequence designed  to  encode  the  amino  acid  sequence of the full‐length S protein from Omicron BA.1 with proline residues at positions 986  and 987 of SEQ ID NO: 49.   In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 51, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  51,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 51, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 49, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 49, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 49. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 51; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 49. The nucleotide  sequence of SEQ ID NO: 51 corresponds to an RNA construct (e.g., comprising a 5’ UTR, a S‐ protein‐encoding  sequence,  a  3’  UTR,  and  a  poly‐A  tail),  which  encodes  the  amino  acid  sequence of the full‐length S protein from Omicron BA.1 with proline residues at positions 986  and 987 of SEQ ID NO: 49.    In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 55,  an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the  amino  acid  sequence  of  SEQ  ID NO:  55,  or  an  immunogenic  fragment  of  the  amino  acid  sequence of SEQ ID NO: 55, or the amino acid sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55. In one embodiment,  a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 55.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 56, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  56,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 56, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 56; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 55, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 55. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 56; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 57, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  57,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 57, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 55, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 55, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 55. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 57; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 55.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 58,  an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the  amino  acid  sequence  of  SEQ  ID NO:  58,  or  an  immunogenic  fragment  of  the  amino  acid  sequence of SEQ ID NO: 58, or the amino acid sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58. In one embodiment,  a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 58.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 59, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  59,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 59, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 59; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 58, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 58, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 58. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 59; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 60, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  60,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 60, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 58, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 58, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 58. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 60; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 58.  In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 61,  an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the  amino  acid  sequence  of  SEQ  ID NO:  61,  or  an  immunogenic  fragment  of  the  amino  acid  sequence of SEQ ID NO: 61, or the amino acid sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61. In one embodiment,  a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 61.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 62, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  62,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 62, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 62; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 61, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 61, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 61. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 62; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 63, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  63,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 63, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 63; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 61, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 61, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 61. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 63; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 61.    In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 52,  an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the  amino  acid  sequence  of  SEQ  ID NO:  52,  or  an  immunogenic  fragment  of  the  amino  acid  sequence of SEQ ID NO: 52, or the amino acid sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52. In one embodiment,  a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 52.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 53, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  53,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 53, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 52, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 52. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 53; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  54,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 54, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 52, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 52, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 52. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 54; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 52.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 83, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  83,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 83, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 83; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 80, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 80, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 80. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 83; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 80.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 103, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID NO:  103,  or  a  fragment  of  the  nucleotide  sequence of SEQ ID NO: 103, or the nucleotide sequence having at least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 103; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ  ID NO: 100, an amino  acid sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino  acid sequence of SEQ ID NO: 100, or an immunogenic fragment of the amino acid sequence of  SEQ ID NO: 100, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or  97%    identity  to  the  amino  acid  sequence  of  SEQ  ID NO:  100.  In  one  embodiment,  RNA  encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 103; and/or  (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 100.  In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of  SEQ ID NO: 98, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity  to  the  nucleotide  sequence  of  SEQ  ID  NO:  98,  or  a  fragment  of  the  nucleotide  sequence of SEQ  ID NO: 98, or the nucleotide sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the nucleotide sequence of SEQ ID NO: 98; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 95, an amino acid  sequence having at  least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%  identity to the amino acid  sequence of SEQ ID NO: 95, or an immunogenic fragment of the amino acid sequence of SEQ  ID NO: 95, or the amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97%   identity to the amino acid sequence of SEQ ID NO: 95. In one embodiment, RNA encoding a  vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 98; and/or (ii) encodes  an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 95.    In one embodiment, the vaccine antigens described above comprise a contiguous sequence  of SARS‐CoV‐2 coronavirus spike (S) protein that consists of or essentially consists of the above  described  amino  acid  sequences  derived  from  SARS‐CoV‐2  S  protein  or  immunogenic  fragments  thereof  (antigenic  peptides  or  proteins)  comprised  by  the  vaccine  antigens  described  above.  In  one  embodiment,  the  vaccine  antigens  described  above  comprise  a  contiguous sequence of SARS‐CoV‐2 coronavirus spike (S) protein of no more than 220 amino  acids, 215 amino acids, 210 amino acids, or 205 amino acids.   In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2), or  BNT162b3  (e.g.,  BNT162b3c).  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA)  described  herein  as  RBP020.2.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA) described herein as BNT162b3 (e.g., BNT162b3c).  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprising the amino  acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  5.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ  ID NO: 19, or 20, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ  ID NO: 19, or 20, and/or  (ii) encodes an amino acid sequence  comprising the amino acid sequence of SEQ  ID NO: 7, or an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ  ID  NO:  7.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20;  and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ  ID  NO: 7.   In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20, a nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprising the amino  acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%,  95%,  90%,  85%,  or  80%  identity  to  the  amino  acid  sequence  of  SEQ  ID  NO:  7.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising the amino  acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 99%, 98%, 97%,  96%, 95%, 90%, 85%, or 80%  identity to the amino acid sequence of SEQ  ID NO: 29.  In one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 50, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  50,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 49, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  50;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 49.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 51, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  51,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 49, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 49. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  51;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 49.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 57, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  57,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 55, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 55. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  57;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 55.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 60, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  60,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 58, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 58. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  60;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 58.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 63, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  63,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 61, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 61. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  63;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 61.    In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 53, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  53,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 52, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  53;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 52.  In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 54, a nucleotide sequence  having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence of  SEQ  ID  NO:  54,  and/or  (ii)  encodes  an  amino  acid  sequence  comprising  the  amino  acid  sequence of SEQ  ID NO: 52, or an amino acid sequence having at  least 99.5%, 99%, 98.5%,  98%, 98.5% or 97% identity to the amino acid sequence of SEQ ID NO: 52. In one embodiment,  RNA encoding  a  vaccine antigen  is nucleoside modified messenger RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  54;  and/or  (ii)  encodes  an  amino  acid  sequence comprising the amino acid sequence of SEQ ID NO: 52.  As used herein, the term "vaccine" refers to a composition that induces an immune response  upon  inoculation  into  a  subject.  In  some  embodiments,  the  induced  immune  response  provides protective immunity.  In one embodiment, the RNA encoding the antigen molecule is expressed in cells of the subject  to provide the antigen molecule. In one embodiment, expression of the antigen molecule is at  the cell surface or  into the extracellular space. In one embodiment, the antigen molecule is  presented in the context of MHC. In one embodiment, the RNA encoding the antigen molecule  is transiently expressed in cells of the subject. In one embodiment, after administration of the  RNA encoding the antigen molecule,  in particular after  intramuscular administration of  the  RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in  muscle occurs.  In one embodiment, after administration of  the RNA encoding  the antigen  molecule,  expression  of  the RNA  encoding  the  antigen molecule  in  spleen  occurs.  In  one  embodiment, after administration of the RNA encoding the antigen molecule, expression of  the RNA encoding the antigen molecule  in antigen presenting cells, preferably professional  antigen presenting cells occurs. In one embodiment, the antigen presenting cells are selected  from the group consisting of dendritic cells, macrophages and B cells.  In one embodiment,  after administration of the RNA encoding the antigen molecule, no or essentially no expression  of the RNA encoding the antigen molecule  in  lung and/or  liver occurs.  In one embodiment,  after  administration  of  the  RNA  encoding  the  antigen  molecule,  expression  of  the  RNA  encoding the antigen molecule in spleen is at least 5‐fold the amount of expression in lung.   In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following  administration,  in particular  following  intramuscular  administration,  to  a  subject  result  in delivery of  the RNA encoding a vaccine antigen  to  lymph nodes and/or spleen.  In  some embodiments, RNA encoding a vaccine antigen  is detectable  in  lymph nodes and/or  spleen 6 hours or later following administration and preferably up to 6 days or longer.   In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following  administration,  in particular  following  intramuscular  administration,  to  a  subject  result in delivery of the RNA encoding a vaccine antigen to B cell follicles, subcapsular sinus,  and/or T cell zone, in particular B cell follicles and/or subcapsular sinus of lymph nodes.   In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following  administration,  in particular  following  intramuscular  administration,  to  a  subject  result in delivery of the RNA encoding a vaccine antigen to B cells (CD19+), subcapsular sinus  macrophages  (CD169+) and/or dendritic cells  (CD11c+)  in  the T cell zone and  intermediary  sinus of lymph nodes, in particular to B cells (CD19+) and/or subcapsular sinus macrophages  (CD169+) of lymph nodes.  In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following  administration,  in particular  following  intramuscular  administration,  to  a  subject  result in delivery of the RNA encoding a vaccine antigen to white pulp of spleen.   In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following  administration,  in particular  following  intramuscular  administration,  to  a  subject  result in delivery of the RNA encoding a vaccine antigen to B cells, DCs (CD11c+), in particular  those surrounding the B cells, and/or macrophages of spleen,  in particular to B cells and/or  DCs (CD11c+).  In  one  embodiment,  the  vaccine  antigen  is  expressed  in  lymph  node  and/or  spleen,  in  particular in the cells of lymph node and/or spleen described above.  The peptide and protein antigens suitable for use according to the disclosure typically include  a peptide or protein comprising an epitope of SARS‐CoV‐2 S protein or a  functional variant  thereof for inducing an immune response. The peptide or protein or epitope may be derived  from a target antigen, i.e. the antigen against which an immune response is to be elicited. For  example,  the  peptide  or  protein  antigen  or  the  epitope  contained within  the  peptide  or  protein antigen may be a target antigen or a fragment or variant of a target antigen. The target  antigen may be a coronavirus S protein, in particular SARS‐CoV‐2 S protein.  The antigen molecule or a procession product thereof, e.g., a fragment thereof, may bind to  an antigen receptor such as a BCR or TCR carried by immune effector cells, or to antibodies.   A  peptide  and  protein  antigen  which  is  provided  to  a  subject  according  to  the  present  disclosure by  administering RNA encoding  the peptide  and protein  antigen,  i.e., a vaccine  antigen, preferably results  in the  induction of an  immune response, e.g., a humoral and/or  cellular immune response in the subject being provided the peptide or protein antigen. Said  immune response  is preferably directed against a target antigen, in particular coronavirus S  protein, in particular SARS‐CoV‐2 S protein. Thus, a vaccine antigen may comprise the target  antigen,  a  variant  thereof,  or  a  fragment  thereof.  In  one  embodiment,  such  fragment  or  variant  is  immunologically  equivalent  to  the  target  antigen.  In  the  context of  the present  disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which  results in the induction of an immune response which immune response targets the antigen,  i.e. a target antigen. Thus, the vaccine antigen may correspond to or may comprise the target  antigen, may  correspond  to  or may  comprise  a  fragment  of  the  target  antigen  or may  correspond to or may comprise an antigen which  is homologous to the target antigen or a  fragment  thereof.  Thus,  according  to  the  disclosure,  a  vaccine  antigen may  comprise  an  immunogenic fragment of a target antigen or an amino acid sequence being homologous to  an  immunogenic  fragment of a  target antigen. An  "immunogenic  fragment of an antigen"  according to the disclosure preferably relates to a fragment of an antigen which is capable of  inducing  an  immune  response  against  the  target  antigen.  The  vaccine  antigen may  be  a  recombinant antigen.  The term "immunologically equivalent" means that the immunologically equivalent molecule  such as the immunologically equivalent amino acid sequence exhibits the same or essentially  the  same  immunological  properties  and/or  exerts  the  same  or  essentially  the  same  immunological  effects,  e.g., with  respect  to  the  type  of  the  immunological  effect.  In  the  context of the present disclosure, the term "immunologically equivalent"  is preferably used  with respect to the immunological effects or properties of antigens or antigen variants used  for  immunization. For example, an amino acid sequence  is  immunologically equivalent to a  reference amino acid sequence  if said amino acid sequence when exposed  to  the  immune  system of  a  subject  induces  an  immune  reaction having  a  specificity  of  reacting with  the  reference amino acid sequence.   "Activation" or "stimulation", as used herein, refers to the state of an  immune effector cell  such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation.  Activation  can  also  be  associated with  initiation  of  signaling  pathways,  induced  cytokine  production, and detectable effector  functions. The  term  "activated  immune effector  cells"  refers to, among other things, immune effector cells that are undergoing cell division.   The term "priming" refers to a process wherein an immune effector cell such as a T cell has its  first  contact with  its  specific  antigen  and  causes differentiation  into  effector  cells  such  as  effector T cells.  The term "clonal expansion" or "expansion" refers to a process wherein a specific entity  is  multiplied. In the context of the present disclosure, the term is preferably used in the context  of an  immunological response in which  immune effector cells are stimulated by an antigen,  proliferate,  and  the  specific  immune  effector  cell  recognizing  said  antigen  is  amplified.  Preferably, clonal expansion leads to differentiation of the immune effector cells.   The  term  "antigen"  relates  to  an  agent  comprising  an  epitope  against which  an  immune  response can be generated. The term "antigen" includes, in particular, proteins and peptides.  In one embodiment, an antigen is presented by cells of the immune system such as antigen  presenting  cells  like  dendritic  cells  or macrophages.  An  antigen  or  a  procession  product  thereof such as a T‐cell epitope is in one embodiment bound by a T‐ or B‐cell receptor, or by  an  immunoglobulin molecule such as an antibody. Accordingly, an antigen or a procession  product  thereof may  react  specifically  with  antibodies  or  T lymphocytes  (T cells).  In  one  embodiment, an antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS‐CoV‐2 S  protein, and an epitope is derived from such antigen.  The term "viral antigen" refers to any viral component having antigenic properties, i.e. being  able to provoke an immune response in an individual. The viral antigen may be coronavirus S  protein, e.g., SARS‐CoV‐2 S protein.  The term "expressed on the cell surface" or "associated with the cell surface" means that a  molecule such as an antigen is associated with and located at the plasma membrane of a cell,  wherein  at  least  a  part  of  the molecule  faces  the  extracellular  space  of  said  cell  and  is  accessible  from  the outside of said cell, e.g., by antibodies  located outside  the cell.  In  this  context,  a part  is preferably  at  least 4, preferably  at  least 8, preferably  at  least 12, more  preferably at least 20 amino acids. The association may be direct or indirect. For example, the  association may be by one or more transmembrane domains, one or more lipid anchors, or by  the interaction with any other protein, lipid, saccharide, or other structure that can be found  on the outer  leaflet of the plasma membrane of a cell. For example, a molecule associated  with the surface of a cell may be a transmembrane protein having an extracellular portion or  may be a protein associated with the surface of a cell by interacting with another protein that  is a transmembrane protein.     "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art,  and thus includes the outside of the cell which is accessible to binding by proteins and other  molecules. An antigen is expressed on the surface of cells if it is located at the surface of said  cells and is accessible to binding by e.g. antigen‐specific antibodies added to the cells.   The term "extracellular portion" or "exodomain" in the context of the present disclosure refers  to a part of a molecule such as a protein that  is facing the extracellular space of a cell and  preferably  is  accessible  from  the  outside  of  said  cell,  e.g.,  by  binding molecules  such  as  antibodies  located outside the cell. Preferably, the term refers to one or more extracellular  loops or domains or a fragment thereof.  The  term  "epitope"  refers  to a part or  fragment of a molecule  such as  an  antigen  that  is  recognized by the immune system. For example, the epitope may be recognized by T cells, B  cells  or  antibodies.  An  epitope  of  an  antigen may  include  a  continuous  or  discontinuous  portion of the antigen and may be between about 5 and about 100, such as between about 5  and  about 50, more preferably between  about 8 and about 30, most preferably between  about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10,  11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25  amino  acids  in  length.  In one  embodiment, an epitope is between about 10 and about 25 amino acids in length. The term  "epitope" includes T cell epitopes.   The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell  when  presented  in  the  context  of  MHC  molecules.  The  term  "major  histocompatibility  complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and  relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules  are important for signaling between lymphocytes and antigen presenting cells or diseased cells  in  immune  reactions, wherein  the MHC  proteins  or molecules  bind  peptide  epitopes  and  present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC  are expressed on the surface of cells, and display both self‐antigens (peptide fragments from  the cell itself) and non‐self‐antigens (e.g., fragments of invading microorganisms) to a T cell.  In the case of class  I MHC/peptide complexes, the binding peptides are typically about 8 to  about 10 amino acids long although longer or shorter peptides may be effective. In the case  of class  II MHC/peptide complexes, the binding peptides are typically about 10 to about 25  amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer  and shorter peptides may be effective.  The peptide and protein antigen can be 2‐100 amino acids,  including  for example, 5 amino  acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35  amino  acids,  40  amino  acids,  45  amino  acids,  or  50  amino  acids  in  length.  In  some  embodiments,  a peptide  can be  greater  than 50  amino  acids.  In  some  embodiments,  the  peptide can be greater than 100 amino acids.   The peptide or protein antigen can be any peptide or protein that can induce or increase the  ability of  the  immune system to develop antibodies and T cell responses to the peptide or  protein.   In one embodiment, vaccine antigen is recognized by an immune effector cell. Preferably, the  vaccine antigen if recognized by an immune effector cell is able to induce in the presence of  appropriate  co‐stimulatory  signals,  stimulation,  priming  and/or  expansion  of  the  immune  effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the  embodiments of the present disclosure, the vaccine antigen is preferably presented or present  on the surface of a cell, preferably an antigen presenting cell. In one embodiment, an antigen  is presented by a diseased cell such as a virus‐infected cell. In one embodiment, an antigen  receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In  one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an  antigen presented by  cells  such  as  antigen presenting  cells  results  in  stimulation, priming  and/or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T  cells and/or present on T cells to an antigen presented on diseased cells results  in cytolysis  and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors,  e.g. perforins and granzymes.   In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an  epitope  in an antigen.  In one embodiment, an antibody or B  cell  receptor binds  to native  epitopes of an antigen.   Bivalent Vaccine Combinations   Multiple various spike protein (S) of SARS‐CoV‐2 variants as described herein may be delivered  in combination, for example by a bivalent RNA vaccine comprising at least one RNA encoding  two or more spike proteins (S) or any variants thereof (e.g., as described herein).  Exemplary  combinations of spike proteins are described herein and shown, e.g., in Tables below.  Bivalent  vaccines may include any of these described combinations in either spike protein encoded by  the RNA vaccine.  Additionally, mutations described herein (e.g., in Tables 2A, 2B, and 2C) may  be included in any of the various coronavirus strains described herein, and additionally, any  additional known coronavirus strains (see e.g., the World Health Organization data base for  tracking  of  SARS‐CoV‐2  variants  a
Figure imgf000176_0001
variants).    Exemplary spike protein variants  (where mutations described herein are applied to various  strains of coronavirus spike protein sequences are shown in Table 7 below.   Table 7 
Figure imgf000176_0002
Figure imgf000177_0001
Figure imgf000178_0001
According to the present disclosure, in some embodiments, an RNA vaccine comprises at least  one  RNA  encoding  one  or more  coronavirus  spike  proteins  (e.g.,  a  spike  protein  variant  described in Table 7). In some embodiments, an RNA vaccine comprises at least two RNA each  encoding a distinct coronavirus spike protein (e.g., a spike protein variant described in Table  7).  Coronavius  spike  protein  antigens may  be  administered  as  single‐stranded,  5'  capped  mRNA  that  is  translated  into  the  respective protein upon entering  cells of a  subject being  administered  the  RNA.  Preferably,  the  RNA  contains  structural  elements  optimized  for  maximal efficacy of  the RNA with respect to stability and translational efficiency  (5' cap, 5'  UTR, 3' UTR, poly(A) sequence).   In one embodiment, beta‐S‐ARCA(D1) is utilized as specific capping structure at the 5'‐end of  the  RNA.  In  one  embodiment, m27,3’‐OGppp(m12’‐O)ApG  is  utilized  as  specific  capping  structure at the 5'‐end of the RNA. In one embodiment, the 5'‐UTR sequence is derived from  the human alpha‐globin mRNA and optionally has an optimized ʻKozak sequenceʼ to increase  translational  efficiency.  In one embodiment,  a  combination of  two  sequence elements  (FI  element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the  mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence  and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence  of  the mRNA.  In one embodiment,  two  re‐iterated 3'‐UTRs derived  from  the human beta‐ globin mRNA are placed between the coding sequence and the poly(A) sequence to assure  higher maximum protein levels and prolonged persistence of the mRNA. In one embodiment,  a  poly(A)  sequence  measuring  110  nucleotides  in  length,  consisting  of  a  stretch  of  30  adenosine residues, followed by a 10 nucleotide  linker sequence and another 70 adenosine  residues  is  used.  This  poly(A)  sequence  was  designed  to  enhance  RNA  stability  and  translational efficiency.    RNA vaccines encoding any of the coronavirus spike protein variants described herein  (and  e.g.,  in Table 7) may  include any of  the other nucleic acid modification and RNA construct  components described herein.  In some embodiments, RNA moelcules may be formulated in the lipid nanoparticles (LNPs) to  form a bivalent vaccine (e.g., two populations of RNAs are mixed prior to LNP formulation; or  each RNA is formulated in a separate LNP composition, followed by mixing together).  Combinations of exemplary spike protein variants described herein (e.g., as shown in Table 7)  may be utilized in a bivalent RNA vaccine.  Exemplary combinations of spike proteins that can  be utilized in a bivalent RNA vaccine are shown in Table 8 below.  
Figure imgf000180_0001
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Figure imgf000210_0001
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Nucleic acids  The term "polynucleotide" or "nucleic acid", as used herein, is intended to include DNA and  RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized  molecules. A nucleic acid may be single‐stranded or double‐stranded. RNA  includes  in vitro  transcribed  RNA  (IVT  RNA)  or  synthetic  RNA.  According  to  the  present  disclosure,  a  polynucleotide is preferably isolated.  Nucleic acids may be comprised  in a vector. The term "vector" as used herein  includes any  vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors  such as  lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or  artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial  chromosomes (YAC), or P1 artificial chromosomes (PAC). Said vectors  include expression as  well  as  cloning  vectors.  Expression  vectors  comprise plasmids  as well  as  viral  vectors  and  generally contain a desired coding sequence and appropriate DNA sequences necessary for  the expression of  the operably  linked  coding  sequence  in a particular host organism  (e.g.,  bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are  generally  used  to  engineer  and  amplify  a  certain  desired  DNA  fragment  and  may  lack  functional sequences needed for expression of the desired DNA fragments.  In one embodiment of all aspects of  the present disclosure,  the RNA encoding  the vaccine  antigen is expressed in cells such as antigen presenting cells of the subject treated to provide  the vaccine antigen.   The nucleic acids described herein may be recombinant and/or isolated molecules.  In the present disclosure, the term "RNA" relates to a nucleic acid molecule which  includes  ribonucleotide  residues.  In preferred  embodiments,  the RNA  contains  all or  a majority of  ribonucleotide  residues.  As  used  herein,  "ribonucleotide"  refers  to  a  nucleotide  with  a  hydroxyl group at  the 2'‐position of a  β‐D‐ribofuranosyl group. RNA encompasses without  limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified  RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified  RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or  alteration  of  one  or  more  nucleotides.  Such  alterations  may  refer  to  addition  of  non‐ nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated  herein  that  nucleotides  in  RNA  may  be  non‐standard  nucleotides,  such  as  chemically  synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs  are considered analogs of naturally‐occurring RNA.   In certain embodiments of the present disclosure, the RNA  is messenger RNA  (mRNA) that  relates to a RNA transcript which encodes a peptide or protein. As established in the art, mRNA  generally  contains  a  5'  untranslated  region  (5'‐UTR),  a  peptide  coding  region  and  a  3'  untranslated  region  (3'‐UTR).  In  some  embodiments,  the  RNA  is  produced  by  in  vitro  transcription or chemical synthesis.  In one embodiment,  the mRNA  is produced by  in vitro  transcription  using  a  DNA  template  where  DNA  refers  to  a  nucleic  acid  that  contains  deoxyribonucleotides.   In one embodiment, RNA is in vitro transcribed RNA (IVT‐RNA) and may be obtained by in vitro  transcription of an appropriate DNA template. The promoter for controlling transcription can  be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be  obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate  vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.  In  certain  embodiments of  the present  disclosure,  the RNA  is  "replicon RNA"  or  simply  a  "replicon",  in particular  "self‐replicating RNA" or  "self‐amplifying RNA".  In one particularly  preferred  embodiment,  the  replicon  or  self‐replicating  RNA  is  derived  from  or  comprises  elements derived from a ssRNA virus, in particular a positive‐stranded ssRNA virus such as an  alphavirus.  Alphaviruses  are  typical  representatives  of  positive‐stranded  RNA  viruses.  Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle  see José et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many  alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA  typically has a 5’‐cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non‐structural  proteins  (involved  in transcription, modification and replication of viral RNA and  in protein  modification) and structural proteins (forming the virus particle). There are typically two open  reading  frames  (ORFs)  in  the  genome.  The  four  non‐structural  proteins  (nsP1–nsP4)  are  typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while  alphavirus  structural  proteins  are  encoded  together  by  a  second  ORF  which  is  found  downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the  first ORF  is  larger  than  the second ORF,  the ratio being roughly 2:1.  In cells  infected by an  alphavirus, only the nucleic acid sequence encoding non‐structural proteins is translated from  the genomic RNA, while the genetic information encoding structural proteins is translatable  from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger  RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124). Following infection, i.e. at  early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger  RNA for the translation of the open reading frame encoding the non‐structural poly‐protein  (nsP1234). Alphavirus‐derived  vectors  have  been  proposed  for  delivery  of  foreign  genetic  information  into  target  cells or  target organisms.  In  simple  approaches,  the open  reading  frame encoding alphaviral structural proteins is replaced by an open reading frame encoding  a protein of interest. Alphavirus‐based trans‐replication systems rely on alphavirus nucleotide  sequence  elements  on  two  separate  nucleic  acid  molecules:  one  nucleic  acid  molecule  encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by  said  replicase  in  trans  (hence  the  designation  trans‐replication  system).  Trans‐replication  requires the presence of both these nucleic acid molecules  in a given host cell. The nucleic  acid molecule  capable of being  replicated by  the  replicase  in  trans must  comprise  certain  alphaviral  sequence  elements  to  allow  recognition  and  RNA  synthesis  by  the  alphaviral  replicase.  In  one  embodiment,  the  RNA  described  herein may  have modified  nucleosides.  In  some  embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every)  uridine.  The  term  "uracil," as used herein, describes one of  the nucleobases  that  can occur  in  the  nucleic acid of RNA. The structure of uracil is: 
Figure imgf000217_0001
The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA.  The structure of uridine is:  
Figure imgf000218_0004
UTP (uridine 5’‐triphosphate) has the following structure: 
Figure imgf000218_0001
Pseudo‐UTP (pseudouridine 5’‐triphosphate) has the following structure: 
Figure imgf000218_0002
"Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where  the uracil  is attached  to  the pentose  ring via a carbon‐carbon bond  instead of a nitrogen‐ carbon glycosidic bond.   Another exemplary modified nucleoside  is N1‐methyl‐pseudouridine  (m1Ψ), which has  the  structure: 
Figure imgf000218_0003
N1‐methyl‐pseudo‐UTP has the following structure: 
Figure imgf000219_0002
Another exemplary modified nucleoside is 5‐methyl‐uridine (m5U), which has the structure: 
Figure imgf000219_0001
In  some embodiments, one or more uridine  in  the RNA described herein  is  replaced by a  modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.  In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine.  In some embodiments, RNA comprises a modified nucleoside in place of each uridine.   In some embodiments, the modified nucleoside is independently selected from pseudouridine  (ψ), N1‐methyl‐pseudouridine  (m1ψ), and 5‐methyl‐uridine  (m5U).  In  some embodiments,  the modified nucleoside comprises pseudouridine (ψ).  In some embodiments, the modified  nucleoside comprises N1‐methyl‐pseudouridine (m1ψ). In some embodiments, the modified  nucleoside  comprises  5‐methyl‐uridine  (m5U).  In  some  embodiments,  RNA may  comprise  more than one type of modified nucleoside, and the modified nucleosides are independently  selected  from  pseudouridine  (ψ),  N1‐methyl‐pseudouridine  (m1ψ),  and  5‐methyl‐uridine  (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1‐ methyl‐pseudouridine  (m1ψ).  In  some  embodiments,  the modified  nucleosides  comprise  pseudouridine  (ψ)  and  5‐methyl‐uridine  (m5U).  In  some  embodiments,  the  modified  nucleosides comprise N1‐methyl‐pseudouridine (m1ψ) and 5‐methyl‐uridine (m5U). In some  embodiments,  the  modified  nucleosides  comprise  pseudouridine  (ψ),  N1‐methyl‐ pseudouridine (m1ψ), and 5‐methyl‐uridine (m5U).  In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the  RNA may be any one or more of 3‐methyl‐uridine  (m3U), 5‐methoxy‐uridine  (mo5U), 5‐aza‐ uridine, 6‐aza‐uridine, 2‐thio‐5‐aza‐uridine, 2‐thio‐uridine  (s2U), 4‐thio‐uridine  (s4U), 4‐thio‐ pseudouridine, 2‐thio‐pseudouridine, 5‐hydroxy‐uridine (ho5U), 5‐aminoallyl‐uridine, 5‐halo‐ uridine (e.g., 5‐iodo‐uridine or 5‐bromo‐uridine), uridine 5‐oxyacetic acid (cmo5U), uridine 5‐ oxyacetic  acid methyl  ester  (mcmo5U),  5‐carboxymethyl‐uridine  (cm5U),  1‐carboxymethyl‐ pseudouridine,  5‐carboxyhydroxymethyl‐uridine  (chm5U),  5‐carboxyhydroxymethyl‐uridine  methyl  ester  (mchm5U),  5‐methoxycarbonylmethyl‐uridine  (mcm5U),  5‐ methoxycarbonylmethyl‐2‐thio‐uridine  (mcm5s2U),  5‐aminomethyl‐2‐thio‐uridine  (nm5s2U),  5‐methylaminomethyl‐uridine  (mnm5U),  1‐ethyl‐pseudouridine,  5‐methylaminomethyl‐2‐ thio‐uridine  (mnm5s2U),  5‐methylaminomethyl‐2‐seleno‐uridine  (mnm5se2U),  5‐ carbamoylmethyl‐uridine  (ncm5U),  5‐carboxymethylaminomethyl‐uridine  (cmnm5U),  5‐ carboxymethylaminomethyl‐2‐thio‐uridine  (cmnm5s2U),  5‐propynyl‐uridine,  1‐propynyl‐ pseudouridine,  5‐taurinomethyl‐uridine  (τm5U),  1‐taurinomethyl‐pseudouridine,  5‐ taurinomethyl‐2‐thio‐uridine(τm5s2U),  1‐taurinomethyl‐4‐thio‐pseudouridine),  5‐methyl‐2‐ thio‐uridine (m5s2U), 1‐methyl‐4‐thio‐pseudouridine (m1s4ψ), 4‐thio‐1‐methyl‐pseudouridine,  3‐methyl‐pseudouridine  (m3ψ),  2‐thio‐1‐methyl‐pseudouridine,  1‐methyl‐1‐deaza‐ pseudouridine,  2‐thio‐1‐methyl‐1‐deaza‐pseudouridine,  dihydrouridine  (D),  dihydropseudouridine,  5,6‐dihydrouridine,  5‐methyl‐dihydrouridine  (m5D),  2‐thio‐ dihydrouridine,  2‐thio‐dihydropseudouridine,  2‐methoxy‐uridine,  2‐methoxy‐4‐thio‐uridine,  4‐methoxy‐pseudouridine, 4‐methoxy‐2‐thio‐pseudouridine, N1‐methyl‐pseudouridine, 3‐(3‐ amino‐3‐carboxypropyl)uridine (acp3U), 1‐methyl‐3‐(3‐amino‐3‐carboxypropyl)pseudouridine  (acp3  ψ),  5‐(isopentenylaminomethyl)uridine  (inm5U),  5‐(isopentenylaminomethyl)‐2‐thio‐ uridine (inm5s2U), α‐thio‐uridine, 2′‐O‐methyl‐uridine (Um), 5,2′‐O‐dimethyl‐uridine (m5Um),  2′‐O‐methyl‐pseudouridine  (ψm),  2‐thio‐2′‐O‐methyl‐uridine  (s2Um),  5‐ methoxycarbonylmethyl‐2′‐O‐methyl‐uridine  (mcm5Um),  5‐carbamoylmethyl‐2′‐O‐methyl‐ uridine  (ncm5Um),  5‐carboxymethylaminomethyl‐2′‐O‐methyl‐uridine  (cmnm5Um),  3,2′‐O‐ dimethyl‐uridine (m3Um), 5‐(isopentenylaminomethyl)‐2′‐O‐methyl‐uridine (inm5Um), 1‐thio‐ uridine,  deoxythymidine,  2′‐F‐ara‐uridine,  2′‐F‐uridine,  2′‐OH‐ara‐uridine,  5‐(2‐ carbomethoxyvinyl) uridine, 5‐[3‐(1‐E‐propenylamino)uridine, or any other modified uridine  known in the art.  In  one  embodiment,  the RNA  comprises  other modified  nucleosides or  comprises  further  modified nucleosides, e.g., modified cytidine. For example, in one embodiment, in the RNA 5‐ methylcytidine  is substituted partially or completely, preferably completely,  for cytidine.  In  one  embodiment,  the  RNA  comprises  5‐methylcytidine  and  one  or  more  selected  from  pseudouridine  (ψ),  N1‐methyl‐pseudouridine  (m1ψ),  and  5‐methyl‐uridine  (m5U).  In  one  embodiment, the RNA comprises 5‐methylcytidine and N1‐methyl‐pseudouridine  (m1ψ).  In  some embodiments, the RNA comprises 5‐methylcytidine  in place of each cytidine and N1‐ methyl‐pseudouridine (m1ψ) in place of each uridine.  In some embodiments, the RNA according to the present disclosure comprises a 5’‐cap. In one  embodiment, the RNA of the present disclosure does not have uncapped 5'‐triphosphates. In  one embodiment, the RNA may be modified by a 5'‐ cap analog. The term "5'‐cap" refers to a  structure  found on the 5'‐end of an RNA  (e.g., mRNA) molecule and generally consists of a  guanosine nucleotide connected to the RNA (e.g., mRNA) via a 5'‐ to 5'‐triphosphate linkage.  In one embodiment, this guanosine is methylated at the 7‐position. Providing an RNA with a  5'‐cap or 5'‐cap analog may be achieved by  in vitro transcription,  in which the 5'‐cap  is co‐ transcriptionally  expressed  into  the  RNA  strand,  or  may  be  attached  to  RNA  post‐ transcriptionally using capping enzymes.   In some embodiments, RNA (e.g., mRNA) comprises a cap0, cap1, or cap2, preferably cap1 or  cap2, more preferably cap1. According to the present disclosure, the term "cap0" comprises  the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'.  According to the present disclosure, the term "cap1" comprises the structure "m7GpppNm",  wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present  disclosure,  the  term "cap2" comprises  the structure "m7GpppNmNm", wherein each Nm  is  independently any nucleoside bearing an OCH3 moiety at position 2'.  In  some  embodiments,  the  building  block  cap  for  RNA  is  m2 7,3’‐OGppp(m1 2’‐O)ApG  (also  sometimes referred to as m2 7,3`OG(5’)ppp(5’)m2’‐OApG), which has the following structure: 
  
Figure imgf000222_0001
.   Below is an exemplary Cap1 RNA, which comprises RNA and m2 7,3`OG(5’)ppp(5’)m2’‐OApG: 
Figure imgf000222_0002
Below is another exemplary Cap1 RNA (no cap analog):  
Figure imgf000222_0003
.  In some embodiments, the RNA is modified with "Cap0" structures using, in one embodiment,  the cap analog anti‐reverse cap (ARCA Cap (m2 7,3`OG(5’)ppp(5’)G)) with the structure:   
Figure imgf000223_0001
.  Below is an exemplary Cap0 RNA comprising RNA and m2 7,3`OG(5’)ppp(5’)G:   
Figure imgf000223_0002
  In some embodiments, the "Cap0" structures are generated using the cap analog Beta‐S‐ARCA  (m2 7,2`OG(5’)ppSp(5’)G) with the structure: 
Figure imgf000223_0003
Below is an exemplary Cap0 RNA comprising Beta‐S‐ARCA (m2 7,2`OG(5’)ppSp(5’)G) and RNA: 
Figure imgf000224_0001
The "D1" diastereomer of beta‐S‐ARCA or "beta‐S‐ARCA(D1)"  is the diastereomer of beta‐S‐ ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta‐S‐ARCA  (beta‐S‐ARCA(D2)) and thus exhibits a shorter retention time (cf., WO 2011/015347, herein  incorporated by reference).  A particularly preferred cap is beta‐S‐ARCA(D1) (m2 7,2'‐OGppSpG) or m2 7,3’‐OGppp(m1 2’‐O)ApG.  In some embodiments, RNA according to the present disclosure comprises a 5'‐UTR and/or a  3'‐UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which  is  transcribed but  is not  translated  into  an amino  acid  sequence, or  to  the  corresponding  region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be  present 5' (upstream) of an open reading frame (5'‐UTR) and/or 3' (downstream) of an open  reading  frame  (3'‐UTR). A 5'‐UTR,  if present,  is  located at the 5' end, upstream of the start  codon of a protein‐encoding region. A 5'‐UTR  is downstream of the 5'‐cap  (if present), e.g.  directly adjacent to the 5'‐cap. A 3'‐UTR,  if present,  is located at the 3' end, downstream of  the termination codon of a protein‐encoding region, but the term "3'‐UTR" does preferably  not  include the poly(A) sequence. Thus, the 3'‐UTR  is upstream of the poly(A) sequence  (if  present), e.g. directly adjacent to the poly(A) sequence.   In some embodiments, RNA comprises a 5’‐UTR comprising the nucleotide sequence of SEQ  ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of SEQ ID NO: 12.   In some embodiments, RNA comprises a 3’‐UTR comprising the nucleotide sequence of SEQ  ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or  80% identity to the nucleotide sequence of SEQ ID NO: 13.  A  particularly  preferred  5’‐UTR  comprises  the  nucleotide  sequence  of  SEQ  ID  NO:  12.  A  particularly preferred 3’‐UTR comprises the nucleotide sequence of SEQ ID NO: 13.  In some embodiments, the RNA according  to  the present disclosure comprises a 3'‐poly(A)  sequence.   As used herein,  the  term "poly(A) sequence" or "poly‐A  tail"  refers  to an uninterrupted or  interrupted sequence of adenylate residues which is typically located at the 3'‐end of an RNA  molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3’‐UTR  in  the  RNAs  described  herein.  An  uninterrupted  poly(A)  sequence  is  characterized  by  consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs  disclosed herein can have a poly(A) sequence attached  to  the  free 3'‐end of  the RNA by a  template‐independent RNA polymerase after transcription or a poly(A) sequence encoded by  DNA and transcribed by a template‐dependent RNA polymerase.   It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial  influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein  that  is  translated  from an open  reading  frame  that  is present upstream  (5’) of  the poly(A)  sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009‐4017).   The  poly(A)  sequence may  be  of  any  length.  In  some  embodiments,  a  poly(A)  sequence  comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80,  or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and,  in particular, about 120 A nucleotides.  In  this context, "essentially consists of" means  that  most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at  least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of  nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides  are nucleotides other  than A nucleotides,  such as U nucleotides  (uridylate), G nucleotides  (guanylate),  or  C  nucleotides  (cytidylate).  In  this  context,  "consists  of"  means  that  all  nucleotides  in  the  poly(A)  sequence,  i.e.,  100%  by  number  of  nucleotides  in  the  poly(A)  sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.  In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during  preparation of  in vitro  transcribed RNA, based on a DNA  template comprising repeated dT  nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA  sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.  In some embodiments, the poly(A) cassette present  in the coding strand of DNA essentially  consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides  (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides  in  length.  Such  a  cassette  is  disclosed  in WO  2016/005324  A1,  hereby  incorporated  by  reference. Any poly(A)  cassette disclosed  in WO  2016/005324 A1 may  be used  in  certain  enbodiments  of  the  present  disclosure.  A  poly(A)  cassette  that  essentially  consists  of  dA  nucleotides, but is interrupted by a random sequence having an equal distribution of the four  nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA  level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with  the beneficial properties with respect to supporting RNA stability and translational efficiency  is encompassed. Consequently, in some embodiments, the poly(A) sequence contained in an  RNA molecule described herein essentially consists of A nucleotides, but  is interrupted by a  random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50,  10 to 30, or 10 to 20 nucleotides in length.  In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its  3'‐end, i.e., the poly(A) sequence is not masked or followed at its 3'‐end by a nucleotide other  than A.  In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40,  at  least 80, or at  least 100 and up  to 500, up  to 400, up  to 300, up  to 200, or up  to 150  nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least  20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up  to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of  at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to  300,  up  to  200,  or  up  to  150  nucleotides.  In  some  embodiments,  the  poly(A)  sequence  comprises at  least 100 nucleotides.  In some embodiments, the poly(A) sequence comprises  about 150 nucleotides.  In  some embodiments,  the poly(A)  sequence  comprises about 120  nucleotides.    In  some  embodiments,  a  poly(A)  sequence  included  in  an  RNA  described  herein  is  a  interrupted poly(A) sequence, e.g., as described  in WO2016/005324,  the entire content of  which  is  incorporated  herein  by  reference  for  purposes  described  herein.  In  some  embodiments,  a  poly(A)  sequence  comprises  a  stretch  of  at  least  20  adenosine  residues  (including, e.g., at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine  residues),  followed  by  a  linker  sequence  (e.g.,  in  some  embodiments  comprising  non‐A  nucleotides) and another stretch of at least 20 adenosine residues (including, e.g., at least 30,  at  least  40,  at  least  50,  at  least  60,  at  least  70,  or more  adenosine  residues).  In  some  embodiments, such a linker sequence may be 3‐50 nucleotides in length, or 5‐25 nucleotides  in length, or 10‐15 nucleotides in length. In some embodiments, a poly(A) sequence comprises  a stretch of about 30 adenosine residues, followed by a  linker sequence having a  length of  about 5‐15 nucleoties (e.g., in some embodiments comprising non‐A nucleotides) and another  stretch of about 70 adenosine residues.  In  some  embodiments,  RNA  comprises  a  poly(A)  sequence  comprising  the  nucleotide  sequence of SEQ  ID NO: 14, or a nucleotide sequence having at  least 99%, 98%, 97%, 96%,  95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 14.  A particularly preferred poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:  14.  According  to  the disclosure, vaccine antigen  is preferably administered as  single‐stranded,  5'‐capped mRNA that is translated into the respective protein upon entering cells of a subject  being administered the RNA. Preferably, the RNA contains structural elements optimized for  maximal  efficacy  of  the  RNA with  respect  to  stability  and  translational  efficiency  (5'‐cap,  5'‐UTR, 3'‐UTR, poly(A) sequence).   In one embodiment, beta‐S‐ARCA(D1) is utilized as specific capping structure at the 5'‐end of  the RNA. In one embodiment, m2 7,3’‐OGppp(m1 2’‐O)ApG is utilized as specific capping structure  at the 5'‐end of the RNA. In one embodiment, the 5'‐UTR sequence is derived from the human  alpha‐globin mRNA and optionally has an optimized  ʻ Kozak sequenceʼ to increase translaƟonal  efficiency. In one embodiment, a combination of two sequence elements (FI element) derived  from  the  "amino  terminal enhancer of  split"  (AES) mRNA  (called F) and  the mitochondrial  encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A)  sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.  In one embodiment, two re‐iterated 3'‐UTRs derived from the human beta‐globin mRNA are  placed between the coding sequence and the poly(A) sequence to assure higher maximum  protein  levels  and  prolonged  persistence  of  the  mRNA.  In  one  embodiment,  a  poly(A)  sequence  measuring  110  nucleotides  in  length,  consisting  of  a  stretch  of  30  adenosine  residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is  used.  This  poly(A)  sequence  was  designed  to  enhance  RNA  stability  and  translational  efficiency.  In one embodiment of all aspects of the present disclosure, RNA encoding a vaccine antigen is  expressed in cells of the subject treated to provide the vaccine antigen. In one embodiment  of all aspects of the present disclosure, the RNA is transiently expressed in cells of the subject.  In one embodiment of all aspects of the present disclosure, the RNA  is  in vitro transcribed  RNA.  In one embodiment of all aspects of the present disclosure, expression of the vaccine  antigen is at the cell surface. In one embodiment of all aspects of the present disclosure, the  vaccine antigen is expressed and presented in the context of MHC. In one embodiment of all  aspects of the present disclosure, expression of the vaccine antigen  is  into the extracellular  space, i.e., the vaccine antigen is secreted.   In the context of the present disclosure, the term "transcription" relates to a process, wherein  the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be  translated into peptide or protein.   According  to  the  present  disclosure,  the  term  "transcription"  comprises  "in  vitro  transcription", wherein the term "in vitro transcription" relates to a process wherein RNA, in  particular mRNA, is in vitro synthesized in a cell‐free system, preferably using appropriate cell  extracts.  Preferably,  cloning  vectors  are  applied  for  the  generation  of  transcripts.  These  cloning vectors are generally designated as  transcription  vectors and are according  to  the  present disclosure encompassed by the term "vector". According to the present disclosure,  the  RNA  used  in  certain  embodiments  of  the  present  disclosure  preferably  is  in  vitro  transcribed RNA (IVT‐RNA) and may be obtained by  in vitro transcription of an appropriate  DNA template. The promoter for controlling transcription can be any promoter for any RNA  polymerase.  Particular  examples  of  RNA  polymerases  are  the  T7,  T3,  and  SP6  RNA  polymerases.  Preferably,  the  in  vitro  transcription  according  to  the  present  disclosure  is  controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained  by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector  for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.   With  respect  to RNA,  the  term  "expression" or  "translation"  relates  to  the process  in  the  ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino  acids to make a peptide or protein.  In one embodiment, after administration of the RNA described herein, e.g., formulated as RNA  lipid particles, at least a portion of the RNA is delivered to a target cell. In one embodiment,  at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment,  the RNA is translated by the target cell to produce the peptide or protein it encodes. In one  embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen  presenting  cell  such  as  a  professional  antigen  presenting  cell  in  the  spleen.  In  one  embodiment, the target cell is a dendritic cell or macrophage. RNA particles such as RNA lipid  particles described herein may be used for delivering RNA to such target cell. Accordingly, the  present disclosure also  relates  to a method  for delivering RNA  to a  target cell  in a subject  comprising  the administration of  the RNA particles described herein  to  the subject.  In one  embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the  RNA is translated by the target cell to produce the peptide or protein encoded by the RNA.   "Encoding"  refers  to  the  inherent  property  of  specific  sequences  of  nucleotides  in  a  polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of  other polymers and macromolecules in biological processes having either a defined sequence  of nucleotides  (i.e., rRNA,  tRNA and mRNA) or a defined sequence of amino acids and  the  biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and  translation  of mRNA  corresponding  to  that  gene  produces  the  protein  in  a  cell  or  other  biological system. Both the coding strand, the nucleotide sequence of which is identical to the  mRNA sequence and is usually provided in sequence listings, and the non‐coding strand, used  as the template for transcription of a gene or cDNA, can be referred to as encoding the protein  or other product of that gene or cDNA.   In one embodiment, the RNA encoding vaccine antigen to be administered according to the  present  disclosure  is  non‐immunogenic.  RNA  encoding  immunostimulant  may  be  administered  according  to  the  present  disclosure  to  provide  an  adjuvant  effect.  The RNA  encoding immunostimulant may be standard RNA or non‐immunogenic RNA.   The  term  "non‐immunogenic  RNA"  as  used  herein  refers  to  RNA  that  does  not  induce  a  response by the immune system upon administration, e.g., to a mammal, or induces a weaker  response than would have been induced by the same RNA that differs only in that it has not  been subjected to the modifications and treatments that render the non‐immunogenic RNA  non‐immunogenic,  i.e.,  than would have been  induced by  standard RNA  (stdRNA).  In one  preferred  embodiment,  non‐immunogenic  RNA,  which  is  also  termed  modified  RNA  (modRNA)  herein,  is  rendered  non‐immunogenic  by  incorporating  modified  nucleosides  suppressing RNA‐mediated activation of innate immune receptors into the RNA and removing  double‐stranded RNA (dsRNA).  For rendering the non‐immunogenic RNA non‐immunogenic by the incorporation of modified  nucleosides,  any  modified  nucleoside  may  be  used  as  long  as  it  lowers  or  suppresses  immunogenicity of  the RNA. Particularly preferred are modified nucleosides  that  suppress  RNA‐mediated  activation  of  innate  immune  receptors.  In  one  embodiment,  the modified  nucleosides comprises a replacement of one or more uridines with a nucleoside comprising a  modified nucleobase. In one embodiment, the modified nucleobase is a modified uracil. In one  embodiment,  the nucleoside comprising a modified nucleobase  is selected  from  the group  consisting of 3‐methyl‐uridine (m3U), 5‐methoxy‐uridine (mo5U), 5‐aza‐uridine, 6‐aza‐uridine,  2‐thio‐5‐aza‐uridine, 2‐thio‐uridine  (s2U), 4‐thio‐uridine  (s4U), 4‐thio‐pseudouridine, 2‐thio‐ pseudouridine,  5‐hydroxy‐uridine  (ho5U),  5‐aminoallyl‐uridine,  5‐halo‐uridine  (e.g.,  5‐iodo‐ uridine or 5‐bromo‐uridine), uridine 5‐oxyacetic acid (cmo5U), uridine 5‐oxyacetic acid methyl  ester  (mcmo5U),  5‐carboxymethyl‐uridine  (cm5U),  1‐carboxymethyl‐pseudouridine,  5‐ carboxyhydroxymethyl‐uridine  (chm5U),  5‐carboxyhydroxymethyl‐uridine  methyl  ester  (mchm5U),  5‐methoxycarbonylmethyl‐uridine  (mcm5U),  5‐methoxycarbonylmethyl‐2‐thio‐ uridine  (mcm5s2U),  5‐aminomethyl‐2‐thio‐uridine  (nm5s2U),  5‐methylaminomethyl‐uridine  (mnm5U),  1‐ethyl‐pseudouridine,  5‐methylaminomethyl‐2‐thio‐uridine  (mnm5s2U),  5‐ methylaminomethyl‐2‐seleno‐uridine  (mnm5se2U),  5‐carbamoylmethyl‐uridine  (ncm5U),  5‐ carboxymethylaminomethyl‐uridine  (cmnm5U), 5‐carboxymethylaminomethyl‐2‐thio‐uridine  (cmnm5s2U), 5‐propynyl‐uridine, 1‐propynyl‐pseudouridine, 5‐taurinomethyl‐uridine (τm5U),  1‐taurinomethyl‐pseudouridine, 5‐taurinomethyl‐2‐thio‐uridine(τm5s2U), 1‐taurinomethyl‐4‐ thio‐pseudouridine),  5‐methyl‐2‐thio‐uridine  (m5s2U),  1‐methyl‐4‐thio‐pseudouridine  (m1s4ψ),  4‐thio‐1‐methyl‐pseudouridine,  3‐methyl‐pseudouridine  (m3ψ),  2‐thio‐1‐methyl‐ pseudouridine,  1‐methyl‐1‐deaza‐pseudouridine,  2‐thio‐1‐methyl‐1‐deaza‐pseudouridine,  dihydrouridine  (D),  dihydropseudouridine,  5,6‐dihydrouridine,  5‐methyl‐dihydrouridine  (m5D), 2‐thio‐dihydrouridine, 2‐thio‐dihydropseudouridine, 2‐methoxy‐uridine, 2‐methoxy‐4‐ thio‐uridine,  4‐methoxy‐pseudouridine,  4‐methoxy‐2‐thio‐pseudouridine,  N1‐methyl‐ pseudouridine,  3‐(3‐amino‐3‐carboxypropyl)uridine  (acp3U),  1‐methyl‐3‐(3‐amino‐3‐ carboxypropyl)pseudouridine  (acp3  ψ),  5‐(isopentenylaminomethyl)uridine  (inm5U),  5‐ (isopentenylaminomethyl)‐2‐thio‐uridine (inm5s2U), α‐thio‐uridine, 2′‐O‐methyl‐uridine (Um),  5,2′‐O‐dimethyl‐uridine (m5Um), 2′‐O‐methyl‐pseudouridine (ψm), 2‐thio‐2′‐O‐methyl‐uridine  (s2Um), 5‐methoxycarbonylmethyl‐2′‐O‐methyl‐uridine (mcm5Um), 5‐carbamoylmethyl‐2′‐O‐ methyl‐uridine  (ncm5Um),  5‐carboxymethylaminomethyl‐2′‐O‐methyl‐uridine  (cmnm5Um),  3,2′‐O‐dimethyl‐uridine (m3Um), 5‐(isopentenylaminomethyl)‐2′‐O‐methyl‐uridine (inm5Um),  1‐thio‐uridine,  deoxythymidine,  2′‐F‐ara‐uridine,  2′‐F‐uridine,  2′‐OH‐ara‐uridine,  5‐(2‐ carbomethoxyvinyl)  uridine,  and  5‐[3‐(1‐E‐propenylamino)uridine.  In  one  particularly  preferred embodiment,  the nucleoside comprising a modified nucleobase  is pseudouridine  (ψ),  N1‐methyl‐pseudouridine  (m1ψ)  or  5‐methyl‐uridine  (m5U),  in  particular  N1‐methyl‐ pseudouridine.  In one embodiment, the replacement of one or more uridines with a nucleoside comprising a  modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least  4%, at  least 5%, at least 10%, at least 25%, at  least 50%, at  least 75%, at  least 90%, at least  95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.  During synthesis of RNA (e.g., mRNA) by in vitro transcription (IVT) using T7 RNA polymerase  significant  amounts  of  aberrant  products,  including  double‐stranded  RNA  (dsRNA)  are  produced  due  to  unconventional  activity  of  the  enzyme.  dsRNA  induces  inflammatory  cytokines and activates effector enzymes  leading to protein synthesis  inhibition. dsRNA can  be removed from RNA such as IVT RNA, for example, by ion‐pair reversed phase HPLC using a  non‐porous  or  porous  C‐18  polystyrene‐divinylbenzene  (PS‐DVB) matrix.  Alternatively,  an  enzymatic based method using  E.  coli RNaseIII  that  specifically hydrolyzes dsRNA but not  ssRNA,  thereby  eliminating dsRNA  contaminants  from  IVT RNA preparations  can be used.  Furthermore,  dsRNA  can  be  separated  from  ssRNA  by  using  a  cellulose material.  In  one  embodiment,  an RNA preparation  is  contacted with a  cellulose material and  the  ssRNA  is  separated from the cellulose material under conditions which allow binding of dsRNA to the  cellulose material and do not allow binding of ssRNA to the cellulose material.  As the term is used herein, "remove" or "removal" refers to the characteristic of a population  of first substances, such as non‐immunogenic RNA, being separated from the proximity of a  population of second substances, such as dsRNA, wherein the population of first substances  is not necessarily devoid of the second substance, and the population of second substances is  not  necessarily  devoid  of  the  first  substance.  However,  a  population  of  first  substances  characterized by the removal of a population of second substances has a measurably lower  content of second substances as compared to the non‐separated mixture of first and second  substances.  In one embodiment, the removal of dsRNA from non‐immunogenic RNA comprises a removal  of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less  than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non‐immunogenic  RNA  composition  is  dsRNA.  In  one  embodiment,  the  non‐immunogenic  RNA  is  free  or  essentially  free of dsRNA.  In  some embodiments,  the non‐immunogenic RNA  composition  comprises a purified preparation of single‐stranded nucleoside modified RNA. For example, in  some embodiments, the purified preparation of single‐stranded nucleoside modified RNA is  substantially  free  of  double  stranded  RNA  (dsRNA).  In  some  embodiments,  the  purified  preparation is at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%,  at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single  stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA,  etc.).  In one embodiment, the non‐immunogenic RNA  is translated  in a cell more efficiently than  standard RNA with  the  same  sequence.  In one embodiment,  translation  is enhanced by a  factor  of  2‐fold  relative  to  its  unmodified  counterpart.  In  one  embodiment,  translation  is  enhanced by a 3‐fold factor. In one embodiment, translation is enhanced by a 4‐fold factor. In  one embodiment, translation is enhanced by a 5‐fold factor. In one embodiment, translation  is enhanced by a 6‐fold factor. In one embodiment, translation is enhanced by a 7‐fold factor.  In  one  embodiment,  translation  is  enhanced  by  an  8‐fold  factor.  In  one  embodiment,  translation is enhanced by a 9‐fold factor. In one embodiment, translation is enhanced by a  10‐fold  factor.  In  one  embodiment,  translation  is  enhanced  by  a  15‐fold  factor.  In  one  embodiment, translation is enhanced by a 20‐fold factor. In one embodiment, translation is  enhanced by a 50‐fold factor. In one embodiment, translation is enhanced by a 100‐fold factor.  In  one  embodiment,  translation  is  enhanced  by  a  200‐fold  factor.  In  one  embodiment,  translation is enhanced by a 500‐fold factor. In one embodiment, translation is enhanced by a  1000‐fold factor.  In one embodiment, translation  is enhanced by a 2000‐fold factor.  In one  embodiment, the factor is 10‐1000‐fold. In one embodiment, the factor is 10‐100‐fold. In one  embodiment, the factor is 10‐200‐fold. In one embodiment, the factor is 10‐300‐fold. In one  embodiment, the factor is 10‐500‐fold. In one embodiment, the factor is 20‐1000‐fold. In one  embodiment, the factor is 30‐1000‐fold. In one embodiment, the factor is 50‐1000‐fold. In one  embodiment, the factor is 100‐1000‐fold. In one embodiment, the factor is 200‐1000‐fold. In  one  embodiment,  translation  is  enhanced  by  any  other  significant  amount  or  range  of  amounts.  In  one  embodiment,  the  non‐immunogenic  RNA  exhibits  significantly  less  innate  immunogenicity than standard RNA with the same sequence. In one embodiment, the non‐ immunogenic RNA exhibits an innate immune response that is 2‐fold less than its unmodified  counterpart. In one embodiment, innate immunogenicity is reduced by a 3‐fold factor. In one  embodiment, innate immunogenicity is reduced by a 4‐fold factor. In one embodiment, innate  immunogenicity is reduced by a 5‐fold factor. In one embodiment, innate immunogenicity is  reduced by a 6‐fold factor. In one embodiment, innate immunogenicity is reduced by a 7‐fold  factor.  In  one  embodiment,  innate  immunogenicity  is  reduced  by  a  8‐fold  factor.  In  one  embodiment, innate immunogenicity is reduced by a 9‐fold factor. In one embodiment, innate  immunogenicity is reduced by a 10‐fold factor. In one embodiment, innate immunogenicity is  reduced by a 15‐fold factor. In one embodiment, innate immunogenicity is reduced by a 20‐ fold factor. In one embodiment, innate immunogenicity is reduced by a 50‐fold factor. In one  embodiment,  innate  immunogenicity  is  reduced by a 100‐fold  factor.  In one embodiment,  innate  immunogenicity  is  reduced  by  a  200‐fold  factor.  In  one  embodiment,  innate  immunogenicity is reduced by a 500‐fold factor. In one embodiment, innate immunogenicity  is reduced by a 1000‐fold factor. In one embodiment, innate immunogenicity is reduced by a  2000‐fold factor.   The term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in  innate  immunogenicity.  In  one  embodiment,  the  term  refers  to  a  decrease  such  that  an  effective  amount  of  the  non‐immunogenic  RNA  can  be  administered without  triggering  a  detectable innate immune response. In one embodiment, the term refers to a decrease such  that the non‐immunogenic RNA can be repeatedly administered without eliciting an  innate  immune response sufficient to detectably reduce production of the protein encoded by the  non‐immunogenic RNA. In one embodiment, the decrease is such that the non‐immunogenic  RNA can be repeatedly administered without eliciting an innate immune response sufficient  to eliminate detectable production of the protein encoded by the non‐immunogenic RNA.  "Immunogenicity"  is the ability of a foreign substance, such as RNA, to provoke an  immune  response in the body of a human or other animal. The innate immune system is the component  of  the  immune  system  that  is  relatively  unspecific  and  immediate.  It  is  one  of  two main  components of the vertebrate immune system, along with the adaptive immune system.  As used herein "endogenous" refers to any material from or produced inside an organism, cell,  tissue or system.   As used herein,  the  term "exogenous" refers  to any material  introduced  from or produced  outside an organism, cell, tissue or system.   The term "expression" as used herein is defined as the transcription and/or translation of a  particular nucleotide sequence.   As used herein, the terms "linked," "fused", or "fusion" are used interchangeably. These terms  refer to the joining together of two or more elements or components or domains.     Codon‐optimization / Increase in G/C content  In  some  embodiment,  the  amino  acid  sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic variant thereof, or an  immunogenic fragment of the SARS‐CoV‐2 S protein or  the immunogenic variant thereof described herein is encoded by a coding sequence which is  codon‐optimized and/or the G/C content of which is increased compared to wild type coding  sequence. This also  includes embodiments, wherein one or more  sequence  regions of  the  coding sequence are codon‐optimized and/or increased in the G/C content compared to the  corresponding sequence regions of the wild type coding sequence. In one embodiment, the  codon‐optimization and/or the  increase  in the G/C content preferably does not change the  sequence of the encoded amino acid sequence.  The term "codon‐optimized" refers to the alteration of codons in the coding region of a nucleic  acid molecule to reflect the typical codon usage of a host organism without preferably altering  the amino acid sequence encoded by  the nucleic acid molecule. Within  the context of  the  present disclosure, coding regions are preferably codon‐optimized for optimal expression in a  subject to be treated using the RNA molecules described herein. Codon‐optimization is based  on the finding that the translation efficiency is also determined by a different frequency in the  occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for  which frequently occurring tRNAs are available are inserted in place of "rare codons".   In some embodiments of the present disclosure, the guanosine/cytosine (G/C) content of the  coding region of the RNA described herein is increased compared to the G/C content of the  corresponding  coding  sequence  of  the wild  type  RNA, wherein  the  amino  acid  sequence  encoded by the RNA is preferably not modified compared to the amino acid sequence encoded  by the wild type RNA. This modification of the RNA sequence  is based on the  fact that the  sequence of any RNA region to be translated is important for efficient translation of that RNA  (e.g., mRNA). Sequences having an  increased G  (guanosine)/C  (cytosine) content are more  stable than sequences having an increased A (adenosine)/U (uracil) content. In respect to the  fact that several codons code for one and the same amino acid (so‐called degeneration of the  genetic  code),  the most  favourable  codons  for  the  stability  can  be  determined  (so‐called  alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are  various  possibilities  for  modification  of  the  RNA  sequence,  compared  to  its  wild  type  sequence.  In particular, codons which  contain A and/or U nucleotides  can be modified by  substituting these codons by other codons, which code for the same amino acids but contain  no A and/or U or contain a lower content of A and/or U nucleotides.   In various embodiments, the G/C content of the coding region of the RNA described herein is  increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%,  or even more compared to the G/C content of the coding region of the wild type RNA.  In some  embodiments, G/C content of a coding region is increased by about 10% to about 60% (e.g.,  by about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to  about 60%, or by about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about  57%, about 58%, about 59%, or about 60%) compared to the G/C content of the coding region  of the wild type RNA.   In some embodiments, RNA disclosed herein comprises a sequence disclosed herein (e.g., SEQ  ID NO: 9), that has been modified to encode one or more mutations characteristic of a SARS‐ CoV‐2 varaint (e.g., a BA.2 or a BA.4/5 Omicron variant).  In some embodiments, RNA can be  modified to encode one or more mutations characteristic of a SARS‐CoV‐2 variant by making  as few nucleotide changes as possible.  In some embodiments, RNA can be modified to encode  one  or  more  mutations  that  are  characteristic  of  a  SARS‐CoV‐2  variant    by  introducing  mutations that result in high codon‐optimization and/or increased G/C content.  In  some embodiments, one or more mutations  characteristic of  a  SARS‐CoV‐2  variant  are  introduced onto a full‐length S protein (e.g., an S protein comprising SEQ ID NO: 1).  In some  embodiments one or more mutations characteristic of a SARS‐CoV‐2 variant are  introduced  onto a full‐length S protein having one or more proline mutations that increase stability of a  prefusion confirmation. For example, in some embodiments, proline substitutions are made  at positions corresponding to positions 986 and 987 of SEQ ID NO: 1.  In some embodiments,  proline substitutions are made at positions corresponding to positions 985 and 987 of SEQ ID  NO:  1.    In  some  embodiments,  at  least  4  proline  substitutions  are  made.  In  some  embodiments,  at  least  four  of  such  proline  mutations  include  mutations  at  positions  corresponding to residues 817, 892, 899, and 942 of SEQ  ID NO: 1.    In some embodiments,  such  a  SARS‐CoV‐2  protein  comprising  proline  substitutions  at  positions  corresponding  to  residues 817, 892, 899, and 942 of SEQ ID NO: 1, may further comprise proline substitutions  at positions corresponding to residues 986 and 987 of SEQ ID NO: 1.  In some embodiments,  such  a  SARS‐CoV‐2  protein  comprising  proline  substitutions  at  positions  corresponding  to  residues 817, 892, 899, and 942 of SEQ ID NO: 1, may further comprise proline substitutions  at positions corresponding to residues 985 and 987 of SEQ ID NO: 1.  In some embodiments,  one  or  more  mutations  characteristic  of  a  SARS‐CoV‐2  variant  are  introduced  onto  an  immunogenic fragment of an S protein (e.g., the RBD).    Embodiments of administered RNAs  In some embodiments, the present disclosure provides an RNA (e.g., mRNA) comprising an  open reading frame encoding a polypeptide that comprises at least a portion of a SARS‐CoV‐2  S  protein.  The  RNA  is  suitable  for  intracellular  expression  of  the  polypeptide.  In  some  embodiments,  such  an  encoded  polypeptide  comprises  a  sequence  corresponding  to  the  complete S protein. In some embodiments, such an encoded polypeptide does not comprise  a sequence corresponding  to  the complete S protein.  In some embodiments,  the encoded  polypeptide comprises a sequence that corresponds to the receptor binding domain (RBD).  In  some embodiments, the encoded polypeptide comprises a sequence that corresponds to the  RBD, and further comprises a trimerization domain (e.g., a trimerization domain as disclosed  herein, such as a fibritin domain).  In some embodiments an RBD comprises a signaling domain  (e.g., a  signaling domain as disclosed herein).    In  some embodiments an RBD  comprises a  transmembrane  domain  (e.g.,  a  transmembrane  domain  as  disclosed  herein).    In  some  embodiments, an RBD comprises a signaling domain and a  trimerization domain.    In some  embodiments,  an  RBD  comprises  a  signaling  domain,  a  trimerization  domain,  and  transmembrane domain.  In some embodiments, the encoded polypeptide comprises a sequence that corresponds to  two receptor binding domains.  In some embodiments, the encoded polypeptide comprises a  sequence that corresponds to two receptor binding domains in tandem in an amino acid chain,  e.g., as disclosed in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against  COVID‐19,  MERS,  and  SARS,"  Cell  182.3  (2020):  722‐733,  the  contents  of  which  are  incorporated by reference herein in their entirety.  In some embodiments, a SARS‐CoV‐2 S protein, or an immunogenic fragment thereof  comprises one or more mutations to alter, add, or remove a glycosylation site, e.g., as  described in WO2022221835A2, US20220323574A1, WO2022266012A1, or  WO2022195351A1.  In some embodiments, compositions or medical preparations described herein comprise RNA  encoding an amino acid sequence comprising SARS‐CoV‐2 S protein, an immunogenic variant  thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant  thereof. Likewise, methods described herein comprise administration of such RNA.   The active platform for use herein is based on an antigen‐coding RNA vaccine to induce robust  neutralising antibodies and accompanying/concomitant T cell response to achieve protective  immunization with preferably minimal vaccine doses. The RNA administered is preferably in‐ vitro transcribed RNA.  Three  different  RNA  platforms  are  particularly  preferred,  namely  non‐modified  uridine  containing mRNA  (uRNA),  nucleoside modified mRNA  (modRNA)  and  self‐amplifying  RNA  (saRNA).  In one particularly preferred embodiment, the RNA  is  in vitro transcribed RNA.  In  some embodiments, uRNA is mRNA. In some embodiments, modRNA is mRNA.  In the following, embodiments of these three different RNA platforms are described, wherein  certain terms used when describing elements thereof have the following meanings:    S1S2 protein/S1S2 RBD: Sequences encoding the respective antigen of SARS‐CoV‐2.  nsP1, nsP2, nsP3, and nsP4: Wildtype sequences encoding the Venezuelan equine encephalitis  virus  (VEEV)  RNA‐dependent  RNA  polymerase  replicase  and  a  subgenomic  promotor  plus  conserved sequence elements supporting replication and translation.  virUTR:  Viral  untranslated  region  encoding  parts  of  the  subgenomic  promotor  as well  as  replication and translation supporting sequence elements.  hAg‐Kozak:  5'‐UTR  sequence  of  the  human  alpha‐globin mRNA with  an  optimized  ʻKozak  sequenceʼ to increase translaƟonal efficiency.  Sec: Sec corresponds  to a secretory signal peptide  (sec), which guides  translocation of  the  nascent polypeptide chain into the endoplasmatic reticulum.  In some embodiments, such a  secretory signal peptide includes the intrinsic S1S2 secretory signal peptide of a SARS‐CoV‐2 S  protein. In some embodiments, such a secretory signal peptide is a secretory signal peptide  from a non‐S1S2 protein. For example, an immunoglobulin secretory signal peptide (aa 1‐22),  an HSV‐1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV‐2 gD signal peptide  (MGRLTSGVGTAALLVVAVGLRVVCA); a human SPARC signal peptide, a human insulin isoform  1 signal peptide, a human albumin signal peptide, or any other signal peptide described herein.  Glycine‐serine  linker  (GS):  Sequences  coding  for  short  linker  peptides  predominantly  consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.  Fibritin: Partial sequence of T4 fibritin (foldon), used as artificial trimerization domain.  TM: TM sequence corresponds to the transmembrane part of a protein. A transmembrane  domain  can  be  N‐terminal,  C‐terminal,  or  internal  to  an  encoded  polypeptide.  A  coding  sequence of a transmembrane element is typically placed in frame (i.e., in the same reading  frame),  5',  3',  or  internal  to  coding  sequences  of  sequences  (e.g.,  sequences  encoding  polypeptide(s)) with which it is to be linked. In some embodiments, a transmembrane domain  comprises or is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV‐  1, equine  infectious anaemia virus  (EIAV), murine  leukaemia virus  (MLV), mouse mammary  tumor  virus,  G  protein  of  vesicular  stomatitis  virus  (VSV),  Rabies  virus,  or  a  seven  transmembrane  domain  receptor.  In  some  embodiments,  the  transmembrane  part  of  a  protein is from the S1S2 protein.  FI element: The 3'‐UTR is a combination of two sequence elements derived from the “amino  terminal  enhancer  of  split”  (AES)  mRNA  (called  F)  and  the  mitochondrial  encoded  12S  ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences  that confer RNA stability and augment total protein expression.  A30L70:  A  poly(A)‐tail  measuring  110  nucleotides  in  length,  consisting  of  a  stretch  of  30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine  residues designed to enhance RNA stability and translational efficiency in dendritic cells.    In general, vaccine RNA described herein may comprise, from 5' to 3', one of the following  structures:  Cap‐5'‐UTR‐Vaccine Antigen‐Encoding Sequence‐3'‐UTR‐Poly(A)  or   Cap‐ hAg‐Kozak‐Vaccine Antigen‐Encoding Sequence‐FI‐A30L70.    In some embodiments, a vaccine antigen described herein may comprise a full‐length S protein  or  an  immunogenic  fragment  thereof  (e.g., RBD).  In  some  embodiments where  a  vaccine  antigen comprises a full‐length S protein, its secretory signal peptide and/or transmembrane  domain may be replaced by a heterologous secretory signal peptide (e.g., as described herein)  and/or a heterologous transmembrane domain (e.g., as described herein).  In some embodiments, a vaccine antigen described herein may comprise, from N‐terminus to  C‐terminus, one of the following structures:  Signal Sequence‐RBD‐Trimerization Domain  or  Signal Sequence‐RBD‐Trimerization Domain‐Transmembrane Domain.    RBD and Trimerization Domain may be separated by a linker, in particular a GS linker such as  a  linker  having  the  amino  acid  sequence  GSPGSGSGS.  Trimerization  Domain  and  Transmembrane Domain may be separated by a linker, in particular a GS linker such as a linker  having the amino acid sequence GSGSGS.  Signal Sequence may be a signal sequence as described herein. RBD may be a RBD domain as  described herein. Trimerization Domain may be a trimerization domain as described herein.  Transmembrane Domain may be a transmembrane domain as described herein.  In one embodiment,   Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19  of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or an  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  this amino acid sequence,  RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or  an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity  to this amino acid sequence,  Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of  SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having  at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence; and  Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to  1254 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to this amino acid sequence.  In one embodiment,   Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19  of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31,  RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1,  Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of  SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10; and  Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to  1254 of SEQ ID NO: 1.  In  some  embodiments,  an  RNA  polynucleotide  comprising  a  sequence  encoding  a  vaccine  antigen  (e.g.,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof) or  comprising an open reading frame encoding a vaccine antigen (e.g., a SARS‐CoV‐2 S protein,  an immunogenic variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or  the immunogenic variant thereof) such as the nucleotide sequence of SEQ ID NO: 50 or the  nucleotide sequence of SEQ ID NO: 53, a variant or fragment thereof, further comprises a 5’  cap, e.g., a 5’ cap comprising a Cap1 structure, a 5’ UTR sequence, e.g., a 5’ UTR sequence  comprising  the nucleotide  sequence of  SEQ  ID NO: 12,  a 3’ UTR  sequence,  e.g.,  a  3’ UTR  sequence comprising the nucleotide sequence of SEQ ID NO: 13, and polyA sequence, e.g., a  polyA sequence comprising the nucleotide sequence of SEQ ID NO: 14. In some embodiments,  the  RNA  polynucleotide  is  formulated  in  a  composition  comprising  ((4‐ hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  cholesterol,  distearoylphosphatidylcholine,  and  (2‐[(polyethylene  glycol)‐2000]‐N,N‐ ditetradecylacetamide).  The RNA described herein or RNA encoding the vaccine antigen described herein may be non‐ modified  uridine  containing mRNA  (uRNA),  nucleoside modified mRNA  (modRNA)  or  self‐ amplifying RNA (saRNA). In one embodiment, the RNA described herein or RNA encoding the  vaccine antigen described herein is nucleoside modified mRNA (modRNA).   Variant Specific Vaccines  In some embodiments, RNA disclosed herein encodes an S protein comprising one or more  mutations  that  are  characteristic  of  a  SARS‐CoV‐2  variant.    In  some  embodiments,  RNA  encodes a SARS‐CoV‐2 S protein comprising one or more mutations characteristic of an Alpha  variant.  In some embodiments, RNA encodes a SARS‐CoV‐2 S protein comprising one or more  mutations characteristic of a Beta variant.  In some embodiments, RNA encodes a SARS‐CoV‐ 2  S protein  comprising one or more mutations  characteristic of  a Delta  variant.    In  some  embodiments,  RNA  encodes  a  SARS‐CoV‐2  S  protein  comprising  one  or more mutations  characteristic of an Omicron variant  (e.g., an S protein comprising one or more mutations  characteristic  of  a  BA.1,  BA.2,  or  BA.4/5  Omicron  variant).    In  some  embodiments,  RNA  encodes a SARS‐CoV‐2 S protein comprising one or more mutations characteristic of an BA.1  Omicron variant. In some embodiments, RNA encodes a SARS‐CoV‐2 S protein comprising one  or more mutations  characteristic of an BA.2 Omicron variant.  In  some embodiments, RNA  encodes  a  SARS‐CoV‐2  S  protein  comprising  one  or more mutations  characteristic  of  an  BA.2.12.1 Omicron  variant.  In  some  embodiments,  RNA  encodes  a  SARS‐CoV‐2  S  protein  comprising  one  or  more  mutations  characteristic  of  a  BA.3  Omicron  variant.  In  some  embodiments,  RNA  encodes  a  SARS‐CoV‐2  S  protein  comprising  one  or more mutations  characteristic of a BA.4 Omicron variant. In some embodiments, RNA encodes a SARS‐CoV‐2 S  protein comprising one or more mutations characteristic of a BA.5 Omicron variant.    Non‐modified uridine messenger RNA (uRNA)  The active principle of the non‐modified messenger RNA (uRNA) drug substance  is a single‐ stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding  the  coronavirus  vaccine  antigen  (i.e. open  reading  frame), each uRNA preferably  contains  common  structural  elements  optimized  for maximal  efficacy  of  the  RNA with  respect  to  stability and translational efficiency (5′‐cap, 5′‐UTR, 3′‐UTR, poly(A)‐tail). The preferred 5’ cap  structure is beta‐S‐ARCA(D1) (m2 7,2'‐OGppSpG). The preferred 5′‐UTR and 3′‐UTR comprise the  nucleotide  sequence  of  SEQ  ID  NO:  12  and  the  nucleotide  sequence  of  SEQ  ID  NO:  13,  respectively. The preferred poly(A)‐tail comprises the sequence of SEQ ID NO: 14.    Different embodiment of this platform are as follows:     RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7)  Structure  beta‐S‐ARCA(D1)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant)  RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7)  Structure  beta‐S‐ARCA(D1)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant)  BNT162a1; RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5)  Structure  beta‐S‐ARCA(D1)‐hAg‐Kozak‐RBD‐GS‐Fibritin‐FI‐A30L70  Encoded antigen  Viral spike protein (S protein) of the SARS‐CoV‐2 (partial sequence,  Receptor Binding Domain (RBD) of S1S2 protein)    Figure 3 schematizes the general structure of the antigen‐encoding RNAs.    Nucleoside modified messenger RNA (modRNA)  The active principle of the nucleoside modified messenger RNA (modRNA) drug substance is  as well a  single‐stranded mRNA  that  is  translated upon entering a  cell.  In  addition  to  the  sequence encoding the coronavirus vaccine antigen (i.e. open reading frame), each modRNA  contains common structural elements optimized for maximal efficacy of the RNA as the uRNA  (5′‐cap,  5′‐UTR,  3′‐UTR, poly(A)‐tail).  Compared  to  the  uRNA, modRNA  contains  1‐methyl‐ pseudouridine  instead of uridine. The preferred 5’ cap structure  is m2 7,3’‐OGppp(m1 2’‐O)ApG.  The preferred 5′‐UTR and 3′‐UTR comprise the nucleotide sequence of SEQ ID NO: 12 and the  nucleotide sequence of SEQ ID NO: 13, respectively. The preferred poly(A)‐tail comprises the  sequence of SEQ ID NO: 14. An additional purification step is applied for modRNA to reduce  dsRNA contaminants generated during the in vitro transcription reaction.    Different embodiments of this platform are as follows:     BNT162b2; RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant)    BNT162b2; RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant)    BNT162b1; RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐RBD‐GS‐Fibritin‐FI‐A30L70  Encoded antigen  Viral spike protein  (S1S2 protein) of  the SARS‐CoV‐2  (partial sequence,  Receptor Binding Domain (RBD) of S1S2 protein fused to fibritin)    Figure 4 schematizes the general structure of the antigen‐encoding RNAs.    BNT162b3c (SEQ ID NO: 29; SEQ ID NO: 30)  Structure   m2 7,3’‐OGppp(m1 2’‐O)ApG‐hAg‐Kozak‐RBD‐GS‐Fibritin‐GS‐TM‐FI‐A30L70  Encoded antigen   Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (partial sequence,  Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused to Transmembrane  Domain (TM) of S1S2 protein); intrinsic S1S2 protein secretory signal peptide (aa 1‐19) at the  N‐terminus of the antigen sequence BNT162b3d (SEQ ID NO: 31; SEQ ID NO: 32)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG‐hAg‐Kozak‐RBD‐GS‐Fibritin‐GS‐TM‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (partial sequence,  Receptor Binding Domain  (RBD) of S1S2 protein  fused  to Fibritin  fused  to Transmembrane  Domain (TM) of S1S2 protein);  immunoglobulin secretory signal peptide (aa 1‐22) at the N‐ terminus of the antigen sequence.    BNT162b2 – Beta variant; RBP020.11 (SEQ ID NO: 57; SEQ ID NO: 55)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant), comprising mutations characteristic of the  Beta variant of SARS‐CoV‐2  BNT162b2 – Alpha variant; RBP020.14 (SEQ ID NO: 60; SEQ ID NO: 58)  Structure  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral spike protein (S1S2 protein) of the SARS‐CoV‐2 (S1S2 full‐length  protein, sequence variant), comprising mutations characteristic of the  Alpha variant of SARS‐CoV‐2  BNT162b2 – Delta variant; RBP020.16 (SEQ ID NO: 63; SEQ ID NO: 61)  Structure  m27,3’‐OGppp(m12’‐O)ApG)‐hAg‐Kozak‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral  spike  protein  (S1S2  protein)  of  the  SARS‐CoV‐2  (S1S2  full‐length  protein,  sequence  variant),  comprising mutations  characteristic  of  the  Delta variant of SARS‐CoV‐2   
Nucleotide Sequence of RBP020.11 (Beta‐specific vaccine)  Nucleotide sequence is shown with individual sequence elements as indicated in bold letters.  In addition, the sequence of the translated protein is shown in italic letters below the coding  nucleotide sequence (* = stop codon). Red text  indicates point mutations  in the nucleotide and  amino acid sequences.                                                       
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
 
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
  Sequences of RBP020.11 are also shown in Table 9.  Additional sequences of exemplary RNA  constructs encoding SARS‐CoV‐2 spike sequence variants are shown in Tables 8‐18.  For each  variant,  the  spike  protein  sequence  and  encoding  DNA  and  RNA  sequence  are  provided.   Additionally,  exemplary  full  length  RNA  vaccine  and  corresponding  DNA  sequences  are  provided.  In the full length sequences provided in these Tables (7‐18a) and other DNA or RNA  sequences provided herein, “U” may  represent a naturally‐occurring uridine or a modified  uridine,  e.g.,  pseudouridine.  Additionally,  it  is  noted  that  in  the  full‐length  RNA  vaccine  sequences and their corresponding DNA sequences provided in Tables 7‐18a, a poly‐A tail is  included in the sequence.  According to the present disclosure herein,  in some embodiments,  RNA and DNA sequences described herein may  include a polyA tail that is shorter or longer  than what is shown, e.g., by at least 1, at least 2, at least 3, at least 4 nucletodides and up to  at least 10 “A” nucleotides.  In some embodiments, an RNA construct encoding a spike protein from a coronavirus variant  as described in Tables 7 ‐18a has a structure as shown below:  m2 7,3’‐OGppp(m1 2’‐O)ApG)‐hAg‐Kozak‐Antigen‐FI‐A30L70, wherein the encoded “Antigen” is  the viral spike protein (S1S2 protein) of the  SARS‐CoV‐2 (S1S2 full‐length protein) as  indicated in Tables 7‐18a.             
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Nucleotide Sequence of RBP020.14 (Alpha‐specific vaccine)  Nucleotide sequence is shown with individual sequence elements as indicated in bold letters. In  addition,  the  sequence  of  the  translated  protein  is  shown  in  italic  letters  below  the  coding  nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and  amino acid sequences. 
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Sequences of RBP020.14 are also shown in Table 10. 
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Nucleotide Sequence of RBP020.16 (Delta‐specific vaccine)  Nucleotide sequence is shown with individual sequence elements as indicated in bold letters. In  addition,  the  sequence  of  the  translated  protein  is  shown  in  italic  letters  below  the  coding  nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and  amino acid sequences. 
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
I S S V L N D I mut4  I R A A E I mut4 
Figure imgf000274_0001
Figure imgf000275_0001
y
Figure imgf000276_0001
Sequences of RBP020.14 are also shown in Table 11. 
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
Figure imgf000287_0001
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Figure imgf000292_0001
Figure imgf000293_0001
Figure imgf000294_0001
Figure imgf000295_0001
Figure imgf000296_0001
Figure imgf000297_0001
Figure imgf000298_0001
Figure imgf000299_0001
Figure imgf000300_0001
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
Figure imgf000309_0001
Figure imgf000310_0001
Figure imgf000311_0001
Figure imgf000312_0001
Figure imgf000313_0001
Figure imgf000314_0001
Figure imgf000315_0001
Figure imgf000316_0001
Figure imgf000317_0001
Figure imgf000318_0001
Figure imgf000319_0001
Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
Figure imgf000324_0001
Figure imgf000325_0001
Figure imgf000326_0001
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
Figure imgf000330_0001
Figure imgf000331_0001
Figure imgf000332_0001
Figure imgf000333_0001
Figure imgf000334_0001
Figure imgf000335_0001
Figure imgf000336_0001
Figure imgf000337_0001
Figure imgf000338_0001
Figure imgf000339_0001
Figure imgf000340_0001
Figure imgf000341_0001
Figure imgf000342_0001
Figure imgf000343_0001
Figure imgf000344_0001
Figure imgf000345_0001
Figure imgf000346_0001
Figure imgf000347_0001
Figure imgf000348_0001
Figure imgf000349_0001
Figure imgf000350_0001
Figure imgf000351_0001
Figure imgf000352_0001
Figure imgf000353_0001
Figure imgf000354_0001
Figure imgf000355_0001
Table 25: Description of sequences of RBP020.23 (Omicron BA.4/BA.5-specific RNA vaccine) as described in Table 24 above
Figure imgf000356_0001
Figure imgf000357_0001
Figure imgf000358_0001
Figure imgf000359_0001
Figure imgf000360_0001
Figure imgf000361_0001
Figure imgf000362_0001
Figure imgf000363_0001
Figure imgf000364_0001
Figure imgf000365_0001
Figure imgf000366_0001
Figure imgf000367_0001
Figure imgf000368_0001
Figure imgf000369_0001
Figure imgf000370_0001
Figure imgf000371_0001
Figure imgf000372_0001
Self‐amplifying RNA (saRNA)  The active principle of the self‐amplifying mRNA (saRNA) drug substance is a single‐stranded  RNA,  which  self‐amplifies  upon  entering  a  cell,  and  the  coronavirus  vaccine  antigen  is  translated  thereafter.  In  contrast  to uRNA  and modRNA  that preferably  code  for  a  single  protein,  the coding  region of saRNA contains  two open  reading  frames  (ORFs). The 5’‐ORF  encodes the RNA‐dependent RNA polymerase such as Venezuelan equine encephalitis virus  (VEEV) RNA‐dependent RNA polymerase  (replicase). The  replicase ORF  is  followed 3’ by  a  subgenomic promoter and a  second ORF encoding  the antigen. Furthermore,  saRNA UTRs  contain 5’ and 3’  conserved  sequence elements  (CSEs)  required  for  self‐amplification. The  saRNA contains common structural elements optimized for maximal efficacy of the RNA as the  uRNA  (5′‐cap,  5′‐UTR,  3′‐UTR,  poly(A)‐tail).  In  some  embodiments,  the  saRNA  preferably  contains  uridine.    In  some  embodiments,  the  saRNA  comprises  one  or more  nucleoside  modifications as described herein. The preferred 5’ cap structure is beta‐S‐ARCA(D1) (m2 7,2'‐ OGppSpG).  In some embodiments, an saRNA described herein encodes a single antigen (e.g., one SARS‐ CoV‐2  S  polypeptide).    In  some  embodiments,  an  saRNA  utilized  in  accordance with  the  present  disclosure  encodes  two  or  more  antigens  (e.g.,  two  or  more  SARS‐CoV‐2  S  polypeptides,).    In some embodiments, an saRNA encodes two S polypeptides, each from a  different SARS‐CoV‐2 variant.    In some embodiments, an saRNA platform can provide certain advantages as compared  to  other RNA platforms.    For  example,  in  some  embodiments,  saRNA  can  provide  increased  duration  of  expression  of  an  antigen,  lower  dose  levels,  improved  tolerability,  and/or  increased antigen capacity, while maintaining a robust antibody and T cell response.  Cytoplasmic delivery of saRNA initiates an alphavirus‐like life cycle. However, the saRNA does  not encode for alphaviral structural proteins that are required for genome packaging or cell  entry,  therefore  generation of  replication  competent  viral particles  is  very unlikely  to not  possible.  Replication  does  not  involve  any  intermediate  steps  that  generate  DNA.  The  use/uptake of  saRNA  therefore  poses  no  risk  of  genomic  integration or  other  permanent  genetic  modification  within  the  target  cell.  Furthermore,  the  saRNA  itself  prevents  its  persistent  replication  by  effectively  activating  innate  immune  response  via  recognition  of  dsRNA intermediates.    Different embodiments of this platform are as follows:     RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7)  Structure  beta‐S‐ARCA(D1)‐replicase‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral  spike  protein  (S  protein)  of  the  SARS‐CoV‐2  (S1S2  full‐length  protein, sequence variant)    RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7)  Structure  beta‐S‐ARCA(D1)‐replicase‐S1S2‐PP‐FI‐A30L70  Encoded antigen  Viral  spike  protein  (S  protein)  of  the  SARS‐CoV‐2  (S1S2  full‐length  protein, sequence variant)    BNT162c1; RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5)  Structure    beta‐S‐ARCA(D1)‐replicase‐RBD‐GS‐Fibritin‐FI‐A30L70  Encoded antigen  Viral  spike  protein  (S  protein)  of  the  SARS‐CoV‐2  (partial  sequence,  Receptor Binding Domain (RBD) of S1S2 protein)    RBS004.4 (SEQ ID NO: 27; SEQ ID NO: 28)  Structure    beta‐S‐ARCA(D1)‐replicase‐RBD‐GS‐Fibritin‐TM‐FI‐A30L70  Encoded antigen  Viral  spike  protein  (S  protein)  of  the  SARS‐CoV‐2  (partial  sequence,  Receptor Binding Domain (RBD) of S1S2 protein)    Figure 5 schematizes the general structure of the antigen‐encoding RNAs.      In  some  embodiments,  vaccine  RNA  described  herein  comprises  a  nucleotide  sequence  selected from the group consisting of SEQ ID NO: 15, 16, 17, 19, 20, 21, 24, 25, 26, 27, 30, and  32. A particularly preferred vaccine RNA described herein comprises a nucleotide sequence  selected from the group consisting of SEQ  ID NO: 15, 17, 19, 21, 25, 26, 30, and 32 such as  selected from the group consisting of SEQ ID NO: 17, 19, 21, 26, 30, and 32.    In some embodiments, RNA described herein  is  formulated  in  lipid nanoparticles,  lipoplex,  polyplexes (PLX),  lipidated polyplexes (LPLX),  liposomes, or polysaccharide nanoparticles.  In  some  embodiments,  RNA  described  herein  is  preferably  formulated  in  lipid  nanoparticles  (LNP).  In  one  embodiment,  the  LNP  comprise  a  cationic  lipid,  a  neutral  lipid,  a  steroid,  a  polymer conjugated lipid; and the RNA. In one embodiment, the cationic lipid is ALC‐0315, the  neutral lipid is DSPC, the steroid is cholesterol, and the polymer conjugated lipid is ALC‐0159.  The preferred mode of  administration  is  intramuscular  administration, more preferably  in  aqueous  cryoprotectant  buffer  for  intramuscular  administration.  The  drug  product  is  a  preferably  a  preservative‐free,  sterile  dispersion  of  RNA  formulated  in  lipid  nanoparticles  (LNP) in aqueous cryoprotectant buffer for intramuscular administration.     In  different  embodiments,  the  drug  product  comprises  the  components  shown  below,  preferably at the proportions or concentrations shown below:   
Figure imgf000375_0001
Figure imgf000376_0005
  [1] ALC‐0315 = ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate) / 6‐[N‐6‐(2‐ hexyldecanoyloxy)hexyl‐N‐(4‐hydroxybutyl)amino]hexyl 2‐hexyldecanoate  [2] ALC‐0159 = 2‐[(polyethylene glycol)‐2000]‐N,N‐ditetradecylacetamide / 2‐[2‐(ω‐methoxy  (polyethyleneglycol2000) ethoxy]‐N,N‐ditetradecylacetamide  [3] DSPC = 1,2‐Distearoyl‐sn‐glycero‐3‐phosphocholine   q.s. = quantum satis (as much as may suffice)    ALC‐0315:  
Figure imgf000376_0001
  ALC‐0159:   
Figure imgf000376_0002
    DSPC: 
Figure imgf000376_0003
  Cholesterol: 
Figure imgf000376_0004
  In some embodiments, particles disclosed herein are formulated in a solution comprising 10  mM Tris and 10% sucrose, and optionally having a pH of about 7.4.  In some embodiments,  particles disclosed herein are formulated in a solution comprising about 103 mg/ml sucrose,  about 0.20 mg/ml tromethamine (Tris base), and about 1.32 mg/ml Tris.  In some embodiments, a composition comprises:  (a) about 0.1 mg/mL RNA  comprising an open  reading  frame encoding a polypeptide  that  comprises a SARS‐CoV‐2 protein or an immunogenic fragment or variant thereof,  (b) about 1.43 mg/ml ALC‐0315,  (c) about 0.18 mg/ml ALC‐0159  (d) about 0.31 mg/ml DSPC,  (e) about 0.62 mg/ml cholesterol,  (f) about 103 mg/ml sucrose,  (g) about 0.20 mg/ml tromethamine (Tris base),  (h) about 1.32 mg/ml Tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl), and  (i) q.s. water.    In one embodiment, the ratio of RNA (e.g., mRNA) to total lipid (N/P) is between 6.0 and 6.5  such as about 6.0 or about 6.3.    Nucleic acid containing particles  Nucleic acids described herein such as RNA encoding a vaccine antigen may be administered  formulated as particles.  In  the  context of  the present disclosure,  the  term  "particle"  relates  to a  structured entity  formed by molecules or molecule complexes. In one embodiment, the term "particle" relates  to  a  micro‐  or  nano‐sized  structure,  such  as  a  micro‐  or  nano‐sized  compact  structure  dispersed in a medium. In one embodiment, a particle is a nucleic acid containing particle such  as a particle comprising DNA, RNA or a mixture thereof.  Electrostatic  interactions between positively charged molecules such as polymers and  lipids  and  negatively  charged  nucleic  acid  are  involved  in  particle  formation.  This  results  in  complexation  and  spontaneous  formation of nucleic  acid particles.  In one embodiment,  a  nucleic acid particle is a nanoparticle.  As  used  in  the  present  disclosure,  "nanoparticle"  refers  to  a  particle  having  an  average  diameter suitable for parenteral administration.   A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g.,  cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one  cationic or cationically ionizable lipid or lipid‐like material, at least one cationic polymer such  as protamine, or a mixture thereof and nucleic acid. In some embodiments, exemplary  nucleic acid particles include lipid nanoparticles, polyplexes (PLX), lapidated polyplexes  (LPLX),  (LNP)‐based and lipoplex (LPX)‐based formulations, liposomes, or polysaccharide  nanoparticles.  In some embodiments, RNA encoding an amino acid sequence comprising a  SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the  SARS‐CoV‐2 S protein or the immunogenic variant thereof is formulated as LNPs.  In some  embodiments, LNPs comprise one or more cationically ionizable lipids; one or more neutral  lipids (e.g., in some embodiments a sterol such as, e.g., cholesterol; and/or phospholipids),  and one or more polymer‐conjugated lipids. In some embodiments, the formulation  comprises ALC‐0315 (4‐ hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  ALC‐0159 (2‐[(polyethylene glycol)‐2000]‐N,N‐ditetradecylacetamide), DSPC (1,2‐distearoyl‐ sn‐glycero‐3‐ phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol  hydrochloride and water.  RNA particles described herein include nanoparticles. In some  embodiments, exemplary nanoparticles include lipid nanoparticles, lipoplex, polyplexes  (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles.  Polyplexes  (PLX), polysaccharide nanoparticles, and liposomes, are all delivery technologies that are  well known to a person of skill in the art. See, e.g., Lächelt, Ulrich, and Ernst Wagner.  "Nucleic acid therapeutics using polyplexes: a journey of 50 years (and beyond)" Chemical  reviews 115.19 (2015): 11043‐11078; Plucinski, Alexander, Zan Lyu, and Bernhard VKJ  Schmidt, "Polysaccharide nanoparticles: from fabrication to applications." Journal of  Materials Chemistry B (2021); and Tenchov, Rumiana, et al. "Lipid Nanoparticles─ From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement,"  ACS nanooo15.11 (2021): 16982‐17015, respectively, the contents of each of which are  hereby incorporated by reference herein in their entirety.  In some embodiments, the  concentration of RNA in a pharmaceutical RNA preparation is about 0.1 mg/ml. In some  embodiments, the concentration of RNA in a pharmaceutical RNA preparation is about 30  µg/ml to about 100 µg/ml. In some embodiments, the concentration of RNA in a  pharmaceutical RNA preparation is about 50 µg/ml to about 100 µg/ml.  Without  intending to be bound by any theory,  it  is believed that the cationic or cationically  ionizable  lipid or  lipid‐like material and/or the cationic polymer combine together with the  nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.   In one embodiment, particles described herein further comprise at least one lipid or lipid‐like  material other than a cationic or cationically ionizable lipid or lipid‐like material, at least one  polymer other than a cationic polymer, or a mixture thereof  In  some embodiments, nucleic acid particles comprise more  than one  type of nucleic acid  molecules, where the molecular parameters of the nucleic acid molecules may be similar or  different from each other, like with respect to molar mass or fundamental structural elements  such as molecular architecture, capping, coding regions or other features.  Nucleic acid particles described herein may have an average diameter that in one embodiment  ranges from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about  70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about  300 nm.   Nucleic acid particles described herein may exhibit a polydispersity index less than about 0.5,  less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic  acid particles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about  0.2 to about 0.3.  With respect to RNA lipid particles, the N/P ratio gives the ratio of the nitrogen groups in the  lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the  nitrogen  atoms  (depending  on  the  pH)  are  usually  positively  charged  and  the  phosphate  groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on  the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve,  because positively charged nanoparticles are considered  favorable  for  transfection.  In  that  case, RNA is considered to be completely bound to nanoparticles.  Nucleic acid particles described herein can be prepared using a wide range of methods that  may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid‐ like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to  obtain nucleic acid particles.   The term "colloid" as used herein relates to a type of homogeneous mixture in which dispersed  particles do not settle out. The insoluble particles in the mixture are microscopic, with particle  sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal  suspension. Sometimes the term "colloid" only refers to the particles in the mixture and not  the entire suspension.  For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or  lipid‐like material and/or at least one cationic polymer methods are applicable herein that are  conventionally used for preparing liposomal vesicles and are appropriately adapted. The most  commonly used methods  for preparing  liposomal vesicles share  the  following  fundamental  stages: (i)  lipids dissolution  in organic solvents,  (ii) drying of the resultant solution, and  (iii)  hydration of dried lipid (using various aqueous media).   In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried  down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using  an  appropriate  aqueous  medium  to  produce  a  liposomal  dispersion.  Furthermore,  an  additional downsizing step may be included.   Reverse  phase  evaporation  is  an  alternative method  to  the  film  hydration  for  preparing  liposomal vesicles  that  involves  formation of a water‐in‐oil emulsion between an aqueous  phase and an organic phase containing lipids. A brief sonication of this mixture is required for  system homogenization. The removal of the organic phase under reduced pressure yields a  milky gel that turns subsequently into a liposomal suspension.   The  term  "ethanol  injection  technique"  refers  to  a  process,  in which  an  ethanol  solution  comprising  lipids  is rapidly  injected  into an aqueous solution  through a needle. This action  disperses  the  lipids  throughout  the  solution  and  promotes  lipid  structure  formation,  for  example  lipid  vesicle  formation  such  as  liposome  formation.  Generally,  the  RNA  lipoplex  particles described herein are obtainable by adding RNA to a colloidal  liposome dispersion.  Using  the  ethanol  injection  technique,  such  colloidal  liposome  dispersion  is,  in  one  embodiment, formed as follows: an ethanol solution comprising lipids, such as cationic lipids  and additional lipids, is injected into an aqueous solution under stirring. In one embodiment,  the RNA lipoplex particles described herein are obtainable without a step of extrusion.  The term "extruding" or "extrusion" refers to the creation of particles having a fixed, cross‐ sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is  forced through filters with defined pores.   Other methods having organic solvent free characteristics may also be used according to the  present disclosure for preparing a colloid.  LNPs  typically  comprise  four  components:  ionizable  cationic  lipids,  neutral  lipids  such  as  phospholipids,  a  steroid  such  as  cholesterol,  and  a  polymer  conjugated  lipid  such  as  polyethylene glycol (PEG)‐lipids. Each component is responsible for payload protection, and  enables effective  intracellular delivery. LNPs may be prepared by mixing  lipids dissolved  in  ethanol rapidly with nucleic acid in an aqueous buffer.  The  term  "average  diameter"  refers  to  the mean  hydrodynamic  diameter  of  particles  as  measured  by  dynamic  laser  light  scattering  (DLS)  with  data  analysis  using  the  so‐called  cumulant algorithm, which provides as results the so‐called Zaverage with the dimension of a  length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57,  1972, pp 4814‐4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles  is used synonymously with this value of the Zaverage.  The  "polydispersity  index"  is  preferably  calculated  based  on  dynamic  light  scattering  measurements  by  the  so‐called  cumulant  analysis  as mentioned  in  the  definition  of  the  "average diameter". Under  certain prerequisites,  it  can be  taken as a measure of  the  size  distribution of an ensemble of nanoparticles.  Different  types  of  nucleic  acid  containing  particles  have  been  described  previously  to  be  suitable  for delivery of nucleic  acid  in particulate  form  (e.g.  Kaczmarek,  J. C.  et  al.,  2017,  Genome  Medicine  9,  60).  For  non‐viral  nucleic  acid  delivery  vehicles,  nanoparticle  encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending  on the specific chemistry, can aid in cellular uptake and endosomal escape.  The present disclosure describes particles  comprising nucleic acid, at  least one  cationic or  cationically  ionizable  lipid or  lipid‐like material, and/or at  least one cationic polymer which  associate with nucleic acid to form nucleic acid particles and compositions comprising such  particles. The nucleic acid particles may comprise nucleic acid which is complexed in different  forms by non‐covalent interactions to the particle. The particles described herein are not viral  particles, in particular infectious viral particles, i.e., they are not able to virally infect cells.   Suitable cationic or cationically ionizable lipids or lipid‐like materials and cationic polymers are  those  that  form  nucleic  acid  particles  and  are  included  by  the  term  "particle  forming  components"  or  "particle  forming  agents".  The  term  "particle  forming  components"  or  "particle forming agents" relates to any components which associate with nucleic acid to form  nucleic acid particles. Such components include any component which can be part of nucleic  acid particles.  In  some  embodiments,  a  nucleic  acid  containing  particle  (e.g.,  a  lipid  nanoparticle  (LNP))  comprises two or more RNA molecules, each comprising a different nucleic acid sequence.  In  some embodiments, a nucleic acid containing particle comprises two or more RNA molecules,  each encoding a different  immunogenic polypeptide or  immunogenic  fragment  thereof.  In  some embodiments, two or more RNA molecules present in a nucleic acid containing particle  comprise:  a  first  RNA  molecule  encodes  an  immunogenic  polypeptide  or  immunogenic  fragment thereof from a coronavirus and a second RNA molecule encodes an immunogenic  polypeptide  or  immunogenic  fragment  thereof  from  an  infectious  disease  pathogen  (e.g.,  virus,  bacteria,  parasite,  etc.).  For  example,  in  some  embodiments,  two  or  more  RNA  molecules present in a nucleic acid containing particle comprise: a first RNA molecule encoding  an immunogenic polypeptide or immunogenic fragment thereof from a coronavirus (e.g.,  in  some embodiments SARS‐CoV‐2 Wuhan strain or a variant thereof, e.g., a SARS‐CoV‐2 having  one or more mutations  characteristic of an Omicron  variant)  and a  second RNA molecule  encoding an immunogenic polypeptide or immunogenic fragment thereof from an influenza  virus.    In  some  embodiments,  a  nucleic  acid  containing  particle  comprises:  a  first RNA molecule  encoding  an  immunogenic  polypeptide  or  immunogenic  fragment  thereof  from  a  first  coronavirus (e.g., as described herein) and a second RNA molecule encoding an immunogenic  polypeptide or immunogenic fragment thereof from a second coronavirus (e.g., as described  herein). In some embodiments, a first coronavirus is different from a second coronavirus. In  some embodiments, a  first and/or second coronavirus  is  independently  from a SARS‐CoV‐2  Wuhan  strain  or  a  variant  thereof,  e.g.,  a  SARS‐CoV‐2  having  one  or  more  mutations  characteristic of an Omicron variant.    In  some embodiments,  two or more RNA molecules  present in a nucleic acid containing particle each encode an immunogenic polypeptide or an  immunogenic  fragment  thereof  from  the  same  and/or different  strains  and/or  variants of  coronavirus (e.g., in some embodiments SARS‐CoV‐2 strains or variants).  For example, in some  embodiments, two or more RNA molecules present in a nucleic acid containing particle each  encode  a  different  immunogenic  polypeptide  or  immunogenic  fragment  thereof  from  a  coronavirus  membrane  protein,  a  coronavirus  nucleocapsid  protein,  a  coronavirus  spike  protein, a coronavirus non‐structural protein and/or a coronavirus accessory protein. In some  embodiments, such  immunogenic polypeptides or  immunogenic  fragments thereof may be  from the same or a different coronavirus (e.g., in some embodiments a SARS‐CoV‐2 Wuhan  strain or variants thereof, for example, in some embodiments a variant having one or more  mutations  characteristic  of  a  prevalent  variant  such  as  an  Omicron  variant).    In  some  embodiments, a nucleic acid containing particle comprises a first RNA molecule encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof from a first strain or variant, and  a second RNA molecule encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  from a second strain or variant, wherein the second strain or variant is different from the first  strain or variant.    In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises a first RNA molecule encoding a SARS‐CoV‐2 S protein from a  Wuhan strain and a second RNA molecule encoding a SARS‐CoV‐2 S protein comprising one or  more mutations that are characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.3, BA.4,  or BA.5 Omicron variant).  In some embodiments, a nucleic acid containing particle comprises  a first RNA molecule encoding a SARS‐CoV‐2 S protein from a Wuhan strain and a second RNA  molecule  encoding  a  SARS‐CoV‐2  S  protein  comprising  one  or more mutations  that  are  characteristic of an Omicron BA.1 variant.    In some embodiments, a nucleic acid containing  particle comprises a first RNA molecule encoding a SARS‐CoV‐2 S protein from a Wuhan strain  and  a  second  RNA molecule  encoding  a  SARS‐CoV‐2  S  protein  comprising  one  or more  mutations that are characteristic of an Omicron BA.2 variant.  In some embodiments, a nucleic  acid containing particle comprises a first RNA molecule encoding a SARS‐CoV‐2 S protein from  a Wuhan strain and a second RNA molecule encoding a SARS‐CoV‐2 S protein comprising one  or more mutations that are characteristic of an Omicron BA.3 variant.  In some embodiments,  a nucleic acid containing particle comprises a  first RNA molecule encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and a second RNA molecule encoding a SARS‐CoV‐2 S protein  comprising one or more mutations that are characteristic of an Omicron BA.4 or BA.5 variant.    In  some  embodiments,  a  nucleic  acid  containing  particle  comprises  a  first  RNA molecule  encoding a SARS‐CoV‐2 S protein  from a  first Omicron variant and a second RNA molecule  encoding a SARS‐CoV‐2 S protein comprising one or more mutations that are characteristic of  a second Omicron variant.  In some embodiments, a nucleic acid containing particle comprises  a  first RNA molecule encoding a SARS‐CoV‐2 S protein  from a BA.1 Omicron variant and a  second RNA molecule encoding a SARS‐CoV‐2 S protein comprising one or more mutations  that  are  characteristic  of  a  BA.2 Omicron  variant.    In  some  embodiments,  a  nucleic  acid  containing particle comprises a first RNA molecule encoding a SARS‐CoV‐2 S protein from a  BA.1 Omicron variant and a second RNA molecule encoding a SARS‐CoV‐2 S protein comprising  one  or  more  mutations  that  are  characteristic  of  a  BA.3  Omicron  variant.    In  some  embodiments, a nucleic acid containing particle comprises a first RNA molecule encoding a  SARS‐CoV‐2 S protein from a BA.1 Omicron variant and a second RNA molecule encoding a  SARS‐CoV‐2 S protein comprising one or more mutations that are characteristic of a BA.4 or  BA.5 Omicron variant.  In some embodiments, a nucleic acid containing particle comprises a  first RNA molecule encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant and a second  RNA molecule encoding a SARS‐CoV‐2 S protein comprising one or more mutations that are  characteristic of a BA.3 Omicron variant.    In some embodiments, a nucleic acid containing  particle comprises a first RNA molecule encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron  variant and a second RNA molecule encoding a SARS‐CoV‐2 S protein comprising one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a  nucleic  acid  containing  particle  comprises  a  first  RNA molecule  encoding  a  SARS‐CoV‐2  S  protein from a BA.3 Omicron variant and a second RNA molecule encoding a SARS‐CoV‐2 S  protein comprising one or more mutations that are characteristic of a BA.4 or BA.5 Omicron  variant.    In some embodiments, a first RNA molecule encoding a SARS‐CoV‐2 S protein from a Wuhan  strain comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:  7. In some embodiments, a first RNA molecule encoding a SARS‐CoV‐2 S protein from a Wuhan  strain comprises a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least  97%, at  least 98%, or at  least 99%  identical) to SEQ ID NO: 9.  In some embodiments, a first  RNA molecule encoding a SARS‐COV‐2 S protein from a Wuhan strain comprises a nucleotide  sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least 91%, at least  92%, at least 93%, at least 94%, at  least 95%, at  least 96%, at least 97%, at least 98%, or at  least 99% identical to) SEQ ID NO: 20. In some embodiments, a first RNA molecule encoding a  SARS‐COV‐2 S protein from a Wuhan strain comprises a nucleotide sequence that encodes an  amino acid sequence that is at least 80% identical to (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, or at least 99% identical to) SEQ ID NO: 7.  In some embodiments, a second RNA molecule  encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of an  Omicron variant comprises a nucleotide sequence that encodes the amino acid sequence of  SEQ  ID NO: 49.    In  some embodiments, a  second RNA molecule encoding a SARS‐CoV‐2 S  protein comprising one or more mutations characteristic of an Omicron variant comprises a  nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 91%,  at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,  or at  least 99%  identical) to SEQ ID NO: 50.  In some embodiments, a second RNA molecule  encoding  a  SARS‐COV‐2  S  protein  comprising  one  or more mutations  characteristic  of  an  Omicron variant comprises a nucleotide sequence that is at least 80% identical to (e.g., at least  85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least  96%,  at  least  97%,  at  least  98%,  or  at  least  99%  identical  to)  SEQ  ID  NO:  51.  In  some  embodiments, a second RNA molecule encoding a SARS‐COV‐2 S protein comprising one or  more mutations characteristic of an Omicron variant comprises a nucleotide sequence that  encodes an amino acid sequence that is at least 80% identical to (e.g., at least 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least  97%, at least 98%, or at least 99% identical to) SEQ ID NO: 49.    In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that  encodes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 7); and a second RNA molecule comprising a nucleotide sequence that encodes the amino  acid sequence of SEQ ID NO: 49 or an amino acid sequence that is at least 80% (e.g., at least  85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least  96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 49.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 9); and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at  least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at  least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 50.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 20; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence that is at least 80% (e.g., at  least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at  least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 51.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that  encodes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 7); and a second RNA molecule comprising a nucleotide sequence that encodes the amino  acid sequence of SEQ ID NO: 55, 58, or 61 or an amino acid sequence that is at least 80% (e.g.,  at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,  at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 55, 58,  or 61.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 9; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 56, 59, or 62  or a nucleotide sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 56, 59, or 62.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 20 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 20; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 57, 60, or 63 or a nucleotide sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 57, 60, or 63.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that  encodes the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 58; and a second RNA molecule comprising a nucleotide sequence that encodes an amino  acid sequence of SEQ ID NO: 49, 55, or 61 or an amino acid sequence that is at least 80% (e.g.,  at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,  at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 49, 55,  or 61.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 59 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 59; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 50, 56, or 62, or a nucleotide sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 50, 56, or 62.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 60 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 60; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 51, 57, or 63, or a nucleotide sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 51, 57, or 63.    In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that  encodes the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 49; and a second RNA molecule comprising a nucleotide sequence that encodes the amino  acid sequence of SEQ ID NO: 55 or 61 or an amino acid sequence that is at least 80% (e.g., at  least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at  least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 55 or 61.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 50; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 56 or 62 or a nucleotide sequence that is at least 80%  (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least  95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO:  56 or 62.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 51 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 51; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 57 or 63 or a nucleotide sequence that is at least 80%  (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least  95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO:  57 or 63.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence that  encodes the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence that is at least  80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at  least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID  NO: 55; and a second RNA molecule comprising a nucleotide sequence that encodes the amino  acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 80% (e.g., at least  85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least  96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 61.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 56 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 56; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence that is at least 80% (e.g., at  least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at  least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 62.  In some embodiments, a nucleic acid containing particle (e.g., in some embodiments an LNP  as described herein) comprises: a first RNA molecule comprising a nucleotide sequence of SEQ  ID NO: 57 or a nucleotide sequence that is at least 80% (e.g., at least 85%, at least 90%, at least  91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least  98%, at least 99% or higher) identical to SEQ ID NO: 57; and a second RNA molecule comprising  a nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence that is at least 80% (e.g., at  least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at  least 96%, at least 97%, at least 98%, at least 99% or higher) identical to SEQ ID NO: 63.  In some embodiments, a particle (e.g., in some embodiments an LNP) containing nucleic acids  (e.g., RNAs) encoding different polypeptides can be formed by mixing a plurality of (e.g., at  least  two, at  least  three, or more) RNA molecules with particle‐forming components  (e.g.,  lipids). In some embodiments, nucleic acids (e.g., RNAs) encoding different polypeptides can  be  mixed  (e.g.,  in  some  embodiments  in  substantially  equal  proportions,  e.g.,  in  some  embodiments  at  a  1:1  ratio when  two  RNA molecules  are  present)  prior  to mixing with  particle‐forming components (e.g., lipids).    In some embodiments, two or more RNA molecules each encoding a different polypeptide  (e.g., as described herein)  can be mixed with particle‐forming agents  to  form nucleic acid  containing  particles  as  described  above.  In  alternative  embodiments,  two  or  more  RNA  molecules each encoding a different polypeptide (e.g., as described herein) can be formulated  into separate particle compositions, which are  then mixed  together. For example,  in some  embodiments,  individual  populations  of  nucleic  acid  containing  particles,  each  population  comprising an RNA molecule encoding a different immunogenic polypeptide or immunogenic  fragment  thereof  (e.g.,  as  described  herein),  can  be  separately  formed  and  then mixed  together, for example, prior to filling into vials during a manufacturing process, or immediately  prior to administration (e.g., by an administering health‐care professional)).  Accordingly,  in  some embodiments, described herein is a composition comprises two or more populations of  particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least  one RNA molecule encoding a different immunogenic polypeptide or immunogenic fragment  thereof (e.g., a SARS‐CoV‐2 S protein, or fragments thereof, from a different variant). In some  embodiments, each population may be provided in a composition at a desirable proportion  (e.g., in some embodiments, each population may be provided in a composition in an amount  that provides the same amount of RNA molecules).      Cationic polymer  Given  their high degree of  chemical  flexibility, polymers are  commonly used materials  for  nanoparticle‐based  delivery.  Typically,  cationic  polymers  are  used  to  electrostatically  condense  the negatively  charged nucleic acid  into nanoparticles. These positively  charged  groups often consist of amines that change their state of protonation in the pH range between  5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers  such as poly‐L‐lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally  occurring polymers such as chitosan have all been applied  to nucleic acid delivery and are  suitable  as  cationic  polymers  herein.  In  addition,  some  investigators  have  synthesized  polymers specifically for nucleic acid delivery. Poly(β‐amino esters), in particular, have gained  widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability.  Such synthetic polymers are also suitable as cationic polymers herein.  A  "polymer,"  as  used  herein,  is  given  its  ordinary  meaning,  i.e.,  a  molecular  structure  comprising one or more repeat units (monomers), connected by covalent bonds. The repeat  units can all be  identical, or in some cases, there can be more than one type of repeat unit  present  within  the  polymer.  In  some  cases,  the  polymer  is  biologically  derived,  i.e.,  a  biopolymer such as a protein. In some cases, additional moieties can also be present  in the  polymer, for example targeting moieties such as those described herein.  If more than one type of repeat unit is present within the polymer, then the polymer is said to  be a "copolymer."  It  is to be understood that the polymer being employed herein can be a  copolymer.  The  repeat  units  forming  the  copolymer  can  be  arranged  in  any  fashion.  For  example, the repeat units can be arranged in a random order, in an alternating order, or as a  "block" copolymer,  i.e., comprising one or more regions each comprising a first repeat unit  (e.g., a  first block), and one or more  regions each comprising a second  repeat unit  (e.g., a  second block), etc. Block copolymers can have  two  (a diblock copolymer),  three  (a  triblock  copolymer), or more numbers of distinct blocks.  In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers  that  typically do not  result  in  significant  cell death at moderate  concentrations.  In  certain  embodiments,  the  biocompatible  polymer  is  biodegradable,  i.e.,  the  polymer  is  able  to  degrade, chemically and/or biologically, within a physiological environment, such as within the  body.   In  certain  embodiments,  polymer  may  be  protamine  or  polyalkyleneimine,  in  particular  protamine.  The  term  "protamine"  refers  to  any  of  various  strongly  basic  proteins  of  relatively  low  molecular weight that are rich  in arginine and are  found associated especially with DNA  in  place of somatic histones in the sperm cells of various animals (as fish). In particular, the term  "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water,  are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they  are used in a long‐acting formulation of insulin and to neutralize the anticoagulant effects of  heparin.  According to the disclosure, the term "protamine" as used herein  is meant to comprise any  protamine  amino  acid  sequence  obtained  or  derived  from  natural  or  biological  sources  including fragments thereof and multimeric forms of said amino acid sequence or fragment  thereof as well as (synthesized) polypeptides which are artificial and specifically designed for  specific purposes and cannot be isolated from native or biological sources.  In  one  embodiment,  the  polyalkyleneimine  comprises  polyethylenimine  and/or  polypropylenimine,  preferably  polyethyleneimine.  A  preferred  polyalkyleneimine  is  polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75∙102 to 107 Da,  preferably 1000  to 105 Da, more preferably 10000  to 40000 Da, more preferably 15000  to  30000 Da, even more preferably 20000 to 25000 Da.  Preferred  according  to  the  disclosure  is  linear  polyalkyleneimine  such  as  linear  polyethyleneimine (PEI).   Cationic polymers (including polycationic polymers) contemplated for use herein include any  cationic polymers which are able to electrostatically bind nucleic acid.  In one embodiment,  cationic  polymers  contemplated  for  use  herein  include  any  cationic  polymers with which  nucleic acid can be associated, e.g. by  forming complexes with  the nucleic acid or  forming  vesicles in which the nucleic acid is enclosed or encapsulated.  Particles described herein may also comprise polymers other than cationic polymers, i.e., non‐ cationic polymers  and/or  anionic polymers. Collectively,  anionic  and neutral polymers  are  referred to herein as non‐cationic polymers.     Lipid and lipid‐like material  The  terms  "lipid"  and  "lipid‐like material"  are  broadly  defined  herein  as molecules which  comprise  one  or more  hydrophobic moieties  or  groups  and  optionally  also  one  or more  hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic  moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water.  In an aqueous environment, the amphiphilic nature allows the molecules to self‐assemble into  organized structures and different phases. One of those phases consists of  lipid bilayers, as  they  are  present  in  vesicles,  multilamellar/unilamellar  liposomes,  or  membranes  in  an  aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that  include, but are not  limited to,  long‐chain saturated and unsaturated aliphatic hydrocarbon  groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic  group(s).  The  hydrophilic  groups may  comprise  polar  and/or  charged  groups  and  include  carbohydrates, phosphate, carboxylic,  sulfate, amino,  sulfhydryl, nitro, hydroxyl, and other  like groups.  As used herein, the term "amphiphilic" refers to a molecule having both a polar portion and a  non‐polar  portion. Often,  an  amphiphilic  compound  has  a  polar  head  attached  to  a  long  hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non‐ polar portion  is  insoluble  in water.  In addition, the polar portion may have either a  formal  positive charge, or a formal negative charge. Alternatively, the polar portion may have both a  formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the  disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural  or non‐natural lipids and lipid‐like compounds.   The  term  "lipid‐like  material",  "lipid‐like  compound"  or  "lipid‐like  molecule"  relates  to  substances that structurally and/or functionally relate to lipids but may not be considered as  lipids  in  a  strict  sense.  For  example,  the  term  includes  compounds  that  are  able  to  form  amphiphilic  layers  as  they  are  present  in  vesicles, multilamellar/unilamellar  liposomes,  or  membranes in an aqueous environment and includes surfactants, or synthesized compounds  with  both  hydrophilic  and  hydrophobic moieties.  Generally  speaking,  the  term  refers  to  molecules, which  comprise hydrophilic and hydrophobic moieties with different  structural  organization, which may or may not be similar to that of lipids. As used herein, the term "lipid"  is  to be  construed  to  cover both  lipids  and  lipid‐like materials unless otherwise  indicated  herein or clearly contradicted by context.  Specific examples of amphiphilic  compounds  that may be  included  in an amphiphilic  layer  include, but are not limited to, phospholipids, aminolipids and sphingolipids.  In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group  of organic compounds that are characterized by being insoluble in water, but soluble in many  organic  solvents.  Generally,  lipids  may  be  divided  into  eight  categories:  fatty  acids,  glycerolipids, glycerophospholipids,  sphingolipids,  saccharolipids, polyketides  (derived  from  condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation  of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats  are a subgroup of  lipids called  triglycerides. Lipids also encompass molecules such as  fatty  acids and their derivatives (including tri‐, di‐, monoglycerides, and phospholipids), as well as  sterol‐containing metabolites such as cholesterol.   Fatty acids, or  fatty acid residues are a diverse group of molecules made of a hydrocarbon  chain  that  terminates with a carboxylic acid group;  this arrangement confers  the molecule  with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The  carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated,  and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur.  If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric  isomerism, which significantly affects the molecule's configuration. Cis‐double bonds cause  the  fatty acid chain to bend, an effect that  is compounded with more double bonds  in the  chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.   Glycerolipids are composed of mono‐, di‐, and tri‐substituted glycerols, the best‐known being  the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes  used  synonymously with  "triglyceride".  In  these  compounds,  the  three hydroxyl groups of  glycerol  are  each  esterified,  typically  by  different  fatty  acids.  Additional  subclasses  of  glycerolipids are represented by glycosylglycerols, which are characterized by the presence of  one or more sugar residues attached to glycerol via a glycosidic linkage.   The  glycerophospholipids  are  amphipathic  molecules  (containing  both  hydrophobic  and  hydrophilic regions) that contain a glycerol core linked to two fatty acid‐derived "tails" by ester  linkages  and  to  one  "head"  group  by  a  phosphate  ester  linkage.  Examples  of  glycerophospholipids, usually referred  to as phospholipids  (though sphingomyelins are also  classified as phospholipids) are phosphatidylcholine  (also known as PC, GPCho or  lecithin),  phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).   Sphingolipids are a complex family of compounds that share a common structural feature, a  sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as  sphingosine.  Ceramides  (N‐acyl‐sphingoid  bases)  are  a major  subclass  of  sphingoid  base  derivatives with an amide‐linked fatty acid. The fatty acids are typically saturated or mono‐ unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids  of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly  ceramide  phosphoethanolamines  and  fungi  have  phytoceramide  phosphoinositols  and  mannose‐containing headgroups. The  glycosphingolipids  are  a diverse  family of molecules  composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base.  Examples of these are the simple and complex glycosphingolipids such as cerebrosides and  gangliosides.  Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an  important  component  of  membrane  lipids,  along  with  the  glycerophospholipids  and  sphingomyelins.   Saccharolipids  describe  compounds  in  which  fatty  acids  are  linked  directly  to  a  sugar  backbone,  forming  structures  that  are  compatible  with  membrane  bilayers.  In  the  saccharolipids,  a  monosaccharide  substitutes  for  the  glycerol  backbone  present  in  glycerolipids  and  glycerophospholipids.  The most  familiar  saccharolipids  are  the  acylated  glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram‐negative  bacteria. Typical  lipid A molecules are disaccharides of glucosamine, which are derivatized  with as many as seven fatty‐acyl chains. The minimal lipopolysaccharide required for growth  in E. coli is Kdo2‐Lipid A, a hexa‐acylated disaccharide of glucosamine that is glycosylated with  two 3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residues.  Polyketides  are  synthesized  by  polymerization  of  acetyl  and  propionyl  subunits  by  classic  enzymes as well as iterative and multimodular enzymes that share mechanistic features with  the fatty acid synthases. They comprise a large number of secondary metabolites and natural  products from animal, plant, bacterial, fungal and marine sources, and have great structural  diversity. Many polyketides are cyclic molecules whose backbones are often further modified  by glycosylation, methylation, hydroxylation, oxidation, or other processes.   According to the disclosure, lipids and lipid‐like materials may be cationic, anionic or neutral.  Neutral  lipids or  lipid‐like materials exist  in an uncharged or neutral zwitterionic  form at a  selected pH.     Cationic or cationically ionizable lipids or lipid‐like materials  The nucleic acid particles described herein may comprise at least one cationic or cationically  ionizable lipid or lipid‐like material as particle forming agent. Cationic or cationically ionizable  lipids or  lipid‐like materials contemplated for use herein  include any cationic or cationically  ionizable  lipids or  lipid‐like materials which are able to electrostatically bind nucleic acid.  In  one embodiment, cationic or cationically ionizable lipids or lipid‐like materials contemplated  for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic  acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.  As used herein, a "cationic  lipid" or "cationic lipid‐like material" refers to a lipid or lipid‐like  material having a net positive  charge. Cationic  lipids or  lipid‐like materials bind negatively  charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic  moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the  lipid typically carries the positive charge.   In certain embodiments, a cationic lipid or lipid‐like material has a net positive charge only at  certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably  has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH.  This ionizable behavior is thought to enhance efficacy through helping with endosomal escape  and reducing toxicity as compared with particles that remain cationic at physiological pH.  For  purposes  of  the  present  disclosure,  such  "cationically  ionizable"  lipids  or  lipid‐like  materials are comprised by the term "cationic lipid or lipid‐like material" unless contradicted  by the circumstances.   In one embodiment, the cationic or cationically ionizable lipid or lipid‐like material comprises  a head  group which  includes  at  least one nitrogen  atom  (N) which  is positive  charged or  capable of being protonated.  Examples of cationic lipids include, but are not limited to 1,2‐dioleoyl‐3‐trimethylammonium  propane (DOTAP); N,N‐dimethyl‐2,3‐dioleyloxypropylamine (DODMA), 1,2‐di‐O‐octadecenyl‐ 3‐trimethylammonium  propane  (DOTMA),  3‐(N—(N′,N′‐dimethylaminoethane)‐ carbamoyl)cholesterol  (DC‐Chol),  dimethyldioctadecylammonium  (DDAB);  1,2‐dioleoyl‐3‐ dimethylammonium‐propane  (DODAP);  1,2‐diacyloxy‐3‐dimethylammonium  propanes;  1,2‐ dialkyloxy‐3‐dimethylammonium  propanes;  dioctadecyldimethyl  ammonium  chloride  (DODAC),  1,2‐distearyloxy‐N,N‐dimethyl‐3‐aminopropane  (DSDMA),  2,3‐ di(tetradecoxy)propyl‐(2‐hydroxyethyl)‐dimethylazanium  (DMRIE),  1,2‐dimyristoyl‐sn‐ glycero‐3‐ethylphosphocholine  (DMEPC),  l,2‐dimyristoyl‐3‐trimethylammonium  propane  (DMTAP),  1,2‐dioleyloxypropyl‐3‐dimethyl‐hydroxyethyl  ammonium  bromide  (DORIE),  and  2,3‐dioleoyloxy‐  N‐[2(spermine  carboxamide)ethyl]‐N,N‐dimethyl‐l‐propanamium  trifluoroacetate  (DOSPA),  1,2‐dilinoleyloxy‐N,N‐dimethylaminopropane  (DLinDMA),  1,2‐ dilinolenyloxy‐N,N‐dimethylaminopropane  (DLenDMA),  dioctadecylamidoglycyl  spermine  (DOGS),  3‐dimethylamino‐2‐(cholest‐5‐en‐3‐beta‐oxybutan‐4‐oxy)‐1‐(cis,cis‐9,12‐oc‐ tadecadienoxy)propane  (CLinDMA),  2‐[5′‐(cholest‐5‐en‐3‐beta‐oxy)‐3′‐oxapentoxy)‐3‐ dimethyl‐1‐(cis,cis‐9′,12′‐octadecadienoxy)propane  (CpLinDMA),  N,N‐dimethyl‐3,4‐ dioleyloxybenzylamine  (DMOBA),  1,2‐N,N′‐dioleylcarbamyl‐3‐dimethylaminopropane  (DOcarbDAP),  2,3‐Dilinoleoyloxy‐N,N‐dimethylpropylamine  (DLinDAP),  1,2‐N,N′‐ Dilinoleylcarbamyl‐3‐dimethylaminopropane  (DLincarbDAP),  1,2‐Dilinoleoylcarbamyl‐3‐ dimethylaminopropane  (DLinCDAP),  2,2‐dilinoleyl‐4‐dimethylaminomethyl‐[1,3]‐dioxolane  (DLin‐K‐DMA),  2,2‐dilinoleyl‐4‐dimethylaminoethyl‐[1,3]‐dioxolane  (DLin‐K‐XTC2‐DMA),  2,2‐ dilinoleyl‐4‐(2‐dimethylaminoethyl)‐[1,3]‐dioxolane  (DLin‐KC2‐DMA),  heptatriaconta‐ 6,9,28,31‐tetraen‐19‐yl‐4‐(dimethylamino)butanoate  (DLin‐MC3‐DMA),  N‐(2‐Hydroxyethyl)‐ N,N‐dimethyl‐2,3‐bis(tetradecyloxy)‐1‐propanaminium  bromide  (DMRIE),  (±)‐N‐(3‐ aminopropyl)‐N,N‐dimethyl‐2,3‐bis(cis‐9‐tetradecenyloxy)‐1‐propanaminium  bromide  (GAP‐ DMORIE),  (±)‐N‐(3‐aminopropyl)‐N,N‐dimethyl‐2,3‐bis(dodecyloxy)‐1‐propanaminium  bromide  (GAP‐DLRIE),  (±)‐N‐(3‐aminopropyl)‐N,N‐dimethyl‐2,3‐bis(tetradecyloxy)‐1‐ propanaminium  bromide  (GAP‐DMRIE),  N‐(2‐Aminoethyl)‐N,N‐dimethyl‐2,3‐ bis(tetradecyloxy)‐1‐propanaminium  bromide  (βAE‐DMRIE),  N‐(4‐carboxybenzyl)‐N,N‐ dimethyl‐2,3‐bis(oleoyloxy)propan‐1‐aminium  (DOBAQ),  2‐({8‐[(3β)‐cholest‐5‐en‐3‐ yloxy]octyl}oxy)‐N,N‐dimethyl‐3‐[(9Z,12Z)‐octadeca‐9,12‐dien‐1‐yloxy]propan‐1‐amine  (Octyl‐CLinDMA),  1,2‐dimyristoyl‐3‐dimethylammonium‐propane  (DMDAP),  1,2‐dipalmitoyl‐ 3‐dimethylammonium‐propane  (DPDAP),  N1‐[2‐((1S)‐1‐[(3‐aminopropyl)amino]‐4‐[di(3‐ amino‐propyl)amino]butylcarboxamido)ethyl]‐3,4‐di[oleyloxy]‐benzamide  (MVL5),  1,2‐ dioleoyl‐sn‐glycero‐3‐ethylphosphocholine (DOEPC), 2,3‐bis(dodecyloxy)‐N‐(2‐hydroxyethyl)‐ N,N‐dimethylpropan‐1‐amonium  bromide  (DLRIE),  N‐(2‐aminoethyl)‐N,N‐dimethyl‐2,3‐ bis(tetradecyloxy)propan‐1‐aminium  bromide  (DMORIE),  di((Z)‐non‐2‐en‐1‐yl)  8,8'‐ ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate  (ATX),  N,N‐dimethyl‐2,3‐ bis(dodecyloxy)propan‐1‐amine  (DLDMA),  N,N‐dimethyl‐2,3‐bis(tetradecyloxy)propan‐1‐ amine  (DMDMA),  Di((Z)‐non‐2‐en‐1‐yl)‐9‐((4‐ (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N‐Dodecyl‐3‐((2‐dodecylcarbamoyl‐ ethyl)‐{2‐[(2‐dodecylcarbamoyl‐ethyl)‐2‐{(2‐dodecylcarbamoyl‐ethyl)‐[2‐(2‐ dodecylcarbamoyl‐ethylamino)‐ethyl]‐amino}‐ethylamino)propionamide (lipidoid 98N12‐5), 1‐ [2‐[bis(2‐hydroxydodecyl)amino]ethyl‐[2‐[4‐[2‐[bis(2  hydroxydodecyl)amino]ethyl]piperazin‐ 1‐yl]ethyl]amino]dodecan‐2‐ol (lipidoid C12‐200).   In some embodiments, the cationic lipid may comprise from about 10 mol % to about 100 mol  %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol %  to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the  particle.     Additional lipids or lipid‐like materials  Particles described herein may also comprise lipids or lipid‐like materials other than cationic  or  cationically  ionizable  lipids  or  lipid‐like  materials,  i.e.,  non‐cationic  lipids  or  lipid‐like  materials  (including  non‐cationically  ionizable  lipids  or  lipid‐like  materials).  Collectively,  anionic and neutral lipids or lipid‐like materials are referred to herein as non‐cationic lipids or  lipid‐like materials. Optimizing the formulation of nucleic acid particles by addition of other  hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid  or lipid‐like material may enhance particle stability and efficacy of nucleic acid delivery.  An additional lipid or lipid‐like material may be incorporated which may or may not affect the  overall charge of  the nucleic acid particles.  In certain embodiments,  the additional  lipid or  lipid‐like material  is  a  non‐cationic  lipid  or  lipid‐like material.  The  non‐cationic  lipid may  comprise, e.g., one or more anionic  lipids and/or neutral  lipids. As used herein, an "anionic  lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, a "neutral  lipid" refers to any of a number of  lipid species that exist either  in an uncharged or neutral  zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises  one  of  the  following  neutral  lipid  components:  (1)  a  phospholipid,  (2)  cholesterol  or  a  derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.  Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone,  cholestenone,  coprostanol,  cholesteryl‐2'‐hydroxyethyl  ether,  cholesteryl‐4'‐  hydroxybutyl  ether, tocopherol and derivatives thereof, and mixtures thereof.   Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines,  phosphatidylethanolamines, phosphatidylglycerols, phosphatidic  acids, phosphatidylserines  or sphingomyelin. Such phospholipids  include  in particular diacylphosphatidylcholines, such  as  distearoylphosphatidylcholine  (DSPC),  dioleoylphosphatidylcholine  (DOPC),  dimyristoylphosphatidylcholine  (DMPC),  dipentadecanoylphosphatidylcholine,  dilauroylphosphatidylcholine,  dipalmitoylphosphatidylcholine  (DPPC),  diarachidoylphosphatidylcholine  (DAPC),  dibehenoylphosphatidylcholine  (DBPC),  ditricosanoylphosphatidylcholine  (DTPC),  dilignoceroylphatidylcholine  (DLPC),  palmitoyloleoyl‐phosphatidylcholine  (POPC),  1,2‐di‐O‐octadecenyl‐sn‐glycero‐3‐ phosphocholine  (18:0  Diether  PC),  1‐oleoyl‐2‐cholesterylhemisuccinoyl‐sn‐glycero‐3‐ phosphocholine  (OChemsPC),  1‐hexadecyl‐sn‐glycero‐3‐phosphocholine  (C16  Lyso  PC)  and  phosphatidylethanolamines,  in  particular  diacylphosphatidylethanolamines,  such  as  dioleoylphosphatidylethanolamine  (DOPE),  distearoyl‐phosphatidylethanolamine  (DSPE),  dipalmitoyl‐phosphatidylethanolamine  (DPPE),  dimyristoyl‐phosphatidylethanolamine  (DMPE), dilauroyl‐phosphatidylethanolamine (DLPE), diphytanoyl‐phosphatidylethanolamine  (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.  In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.  In  certain  embodiments,  the  nucleic  acid  particles  include  both  a  cationic  lipid  and  an  additional lipid.   In one embodiment, particles described herein include a polymer conjugated lipid such as a  pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion  and a polyethylene glycol portion. Pegylated lipids are known in the art.  Without wishing to be bound by theory, the amount of the at least one cationic lipid compared  to the amount of the at least one additional lipid may affect important nucleic acid particle  characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the  nucleic acid. Accordingly, in some embodiments, the molar ratio of the at least one cationic  lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2,  or about 3:1 to about 1:1.  In some embodiments,  the non‐cationic  lipid,  in particular neutral  lipid,  (e.g., one or more  phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from  about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol  % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in  the particle.     Lipoplex Particles  In certain embodiments of the present disclosure, the RNA described herein may be present  in RNA lipoplex particles.   In the context of the present disclosure, the term "RNA lipoplex particle" relates to a particle  that contains  lipid,  in particular  cationic  lipid, and RNA. Electrostatic  interactions between  positively  charged  liposomes  and  negatively  charged  RNA  results  in  complexation  and  spontaneous  formation  of  RNA  lipoplex  particles.  Positively  charged  liposomes  may  be  generally  synthesized using a  cationic  lipid,  such as DOTMA, and additional  lipids,  such as  DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.  In  certain  embodiments,  the  RNA  lipoplex  particles  include  both  a  cationic  lipid  and  an  additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional  lipid is DOPE.   In some embodiments, the molar ratio of the at  least one cationic  lipid to the at  least one  additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about  1:1.  In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1,  about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary  embodiment, the molar ratio of the at least one cationic lipid to the at least one additional  lipid is about 2:1.  RNA  lipoplex particles described herein have an average diameter that  in one embodiment  ranges  from about 200 nm  to about 1000 nm,  from about 200 nm  to about 800 nm,  from  about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about  500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex  particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275  nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425  nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575  nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750  nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900  nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the  RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700  nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges  from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles  have an average diameter of about 400 nm.  The  RNA  lipoplex  particles  and  compositions  comprising  RNA  lipoplex  particles  described  herein are useful  for delivery of RNA  to a  target  tissue after parenteral administration,  in  particular after intravenous administration. The RNA lipoplex particles may be prepared using  liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a  suitable aqueous phase.  In one embodiment,  the aqueous phase has an acidic pH.  In one  embodiment, the aqueous phase comprises acetic acid, e.g.,  in an amount of about 5 mM.  Liposomes may be used  for preparing RNA  lipoplex particles by mixing  the  liposomes with  RNA.  In one embodiment,  the  liposomes and RNA  lipoplex particles comprise at  least one  cationic lipid and at  least one additional  lipid. In one embodiment, the at least one cationic  lipid  comprises  1,2‐di‐O‐octadecenyl‐3‐trimethylammonium  propane  (DOTMA)  and/or  1,2‐ dioleoyl‐3‐trimethylammonium‐propane  (DOTAP).  In  one  embodiment,  the  at  least  one  additional  lipid  comprises  1,2‐di‐(9Z‐octadecenoyl)‐sn‐glycero‐3‐phosphoethanolamine  (DOPE),  cholesterol  (Chol)  and/or 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine  (DOPC).  In one  embodiment,  the  at  least  one  cationic  lipid  comprises  1,2‐di‐O‐octadecenyl‐3‐ trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2‐di‐ (9Z‐octadecenoyl)‐sn‐glycero‐3‐phosphoethanolamine  (DOPE).  In  one  embodiment,  the  liposomes and RNA  lipoplex particles comprise 1,2‐di‐O‐octadecenyl‐3‐trimethylammonium  propane (DOTMA) and 1,2‐di‐(9Z‐octadecenoyl)‐sn‐glycero‐3‐phosphoethanolamine (DOPE).  Spleen  targeting  RNA  lipoplex  particles  are  described  in  WO  2013/143683,  herein  incorporated by reference. It has been found that RNA lipoplex particles having a net negative  charge may be used  to preferentially  target  spleen  tissue or  spleen  cells  such  as antigen‐ presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA  lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA  lipoplex  particles  of  the  disclosure may  be  used  for  expressing  RNA  in  the  spleen.  In  an  embodiment,  after  administration of  the RNA  lipoplex particles, no or  essentially no RNA  accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, after  administration of  the RNA  lipoplex particles, RNA  accumulation  and/or RNA expression  in  antigen presenting cells, such as professional antigen presenting cells  in the spleen occurs.  Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen  presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or  macrophages.    Lipid nanoparticles (LNPs)  In one embodiment, nucleic acid such as RNA described herein is administered in the form of  lipid nanoparticles (LNPs). The LNP may comprise any  lipid capable of  forming a particle to  which  the one or more nucleic acid molecules are attached, or  in which  the one or more  nucleic acid molecules are encapsulated.  In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing  lipids. Stabilizing lipids include neutral lipids and pegylated lipids.  In one embodiment, the LNP comprises a cationic  lipid, a neutral  lipid, a steroid, a polymer  conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.  In one embodiment, the LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent,  from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to  48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol  percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In  one embodiment, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8,  47.9 or 48.0 mol percent of the cationic lipid.   In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 15 mol  percent,  from 7  to 13 mol percent, or  from 9  to 11 mol percent.  In one embodiment,  the  neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.   In one  embodiment,  the  steroid  is present  in  a  concentration  ranging  from  30  to  50 mol  percent, from 35 to 45 mol percent or from 38 to 43 mol percent. In one embodiment, the  steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.   In one embodiment, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or  from 1 to 2.5 mol percent of the polymer conjugated lipid.  In one embodiment, the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to  15 mol percent of a neutral  lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol  percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with  the lipid nanoparticle.  In one embodiment, the mol percent is determined based on total mol of lipid present in the  lipid nanoparticle.   In one embodiment,  the neutral  lipid  is selected  from  the group consisting of DSPC, DPPC,  DMPC,  DOPC,  POPC,  DOPE,  DOPG,  DPPG,  POPE,  DPPE,  DMPE,  DSPE,  and  SM.  In  one  embodiment, the neutral  lipid  is selected  from the group consisting of DSPC, DPPC, DMPC,  DOPC, POPC, DOPE and SM. In one embodiment, the neutral lipid is DSPC.  In one embodiment, the steroid is cholesterol.  In one embodiment, the polymer conjugated lipid is a pegylated lipid. In one embodiment, the  pegylated  lipid  has  the  following  structure: 
Figure imgf000403_0001
  or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:   R12 and R13 are each independently a straight or branched, saturated or unsaturated alkyl chain  containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by  one or more ester bonds; and w has a mean value ranging from 30 to 60. In one embodiment,  R12 and R13 are each independently straight, saturated alkyl chains containing from 12 to 16  carbon  atoms.  In  one  embodiment, w  has  a mean  value  ranging  from  40  to  55.  In  one  embodiment,  the  average  w  is  about  45.  In  one  embodiment,  R12  and  R13  are  each  independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has  a mean value of about 45.  In one embodiment, the pegylated lipid is DMG‐PEG 2000, e.g., having the following structure: 
Figure imgf000404_0001
  In some embodiments, the cationic lipid component of the LNPs has the structure of Formula  (III): 
Figure imgf000404_0002
  (III)  or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:  one of L1 or L2 is –O(C=O)‐, ‐(C=O)O‐, ‐C(=O)‐, ‐O‐, ‐S(O)x‐, ‐S‐S‐, ‐C(=O)S‐, SC(=O)‐, ‐NRaC(=O)‐,  ‐C(=O)NRa‐, NRaC(=O)NRa‐, ‐OC(=O)NRa‐ or ‐NRaC(=O)O‐, and the other of L1 or L2 is –O(C=O)‐,  ‐(C=O)O‐, ‐C(=O)‐, ‐O‐, ‐S(O)x‐, ‐S‐S‐, ‐C(=O)S‐, SC(=O)‐, ‐NRaC(=O)‐, ‐C(=O)NRa‐, NRaC(=O)NRa‐,  ‐OC(=O)NRa‐ or ‐NRaC(=O)O‐ or a direct bond;  G1 and G2 are each independently unsubstituted C1‐C12 alkylene or C1‐C12 alkenylene;  G3 is C1‐C24 alkylene, C1‐C24 alkenylene, C3‐C8 cycloalkylene, C3‐C8 cycloalkenylene;  Ra is H or C1‐C12 alkyl;  R1 and R2 are each independently C6‐C24 alkyl or C6‐C24 alkenyl;   R3 is H, OR5, CN, ‐C(=O)OR4, ‐OC(=O)R4 or –NR5C(=O)R4;   R4 is C1‐C12 alkyl;  R5 is H or C1‐C6 alkyl; and  x is 0, 1 or 2.  In  some of  the  foregoing embodiments of Formula  (III),  the  lipid has one of  the  following  structures (IIIA) or (IIIB): 
Figure imgf000404_0003
(IIIA)        (IIIB)  wherein:  A is a 3 to 8‐membered cycloalkyl or cycloalkylene ring;  R6 is, at each occurrence, independently H, OH or C1‐C24 alkyl;  n is an integer ranging from 1 to 15.  In some of the foregoing embodiments of Formula (III), the  lipid has structure (IIIA), and  in  other embodiments, the lipid has structure (IIIB).  In other embodiments of Formula (III), the  lipid has one of the following structures (IIIC) or  (IIID): 
Figure imgf000405_0001
      (IIIC)        (IIID)  wherein y and z are each independently integers ranging from 1 to 12.  In any of the foregoing embodiments of Formula (III), one of L1 or L2 is ‐O(C=O)‐. For example,  in some embodiments each of L1 and L2 are ‐O(C=O)‐. In some different embodiments of any  of the foregoing, L1 and L2 are each independently ‐(C=O)O‐ or ‐O(C=O)‐. For example, in some  embodiments each of L1 and L2 is ‐(C=O)O‐.  In some different embodiments of Formula (III), the lipid has one of the following structures  (IIIE) or (IIIF): 
Figure imgf000405_0002
(IIIE)                (IIIF)  In  some of  the  foregoing embodiments of Formula  (III),  the  lipid has one of  the  following  structures (IIIG), (IIIH), (IIII), or (IIIJ): 
Figure imgf000405_0003
(IIIG)           (IIIH) 
Figure imgf000406_0001
    .  (IIII)           (IIIJ)  In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12,  for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In  some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some  embodiments, n is 6.  In some other of the foregoing embodiments of Formula (III), y and z are each independently  an  integer  ranging  from  2  to  10.  For  example,  in  some  embodiments,  y  and  z  are  each  independently an integer ranging from 4 to 9 or from 4 to 6.   In  some of  the  foregoing embodiments of  Formula  (III), R6  is H.  In other of  the  foregoing  embodiments, R6 is C1‐C24 alkyl. In other embodiments, R6 is OH.  In  some  embodiments  of  Formula  (III), G3  is  unsubstituted.  In  other  embodiments, G3  is  substituted.  In  various different  embodiments, G3  is  linear C1‐C24  alkylene or  linear  C1‐C24  alkenylene.  In some other foregoing embodiments of Formula (III), R1 or R2, or both, is C6‐C24 alkenyl. For  example, in some embodiments, R1 and R2 each, independently have the following structure: 
Figure imgf000406_0002
,  wherein:  R7a and R7b are, at each occurrence, independently H or C1‐C12 alkyl; and  a is an integer from 2 to 12,  wherein R7a, R7b and a are each selected such that R1 and R2 each  independently comprise  from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5  to 9 or from 8 to 12.  In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For  example, in some embodiments, R7a is H at each occurrence. In other different embodiments  of  the  foregoing,  at  least  one  occurrence  of  R7b  is  C1‐C8  alkyl.  For  example,  in  some  embodiments, C1‐C8 alkyl is methyl, ethyl, n‐propyl, iso‐propyl, n‐butyl, iso‐butyl, tert‐butyl, n‐ hexyl or n‐octyl.  In different embodiments of Formula (III), R1 or R2, or both, has one of the following structures: 
Figure imgf000407_0001
In some of the foregoing embodiments of Formula (III), R3 is OH, CN, ‐C(=O)OR4, ‐OC(=O)R4 or  –NHC(=O)R4. In some embodiments, R4 is methyl or ethyl.  In various different embodiments, the cationic lipid of Formula (III) has one of the structures  set forth in the table below.    Table 26: Representative Compounds of Formula (III).   
Figure imgf000407_0002
Figure imgf000408_0001
Figure imgf000409_0001
Figure imgf000410_0001
Figure imgf000411_0001
Figure imgf000412_0001
  In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid  and a pegylated  lipid.  In some embodiments, the  lipid of Formula (III)  is compound  III‐3.  In  some  embodiments,  the  neutral  lipid  is  DSPC.  In  some  embodiments,  the  steroid  is  cholesterol. In some embodiments, the pegylated lipid is ALC‐0159.  In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to  about 50 mole percent.  In one  embodiment,  the neutral  lipid  is present  in  the  LNP  in  an  amount from about 5 to about 15 mole percent. In one embodiment, the steroid is present in  the  LNP  in an amount  from about 35  to about 45 mole percent.  In one embodiment,  the  pegylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent.   In some embodiments,  the LNP comprises compound  III‐3  in an amount  from about 40  to  about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol  in an amount from about 35 to about 45 mole percent, and ALC‐0159 in an amount from about  1 to about 10 mole percent.  In some embodiments, the LNP comprises compound III‐3 in an amount of about 47.5 mole  percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7  mole percent, and ALC‐0159 in an amount of about 1.8 mole percent.  In various different embodiments, the cationic lipid has one of the structures set forth in the  table below.    Table 27: Representative cationic lipids. 
Figure imgf000413_0001
  In some embodiments, the LNP comprises a cationic  lipid shown  in the above table, e.g., a  cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a  neutral lipid, a steroid and a pegylated lipid. In some embodiments, the neutral lipid is DSPC.  In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is  DMG‐PEG 2000.  In one embodiment, the LNP comprises a cationic lipid that is an ionizable lipid‐like material  (lipidoid). In one embodiment, the cationic lipid has the following structure: 
Figure imgf000414_0001
  The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from  4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.  LNP described herein may have an average diameter that  in one embodiment ranges  from  about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.    RNA Targeting  Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein (e.g.,  RNA encoding vaccine antigens and/or immunostimulants).   In  one  embodiment,  the  disclosure  involves  targeting  lung.  Targeting  lung  is  in  particular  preferred if the RNA administered is RNA encoding vaccine antigen. RNA may be delivered to  lung,  for  example,  by  administering  the  RNA  which  may  be  formulated  as  particles  as  described herein, e.g., lipid particles, by inhalation.  In  one  embodiment,  the  disclosure  involves  targeting  the  lymphatic  system,  in  particular  secondary  lymphoid  organs, more  specifically  spleen.  Targeting  the  lymphatic  system,  in  particular secondary lymphoid organs, more specifically spleen is in particular preferred if the  RNA administered is RNA encoding vaccine antigen.   In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an  antigen presenting cell such as a professional antigen presenting cell  in  the spleen.  In one  embodiment, the target cell is a dendritic cell in the spleen.  The "lymphatic system" is part of the circulatory system and an important part of the immune  system, comprising a network of  lymphatic vessels that carry  lymph. The  lymphatic system  consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating  lymph.  The  primary  or  central  lymphoid  organs  generate  lymphocytes  from  immature  progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs.  Secondary  or  peripheral  lymphoid  organs,  which  include  lymph  nodes  and  the  spleen,  maintain mature naïve lymphocytes and initiate an adaptive immune response.  RNA may be delivered to spleen by so‐called lipoplex formulations, in which the RNA is bound  to  liposomes comprising a cationic  lipid and optionally an additional or helper  lipid to form  injectable nanoparticle formulations. The liposomes may be obtained by injecting a solution  of the lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles may be  prepared  by mixing  the  liposomes with  RNA.  Spleen  targeting  RNA  lipoplex  particles  are  described in WO 2013/143683, herein incorporated by reference. It has been found that RNA  lipoplex particles having a net negative charge may be used  to preferentially  target spleen  tissue or spleen cells such as antigen‐presenting cells, in particular dendritic cells. Accordingly,  following  administration  of  the  RNA  lipoplex  particles,  RNA  accumulation  and/or  RNA  expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for  expressing RNA  in  the spleen.  In an embodiment, after administration of  the RNA  lipoplex  particles, no or essentially no RNA accumulation and/or RNA expression  in the  lung and/or  liver  occurs.  In  one  embodiment,  after  administration  of  the  RNA  lipoplex  particles,  RNA  accumulation and/or RNA expression in antigen presenting cells, such as professional antigen  presenting cells  in the spleen occurs. Thus, RNA  lipoplex particles of the disclosure may be  used  for expressing RNA  in such antigen presenting cells.  In one embodiment,  the antigen  presenting cells are dendritic cells and/or macrophages.  The electric charge of the RNA  lipoplex particles of the present disclosure  is the sum of the  electric charges present  in the at  least one cationic  lipid and the electric charges present in  the RNA. The charge ratio is the ratio of the positive charges present in the at least one cationic  lipid  to  the negative  charges present  in  the RNA. The  charge  ratio of  the positive  charges  present  in  the  at  least  one  cationic  lipid  to  the  negative  charges  present  in  the  RNA  is  calculated by the following equation: charge ratio=[(cationic lipid concentration (mol)) * (the  total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (the total  number of negative charges in RNA)].   The spleen targeting RNA  lipoplex particles described herein at physiological pH preferably  have a net negative charge such as a charge ratio of positive charges to negative charges from  about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific  embodiments,  the charge ratio of positive charges to negative charges  in  the RNA  lipoplex  particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about  1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.   Immunostimulants may be provided to a subject by administering to the subject RNA encoding  an immunostimulant in a formulation for preferential delivery of RNA to liver or liver tissue.  The delivery of RNA to such target organ or tissue is preferred, in particular, if it is desired to  express  large  amounts  of  the  immunostimulant  and/or  if  systemic  presence  of  the  immunostimulant, in particular in significant amounts, is desired or required.  RNA delivery systems have an  inherent preference to the  liver. This pertains to  lipid‐based  particles,  cationic  and  neutral  nanoparticles,  in  particular  lipid  nanoparticles  such  as  liposomes, nanomicelles and lipophilic ligands in bioconjugates. Liver accumulation is caused  by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and  lipid or cholesterol conjugates).  For in vivo delivery of RNA to the liver, a drug delivery system may be used to transport the  RNA  into  the  liver  by  preventing  its  degradation.  For  example,  polyplex  nanomicelles  consisting of a poly(ethylene glycol) (PEG)‐coated surface and an RNA (e.g., mRNA)‐containing  core is a useful system because the nanomicelles provide excellent in vivo stability of the RNA,  under physiological conditions. Furthermore, the stealth property provided by the polyplex  nanomicelle  surface,  composed  of  dense  PEG  palisades,  effectively  evades  host  immune  defenses.  Examples of  suitable  immunostimulants  for  targeting  liver are  cytokines  involved  in T  cell  proliferation and/or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments  and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as  extended‐PK cytokines.  In  another  embodiment,  RNA  encoding  an  immunostimulant  may  be  administered  in  a  formulation for preferential delivery of RNA to the lymphatic system, in particular secondary  lymphoid organs, more specifically spleen. The delivery of an immunostimulant to such target  tissue is preferred, in particular, if presence of the immunostimulant in this organ or tissue is  desired (e.g., for inducing an immune response, in particular in case immunostimulants such  as cytokines are  required during T‐cell priming or  for activation of  resident  immune cells),  while  it  is  not  desired  that  the  immunostimulant  is  present  systemically,  in  particular  in  significant amounts (e.g., because the immunostimulant has systemic toxicity).  Examples of suitable immunostimulants are cytokines involved in T cell priming. Examples of  suitable  cytokines  include  IL12,  IL15,  IFN‐α, or  IFN‐β,  fragments and  variants  thereof,  and  fusion proteins of these cytokines, fragments and variants, such as extended‐PK cytokines.     Immunostimulants  In one embodiment, the RNA encoding vaccine antigen may be non‐immunogenic. In this and  other  embodiments,  the  RNA  encoding  vaccine  antigen may  be  co‐administered with  an  immunostimulant or RNA encoding an immunostimulant. The methods and agents described  herein  are  particularly  effective  if  the  immunostimulant  is  attached  to  a  pharmacokinetic  modifying  group  (hereafter  referred  to  as  "extended‐pharmacokinetic  (PK)"  immunostimulant). The methods and agents described herein are particularly effective if the  immunostimulant is administered in the form of RNA encoding an immunostimulant. In one  embodiment,  said  RNA  is  targeted  to  the  liver  for  systemic  availability.  Liver  cells  can  be  efficiently transfected and are able to produce large amounts of protein.  An  “immunostimulant”  is  any  substance  that  stimulates  the  immune  system  by  inducing  activation or  increasing  activity of  any of  the  immune  system's  components,  in particular  immune effector cells. The immunostimulant may be pro‐inflammatory.  According to one aspect, the immunostimulant is a cytokine or a variant thereof. Examples of  cytokines  include  interferons, such as  interferon‐alpha (IFN‐α) or  interferon‐gamma (IFN‐γ),  interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M‐CSF and  GM‐CSF,  and  tumor  necrosis  factor.  According  to  another  aspect,  the  immunostimulant  includes an adjuvant‐type immunostimulatory agent such as APC Toll‐like Receptor agonists  or costimulatory/cell adhesion membrane proteins. Examples of Toll‐like Receptor agonists  include costimulatory/adhesion proteins such as CD80, CD86, and ICAM‐1.  Cytokines are a category of small proteins  (~5–20 kDa) that are  important  in cell signaling.  Their release has an effect on the behavior of cells around them. Cytokines are  involved  in  autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents.  Cytokines  include chemokines,  interferons,  interleukins,  lymphokines, and  tumour necrosis  factors  but  generally  not  hormones  or  growth  factors  (despite  some  overlap  in  the  terminology). Cytokines are produced by a broad range of cells,  including  immune cells  like  macrophages,  B  lymphocytes,  T  lymphocytes  and mast  cells,  as well  as  endothelial  cells,  fibroblasts, and various stromal cells. A given cytokine may be produced by more than one  type of  cell. Cytokines act  through  receptors, and are especially  important  in  the  immune  system; cytokines modulate the balance between humoral and cell‐based immune responses,  and they regulate the maturation, growth, and responsiveness of particular cell populations.  Some cytokines enhance or inhibit the action of other cytokines in complex ways.  According to the disclosure, a cytokine may be a naturally occurring cytokine or a functional  fragment or variant thereof. A cytokine may be human cytokine and may be derived from any  vertebrate, especially any mammal. One particularly preferred cytokine is interferon‐α.    Interferons  Interferons (IFNs) are a group of signaling proteins made and released by host cells in response  to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells.  In  a  typical  scenario,  a  virus‐infected  cell will  release  interferons  causing  nearby  cells  to  heighten their anti‐viral defenses.  Based on  the  type of  receptor  through which  they  signal,  interferons are  typically divided  among three classes: type I interferon, type II interferon, and type III interferon.   All type  I  interferons bind to a specific cell surface receptor complex known as the  IFN‐α/β  receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.   The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ and IFNω. In general, type  I  interferons are produced when  the body  recognizes a virus  that has  invaded  it. They are  produced by  fibroblasts and monocytes. Once  released,  type  I  interferons bind  to  specific  receptors on target cells, which leads to expression of proteins that will prevent the virus from  producing and replicating its RNA and DNA.  The IFNα proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly  involved in innate immunity against viral infection. The genes responsible for their synthesis  come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10,  IFNA13,  IFNA14,  IFNA16,  IFNA17,  IFNA21. These genes are  found  together  in  a  cluster on  chromosome 9.  The IFNβ proteins are produced in large quantities by fibroblasts. They have antiviral activity  that is involved mainly in innate immune response. Two types of IFNβ have been described,  IFNβ1 and  IFNβ3. The natural and recombinant forms of  IFNβ1 have antiviral, antibacterial,  and anticancer properties.   Type  II  interferon (IFNγ  in humans)  is also known as  immune  interferon and  is activated by  IL12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells.  Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2‐ 4) and IFNLR1 (also called CRF2‐12). Although discovered more recently than type I and type  II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus  or fungal infections.  In general, type I and II interferons are responsible for regulating and activating the immune  response.   According to the disclosure, a type  I  interferon  is preferably  IFNα or  IFNβ, more preferably  IFNα.  According  to  the  disclosure,  an  interferon may  be  a  naturally  occurring  interferon  or  a  functional fragment or variant thereof. An interferon may be human interferon and may be  derived from any vertebrate, especially any mammal.     Interleukins  Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be  divided  into  four major groups based on distinguishing  structural  features. However,  their  amino acid sequence similarity is rather weak (typically 15–25% identity). The human genome  encodes more than 50 interleukins and related proteins.   According  to  the  disclosure,  an  interleukin may  be  a  naturally  occurring  interleukin  or  a  functional fragment or variant thereof. An interleukin may be human interleukin and may be  derived from any vertebrate, especially any mammal.     Extended‐PK group  Immunostimulant    polypeptides  described  herein  can  be  prepared  as  fusion  or  chimeric  polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e.,  a polypeptide  that  is not an  immunostimulant). The  immunostimulant may be  fused  to an  extended‐PK group, which increases circulation half‐life. Non‐limiting examples of extended‐ PK groups are described infra. It should be understood that other PK groups that increase the  circulation  half‐life  of  immunostimulants  such  as  cytokines,  or  variants  thereof,  are  also  applicable  to  the present disclosure.  In  certain embodiments,  the extended‐PK  group  is  a  serum albumin domain (e.g., mouse serum albumin, human serum albumin).   As  used  herein,  the  term  "PK"  is  an  acronym  for  "pharmacokinetic"  and  encompasses  properties of a compound including, by way of example, absorption, distribution, metabolism,  and elimination by a  subject. As used herein, an  "extended‐PK group"  refers  to a protein,  peptide, or moiety that increases the circulation half‐life of a biologically active molecule when  fused  to  or  administered  together with  the  biologically  active molecule.  Examples  of  an  extended‐PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and  variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders  (as disclosed  in U.S. Publication Nos. 2005/0287153 and 2007/0003549). Other exemplary  extended‐PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903‐ 15 which is herein incorporated by reference in its entirety. As used herein, an "extended‐PK"  immunostimulant refers to an immunostimulant moiety in combination with an extended‐PK  group. In one embodiment, the extended‐PK immunostimulant is a fusion protein in which an  immunostimulant moiety is linked or fused to an extended‐PK group.   In certain embodiments, the serum half‐life of an extended‐PK immunostimulant is increased  relative to the immunostimulant alone (i.e., the immunostimulant not fused to an extended‐ PK group). In certain embodiments, the serum half‐life of the extended‐PK immunostimulant  is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer relative to  the serum half‐life of the immunostimulant alone. In certain embodiments, the serum half‐life  of the extended‐PK  immunostimulant  is at  least 1.5‐fold, 2‐fold, 2.5‐fold, 3‐fold, 3.5 fold, 4‐ fold, 4.5‐fold, 5‐fold, 6‐fold, 7‐fold, 8‐fold, 10‐ fold, 12‐fold, 13‐fold, 15‐fold, 17‐fold, 20‐fold,  22‐ fold, 25‐fold, 27‐fold, 30‐fold, 35‐fold, 40‐fold, or 50‐fold greater than the serum half‐life  of the immunostimulant alone. In certain embodiments, the serum half‐life of the extended‐ PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40  hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130  hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.   As used herein, "half‐life" refers to the time taken for the serum or plasma concentration of a  compound  such  as  a  peptide  or  protein  to  reduce  by  50%,  in  vivo,  for  example  due  to  degradation  and/or  clearance  or  sequestration  by  natural  mechanisms.  An  extended‐PK  immunostimulant suitable for use herein is stabilized in vivo and its half‐life increased by, e.g.,  fusion  to serum albumin  (e.g., HSA or MSA), which  resist degradation and/or clearance or  sequestration.  The  half‐life  can  be  determined  in  any manner  known  per  se,  such  as  by  pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and  may for example generally involve the steps of suitably administering a suitable dose of the  amino acid sequence or compound to a subject; collecting blood samples or other samples  from said subject at regular intervals; determining the level or concentration of the amino acid  sequence or compound in said blood sample; and calculating, from (a plot of) the data thus  obtained, the time until the level or concentration of the amino acid sequence or compound  has  been  reduced  by  50%  compared  to  the  initial  level  upon  dosing.  Further  details  are  provided  in,  e.g.,  standard  handbooks,  such  as  Kenneth,  A.  et  al.,  Chemical  Stability  of  Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis:  A Practical Approach (1996). Reference  is also made to Gibaldi, M. et al., Pharmacokinetics,  2nd Rev. Edition, Marcel Dekker (1982).   In  certain  embodiments,  the  extended‐PK  group  includes  serum  albumin,  or  fragments  thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of  the present disclosure are comprised by the term "albumin"). Polypeptides described herein  may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins.  Such albumin fusion proteins are described in U.S. Publication No. 20070048282.   As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least  one molecule of albumin  (or a  fragment or variant  thereof)  to at  least one molecule of a  protein such as a therapeutic protein, in particular an immunostimulant. The albumin fusion  protein may be generated by translation of a nucleic acid in which a polynucleotide encoding  a  therapeutic  protein  is  joined  in‐frame with  a  polynucleotide  encoding  an  albumin.  The  therapeutic  protein  and  albumin,  once  part  of  the  albumin  fusion  protein, may  each  be  referred  to  as  a  “portion”,  “region”  or  “moiety”  of  the  albumin  fusion  protein  (e.g.,  a  “therapeutic  protein  portion”  or  an  “albumin  protein  portion”).  In  a  highly  preferred  embodiment, an albumin  fusion protein  comprises at  least one molecule of a  therapeutic  protein (including, but not limited to a mature form of the therapeutic protein) and at least  one molecule of albumin  (including but not  limited  to  a mature  form of  albumin).  In one  embodiment, an albumin fusion protein is processed by a host cell such as a cell of the target  organ for administered RNA, e.g. a liver cell, and secreted into the circulation. Processing of  the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used  for expression of the RNA may include, but is not limited to signal peptide cleavage; formation  of  disulfide  bonds;  proper  folding;  addition  and  processing  of  carbohydrates  (such  as  for  example, N‐ and O‐linked glycosylation); specific proteolytic cleavages; and/or assembly into  multimeric  proteins.  An  albumin  fusion  protein  is  preferably  encoded  by  RNA  in  a  non‐ processed  form  which  in  particular  has  a  signal  peptide  at  its  N‐terminus  and  following  secretion by a cell is preferably present in the processed form wherein in particular the signal  peptide has been cleaved off.  In a most preferred embodiment, the “processed form of an  albumin fusion protein” refers to an albumin fusion protein product which has undergone N‐ terminal signal peptide cleavage, herein also referred to as a “mature albumin fusion protein”.  In preferred embodiments, albumin fusion proteins comprising a therapeutic protein have a  higher plasma stability compared to the plasma stability of the same therapeutic protein when  not fused to albumin. Plasma stability typically refers to the time period between when the  therapeutic protein  is administered  in vivo and carried  into the bloodstream and when the  therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the  kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability  is calculated in terms of the half‐life of the therapeutic protein in the bloodstream. The half‐ life of  the  therapeutic protein  in  the bloodstream  can be  readily determined by  common  assays known in the art.  As used herein, “albumin” refers collectively to albumin protein or amino acid sequence, or  an  albumin  fragment  or  variant,  having  one  or more  functional  activities  (e.g.,  biological  activities)  of  albumin.  In  particular,  “albumin”  refers  to  human  albumin  or  fragments  or  variants  thereof  especially  the  mature  form  of  human  albumin,  or  albumin  from  other  vertebrates or fragments thereof, or variants of these molecules. The albumin may be derived  from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non‐ mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of  the  albumin  fusion  protein may  be  from  a  different  animal  than  the  therapeutic  protein  portion.  In certain embodiments, the albumin is human serum albumin (HSA), or fragments or variants  thereof, such as those disclosed  in US 5,876,969, WO 2011/124718, WO 2013/075066, and  WO 2011/0514789.  The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably  herein. The terms, “albumin and “serum albumin” are broader, and encompass human serum  albumin  (and  fragments and  variants  thereof) as well as albumin  from other  species  (and  fragments and variants thereof).  As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma  stability of  the  therapeutic protein  refers  to  a  fragment of  albumin  sufficient  in  length or  structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so  that the plasma stability of the therapeutic protein portion of the albumin fusion protein  is  prolonged or extended compared to the plasma stability in the non‐fusion state.   The  albumin  portion  of  the  albumin  fusion  proteins may  comprise  the  full  length  of  the  albumin  sequence,  or may  include  one  or more  fragments  thereof  that  are  capable  of  stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be of  10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous  amino  acids  from  the albumin  sequence or may  include part or all of  specific domains of  albumin. For instance, one or more fragments of HSA spanning the first two immunoglobulin‐ like domains may be used. In a preferred embodiment, the HSA fragment is the mature form  of HSA.  Generally  speaking, an albumin  fragment or variant will be at  least 100 amino acids  long,  preferably at least 150 amino acids long.  According  to  the disclosure, albumin may be naturally occurring albumin or a  fragment or  variant thereof. Albumin may be human albumin and may be derived  from any vertebrate,  especially any mammal.   Preferably,  the albumin  fusion protein comprises albumin as  the N‐terminal portion, and a  therapeutic  protein  as  the  C‐terminal  portion.  Alternatively,  an  albumin  fusion  protein  comprising albumin as  the C‐terminal portion, and a  therapeutic protein as  the N‐terminal  portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic  protein  fused  to  both  the  N‐terminus  and  the  C‐terminus  of  albumin.  In  a  preferred  embodiment, the therapeutic proteins fused at the N‐ and C‐termini are the same therapeutic  proteins. In another preferred embodiment, the therapeutic proteins fused at the N‐ and C‐ termini  are  different  therapeutic  proteins.  In  one  embodiment,  the  different  therapeutic  proteins are both cytokines.   In  one  embodiment,  the  therapeutic protein(s)  is  (are)  joined  to  the  albumin  through  (a)  peptide linker(s). A linker peptide between the fused portions may provide greater physical  separation  between  the moieties  and  thus maximize  the  accessibility  of  the  therapeutic  protein  portion,  for  instance,  for  binding  to  its  cognate  receptor.  The  linker  peptide may  consist  of  amino  acids  such  that  it  is  flexible  or more  rigid.  The  linker  sequence may  be  cleavable by a protease or chemically.  As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed  by the respective Fc domains (or Fc moieties) of its two heavy chains. As used herein, the term  "Fc  domain"  refers  to  a  portion  or  fragment  of  a  single  immunoglobulin  (Ig)  heavy  chain  wherein  the  Fc  domain  does  not  comprise  an  Fv  domain.  In  certain  embodiments,  an  Fc  domain begins in the hinge region just upstream of the papain cleavage site and ends at the  C‐terminus of  the antibody. Accordingly, a complete Fc domain comprises at  least a hinge  domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises  at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain,  a  CH3  domain,  a  CH4  domain,  or  a  variant,  portion,  or  fragment  thereof.  In  certain  embodiments, an Fc domain comprises a complete Fc domain  (i.e., a hinge domain, a CH2  domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain  (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an  Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion  thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof.  In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a  CH3 domain  (or portion  thereof).  In certain embodiments, an Fc domain consists of a CH2  domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists  of a hinge domain  (or portion  thereof) and  a CH2 domain  (or portion  thereof).  In  certain  embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2  domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the  Fc domain of an immunoglobulin heavy‐chain. This includes, but is not limited to, polypeptides  comprising  the entire CH1, hinge, CH2, and/or CH3 domains as well as  fragments of  such  peptides  comprising  only,  e.g.,  the  hinge,  CH2,  and  CH3  domain.  The  Fc  domain may  be  derived from an immunoglobulin of any species and/or any subtype, including, but not limited  to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses  native Fc and Fc variant molecules. As set forth herein, it will be understood by one of ordinary  skill in the art that any Fc domain may be modified such that it varies in amino acid sequence  from  the  native  Fc  domain  of  a  naturally  occurring  immunoglobulin molecule.  In  certain  embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).   The  Fc  domains  of  a  polypeptide  described  herein  may  be  derived  from  different  immunoglobulin molecules. For example, an Fc domain of a polypeptide may comprise a CH2  and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3  molecule. In another example, an Fc domain can comprise a chimeric hinge region derived, in  part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, an Fc  domain can comprise a chimeric hinge derived, in part, from an IgG1 molecule and,  in part,  from an IgG4 molecule.   In certain embodiments, an extended‐PK group includes an Fc domain or fragments thereof  or variants of the Fc domain or fragments thereof (all of which for the purpose of the present  disclosure are comprised by the term "Fc domain"). The Fc domain does not contain a variable  region  that binds  to antigen. Fc domains suitable  for use  in  the present disclosure may be  obtained from a number of different sources. In certain embodiments, an Fc domain is derived  from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgG1  constant  region.  It  is  understood,  however,  that  the  Fc  domain may  be  derived  from  an  immunoglobulin  of  another mammalian  species,  including  for  example,  a  rodent  (e.g.  a  mouse, rat, rabbit, guinea pig) or non‐ human primate (e.g. chimpanzee, macaque) species.   Moreover,  the  Fc  domain  (or  a  fragment  or  variant  thereof) may  be  derived  from  any  immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype,  including IgG1, IgG2, IgG3, and IgG4.   A  variety  of  Fc  domain  gene  sequences  (e.g.,  mouse  and  human  constant  region  gene  sequences) are available in the form of publicly accessible deposits. Constant region domains  comprising  an  Fc  domain  sequence  can  be  selected  lacking  a  particular  effector  function  and/or  with  a  particular  modification  to  reduce  immunogenicity.  Many  sequences  of  antibodies  and  antibody‐encoding  genes  have  been  published  and  suitable  Fc  domain  sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be  derived from these sequences using art recognized techniques.   In certain embodiments, the extended‐PK group is a serum albumin binding protein such as  those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422,  US2010/0113339, WO2009/083804, and WO2009/133208, which are herein incorporated by  reference in their entirety. In certain embodiments, the extended‐PK group is transferrin, as  disclosed  in US 7,176,278 and US 8,158,579, which are herein  incorporated by reference  in  their entirety.  In certain embodiments,  the extended‐PK group  is a serum  immunoglobulin  binding  protein  such  as  those  disclosed  in  US2007/0178082,  US2014/0220017,  and  US2017/0145062, which  are herein  incorporated by  reference  in  their  entirety.  In  certain  embodiments, the extended‐PK group is a fibronectin (Fn)‐based scaffold domain protein that  binds  to  serum  albumin,  such  as  those  disclosed  in  US2012/0094909,  which  is  herein  incorporated  by  reference  in  its  entirety. Methods  of making  fibronectin‐based  scaffold  domain proteins are also disclosed in US2012/0094909. A non‐limiting example of a Fn3‐based  extended‐PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.   In  certain  aspects,  the  extended‐PK  immunostimulant,  suitable  for  use  according  to  the  disclosure, can employ one or more peptide linkers. As used herein, the term "peptide linker"  refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the  extended‐PK moiety and an  immunostimulant moiety)  in a  linear amino acid sequence of a  polypeptide chain. For example, peptide linkers may be used to connect an immunostimulant  moiety to a HSA domain.   Linkers suitable for fusing the extended‐PK group to e.g. an immunostimulant are well known  in  the  art.  Exemplary  linkers  include  glycine‐serine‐polypeptide  linkers,  glycine‐proline‐ polypeptide  linkers,  and  proline‐alanine  polypeptide  linkers.  In  certain  embodiments,  the  linker is a glycine‐serine‐polypeptide linker, i.e., a peptide that consists of glycine and serine  residues.   In  addition  to,  or  in  place  of,  the  heterologous  polypeptides  described  above,  an  immunostimulant polypeptide described herein can contain sequences encoding a "marker"  or "reporter". Examples of marker or reporter genes  include β‐lactamase, chloramphenicol  acetyltransferase  (CAT),  adenosine  deaminase  (ADA),  aminoglycoside  phosphotransferase,  dihydrofolate  reductase  (DHFR),  hygromycin‐B‐hosphotransferase  (HPH),  thymidine  kinase  (TK), β‐galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).     Pharmaceutical compositions  The  agents  described  herein  may  be  administered  in  pharmaceutical  compositions  or  medicaments  and  may  be  administered  in  the  form  of  any  suitable  pharmaceutical  composition.   In one embodiment,  the pharmaceutical  composition described herein  is an  immunogenic  composition for inducing an immune response against coronavirus in a subject. For example,  in one embodiment, the immunogenic composition is a vaccine.   In one embodiment of all aspects of the present disclosure, the components described herein  such as RNA encoding a vaccine antigen may be administered in a pharmaceutical composition  which may comprise a pharmaceutically acceptable carrier and may optionally comprise one  or more adjuvants, stabilizers etc. In one embodiment, the pharmaceutical composition is for  therapeutic or prophylactic treatments, e.g., for use  in treating or preventing a coronavirus  infection.  The term "pharmaceutical composition" relates to a formulation comprising a therapeutically  effective  agent,  preferably  together  with  pharmaceutically  acceptable  carriers,  diluents  and/or  excipients.  Said  pharmaceutical  composition  is  useful  for  treating,  preventing,  or  reducing  the  severity  of  a  disease  or  disorder  by  administration  of  said  pharmaceutical  composition  to  a  subject.  A  pharmaceutical  composition  is  also  known  in  the  art  as  a  pharmaceutical formulation.   The  pharmaceutical  compositions  of  the  present  disclosure  may  comprise  one  or  more  adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to  a  compound  which  prolongs,  enhances  or  accelerates  an  immune  response.  Adjuvants  comprise  a  heterogeneous  group  of  compounds  such  as  oil  emulsions  (e.g.,  Freund's  adjuvants),  mineral  compounds  (such  as  alum),  bacterial  products  (such  as  Bordetella  pertussis toxin), or  immune‐stimulating complexes. Examples of adjuvants  include, without  limitation,  LPS,  GP96,  CpG  oligodeoxynucleotides,  growth  factors,  and  cytokines,  such  as  monokines, lymphokines, interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5,  IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM‐CSF, LT‐a. Further known adjuvants are aluminium  hydroxide, Freund's adjuvant or oil such as Montanide®  ISA51. Other suitable adjuvants for  use in the present disclosure include lipopeptides, such as Pam3Cys.   The pharmaceutical compositions according to the present disclosure are generally applied in  a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".  The term "pharmaceutically acceptable" refers to the non‐toxicity of a material which does  not interact with the action of the active component of the pharmaceutical composition.  The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to  the  amount which  achieves  a desired  reaction  or  a  desired  effect  alone or  together with  further  doses.  In  the  case  of  the  treatment  of  a  particular  disease,  the  desired  reaction  preferably relates to inhibition of the course of the disease. This comprises slowing down the  progress of the disease and, in particular, interrupting or reversing the progress of the disease.  The desired reaction in a treatment of a disease may also be delay of the onset or a prevention  of  the  onset  of  said  disease  or  said  condition.  An  effective  amount  of  the  compositions  described herein will depend on the condition to be treated, the severeness of the disease,  the  individual  parameters  of  the  patient,  including  age,  physiological  condition,  size  and  weight,  the duration of  treatment,  the  type of  an  accompanying  therapy  (if present),  the  specific route of administration and similar factors. Accordingly, the doses administered of the  compositions described herein may depend on various of such parameters. In the case that a  reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses  achieved by a different, more localized route of administration) may be used.  The  pharmaceutical  compositions  of  the  present  disclosure  may  contain  salts,  buffers,  preservatives,  and  optionally  other  therapeutic  agents.  In  one  embodiment,  the  pharmaceutical  compositions  of  the  present  disclosure  comprise  one  or  more  pharmaceutically acceptable carriers, diluents and/or excipients.   Suitable preservatives for use  in the pharmaceutical compositions of the present disclosure  include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.   The  term  "excipient"  as  used  herein  refers  to  a  substance  which  may  be  present  in  a  pharmaceutical  composition  of  the  present  disclosure  but  is  not  an  active  ingredient.  Examples  of  excipients,  include  without  limitation,  carriers,  binders,  diluents,  lubricants,  thickeners,  surface  active  agents,  preservatives,  stabilizers,  emulsifiers,  buffers,  flavoring  agents, or colorants.  The  term  "diluent"  relates a diluting and/or  thinning agent. Moreover,  the  term  "diluent"  includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of  suitable diluents include ethanol, glycerol and water.  The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic  in  which  the  active  component  is  combined  in  order  to  facilitate,  enhance  or  enable  administration of  the pharmaceutical composition. A carrier as used herein may be one or  more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable  for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer,  Ringer  lactate,  sterile  sodium  chloride  solution,  isotonic  saline,  polyalkylene  glycols,  hydrogenated  naphthalenes  and,  in  particular,  biocompatible  lactide  polymers,  lactide/glycolide  copolymers  or  polyoxyethylene/polyoxy‐propylene  copolymers.  In  one  embodiment,  the  pharmaceutical  composition  of  the  present  disclosure  includes  isotonic  saline.  Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known  in  the pharmaceutical art, and are described,  for example,  in Remington's Pharmaceutical  Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).   Pharmaceutical carriers, excipients or diluents can be selected with regard to the  intended  route of administration and standard pharmaceutical practice.   In  one  embodiment,  pharmaceutical  compositions  described  herein may  be  administered  intravenously,  intraarterially,  subcutaneously,  intradermally  or  intramuscularly.  In  certain  embodiments,  the  pharmaceutical  composition  is  formulated  for  local  administration  or  systemic administration. Systemic administration may  include enteral administration, which  involves absorption through the gastrointestinal tract, or parenteral administration. As used  herein,  "parenteral administration"  refers  to  the administration  in any manner other  than  through  the  gastrointestinal  tract,  such  as  by  intravenous  injection.  In  a  preferred  embodiment, the pharmaceutical composition is formulated for intramuscular administration.  In  another  embodiment,  the  pharmaceutical  composition  is  formulated  for  systemic  administration, e.g., for intravenous administration.   The term "co‐administering" as used herein means a process whereby different compounds  or compositions (e.g., RNA encoding an antigen and RNA encoding an immunostimulant) are  administered  to  the  same  patient.  The  different  compounds  or  compositions  may  be  administered simultaneously, at essentially the same time, or sequentially.   The pharmaceutical compositions and products described herein may be provided as a frozen  concentrate  for  solution  for  injection,  e.g.,  at  a  concentration  of  0.50  mg/mL.  In  one  embodiment, for preparation of solution for injection, a drug product is thawed and diluted  with  isotonic sodium chloride solution  (e.g., 0.9% NaCl, saline), e.g., by a one‐step dilution  process.  In  some  embodiments,  bacteriostatic  sodium  chloride  solution  (e.g.,  0.9%  NaCl,  saline) cannot be used as a diluent. In some embodiments, a diluted drug product is an off‐ white suspension. The concentration of the final solution for injection varies depending on the  respective dose level to be administered.   In one embodiment, administration is performed within 6 h after begin of preparation due to  the  risk  of  microbial  contamination  and  considering  the  multiple‐dose  approach  of  the  preparation process.  In one embodiment,  in this period of 6 h, two conditions are allowed:  room temperature for preparation, handling and transfer as well as 2 to 8°C for storage.  Compositions described herein may be shipped and/or stored under temperature‐controlled  conditions,  e.g.,  temperature  conditions  of  about  4‐5oC  or  below,  about  ‐20oC  or  below,  ‐ 70°C±10°C (e.g., ‐80°C to ‐60°C), e.g., utilizing a cooling system (e.g., that may be or include  dry  ice) to maintain  the desired  temperature.  In one embodiment, compositions described  herein are shipped in temperature‐controlled thermal shippers. Such shippers may contain a  GPS‐enabled  thermal  sensor  to  track  the  location and  temperature of each  shipment. The  compositions can be stored by refilling with, e.g., dry ice.     Treatments  In one aspect, the present disclosure provides methods and agents for inducing an adaptive  immune  response  against  coronavirus  in  a  subject  comprising  administering  an  effective  amount of a composition comprising RNA encoding a coronavirus vaccine antigen described  herein.   In one embodiment, the methods and agents described herein provide immunity in a subject  to coronavirus, coronavirus infection, or to a disease or disorder associated with coronavirus.  In another aspect, the present disclsoure thus provides methods and agents for treating or  preventing the infection, disease, or disorder associated with coronavirus.   In one embodiment, the methods and agents described herein are administered to a subject  having an infection, disease, or disorder associated with coronavirus. In one embodiment, the  methods and agents described herein are administered to a subject at risk for developing the  infection, disease, or disorder associated with coronavirus. For example,  the methods and  agents described herein may be administered to a subject who is at risk for being in contact  with  coronavirus.  In  one  embodiment,  the  methods  and  agents  described  herein  are  administered to a subject who  lives  in, traveled to, or  is expected to travel to a geographic  region  in  which  coronavirus  is  prevalent.  In  one  embodiment,  the methods  and  agents  described herein are administered to a subject who  is  in contact with or expected to be  in  contact with another person who lives in, traveled to, or is expected to travel to a geographic  region  in  which  coronavirus  is  prevalent.  In  one  embodiment,  the methods  and  agents  described  herein  are  administered  to  a  subject  who  has  knowingly  been  exposed  to  coronavirus through their occupation, or other contact. In one embodiment, a coronavirus is  SARS‐CoV‐2.  In some embodiments, methods and agents described herein are administered  to a subject with evidence of prior exposure to and/or infection with SARS‐CoV‐2 and/or an  antigen or epitope thereof or cross‐reactive therewith.  For example, in some embodiments,  methods and agents described herein are administered to a subject  in whom antibodies, B  cells, and/or T cells  reactive with one or more epitopes of a SARS‐CoV‐2 spike protein are  detectable and/or have been detected.  For a composition to be useful as a vaccine, the composition must induce an immune response  against  the  coronavirus  antigen  in  a  cell,  tissue  or  subject  (e.g.,  a  human).  In  some  embodiments, the composition induces an immune response against the coronavirus antigen  in a cell, tissue or subject (e.g., a human). In some instances, the vaccine induces a protective  immune response in a mammal. The therapeutic compounds or compositions of the present  disclosure may be  administered prophylactically  (i.e.,  to prevent  a disease or disorder) or  therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or  susceptible  to)  developing  a  disease  or  disorder.  Such  subjects  may  be  identified  using  standard  clinical  methods.  In  the  context  of  the  present  disclosure,  prophylactic  administration occurs prior to the manifestation of overt clinical symptoms of disease, such  that  a  disease  or  disorder  is  prevented  or  alternatively  delayed  in  its  progression.  In  the  context of the field of medicine, the term "prevent" encompasses any activity, which reduces  the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary  and tertiary prevention levels. While primary prevention avoids the development of a disease,  secondary  and  tertiary  levels  of  prevention  encompass  activities  aimed  at  preventing  the  progression of a disease and the emergence of symptoms as well as reducing the negative  impact of an already established disease by restoring function and reducing disease‐related  complications.  The  term "dose" as used herein  refers  in general  to a "dose amount" which  relates  to  the  amount of RNA administered per administration, i.e., per dosing.   In  some  embodiments,  administration  of  an  immunogenic  composition  or  vaccine  of  the  present  disclosure  may  be  performed  by  single  administration  or  boosted  by  multiple  administrations.   In  some  embodiments,  a  regimen  described  herein  includes  at  least  one  dose.  In  some  embodiments, a  regimen  includes a  first dose and at  least one  subsequent dose.  In  some  embodiments, the first dose  is the same amount as at  least one subsequent dose.  In some  embodiments,  the  first  dose  is  the  same  amount  as  all  subsequent  doses.  In  some  embodiments, the first dose is a different amount as at least one subsequent dose. In some  embodiments,  the  first  dose  is  a  different  amount  than  all  subsequent  doses.  In  some  embodiments, a regimen comprises two doses.  In some embodiments, a provided regimen  consists of two doses. In some embodiments, a regimen comprises three doses.   In one embodiment, the present disclosure envisions administration of a single dose. In one  embodiment, the present disclosure envisions administration of a priming dose followed by  one or more booster doses. The booster dose or the first booster dose may be administered  7  to  28  days  or  14  to  24  days  following  administration  of  the  priming  dose.  In  some  embodiments, a first booster dose may be administered 1 week to 3 months (e.g., 1 week, 2  weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks,  12 weeks) following administration of a priming dose. In some embodiments, a subsequent  booster dose may be adminsitered at least 1 week or longer, including, e.g., at least 2 weeks,  at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least  8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer,  following  a preceding booster dose. In some embodiments, subsequent booster doses may  be administered about 5‐9 weeks or 6‐8 weeks apart.  In  some embodiments, at  least one  subsequent booster dose  (e.g.,  after  a  first booster dose) may be  administered at  least 3  months or longer, including, e.g., at least 4 months, at least 5 months, at least 6 months, at  least 7 months, at least 8 months, at least 9 months, at least 10 months, or longer, following  a preceding dose.   In some embodiments, a subsequent dose given to an  individual  (e.g., as part of a primary  regimen or booster regimen) can have the same amount of RNA as previously given to the  individual. In some embodiments, a subsequent dose given to an individual (e.g., as part of a  primary regimen or booster regimen) can differ  in the amount of RNA, as compared to the  amount previously given to the individual. For example, in some embodiments, a subsequent  dose  can be higher or  lower  than  the prior dose,  for example, based on  consideration of  various  factors,  including, e.g.,  immunogenicity and/or  reactogenicity  induced by  the prior  dose, prevalence of the disease, etc.  In some embodiments, a subsequent dose can be higher  than a prior dose by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least  80%, at least 90%, or higher. In some embodiments, a subsequent dose can be higher than a  prior dose by at  least 1.5‐fold, at  least 2‐fold, at  least 2.5  fold, at  least 3‐fold, or higher.  In  some embodiments, a subsequent dose can be higher than a prior dose by at  least 30%, at  least 40%, at  least 50%, at  least 60%, at  least 70%, at  least 80%, at  least 90%, or higher.  In  some embodiments, a subsequent dose can be  lower than a prior dose by at  least 10%, at  least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or lower.In some  embodiments, an amount the RNA described herein from 0.1 µg to 300 µg, 0.5 µg to 200 µg,  or 1 µg to 100 µg, such as about 1 µg, about 2 µg, about 3 µg, about 4 µg, about 5 µg, about 6  µg, about 7 µg, about 8 µg, about 9 µg, about 10 µg, about 15 µg, about 20 µg, about 25 µg,  about 30 µg, about 35 µg, about 40 µg, about 45 µg, about 50 µg, about 55 µg, about 60 µg,  about 70 µg, about 80 µg, about 90 µg, or about 100 µg may be administered per dose (e.g.,  in a given dose).   In some embodiments, an amount of the RNA described herein of 60 µg or  lower, 55 µg or  lower, 50 µg or lower, 45 µg or lower, 40 µg or lower, 35 µg or lower, 30 µg or lower, 25 µg or  lower, 20 µg or lower, 15 µg or lower, 10 µg or lower, 5 µg or lower, 3 µg or lower, 2.5 µg or  lower, or 1 µg or lower may be administered per dose (e.g., in a given dose).   In some embodiments, an amount of the RNA described herein of at least 0.25 µg, at least 0.5  µg, at least 1 µg, at least 2 µg, at least 3 µg, at least 4 µg, at least 5 µg, at least 10 µg, at least  15 µg, at least 20 µg, at least 25 µg, at least 30 µg, at least 40 µg, at least 50 µg, or at least 60  µg may be administered per dose (e.g., in a given dose). In some embodiments, an amount of  the RNA described herein of at least 3 ug may be administered in at least one of given doses.  In  some embodiments, an  amount of  the RNA described herein of  at  least 10 ug may be  administered  in at  least one of given doses.  In some embodiments, an amount of the RNA  described herein of at least 15 ug may be administered in at least one of given doses. In some  embodiments, an amount of the RNA described herein of at least 20 ug may be administered  in at least one of given doses. In some embodiments, an amount of the RNA described herein  of at least 25 ug may be administered in at least one of given doses. In some embodiments,  an amount of the RNA described herein of at least 30 ug may be administered in at least one  of given doses. In some embodiments, an amount of the RNA described herein of at least 50  ug may be administered in at least one of given doses. In some embodiments, an amount of  the RNA described herein of at least 60 ug may be administered in at least one of given doses.  In some embodiments, combinations of aforementioned amounts may be administered in a  regimen comprising two or more doses (e.g., a prior dose and a subsequent dose can be of  different  amounts  as  described  herein).  In  some  embodiments,  combinations  of  aforementioned amounts may be administered in a primary regimen and a booster regimen  (e.g., different doses can be given in a primary regimen and a booster regimen).  In some embodiments, an amount of the RNA described herein of 0.25 µg to 60 µg, 0.5 µg to  55 µg, 1 µg to 50 µg, 5 µg to 40 µg, or 10 µg to 30 µg may be administered per dose. In some  embodiments, an amount of the RNA described herein of 3 µg to 30 µg may be administered  in at least one of given doses.  In some embodiments, an amount of the RNA described herein  of 3 µg to 20 µg may be administered in at least one of given doses. In some embodiments, an  amount of the RNA described herein of 3 µg to 15 µg may be administered in at least one of  given doses. In some embodiments, an amount of the RNA described herein of 3 µg to 10 µg  may be administered in at least one of given doses. In some embodiments, an amount of the  RNA described herein of 10 µg to 30 µg may be administered in at least one of given doses.  In some embodiments, a regimen administered to a subject may comprise a plurality of doses  (e.g., at  least  two doses, at  least  three doses, or more).  In some embodiments, a  regimen  administered to a subject may comprise a first dose and a second dose, which are given at  least  2  weeks  apart,  at  least  3  weeks  apart,  at  least  4  weeks  apart,  or more.  In  some  embodiments, such doses may be at least 1 month, at least 2 months, at least 3 months, at  least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at  least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart. In  some embodiments, doses may be administered days apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10,  11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,  36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or  more days apart. In some embodiments, doses may be administered about 1 to about 3 weeks  apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to  about 6 weeks apart, or about 1 to more than 6 weeks apart. In some embodiments, doses  may be separated by a period of about 7 to about 60 days, such as for example about 14 to  about 48 days, etc. In some embodiments, a minimum number of days between doses may be  about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some  embodiments, a maximum number of days between doses may be about 60, 59, 58, 57, 56,  55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31,  30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or fewer. In some embodiments, doses may be about 21  to about 28 days apart. In some embodiments, doses may be about 19 to about 42 days apart.  In some embodiments, doses may be about 7 to about 28 days apart. In some embodiments,  doses may be about 14 to about 24 days. In some embodiments, doses may be about 21 to  about 42 days.  In some embodiments, a vaccination regimen comprises a first dose and a second dose.  In  some embodiments, a first dose and a second dose are administered by at least 21 days apart.  In some embodiments, a first dose and a second dose are administered by at  least 28 days  apart.   In  some  embodiments,  a  vaccination  regimen  comprises  a  first  dose  and  a  second  dose,  wherein the amount of RNA administered in the first dose is the same as the amount of RNA  administered in the second dose. In some embodiments, a vaccination regimen comprises a  first dose and a second dose wherein the amount of RNA administered in the first dose differs  from that administered in the second dose.   In  some  embodiments,  a  vaccination  regimen  comprises  a  first  dose  and  a  second  dose,  wherein the amount of RNA administered in the first dose is less than that administered in the  second dose.  In some embodiments, the amount of RNA administered in the first dose is 10%‐ 90% of the second dose. In some embodiments, the amount of RNA administered in the first  dose is 10%‐50% of the second dose.  In some embodiments, the amount of RNA administered  in the first dose is 10%‐20% of the second dose.  In some embodiments, the first dose and the  second dose are administered at least 2 weeks apart, including, at least 3 weeks apart, at least  4 weeks apart, at least 5 weeks apart, at least 6 weeks apart or longer. In some embodiments,  the first dose and the second dose are administered at least 3 weeks apart.  In some embodiments, a first dose comprises less than about 30 ug of RNA and a second dose  comprises at least about 30 ug of RNA.  In some embodiments, a first dose comprises about 1  to less than about 30 ug of RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about  15, about 20, about 25, or less than about 30 ug of RNA) and a second dose comprises about  30 to about 100 ug of RNA (e.g., about 30, about 40, about 50, or about 60 ug of RNA).    In  some embodiments, a first dose comprises about 1 to about 20 ug of RNA, about 1 to about  10 ug of RNA, or about 1 to about 5 ug of RNA and a second dose comprises about 30 to about  60 ug of RNA.    In some embodiments, a first dose comprises about 1 to about 10 ug of RNA (e.g., about 1,  about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 ug of RNA)  and a second dose comprises about 30 to about 60 ug of RNA (e.g., about 30, about 35, about  40, about 45, about 50, about 55, or about 60 ug of RNA).    In some embodiments, a first dose comprises about 1 ug of RNA and a second dose comprises  about 30 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a  second dose comprises about 30 ug of RNA.  In some embodiments, a  first dose comprises  about 5 ug of RNA and a second dose comprises about 30 ug of RNA. In some embodiments,  a first dose comprises about 10 ug of RNA and a second dose comprises about 30 ug of RNA.   In some embodiments, a first dose comprises about 15 ug of RNA and a second dose comprises  about 30 ug of RNA.   In some embodiments, a first dose comprises about 1 ug of RNA and a second dose comprises  about 60 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a  second dose comprises about 60 ug of RNA.  In some embodiments, a  first dose comprises  about 5 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments,  a first dose comprises about 6 ug of RNA and a second dose comprises about 60 ug of RNA. In  some embodiments, a first dose comprises about 10 ug of RNA and a second dose comprises  about 60 ug of RNA. In some embodiments, a first dose comprises about 15 ug of RNA and a  second dose comprises about 60 ug of RNA.  In some embodiments, a  first dose comprises  about 20 ug of RNA and a second dose comprises about 60 ug of RNA. In some embodiments,  a first dose comprises about 25 ug of RNA and a second dose comprises about 60 ug of RNA.  In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises  about 60 ug of RNA.  In some embodiments, a first dose comprises less than about 10 ug of RNA and a second dose  comprises at least about 10 ug of RNA.  In some embodiments, a first dose comprises about  0.1 to less than about 10 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about  4, about 5, about 6, about 7, about 8, or  less than about 10 ug of RNA) and a second dose  comprises about 10 to about 30 ug of RNA (e.g., about 10, about 15, about 20, about 25, or  about 30 ug of RNA).  In some embodiments, a first dose comprises about 0.1 to about 10 ug  of RNA, about 1 to about 5 ug of RNA, or about 0.1 to about 3 ug of RNA and a second dose  comprises about 10 to about 30 ug of RNA.    In some embodiments, a first dose comprises about 0.1 to about 5 ug of RNA (e.g., about 0.1,  about 0.5, about 1, about 2, about 3, about 4, about 5ug of RNA) and a second dose comprises  about 10 to about 20 ug of RNA (e.g., about 10, about 12, about 14, about 16, about 18, about  20ug of RNA).    In  some  embodiments,  a  first  dose  comprises  about  0.1  ug  of  RNA  and  a  second  dose  comprises about 10 ug of RNA. In some embodiments, a first dose comprises about 0.3 ug of  RNA and a second dose comprises about 10 ug of RNA.  In some embodiments, a first dose  comprises about 1 ug of RNA and a  second dose comprises about 10 ug of RNA.  In  some  embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises about  10 ug of RNA.   In some embodiments, a first dose comprises less than about 3 ug of RNA and a second dose  comprises at least about 3 ug of RNA.  In some embodiments, a first dose comprises about 0.1  to  less than about 3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.5, about 0.6,  about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, or about 2.5 ug of RNA) and  a second dose comprises about 3 to about 10 ug of RNA (e.g., about 3, about 4, about 5, about  6, or about 7, about 8, about 9, or about 10 ug of RNA).  In some embodiments, a first dose  comprises about 0.1 to about 3 ug of RNA, about 0.1 to about 1 ug of RNA, or about 0.1 to  about 0.5 ug of RNA and a second dose comprises about 3 to about 10 ug of RNA.    In some embodiments, a first dose comprises about 0.1 to about 1.0 ug of RNA (e.g., about  0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or  about 1.0 ug of RNA) and a second dose comprises about 1 to about 3 ug of RNA (e.g., about  1.0, about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA).    In  some  embodiments,  a  first  dose  comprises  about  0.1  ug  of  RNA  and  a  second  dose  comprises about 3 ug of RNA. In some embodiments, a first dose comprises about 0.3 ug of  RNA and a second dose comprises about 3 ug of RNA.  In some embodiments, a  first dose  comprises about 0.5 ug of RNA and a second dose comprises about 3 ug of RNA.  In some  embodiments, a first dose comprises about 1 ug of RNA and a second dose comprises about 3  ug of RNA.   In  some  embodiments,  a  vaccination  regimen  comprises  a  first  dose  and  a  second  dose,  wherein the amount of RNA administered in the first dose is greater than that administered  in the second dose.  In some embodiments, the amount of RNA administered in the second  dose is 10%‐90% of the first dose. In some embodiments, the amount of RNA administered in  the second dose  is 10%‐50% of  the  first dose.    In some embodiments, the amount of RNA  administered in the second dose is 10%‐20% of the first dose.  In some embodiments, the first  dose and the second dose are administered at least 2 weeks apart, including, at least 3 weeks  apart, at least 4 weeks apart, at least 5 weeks apart, at least 6 weeks apart or longer. In some  embodiments, the first dose and the second dose are administered at least 3 weeks apart  In some embodiments, a first dose comprises at least about 30 ug of RNA and a second dose  comprises less than about 30 ug of RNA.  In some embodiments, a first dose comprises about  30 to about 100 ug of RNA (e.g., about 30, about 40, about 50, or about 60 ug of RNA) and a  second dose comprises about 1 to about 30 ug of RNA (e.g., about 0.1, about 1, about 3, about  5, about 10, about 15, about 20, about 25, or about 30 ug of RNA).  In some embodiments, a  second dose comprises about 1 to about 20 ug of RNA, about 1 to about 10 ug of RNA, or  about 1 to 5 ug of RNA.  In some embodiments, a first dose comprises about 30 to about 60  ug of RNA and a second dose comprises about 1 to about 20 ug of RNA, about 1 to about 10  ug of RNA, or about 0.1 to about 3 ug of RNA.    In some embodiments, a first dose comprises about 30 to about 60 ug of RNA (e.g., about 30,  about 35, about 40, about 45, about 50, about 55, or about 60 ug of RNA) and a second dose  comprises about 1 to about 10 ug of RNA (e.g., about 1, about 2, about 3, about 4, about 5,  about 6, about 7, about 8, about 9, or about 10 ug of RNA).    In some embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises  about 1 ug of RNA. In some embodiments, a first dose comprises about 30 ug of RNA and a  second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about  30 ug of RNA and a second dose comprises about 5 ug of RNA. In some embodiments, a first  dose comprises about 30 ug of RNA and a second dose comprises about 10 ug of RNA. In some  embodiments, a first dose comprises about 30 ug of RNA and a second dose comprises about  15 ug of RNA.     In some embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises  about 1 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a  second dose comprises about 3 ug of RNA. In some embodiments, a first dose comprises about  60 ug of RNA and a second dose comprises about 5 ug of RNA. In some embodiments, a first  dose comprises about 60 ug of RNA and a second dose comprises about 6 ug of RNA. In some  embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about  10 ug of RNA. In some embodiments, a first dose comprises about 60 ug of RNA and a second  dose comprises about 15 ug of RNA. In some embodiments, a first dose comprises about 60  ug of RNA and a second dose comprises about 20 ug of RNA.  In some embodiments, a first  dose comprises about 60 ug of RNA and a second dose comprises about 25 ug of RNA. In some  embodiments, a first dose comprises about 60 ug of RNA and a second dose comprises about  30 ug of RNA.  In some embodiments, a first dose comprises at least about 10 ug of RNA and a second dose  comprises less than about 10 ug of RNA.  In some embodiments, a first dose comprises about  10 to about 30 ug of RNA (e.g., about 10, about 15, about 20, about 25, or about 30 ug of RNA)  and a second dose comprises about 0.1 to less than about 10 ug of RNA (e.g., about 0.1, about  0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, or less than about  10 ug of RNA).  In some embodiments, a first dose comprises about 10 to about 30 ug of RNA,  or about 0.1 to about 3 ug of RNA and a second dose comprises about 1 to about 10 ug of  RNA, or about 1 to about 5 ug of RNA.    In some embodiments, a first dose comprises about 10 to about 20 ug of RNA (e.g., about 10,  about 12, about 14, about 16, about 18, about 20 ug of RNA) and a second dose comprises  about 0.1 to about 5 ug of RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4,  or about 5 ug of RNA).    In some embodiments, a first dose comprises about 10 ug of RNA and a second dose comprises  about 0.1 ug of RNA. In some embodiments, a first dose comprises about 10 ug of RNA and a  second dose comprises about 0.3 ug of RNA.  In some embodiments, a first dose comprises  about 10 ug of RNA and a second dose comprises about 1 ug of RNA. In some embodiments,  a first dose comprises about 10 ug of RNA and a second dose comprises about 3 ug of RNA.   In some embodiments, a first dose comprises at least about 3 ug of RNA and a second dose  comprises less than about 3 ug of RNA.  In some embodiments, a first dose comprises about 3  to about 10 ug of RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9,  or about 10 ug of RNA) and a second dose comprises 0.1 to less than about 3 ug of RNA (e.g.,  about 0.1, about 0.2, about 0.3, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about  1.0,  about  1.5  about  2.0,  or  about  2.5  ug  of  RNA).    In  some  embodiments,  a  first  dose  comprises about 3 to about 10 ug of RNA and a second dose comprises about 0.1 to about 3  ug of RNA, about 0.1 to about 1 ug of RNA, or about 0.1 to about 0.5 ug of RNA.    In some embodiments, a  first dose comprises about 1 to about 3 ug of RNA (e.g., about 1,  about 1.5, about 2.0, about 2.5, or about 3.0 ug of RNA) and a second dose comprises about  0.1 to 0.3 ug of RNA (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6,  about 0.7, about 0.8, about 0.9, or about 1.0 ug of RNA).    In some embodiments, a first dose comprises about 3 ug of RNA and a second dose comprises  about 0.1 ug of RNA. In some embodiments, a first dose comprises about 3 ug of RNA and a  second dose comprises about 0.3 ug of RNA.  In some embodiments, a first dose comprises  about 3 ug of RNA and a second dose comprises about 0.6 ug of RNA. In some embodiments,  a first dose comprises about 3 ug of RNA and a second dose comprises about 1 ug of RNA.    In some embodiments, a vaccination regimen comprises at least two doses, including, e.g., at  least three doses, at least four doses or more. In some embodiments, a vaccination regimen  comprises three doses. In some embodiments, the time interval between the first dose and  the second dose can be the same as the time interval between the second dose and the third  dose.  In some embodiments, the time  interval between the first dose and the second dose  can be longer than the time interval between the second dose and the third dose, e.g., by days  or weeks (including, e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least  1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks,  or  longer).  In some embodiments, the time  interval between the  first dose and the second  dose can be shorter than the time interval between the second dose and the third dose, e.g.,  by days or weeks (including, e.g., at least 3 days, at least 4 days, at least 5 days, at least 6 days,  at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least  6 weeks, or longer).  In some embodiments, the time interval between the first dose and the  second dose can be shorter than the  time  interval between the second dose and the third  dose, e.g., by at least 1 month (including, e.g., at least 2 months, at least 3 months, at least 4  months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9  months, at least 10 months, at least 11 months, at least 12 months, or longer).   In some embodiments, a last dose of a primary regimen and a first dose of a booster regimen   are given at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6  months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least  11 months, at least 12 months, or more apart. In some embodiments, a primary regimen may  comprises two doses. In some embodiments, a primary regimen may comprises three doses.  In some embodiments, a first dose and a second dose (and/or other subsequent dose) may be  administered by intramuscular injection. In some embodiments, a first dose and a second dose  (and/or  other  subsequent  dose)  may  be  administered  in  the  deltoid  muscle.  In  some  embodiments,  a  first  dose  and  a  second  dose  (and/or  other  subsequent  dose) may  be  administered in the same arm.   In some embodiments, an RNA  (e.g., mRNA) composition described herein  is administered  (e.g., by intramuscular injection) as a series of two doses (e.g., 0.3 mL each) 21 days apart. In  some embodiments, an RNA (e.g., mRNA) composition described herein is administered (e.g.,  by intramuscular injection) as a series of two doses (e.g., 0.2 mL each) 21 days apart. In some  embodiments, an RNA  (e.g., mRNA) composition described herein  is administered  (e.g., by  intramuscular  injection) as a series of three doses (e.g., 0.3 mL or  lower  including, e.g., 0.2  mL), wherein doses are given at  least 3 weeks apart.  In  some embodiments,  the  first and  second doses may be administered 3 weeks apart, while the second and third doses may be  administered at a longer time interval than that between the first and the second doses, e.g.,  at least 4 weeks apart or longer (including, at least 5 weeks, at least 6 weeks, at least 7 weeks,  at least 8 weeks, at least 9 weeks, or longer). In some embodiments, each dose is about 60 ug.  In some embodiments, each dose is about 50 ug. In some embodiments, each dose is about  30 ug. In some embodiments, each dose is about 25 ug. In some embodiments, each dose is  about 20 ug. In some embodiments, each dose is about 15 ug.  In some embodiments, each  dose is about 10 ug. In some embodiments, each dose is about 3 ug.   In  some  embodiments,  at  least  one  dose  given  in  a  vaccination  regimen  (e.g.,  a  primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  60  ug.  In  some  embodiments, at  least one dose given  in a vaccination regimen (e.g., a primary vaccination  regimen and/or a booster vaccination regimen) is about 50 ug. In some embodiments, at least  one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster  vaccination  regimen)  is  about  30 ug.  In  some  embodiments,  at  least one dose  given  in  a  vaccination  regimen  (e.g.,  a  primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  25 ug.  In  some  embodiments,  at  least one  dose  given  in  a  vaccination  regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about  20 ug.  In some embodiments, at least one dose given in a vaccination regimen (e.g., a primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  15  ug.  In  some  embodiments, at  least one dose given  in a vaccination regimen (e.g., a primary vaccination  regimen and/or a booster vaccination regimen) is about 10 ug. In some embodiments, at least  one dose given in a vaccination regimen (e.g., a primary vaccination regimen and/or a booster  vaccination regimen) is about 3 ug.  In one embodiment, an amount of the RNA described herein of about 60 µg is administered  per dose.  In one embodiment,  an amount of  the RNA described herein of about 50 µg  is  administered per dose. In one embodiment, an amount of the RNA described herein of about  30 µg is administered per dose. In one embodiment, an amount of the RNA described herein  of about 25 µg is administered per dose. In one embodiment, an amount of the RNA described  herein of about 20 µg is administered per dose. In one embodiment, an amount of the RNA  described herein of about 15 µg is administered per dose. In one embodiment, an amount of  the RNA described herein of about 10 µg  is administered per dose.  In one embodiment, an  amount  of  the  RNA  described  herein  of  about  5  µg  is  administered  per  dose.  In  one  embodiment, an amount of the RNA described herein of about 3 µg is administered per dose.  In one embodiment, at least two of such doses are administered. For example, a second dose  may be administered about 21 days following administration of the first dose.   In some embodiments, the efficacy of the RNA vaccine described herein (e.g., administered in  two  doses,  wherein  a  second  dose  may  be  administered  about  21  days  following  administration of the first dose, and administered, for example, in an amount of about 30 µg  per dose)  is at  least 70%, at  least 80%, at  least 90, or at  least 95% beginning 7 days after  administration of  the second dose  (e.g., beginning 28 days after administration of  the  first  dose  if a second dose  is administered 21 days following administration of the first dose).  In  some embodiments, such efficacy is observed in populations of age of at least 50, at least 55,  at least 60, at  least 65, at  least 70, or older. In some embodiments, the efficacy of the RNA  vaccine described herein  (e.g., administered  in  two doses, wherein a  second dose may be  administered about 21 days following administration of the first dose, and administered, for  example, in an amount of about 30 µg per dose) beginning 7 days after administration of the  second dose (e.g., beginning 28 days after administration of the first dose if a second dose is  administered 21 days following administration of the first dose)  in populations of age of at  least 65, such as 65 to 80, 65 to 75, or 65 to 70, is at least 90%, at least 91%, at least 92%, at  least 93%, at least 94%, or at least 95%. Such efficacy may be observed over time periods of  up to 1 month, 2 months, 3 months, 6 months or even longer.  In one embodiment, vaccine efficacy  is defined as  the percent  reduction  in  the number of  subjects with evidence of infection (vaccinated subjects vs. non‐vaccinated subjects).  In one embodiment, efficacy is assessed through surveillance for potential cases of COVID‐19.  If, at any time, a patient develops acute respiratory illness, for the purposes herein, the patient  can be considered to potentially have COVID‐19 illness. The assessments can include a nasal  (midturbinate)  swab, which may be  tested using a  reverse  transcription‐polymerase  chain  reaction (RT‐PCR) test to detect SARS‐CoV‐2. In addition, clinical information and results from  local standard‐of‐care tests can be assessed.   In some embodiments, efficacy assessments may utilize a definition of SARS‐CoV‐2‐related  cases wherein:  • Confirmed COVID‐19: presence of at  least 1 of  the  following  symptoms and SARS‐CoV‐2  NAAT (nucleic acid amplification‐based test) positive during, or within 4 days before or after,  the symptomatic period: fever; new or increased cough; new or increased shortness of breath;  chills; new or increased muscle pain; new loss of taste or smell; sore throat; diarrhea; vomiting.  Alternatively  or  additionally,  in  some  embodiments,  efficacy  assessments  may  utilize  a  definition  of  SARS‐CoV‐2‐related  cases  wherein  one  or more  of  the  following  additional  symptoms defined by  the CDC  can be  considered:  fatigue; headache; nasal  congestion or  runny nose; nausea.  In some embodiments, efficacy assessments may utilize a definition of SARS‐CoV‐2‐related  severe cases  • Confirmed severe COVID‐19: confirmed COVID‐19 and presence of at least 1 of the following:  clinical signs at rest indicative of severe systemic illness (e.g., RR ≥30 breaths per minute, HR  ≥125  beats  per  minute,  SpO2≤93%  on  room  air  at  sea  level,  or  PaO2/FiO2<300mm  Hg);  respiratory failure (which can be defined as needing high‐flow oxygen, noninvasive ventilation,  mechanical ventilation, or ECMO); evidence of shock (e.g., SBP <90 mm Hg, DBP <60 mm Hg,  or  requiring  vasopressors);  significant  acute  renal,  hepatic,  or  neurologic  dysfunction;  admission to an ICU; death.   Alternatively or additionally,  in some embodiments a serological definition can be used  for  patients without clinical presentation of COVID‐19: e.g., confirmed seroconversion to SARS‐ CoV‐2 without confirmed COVID‐19: e.g., positive N‐binding antibody result in a patient with  a prior negative N‐binding antibody result.   In some embodiments, any or all of the following assays can be performed on serum samples:  SARS‐CoV‐2 neutralization assay; S1‐binding IgG level assay; RBD‐binding IgG level assay; N‐ binding antibody assay.  In one embodiment, methods and agents described herein are administered to a paediatric  population.  In  various  embodiments,  the  paediatric  population  comprises  or  consists  of  subjects under 18 years, e.g., 5 to less than 18 years of age, 12 to less than 18 years of age, 16  to less than 18 years of age, 12 to less than 16 years of age, or 5 to less than 12 years of age.  In various embodiments, the paediatric population comprises or consists of subjects under 5  years, e.g., 2 to less than 5 years of age, 12 to less than 24 months of age, 7 to less than 12  months of age, or less than 6 months of age. In some such embodiments, an RNA (e.g., mRNA)  composition described herein is administered to subjects of less than 2 years old, for example,  6  months  to  less  than  2  years  old.  In  some  such  embodiments,  an  RNA  (e.g.,  mRNA)  composition  described  herein  is  administered  to  subjects  of  less  than  6 months  old,  for  example, 1 month to less than 4 months old.  In some embodiments, a dosing regimen (e.g.,  doses and/or dosing schedule) for a paediatric population may vary for different age groups.  For  example,  in  some  embodiments,  a  subject  6 months  through  4  years  of  age may  be  administered according to a primary regimen comprising at  least three doses,  in which the  initial two doses are adminsitered at least 3 weeks (including, e.g., at least 4 weeks, at least 5  weeks, at least 6 weeks, or longer) apart followed by a third dose administered at least 8 weeks  (including, e.g., at  least 9 weeks, at least 10 weeks, at  least 11 weeks, at least 12 weeks, or  longer) after the second dose. In some such embodiments, at least one dose administered is  3 ug RNA described herein.  In some embodiments, a subject 5  years of age and older may be  administered according to a primary regimen comprising at least two doses, in which the two  doses are administered at least 3 weeks (including, e.g., at least 3 weeks, at least 4 weeks, at  least 5 weeks, at least 6 weeks, or longer) apart.  In some such embodiments, at least one dose  administered is 10 ug RNA described herein. In some embodiments, a subject 5 years of age  and  older who  are  immunocompromised  (e.g.,  in  some  embodiments  subjects who  have  undergone  solid  organ  transplantation,  or  who  are  diagnosed  with  conditions  that  are  considered to have an equivalent of immunocompromise) may be administered according to  a  primary  regimen  comprising  at  least  three  doses,  in  which  the  initial  two  doses  are  administered at  least 3 weeks  (including, e.g., at  least 3 weeks, at  least 4 weeks, at  least 5  weeks, at  least 6 weeks, or  longer) apart,  followed by a  third dose administered at  least 4  weeks (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at  least 9 weeks, at  least 10 weeks, at  least 11 weeks, at  least 12 weeks, or  longer) after  the  second dose.  In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to  subjects of age 12 or older and each dose is about 30 ug.  In some embodiments, an RNA (e.g.,  mRNA) composition described herein is administered to subjects of age 12 or older (including,  e.g., age 18 or older) and each dose is higher than 30 ug, including, e.g., 35 ug, 40 ug, 45 ug,  50 ug, 55 ug, 60 ug, 65 ug , 70 ug, or higher. In some such embodiments,  an RNA (e.g., mRNA)  composition described herein is administered to subjects of age 12 or older and each dose is  about 60 ug.  In some such embodiments, an RNA (e.g., mRNA) composition described herein  is  administered  to  subjects  of  age  12  or  older  and  each  dose  is  about  50  ug.    In  one  embodiment, the paediatric population comprises or consists of subjects 12 to  less than 18  years of age including subjects 16 to less than 18 years of age and/or subjects 12 to less than  16 years of age. In this embodiment, treatments may comprise 2 vaccinations 21 days apart,  wherein, in one embodiment, the vaccine is administered in an amount of 30 µg RNA per dose,  e.g., by intramuscular administration. In some embodiments, higher doses are administered  to older pediatric patients and adults, e.g., to patients 12 years or older, compared to younger  children or infants, e.g. 2 to less than 5 years old, 6 months to less than 2 years old, or less  than 6 months old.  In some embodiments, higher doses are administered to children who are  2 to less than 5 years old, as compared to toddlers and/or infants, e.g., who are 6 months to  less than 2 years old, or less than 6 months old.  In one embodiment, the paediatric population comprises or consists of subjects 5 to less than  18 years of age including subjects 12 to less than 18 years of age and/or subjects 5 to less than  12 years of age. In this embodiment, treatments may comprise 2 vaccinations 21 days apart,  wherein, in various embodiments, the vaccine is administered in an amount of 10 µg, 20 µg,  or 30 µg RNA per dose, e.g., by intramuscular administration. In some such embodiments, an  RNA (e.g., mRNA) composition described herein is administered to subjects of age 5 to 11 and  each dose  is about 10 ug. In some embodiments, each dose comprises about 5 ug of RNA  encoding a SARS‐CoV‐2 S protein of a first variant and about 5 ug of RNA encoding a SARS‐ CoV‐2 S protein of a second variant. In some embodiments, each dose comprises about 5 ug  of RNA encoding a SARS‐CoV‐ 2 S protein of a Wuhan strain and about 5 ug of RNA encoding  a SARS‐CoV‐2 S protein of an Omicron variant. In some embodiments, each dose comprises  about 5 ug of RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising  SEQ  ID NO: 20) and about 5 ug of RNA encoding a SARS‐CoV‐2 S protein of a BA.1 Omicron  variant  (e.g., RNA comprising SEQ  ID NO: 93).  In some embodiments, each dose comprises  about 5 ug of RNA encoding a SARS‐ CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising  SEQ ID NO: 20) and about 5 ug of RNA encoding a SARS‐CoV‐2 S protein of a BA.4/5 Omicron  variant (e.g., RNA comprising SEQ ID NO: 103).  In one embodiment, the paediatric population comprises or consists of subjects  less than 5  years of age including subjects 2 to less than 5 years of age, subjects 12 to less than 24 months  of age, subjects 7 to  less than 12 months of age, subjects 6 to  less  than 12 months of age  and/or subjects less than 6 months of age.   In some embodiments, pediatric patients can be administered a dose  (e.g., a  first, second,  third, fourth, or fifth dose) comprising about 3 µg, about 6 µg, about 10 µg, about 20 µg, or  about 30 µg of RNA (e.g., monovalent or multivalent RNA).  In some embodiments,  a pediatric  patient is administered a multivalent vaccine comprising two or more RNAs, each encoding a  SARS‐CoV‐2 S protein associated with a different variant (e.g., a bivalent vaccine comprising  about 3 µg, about 6 µg, about 10 µg, about 20 µg, or about 30 µg of  total RNA).  In some  embodiments,  a pediatric patient is administered a multivalent vaccine comprising two RNAs,  each encoding  a  SARS‐CoV‐2  S protein  associated with  a different  variant  (e.g., a bivalent  vaccine comprising about 1.5 µg, about 3 µg, about 5 µg, about 10 µg, or about 15 µg of each  RNA).    In  some embodiments, a pediatric dose  is administered a  subsequent dose  (e.g., a  second, third, fourth, or fifth dose) that comprises a higher amount of RNA than a previous  dose.    For  example,  in  some  such  embodiments,  a  pediatric  subject  is  administered  a  subsequent dose (e.g., a third dose, administered as a booster) that is 1‐10x that of a previous  dose (e.g., 1x‐5x, 2x‐5x, 2x‐4x, about 1.5x, about 2x, about 2.5x, about 3x, about 3.5x, about  4x, about 4.5x, about 5x, about 5.5x, about 6x, about 6.5x, about 7.5x, about 8x, about 8.5x,  about 9x, about 9.5x, or about 10x a previous dose).    In this embodiment, treatments may comprise 2 vaccinations, e.g., 21 to 42 days apart, e.g.,  21 days apart, wherein,  in various embodiments, vaccine  is administered  in an amount of  about 3 µg, about 6 µg, about 10 µg, about 20 µg, or about 30 µg RNA per dose, e.g., by  intramuscular administration. In some such embodiments, an RNA (e.g., mRNA) composition  described herein is administered to subjects of age 2 to less than 5 and each dose is about 3  ug. In  some  such  embodiments,  an  RNA  (e.g.,  mRNA)  composition  described  herein  is  administered to subjects of about 6 months to less than about 5 years and each dose is about  3 ug.  In some embodiments, each dose comprises about 1.5 ug of RNA encoding a SARS‐CoV‐ 2 S protein of a first variant and about 1.5 ug of RNA encoding a SARS‐CoV‐2 S protein of a  second variant. In some embodiments, each dose comprises about 1.5 ug of RNA encoding a  SARS‐CoV‐2 S protein of a Wuhan strain and about 1.5 ug of RNA encoding a SARS‐CoV‐2 S  protein of an Omicron variant. In some embodiments, each dose comprises about 1.5 ug of  RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83)  and about 1.5 ug of RNA encoding a SARS‐CoV‐2 S protein of a BA.2 Omicron variant (e.g., RNA  comprising SEQ ID NO: 98). In some embodiments, each dose comprises about 1.5 ug of RNA  encoding a SARS‐CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and  about 1.5 ug of RNA encoding a SARS‐CoV‐2 S protein of a BA.4/5 Omicron variant (e.g., RNA  comprising SEQ ID NO: 103).  In  some  such  embodiments,  an  RNA  (e.g.,  mRNA)  composition  described  herein  is  administered to subjects of about 6 months to less than about 5 years and each dose is about  6 ug.  In some embodiments, each dose comprises about 3 ug of RNA encoding a SARS‐CoV‐2  S protein of a first variant and about 3 ug of RNA encoding a SARS‐CoV‐2 S protein of a second  variant. In some embodiments, each dose comprises about 3 ug of RNA encoding a SARS‐CoV‐ 2 S protein of a Wuhan strain and about 3 ug of RNA encoding a SARS‐CoV‐2 S protein of an  Omicron variant. In some embodiments, each dose comprises about 3 ug of RNA encoding a  SARS‐CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and about 3 ug  of RNA encoding a SARS‐CoV‐2 S protein of a BA.2 Omicron variant (e.g., RNA comprising SEQ  ID NO: 98). In some embodiments, each dose comprises about 3 ug of RNA encoding a SARS‐ CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and about 3 ug of RNA  encoding a SARS‐CoV‐2 S protein of a BA.4/5 Omicron variant (e.g., RNA comprising SEQ  ID  NO: 103).  In  some  such  embodiments,  an  RNA  (e.g.,  mRNA)  composition  described  herein  is  administered to subjects of about 6 months to less than about 5 years and each dose is about  10 ug.  In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS‐CoV‐ 2 S protein of a first variant and about 5 ug of RNA encoding a SARS‐CoV‐2 S protein of a second  variant. In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS‐CoV‐ 2 S protein of a Wuhan strain and about 5 ug of RNA encoding a SARS‐CoV‐2 S protein of an  Omicron variant. In some embodiments, each dose comprises about 5 ug of RNA encoding a  SARS‐CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and about 5 ug  of RNA encoding a SARS‐CoV‐2 S protein of a BA.2 Omicron variant (e.g., RNA comprising SEQ  ID NO: 98). In some embodiments, each dose comprises about 5 ug of RNA encoding a SARS‐ CoV‐2 S protein of a Wuhan strain (e.g., RNA comprising SEQ ID NO: 83) and about 5 ug of RNA  encoding a SARS‐CoV‐2 S protein of a BA.4/5 Omicron variant (e.g., RNA comprising SEQ  ID  NO: 103).    In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to  subjects of age 12 or older and at least one dose given in a vaccination regimen (e.g., a primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  60  ug.  In  some  embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects  of  age  12  or  older  and  at  least  one  dose  given  in  a  vaccination  regimen  (e.g.,  a  primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  30  ug.  In  some  embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects  of  age  12  or  older  and  at  least  one  dose  given  in  a  vaccination  regimen  (e.g.,  a  primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  15  ug.    In  some  embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects  of age 5 to less than 12 years of age and at least one dose given in a vaccination regimen (e.g.,  a primary vaccination regimen and/or a booster vaccination regimen) is about 10 ug. In some  embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects  of age 2 to less than 5 and at least one dose given in a vaccination regimen (e.g., a primary  vaccination  regimen  and/or  a  booster  vaccination  regimen)  is  about  3  ug.  In  some  embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects  of 6 months to  less than age 2 and at least one dose given in a vaccination regimen (e.g., a  primary vaccination regimen and/or a booster vaccination regimen)  is about 3 ug or  lower,  including, e.g., 2 ug, 1 ug, or lower). In some embodiments, an RNA (e.g., mRNA) composition  described herein is administered to infants of less than 6 months and at least one dose given  in a vaccination  regimen  (e.g., a primary vaccination regimen and/or a booster vaccination  regimen) is about 3 ug or lower, including, e.g., 2 ug, 1 ug, 0.5 ug, or lower).  In some embodiments, an RNA  (e.g., mRNA) composition described herein  is administered  (e.g.,  by  intramuscular  injection)  as  a  single  dose.  In  some  embodiments,  a  single  dose  comprise a single RNA encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  (e.g.,  an  RBD  domain).  In  some  embodiments,  a  single  dose  comprise  at  least  two  RNAs  described herein, for example, each RNA encoding a SARS‐CoV‐2 S protein or an immunogenic  fragment thereof (e.g., an RBD domain) from different strains. In some embodiments, such at  least two RNAs described herein can be administered as a single mixture. For example, in some  such  embodiments,  two  separate  RNA  compositions  described  herein  can  be  mixed  to  generate a single mixture prior to  injection.  In some embodiments, such at  least two RNAs  described herein can be administered as two separate compositions, which, for example, can  be administered at different  injection sites (e.g., on different arms, or different sites on the  same arm).  In  some  embodiments,  a  dose  administered  to  subjects  in  need  thereof  may  comprise  administration of a single RNA (e.g., mRNA) composition described herein.   In  some  embodiments,  a  dose  administered  to  subjects  in  need  thereof  may  comprise  administration of at least two or more (including, e.g., at least three or more) different drug  products/formulations. For example,  in some embodiments, at  least two or more different  drug  products/formulations  may  comprise  at  least  two  different  RNA  (e.g.,  mRNA)  compositions described herein (e.g., in some embodiments each comprising a different RNA  construct).   In  some  embodiments,  an  RNA  (e.g.,  mRNA)  composition  disclosed  herein  may  be  administered  in conjunction with a vaccine  targeting a different  infectious agent.    In some  embodiments, the different infectious agent is one that increases the likelihood of a subject  experiencing  deleterious  symptoms when  coinfected with  SARS‐CoV‐2  and  the  infectious  agent.  In some embodiments, the infectious agent is one that increases the infectivity of SARS‐ CoV‐2 when  a  subject  is  coinfected with  SARS‐CoV‐2  and  the  infectious  agent.    In  some  embodiments,  at  least  one  RNA  (e.g.,  mRNA)  composition  described  herein  may  be  administered in combination with a vaccine that targets influenza.  In some embodiments, at  least two or more different drug products/formulations may comprise at least one RNA (e.g.,  mRNA) composition described herein and a vaccine targeting a different infectious agent (e.g.,  an  influenza  vaccine).  In  some  embodiments,  different  drug  products/  formulations  are  separately administered. In some embodiments, such different drug product/formulations are  separately adminsitered at the same time (e.g., at the same vaccination session) at different  sites of a subject (e.g., at different arms of the subject).   In one embodiment, at least two doses are administered. For example, a second dose may be  administered about 21 days following administration of the first dose.   In some embodiments, at least one single dose is administered.  In some embodiments, such  single dose is administered to subjects, for example, who may have previously received one  or more doses of, or a complete regimen of, a SARS‐CoV‐2 vaccine (e.g., of a BNT162b2 vaccine  [including,  e.g.,  as  described  herein],  an mRNA‐1273  vaccine,  an  Ad26.CoV2.S  vaccine,  a  ChAdxOx1 vaccine, an NVX‐CoV2373 vaccine, a CvnCoV vaccine, a GAM‐COVID0Vac vaccine, a  CoronaVac vaccine, a BBIBP‐CorV vaccine, an Ad5‐nCoV vaccine, a zf2001 vaccine, a SCB‐2019  vaccine, a JNJ 78436735 vaccine,or other approved RNA (e.g., mRNA) or adenovector vaccines,  etc.   Alternatively or additionally,  in  some embodiments, a  single dose  is  administered  to  subjects who have been exposed to and/or infected by SARS‐CoV‐2.  In some embodiments,  at least one single dose is administered to subjects who both have received one or more doses  of, or a complete regimen of, a SARS‐CoV‐2 vaccine and have been exposed to and/or infected  with SARS‐CoV‐2.  In some particular embodiments where at  least one single dose  is administered to subjects  who have received one or more doses of a prior SARS‐CoV‐2 vaccine, such prior SARS‐CoV‐2  vaccine is a different vaccine, or a different form (e.g., formulation) and/or dose of a vaccine  with the same active (e.g., BNT162b2);  in some such embodiments, such subjects have not  received a  complete  regimen of  such prior vaccine and/or have experienced one or more  undesirable reactions to or effects of one or more received doses of such prior vaccine.    In  some particular embodiments, such prior vaccine is or comprises higher dose(s) of the same  active  (e.g.,  BNT162b2).    Alternatively  or  additionally,  in  some  such  embodiments,  such  subjects were exposed to and/or  infected by SARS‐CoV‐2 prior to completion (but,  in some  embodiments, after initiation) of a full regimen of such prior vaccine..  In one embodiment, at least two doses are administered. For example, a second dose may be  administered about 21 days following administration of the first dose.   In one embodiment, at least three doses are administered.  In some embodiments, such third  dose is administered a period of time after the second dose that is comparable to (e.g., the  same  as)  the  period  of  time  between  the  first  and  second  doses.    For  example,  in  some  embodiments, a third dose may be administered about 21 days following administration of  the second dose.  In some embodiments, a third dose is administered after a longer period of  time relative to the second dose than the second dose was relative to the first dose.  In some  embodiments, a three‐dose regimen is administered to an immunocompromised patient, e.g.,  a  cancer  patient,  an  HIV  patient,  a  patient  who  has  received  and/or  is  receiving  immunosuppressant therapy (e.g., an organ transplant patient).  In some embodiments, the  length of time between the second and third dose (e.g., a second and third dose administered  to an immunocompromised patient) is at least about 21 days (e.g., at least about 28 days).  In some embodiments, a vaccination regimen comprises administering the same amount of  RNA in different doses (e.g., in first and/or second and/or third and/or subsequent doses).  In  some embodiments, a vaccination regimen comprises administering different amounts of RNA  in different doses.  In some embodiments, one or more later doses is larger than one or more  earlier doses  (e.g.,  in situations where waning of vaccine efficacy  from one or more earlier  doses  is observed and/or  immune escape by a  variant  (e.g., one described herein)  that  is  prevalent  or  rapidly  spreading  is  observed  in  a  relevant  jurisdiction  at  the  time  of  administration  is observed).  In some embodiments, one or more  later doses may be  larger  than one or more earlier doses by at least 30%, at least 40%, at least 50%, at least 60%, at least  70%, at least 80%, at least 90%, at least 95%, or higher, provided that safety and/or tolerability  of such a dose is clinically acceptable.  In some embodiments, one or more later doses may be  larger than one or more earlier doses by at least 1.1‐fold, at least 1.5‐fold, at least 2‐fold, at  least 3‐fold, at least 4‐fold, or higher provided that safety and/or tolerability of such a dose is  clinically acceptable.  In some embodiments, one or more later doses is smaller than one or  more earlier doses  (e.g.,  in a negative  reaction was experienced after one or more earlier  doses and/or  if exposure to and/or  infection by SARS‐CoV‐2 between an earlier dose and a  subsequent dose).  In some embodiments, one or more later doses may be smaller than one  or more earlier doses by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at  least  60%,  or  higher.  In  some  embodiments, where  different  doses  are  utilized,  they  are  related to one another by identity with and/or dilution of a common stock as described herein.   In some embodiments, where at least two or more doses are administered (e.g., at least two  doses  administered  in  a  primary  regimen,  at  least  two  doses  administered  in  a  booster  regimen, or at least one dose administered  in a primary regimen and at least one dose  in a  booster regimen), the same RNA compositions described herein may be administered in such  doses and each of such doses can be  the same or different  (as described herein).  In some  embodiments, where at  least two or more doses are administered (e.g., at  least two doses  administered in a primary regimen, at least two doses administered in a booster regimen, or  at  least  one  dose  administered  in  a  primary  regimen  and  at  least  one  dose  in  a  booster  regimen),  different  RNA  compositions  described  herein  (e.g.,  different  encoded  viral  polypeptides, e.g.,  from different coronavirus clades, or  from different strains of  the same  coronavirus clade; different construct elements such as 5’ cap, 3’ UTR, 5’ UTR, etc.; different  formulations,  e.g.,  different  excipients  and/or  buffers  (e.g.,  PBS  vs.  Tris);  different  LNP  compositions; or combinations thereof) may be administered in such doses and each of such  doses can be the same or different (e.g., as described herein).     In some embodiments, a subject is administered two or more RNAs (e.g., as part of either a  primary regimen or a booster regimen), wherein the two or more RNAs are administered on  the same day or same visit.  In some embodiments, the two or more RNAs are administered  in separate compositions, e.g., by administering each RNA to a separate part of the subject  (e.g., by intramuscular administration to different arms of the subject or to different sites of  the same arm of the subject).  In some embodiments, the two or more RNAs are mixed prior  to administration (e.g., mixed immediately prior to administration, e.g., by the administering  practitioner).  In some embodiments, the two or more RNAs are formulated together (e.g., by  (a) mixing separate populations of LNPs, each population comprising a different RNA; or (b) by  mixing two or more RNAs prior to LNP formulation, so that each LNP comprises two or more  RNAs).  In  some  embodiments,  the  two  or more  RNAs  comprise  an  RNA  that  encode  a  coronavirus  S protein or immunogenic fragment thereof (e.g., RBD or other relevant domains)  from one strain (e.g., Wuhan strain) and a variant that is prevalent or rapidly spreading in a  relevant jurisdiction at the time of administration (e.g., a variant described herein). In some  embodiments, such a variant is an Omicron variant (e.g., a BA.1, BA.2, or BA.3 variant). In some  embodiments, the two or more RNAs comprise a first RNA and a second RNA that have been  shown to elicit a broad immune response in subject.  In some embodiments the two or more  RNAs comprise an RNA encoding a SARS‐CoV‐2 S protein  from a Wuhan strain and an RNA  encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant.  In some embodiments the two  or more RNAs comprise an RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and an  RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant. In some embodiments the  two or more RNAs comprise an RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain  and an RNA encoding a SARS‐CoV‐2 S protein from a BA.4 or BA.5 Omicron variant. In some  embodiments the two or more RNAs comprise an RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and an RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron  variant.      In some embodiments the two or more RNAs comprise an RNA encoding a SARS‐ CoV‐2 S protein from a BA.1 Omicron variant and an RNA encoding a SARS‐CoV‐2 S protein  from a BA.4 or BA.5 Omicron variant.  In some embodiments the two or more RNAs comprise  an RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant and an RNA encoding a  SARS‐CoV‐2 S protein from a BA.4 or 5 Omicron variant.    In some embodiments the two or  more RNAs comprise an RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, an alpha  variant,  a beta  variant,  or  a  delta  variant, or  sublineages  derived  therefrom;  and  an RNA  encoding  a  SARS‐CoV‐2  S  protein  from  a BA.2, BA.4 or  5 Omicron  variant,  or  sublineages  derived therefrom.        In  some  embodiments,  a  subject  may  be  administered  a  dose  comprising  any  one  of  combinations 1 to 66, listed in the below table. In some embodiments, such combinations can  be  administered  using  an  LNP  formulation, where  the  first  RNA  and  the  second  RNA  are  encapsulated in the same LNP or in separate LNPs. In some embodiments, such combinations  can be administered as separate LNP formulations (e.g., by administering at separate sites to  a subject).   
Figure imgf000454_0001
Figure imgf000455_0001
Figure imgf000456_0001
Figure imgf000457_0001
1Listed RNAs encode a SARS‐CoV‐2 S protein having mutations characteristic of the indicated  SARS‐CoV‐2 variant.  In some embodiments, a vaccination regimen comprises a first vaccination regimen (e.g., a  primary regimen) that includes at least two doses of an RNA composition as described herein,  e.g., wherein the second dose may be administered about 21 days following administration of  the first dose, and a second vaccination (e.g., a booster regimen) that comprises a single dose  or multiple  doses,  e.g.,  two  doses,  of  an  RNA  composition  as  described  herein.  In  some  embodiments,  doses  of  a  booster  regimen  are  related  to  those  of  a  primary  regimen  by  identity with or dilution from a common stock as described herein.  In various embodiments,  a booster regimen is administered (e.g., is initiated) at least 1 week, at least 2 weeks, at least  3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5  months, or at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least  10 months, at least 11 months, at least 12 months, or longer, after administration of a primary  regimen, e.g., after completion of a primary regimen comprising at least two doses. In various  embodiments, a booster regimen is administered (e.g., is initiated) 1‐12 months, 2‐12 months,  3‐12 months, 4‐12 months, 6‐12 months, 1‐6 months, 1‐5 months, 1‐4 months, 1‐3 months,  or 2‐3 months after administration of a primary regimen, e.g., after completion of a primary  regimen  comprising  at  least  two  doses.  In  various  embodiments,  a  booster  regimen  is  administered  (e.g.,  is  initiated) 1  to 60 months, 2  to 48 months, 2  to 24 months, 3  to 24  months, 6 to 18 months, 6 to 12 months, or 5 to 7 months after administration of a primary  regimen, e.g., after completion of a two‐dose primary regimen. In some embodiments, each  dose of a primary regimen  is about 60 µg per dose.  In some embodiments, each dose of a  primary  regimen  is about 50 µg per dose.  In  some embodiments, each dose of a primary  regimen is about 30 µg per dose. In some embodiments, each dose of a primary regimen  is  about 25 µg per dose. In some embodiments, each dose of a primary regimen is about 20 µg  per dose. In some embodiments, each dose of a primary regimen is about 15 µg per dose. In  some  embodiments,  each  dose  of  a  primary  regimen  is  about  10  µg  per  dose.  In  some  embodiments, each dose of a primary regimen is about 3 µg per dose.  In some embodiments,  each  dose  of  a  booster  regimen  is  the  same  as  that  of  the  primary  regimen.  In  some  embodiments, each dose of a booster regimen comprises the same amount of RNA as a dose  administered  in a primary  regimen.  In  some embodiments, at  least one dose of a booster  regimen is the same as that of the primary regimen. In some embodiments, at least one dose  of a booster regimen comprises the same amount of RNA as at least one dose of a primary  regimen. In some embodiments, at least one dose of a booster regimen is lower than that of  the primary regimen. In some embodiments, at least one dose of a booster regimen comprises  an amount of RNA that is lower than that of a primary regimen. In some embodiments, at least  one  dose  of  a  booster  regimen  is  higher  than  that  of  the  primary  regimen.  In  some  embodiments, at  least one dose of a booster regimen comprises an amount of RNA that  is  higher than that of a primary regimen.  In  some embodiments,  a booster  regimen  (e.g.,  as described herein)  is  administered  to  a  pediatric patient (e.g., a patient aged 2 through 5 years old, a patient aged 5 through 11 years  old, or a patient aged 12 through 15 years old). In some embodiments, a booster regimen is  administered  to a pediatric patient who  is 6 months old  to  less  than 2 years old.  In  some  embodiments, a booster  regimen  is administered  to a pediatric patient who  is  less  than 6  months old. In some embodiments, a booster regimen is administered to a pediatric patient  who  is 6 months old  to  less  than 5 years old.  In some embodiments, a booster  regimen  is  administered  to  a  pediatric  patient who  is  2  years  old  to  less  than  5  years  old.  In  some  embodiments, a booster regimen is administered to a pediatric patient who is 5 years old to  less than 12 years old. In some embodiments, a booster regimen is administered to a pediatric  patient who is 12 years old to less than 16 years old.    In some embodiments, each dose of a  pediatric booster regimen comprises about 3 µg of RNA.  In some embodiments, each dose of  a pediatric booster regimen comprises about 6 µg of RNA.  In some embodiments, each dose  of a pediatric booster regimen comprises about 10 µg of RNA.  In some embodiments, each  dose of a pediatric booster regimen comprises about 15 µg of RNA.  In some embodiments,  each  dose  of  a  pediatric  booster  regimen  comprises  about  20  µg  of  RNA.  In  some  embodiments, each dose of a pediatric booster regimen comprises about 25 µg of RNA.  In  some embodiments, each dose of a pediatric booster regimen comprises about 30 µg of RNA.  In some embodiments, a booster regimen is administered to a non‐pediatric patient (e.g., a  patient 16 years or older, a patient aged 18 through 64 years old, and/or a patient 65 years  and older).  In some embodiments, each dose of a non‐pediatric booster regimen comprises  about 3 ug of RNA, about 10 ug of RNA, about 25 µg or RNA, about 30 µg of RNA, about 40 µg  of RNA, about 45 µg of RNA, about 50 µg of RNA, about 55 µg of RNA, or about 60 µg of RNA  .   In some embodiments, the same booster regimen may be administered to both pediatric  and non‐pediatric patients  (e.g.,  to a patient 12 years or older).    In  some embodiments, a  booster  regimen  that  is  administered  to  a  non‐pediatric  patient  is  administered  in  a  formulation and dose that is related to that of a primary regimen previously received by the  patient by  identity with or by dilution as described herein.    In some embodiments, a non‐ pediatric patient who receives a booster regimen at a lower dose than a primary regimen may  have experienced an adverse reaction to one or more doses of such primary regimen and/or  may have been exposed to and/or infected by SARS‐CoV‐2 between such primary regimen and  such booster  regimen, or between doses of  such primary  regimen and/or of  such booster  regimen. In some embodiments, pediatric and non‐pediatric patients may receive a booster  regimen at a higher dose than a primary regimen when waning of vaccine efficacy at  lower  doses is observed, and/or when immune escape of a variant that is prevalent and/or spreading  rapidly at a relevant jurisdiction at the time of administration is observed.  In some embodiments one or more doses of a booster regimen differs from that of a primary  regimen.   For example,  in some embodiments, an administered dose may correspond  to a  subject’s  age  and  a  patient may  age  out  of  one  treatment  age  group  and  into  a  next.   Alternatively or additionally, in some embodiments, an administered dose may correspond to  a patient’s condition (e.g., immunocompromised state) and a different dose may be selected  for  one  or more  doses  of  a  booster  regimen  than  for  a  primary  regimen  (e.g.,  due  to  intervening cancer treatment, infection with HIV, receipt of immunosuppressive therapy, for  example associated with an organ transplant.  In some embodiments, at least one dose of a  booster  regimen may  comprise  an  amount  of  RNA  that  is  higher  than  at  least  one  dose  administered in a primary regimen (e.g., in situations where waning of vaccine efficacy from  one or more earlier doses is observed and/or immune escape by a variant (e.g., one described  herein) that is prevalent or rapidly spreading is observed in a relevant jurisdiction at the time  of administration).    In some embodiments, a primary regimen may involve one or more 3 ug doses and a booster  regimen may  involve one or more 10 ug doses, and/or one or more 20 ug doses, or one or  more 30 ug doses.  In some embodiments, a primary regimen may involve one or more 3 ug  doses and a booster regimen may involve one or more 3 ug doses. In some embodiments, a  primary regimen may involve two or more 3 ug doses (e.g., at least two doses, each comprising  3 ug of RNA, and administered about 21 days after one another) and a booster regimen may  involve one or more 3 ug doses. In some embodiments, a primary regimen may involve one or  more 10 ug doses and a booster regimen may involve one or more 20 ug doses, and/or one or  more 30 ug doses. In some embodiments, a primary regimen may involve one or more 10 ug  doses and a booster regimen may involve one or more 10 ug doses.  In some embodiments, a  primary regimen may involve two or more 10 ug doses (e.g., two doses, each comprising 10  ug of RNA, administered about 21 days apart) and a booster regimen may involve one or more  10 ug doses.  In some embodiments, a primary regimen may involve one or more 20 ug doses  and a booster regimen may involve one or more 30 ug doses.  In some embodiments, a primary  regimen may involve one or more 20 ug doses and a booster regimen may involve one or more  20 ug doses.  In some embodiments, a primary regimen may involve one or more 30 ug doses,  and a booster regimen may also involve one or more 30 ug doses. In some embodiments, a  primary regimen may involve two or more 30 ug doses (e.g., two doses, each comprising 30  ug of RNA, administered about 21 days apart), and a booster regimen may also involve one or  more 30 ug doses. In some embodiments, a primary regimen may involve two or more 30 ug  doses (e.g., two doses, each comprising 30 ug of RNA, administered about 21 days apart), and  a booster regimen may  involve one or more 50 ug doses. In some embodiments, a primary  regimen may involve two or more 30 ug doses (e.g., two doses, each comprising 30 ug of RNA,  administered about 21 days apart), and a booster regimen may  involve one or more 60 ug  doses.   In some embodiments, a subject is administered a booster regimen comprising at least one 30  ug  dose  of  RNA.    In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising at  least one 30 ug dose of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain of SARS‐CoV‐2  (e.g., BNT162b2).    In some embodiments, a subject  is administered a  booster regimen comprising at least one dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein  having one or more mutations that are characteristic of a SARS‐CoV‐2 variant (e.g., a variant  described  herein).    In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising at least one dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.3, or BA.4  or BA.5 Omicron variant).  In some embodiments, a subject is administered a booster regimen  comprising at  least one dose of 30 ug of RNA, wherein  the 30 ug of RNA  comprises RNA  encoding a SARS‐CoV‐2 S protein  from a Wuhan  strain and RNA encoding a SARS‐CoV‐2 S  protein comprising mutations that are characteristic of a SARS‐CoV‐2 variant  (e.g.,  in some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 15 ug of  RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of  an Omicron variant).    In  some embodiments, a  subject  is administered a booster  regimen  comprising at least one dose comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan  strain  and  15  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations that are characteristic of a BA.1 Omicron variant.  In some embodiments, a subject  is administered a booster  regimen  comprising at  least one dose  comprising 15 ug of RNA  encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐ 2 S protein having one or more mutations that are characteristic of a BA.2 Omicron variant. In  some embodiments, a subject is administered a booster regimen comprising at least one dose  comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 15 ug of  RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of  a BA.3 Omicron variant. In some embodiments, a subject is administered a booster regimen  comprising at least one dose comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan  strain  and  15  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant  and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered a  booster regimen comprising at least one dose comprising 15 ug of RNA encoding a SARS‐CoV‐ 2 S protein from a BA.1 Omicron variant and 15 ug of RNA encoding a SARS‐CoV‐2 S protein  having one or more mutations  that are  characteristic of  a BA.3 Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant and  15  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more mutations  that  are  characteristic of a BA.4 or BA.5 Omicron variant.   In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant  and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic  of  a  BA.4  or  BA.5  Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising  15  ug  of  RNA  encoding a SARS‐CoV‐2 S protein from a BA.3 Omicron variant and 15 ug of RNA encoding a  SARS‐CoV‐2 S protein having one or more mutations that are characteristic of a BA.4 or BA.5  Omicron variant.  In some embodiments, a subject is administered a booster regimen comprising two or more  doses of 30 ug of RNA, administered at least two months apart from each other.  For example,  in some embodiments, subjects are administered a booster regimen comprising two doses of  30  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more mutations  that  are  characteristic of an Omicron variant (e.g., a BA.1, BA.2, or BA.4 or BA.5 Omicron variant).    In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of an Omicron variant of SARS‐CoV‐2 (e.g., a BA.1, BA.2, BA.3, BA.4, or  BA.5 Omicron  variant), wherein  the booster  regimen  is administered at  least  two months  (including, e.g., at least three months, at least four months, at least five months, at least six  months, or more) after completion of the primary regimen. In some embodiments, a subject  is administered (i) a primary regimen comprising at least two 30 ug doses of RNA encoding a  SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two doses are administered at least  approximately  21 days  apart,  and  (ii)  a booster  regimen  comprising  a  30 ug dose of RNA  comprising 15 ug RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 15 ug of RNA  encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of an  Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.1 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 30 ug dose of RNA  comprising 15 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.1 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.2 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 30 ug dose of RNA  comprising 15 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.4 or BA.5 Omicron variant of SARS‐CoV‐2, wherein the booster  regimen is administered at least two months (including, e.g., at least three months, at least  four months, at least five months, at least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 30 ug dose of RNA  comprising 15 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 30 ug dose of RNA comprising 15 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 30 ug dose of RNA comprising 15 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 30 ug dose of RNA comprising 15 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.2 Omicron variant and 15 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising  at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising at least one 30 ug dose of RNA encoding a SARS‐CoV‐2 S protein from a non‐BA.1  Omicron variant.    In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug  doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two doses  are administered at least approximately 21 days apart, and (ii) a booster regimen comprising  at  least  two  30  ug  doses  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations characteristic of an Omicron variant, wherein the booster regimen is administered  at least two months (including, e.g., at least three months, at least four months, at least five  months, at least six months, or more) after completion of the primary regimen, and the two  booster doses are administered at least two months apart from each other.  In some embodiments, a subject is administered a booster regimen comprising at least one 50  ug  dose  of  RNA.    In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising at least one dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain (e.g., BNT162b2).  In some embodiments, a subject is administered a booster regimen  comprising at least one dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations  that  are  characteristic  of  a  SARS‐CoV‐2  variant  (e.g.,  a  variant  described  herein).    In some embodiments, a subject  is administered a booster regimen comprising at  least one dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations  that are characteristic of an Omicron variant.  In some embodiments, a subject is administered  a booster  regimen  comprising at  least one 50 ug dose of RNA, wherein  the 50 ug of RNA  comprises RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and RNA encoding a  SARS‐CoV‐2 S protein comprising mutations  that are characteristic of a SARS‐CoV‐2 variant  (e.g., in some embodiments, a subject is administered a booster regimen comprising a 50 ug  dose of RNA comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain  and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of an Omicron variant).  In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 25  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or  more  mutations  that  are  characteristic of a BA.1 Omicron variant.  In some embodiments, a subject is administered a  booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS‐CoV‐ 2 S protein from a Wuhan strain and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having  one  or  more  mutations  that  are  characteristic  of  a  BA.2  Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 25 ug of  RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of  a BA.3 Omicron variant. In some embodiments, a subject is administered a booster regimen  comprising at least one dose comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan  strain  and  25  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant  and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered a  booster regimen comprising at least one dose comprising 25 ug of RNA encoding a SARS‐CoV‐ 2 S protein from a BA.1 Omicron variant and 25 ug of RNA encoding a SARS‐CoV‐2 S protein  having one or more mutations  that are  characteristic of  a BA.3 Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant and  25  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more mutations  that  are  characteristic of a BA.4 or BA.5 Omicron variant.   In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant  and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic  of  a  BA.4  or  BA.5  Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising  25  ug  of  RNA  encoding a SARS‐CoV‐2 S protein from a BA.3 Omicron variant and 25 ug of RNA encoding a  SARS‐CoV‐2 S protein having one or more mutations that are characteristic of a BA.4 or BA.5  Omicron variant.    In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug  doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two doses  are administered approximately 21 days apart, and (ii) a booster regimen comprising at least  one 50 ug dose of RNA encoding a SARS‐CoV‐2 S protein having one or mutations that are  characteristic of an Omicron variant of SARS‐CoV‐2 (e.g., a BA.1, BA.2, BA.4 or BA.5 Omicron  variant), wherein the booster regimen is administered at least two months (including, e.g., at  least three months, at least four months, at least five months, at least six months, or more)  after completion of the primary regimen.  In some embodiments, a subject is administered (i)  a primary regimen comprising two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan strain, wherein the two doses are administered approximately 21 days apart, and (ii)  a booster  regimen  comprising at  least one 50 ug dose of RNA, wherein  the 50 ug of RNA  comprises 25 ug of RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain and 25 ug of RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or mutations  that  are  characteristic  of  an  Omicron variant  (e.g., a BA.1, BA.2, BA.4, or BA.5 variant), wherein  the booster regimen  is  administered at least two months (including, e.g., at least three months, at least four months,  at least five months, at least six months, or more) after completion of a first booster regimen.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.1 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 50 ug dose of RNA  comprising 25 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.1 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.2 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 50 ug dose of RNA  comprising 25 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.4 or BA.5 Omicron variant of SARS‐CoV‐2, wherein the booster  regimen is administered at least two months (including, e.g., at least three months, at least  four months, at least five months, at least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 50 ug dose of RNA  comprising 25 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 50 ug dose of RNA comprising 25 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 50 ug dose of RNA comprising 25 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 50 ug dose of RNA comprising 25 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.2 Omicron variant and 25 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.    In some embodiments, a subject is administered a booster regimen comprising at least one 60  ug  dose  of  RNA.    In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising 60 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan variant.    In some  embodiments, a subject is administered 60 ug of RNA encoding a SARS‐CoV‐2 S protein having  one or more mutations that are characteristic of a SARS‐CoV‐2 variant (e.g., a variant described  herein).  In some embodiments, a subject is administered a booster regimen comprising 60 ug  of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic  of  an  Omicron  variant  (e.g.,  a  BA.1,  BA.2,  BA.4,  or  BA.5  Omicron  variant).    In  some  embodiments, a subject is administered a booster regimen comprising 60 ug of RNA, wherein  the RNA comprises a first RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, and at  least one  additional RNA encoding  a  SARS‐CoV‐2  S protein  comprising mutations  that are  characteristic of a SARS‐CoV‐2 variant (e.g., in some embodiments, a subject is  administered  a booster regimen comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that  are characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 variant).    In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 30  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or  more  mutations  that  are  characteristic of a BA.1 Omicron variant.  In some embodiments, a subject is administered a  booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS‐CoV‐ 2 S protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having  one  or  more  mutations  that  are  characteristic  of  a  BA.2  Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 30 ug of  RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of  a BA.3 Omicron variant. In some embodiments, a subject is administered a booster regimen  comprising at least one dose comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan  strain  and  30  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant  and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered a  booster regimen comprising at least one dose comprising 30 ug of RNA encoding a SARS‐CoV‐ 2 S protein from a BA.1 Omicron variant and 30 ug of RNA encoding a SARS‐CoV‐2 S protein  having one or more mutations  that are  characteristic of  a BA.3 Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.1 Omicron variant and  30  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more mutations  that  are  characteristic of a BA.4 or BA.5 Omicron variant.   In some embodiments, a subject  is administered a booster regimen comprising at least one  dose comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a BA.2 Omicron variant  and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic  of  a  BA.4  or  BA.5  Omicron  variant.  In  some  embodiments,  a  subject  is  administered  a  booster  regimen  comprising  at  least  one  dose  comprising  30  ug  of  RNA  encoding a SARS‐CoV‐2 S protein from a BA.3 Omicron variant and 30 ug of RNA encoding a  SARS‐CoV‐2 S protein having one or more mutations that are characteristic of a BA.4 or BA.5  Omicron variant.    In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug  doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain of SARS‐CoV‐2, wherein  the  two  doses  are  administered  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising at  least one 60 ug dose of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain  of  SARS‐CoV‐2, wherein  the  booster  regimen  is  administered  at  least  two months  (including, e.g., at least three months, at least four months, at least five months, at least six  months, or more) after completion of the primary regimen.    In some embodiments, a subject is administered (i) a primary regimen comprising two 30 ug  doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two doses  are administered approximately 21 days apart, and (ii) a booster regimen comprising at least  one 60 ug dose of RNA encoding a SARS‐CoV‐2 S protein having one or mutations that are  characteristic of an Omicron variant of SARS‐CoV‐2 (e.g., a BA.1, BA.2, BA.4 or BA.5 Omicron  variant), wherein the booster regimen is administered at least two months (including, e.g., at  least three months, at least four months, at least five months, at least six months, or more)  after completion of the primary regimen. In some embodiments, a subject is administered (i)  a primary regimen comprising two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan strain, wherein the two doses are administered approximately 21 days apart, and  (iii) a booster regimen comprising at  least one 60 ug dose of RNA comprising 30 ug of RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or mutations  that  are  characteristic  of  an  Omicron variant of SARS‐CoV‐2 and 30 ug of RNA encoding a SARS‐CoV‐2 S protein  from a  Wuhan  strain,  wherein  a  second  booster  regimen  is  administered  at  least  two  months  (including, e.g., at least three months, at least four months, at least five months, at least six  months, or more) after completion of a first booster regimen.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 60 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.1 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 60 ug dose of RNA  comprising 30 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.1 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 60 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.2 Omicron variant of SARS‐CoV‐2, wherein the booster regimen  is  administered  at  least  two months  (including,  e.g.,  at  least  three months,  at  least  four  months, at  least five months, at  least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 60 ug dose of RNA  comprising 30 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a dose of 60 ug of RNA encoding a SARS‐CoV‐2 S protein having one or mutations  that are characteristic of a BA.4 or BA.5 Omicron variant of SARS‐CoV‐2, wherein the booster  regimen is administered at least two months (including, e.g., at least three months, at least  four months, at least five months, at least six months, or more) after completion of the primary  regimen. In some embodiments, a subject is administered (i) a primary regimen comprising at  least two 30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein  the  two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen comprising a 60 ug dose of RNA  comprising 30 ug RNA encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or  more mutations that are characteristic of a BA.4/5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 60 ug dose of RNA comprising 30 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.2 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 60 ug dose of RNA comprising 30 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.1 Omicron variant and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.  In some embodiments, a subject is administered (i) a primary regimen comprising at least two  30 ug doses of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, wherein the two  doses  are  administered  at  least  approximately  21  days  apart,  and  (ii)  a  booster  regimen  comprising a 60 ug dose of RNA comprising 30 ug RNA encoding a SARS‐CoV‐2 S protein from  a BA.2 Omicron variant and 30 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more  mutations that are characteristic of a BA.4 or BA.5 Omicron variant.    In  some embodiments, a patient  is administered a primary  regimen comprising  two 30 ug  doses, administered approximately 21 days apart, and a booster regimen comprising at least  one 60 ug dose of RNA (e.g., 60 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain, 60 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of an Omicron variant (e.g., a BA.1, BA.2, BA.4, or BA.5 Omicron variant), or 30  ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 30 ug of RNA encoding  a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of an Omicron  variant). In some embodiments, a patient is administered a primary regimen comprising two  30 ug doses, administered approximately 21 days apart, and a booster regimen comprising at  least one 50 ug dose of RNA (e.g., 50 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain, 50 ug of RNA encoding a SARS‐CoV‐2 S protein having one or more mutations that are  characteristic of an Omicron variant, or 25 ug of RNA encoding a SARS‐CoV‐2 S protein from a  Wuhan  strain  and  25  ug  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  one  or more  mutations that are characteristic of an Omicron variant). In some embodiments, a patient is  administered a primary regimen comprising two 30 ug doses, administered approximately 21  days apart, and a booster regimen comprising at least one 30 ug dose of RNA (e.g., 30 ug of  RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, 30 ug of RNA encoding a SARS‐ CoV‐2 S protein having one or more mutations that are characteristic of an Omicron variant,  or 15 ug of RNA encoding a SARS‐CoV‐2 S protein  from a Wuhan strain and 15 ug of RNA  encoding a SARS‐CoV‐2 S protein having one or more mutations that are characteristic of an  Omicron variant).  In some embodiments, a primary regimen may involve one or more 30 ug doses and a booster  regimen may involve one or more 20 ug doses, one or more 10 ug doses, and/or one or more  3 ug doses.  In some embodiments, a primary regimen may involve one or more 20 ug doses  and a booster regimen may involve one or more 10 ug doses, and/or one or more 3 ug doses.   In some embodiments, a primary regimen may involve one or more 10 ug doses and a booster  regimen may involve one or more 3 ug doses.  In some embodiments, a primary regimen may  involve one or more 3 ug doses, and a booster regimen may also  involve one or more 3 ug  doses..  In  some embodiments,  a booster  regimen  comprises  a  single dose, e.g.,  for patients who  experienced an adverse reaction while receiving the primary regimen.    In  some embodiments,  the  same RNA as used  in a primary  regimen  is used  in a   booster  regimen. In some embodiment, an RNA used in primary and booster regimens is BNT162b2.   In some embodiments, a different RNA is used in a booster regimen relative to that used in a  primary regimen administered to the same subject. In some embodiments, BNT162b2 is used  in a primary regimen but not in a booster regimen.  In some embodiments, BNT162b2 is used  in a booster regimen but not in a primary regimen. In some embodiments, a similar BNT162b2  construct can be used  in a primary regimen and  in a booster regimen, except that the RNA  constructs used  in the primary and booster regimens encode a SARS‐CoV‐2 S protein (or an  immunogenic portion thereof) of different SARS‐CoV‐2 strains (e.g., as described herein).  In some embodiments, where BNT162b2 is used for a primary regimen or a booster regimen  but not both, and a different RNA  is used  in the other, such different RNA may be an RNA  encoding  the  same  SARS‐CoV‐2  S  protein  but with  different  codon  optimization  or  other  different RNA sequence.  In some embodiments, such different RNA may encode a SARS‐CoV‐ 2 S protein (or an  immunogenic portion thereof) of a different SARS‐CoV‐2 strain, e.g., of a  variant  strain  discussed  herein.  In  some  such  embodiments,  such  variant  strain  that  is  prevalent or rapidly spreading in a relevant jurisdiction. In some embodiments, such different  RNA may be an RNA encoding a SARS‐CoV‐2 S protein or variant  thereof  (or  immunogenic  portion of either) comprising one or more mutations described herein for S protein variants  such as SARS‐CoV‐2 S protein variants, in particular naturally occurring S protein variants; in  some such embodiments, a SARS‐CoV‐2 variant may be selected from the group consisting of  VOC‐202012/01, 501.V2, Cluster 5 and B.1.1.248. In some embodiments, a SARS‐CoV‐2 variant  may  be  selected  from  the  group  consisting  of    VOC‐202012/01,  501.V2,  Cluster  5  and  B.1.1.248,  B.1.1.7,  B.1.617.2,  and  B.1.1.529.    In  some  embodiments,  a  booster  regimen  comprises at least one dose of RNA that encodes a SARS‐CoV‐2 S protein (or an immunogenic  fragment thereof) of a variant that is spreading rapidly in a relevant jurisdiction at the time of  administration. In some such embodiments, a variant that is encoded by RNA administered in  a booster  regimen may be different  from  that encoded by RNA administered  in a primary  regimen.  In some embodiments, a booster regimen comprises administering (i) a dose of RNA encoding  the  same  SARS‐CoV‐2  S  protein  (or  an  immunogenic  fragment  thereof)  as  the  RNA  administered in the primary regimen (e.g., an RNA encoding a SARS‐CoV‐2the S protein (or an  immunogenic fragment thereof) from the SARS‐CoV‐2 Wuhan strain) and (ii) a dose of RNA  encoding a SARS‐CoV‐2 S protein (or an  immunogenic fragment thereof) of a variant that  is  spreading rapidly in a relevant jurisdiction at the time of administration (e.g., a SARS‐CoV‐2 S  protein (or an immunogenic fragment thereof) from one of the SARS‐CoV‐2 variants discussed  herein).    In some embodiments, a booster regimen comprises multiple doses (e.g., at least two doses,  at least three doses, or more). For example, in some embodiments, a first dose of a booster  regimen may comprise an RNA encoding the same SARS‐CoV‐2 S protein (or an immunogenic  fragment  thereof)  administered  in  the  primary  regimen  and  a  second  dose  of  a  booster  regimen may comprise the RNA encoding a SARS‐CoV‐2 S protein of a variant that is spreading  rapidly in a relevant jurisdiction at the time of administration.  In some embodiments, a first  dose  of  a  booster  regimen may  comprise  RNA  encoding  a  SARS‐CoV‐2  S  protein  (or  an  immunogenic fragment thereof) of a variant that is spreading rapidly in a relevant jurisdiction  at  the  time of administration and a second dose of a booster  regimen may comprise RNA  encoding the same SARS‐CoV‐2 S protein (or an immunogenic fragment thereof) administered  in the primary regimen. In some embodiments, the booster regimen comprises multiple doses,  and  the  RNA  encoding  the  S  protein  of  a  variant  that  is  spreading  rapidly  in  a  relevant  jurisdiction is administered in a first dose and the RNA encoding the S protein administered in  the primary regimen is administered in a second dose.    In some embodiments, doses (e.g., a first and a second dose or any two consecutive doses) in  a booster regimen are administered at least 2 weeks apart, including, e.g., at least 3 weeks, at  least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 week, at least 8 weeks, at least 9  weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14  weeks, at  least 15 weeks, at  least 16 weeks, or  longer, apart.  In some embodiments, doses  (e.g.,  a  first  and  a  second  dose  or  any  two  consecutive  doses)  in  a  booster  regimen  are  administered approximately 2 to 168 weeks apart. In some embodiments, doses (e.g., a first  and  a  second dose or  any  two  consecutive doses)  in  a booster  regimen are administered  approximately 3 to 12 weeks apart. In some embodiments, doses (e.g., a first and a second  dose or any two consecutive doses) in a booster regimen are administered approximately 4 to  10 weeks apart.  In  some embodiments, doses  (e.g., a  first and a  second dose or any  two  consecutive doses) in a booster regimen are administered approximately 6 to 8 weeks apart.   (e.g., about 21 days apart, or about 6 to 8 weeks apart).  In some embodiments, the first and  second dose are administered on the same day (e.g., by intramuscular injection at different  sites on the subject).    In such embodiments, the booster regimen can optionally further comprise a third and fourth  dose, administered approximately 2 to 8 weeks after the first and second dose (e.g., about 21  days after the first and second dose, or about 6 weeks to about 8 weeks after the first and  second dose), where the third and fourth dose are also administered on the same day (e.g.,  by  intramuscular  injection at different  sites on  the  subject), and  comprise  the  same RNAs  administered in the first and second doses of the booster regimen.  In  some  embodiments,  multiple  booster  regimens  may  be  administered.    In  some  embodiments,  a  booster  regimen  is  administered  to  a  patient  who  has  previously  been  administered a booster regimen.   In  some  embodiments,  a  second  booster  regimen  is  administered  to  a  patient who  has  previously received a first booster regimen, and the amount of RNA administered in at least  one dose of a second booster regimen is higher than the amount of RNA administered in at  least one dose of a first booster regimen.   In some embodiments, a second booster regimen comprises administering at least one dose  of 3 ug of RNA.  In some embodiments, a second booster regimen comprises administering at  least one dose of 5 ug of RNA.  In some embodiments, a second booster regimen comprises  administering at  least one dose of 10 ug of RNA.    In some embodiments, a second booster  regimen comprises administering at least one dose of 15 ug of RNA.  In some embodiments, a  second booster regimen comprises administering at least one dose of 20 ug of RNA.  In some  embodiments, a second booster regimen comprises administering at least one dose of 25 ug  of RNA.  In some embodiments, a second booster regimen comprises administering at least  one  dose of  30 ug  of RNA.    In  some  embodiments,  a  second  booster  regimen  comprises  administering at  least one dose of 50 ug of RNA.    In some embodiments, a second booster  regimen comprises administering at least one dose of 60 ug of RNA.    In some embodiments, a subject is administered a primary regimen that comprises two doses  of 30 ug of RNA, administered approximately 21 days apart, and a booster regimen comprising  at  least  one  dose  of  approximately  30  ug  of  RNA.  In  some  embodiments,  a  subject  is  administered a primary  regimen  that  comprises  two doses of 30 ug of RNA, administered  approximately  21  days  apart,  and  a  booster  regimen  comprising  at  least  one  dose  of  approximately  50  ug  of  RNA.  In  some  embodiments,  a  subject  is  administered  a  primary  regimen that comprises two doses of 30 ug of RNA, administered approximately 21 days apart,  and a booster regimen comprising at least one dose of approximately 60 ug of RNA.  In some embodiments, a subject is administered a primary regimen that comprises two doses  of 30 ug of RNA, administered approximately 21 days apart, a first booster regimen comprising  at least one dose of approximately 30 ug of RNA, and a second booster regimen comprising at  least one dose of approximately 30 ug of RNA. In some embodiments, a subject is administered  a primary regimen that comprises two doses of 30 ug of RNA, administered approximately 21  days apart, a  first booster regimen comprising at  least one dose of approximately 30 ug of  RNA, and a second booster regimen comprising at least one dose of approximately 50 ug of  RNA. In some embodiments, a subject is administered a primary regimen that comprises two  doses of 30 ug of RNA, administered approximately 21 days apart, a  first booster  regimen  comprising at least one dose of approximately 30 ug of RNA, and a second booster regimen  comprising at  least one dose of approximately 60 ug of RNA.  In some embodiments, a first  booster regimen comprises two doses of RNA, wherein each dose comprises an RNA encoding  a Spike protein from a different SARS‐CoV‐2 variant.   In some embodiments, a first booster  regimen comprises two doses of RNA, wherein each dose comprises an RNA encoding a Spike  protein  from  a  different  SARS‐CoV‐2  variant,  and  wherein  the  two  doses  of  RNA  are  administered on the same day.  In some embodiments, the two doses of RNA are administered  in a single composition (e.g., by mixing a first composition comprising an RNA encoding a Spike  protein  from  a  first  SARS‐CoV‐2  variant  with  a  second  composition  comprising  an  RNA  encoding a Spike protein from a second SARS‐CoV‐2 variant).    In some embodiments, a subject  is administered a booster regimen comprising a first dose  comprising an RNA that encodes a Spike protein from a Wuhan strain of SARS‐CoV‐2 and a  second dose comprising an RNA that encodes a Spike protein comprising mutations from a  variant  that  is prevalent  and/or  rapidly  spreading  in  a  relevant  jurisdiction  at  the  time of  administering the booster regimen, wherein the first dose and the second dose of RNA may  be administered on the same day.  In some embodiments, a subject is administered a booster  regimen comprising a first dose comprising an RNA that encodes a Spike protein from a Wuhan  strain of  SARS‐CoV‐2  and a  second dose  comprising an RNA  that encodes  a  Spike protein  comprising mutations  from an alpha variant of SARS‐CoV‐2, wherein the  first dose and  the  second dose may be  administered on  the  same day.    In  some  embodiments,  a  subject  is  administered a booster regimen comprising a  first dose comprising an RNA that encodes a  Spike protein from a Wuhan strain of SARS‐CoV‐2 and a second dose comprising an RNA that  encodes a Spike protein comprising mutations from a beta variant of SARS‐CoV‐2, wherein the  first dose and the second dose may be administered on the same day.  In some embodiments,  a subject is administered a booster regimen comprising a first dose comprising an RNA that  encodes a Spike protein from a Wuhan strain of SARS‐CoV‐2 and a second dose comprising an  RNA that encodes a Spike protein comprising mutations from a delta variant of SARS‐CoV‐2,  wherein the first dose and the second dose may be administered on the same day.  In some  embodiments, a subject is administered a booster regimen comprising a first dose comprising  an RNA that encodes a Spike protein from a Wuhan strain of SARS‐CoV‐2 and a second dose  comprising  an  RNA  that  encodes  a  Spike  protein  comprising mutations  from  an Omicron  variant of SARS‐CoV‐2, wherein the first dose and the second dose may be administered on  the  same day.    Such booster  regimens may be  administered,  e.g.,  to  a  subject previously  administered a primary dosing regimen and/or to a subject previously administered a primary  dosing regimen and a booster regimen.  In some embodiments, a subject is administered a first booster regimen comprising a first dose  of 15 ug of RNA encoding a Spike protein from a Wuhan variant and a second dose of 15 ug of  RNA encoding a Spike protein from an Omicron variant of SARS‐CoV‐2, where the first and the  second dose are administered on the same day (e.g., wherein compositions comprising the  RNA are mixed prior to administration, and the mixture is then administered to a patient). In  some embodiments, a subject is administered a first booster regimen comprising a first dose  of 25 ug of RNA encoding a Spike protein from a Wuhan variant and a second dose of 25 ug of  RNA encoding a Spike protein from an Omicron variant of SARS‐CoV‐2. In some embodiments,  the  first  and  the  second  doses  are  optionally  administered  on  the  same  day.    In  some  embodiments, a subject is administered a first booster regimen comprising a first dose of 25  ug of RNA encoding a Spike protein from a Wuhan variant and a second dose of 25 ug of RNA  encoding a Spike protein from an Omicron variant of SARS‐CoV‐2. In some embodiments, the  first and the second doses are administered on the same day.  In some embodiments, a subject  is administered a  first booster regimen comprising a  first dose of 30 ug of RNA encoding a  Spike protein  from a Wuhan variant and a second dose of 30 ug of RNA encoding a Spike  protein from an Omicron variant of SARS‐CoV‐2, wherein the first and the second dose are  optionally  administered  on  the  same  day  (e.g.,  in  separate  administrations  or  as  administration of a multivalent vaccine).  In some embodiments, such a first booster regimen  is administered to a subject previously administered a primary regimen comprising two doses  of  30  ug  of  RNA,  administered  about  21  days  apart wherein  the  first  booster  regimen  is  administered  at  least  3 months  (e.g.,  at  least  4,  at  least  5,  or  at  least  6 months)  after  administration of a primary regimen.  In some embodiments, a subject is administered a second booster regimen comprising a first  dose of 15 ug of RNA encoding a Spike protein from a Wuhan variant and a second dose of 15  ug of RNA encoding a Spike protein from an Omicron variant of SARS‐CoV‐2, where the first  and  the  second  dose  are  administered  on  the  same  day  (e.g.,  wherein  compositions  comprising the RNA are mixed prior to administration to form a multivalent vaccine, and the  mixture is then administered to a patient). In some embodiments, a subject is administered a  second booster regimen comprising a first dose of 25 ug of RNA encoding a Spike protein from  a Wuhan variant and a second dose of 25 ug of RNA encoding a Spike protein from an Omicron  variant of SARS‐CoV‐2, wherein the first dose and the second dose are optionally administered  on  the  same day  (e.g., via administration of a multivalent vaccine or via administration of  separate compositions).  In some embodiments, a subject is administered a second booster  regimen comprising a first dose of 25 ug of RNA encoding a Spike protein from a Wuhan variant  and a second dose of 25 ug of RNA encoding a Spike protein from an Omicron variant of SARS‐ CoV‐2.  In some embodiments, a subject is administered a second booster regimen comprising  a first dose of 30 ug of RNA encoding a Spike protein from a Wuhan variant and a second dose  of 30 ug of RNA encoding a Spike protein from an Omicron variant of SARS‐CoV‐2, wherein the  first  dose  and  the  second  dose  are  optionally  administered  on  the  same  day  (e.g.,  via  administration of a multivalent vaccine or via administration of separate compositions).    In  some embodiments, such a second booster regimen is administered to a subject previously  administered a primary regimen comprising two doses of 30 ug of RNA, administered about  21 days apart.  In some embodiments, such a second booster regimen  is administered  to a  subject previously administered a primary regimen comprising  two doses of 30 ug of RNA,  administered about 21 days apart, and a first booster regimen comprising a dose of 30 ug of  RNA, wherein the second booster regimen is administered at least 3 months (e.g., at least 4,  at least 5, or at least 6 months) after administration of a first booster regimen.  In some embodiments, patients receiving dose(s) of RNA compositions as described herein are  monitored for one or more particular conditions, e.g., following administration of one or more  doses.    In  some  embodiments,  such  condition(s) may  be  or  comprise  allergic  reaction(s)  (particularly in subject(s) with a history of relevant allergies or allergic reactions), myocarditis  (inflammation of the heart muscle, particularly where the subject is a young male and/or may  have experienced prior such  inflammation), pericarditis  (inflammation of  the  lining outside  the heart, particularly where the subject is a young males and/or may have experienced prior  such  inflammation),  fever,  bleeding  (particularly  where  the  subject  is  known  to  have  a  bleeding  disorder  or  to  be  receiving  therapy  with  a  blood  thinner).    Alternatively  or  additionally, patients who may receive closer monitoring may be or include patients who are  immunocompromised  or  are  receiving  therapy with  a medicine  that  affects  the  immune  system,  are  pregnant  or  planning  to  become  pregnant,  are  breastfeeding,  have  received  another COVID‐19 vaccine, and/or have ever fainted in association with an injection.  In some  embodiments, patients are monitored for myocarditis following administration of one of the  compositions disclosed herein.  In some embodiments, patients are monitored for pericarditis  following  administration  of  one  of  the  compositions  disclosed  herein.    Patients may  be  monitored and/or treated for the condition using current standards of care.  In  some  embodiments,  efficacy  for RNA  (e.g., mRNA)  compositions  described  in  pediatric  populations  (e.g.,  described  herein) may  be  assessed  by  various metrics  described  herein  (including, e.g., but not limited to COVID‐19 incidence per 1000 person‐years in subjects with  no  serological or  virological  evidence of  past  SARS‐CoV‐2  infection;  geometric mean  ratio  (GMR) of SARS CoV‐2 neutralizing titers measured, e.g., 7 days after a second dose; etc.)   In some embodiments, pediatric populations described herein (e.g., from 12 to less than 16  years of age) may be monitored for occurrence of multisystem inflammatory syndrome (MIS)  (e.g., inflammation in different body parts such as, e.g., heart, lung, kidneys, brain, skin ,eyes,  and/or  gastrointestinal  organs),  after  administration  of  an  RNA  composition  (e.g., mRNA)  described herein. Exemplary symptoms of MIS in children may include, but are not limited to  fever, abdominal pain, vomiting, diarrhea, neck pain, rash, bloodshot eyes, feeling extra tried,  and combinations thereof.  In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2),  or BNT162b3 (e.g., BNT162b3c). In one embodiment, RNA administered as described above is  nucleoside  modified  messenger  RNA  (modRNA)  described  herein  as  RBP020.2.  In  one  embodiment,  RNA  encoding  a  vaccine  antigen  is  nucleoside  modified  messenger  RNA  (modRNA) described herein as BNT162b3 (e.g., BNT162b3c).  In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA  (modRNA) and  (i) comprises  the nucleotide  sequence of SEQ  ID NO: 21, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one  embodiment, RNA administered as described above  is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.  In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID  NO:  19,  or  20,  a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acid sequence  comprising the amino acid sequence of SEQ  ID NO: 7, or an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ  ID NO: 7. In one embodiment, RNA administered as described above is nucleoside modified  messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20;  and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ  ID  NO: 7.   In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA  (modRNA) and  (i) comprises  the nucleotide  sequence of SEQ  ID NO: 20, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one  embodiment, RNA administered as described above  is nucleoside modified messenger RNA  (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an  amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.  In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA  (modRNA) and  (i) comprises  the nucleotide  sequence of SEQ  ID NO: 30, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ ID NO: 30, and/or (ii) encodes an amino acid sequence comprising  the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 99%,  98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29.  In one embodiment, RNA administered as described above is nucleoside modified messenger  RNA  (modRNA)  and  (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  30;  and/or  (ii)  encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.  In one embodiment, RNA administered is nucleoside modified messenger RNA (modRNA), (i)  comprises  the  nucleotide  sequence  of  SEQ  ID NO:  20;  and/or  (ii)  encodes  an  amino  acid  sequence comprising  the amino acid  sequence of SEQ  ID NO: 7, and  is administered  in an  amount  of  about  30  µg  per  dose.  In  one  embodiment,  at  least  two  of  such  doses  are  administered.  For  example,  a  second  dose may  be  administered  about  21  days  following  administration of the first dose.  In  some  embodiments,  populations  to  be  treated  with  RNA  described  herein  comprise,  essentially consist of, or consist of subjects of age of at least 50, at least 55, at least 60, or at  least  65.  In  some  embodiments,  populations  to  be  treated  with  RNA  described  herein  comprise, essentially consist of, or consist of subjects of age of between 55 to 90, 60 to 85, or  65 to 85.  In some embodiments, the period of time between the doses administered is at least 7 days,  at least 14 days, or at least 21 days. In some embodiments, the period of time between the  doses administered is between 7 days and 28 days such as between 14 days and 23 days.  In some embodiments, no more than 5 doses, no more than 4 doses, or no more than 3 doses  of the RNA described herein may be administered to a subject.  In  some  embodiments,  the methods  and  agents  described  herein  are  administered  (in  a  regimen, e.g., at a dose, frequency of doses and/or number of doses) such that adverse events  (AE),  i.e.,  any  unwanted  medical  occurrence  in  a  patient,  e.g.,  any  unfavourable  and  unintended sign, symptom, or disease associated with the use of a medicinal product, whether  or  not  related  to  the  medicinal  product,  are  mild  or  moderate  in  intensity.  In  some  embodiments, the methods and agents described herein are administered such that adverse  events (AE) can be managed with interventions such as treatment with, e.g., paracetamol or  other  drugs  that  provide  analgesic,  antipyretic  (fever‐reducing)  and/or  anti‐inflammatory  effects,  e.g.,  nonsteroidal  anti‐inflammatory  drugs  (NSAIDs),  e.g.,  aspirin,  ibuprofen,  and  naproxen. Paracetamol or "acetaminophen" which  is not classified as a NSAID exerts weak  anti‐inflammatory  effects  and  can  be  administered  as  analgesic  according  to  the  present  disclosure.  In some embodiments, the methods and agents described herein provide a neutralizing effect  in a subject to coronavirus, coronavirus infection, or to a disease or disorder associated with  coronavirus.  In some embodiments, the methods and agents described herein following administration to  a subject induce an immune response that blocks or neutralizes coronavirus in the subject. In  some embodiments, the methods and agents described herein following administration to a  subject  induce the generation of antibodies such as  IgG antibodies that block or neutralize  coronavirus in the subject. In some embodiments, the methods and agents described herein  following administration to a subject  induce an immune response that blocks or neutralizes  coronavirus S protein binding to ACE2 in the subject. In some embodiments, the methods and  agents  described  herein  following  administration  to  a  subject  induce  the  generation  of  antibodies that block or neutralize coronavirus S protein binding to ACE2 in the subject.  In some embodiments, the methods and agents described herein following administration to  a subject  induce geometric mean concentrations (GMCs) of RBD domain‐binding antibodies  such as IgG antibodies of at  least 500 U/ml, 1000 U/ml, 2000 U/ml, 3000 U/ml, 4000 U/ml,  5000 U/ml, 10000 U/ml, 15000 U/ml, 20000 U/ml, 25000 U/ml, 30000 U/ml or even higher. In  some embodiments, the elevated GMCs of RBD domain‐binding antibodies persist for at least  14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months or even longer.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  geometric  mean  titers  (GMTs)  of  neutralizing  antibodies  such  as  IgG  antibodies of at least 100 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 1000 U/ml, 1500  U/ml, or even higher.  In some embodiments, the elevated GMTs of neutralizing antibodies  persist for at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months or even  longer.  As used herein, the term "neutralization" refers to an event in which binding agents such as  antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby  inhibiting the viral infection of cells. As used herein, the term "neutralization" with respect to  coronavirus,  in particular coronavirus S protein, refers to an event  in which binding agents  such  as  antibodies  bind  to  the  RBD  domain  of  the  S  protein,  thereby  inhibiting  the  viral  infection of cells. In particular, the term "neutralization" refers to an event in which binding  agents eliminate or significantly reduce virulence (e.g. ability of  infecting cells) of viruses of  interest.  The type of immune response generated in response to an antigenic challenge can generally  be  distinguished  by  the  subset  of  T  helper  (Th)  cells  involved  in  the  response.  Immune  responses  can be broadly divided  into  two  types: Th1  and  Th2.  Th1  immune  activation  is  optimized  for  intracellular  infections  such  as  viruses, whereas  Th2  immune  responses  are  optimized  for humoral  (antibody)  responses.  Th1  cells produce  interleukin  2  (IL‐2),  tumor  necrosis factor (TNFα) and interferon gamma (IFNγ). Th2 cells produce IL‐4, IL‐5, IL‐6, IL‐9, IL‐ 10 and  IL‐13. Th1  immune activation  is  the most highly desired  in many clinical situations.  Vaccine compositions specialized in eliciting Th2 or humoral immune responses are generally  not effective against most viral diseases.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  or  promote  a  Th1‐mediated  immune  response  in  the  subject.  In  some  embodiments, the methods and agents described herein following administration to a subject  induce or promote a cytokine profile that is typical for a Th1‐mediated immune response in  the  subject.  In  some  embodiments,  the methods  and  agents  described  herein  following  administration to a subject  induce or promote the production of  interleukin 2 (IL‐2), tumor  necrosis factor (TNFα) and/or interferon gamma (IFNγ) in the subject. In some embodiments,  the methods  and  agents described herein  following  administration  to  a  subject  induce or  promote the production of interleukin 2 (IL‐2) and interferon gamma (IFNγ) in the subject. In  some embodiments, the methods and agents described herein following administration to a  subject do not induce or promote a Th2‐mediated immune response in the subject, or induce  or promote a Th2‐mediated  immune  response  in  the  subject  to a  significant  lower extent  compared  to  the  induction  or  promotion  of  a  Th1‐mediated  immune  response.  In  some  embodiments, the methods and agents described herein following administration to a subject  do  not  induce  or  promote  a  cytokine  profile  that  is  typical  for  a  Th2‐mediated  immune  response  in  the  subject, or  induce or promote a  cytokine profile  that  is  typical  for a Th2‐ mediated  immune  response  in  the  subject  to  a  significant  lower  extent  compared  to  the  induction  or  promotion  of  a  cytokine  profile  that  is  typical  for  a  Th1‐mediated  immune  response.  In  some  embodiments,  the  methods  and  agents  described  herein  following  administration to a subject do not induce or promote the production of IL‐4, IL‐5, IL‐6, IL‐9, IL‐ 10 and/or IL‐13, or induce or promote the production of IL‐4, IL‐5, IL‐6, IL‐9, IL‐10 and/or IL‐13  in  the  subject  to  a  significant  lower  extent  compared  to  the  induction  or  promotion  of  interleukin  2  (IL‐2),  tumor  necrosis  factor  (TNFα)  and/or  interferon  gamma  (IFNγ)  in  the  subject.  In  some  embodiments,  the  methods  and  agents  described  herein  following  administration  to a  subject do not  induce or promote  the production of  IL‐4, or  induce or  promote the production of IL‐4 in the subject to a significant lower extent compared to the  induction or promotion of interleukin 2 (IL‐2) and interferon gamma (IFNγ) in the subject.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  panel  of  different  S  protein  variants  such  as  SARS‐CoV‐2  S  protein  variants, in particular naturally occurring S protein variants. In some embodiments, the panel  of different S protein variants comprises at  least 5, at  least 10, at  least 15, or even more S  protein  variants.  In  some  embodiments,  such  S  protein  variants  comprise  variants  having  amino acid modifications in the RBD domain and/or variants having amino acid modifications  outside the RBD domain. In one embodiment, such S protein variant comprises SARS‐CoV‐2 S  protein  or  a  naturally occurring  variant  thereof wherein  the  amino  acid  corresponding  to  position 321 (Q)  in SEQ ID NO: 1  is S. In one embodiment, such S protein variant comprises  SARS‐CoV‐2  S  protein  or  a  naturally  occurring  variant  thereof  wherein  the  amino  acid  corresponding to position 321 (Q)  in SEQ  ID NO: 1  is L.  In one embodiment, such S protein  variant comprises SARS‐CoV‐2 S protein or a naturally occurring variant thereof wherein the  amino acid corresponding to position 341 (V) in SEQ ID NO: 1 is I. In one embodiment, such S  protein  variant  comprises  SARS‐CoV‐2  S  protein  or  a  naturally  occurring  variant  thereof  wherein  the  amino  acid  corresponding  to  position  348  (A)  in  SEQ  ID  NO:  1  is  T.  In  one  embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a naturally occurring  variant thereof wherein the amino acid corresponding to position 354 (N) in SEQ ID NO: 1 is  D. In one embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a naturally  occurring variant thereof wherein the amino acid corresponding to position 359 (S) in SEQ ID  NO: 1 is N. In one embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a  naturally occurring variant thereof wherein the amino acid corresponding to position 367 (V)  in SEQ  ID NO: 1  is F.  In one embodiment,  such  S protein  variant  comprises  SARS‐CoV‐2  S  protein  or  a  naturally occurring  variant  thereof wherein  the  amino  acid  corresponding  to  position 378 (K)  in SEQ  ID NO: 1  is S.  In one embodiment, such S protein variant comprises  SARS‐CoV‐2  S  protein  or  a  naturally  occurring  variant  thereof  wherein  the  amino  acid  corresponding to position 378 (K)  in SEQ ID NO: 1  is R.   In one embodiment, such S protein  variant comprises SARS‐CoV‐2 S protein or a naturally occurring variant thereof wherein the  amino acid corresponding to position 408 (R) in SEQ ID NO: 1 is I. In one embodiment, such S  protein  variant  comprises  SARS‐CoV‐2  S  protein  or  a  naturally  occurring  variant  thereof  wherein  the  amino  acid  corresponding  to  position  409  (Q)  in  SEQ  ID NO:  1  is  E.  In  one  embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a naturally occurring  variant thereof wherein the amino acid corresponding to position 435 (A) in SEQ ID NO: 1 is S.  In one embodiment,  such S protein variant comprises SARS‐CoV‐2 S protein or a naturally  occurring variant thereof wherein the amino acid corresponding to position 439 (N) in SEQ ID  NO: 1 is K.  In one embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a  naturally occurring variant thereof wherein the amino acid corresponding to position 458 (K)  in SEQ  ID NO: 1  is R.  In one embodiment,  such S protein variant  comprises SARS‐CoV‐2 S  protein  or  a  naturally occurring  variant  thereof wherein  the  amino  acid  corresponding  to  position 472 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, such S protein variant comprises  SARS‐CoV‐2  S  protein  or  a  naturally  occurring  variant  thereof  wherein  the  amino  acid  corresponding to position 476 (G)  in SEQ  ID NO: 1  is S.  In one embodiment, such S protein  variant comprises SARS‐CoV‐2 S protein or a naturally occurring variant thereof wherein the  amino acid corresponding to position 477 (S) in SEQ ID NO: 1 is N.   In one embodiment, such  S protein  variant  comprises  SARS‐CoV‐2  S protein or  a naturally occurring  variant  thereof  wherein  the  amino  acid  corresponding  to  position  483  (V)  in  SEQ  ID  NO:  1  is  A.  In  one  embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a naturally occurring  variant thereof wherein the amino acid corresponding to position 508 (Y) in SEQ ID NO: 1 is H.  In one embodiment,  such S protein variant comprises SARS‐CoV‐2 S protein or a naturally  occurring variant thereof wherein the amino acid corresponding to position 519 (H) in SEQ ID  NO: 1  is P. In one embodiment, such S protein variant comprises SARS‐CoV‐2 S protein or a  naturally occurring variant thereof wherein the amino acid corresponding to position 614 (D)  in SEQ ID NO: 1 is G.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring S protein variant comprising a mutation at a position corresponding  to  position 501 (N) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position  501 (N) in SEQ ID NO: 1 is Y.   Said S protein variant comprising a mutation at a position corresponding to position 501 (N)  in SEQ  ID NO: 1 may  comprise one or more  further mutations.  Such one or more  further  mutations  may  be  selected  from  mutations  at  positions  corresponding  to  the  following  positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118  (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), and 244 (L). In  one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted.  In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted.  In one embodiment,  the amino  acid  corresponding  to position 144  (Y)  in  SEQ  ID NO: 1  is  deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO:  1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1  is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is  H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I.  In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In  one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In  one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one  embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one  embodiment, the amino acid corresponding to position 484 (E)  in SEQ  ID NO: 1  is K.  In one  embodiment, the amino acid corresponding to position 701 (A)  in SEQ ID NO: 1 is V. In one  embodiment,  the amino acid corresponding  to position 18  (L)  in SEQ  ID NO: 1  is F.  In one  embodiment, the amino acid corresponding to position 246 (R)  in SEQ  ID NO: 1  is  I.  In one  embodiment, the amino acid corresponding to position 417 (K)  in SEQ ID NO: 1 is N. In one  embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In  one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted.  In one embodiment,  the  amino  acid  corresponding  to position 244  (L)  in  SEQ  ID NO: 1  is  deleted.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets VOC‐202012/01.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: deletion 69‐70, deletion 144, N501Y,  A570D, D614G, P681H, T716I, S982A, and D1118H.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: D80A, D215G, E484K, N501Y and  A701V, and optionally: L18F, R246I, K417N, and deletion 242‐244. Said S protein variant may  also comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S protein  variant  comprising  a deletion  at  a position  corresponding  to  positions 69 (H) and 70 (V) in SEQ ID NO: 1.   In some embodiments, a S protein variant comprising a deletion at a position corresponding  to positions 69 (H) and 70 (V) in SEQ ID NO: 1 may comprise one or more further mutations.  Such  one  or  more  further  mutations  may  be  selected  from  mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: 144 (Y), 501 (N), 570 (A), 614 (D),  681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242  (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino  acid corresponding to position 144  (Y)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 501 (N)  in SEQ ID NO: 1  is Y. In one embodiment, the  amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the  amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the  amino acid corresponding to position 681 (P)  in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the  amino acid corresponding to position 982 (S)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the  amino acid corresponding to position 484 (E)  in SEQ  ID NO: 1  is K.  In one embodiment, the  amino acid corresponding to position 701 (A)  in SEQ ID NO: 1  is V. In one embodiment, the  amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino  acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid  corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 243 (A)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment,  the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the  amino acid corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the  amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the  amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets VOC‐202012/01.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: deletion 69‐70, deletion 144, N501Y,  A570D, D614G, P681H, T716I, S982A, and D1118H.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "Cluster 5".  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding  to  the  following  positions  in  SEQ  ID  NO:  1:  deletion  69‐70,  Y453F,  I692V,  M1229I, and optionally S1147L.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring S protein variant comprising a mutation at a position corresponding  to  position 614 (D) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position  614 (D) in SEQ ID NO: 1 is G.   In some embodiments, a S protein variant comprising a mutation at a position corresponding  to position 614 (D) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or  more  further mutations may be selected  from mutations at positions corresponding to the  following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 681 (P), 716 (T),  982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244  (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding  to  position  69  (H)  in  SEQ  ID  NO:  1  is  deleted.  In  one  embodiment,  the  amino  acid  corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 144  (Y)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 501 (N)  in SEQ ID NO: 1  is Y. In one embodiment, the  amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the  amino acid corresponding to position 681 (P)  in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the  amino acid corresponding to position 982 (S)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the  amino acid corresponding to position 484 (E)  in SEQ  ID NO: 1  is K.  In one embodiment, the  amino acid corresponding to position 701 (A)  in SEQ ID NO: 1  is V. In one embodiment, the  amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino  acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid  corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 243 (A)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment,  the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the  amino acid corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the  amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the  amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets VOC‐202012/01.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: deletion 69‐70, deletion 144, N501Y,  A570D, D614G, P681H, T716I, S982A, and D1118H.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, D614G  and A701V, and optionally: L18F, R246I, K417N, and deletion 242‐244.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S  protein  variant  comprising  a mutation  at  positions  corresponding  to  positions  501  (N)  and  614  (D)  in  SEQ  ID  NO:  1.  In  one  embodiment,  the  amino  acid  corresponding to position 501 (N) in SEQ ID NO: 1 is Y and the amino acid corresponding to  position 614 (D) in SEQ ID NO: 1 is G.   In some embodiments, a S protein variant comprising a mutation at positions corresponding  to positions 501 (N) and 614 (D) in SEQ ID NO: 1 may comprise one or more further mutations.  Such  one  or  more  further  mutations  may  be  selected  from  mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 681  (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L),  243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid  corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 144  (Y)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the  amino acid corresponding to position 681 (P)  in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the  amino acid corresponding to position 982 (S)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the  amino acid corresponding to position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the  amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the  amino acid corresponding to position 484 (E)  in SEQ  ID NO: 1  is K.  In one embodiment, the  amino acid corresponding to position 701 (A)  in SEQ ID NO: 1  is V. In one embodiment, the  amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino  acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid  corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 243 (A)  in SEQ  ID NO: 1  is deleted.  In one embodiment, the  amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment,  the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the  amino acid corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the  amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the  amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets VOC‐202012/01.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: deletion 69‐70, deletion 144, N501Y,  A570D, D614G, P681H, T716I, S982A, and D1118H.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, D614G  and A701V, and optionally: L18F, R246I, K417N, and deletion 242‐244.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring S protein variant comprising a mutation at a position corresponding  to  position 484 (E) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position  484 (E) in SEQ ID NO: 1 is K.   In some embodiments, a S protein variant comprising a mutation at a position corresponding  to position 484 (E) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or  more  further mutations may be selected  from mutations at positions corresponding to the  following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P),  716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244  (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027  (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ  ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in  SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y)  in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501  (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A)  in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in  SEQ ID NO: 1  is G. In one embodiment, the amino acid corresponding to position 681 (P) in  SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to position 716 (T) in  SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ  ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ  ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID  NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID  NO: 1 is G. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID  NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO:  1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1  is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is  N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1  is  deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO:  1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID  NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ  ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID  NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID  NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID  NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO:  1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is  S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y.  In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In  one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T. In one  embodiment, the amino acid corresponding to position 655 (H)  in SEQ ID NO: 1  is Y. In one  embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one  embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: D80A, D215G, E484K, N501Y and  A701V, and optionally: L18F, R246I, K417N, and deletion 242‐244. Said S protein variant may  also comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "B.1.1.28".   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "B.1.1.248".  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: L18F, T20N, P26S, D138Y, R190S,  K417T, E484K, N501Y, H655Y, T1027I, and V1176F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S  protein  variant  comprising  a mutation  at  positions  corresponding  to  positions  501  (N)  and  484  (E)  in  SEQ  ID  NO:  1.  In  one  embodiment,  the  amino  acid  corresponding to position 501 (N) in SEQ ID NO: 1 is Y and the amino acid corresponding to  position 484 (E) in SEQ ID NO: 1 is K.   In some embodiments, a S protein variant comprising a mutation at positions corresponding  to positions 501 (N) and 484 (E) in SEQ ID NO: 1 may comprise one or more further mutations.  Such  one  or  more  further  mutations  may  be  selected  from  mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614  (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L),  243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655  (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69  (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position  70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position  144  (Y)  in  SEQ  ID NO: 1  is deleted.  In one embodiment,  the amino acid  corresponding  to  position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to  position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 681 (P)  in SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to  position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 982 (S)  in SEQ ID NO: 1  is A. In one embodiment, the amino acid corresponding to  position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to  position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the amino acid corresponding to  position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 701 (A) in SEQ ID NO: 1  is V. In one embodiment, the amino acid corresponding to  position 18  (L)  in SEQ  ID NO: 1  is F.  In one embodiment,  the amino acid corresponding  to  position 246 (R)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 417 (K)  in SEQ ID NO: 1  is N. In one embodiment, the amino acid corresponding to  position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding  to  position  243  (A)  in  SEQ  ID  NO:  1  is  deleted.  In  one  embodiment,  the  amino  acid  corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid  corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the amino acid  corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid  corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 20  (T)  in SEQ  ID NO: 1  is N.  In one embodiment, the amino acid  corresponding  to position 26  (P)  in SEQ  ID NO: 1  is S.  In one embodiment,  the amino acid  corresponding to position 138 (D)  in SEQ  ID NO: 1  is Y.  In one embodiment, the amino acid  corresponding to position 190 (R)  in SEQ  ID NO: 1  is S.  In one embodiment, the amino acid  corresponding to position 417 (K)  in SEQ  ID NO: 1  is T.  In one embodiment, the amino acid  corresponding to position 655 (H) in SEQ ID NO: 1  is Y. In one embodiment, the amino acid  corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 1176 (V) in SEQ ID NO: 1 is F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: D80A, D215G, E484K, N501Y and  A701V, and optionally: L18F, R246I, K417N, and deletion 242‐244. Said S protein variant may  also comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "B.1.1.248".  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: L18F, T20N, P26S, D138Y, R190S,  K417T, E484K, N501Y, H655Y, T1027I, and V1176F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S  protein  variant  comprising  a mutation  at  positions  corresponding  to  positions 501 (N), 484 (E) and 614 (D)  in SEQ ID NO: 1. In one embodiment, the amino acid  corresponding  to  position  501  (N)  in  SEQ  ID NO:  1  is  Y,  the  amino  acid  corresponding  to  position 484 (E) in SEQ ID NO: 1 is K and the amino acid corresponding to position 614 (D) in  SEQ ID NO: 1 is G.   In some embodiments, a S protein variant comprising a mutation at positions corresponding  to positions 501 (N), 484 (E) and 614 (D) in SEQ ID NO: 1 may comprise one or more further  mutations. Such one or more further mutations may be selected from mutations at positions  corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 681  (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A),  244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H),  1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H)  in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70  (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position  144  (Y)  in  SEQ  ID NO: 1  is deleted.  In one embodiment,  the amino acid  corresponding  to  position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to  position 681 (P)  in SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to  position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 982 (S)  in SEQ ID NO: 1  is A. In one embodiment, the amino acid corresponding to  position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to  position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the amino acid corresponding to  position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 701 (A) in SEQ ID NO: 1  is V. In one embodiment, the amino acid corresponding to  position 18  (L)  in SEQ  ID NO: 1  is F.  In one embodiment,  the amino acid corresponding  to  position 246 (R)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 417 (K)  in SEQ ID NO: 1  is N. In one embodiment, the amino acid corresponding to  position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding  to  position  243  (A)  in  SEQ  ID  NO:  1  is  deleted.  In  one  embodiment,  the  amino  acid  corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid  corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the amino acid  corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid  corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 20  (T)  in SEQ  ID NO: 1  is N.  In one embodiment, the amino acid  corresponding  to position 26  (P)  in SEQ  ID NO: 1  is S.  In one embodiment,  the amino acid  corresponding to position 138 (D)  in SEQ  ID NO: 1  is Y.  In one embodiment, the amino acid  corresponding to position 190 (R)  in SEQ  ID NO: 1  is S.  In one embodiment, the amino acid  corresponding to position 417 (K)  in SEQ  ID NO: 1  is T.  In one embodiment, the amino acid  corresponding to position 655 (H) in SEQ ID NO: 1  is Y. In one embodiment, the amino acid  corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 1176 (V) in SEQ ID NO: 1 is F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V,  and D614G, and optionally: L18F, R246I, K417N, and deletion 242‐244.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S protein  variant  comprising  a deletion  at  a position  corresponding  to  positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1.   In some embodiments, a S protein variant comprising a deletion at a position corresponding  to positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1 may comprise one or more further  mutations. Such one or more further mutations may be selected from mutations at positions  corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570  (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R),  417 (K), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H),  1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H)  in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70  (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position  144  (Y)  in  SEQ  ID NO: 1  is deleted.  In one embodiment,  the amino acid  corresponding  to  position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to  position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to  position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 681 (P)  in SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to  position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 982 (S)  in SEQ ID NO: 1  is A. In one embodiment, the amino acid corresponding to  position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to  position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the amino acid corresponding to  position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 484 (E)  in SEQ  ID NO: 1  is K.  In one embodiment, the amino acid corresponding to  position 701 (A) in SEQ ID NO: 1  is V. In one embodiment, the amino acid corresponding to  position 18  (L)  in SEQ  ID NO: 1  is F.  In one embodiment,  the amino acid corresponding  to  position 246 (R)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 417 (K)  in SEQ ID NO: 1  is N. In one embodiment, the amino acid corresponding to  position 453 (Y)  in SEQ  ID NO: 1  is F.  In one embodiment, the amino acid corresponding to  position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the amino acid corresponding to  position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to  position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to  position 20  (T)  in SEQ  ID NO: 1  is N.  In one embodiment, the amino acid corresponding to  position 26  (P)  in SEQ  ID NO: 1  is S.  In one embodiment,  the amino acid corresponding  to  position 138 (D)  in SEQ  ID NO: 1  is Y. In one embodiment, the amino acid corresponding to  position 190 (R)  in SEQ  ID NO: 1  is S.  In one embodiment, the amino acid corresponding to  position 417 (K)  in SEQ  ID NO: 1  is T.  In one embodiment, the amino acid corresponding to  position 655 (H)  in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to  position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to  position 1176 (V) in SEQ ID NO: 1 is F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V  and deletion 242‐244, and optionally: L18F, R246I, and K417N. Said S protein variant may also  comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring S protein variant comprising a mutation at a position corresponding  to  position 417 (K) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position  417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position  417 (K) in SEQ ID NO: 1 is T.  In some embodiments, a S protein variant comprising a mutation at a position corresponding  to position 417 (K) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or  more  further mutations may be selected  from mutations at positions corresponding to the  following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P),  716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244  (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and  1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO:  1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID  NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ  ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in  SEQ  ID NO: 1  is Y. In one embodiment, the amino acid corresponding to position 570 (A)  in  SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in  SEQ ID NO: 1  is G. In one embodiment, the amino acid corresponding to position 681 (P) in  SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to position 716 (T) in  SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ  ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ  ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID  NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID  NO: 1  is G. In one embodiment, the amino acid corresponding to position 484 (E)  in SEQ ID  NO: 1  is K. In one embodiment, the amino acid corresponding to position 701 (A)  in SEQ  ID  NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO:  1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1  is I. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is  deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO:  1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID  NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ  ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID  NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID  NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID  NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO:  1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is  S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y.  In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In  one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1  is Y. In  one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In  one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V,  and K417N, and optionally: L18F, R246I, and deletion 242‐244. Said S protein variant may also  comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "B.1.1.248".  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: L18F, T20N, P26S, D138Y, R190S,  K417T, E484K, N501Y, H655Y, T1027I, and V1176F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets a S protein variant  such as SARS‐CoV‐2 S protein variant,  in particular  naturally occurring  S  protein  variant  comprising  a mutation  at  positions  corresponding  to  positions 417 (K) and 484 (E) and/or 501 (N) in SEQ ID NO: 1. In one embodiment, the amino  acid corresponding to position 417 (K) in SEQ ID NO: 1 is N, and the amino acid corresponding  to position 484 (E) in SEQ ID NO: 1 is K and/or the amino acid corresponding to position 501  (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 417 (K)  in SEQ ID NO: 1 is T, and the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K  and/or the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.  In some embodiments, a S protein variant comprising a mutation at positions corresponding  to positions 417 (K) and 484 (E) and/or 501 (N) in SEQ ID NO: 1 may comprise one or more  further mutations. Such one or more further mutations may be selected from mutations at  positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570  (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 242 (L),  243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H),  1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H)  in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70  (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position  144  (Y)  in  SEQ  ID NO: 1  is deleted.  In one embodiment,  the amino acid  corresponding  to  position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to  position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 681 (P)  in SEQ ID NO: 1  is H. In one embodiment, the amino acid corresponding to  position 716  (T)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 982 (S)  in SEQ ID NO: 1  is A. In one embodiment, the amino acid corresponding to  position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to  position 80  (D)  in SEQ  ID NO: 1  is A.  In one embodiment, the amino acid corresponding to  position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to  position 701 (A) in SEQ ID NO: 1  is V. In one embodiment, the amino acid corresponding to  position 18  (L)  in SEQ  ID NO: 1  is F.  In one embodiment,  the amino acid corresponding  to  position 246 (R)  in SEQ  ID NO: 1  is  I.  In one embodiment, the amino acid corresponding to  position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding  to  position  243  (A)  in  SEQ  ID  NO:  1  is  deleted.  In  one  embodiment,  the  amino  acid  corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino  acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid  corresponding to position 692 (I)  in SEQ  ID NO: 1  is V.  In one embodiment, the amino acid  corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid  corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 20  (T)  in SEQ  ID NO: 1  is N.  In one embodiment, the amino acid  corresponding  to position 26  (P)  in SEQ  ID NO: 1  is S.  In one embodiment,  the amino acid  corresponding to position 138 (D)  in SEQ  ID NO: 1  is Y.  In one embodiment, the amino acid  corresponding to position 190 (R)  in SEQ  ID NO: 1  is S.  In one embodiment, the amino acid  corresponding to position 655 (H) in SEQ ID NO: 1  is Y. In one embodiment, the amino acid  corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid  corresponding to position 1176 (V) in SEQ ID NO: 1 is F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets 501.V2.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V,  and K417N and optionally: L18F, R246I, and deletion 242‐244. Said S protein variant may also  comprise a D‐>G mutation at a position corresponding to position 614 in SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets "B.1.1.248".  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject  that  targets  a  S  protein  variant  comprising  the  following mutations  at  positions  corresponding to the following positions  in SEQ  ID NO: 1: L18F, T20N, P26S, D138Y, R190S,  K417T, E484K, N501Y, H655Y, T1027I, and V1176F.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets the Omicron (B.1.1.529) variant.   In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets a S protein variant comprising at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least  29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least  37 of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L,  N440K, G339D, G446S, N856K, N764K,  K417N, D796Y, Q954H,  T95I, A67V,  L981F,  S477N,  G496S,  T478K,  Q498R,  Q493R,  E484A,  N501Y,  S375F,  Y505H,  V143del,  H69del,  V70del,  N211del,  L212I,  ins214EPE, G142D,  Y144del,  Y145del,  L141del,  Y144F,  Y145D, G142del,  as  compared to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets a S protein variant comprising at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, or all of the following mutations: T547K, H655Y, D614G,  N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y,  Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, as compared to SEQ  ID NO: 1. Said S protein variant may include at least 1, at least 2, at least 3, at least 4, at least  5,  or  all  of  the  following mutations:  N501Y,  S375F,  Y505H,  V143del,  H69del,  V70del,  as  compared to SEQ ID NO: 1 and/or may include at least 1, at least 2, at least 3, at least 4, at  least 5, or all of the following mutations: N211del, L212I, ins214EPE, G142D, Y144del, Y145del,  as compared to SEQ  ID NO: 1.  In some embodiments, said S protein variant may  include at  least 1, at least 2, at least 3, or all of the following mutations: L141del, Y144F, Y145D, G142del,  as compared to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets a S protein variant comprising at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least  29, at least 30, at least 31, at least 32, or at least 33 of the following mutations:   A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets a S protein variant comprising the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a subject induce an antibody response, in particular a neutralizing antibody response, in the  subject that targets a S protein variant comprising the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets the Omicron (B.1.1.529) variant.   In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets a S protein variant comprising at  least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at  least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at  least 34, at  least 35, at  least 36, or at  least 37 of  the  following mutations: T547K, H655Y,  D614G, N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N,  D796Y, Q954H,  T95I,  A67V,  L981F,  S477N,  G496S,  T478K, Q498R, Q493R,  E484A,  N501Y,  S375F, Y505H, V143del, H69del, V70del, N211del, L212I, ins214EPE, G142D, Y144del, Y145del,  L141del, Y144F, Y145D, G142del, as compared to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets a S protein variant comprising at  least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, or all of the  following mutations:  T547K, H655Y, D614G, N679K,  P681H, N969K,  S373P,  S371L, N440K,  G339D, G446S, N856K, N764K,  K417N, D796Y, Q954H,  T95I,  A67V,  L981F,  S477N, G496S,  T478K, Q498R, Q493R, E484A, as compared to SEQ ID NO: 1. Said S protein variant may include  at least 1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations: N501Y,  S375F, Y505H, V143del, H69del, V70del, as compared to SEQ ID NO: 1 and/or may include at  least 1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations: N211del,  L212I,  ins214EPE,  G142D,  Y144del,  Y145del,  as  compared  to  SEQ  ID  NO:  1.  In  some  embodiments, said S protein variant may include at least 1, at least 2, at least 3, or all of the  following mutations: L141del, Y144F, Y145D, G142del, as compared to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets a S protein variant comprising at  least 10, at least 15, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at  least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, or at least 33 of  the following mutations:   A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets a S protein variant comprising the  following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ ID NO: 1.  In some embodiments, the methods and agents described herein following administration to  a  subject  induce  an  immune  response  (cellular  and/or  antibody  response,  in  particular  neutralizing antibody response) in the subject that targets a S protein variant comprising the  following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.  The  term "amino acid corresponding  to position…" as used herein  refers  to an amino acid  position number corresponding to an amino acid position number in SARS‐CoV‐2 S protein, in  particular the amino acid sequence shown in SEQ ID NO: 1. The phrase "as compared to SEQ  ID NO: 1" is equivalent to "at positions corresponding to the following positions in SEQ ID NO:  1". Corresponding amino acid positions in other coronavirus S protein variants such as SARS‐ CoV‐2 S protein variants may be found by alignment with SARS‐CoV‐2 S protein, in particular  the amino acid sequence shown in SEQ ID NO: 1. It is considered well‐known in the art how to  align a sequence or segment in a sequence and thereby determine the corresponding position  in  a  sequence  to  an  amino  acid  position  according  to  the  present  disclosure.  Standard  sequence alignment programs such as ALIGN, ClustalW or similar, typically at default settings  may be used.   In some embodiments, the panel of different S protein variants to which an antibody response  is  targeted  comprises  at  least 5,  at  least 10,  at  least  15, or  even more  S protein  variants  selected from the group consisting of the Q321S, V341I, A348T, N354D, S359N, V367F, K378S,  R408I,  Q409E,  A435S,  K458R,  I472V,  G476S,  V483A,  Y508H,  H519P  and  D614G  variants  described above. In some embodiments, the panel of different S protein variants to which an  antibody response is targeted comprises all S protein variants from the group consisting of the  Q321S,  V341I, A348T, N354D,  S359N,  V367F,  K378S,  R408I, Q409E, A435S,  K458R,  I472V,  G476S, V483A, Y508H, H519P and D614G variants described above.  In some embodiments, the panel of different S protein variants to which an antibody response  is  targeted  comprises  at  least 5,  at  least 10,  at  least  15, or  even more  S protein  variants  selected from the group consisting of the Q321L, V341I, A348T, N354D, S359N, V367F, K378R,  R408I, Q409E, A435S, N439K, K458R, I472V, G476S, S477N, V483A, Y508H, H519P and D614G  variants described above. In some embodiments, the panel of different S protein variants to  which  an  antibody  response  is  targeted  comprises  all  S  protein  variants  from  the  group  consisting of the Q321L, V341I, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S,  N439K, K458R,  I472V, G476S, S477N, V483A, Y508H, H519P and D614G variants described  above.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises one or more of the mutations described  herein  for S protein variants  such as SARS‐CoV‐2 S protein variants,  in particular naturally  occurring S protein variants.  In one embodiment, a SARS‐CoV‐2 S protein, an  immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant  thereof, e.g., as encoded by  the RNA described herein,  comprises a mutation at a  position corresponding to position 501 (N)  in SEQ ID NO: 1.  In one embodiment, the amino  acid corresponding to position 501 (N)  in SEQ ID NO: 1  is Y. In some embodiments, a SARS‐ CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS‐ CoV‐2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described  herein, comprises one or more mutations, such as all mutations, of a SARS‐CoV‐2 S protein of  a SARS‐CoV‐2 variant selected from the group consisting of VOC‐202012/01, 501.V2, Cluster 5  and  B.1.1.248.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant  thereof, e.g., as encoded by the RNA described herein, comprises an amino acid sequence with  alanine substitution at position 80, glycine substitution at position 215, lysine substitution at  position  484,  tyrosine  substitution  at  position  501,  valine  substitution  at  position  701,  phenylalanine substitution at position 18, isoleucine substitution at position 246, asparagine  substitution at position 417, glycine substitution at position 614, deletions at positions 242 to  244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least  29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, or at least  37 of the following mutations: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S371L,  N440K, G339D, G446S, N856K, N764K,  K417N, D796Y, Q954H,  T95I, A67V,  L981F,  S477N,  G496S,  T478K,  Q498R,  Q493R,  E484A,  N501Y,  S375F,  Y505H,  V143del,  H69del,  V70del,  N211del,  L212I,  ins214EPE, G142D,  Y144del,  Y145del,  L141del,  Y144F,  Y145D, G142del,  as  compared to SEQ ID NO: 1. In some embodiments, a SARS‐CoV‐2 S protein, an immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant  thereof, e.g., as encoded by  the RNA described herein,  comprising  said mutations  comprises K986P and V987P, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, or all of the following mutations: T547K, H655Y, D614G,  N679K, P681H, N969K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y,  Q954H, T95I, A67V, L981F, S477N, G496S, T478K, Q498R, Q493R, E484A, as compared to SEQ  ID NO: 1. Said SARs‐CoV‐2 S protein, variant, or fragment may include at least 1, at least 2, at  least 3, at least 4, at least 5, or all of the following mutations: N501Y, S375F, Y505H, V143del,  H69del, V70del, as compared to SEQ ID NO: 1 and/or may include at least 1, at least 2, at least  3, at least 4, at least 5, or all of the following mutations: N211del, L212I, ins214EPE, G142D,  Y144del, Y145del, as compared to SEQ  ID NO: 1.  In some embodiments, said SARs‐CoV‐2 S  protein, variant, or fragment may include at least 1, at least 2, at least 3, or all of the following  mutations:  L141del,  Y144F,  Y145D,  G142del,  as  compared  to  SEQ  ID  NO:  1.  In  some  embodiments, a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic  fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g., as encoded  by  the  RNA  described  herein,  comprising  said mutations  comprises  K986P  and V987P,  as  compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises at least 10, at least 15, at least 20, at least  21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least  29, at least 30, at least 31, at least 32, or at least 33 of the following mutations:   A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ  ID NO: 1.  In  some embodiments, a SARS‐CoV‐2 S protein, an  immunogenic variant  thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant  thereof, e.g., as encoded by the RNA described herein, comprising said mutations comprises  K986P and V987P, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared  to SEQ ID NO: 1.   In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by  the RNA described herein, comprising said mutations comprises K986P and  V987P, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by  the RNA described herein, comprising said mutations comprises K986P and  V987P, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, as compared to SEQ ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQ  ID NO: 1.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  In some embodiments, the spike changes in Omicron BA.2 variant include T19I, Δ24‐26, A27S,  G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N,  T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,  Q954H, N969K, K986P, and V987P, as compared to SEQ ID NO: 1.    In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprises the following mutations:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO: 1.  In some embodiments, administration of a variant specific vaccine (e.g., a variant  specific vaccine disclosed herein) may result in an improved immune response in a patient as  compared to administration of vaccine encoding or comprising a SARS‐CoV‐2 S protein from a  Wuhan strain, or an immunogenic fragment thereof.  In some embodiments, administration  of a variant‐specific vaccine may result in induction of a broader immune response in a subject  as  compared  to  a patient  administered  a  vaccine  comprising or  encoding  a  SARS‐CoV‐2  S  protein from a Wuhan strain (or an immunogenic fragment thereof) (e.g., induce a stronger  neutralization  response  against  a  greater  number  of  SARS‐CoV‐2  variants  and/or  a  neutralization response that recognizes epitopes in a greater number of SARS‐CoV‐2 variants).   In  particular  embodiments,  a  broader  immune  response may  be  induced when  a  variant  specific vaccine is administered in combination with a vaccine comprising or encoding a SARS‐ CoV‐2 S protein from a different variant or from a Wuhan strain (e.g., in some embodiments,  a broader immune response may be induced when a variant specific vaccine is administered  in combination with a vaccine comprising or encoding a SARS‐CoV‐2 S protein from a  Wuhan  strain  or  a  vaccine  comprising  or  encoding  a  SARS‐CoV‐2  S  protein  comprising mutations  characteristic of a different SARS‐CoV‐2 variant).  For example, a broader immune response  may be induced when an RNA vaccine encoding a SARS‐CoV‐2 S protein from a Wuhan strain  is administered in combination with an RNA vaccine encoding a SARS‐CoV‐2 S protein having  mutations characteristic of an Omicron variant.   In another embodiment, a broader immune  response may be induced when an RNA vaccine encoding a SARS‐CoV‐2 S protein comprising  one or more mutations characteristic of a delta variant is administered in combination with  an  RNA  vaccine  encoding  a  SARS‐CoV‐2  S  protein  comprising  one  or  more  mutations  characteristic of an Omicron variant.    In such embodiments, a “broader”  immune response  may be defined relative to a patient administered a vaccine comprising or encoding a SARS‐ CoV‐2 S protein from a single variant (e.g., an RNA vaccine encoding a SARS‐CoV‐2 S protein  from a Wuhan strain).  Vaccines comprising or encoding S proteins from different SARS‐CoV‐2  variants,  or  immunogenic  fragments  thereof,  may  be  administered  in  combination  by  administering at different time points (e.g.,  administering a vaccine encoding a SARS‐CoV‐2 S  protein from a Wuhan strain and a vaccine encoding a SARS‐CoV‐2 S protein having one or  more  mutations  characteristic  of  a  variant  strain  at  different  time  points,  e.g.,  both  administered as part of a primary regimen or part of a booster regimen; or one is administered  as part of a primary regimen while another is administered as part of a booster regimen).  In  some embodiments, vaccines comprising or encoding S proteins from different SARS‐CoV‐2  variants,  or  immunogenic  fragments  thereof,  may  be  administered  in  combination  by  administering a multivalent vaccine  (e.g., a composition comprising RNA encoding a SARS‐ CoV‐2  S  protein  from  a Wuhan  strain  and  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  mutations  characteristic of an Omicron variant).    In  some embodiments, a variant  specific  vaccine may  induce  a  superior  immune  response  (e.g.,  inducing  higher  concentrations  of  neutralizing antibodies) against a variant against which the vaccine is specifically designed to  immunize,  and  an  immune  response  against  one  or more  other  variants.  In  some  such  embodiments, an immune response against other variant(s) may be comparable to or higher  than that as observed with a vaccine that encodes or comprises a SARS‐CoV‐2 S protein from  a Wuhan strain.  In some embodiments, the geometric mean ratio (GMR) or geometric mean fold rise (GMFR)  of neutralization antibodies induced by a variant specific vaccine is at least  1.1, 1.2, 1.3, 1.4,  1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 (e.g., 1.1 to 4, 1.1 to 3.5, 1.1 to 3, 1.5 to 3, or 1.1 to 1.5) fold higher  than that induced by a non‐variant specific vaccine (e.g., as measured 1 day to 3 months after  immunization, 7 days to 2 months after administration, about 7 days, or about 1 month after  administration).  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprising said mutations comprises the amino acid  sequence of SEQ ID NO: 49, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%,  98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:  49, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 49, or the amino  acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the amino acid sequence of SEQ  ID NO: 49.  In some embodiments, a SARS‐ CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS‐ CoV‐2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described  herein, comprising said mutations comprises the amino acid sequence of SEQ ID NO: 49.  In  some  embodiments,  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof, e.g.,  as encoded by the RNA described herein, comprising said mutations comprises the amino acid  sequence of SEQ ID NO: 52, an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%,  98.5%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:  52, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 52, or the amino  acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85%, or  80%  identity to the amino acid sequence of SEQ  ID NO: 52.  In some embodiments, a SARS‐ CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS‐ CoV‐2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described  herein, comprising said mutations comprises the amino acid sequence of SEQ ID NO: 52.  In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following administration  to a subject  induce a cell‐mediated  immune  response  (e.g., CD4+  and/or CD8+ T cell response). In some embodiments, T cells are induced that recognize one or  more epitopes  (e.g., MHC class  I‐restricted epitopes) selected  from the group consisting of  LPFNDGVYF,  GVYFASTEK,  YLQPRTFLL,  QPTESIVRF,  CVADYSVLY,  KCYGVSPTK,  NYNYLYRLF,  FQPTNGVGY,  IPFAMQMAY,  RLQSLQTYV,  GTHWFVTQR,  VYDPLQPEL,  QYIKWPWYI,  and  KWPWYIWLGF. In one embodiment, T cells are induced that recognize the epitope YLQPRTFLL.  In  one  embodiment,  T  cells  are  induced  that  recognize  the  epitope  NYNYLYRLF.  In  one  embodiment, T cells are induced that recognize the epitope QYIKWPWYI. In one embodiment,  T cells are  induced  that  recognize the epitope KCYGVSPTK.  In one embodiment, T cells are  induced  that  recognize  the  epitope RLQSLQTYV.  In  some  embodiments,  the methods  and  agents, e.g., mRNA compositions, described herein are administered according to a regimen  which achieves such induction of T cells.  In some embodiments, the methods and agents, e.g., mRNA compositions, described herein  following administration  to a subject  induce a cell‐mediated  immune  response  (e.g., CD4+  and/or CD8+ T cell response) that is detectable 15 weeks or later, 16 weeks or later, 17 weeks  or later, 18 weeks or later, 19 weeks or later, 20 weeks or later, 21 weeks or later, 22 weeks  or later, 23 weeks or later, 24 weeks or later or 25 weeks or later after administration, e.g.,  using two doses of the RNA described herein (wherein the second dose may be administered  about 21 days following administration of the first dose). In some embodiments, the methods  and  agents,  e.g., mRNA  compositions,  described  herein  are  administered  according  to  a  regimen which achieves such induction of a cell‐mediated immune response. In  one  embodiment,  vaccination  against  Coronavirus  described  herein,  e.g.,  using  RNA  described herein which may be administered in the amounts and regimens described herein,  e.g., at two doses of 30 µg per dose e.g. administered 21 days apart, may be repeated after a  certain period of time, e.g., once it is observed that protection against Coronavirus infection  diminishes, using the same or a different vaccine as used for the first vaccination. Such certain  period of time may be at least 6 months, 1 year, two years etc. In one embodiment, the same  RNA as used for the first vaccination is used for the second or further vaccination, however,  at a lower dose or a lower frequency of administration. For example, the first vaccination may  comprise vaccination using a dose of about 30 µg per dose, wherein in one embodiment, at  least two of such doses are administered, (for example, a second dose may be administered  about 21 days following administration of the first dose) and the second or further vaccination  may comprise vaccination using a dose of  less  than about 30 µg per dose, wherein  in one  embodiment, only one of such doses is administered. In one embodiment, a different RNA as  used for the first vaccination is used for the second or further vaccination, e.g., BNT162b2 is  used for the first vaccination and BNT162B1 or BNT162b3  is used for the second or further  vaccination.  In one embodiment, the vaccination regimen comprises a first vaccination using at least two  doses of the RNA described herein, e.g., two doses of the RNA described herein (wherein the  second dose may be administered about 21 days following administration of the first dose),  and a second vaccination using a single dose or multiple doses, e.g., two doses, of the RNA  described herein.  In various embodiments,  the second vaccination  is administered 3  to 24  months, 6 to 18 months, 6 to 12 months, or 5 to 7 months after administration of the first  vaccination, e.g., after the initial two‐dose regimen. The amount of RNA used in each dose of  the second vaccination may be equal or different to the amount of RNA used in each dose of  the first vaccination. In one embodiment, the amount of RNA used in each dose of the second  vaccination is equal to the amount of RNA used in each dose of the first vaccination. In one  embodiment, the amount of RNA used in each dose of the second vaccination and the amount  of RNA used in each dose of the first vaccination is about 30 µg per dose. In one embodiment,  the same RNA as used for the first vaccination is used for the second vaccination.   In one embodiment, the RNA used for the first vaccination and for the second vaccination is  BNT162b2.   In  some  embodiments, when  the  RNA  used  for  the  first  vaccination  and  for  the  second  vaccination is BNT162b2, the aim is to induce an immune response that targets SARS‐CoV‐2  variants  including, but not  limited to, the Omicron (B.1.1.529) variant. Accordingly,  in some  embodiments, when the RNA used for the first vaccination and for the second vaccination is  BNT162b2, the aim is to protect a subject from infection with SARS‐CoV‐2 variants including,  but not limited to, the Omicron (B.1.1.529) variant.  In one embodiment, a different RNA as used for the first vaccination  is used for the second  vaccination. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the  RNA used for the second vaccination is RNA encoding a SARS‐CoV‐2 S protein of a SARS‐CoV‐ 2 variant strain, e.g., a strain discussed herein. In one embodiment, the RNA used for the first  vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS‐ CoV‐2 S protein of a SARS‐CoV‐2 variant strain that  is prevalent or rapidly spreading at the  time of the second vaccination. In one embodiment, the RNA used for the first vaccination is  BNT162b2  and  the RNA used  for  the  second  vaccination  is RNA encoding a  SARS‐CoV‐2  S  protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S  protein  or  the  immunogenic  variant  thereof  comprising  one  or  more  of  the  mutations  described herein  for S protein variants such as SARS‐CoV‐2 S protein variants,  in particular  naturally  occurring  S  protein  variants.  In  one  embodiment,  the  RNA  used  for  the  first  vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS‐ CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS‐ CoV‐2 S protein or the immunogenic variant thereof comprising one or more mutations, such  as all mutations, of a SARS‐CoV‐2 S protein of a SARS‐CoV‐2 variant selected from the group  consisting of VOC‐202012/01, 501.V2, Cluster 5, B.1.1.248, and Omicron (B.1.1.529).   In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID  NO:1 and the RNA used for the second vaccination is RNA encoding a polypeptide comprising  an  amino  acid  sequence  with  alanine  substitution  at  position  80,  glycine  substitution  at  position 215, lysine substitution at position 484, tyrosine substitution at position 501, valine  substitution at position 701, phenylalanine substitution at position 18, isoleucine substitution  at position 246, asparagine substitution at position 417, glycine substitution at position 614,  deletions at positions 242 to 244, and proline substitutions at positions 986 and 987 of SEQ ID  NO:1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID  NO:1 and the RNA used for the second vaccination is RNA encoding a polypeptide comprising  an amino acid sequence with the following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, as compared to SEQ ID NO: 1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with following mutations  in SEQ  ID NO: 1: residue substitutions at  positions 986 and 987 of SEQ  ID NO:1 and the RNA used for the second vaccination  is RNA  encoding a polypeptide comprising an amino acid sequence with the following mutations in  SEQ ID NO:1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ ID NO: 1.    In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID  NO:1 and the RNA used for the second vaccination is RNA encoding a polypeptide comprising  an amino acid sequence with the following mutations in SEQ ID NO:1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO: 1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with following mutations in SEQ ID NO: 1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, and the RNA used  for the second vaccination encodes a polypeptide comprising an  amino acid sequence with the following mutations in SEQ ID NO: 1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ ID NO: 1.    In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with following mutations in SEQ ID NO: 1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, and the RNA used  for the second vaccination encodes a polypeptide comprising an  amino acid sequence with the following mutations in SEQ ID NO: 1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO: 1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with following mutations in SEQ ID NO: 1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ ID NO: 1, and  the RNA used  for  the second vaccination encodes a polypeptide comprising an amino acid  sequence with the following mutations in SEQ ID NO: 1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO: 1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 7 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 49.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  20  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 51.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 7 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 55, 58 or 61.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  20  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 57, 60, or 63.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 58 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 49, 55 or 61.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  60  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 51, 57, or 63.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 49 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 55 or 61.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  51  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 57 or 63.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 55 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 61.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  57  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 63.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID  NO:1 and the RNA used for the second vaccination is RNA encoding a polypeptide comprising  an amino acid sequence with the following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQ  ID NO: 1.  In some embodiments,  the polypeptide encoded by  the RNA used  in  the second  vaccination further comprises proline residue substitutions at positions corresponding to 986  and 987 of SEQ ID NO:1.  In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising  the amino acid sequence of SEQ ID NO: 7 and the RNA used for the second vaccination is RNA  encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 52.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 30 ug of RNA comprising  a  sequence encoding a polypeptide comprising  the amino acid  sequence of SEQ  ID NO: 7,  wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at least  4 months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 50 ug of RNA comprising  a  sequence encoding a polypeptide comprising  the amino acid  sequence of SEQ  ID NO: 7,  wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at least  4 months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 60 ug of RNA comprising  a  sequence encoding a polypeptide comprising  the amino acid  sequence of SEQ  ID NO: 7,  wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at least  4 months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 30 ug of RNA comprising  a sequence encoding a polypeptide comprising the amino acid sequence of SEQ  ID NO: 49,  wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at least  4 months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising at least two 30  ug doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid  sequence of SEQ ID NO: 7, and a booster regimen comprising at least two doses of 30 ug of  RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ  ID NO: 49, wherein  in  some embodiments  the  two doses of  the booster  regimen are  administered  at  least 2 months  apart  from  each other  (e.g.,  at  least 3 months,  at  least 4  months, at least 5 months, or at least 6 months apart from each other). In some embodiments,  such a  subject may have previously been administered a 30 ug dose of RNA comprising a  sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7 as a  booster dose.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 50 ug of RNA comprising  a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49, wherein the  booster regimen is administered at least 2 months (e.g., at least 3 months, at least 4 months,  at  least 5 months, or at  least 6 months) after administration of  the primary  regimen, and  wherein  the  subject  has  optionally  previously  been  administered  a  first  booster  regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 60 ug of RNA comprising  a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 49, wherein the  booster regimen is administered at least 2 months (e.g., at least 3 months, at least 4 months,  at  least 5 months, or at  least 6 months) after administration of  the primary  regimen, and  wherein  the  subject  has  optionally  previously  been  administered  a  first  booster  regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In some embodiments, a subject is administered a primary regimen comprising two doses  of  30 ug of RNA (administered, e.g., about 21 days after one another), wherein each 30 ug dose  of RNA comprises 15 ug of RNA comprising a nucleotide sequence encoding the amino acid  sequence of SEQ ID NO: 7 and 15 ug of RNA comprising a nucleotide sequence encoding the  amino acid sequence of SEQ  ID NO: 49.    In some embodiments, such a primary  regimen  is  administered to a vaccine naive subject.  In some embodiments, a subject is administered a primary regimen comprising two doses  of  30 ug of RNA (administered, e.g., about 21 days after one another), wherein each 30 ug dose  of RNA comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49.   In some embodiments, such a primary regimen is administered to a vaccine naive subject.    In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose  of 30 ug of RNA, wherein  the 30 ug of RNA comprises 15 ug of RNA comprising a nucleotide sequence encoding  the  amino acid sequence of SEQ  ID NO: 7 and 15 ug of RNA comprising a nucleotide sequence  encoding the amino acid sequence of SEQ  ID NO: 49, wherein the two RNAs are optionally  administered in the same composition, and wherein the booster regimen is administered at  least 2 months  (e.g., at  least 3 months, at  least 4 months, at  least 5 months, or at  least 6  months) after administration of the primary regimen, and wherein the subject has optionally  previously  been  administered  a  first  booster  regimen  comprising  a  30  ug  dose  of  RNA  comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID  NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 50 ug of RNA, wherein  the 50 ug of RNA comprises 25 ug of RNA comprising a nucleotide sequence encoding  the  amino acid sequence of SEQ  ID NO: 7 and 25 ug of RNA comprising a nucleotide sequence  encoding the amino acid sequence of SEQ  ID NO: 49, wherein the two RNAs are optionally  administered  in  the  same  composition  (e.g.,  a  formulation  comprising  both  RNAs),  and  wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at least  4 months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a sequence encoding a polypeptide comprising the  amino acid sequence of SEQ ID NO: 7.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of  SEQ ID NO: 7, and a booster regimen comprising at least one dose of 60 ug of RNA, wherein  the 60 ug of RNA comprises 30 ug of RNA comprising a nucleotide sequence encoding  the  amino acid sequence of SEQ ID NO: 7 and 30 ug of an RNA comprising a nucleotide sequence  encoding the amino acid sequence of SEQ  ID NO: 49, wherein the two RNAs are optionally  administered in the same composition, and wherein the booster regimen is administered at  least 2 months  (e.g., at  least 3 months, at  least 4 months, at  least 5 months, or at  least 6  months) after administration of the primary regimen, and wherein the subject has optionally  previously  been  administered  a  first  booster  regimen  comprising  a  30  ug  dose  of  RNA  comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID  NO: 7.  In one embodiment, the RNA used for the first vaccination comprises the nucleotide sequence  of  SEQ  ID  NO:  20  and  the  RNA  used  for  the  second  vaccination  is  RNA  comprising  the  nucleotide sequence of SEQ ID NO: 54.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 50 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20..  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 60 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 20, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 50 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 60 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 51, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA, wherein the 30 ug of RNA comprises 15 ug of RNA comprising  a nucleotide sequence of SEQ ID NO: 20 and 15 ug of RNA comprising a nucleotide sequence  of SEQ ID NO: 51, wherein the two RNAs are optionally administered in the same composition,  and wherein the booster regimen is administered at least 2 months (e.g., at least 3 months, at  least 4 months, at least 5 months, or at  least 6 months) after administration of the primary  regimen, and wherein the subject has optionally previously been administered a first booster  regimen comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose comprising 50 ug of a RNA, wherein the 50 ug of RNA comprises 25 ug of  RNA  comprising  a nucleotide  sequence of  SEQ  ID NO: 20  and 25 ug of RNA  comprising  a  nucleotide sequence of SEQ ID NO: 51, wherein the two RNAs are optionally administered in  the same composition, and wherein the booster regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4 months, at  least 5 months, or at  least 6 months) after  administration of the primary regimen, and wherein the subject has optionally previously been  administered a first booster regimen comprising a 30 ug dose of RNA comprising a nucleotide  sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose comprising 60 ug of RNA, wherein the 60 ug of RNA comprises 30 ug of an  RNA comprising a nucleotide sequence of SEQ ID NO: 20 and 30 ug of an RNA comprising a  nucleotide sequence of SEQ ID NO: 51, wherein the two RNAs are optionally administered in  the same composition, and wherein the booster regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4 months, at  least 5 months, or at  least 6 months) after  administration of the primary regimen, and wherein the subject has optionally previously been  administered a first booster regimen comprising a 30 ug dose of RNA comprising a nucleotide  sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 50 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 60 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 50 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 60 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 60, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.    In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 30 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 63, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 20, and a booster regimen comprising  at least one dose of 50 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 63, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, a subject is administered a primary regimen comprising two 30 ug doses  of RNA comprising a nucleotide sequence of SEQ ID NO: 63, and a booster regimen comprising  at least one dose of 60 ug of RNA comprising a nucleotide sequence of SEQ ID NO: 57, wherein  the booster  regimen  is administered at  least 2 months  (e.g., at  least 3 months, at  least 4  months, at least 5 months, or at least 6 months) after administration of the primary regimen,  and wherein the subject has optionally previously been administered a first booster regimen  comprising a 30 ug dose of RNA comprising a nucleotide sequence of SEQ ID NO: 20.  In one embodiment, the vaccination regimen comprises a first vaccination using two doses of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence  with  proline  residue  substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and  a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide  comprising an amino acid sequence with proline residue substitutions at positions 986 and  987 of SEQ ID NO:1 administered about 4 to 12 months, 5 to 12 months, or 6 to 12 months  after administration of  the  first vaccination,  i.e., after  the  initial  two‐dose  regimen.  In one  embodiment,  each  RNA  dose  comprises  30  µg  RNA.  In  this  embodiment,  the  aim  in  one  embodiment is to induce an immune response that targets SARS‐CoV‐2 variants including, but  not limited to, the Omicron (B.1.1.529) variant. Accordingly, in this embodiment, the aim in  one embodiment is to protect a subject from infection with SARS‐CoV‐2 variants including, but  not limited to, the Omicron (B.1.1.529) variant.   In one embodiment, the vaccination regimen comprises a first vaccination using two doses of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence  with  proline  residue  substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and  a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide  comprising  an  amino  acid  sequence  with  alanine  substitution  at  position  80,  glycine  substitution  at  position  215,  lysine  substitution  at  position  484,  tyrosine  substitution  at  position 501, valine substitution at position 701, phenylalanine substitution at position 18,  isoleucine  substitution  at  position  246,  asparagine  substitution  at  position  417,  glycine  substitution at position 614, deletions at positions 242 to 244, and proline substitutions at  positions 986 and 987 of SEQ ID NO:1 administered about 6 to 12 months after administration  of the first vaccination, i.e., after the initial two‐dose regimen. In one embodiment, each RNA  dose comprises 30 µg RNA.  In one embodiment, the vaccination regimen comprises a first vaccination using two doses of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence  with  proline  residue  substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and  a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide  comprising an amino acid sequence with the following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, as compared to SEQ  ID NO: 1, administered after, e.g., about 6 to 12 months after  administration  of  the  first  vaccination,  i.e.,  after  the  initial  two‐dose  regimen.  In  one  embodiment, each RNA dose comprises 30 µg RNA.  In one embodiment, the vaccination regimen comprises a first vaccination using two doses of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence  with  proline  residue  substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and  a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide  comprising an amino acid sequence with the following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, K986P and V987P, as compared to SEQ  ID NO:  1,  administered  after,  e.g.,  about  6  to 12 months  after  administration  of  the  first  vaccination,  i.e.,  after  the  initial  two‐dose  regimen.  In  one  embodiment,  each  RNA  dose  comprises 30 µg RNA.    In some embodiments,  the encoded polypeptide  further comprises  proline residue substitutions at positions corresponding to 986 and 987 of SEQ ID NO:1.  In one embodiment, the vaccination regimen comprises a first vaccination involving at least  two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline  residue substitutions at positions 986 and 987 of SEQ ID NO: 1 administered about 21 days  apart and a second vaccination  involving a single dose or multiple doses of RNA encoding a  polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:  1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K, K986P,  and V987P,  as  compared  to  SEQ  ID NO: 1,  administered after, e.g., about 6 to 12 months after administration of the first vaccination, i.e.,  after  the  initial  two‐dose  regimen.  In  one  embodiment,  each  or  at  least  one  RNA  dose  comprises 30 µg RNA.      In one embodiment, the vaccination regimen comprises a first vaccination involving at least  two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline  residue substitutions at positions 986 and 987 of SEQ  ID NO:1 administered about 21 days  apart and a second vaccination  involving a single dose or multiple doses of RNA encoding a  polypeptide comprising an amino acid sequence with the following mutations in SEQ ID NO:1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO:  1  administered  after,  e.g.,  about  6  to  12 months  after  administration  of  the  first  vaccination, i.e., after the initial two‐dose regimen. In one embodiment, each or at least one  RNA dose comprises 30 µg RNA.      In one embodiment, the vaccination regimen comprises a first vaccination involving at least  two  doses  of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence with  the  following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, wherein the two doses of the first vaccination are administered about 21 days apart  and wherein the vaccination regimen comprises a second vaccination involving a single dose  or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the  following mutations in SEQ ID NO:1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K,  K986P,  and V987P,  as  compared  to  SEQ  ID NO:  1  administered after, e.g., about 6 to 12 months after administration of the first vaccination, i.e.,  after  the  initial  two‐dose  regimen.  In  one  embodiment,  each  or  at  least  one  RNA  dose  comprises 30 µg RNA.        In one embodiment, the vaccination regimen comprises a first vaccination involving at least  two  doses  of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence with  the  following mutations in SEQ ID NO:1:  A67V, Δ69‐70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G,  H655Y,  N679K,  P681H,  N764K,  D796Y,  N856K, Q954H,  N969K,  L981F,  K986P  and  V987P, wherein the two doses of the first vaccination are administered about 21 days apart  and wherein the vaccination regimen comprises a second vaccination involving a single dose  or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the  following mutations in SEQ ID NO:1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO:  1  administered  after,  e.g.,  about  6  to  12 months  after  administration  of  the  first  vaccination, i.e., after the initial two‐dose regimen. In one embodiment, each or at least one  RNA dose comprises 30 µg RNA.      In one embodiment, the vaccination regimen comprises a first vaccination involving at least  two  doses  of  RNA  encoding  a  polypeptide  comprising  an  amino  acid  sequence with  the  following mutations in SEQ ID NO:1:  T19I,  Δ24‐26,  A27S,  G142D,  V213G,  G339D,  S371F,  S373P,  S375F,  T376A,  D405N,  R408S,  K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K,  P681H, N764K, D796Y, Q954H, N969K, K986P,  and V987P,  as  compared  to  SEQ  ID NO: 1,  wherein  the  two doses of  the  first  vaccination are  administered  about 21 days  apart  and  wherein the vaccination regimen comprises a second vaccination  involving a single dose or  multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with the  following mutations in SEQ ID NO:1:  T19I,    Δ24‐26, A27S,  Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,  R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G,  H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, K986P, and V987P, as compared to SEQ  ID NO:  1  administered  after,  e.g.,  about  6  to  12 months  after  administration  of  the  first  vaccination, i.e., after the initial two‐dose regimen. In one embodiment, each or at least one  RNA dose comprises 30 µg RNA.        In one embodiment, the second vaccination results in a boosting of the immune response.  In one embodiment, the RNA described herein is co‐administered with other vaccines. In some  embodiments, the RNA described herein  is co‐administered with a composition comprising  one or more T‐cell epitopes of SARS‐CoV‐2 or RNA encoding the same.  In some embodiments,  the RNA described herein  is co‐administered one or more T‐cell epitopes, or RNA encoding  the same, derived from an M protein, an N protein, and/or an ORF1ab protein of SARS‐CoV‐2,  e.g., a composition disclosed  in WO2021188969,  the contents of which  is  incorporated by  reference  herein  in  its  entirety.    In  some  embodiments,  RNA  described  herein  (e.g.,  RNA  encoding  a  SARS‐CoV‐2  S  protein  comprising mutations  characteristic  of  a  BA.1,  BA.2,  or  BA.4/5 Omicron variant, optionally administered with RNA encoding a SARS‐CoV‐2 S protein  of  a  Wuhan  variant)  can  be  co‐administered  with  a  T‐string  construct  described  in  WO2021188969 (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969).  In some  embodiments, RNA described herein and a T‐string construct described in WO2021188969 are  administered  in  a  combination  comprising  up  to  about  100  ug  RNA  total.  In  some  embodiments, subjects are administered at least 2 doses, each comprising one or more RNAs  described herein (e.g., in some embodiments at 15 ug each) and a T‐string construct (e.g., an  RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., two or more RNAs described  herein and an RNA encoding SEQ ID NO: RS C7p2full in a total amount of about 100 ug or less  of RNA.  In some embodiments, each dose comprises 5 ug of a T‐string construct and 3 ug of  RNA  described  herein  (e.g.,  3  ug  of  a  bivalent  RNA  vaccine  described  herein).    In  some  embodiments, each dose comprises 5 ug of a T‐string construct and 6 ug of RNA described  herein (e.g., 3 ug of a bivalent RNA vaccine described herein).  In some embodiments, each  dose comprises 5 ug of a T‐string construct and 10 ug of RNA described herein (e.g., 10 ug of  a bivalent RNA vaccine described herein).  In some embodiments, each dose comprises 5 ug  of a T‐string construct and 30 ug of RNA described herein (e.g., 30 ug of a bivalent RNA vaccine  described herein).    In some embodiments, each dose comprises 10 ug of a T‐string construct and 3 ug of RNA  described  herein  (e.g.,  3  ug  of  a  bivalent  RNA  vaccine  described  herein).    In  some  embodiments, each dose comprises 10 ug of a T‐string construct and 6 ug of RNA described  herein (e.g., 3 ug of a bivalent RNA vaccine described herein).  In some embodiments, each  dose comprises 10 ug of a T‐string construct and 10 ug of RNA described herein (e.g., 10 ug of  a bivalent RNA vaccine described herein).  In some embodiments, each dose comprises 10 ug  of a T‐string construct and 30 ug of RNA described herein (e.g., 30 ug of a bivalent RNA vaccine  described herein).    In some embodiments, each dose comprises 15 ug of a T‐string construct and 3 ug of RNA  described  herein  (e.g.,  3  ug  of  a  bivalent  RNA  vaccine  described  herein).    In  some  embodiments, each dose comprises 15 ug of a T‐string construct and 6 ug of RNA described  herein (e.g., 3 ug of a bivalent RNA vaccine described herein).  In some embodiments, each  dose comprises 10 ug of a T‐string construct and 15 ug of RNA described herein (e.g., 10 ug of  a bivalent RNA vaccine described herein).  In some embodiments, each dose comprises 15 ug  of a T‐string construct and 30 ug of RNA described herein (e.g., 30 ug of a bivalent RNA vaccine  described herein).    In some embodiments, the two doses are administered at least 4 weeks or longer (including,  e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at  least 10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from one another. In  some embodiments, subjects are administered at least 3 doses, each comprising one or more  RNAs described herein (e.g., at 30 ug each) and a T‐string construct (e.g., an RNA encoding  SEQ  ID NO: RS C7p2full of WO2021/188969).    In some embodiments, each of  the 3 doses  comprises up to about 100 ug RNA total.  In some embodiments, the first and the second doses  and  the  second and  third doses are each  independently administered at  least 4 weeks or  longer (including, e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at  least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from  one another.   In some embodiments, RNA described herein and a T‐string construct may be co‐administered  as  separate  formulations  (e.g.,  by  IM  injection  at  separate  injection  sites).  In  some  embodiments, the RNA described herein and the T‐string construct may be co‐administered  as a co‐formulation (e.g., a co‐formulation  in which each RNA  is encapsulated in a separate  LNP, a co‐formulation  in which RNA described herein is encapsulated  in a first LNP and a T‐ string  construct  encapsulated  in  a  second  LNP, or  a  co‐formulation  in which  all RNAs  are  encapsulated together in the same LNP (e.g., by mixing all RNAs prior to LNP formulation)).  In some embodiments, an RNA composition described herein is co‐administered with one or  more vaccines against a non‐SARS‐CoV‐2 disease.  In some embodiments, an RNA composition  described herein is co‐administered with one or more vaccines against a non‐SARS‐COV‐2 viral  disease. In some embodiments, an RNA composition described herein is co‐administered with  one or more vaccines against a non‐SARS‐CoV‐2 respiratory disease. In some embodiments,  the non‐SARS‐CoV‐2 respiratory disease is a non‐SARS‐CoV‐2 Coronavirus, an Influenza virus,  a Pneumoviridae virus, or a Paramyxoviridae virus. In some embodiments, the Pneumoviridae  virus  is  a  Respiratory  syncytial  virus  or  a Metapneumovirus.  In  some  embodiments,  the  Metapneumovirus  is  a  human  metapneumovirus  (hMPV).  In  some  embodiments,  the  Paramyxoviridae virus  is a Parainfluenza virus or a Henipavirus.  In  some embodiments  the  parainfluenzavirus  is  PIV3.  In  some  embodiments,  the  non‐SAR‐CoV‐2  coronavirus  is  a  betacoronavirus (e.g., SARS‐CoV‐1).  In come embodiments the non‐SARS‐CoV‐2 coronavirus  is a Merbecovirus (e.g., a MERS‐CoV virus).  In some embodiments, an RNA composition described herein is co‐administered with an RSV  vaccine (e.g., an RSV A or RSV B vaccine). In some embodiments, the RSV vaccine delivers (e.g.,  comprises or encodes) an RSV fusion protein (F), an RSV attachment protein (G), an RSV small  hydrophobic protein 20 (SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2‐ 1 protein, an RSV Large polymerase (L), and/or an RSV phosphoprotein (P), or an immunogenic  fragment of immunogenic variant thereof, or a nucleic acid (e.g., RNA), encoding any one of  the same.  In some embodiments, the RSV vaccine delivers a prefusion stabilized F protein.  In  some embodiments  the RSV vaccine delivers a bivalent stabilized prefusion F protein  (e.g.,  comprising a sequence based on RSV A and RSV B strains, e.g., RSVpreF, as described in Falsey  A., et al. J. Infect Dis 2022;225(12):2056‐2066; Walsh E., et al. J. Infect Dis 2022;225(8):1357‐ 1366; and/or Baber J., et al. J. Infect Dis 2022 May 11;jiac189, the contents of each of which  are hereby incorporated by reference in their entirety).  In  some  embodiments,  an  RNA  composition  described  herein  is  co‐administered with  an  influenza vaccine. In some embodiments, the influenza vaccine is an alpha influenza virus, a  beta  influenza  virus,  a  gamma  influenza  virus  or  a  delta  influenza  virus  vaccine.  In  some  embodiments the vaccine is an Influenza A virus, an Influenza B virus, an Influenza C virus, or  an Influenza D virus vaccine. In some embodiments, the influenza A virus vaccine comprises a  hemagglutinin selected from H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,  H15, H16, H17, and H18, or an immunogenic fragment or variant of the same, or a nucleic acid  (e.g., RNA) encoding any one of  the  same.  In  some embodiments  the  influenza A  vaccine  comprises or encodes a neuraminidase (NA) selected from N1, N2, N3, N4, N5, N6, N7, N8, N9,  N10, and N11, or an  immunogenic  fragment or variant of the same, or a nucleic acid (e.g.,  RNA) encoding any one of the same. In some embodiments, the influenza vaccine comprises  at  least  one  Influenza  virus  hemagglutinin  (HA),  neuraminidase  (NA),  nucleoprotein  (NP),  matrix protein 1 (M1), matrix protein 2 (M2), non‐structural protein 1 (NS1 ), non‐structural  protein 2  (NS2), nuclear export protein  (NEP), polymerase acidic protein  (PA), polymerase  basic  protein  PB1,  PB1‐F2,  and/or  polymerase  basic  protein  2  (PB2),  or  an  immunogenic  fragment or variant thereof, or a nucleic acid (e.g., RNA) encoding any of one of the same.    Exemplary  influenza vaccines that can be administered  in combination with RNA described  herein  include  commercially  approved  influenza  vaccines,  an  inactivated  influenza  virus  vaccine  (e.g.,  Fluzone®,  Fluzone  high‐dose  quadrivalent®,  Fluzone  quadrivalent®,  Fluzone  intradermal  quardivalent®,  Fluzone  quadrivalent  southern  hemisphere®,  Fluad®,  Fluad  quadrivalent®,  Afluria  Quardivalent®,  Fluarix  Quadrivalent®,  FluLaval  Quadrivalent®,  or  Flucelvax Quadrivalent®), a recombinant influenza vaccine (e.g., Flublok quadrivalent®), a live  attenuated  influenza  vaccine  (e.g.,  FluMist  Quadrivalent®),  an  non‐adjuvanted  influenza  vaccine, an adjuvanted influenza vaccine, or a subunit or split vaccine. In some embodiments,  RNA  disclosed  herein  can  be  administered  in  combination with  one  or more  RNAs,  each  encoding one or more antigenic polypeptides associated with an influenza virus (e.g., an HA  protein or an NA protein).  Exemplary RNA vaccines are known in the art (e.g., as described in  Feldman,  Robert  A.,  et  al.  "mRNA  vaccines  against H10N8  and H7N9  influenza  viruses  of  pandemic  potential  are  immunogenic  and  well  tolerated  in  healthy  adults  in  phase  1  randomized  clinical  trials."  Vaccine  37.25  (2019):  3326‐  3334,  the  contents  of which  are  incorporated by reference herein in their entirety).  In some embodiments, an RNA composition described herein is administered in combination  with an saRNA encoding an  influenza antigen.   In some embodiments, an RNA composition  described herein  is administered  in combination with an saRNA encoding a single  influenza  antigen (e.g., one HA polypeptide, one NA polypeptide, or one SARS‐CoV‐2 S polypeptide).  In  some embodiments, an RNA composition described herein is co‐administered with an saRNA  encoding  two  or  more  antigens  (e.g.,  two  or  more  HA  polypeptides,  two  or  more  NA  polypeptides, one or more HA polypeptides and one or more NA polypeptides,  one or more  NA polypeptides and one or more SARS‐CoV‐2 S polypeptides).  In some embodiments, an RNA  composition  described  herein  is  co‐administered  with  an  saRNA  encoding  two  HA  polypeptides,  each  from  a  different  influenza  strain.    In  some  embodiments,  an  RNA  composition  described  herein  is  co‐administered  with  an  saRNA  encoding  two  NA  polypeptides,  each  from  a  different  influenza  strain.    In  some  embodiments,  an  saRNA  encodes an HA polypeptide and an NA polypeptide, each from the same influenza strain (e.g.,  as described in “Pfizer Near‐Term Launches + High‐Value Pipeline Day”, published December  12,  2022;  chrome‐ extension://efaidnbmnnnibpcajpcglclefindmkaj/https://s28.q4cdn.com/781576035/files/doc _presentation/2022/12/B/Pfizer‐Near‐Term‐Launches‐High‐Value‐Pipeline‐Day‐ Presentation_6pm_v2.pdf).    In some embodiments, a nucleotide sequence encoding an antigenic polypeptide  is  located  downstream  of  saRNA  Replicase  genes.    In  some  embodiments,  an  saRNA  comprises  a  nucleotide  sequence  encoding  an HA  antigen  and  a nucleotide  sequence  encoding  an NA  antigen, where the nucleotide sequence encoding the HA antigen is located upstream of the  nucleotide sequence encoding the NA antigen.  In some embodiments an RNA composition described herein is co‐administered with an RSV  vaccine, an influenza vaccine, or an RSV vaccine and an influenza vaccine.  In some embodiments, an RNA composition provided herein and other injectable vaccine(s)  are administered at different  times.  In  some embodiments, an RNA  composition provided  herein  is  administered  at  the  same  time  as  other  injectable  vaccine(s).  In  some  such  embodiments,  an  RNA  composition  provided  herein  and  at  least  one  another  injectable  vaccine(s)  are  administered  at  different  injection  sites.  In  some  embodiments,  an  RNA  composition provided herein is not mixed with any other vaccine in the same syringe. In some  embodiments, an RNA composition provided herein is not combined with other coronavirus  vaccines as part of vaccination against coronavirus, e.g., SARS‐CoV‐2.  The term "disease" refers to an abnormal condition that affects the body of an individual. A  disease is often construed as a medical condition associated with specific symptoms and signs.  A disease may be  caused by  factors originally  from an external  source,  such as  infectious  disease,  or  it may  be  caused  by  internal  dysfunctions,  such  as  autoimmune  diseases.  In  humans,  "disease"  is often used more broadly  to  refer  to any  condition  that  causes pain,  dysfunction, distress, social problems, or death to the individual afflicted, or similar problems  for those in contact with the individual. In this broader sense, it sometimes includes injuries,  disabilities,  disorders,  syndromes,  infections,  isolated  symptoms,  deviant  behaviors,  and  atypical variations of structure and function, while in other contexts and for other purposes  these may be considered distinguishable categories. Diseases usually affect  individuals not  only physically, but also emotionally, as contracting and  living with many diseases can alter  one's perspective on life, and one's personality.  In the present context, the term "treatment", "treating" or "therapeutic intervention" relates  to the management and care of a subject for the purpose of combating a condition such as a  disease or disorder. The term is intended to include the full spectrum of treatments for a given  condition  from which  the subject  is suffering, such as administration of the therapeutically  effective compound to alleviate the symptoms or complications, to delay the progression of  the  disease,  disorder or  condition,  to  alleviate or  relief  the  symptoms  and  complications,  and/or  to  cure  or  eliminate  the  disease,  disorder  or  condition  as well  as  to  prevent  the  condition,  wherein  prevention  is  to  be  understood  as  the  management  and  care  of  an  individual for the purpose of combating the disease, condition or disorder and  includes the  administration  of  the  active  compounds  to  prevent  the  onset  of  the  symptoms  or  complications.   The term "therapeutic treatment" relates to any treatment which improves the health status  and/or prolongs  (increases) the  lifespan of an  individual. Said treatment may eliminate the  disease in an individual, arrest or slow the development of a disease in an individual, inhibit  or slow the development of a disease in an individual, decrease the frequency or severity of  symptoms in an individual, and/or decrease the recurrence in an individual who currently has  or who previously has had a disease.  The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is  intended  to  prevent  a  disease  from  occurring  in  an  individual.  The  terms  "prophylactic  treatment" or "preventive treatment" are used herein interchangeably.  The terms "individual" and "subject" are used herein interchangeably. They refer to a human  or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate)  that can be afflicted with or is susceptible to a disease or disorder but may or may not have  the  disease  or  disorder.  In many  embodiments,  the  individual  is  a  human  being.  Unless  otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus  encompass  adults,  elderlies,  children,  and  newborns.  In  some  embodiments,  the  term  "subject" includes humans of age of at least 50, at least 55, at least 60, at least 65, at least 70,  or older. In some embodiments, the term "subject" includes humans of age of at least 65, such  as 65 to 80, 65 to 75, or 65 to 70. In embodiments of the present disclosure, the "individual"  or "subject" is a "patient".  The  term  "patient" means  an  individual or  subject  for  treatment,  in particular  a diseased  individual or subject.  In one  embodiment of  the disclosure,  the  aim  is  to provide  an  immune  response  against  coronavirus, and to prevent or treat coronavirus infection.  A pharmaceutical composition comprising RNA encoding a peptide or protein comprising an  epitope may be administered to a subject to elicit an  immune response against an antigen  comprising said epitope in the subject which may be therapeutic or partially or fully protective.  A  person  skilled  in  the  art  will  know  that  one  of  the  principles  of  immunotherapy  and  vaccination is based on the fact that an immunoprotective reaction to a disease is produced  by immunizing a subject with an antigen or an epitope, which is immunologically relevant with  respect  to  the disease  to be  treated. Accordingly, pharmaceutical  compositions described  herein  are  applicable  for  inducing  or  enhancing  an  immune  response.  Pharmaceutical  compositions described herein are thus useful in a prophylactic and/or therapeutic treatment  of a disease involving an antigen or epitope.  As used herein, "immune response" refers to an integrated bodily response to an antigen or a  cell expressing an antigen and refers to a cellular immune response and/or a humoral immune  response. The  immune system  is divided  into a more primitive  innate  immune system, and  acquired or adaptive  immune  system of vertebrates, each of which  contains humoral and  cellular components.  "Cell‐mediated immunity", "cellular immunity", "cellular immune response", or similar terms  are meant to  include a cellular response directed to cells characterized by expression of an  antigen, in particular characterized by presentation of an antigen with class I or class II MHC.  The cellular response relates to immune effector cells, in particular to cells called T cells or T  lymphocytes which act as either "helpers" or "killers". The helper T cells (also termed CD4+ T  cells) play a central role by regulating the immune response and the killer cells (also termed  cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as virus‐infected  cells, preventing the production of more diseased cells.  An  immune  effector  cell  includes  any  cell  which  is  responsive  to  vaccine  antigen.  Such  responsiveness includes activation, differentiation, proliferation, survival and/or indication of  one  or more  immune  effector  functions.  The  cells  include,  in  particular,  cells  with  lytic  potential,  in  particular  lymphoid  cells,  and  are  preferably  T  cells,  in  particular  cytotoxic  lymphocytes,  preferably  selected  from  cytotoxic  T  cells,  natural  killer  (NK)  cells,  and  lymphokine‐activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes  triggers the destruction of target cells. For example, cytotoxic T cells trigger the destruction of  target cells by either or both of  the  following means. First, upon activation T cells  release  cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores  in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm  that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced  via Fas‐Fas ligand interaction between the T cells and target cells.   The term "effector functions" in the context of the present disclosure includes any functions  mediated by components of the immune system that result, for example, in the neutralization  of a pathogenic agent  such as a  virus and/or  in  the  killing of diseased  cells  such as  virus‐ infected  cells.  In  one  embodiment,  the  effector  functions  in  the  context  of  the  present  disclosure are T cell mediated effector  functions. Such  functions comprise  in  the case of a  helper T cell (CD4+ T cell) the release of cytokines and/or the activation of CD8+ lymphocytes  (CTLs) and/or B cells, and in the case of CTL the elimination of cells, i.e., cells characterized by  expression of an antigen, for example, via apoptosis or perforin‐mediated cell lysis, production  of cytokines such as IFN‐γ and TNF‐α, and specific cytolytic killing of antigen expressing target  cells.  The  term  "immune  effector  cell"  or  "immunoreactive  cell"  in  the  context  of  the  present  disclosure  relates  to a cell which exerts effector  functions during an  immune  reaction. An  "immune effector cell" in one embodiment is capable of binding an antigen such as an antigen  presented in the context of MHC on a cell or expressed on the surface of a cell and mediating  an immune response. For example, immune effector cells comprise T cells (cytotoxic T cells,  helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages,  and dendritic  cells. Preferably,  in  the  context of  the present disclosure,  "immune effector  cells" are T cells, preferably CD4+ and/or CD8+ T cells, most preferably CD8+ T cells. According  to  the present  disclosure,  the  term  "immune  effector  cell"  also  includes  a  cell which  can  mature into an immune cell (such as T cell, in particular T helper cell, or cytolytic T cell) with  suitable  stimulation.  Immune  effector  cells  comprise  CD34+  hematopoietic  stem  cells,  immature and mature T cells and immature and mature B cells. The differentiation of T cell  precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of  the immune system.  A "lymphoid cell" is a cell which is capable of producing an immune response such as a cellular  immune  response, or a precursor cell of  such cell, and  includes  lymphocytes, preferably T  lymphocytes, lymphoblasts, and plasma cells. A lymphoid cell may be an immune effector cell  as described herein. A preferred lymphoid cell is a T cell.  The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper  cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells.  The  term  "antigen‐specific T  cell" or  similar  terms  relate  to  a T  cell which  recognizes  the  antigen to which the T cell is targeted and preferably exerts effector functions of T cells.   T cells belong to a group of white blood cells known as lymphocytes, and play a central role in  cell‐mediated  immunity. They can be distinguished from other  lymphocyte types, such as B  cells and natural killer cells by the presence of a special receptor on their cell surface called T  cell receptor (TCR). The thymus is the principal organ responsible for the maturation of T cells.  Several different subsets of T cells have been discovered, each with a distinct function.  T helper cells assist other white blood cells in immunologic processes, including maturation of  B cells  into plasma cells and activation of cytotoxic T cells and macrophages, among other  functions.  These  cells  are  also  known  as  CD4+  T  cells  because  they  express  the  CD4  glycoprotein on their surface. Helper T cells become activated when they are presented with  peptide  antigens by MHC  class  II molecules  that  are  expressed on  the  surface of  antigen  presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called  cytokines that regulate or assist in the active immune response.   Cytotoxic  T  cells  destroy  virally  infected  cells  and  tumor  cells,  and  are  also  implicated  in  transplant rejection. These cells are also known as CD8+ T cells since they express the CD8  glycoprotein  on  their  surface.  These  cells  recognize  their  targets  by  binding  to  antigen  associated with MHC class I, which is present on the surface of nearly every cell of the body.  A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The  TCR  of  a  T  cell  is  able  to  interact with  immunogenic  peptides  (epitopes)  bound  to major  histocompatibility  complex  (MHC) molecules and presented on  the  surface of  target  cells.  Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation  and differentiation into a maturated effector T cell. The actual T cell receptor is composed of  two separate peptide chains, which are produced from the independent T cell receptor alpha  and beta (TCRα and TCRβ) genes and are called α‐ and β‐TCR chains. γδ T cells (gamma delta  T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their  surface. However, in γδ T cells, the TCR is made up of one γ‐chain and one δ‐chain. This group  of T cells is much less common (2% of total T cells) than the αβ T cells.  "Humoral  immunity"  or  "humoral  immune  response"  is  the  aspect  of  immunity  that  is  mediated  by  macromolecules  found  in  extracellular  fluids  such  as  secreted  antibodies,  complement  proteins,  and  certain  antimicrobial  peptides.  It  contrasts with  cell‐mediated  immunity. Its aspects involving antibodies are often called antibody‐mediated immunity.  Humoral immunity refers to antibody production and the accessory processes that accompany  it, including: Th2 activation and cytokine production, germinal center formation and isotype  switching,  affinity maturation  and memory  cell  generation.  It  also  refers  to  the  effector  functions  of  antibodies,  which  include  pathogen  neutralization,  classical  complement  activation, and opsonin promotion of phagocytosis and pathogen elimination.  In humoral  immune  response,  first  the B cells mature  in  the bone marrow and gain B‐cell  receptors (BCR's) which are displayed in large number on the cell surface. These membrane‐ bound protein complexes have antibodies which are specific for antigen detection. Each B cell  has a unique antibody that binds with an antigen. The mature B cells migrate from the bone  marrow  to  the  lymph  nodes  or  other  lymphatic  organs, where  they  begin  to  encounter  pathogens. When a B cell encounters an antigen, the antigen  is bound to the receptor and  taken inside the B cell by endocytosis. The antigen is processed and presented on the B cell's  surface again by MHC‐II proteins. The B cell waits for a helper T cell (TH) to bind to the complex.  This binding will activate the TH cell, which then releases cytokines that induce B cells to divide  rapidly, making thousands of identical clones of the B cell. These daughter cells either become  plasma  cells or memory  cells. The memory B  cells  remain  inactive here;  later when  these  memory B cells encounter the same antigen due to reinfection, they divide and form plasma  cells. On the other hand, the plasma cells produce a  large number of antibodies which are  released free into the circulatory system. These antibodies will encounter antigens and bind  with them. This will either interfere with the chemical interaction between host and foreign  cells,  or  they  may  form  bridges  between  their  antigenic  sites  hindering  their  proper  functioning,  or  their  presence  will  attract  macrophages  or  killer  cells  to  attack  and  phagocytose them.  The term "antibody" includes an immunoglobulin comprising at least two heavy (H) chains and  two  light  (L) chains  inter‐connected by disulfide bonds. Each heavy chain  is comprised of a  heavy chain variable region  (abbreviated herein as VH) and a heavy chain constant  region.  Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a  light chain constant region. The VH and VL regions can be further subdivided into regions of  hypervariability,  termed  complementarity  determining  regions  (CDR),  interspersed  with  regions that are more conserved, termed framework regions (FR). Each VH and VL is composed  of  three  CDRs  and  four  FRs,  arranged  from  amino‐terminus  to  carboxy‐terminus  in  the  following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and  light chains contain a binding domain that interacts with an antigen. The constant regions of  the antibodies may mediate  the binding of  the  immunoglobulin  to host  tissues or  factors,  including various cells of the immune system (e.g., effector cells) and the first component (Clq)  of the classical complement system. An antibody binds, preferably specifically binds with an  antigen.   Antibodies expressed by B cells are  sometimes  referred  to as  the BCR  (B  cell  receptor) or  antigen receptor. The five members  included  in this class of proteins are  IgA,  IgG, IgM, IgD,  and IgE. IgA is the primary antibody that  is present  in body secretions, such as saliva, tears,  breast  milk,  gastrointestinal  secretions  and  mucus  secretions  of  the  respiratory  and  genitourinary  tracts.  IgG  is  the  most  common  circulating  antibody.  IgM  is  the  main  immunoglobulin produced  in the primary  immune response  in most subjects.  It  is the most  efficient  immunoglobulin  in  agglutination,  complement  fixation,  and  other  antibody  responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin  that  has  no  known  antibody  function,  but may  serve  as  an  antigen  receptor.  IgE  is  the  immunoglobulin  that mediates  immediate hypersensitivity by causing  release of mediators  from mast cells and basophils upon exposure to allergen.   An "antibody heavy chain", as used herein, refers to the larger of the two types of polypeptide  chains present in antibody molecules in their naturally occurring conformations.  An "antibody light chain", as used herein, refers to the smaller of the two types of polypeptide  chains present in antibody molecules in their naturally occurring conformations, κ and λ light  chains refer to the two major antibody light chain isotypes.   The present disclosure contemplates an immune response that may be protective, preventive,  prophylactic and/or therapeutic. As used herein, "induces [or inducing] an immune response"  may  indicate  that  no  immune  response  against  a  particular  antigen  was  present  before  induction  or  it may  indicate  that  there was  a  basal  level  of  immune  response  against  a  particular antigen before induction, which was enhanced after induction. Therefore, "induces  [or inducing] an immune response" includes "enhances [or enhancing] an immune response".   The term "immunotherapy" relates to the treatment of a disease or condition by inducing, or  enhancing an immune response. The term "immunotherapy" includes antigen immunization  or antigen vaccination.   The terms "immunization" or "vaccination" describe the process of administering an antigen  to  an  individual  with  the  purpose  of  inducing  an  immune  response,  for  example,  for  therapeutic or prophylactic reasons.  The  term  "macrophage"  refers  to  a  subgroup  of  phagocytic  cells  produced  by  the  differentiation of monocytes. Macrophages which are activated by  inflammation,  immune  cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the  macrophage  by  hydrolytic  and  oxidative  attack  resulting  in  degradation  of  the  pathogen.  Peptides from degraded proteins are displayed on the macrophage cell surface where they  can be  recognized by  T  cells,  and  they  can  directly  interact with  antibodies on  the B  cell  surface, resulting in T and B cell activation and further stimulation of the immune response.  Macrophages  belong  to  the  class  of  antigen  presenting  cells.  In  one  embodiment,  the  macrophages are splenic macrophages.  The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the  class  of  antigen  presenting  cells.  In  one  embodiment,  dendritic  cells  are  derived  from  hematopoietic bone marrow progenitor cells. These progenitor cells  initially transform  into  immature dendritic cells. These immature cells are characterized by high phagocytic activity  and low T cell activation potential. Immature dendritic cells constantly sample the surrounding  environment for pathogens such as viruses and bacteria. Once they have come into contact  with a presentable antigen, they become activated  into mature dendritic cells and begin to  migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens  and degrade their proteins into small pieces and upon maturation present those fragments at  their cell surface using MHC molecules. Simultaneously, they upregulate cell‐surface receptors  that act as co‐receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing  their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces  the dendritic cell to travel through the blood stream to the spleen or through the lymphatic  system to a lymph node. Here they act as antigen‐presenting cells and activate helper T cells  and killer T cells as well as B cells by presenting them antigens, alongside non‐antigen specific  co‐stimulatory  signals.  Thus,  dendritic  cells  can  actively  induce  a  T  cell‐  or  B  cell‐related  immune response. In one embodiment, the dendritic cells are splenic dendritic cells.  The term "antigen presenting cell" (APC)  is a cell of a variety of cells capable of displaying,  acquiring,  and/or presenting  at  least  one  antigen or  antigenic  fragment  on  (or  at)  its  cell  surface. Antigen‐presenting cells can be distinguished in professional antigen presenting cells  and non‐professional antigen presenting cells.   The  term  "professional  antigen presenting  cells"  relates  to  antigen presenting  cells which  constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules  required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule  complex on the membrane of the antigen presenting cell, the antigen presenting cell produces  a co‐stimulatory molecule  inducing activation of  the T cell. Professional antigen presenting  cells comprise dendritic cells and macrophages.   The term "non‐professional antigen presenting cells" relates to antigen presenting cells which  do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines  such  as  interferon‐gamma.  Exemplary,  non‐professional  antigen  presenting  cells  include  fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or  vascular endothelial cells.  "Antigen processing" refers to the degradation of an antigen into procession products, which  are  fragments  of  said  antigen  (e.g.,  the  degradation  of  a  protein  into  peptides)  and  the  association of one or more of  these  fragments  (e.g., via binding) with MHC molecules  for  presentation by cells, such as antigen presenting cells to specific T cells.  The term "disease involving an antigen" refers to any disease which implicates an antigen, e.g.  a  disease which  is  characterized  by  the  presence  of  an  antigen.  The  disease  involving  an  antigen  can be an  infectious disease. As mentioned above,  the antigen may be a disease‐ associated antigen, such as a viral antigen. In one embodiment, a disease involving an antigen  is a disease involving cells expressing an antigen, preferably on the cell surface.  The term "infectious disease" refers to any disease which can be transmitted from individual  to individual or from organism to organism, and is caused by a microbial agent (e.g. common  cold).  Infectious diseases are known  in  the art and  include,  for example, a viral disease, a  bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and  a parasite, respectively. In this regard, the  infectious disease can be, for example, hepatitis,  sexually  transmitted  diseases  (e.g.  chlamydia  or  gonorrhea),  tuberculosis,  HIV/acquired  immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute  respiratory syndrome (SARS), the bird flu, and influenza.   Exemplary Dosing Regimens  In some embodiments, compositions and methods disclosed herein can be used in accordance  with an exemplary vaccination regimen as illustrated in Figure 14.    Primary Dosing Regimens  In some embodiments, subjects are administered a primary dosing regimen.  A primary dosing  regimen can comprise one or more doses. For example,  in  some embodiments, a primary  dosing regimen comprises a single dose (PD1). In some embodiments a primary dosing regimen  comprises a first dose (PD1) and a second dose (PD2). In some embodiments, a primary dosing  regimen comprises a first dose, a second dose, and a third dose (PD3). In some embodiments,  a primary dosing regimen comprises a first dose, a second dose, a third dose, and one or more  additional doses (PDn) of any one of the pharmaceutical compositions described herein.    In  some  embodiments,  PD1  comprises  administering  1  to  100  ug  of  RNA.  In  some  embodiments, PD1 comprises administering 1 to 60 ug of RNA    In some embodiments, PD1  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD1  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD1 comprises administering about  3 ug of RNA.  In some embodiments, PD1 comprises administering about 5 ug of RNA.  In some  embodiments, PD1 comprises administering about 10 ug of RNA.  In some embodiments, PD1  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD1  comprises  administering about 20 ug of RNA.  In some embodiments, PD1 comprises administering about  30 ug of RNA.  In some embodiments, PD1 comprises administering about 50 ug of RNA.  In  some embodiments, PD1 comprises administering about 60 ug of RNA.    In  some  embodiments,  PD2  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PD2 comprises administering 1 to 60 ug of RNA.    In some embodiments, PD2  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD 2  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD2 comprises administering about  3  ug.    In  some  embodiments,  PD2  comprises  administering  about  5  ug  of  RNA.    In  some  embodiments, PD2 comprises administering about 10 ug of RNA.  In some embodiments, PD2  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD2  comprises  administering about 20 ug RNA.  In some embodiments, PD2 comprises administering about  30 ug of RNA.   In some embodiments, PD2 comprises administering about 50 ug of RNA.  In  some embodiments, PD2 comprises administering about 60 ug of RNA.    In  some  embodiments,  PD3  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PD3 comprises administering 1 to 60 ug of RNA.    In some embodiments, PD3  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD3  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD3 comprises administering about  3 ug of RNA.  In some embodiments, PD3 comprises administering about 5 ug of RNA.  In some  embodiments, PD3 comprises administering about 10 ug of RNA.  In some embodiments, PD3  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD3  comprises  administering about 20 ug of RNA.  In some embodiments, PD3 comprises administering about  30 ug of RNA.  In some embodiments, PD3 comprises administering about 50 ug of RNA.  In  some embodiments, PD3 comprises administering about 60 ug of RNA.    In  some  embodiments,  PDn  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PDn comprises administering 1 to 60 ug of RNA.    In some embodiments, PDn  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PDn  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PDn comprises administering about  3 ug of RNA.  In some embodiments, PDn comprises administering about 5 ug of RNA.  In some  embodiments, PDn comprises administering about 10 ug of RNA.  In some embodiments, PDn  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PDn  comprises  administering about 20 ug of RNA.  In some embodiments, PDn comprises administering about  30 ug of RNA.  In some embodiments, PDn comprises administering about 50 ug of RNA.  In  some embodiments, PDn comprises administering about 60 ug of RNA.    In  some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD1 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PD1 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD1 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In  some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD2 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PD2 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD2 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.      In  some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD3 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PD3 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD3 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In  some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PDn comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PDn comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PDn comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In some embodiments, PD1, PD2, PD3, and PDn can each independently comprise a plurality of  (e.g., at least two) RNA (e.g., mRNA) compositions described herein.  In some embodiments  PD1, PD2, PD3, and PDn can each independently comprise a first and a second RNA (e.g., mRNA)  composition.    In  some  embodiments,  at  least  one  of  a  plurality  of  RNA  (e.g.,  mRNA)  compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least  one of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding  a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  from a different SARS‐CoV‐2  variant.  In some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions  comprises an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof from a Wuhan strain of SARS‐CoV‐2.  In some embodiments, at least one of a plurarity  of RNA (e.g., mRNA) compositions comprises an RNA encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising  one  or more mutations  from  a  variant  that  is  prevalent and/or spreading rapidly in a relevant jurisdiction.   In some embodiments, at least  one of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations from an alpha variant.    In some embodiments, at  least one of a plurality of RNA  (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof comprising one or more mutations from a delta variant.    In some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  comprising one or more mutations from an Omicron variant.     In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or  PDn  can  each  independently  comprise  at  least  two  different  RNA  (e.g.,  mRNA)  constructs  (e.g.,  differing  in  at  protein‐encoding  sequences).  For  example,  in  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2, PD3, and/or PDn  can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof  from a Wuhan strain of SARS‐CoV‐2 and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations  from  a  variant  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.  In some embodiments a plurality of RNA (e.g., mRNA) compositions given in PD1,  PD2, PD3, and/or PDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof  derived from a Wuhan strain of SARS‐ CoV‐2 and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof comprising one or more mutations from a variant that is prevalent and/or spreading  rapidly in a relevant jurisdiction.  In some such embodiments, a variant can be an alpha variant.  In some such embodiments, a variant can be a delta variant.  In some such embodiments a  variant can be an Omicron variant.   In some embodiments, each of a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2,  PD3, and/or PDn can independently comprise at least two RNA (e.g., mRNA)s, each encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction.  In  some embodiments, each of a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2,  PD3, and/or PDn can  independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S  protein or an immunogenic fragment thereof  from an alpha variant and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations from a delta variant.  In some embodiments, each of a plurality of RNA (e.g.,  mRNA) compositions given  in PD1, PD2, PD3, and/or PDn can  independently comprise an RNA  (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  from an  alpha variant and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic  fragment  thereof  comprising  one  or more mutations  from  an Omicron  variant.    In  some  embodiments, each of a plurality of RNA  (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or PDn can independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof  from a delta variant and an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations from an Omicron variant.    In some embodiments, PD1, PD2, PD3, and/or PDn each comprise a plurality of RNA (e.g., mRNA)  compositions, wherein each RNA  (e.g., mRNA) composition  is separately administered  to a  subject.  For  example,  in  some  embodiments  each  RNA  (e.g.,  mRNA)  composition  is  administered  via  intramuscular  injection  at different  injection  sites.  For example,  in  some  embodiments, a first and second RNA (e.g., mRNA) composition given in PD1, PD2, PD3, and/or  PDn are separately administered to different arms of a subject via intramuscular injection.   In some embodiments, PD1, PD2, PD3, and/or PDn comprise administering a plurality of RNA  molecules, wherein each RNA molecule encodes a Spike protein comprising mutations from a  different SARS‐CoV‐2 variant, and wherein the plurality of RNA molecules are administered to  the subject in a single formulation.  In some embodiments, the single formulation comprises  an RNA encoding a Spike protein or an immunogenic variant thereof from the Wuhan strain  and  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising one or more mutations from an alpha variant.  In some embodiments, the single  formulation  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or  an immunogenic fragment thereof comprising one or more mutations from a beta variant.  In  some embodiments, the single formulation comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from a delta variant.  In some embodiments, the single formulation comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from an Omicron variant.    In some embodiments, the length of time between PD1 and PD2 (PI1) is at least about 1 week,  at  least  about  2  weeks,  at  least  about  3  weeks,  or  at  least  about  4  weeks.    In  some  embodiments, PI1 is about 1 week to about 12 weeks. In some embodiments, PI1 is about 1  week to about 10 weeks.  In some embodiments, PI1 is about 2 weeks to about 10 weeks.  In  some embodiments, PI1 is about 2 weeks to about 8 weeks.  In some embodiments, PI1 is about  3 weeks to about 8 weeks.  In some embodiments, PI1 is about 4 weeks to about 8 weeks.  In  some embodiments, PI1 is about 6 weeks to about 8 weeks.  In some embodiments PI1 is about  3 to about 4 weeks.  In some embodiments, PI1 is about 1 week.  In some embodiments, PI1 is  about 2 weeks.  In some embodiments, PI1 is about 3 weeks. In some embodiments, PI1 is about  4 weeks.  In some embodiments, PI1 is about 5 weeks.  In some embodiments, PI1 is about 6  weeks.    In some embodiments, PI1 is about 7 weeks.    In some embodiments, PI1 is about 8  weeks.  In some embodiments, PI1 is about 9 weeks.  In some embodiments, PI1 is about 10  weeks.  In some embodiments, PI1 is about 11 weeks.  In some embodiments, PI1 is about 12  weeks.    In some embodiments, the length of time between PD2 and PD3 (PI2) is at least about 1 week,  at least about 2 weeks, or at least about 3 weeks.  In some embodiments, PI2 is about 1 week  to about 12 weeks. In some embodiments, PI2 is about 1 week to about 10 weeks.  In some  embodiments, PI2 is about 2 weeks to about 10 weeks.  In some embodiments, PI2 is about 2  weeks to about 8 weeks.  In some embodiments, PI2 is about 3 weeks to about 8 weeks.  In  some embodiments, PI2 is about 4 weeks to about 8 weeks.  In some embodiments, PI2 is about  6 weeks to about 8 weeks.  In some embodiments PI2 is about 3 to about 4 weeks.  In some  embodiments, PI2  is about 1 week.    In some embodiments, PI2  is about 2 weeks.    In some  embodiments, PI2  is about 3 weeks.  In some embodiments, PI2  is about 4 weeks.    In  some  embodiments, PI2 is about 5 weeks.    In some embodiments, PI2 is about 6 weeks.    In some  embodiments, PI2 is about 7 weeks.    In some embodiments, PI2 is about 8 weeks.    In some  embodiments, PI2 is about 9 weeks.  In some embodiments, PI2 is about 10 weeks.  In some  embodiments, PI 2 is about 11 weeks.  In some embodiments, PI2 is about 12 weeks.    In some embodiments, the length of time between PD3 and a subsequent dose that is part of  the  Primary  Dosing  Regimen,  or  between  doses  for  any  dose  beyond  PD3  (PIn)  is  each  separately and independently selected from: about 1 week or more, about 2 weeks or more,  or about 3 weeks or more.  In some embodiments, PIn is about 1 week to about 12 weeks. In  some embodiments, PIn is about 1 week to about 10 weeks.  In some embodiments, PIn is about  2 weeks to about 10 weeks.  In some embodiments, PIn is about 2 weeks to about 8 weeks.  In  some embodiments, PIn is about 3 weeks to about 8 weeks.  In some embodiments, PIn is about  4 weeks to about 8 weeks.  In some embodiments, PIn is about 6 weeks to about 8 weeks.  In  some embodiments PIn is about 3 to about 4 weeks.    In some embodiments, PI2 is about 1      In some embodiments, PIn  is about 2 weeks.    In  some embodiments, PIn  is about 3  weeks.  In some embodiments, PIn  is about 4 weeks.    In some embodiments, PIn  is about 5  weeks.    In some embodiments, PInis about 6 weeks.    In some embodiments, PIn is about 7  weeks.    In some embodiments, PInis about 8 weeks.    In some embodiments, PIn is about 9  weeks.  In some embodiments, PIn is about 10 weeks.  In some embodiments, PIn is about 11  weeks.  In some embodiments, PIn is about 12 weeks.    In some embodiments, one or more compositions adminstered in PD1 are formulated in a Tris  buffer.  In some embodiments, one or more compositions administered in PD2 are formulated  in a Tris buffer.  In some embodiments, one or more compositions administering in PD3 are  formulated in a Tris buffer.  In some embodiments, one or more compositions adminsitered  in PDn are formulated in a Tris buffer.    In some embodiments, the primary dosing regimen comprises administering two or more RNA  (e.g., mRNA)  compositions  described  herein,  and  at  least  two  of  the  RNA  (e.g.,  mRNA)  compositions have different formulations.  In some embodiments, the primary dosing regimen  comprises PD1 and PD2, where PD1 comprises administering an RNA (e.g., mRNA) formulated  in a Tris buffer and PD2 comprises administering an RNA  (e.g., mRNA)  formulated  in a PBS  buffer.  In some embodiments, the primary dosing regimen comprises PD1 and PD2, where PD1  comprises administering an RNA (e.g., mRNA) formulated in a PBS buffer and PD2 comprises  administering an RNA (e.g., mRNA) formulated in a Tris buffer.    In some embodiments, one or more RNA  (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or PDn can be administered in combination with another vaccine. In some embodiments,  another vaccine is for a disease that is not COVID‐19.  In some embodiments, the disease is  one that  increases deleterious effects of SARS‐CoV‐2 when a subject  is coinfected with the  disease  and  SARS‐CoV‐2.    In  some  embodiments,  the  disease  is  one  that  increases  the  transmission rate of SARS‐CoV‐2 when a subject is coinfected with the disease and SARS‐CoV‐ 2.    In some embodiments, another vaccine  is a different commerically available vaccine.  In  some embodiments, the different commercially available vaccine is an RNA vaccine.  In some  embodiments, the different commercially available vaccine is a polypeptide‐based vaccine. In  some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g.,  mRNA)  compositions  given  in  PD1,  PD2,  PD3,  and/or  PDn  are  separately  administered,  for  example,  in some embodiments via  intramuscular  injection, at different  injection sites. For  example, in some embodiments, an influenza vaccine and one or more SARS‐CoV‐2 RNA (e.g.,  mRNA)  compositions  described  herein  given  in  PD1,  PD2,  PD3,  and/or  PDn  are  separately  administered to different arms of a subject via intramuscular injection.    Booster Dosing Regimens In  some  embodiments,  methods  of  vaccination  disclosed  herein  comprise  one  or  more  Booster Dosing Regimens.   The Booster Dosing Regimens disclosed herein comprise one or  more doses.    In some embodiments, a Booster Dosing Regimen  is administered to patients  who have been administered a Primary Dosing Regimen (e.g., as described herein). In some  embodiments a Booster Dosing Regimen  is administed to patients who have not received a  pharmaceutical  composition  disclosed  herein.  In  some  embodiments  a  Booster  Dosing  Regimen  is administered to patients who have been previously vaccinated with a COVID‐19  vaccine that is different from the vaccine administered in a Primary Dosing Regimen.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks,  at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least  10 weeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4  months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9  months, at  least 10 months, at  least 11 months, or at  least 12 months or  longer.  In  some  embodiments,  the  length  of  time  between  the  Primary Dosing  Regimen  and  the  Booster  Dosing Regimen  is about 1 month.    In some embodiments, the  length of time between the  Primary Dosing Regimen and the Booster Dosing Regimen is at least about 2 months.  In some  embodiments,  the  length  of  time  between  the  Primary Dosing  Regimen  and  the  Booster  Dosing Regimen is at least about 3 months.  In some embodiments, the length of time between  the Primary Dosing Regimen and the Booster Dosing Regimen is at least about 4 months.  In  some embodiments, the length of time between the Primary Dosing Regimen and the Booster  Dosing Regimen is at least about 5 months.  In some embodiments, the length of time between  the Primary Dosing Regimen and the Booster Dosing Regimen is at least about 6 months.  In  some embodiments, the length of time between the Primary Dosing Regimen and the Booster  Dosing Regimen is from about 1 month to about 48 months.  In some embodiments, the length  of time between the Primary Dosing Regimen and the Booster Dosing Regimen is from about  1 month to about 36 months.  In some embodiments, the length of time between the primary  dosing regimen and the Booster Dosing Regimen is from about 1 month to about 24 months.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is from about 2 months to about 24 months.  In some embodiments,  the length of time between the Primary Dosing Regimen and the Booster Dosing Regimen is  from about 3 months to about 24 months.  In some embodiments, the length of time between  the primary dosing regimen and the Booster Dosing Regimen is from about 3 months to about  18 months.  In some embodiments, the length of time between the primary dosing regimen  and  the  Booster Dosing  Regimen  is  from  about  3 months  to  about  12 months.    In  some  embodiments, the length of time between the primary dosing regimen and the Booster Dosing  Regimen is from about 6 months to about 12 months.  In some embodiments, the length of  time between the Primary Dosing Regimen and the Booster Dosing Regimen is from about 3  months to about 9 months.  In some embodiments, the length of time between the Primary  Dosing Regimen and the Booster Dosing Regimen is from about 5 months to about 7 months.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is about 6 months.    In some embodiments, subjects are administered a Booster Dosing Regimen.  A Booster dosing  regimen can comprise one or more doses. For example,  in  some embodiments, a Booster  Dosing  Regimen  comprises  a  single  dose  (BD1).  In  some  embodiments  a  Booster  Dosing  Regimen  comprises  a  first  dose  (BD1)  and  a  second  dose  (BD2).  In  some  embodiments,  a  Booster Dosing Regimen comprises a first dose, a second dose, and a third dose (BD3). In some  embodiments, a Booster Dosing Regimen comprises a first dose, a second dose, a third dose,  and  one  or more  additional  doses  (BDn)  of  any  one  of  the  pharmaceutical  compositions  described herein.    In  some  embodiments,  BD1  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD1 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD1  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD1  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD1 comprises administering about  3 ug of RNA.  In some embodiments, BD1 comprises administering about 5 ug of RNA.  In some  embodiments, BD1 comprises administering about 10 ug of RNA.  In some embodiments, BD1  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD1  comprises  administering about 20 ug of RNA.  In some embodiments, BD1 comprises administering about  30 ug of RNA.  In some embodiments, BD1 comprises administering about 50 ug of RNA.  In  some embodiments, BD1 comprises administering about 60 ug of RNA.    In  some  embodiments,  BD2  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD2 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD2  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD2  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD2 comprises administering about  3  ug.    In  some  embodiments,  BD2  comprises  administering  about  5  ug  of  RNA.    In  some  embodiments, BD2 comprises administering about 10 ug of RNA.  In some embodiments, BD2  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD2  comprises  administering about 20 ug RNA.  In some embodiments, BD2 comprises administering about  30 ug of RNA.  In some embodiments, BD2 comprises administering about 50 ug of RNA.  In  some embodiments, BD2 comprises administering about 60 ug of RNA.    In  some  embodiments,  BD3  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD3 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD3  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD3  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD3 comprises administering about  3 ug of RNA.  In some embodiments, BD3 comprises administering about 5 ug of RNA.  In some  embodiments, BD3 comprises administering about 10 ug of RNA.  In some embodiments, BD3  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD3  comprises  administering about 20 ug of RNA.  In some embodiments, BD3 comprises administering about  30 ug of RNA.  In some embodiments, BD 3 comprises administering about 50 ug of RNA.  In  some embodiments, BD3 comprises administering about 60 ug of RNA.    In  some  embodiments,  BDn  comprises  administering  1  to  100  ug  of  RNA.  In  some  embodiments, BDn comprises administering 1 to 60 ug of RNA.   In some embodiments, BDn  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BDn  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BDn comprises administering about  3 ug of RNA.  In some embodiments, BDn comprises administering about 5 ug of RNA.  In some  embodiments, BDn comprises administering about 10 ug of RNA.  In some embodiments, BDn  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BDn  comprises  administering about 20 ug of RNA.  In some embodiments, BDn comprises administering about  30 ug of RNA.  In some embodiments, BDn comprises administering about 60 ug of RNA.  In  some embodiments, BDn comprises administering about 50 ug of RNA.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD1 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD1 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.    In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD2 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD2 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.      In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD3 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD3 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD 3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.    In  some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BDn comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.    In  some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BDn comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.      In some embodiments, BD1, BD2, BD3, and BDn can each independently comprise a plurality of  (e.g., at least two) RNA (e.g., mRNA) compositions described herein.  In some embodiments  BD1, BD2, BD3, and BDn can each independently comprise a first and a second RNA (e.g., mRNA)  composition.  In some embodiments, BD1, BD2, BD3, and BDn can each independently comprise  a plurality of (e.g., at least two) RNA (e.g., mRNA) compositions, wherein , at least one of the  plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  from a different SARS‐CoV‐2 variant (e.g., a variant that is prevalent or rapidly spreading in a  relevant jurisdiction, e.g., a variant disclosed herein).  In some embodiments, at least one of a  plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof from a Wuhan strain of SARS‐CoV‐2.  In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one  or more mutations  from a variant  that  is prevalent and/or  spreading  rapidly  in a  relevant  jurisdiction.    In  some  embodiments,  at  least  one  of  a  plurality  of  RNA  (e.g.,  mRNA)  compositions  comprises  an  RNA  (e.g.,  mRNA)  encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  comprising one or more mutations from a delta variant.  In some embodiments, at least one  of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from an Omicron variant.     In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can each indendently comprise at least two different RNA (e.g., mRNA) constructs  (e.g., RNA  constructs  having  differing  protein‐encoding  sequences).  For  example,  in  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or BDn  can each indendently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof from a Wuhan strain of SARS‐CoV‐2 and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations  from  a  variant  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.  In some embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1,  BD2, BD3, and/or BDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof derived from a Wuhan strain of SARS‐ CoV‐2 and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof comprising one or more mutations from a variant that is prevalent and/or spreading  rapidly in a relevant jurisdiction.  In some such embodiments, a variant can be an alpha variant.  In some such embodiments, a variant can be a delta variant.  In some such embodiments a  variant can be an Omicron variant.   In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can each independently comprise at least two RNA (e.g., mRNA)s each encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction.  In  some embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or  BDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof from an alpha variant and an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments  a  plurality  of  RNA  (e.g., mRNA)  compositions given in BD1, BD2, BD3, and/or BDn can each independently comprise an RNA (e.g.,  mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof from an alpha  variant  and  an  RNA  (e.g., mRNA)  encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more mutations  from  an Omicron  variant.    In  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or BDn  can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof from a delta variant and an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations from  an Omicron variant.    In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn are separately administered to a subject, for example, in some embodiments via  intramuscular injection, at different injection sites. For example, in some embodiments, a first  and second RNA (e.g., mRNA) composition given  in BD1, BD2, BD3, and/or BDn are separately  administered to different arms of a subject via intramuscular injection.   In some embodiments, the length of time between BD1 and BD2 (BI1) is at least about 1 week,  at  least  about  2  weeks,  at  least  about  3  weeks,  or  at  least  about  4  weeks.    In  some  embodiments, BI1 is about 1 week to about 12 weeks.  In some embodiments, BI1 is about 1  week to about 10 weeks.  In some embodiments, BI1 is about 2 weeks to about 10 weeks.  In  some embodiments, BI1 is about 2 weeks to about 8 weeks.  In some embodiments, BI1 is about  3 weeks to about 8 weeks.  In some embodiments, BI1 is about 4 weeks to about 8 weeks.  In  some embodiments, BI1 is about 6 weeks to about 8 weeks.  In some embodiments BI1 is about  3 to about 4 weeks.  In some embodiments, BI1 is about 1 week.  In some embodiments, BI1 is  about 2 weeks.    In some embodiments, BI1  is about 3 weeks.  In some embodiments, BI1  is  about 4 weeks. In some embodiments, BI1 is about 5 weeks. In some embodiments, BI1 is about  6 weeks.  In some embodiments, BI1 is about 7 weeks.  In some embodiments, BI1 is about 8  weeks.  In some embodiments, BI1 is about 9 weeks.  In some embodiments, BI1 is about 10  weeks.  In some embodiments, the length of time between BD2 and BD3 (BI2) is at least about 1 week,  at least about 2 weeks, or at least about 3 weeks.  In some embodiments, BI2 is about 1 week  to about 12 weeks. In some embodiments, BI2 is about 1 week to about 10 weeks.  In some  embodiments, BI2 is about 2 weeks to about 10 weeks.  In some embodiments, BI2 is about 2  weeks to about 8 weeks.  In some embodiments, BI2 is about 3 weeks to about 8 weeks.  In  some embodiments, BI2 is about 4 weeks to about 8 weeks.  In some embodiments, BI2 is about  6 weeks to about 8 weeks.  In some embodiments BI2 is about 3 to about 4 weeks.  In some  embodiments, BI2  is about 1 week.    In some embodiments, BI2  is about 2 weeks.    In some  embodiments, BI2  is about 3 weeks.  In  some embodiments, BI2  is about 4 weeks.  In  some  embodiments, BI2  is about 5 weeks.  In  some embodiments, BI2  is about 6 weeks.  In  some  embodiments, BI2  is about 7 weeks.  In  some embodiments, BI2  is about 8 weeks.  In  some  embodiments, BI 2 is about 9 weeks. In some embodiments, BI2 is about 10 weeks.  In some embodiments, the length of time between BD3 and a subsequent dose that is part of  the  Booster  Dosing  Regimen,  or  between  doses  for  any  dose  beyond  BD3  (BIn)  is  each  separately and independently selected from: about 1 week or more, about 2 weeks or more,  or about 3 weeks or more.  In some embodiments, BIn is about 1 week to about 12 weeks. In  some embodiments, BIn  is about 1 week  to about 10 weeks.    In some embodiments, BIn  is  about 2 weeks to about 10 weeks.    In some embodiments, BIn  is about 2 weeks to about 8  weeks.  In some embodiments, BIn is about 3 weeks to about 8 weeks.  In some embodiments,  BIn is about 4 weeks to about 8 weeks.  In some embodiments, BIn is about 6 weeks to about  8 weeks.  In some embodiments BIn is about 3 to about 4 weeks.  In some embodiments, BIn is  about 1 week.  In some embodiments, BIn is about 2 weeks.  In some embodiments, BIn is about  3 weeks. In some embodiments, BIn is about 4 weeks. In some embodiments, BIn is about 5  weeks.  In  some embodiments, BIn  is about 6 weeks.  In some embodiments, BIn  is about 7  weeks.  In  some embodiments, BIn  is about 8 weeks.  In some embodiments, BIn  is about 9  weeks. In some embodiments, BIn is about 10 weeks.  In some embodiments, one or more compositions adminstered in BD1 are formulated in a Tris  buffer.  In some embodiments, one or more compositions administered in BD2 are formulated  in a Tris buffer.  In some embodiments, one or more compositions administering in BD3 are  formulated in a Tris buffer.  In some embodiments, one or more compositions adminsitered  in BD3 are formulated in a Tris buffer.    In some embodiments, the Booster dosing regimen comprises administering two or more RNA  (e.g., mRNA)  compositions  described  herein,  and  at  least  two  of  the  RNA  (e.g.,  mRNA)  compositions  have  differnent  formulations.    In  some  embodiments,  the  Booster  dosing  regimen comprises BD1 and BD2, where BD1 comprises administering an RNA  (e.g., mRNA)  formulated in a Tris buffer and BD2 comprises administering an RNA (e.g., mRNA) formulated  in a PBS buffer.  In some embodiments, the Booster dosing regimen comprises BD1 and BD2,  where BD1 comprises administering an RNA (e.g., mRNA) formulated in a PBS buffer and BD2  comprises administering an RNA (e.g., mRNA) formulated in a Tris buffer.    In some embodiments, one or more RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can be administered in combination with another vaccine. In some embodiments,  another vaccine is for a disease that is not COVID‐19.  In some embodiments, the disease is  one that  increases deleterious effects of SARS‐CoV‐2 when a subject  is coinfected with the  disease  and  SARS‐CoV‐2.    In  some  embodiments,  the  disease  is  one  that  increases  the  transmission rate of SARS‐CoV‐2 when a subject is coinfected with the disease and SARS‐CoV‐ 2.    In some embodiments, another vaccine  is a different commerically available vaccine.  In  some embodiments, the different commercially available vaccine is an RNA vaccine.  In some  embodiments, the different commercially available vaccine is a polypeptide‐based vaccine. In  some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g.,  mRNA)  compositions  given  in  BD1,  BD2,  BD3,  and/or  BDn  are  separately  administered,  for  example,  in some embodiments via  intramuscular  injection, at different  injection sites. For  example, in some embodiments, an influenza vaccine and one or more SARS‐CoV‐2 RNA (e.g.,  mRNA)  compositions  described  herein  given  in  BD1,  BD2,  BD3,  and/or  BDn  are  separately  administered to different arms of a subject via intramuscular injection.    Additional Booster Regimens  In some embodiments, methods of vaccination disclosed herein comprise administering more  than one Booster Dosing Regimen.    In some embodiments, more  than one Booster Dosing  Regimen may need to be administered to increase neutralizing antibody response.  In some  embodiments, more than one booster dosing regimen may be needed to counteract a SARS‐ CoV‐2  strain  that has been  shown  to have  a high  likelihood of  evading  immune  response  elicited  by  vaccines  that  a  patient  has  previously  received.    In  some  embodiments,  an  additional Booster Dosing Regimen is administered to a patient who has been determined to  produce low concentrations of neutralizing antibodies.  In some embodiments, an additional  booster dosing regimen is administered to a patient who has been determined to have a high  likelihood of being susceptible to SARS‐CoV‐2 infection, despite previous vaccination (e.g., an  immunocompromised patient, a cancer patient, and/or an organ transplant patient).  The description provided above for the first Booster Dosing Regimen also describes the one or  more additional Booster Dosing Regimens.   The  interval of  time between  the  first Booster  Dosing  Regimen  and  a  second  Booster  Dosing  Regimen,  or  between  subsequent  Booster  Dosing Regimens can be any of the acceptable intervals of time described above between the  Primary Dosing Regimen and the First Booster Dosing Regimen.    In some embodiments, a dosing regimen comprises a primary regimen and a booster regimen,  wherein at least one dose given in the primary regimen and/or the booster regimen comprises  a composition comprising an RNA that encodes a S protein or immungenic fragment thereof  from a variant that is prevalent or is spreading rapidly in a relevant jurisdiction (e.g., Omicron  variant  as  described  herein).  For  example,  in  some  embodiments,  a  primary  regimen  comprises at least 2 doses of BNT162b2 (e.g., encoding a Wuhan strain), for example, given at  least  3 weeks  apart,  and  a  booster  regimen  comprises  at  least  1  dose  of  a  composition  comprising RNA that encodes a S protein or immungenic fragment thereof from a variant that  is prevalent or is spreading rapidly in a relevant jurisdiction (e.g., Omicron variant as described  herein). In some such embodiments, such a dose of a booster regimen may further comprise  an RNA  that encodes a S protein or  immungenic  fragement  thereof  from a Wuhan  strain,  which can be administered with an RNA that encodes a S protein or  immungenic fragment  thereof from a variant that is prevalent or is spreading rapidly in a relevant jurisdiction (e.g.,  Omicron variant as described herein), as a single mixture, or as two separate compositions,  for example, in 1:1 weight ratio. In some embodiments, a booster regimen can also comprise  at least 1 dose of BNT162b2, which can be administered as a first booster dose or a subsequent  booster dose.   In some embodiments, an RNA composition described herein is given as a booster at a dose  that is higher than the doses given during a primary regimen (primary doses) and/or the dose  given for a first booster, if any. For example, in some embodiments, such a dose may be 60  ug; or in some embodiments such a dose may be higher than 30 ug and lower than 60 ug (e.g.,  55 ug, 50 ug, or lower). In some embodiments, an RNA composition described herein is given  as a booster at least 3‐12 months or 4‐12 months, or 5‐12 months, or 6‐12 months after the  last dose (e.g., the  last dose of a primary regimen or a first dose of a booster regimen).    In  some embodiments, the primary doses and/or the first booster dose (if any) may comprise  BNT162b2, for example at 30 ug per dose.  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 49 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  49). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 50 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 50).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 51).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 55 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  55. In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 56 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 56).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 57).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 58 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  58). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 59 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 59).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 60).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 61 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  61). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 62 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 62).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 63 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 63).  In some embodiments, the formulations disclosed herein can be used to carry out any of the  dosing regimens described in Table 28 (below).     
Figure imgf000571_0001
Figure imgf000572_0001
Figure imgf000573_0001
In some embodiments of certain exemplary dosing regimens as described in Table 28 above,  an RNA  composition  described herein  (e.g.,  comprising RNA  encoding  a  variant described  herein)  is  given  in  a  first  dose  of  a  primary  regimen.    In  some  embodiments  of  certain  exemplary dosing regimens as described  in Table 28 above, an RNA composition described  herein (e.g., comprising RNA encoding a variant described herein) is given in a second dose of  a primary regimen.  In some embodiments of certain exemplary dosing regimens as described  in Table 28 above, an RNA composition described herein  (e.g., comprising RNA encoding a  variant described herein) is given in a first dose and a second dose of a primary regimen.  In  some embodiments of certain exemplary dosing regimens as described in Table 28 above, an  RNA composition described herein (e.g., comprising RNA encoding a variant described herein)  is given in a first dose of a booster regimen.  In some embodiments of certain exemplary dosing  regimens  as  described  in  Table  28  above,  an  RNA  composition  described  herein  (e.g.,  comprising RNA encoding a variant described herein)  is given in a second dose of a booster  regimen.  In some embodiments of certain exemplary dosing regimens as described in Table  28 above, an RNA  composition described herein  (e.g.,  comprising RNA encoding a  variant  described herein)  is given  in a first dose and a second dose of a booster regimen.    In some  embodiments of certain exemplary dosing regimens as described in Table 28 above, an RNA  composition described herein (e.g., comprising RNA encoding a variant described herein)  is  given in a first dose and a second dose of a primary regimen and also in at least one dose of a  booster regimen.  In some embodiments of certain exemplary dosing regimens as described  in Table 28 above, an RNA composition described herein  (e.g., comprising RNA encoding a  variant described herein) is given in at least one dose (including, e.g., at least two doses) of a  booster regimen and BNT162b2 is given in a primary regimen. In some embodiments of certain  exemplary dosing regimens as described  in Table 28 above, an RNA composition described  herein (e.g., comprising RNA encoding a variant described herein) is given in a second dose of  a booster regimen and BNT162b2 is given in a primary regimen and in a first dose of a booster  regimen. In some embodiments, an RNA composition described herein (e.g., comprising RNA  encoding a variant described herein) comprises an RNA encoding a polypeptide as set forth in  SEQ ID NO: 49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least  70% or more, including, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,  at least 97%, at least 98%, or higher, identity to SEQ ID NO: 49). In some embodiments, an RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described  herein)  comprises an RNA  that  includes  the  sequence of SEQ  ID NO: 50 or a variant  thereof  (e.g.,  having at least 70% or more, including, e.g., at least 80%, at least 85%, at least 90%, at least  95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO: 50).  In some  embodiments, an RNA composition described herein (e.g., comprising RNA encoding a variant  described herein) comprises an RNA that includes the sequence of SEQ ID NO: 51 or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  51).    In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 55 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  55). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 56 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 56).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 57).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 58 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  58). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 59 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 59).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 60).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 61 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  61). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 62 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 62).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 63 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 63).  In  some  embodiments,  such  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding a variant described herein) can  further comprise RNA encoding a S protein or an  immungenic fragment thereof of a different strain (e.g., a Wuhan strain). By way of example,  in some embodiments, a second dose of a booster regimen of Regimens #9‐11 as described in  Table 28 above  can  comprise an RNA  composition described herein  (e.g.,  comprising RNA  encoding a variant described herein such as Omicron, for example, in one embodiment RNA  as described in this Example) and a BNT162b2 construct, for example, in 1: 1 weight ratio.    In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary  regimen and a  first dose and a  second dose of a booster  regimen each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described herein such as Omicron, for example, in one embodiment RNA as described in this  Example).  In  some  such  embodiments,  a  second  dose  of  a  booster  regimen may  not  be  necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary  regimen and a  first dose and a  second dose of a booster  regimen each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described herein such as Omicron, for example, in one embodiment RNA as described in this  Example).  In  some  such  embodiments,  a  second  dose  of  a  booster  regimen may  not  be  necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary regimen each comprise a BNT162b2 construct, and a first dose and a second  dose  of  a  booster  regimen  each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising RNA encoding a variant described herein such as Omicron,  for example,  in one  embodiment RNA as described in this Example). In some such embodiments, a second dose of  a booster regimen may not be necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary regimen and a first dose of a booster regimen each comprise a BNT162b2  construct, and a second dose of a booster regimen comprises an RNA composition described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described  herein  such  as  Omicron,  for  example, in one embodiment RNA as described in this Example).     Certain Exemplary Embodiments:  1. A composition or medical preparation comprising RNA encoding an amino acid sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof.    2.  The  composition  or medical  preparation  of  embodiment  1,  wherein  an  immunogenic  fragment of the SARS‐CoV‐2 S protein comprises the S1 subunit of the SARS‐CoV‐2 S protein,  or the receptor binding domain (RBD) of the S1 subunit of the SARS‐CoV‐2 S protein .    3. The composition or medical preparation of embodiments 1 or 2, wherein the amino acid  sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof  is  encoded by a coding sequence which is codon‐optimized and/or the G/C content of which is  increased compared to wild type coding sequence, wherein the codon‐optimization and/or  the  increase  in  the G/C content preferably does not change  the sequence of  the encoded  amino acid sequence.    4. The composition or medical preparation of any one of embodiments 1 to 3, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the nucleotide  sequence of nucleotides 979  to 1584 of SEQ  ID NO: 2, 8 or 9, a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of nucleotides 979 to 1584  of SEQ ID NO: 2, 8 or 9, or a fragment of  the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of  amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to  528 of SEQ ID NO: 1.    5. The composition or medical preparation of any one of embodiments 1 to 4, wherein   (i)  the RNA  encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence  of  nucleotides  111  to  986  of  SEQ  ID  NO:  30,  or  a  fragment  of  the  nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or the nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or   (ii) a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the  amino  acid  sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of  amino acids 20 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to  311 of SEQ ID NO: 29..    6. The composition or medical preparation of any one of embodiments 1 to 5, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the  nucleotide  sequence  of nucleotides  49  to  3819 of  SEQ  ID NO:  2,  8  or  9,  a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of  the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having  at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7.    7. The composition or medical preparation of any one of embodiments 1 to 6, wherein the  amino acid sequence comprising a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or  an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof  comprises a secretory signal peptide.    8. The composition or medical preparation of embodiment 7, wherein  the secretory signal  peptide is fused, preferably N‐terminally, to a SARS‐CoV‐2 S protein, an immunogenic variant  thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic variant  thereof.    9. The composition or medical preparation of embodiment 7 or 8, wherein  (i)  the RNA encoding the secretory signal peptide comprises the nucleotide sequence of  nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ  ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or  9; and/or   (ii)  the secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16  of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80%  identity  to  the amino acid  sequence of amino acids 1  to 16 of SEQ  ID NO: 1, or a  functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.    10. The composition or medical preparation of any one of embodiments 1 to 9, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of SEQ  ID NO: 6, a nucleotide sequence having at  least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID  NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 6; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic  fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ  ID NO: 5.    11. The composition or medical preparation of any one of embodiments 1 to 10, wherein  (i)  the RNA  encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide  sequence of nucleotides 54 to 986 of SEQ  ID NO: 30, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the nucleotide sequence of  nucleotides 54 to 986 of SEQ ID NO: 30; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of  amino acids 1 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311  of SEQ ID NO: 29.    12. The composition or medical preparation of any one of embodiments 1 to 10, wherein the  RNA comprises a modified nucleoside in place of uridine, in particular wherein the modified  nucleoside  is  selected  from  pseudouridine  (ψ),  N1‐methyl‐pseudouridine  (m1ψ),  and  5‐ methyl‐uridine  (m5U),  in  particular  wherein  the  modified  nucleoside  is  N1‐methyl‐ pseudouridine (m1ψ).    13. The composition or medical preparation of any one of embodiments 1 to 12, wherein the  RNA comprises a 5’ cap.    14. The composition or medical preparation of any one of embodiments 1 to 13, wherein the  RNA encoding an amino acid sequence comprising a SARS‐CoV‐2 S protein, an immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant thereof comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of SEQ ID NO: 12.    15. The composition or medical preparation of any one of embodiments 1 to 14, wherein the  RNA encoding an amino acid sequence comprising a SARS‐CoV‐2 S protein, an immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant thereof comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of SEQ ID NO: 13.    16. The composition or medical preparation of any one of embodiments 1 to 15, wherein the  RNA encoding an amino acid sequence comprising a SARS‐CoV‐2 S protein, an immunogenic  variant thereof, or an immunogenic fragment of the SARS‐CoV‐2 S protein or the immunogenic  variant thereof comprises a poly‐A sequence.    17. The composition or medical preparation of embodiment 16, wherein the poly‐A sequence  comprises at least 100 nucleotides.    18. The  composition or medical preparation of embodiment 16 or 17, wherein  the poly‐A  sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14.    19. The composition or medical preparation of any one of embodiments 1 to 18, wherein the  RNA is formulated or is to be formulated as a liquid, a solid, or a combination thereof.    20. The composition or medical preparation of any one of embodiments 1 to 19, wherein the  RNA is formulated or is to be formulated for injection.    21. The composition or medical preparation of any one of embodiments 1 to 20, wherein the  RNA is formulated or is to be formulated for intramuscular administration.    22. The composition or medical preparation of any one of embodiments 1 to 21, wherein the  RNA is formulated or is to be formulated as particles.    23.  The composition or medical preparation of embodiment 22, wherein the particles are  lipid nanoparticles (LNP) or lipoplex (LPX) particles.    24.  The composition or medical preparation of embodiment 23, wherein the LNP particles  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  2‐ [(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐sn‐glycero‐3‐ phosphocholine, and cholesterol.    25. The composition or medical preparation of embodiment 23, wherein  the RNA  lipoplex  particles are obtainable by mixing the RNA with liposomes.    26. The composition or medical preparation of any one of embodiments 1 to 25, wherein the  RNA is mRNA or saRNA.    27. The composition or medical preparation of any one of embodiments 1 to 26, which is a  pharmaceutical composition.    28. The composition or medical preparation of any one of embodiments 1 to 27, which is a  vaccine.    29.  The  composition  or  medical  preparation  of  embodiment  27  or  28,  wherein  the  pharmaceutical  composition  further  comprises  one  or more  pharmaceutically  acceptable  carriers, diluents and/or excipients.    30. The composition or medical preparation of any one of embodiments 1 to 26, which is a kit.    31.  The  composition  or  medical  preparation  of  embodiment  30,  wherein  the  RNA  and  optionally the particle forming components are in separate vials.     32.  The  composition or medical preparation of  embodiment  30 or  31,  further  comprising  instructions  for  use  of  the  composition  or medical  preparation  for  inducing  an  immune  response against coronavirus in a subject.    33.  The  composition  or  medical  preparation  of  any  one  of  embodiments  1  to  32  for  pharmaceutical use.    34. The composition or medical preparation of embodiment 33, wherein the pharmaceutical  use comprises inducing an immune response against coronavirus in a subject.    35.  The  composition  or  medical  preparation  of  embodiment  33  or  34,  wherein  the  pharmaceutical  use  comprises  a  therapeutic  or  prophylactic  treatment  of  a  coronavirus  infection.    36. The composition or medical preparation of any one of embodiments 1 to 35, which is for  administration to a human.    37. The composition or medical preparation of any one of embodiments 32 to 36, wherein the  coronavirus is a betacoronavirus.    38. The composition or medical preparation of any one of embodiments 32 to 37, wherein the  coronavirus is a sarbecovirus.    39. The composition or medical preparation of any one of embodiments 32 to 38, wherein the  coronavirus is SARS‐CoV‐2.    40. A method of  inducing an  immune response against coronavirus  in a subject comprising  administering to the subject a composition comprising RNA encoding an amino acid sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof.    41. The method of embodiment 40, wherein an immunogenic fragment of the SARS‐CoV‐2 S  protein comprises the S1 subunit of the SARS‐CoV‐2 S protein, or the receptor binding domain  (RBD) of the S1 subunit of the SARS‐CoV‐2 S protein.    42. The method of  any one of embodiments 40 or 41, wherein  the amino  acid  sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof  is encoded by a  coding  sequence which  is  codon‐optimized  and/or  the G/C  content  of which  is  increased  compared to wild type coding sequence, wherein the codon‐optimization and/or the increase  in  the G/C  content  preferably  does  not  change  the  sequence  of  the  encoded  amino  acid  sequence.    43. The method of any one of embodiments 40 to 42, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the nucleotide  sequence of nucleotides 979  to 1584 of SEQ  ID NO: 2, 8 or 9, a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of nucleotides 979 to 1584  of SEQ ID NO: 2, 8 or 9, or a fragment of  the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of  amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to  528 of SEQ ID NO: 1.    44. The method of any one of embodiments 40 to 43, wherein   (i)  the RNA  encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence  of  nucleotides  111  to  986  of  SEQ  ID  NO:  30,  or  a  fragment  of  the  nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or the nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or   (ii) a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the  amino  acid  sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of  amino acids 20 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to  311 of SEQ ID NO: 29.    45. The method of any one of embodiments 40 to 44, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  the  nucleotide  sequence  of nucleotides  49  to  3819 of  SEQ  ID NO:  2,  8  or  9,  a  nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of  the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid  sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having  at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of  amino acids 17 to 1273 of SEQ ID NO: 1 or 7.    46.  The method of  any one of embodiments 40  to  45, wherein  the  amino  acid  sequence  comprising  a  SARS‐CoV‐2  S  protein,  an  immunogenic  variant  thereof,  or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises  a  secretory signal peptide.    47. The method of embodiment 46, wherein the secretory signal peptide is fused, preferably  N‐terminally, to a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic  fragment of the SARS‐CoV‐2 S protein or the immunogenic variant thereof.    48. The method of embodiment 46 or 47, wherein  (i)  the RNA encoding the secretory signal peptide comprises the nucleotide sequence of  nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48  of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ  ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%,  85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or  9; and/or   (ii)  the secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16  of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80%  identity  to  the amino acid  sequence of amino acids 1  to 16 of SEQ  ID NO: 1, or a  functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the  amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to  the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.    49. The method of any one of embodiments 40 to 48, wherein   (i)  the RNA encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of SEQ  ID NO: 6, a nucleotide sequence having at  least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID  NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide  sequence of SEQ ID NO: 6; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%,  90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic  fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ  ID NO: 5.    50. The method of any one of embodiments 40 to 49, wherein  (i)  the RNA  encoding  a  SARS‐CoV‐2  S protein,  an  immunogenic  variant  thereof, or  an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide  sequence of nucleotides 54 to 986 of SEQ  ID NO: 30, or the nucleotide sequence having at  least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%  identity  to  the nucleotide sequence of  nucleotides 54 to 986 of SEQ ID NO: 30; and/or   (ii)  a SARS‐CoV‐2 S protein, an immunogenic variant thereof, or an immunogenic fragment  of the SARS‐CoV‐2 S protein or the  immunogenic variant thereof comprises the amino acid  sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least  99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino  acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of  amino acids 1 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311  of SEQ ID NO: 29.    51. The method of any one of embodiments 40 to 49, wherein the RNA comprises a modified  nucleoside in place of uridine, in particular wherein the modified nucleoside is selected from  pseudouridine  (ψ),  N1‐methyl‐pseudouridine  (m1ψ),  and  5‐methyl‐uridine  (m5U),  in  particular wherein the modified nucleoside is N1‐methyl‐pseudouridine (m1ψ).    52. The method of any one of embodiments 40 to 51, wherein the RNA comprises a cap.    53. The method of any one of embodiments 40 to 52, wherein the RNA encoding an amino  acid  sequence  comprising a SARS‐CoV‐2 S protein, an  immunogenic  variant  thereof, or an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises a 5’ UTR comprising  the nucleotide sequence of SEQ  ID NO: 12, or a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ ID NO: 12.    54. The method of any one of embodiments 40 to 53, wherein the RNA encoding an amino  acid  sequence  comprising a SARS‐CoV‐2 S protein, an  immunogenic  variant  thereof, or an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises a 3’ UTR comprising  the nucleotide sequence of SEQ  ID NO: 13, or a nucleotide  sequence  having  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identity  to  the  nucleotide sequence of SEQ ID NO: 13.    55. The method of any one of embodiments 40 to 54, wherein the RNA encoding an amino  acid  sequence  comprising a SARS‐CoV‐2 S protein, an  immunogenic  variant  thereof, or an  immunogenic  fragment  of  the  SARS‐CoV‐2  S  protein  or  the  immunogenic  variant  thereof  comprises a poly‐A sequence.    56.  The method of  embodiment 55, wherein  the poly‐A  sequence  comprises  at  least 100  nucleotides.    57. The method of embodiment 55 or 56, wherein the poly‐A sequence comprises or consists  of the nucleotide sequence of SEQ ID NO: 14.    58. The method of any one of embodiments 40 to 57, wherein the RNA  is  formulated as a  liquid, a solid, or a combination thereof.    59. The method of any one of embodiments 40 to 58, wherein the RNA  is administered by  injection.    60. The method of any one of embodiments 40 to 59, wherein the RNA  is administered by  intramuscular administration.    61. The method of any one of embodiments 40  to 60, wherein  the RNA  is  formulated  as  particles.    62. The method of embodiment 61, wherein  the particles are  lipid nanoparticles  (LNP) or  lipoplex (LPX) particles.    63.  The  method  of  embodiment  62,  wherein  the  LNP  particles  comprise  ((4‐ hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐hexyldecanoate),  2‐[(polyethylene  glycol)‐ 2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐sn‐glycero‐3‐phosphocholine,  and  cholesterol.    64.  The method  of  any  one  of  embodiment  62,  wherein  the  RNA  lipoplex  particles  are  obtainable by mixing the RNA with liposomes.    65. The composition or medical preparation of any one of embodiments 40 to 64, wherein the  RNA is mRNA or saRNA.    66. The method of any one of embodiments 40 to 65, which is a method for vaccination against  coronavirus.    67. The method of any one of embodiments 40 to 66, which is a method for therapeutic or  prophylactic treatment of a coronavirus infection.    68. The method of any one of embodiments 40 to 67, wherein the subject is a human.    69.  The  method  of  any  one  of  embodiments  40  to  68,  wherein  the  coronavirus  is  a  betacoronavirus.    70.  The  method  of  any  one  of  embodiments  40  to  69,  wherein  the  coronavirus  is  a  sarbecovirus.    71. The method of any one of embodiments 40 to 70, wherein the coronavirus is SARS‐CoV‐2.    72.  The  method  of  any  one  of  embodiments  40  to  71,  wherein  the  composition  is  a  composition of any one of embodiments 1 to 39.    73. A composition or medical preparation of any one of embodiments 1  to 39  for use  in a  method of any one of embodiments 40 to 72.    74.  An immunogenic composition comprising: a lipid nanoparticle (LNP) comprising an RNA,  wherein the RNA encodes  the polypeptide of SEQ  ID NO: 49 and comprises  the nucleotide  sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 50, and wherein the RNA comprises:  (a) modified uridines;   (b) a 5’ cap; and  wherein  the  LNP  comprises  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    75.  The  immunogenic  composition  of  embodiment  74, wherein  the  nucleotide  sequence  includes modified uridines in place of all uridines.    76. The immunogenic of embodiment 74 or 75, wherein the modified uridines are each N1‐ methyl‐pseudouridine.    77. The  immunogenic composition of any one of embodiments 74  to 76, wherein  the RNA  further comprises at least one, at least two, or all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     78. The immunogenic composition of embodiment 77, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    79.  The  immunogenic  composition  of  embodiment  77,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    80. The  immunogenic composition of any one of embodiments 76  to 79, wherein  the RNA  comprises SEQ ID NO: 51.    81. An  immunogenic composition comprising a  lipid nanoparticle (LNP) comprising an RNA,  wherein the RNA encodes  the polypeptide of SEQ  ID NO: 55 and comprises  the nucleotide  sequence of SEQ ID NO: 56 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 56, and wherein the RNA comprises:  (a) modified uridines;   (b) a 5’ cap; and  wherein  the  LNP  comprises  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    82.  The  immunogenic  composition  of  embodiment  81, wherein  the  nucleotide  sequence  includes modified uridines in place of all uridines.    83. The immunogenic of embodiment 81 or 82, wherein the modified uridines are each N1‐ methyl‐pseudouridine.    84. The  immunogenic composition of any one of embodiments 81  to 83, wherein  the RNA  further comprises at least one, at least two, or all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     85. The immunogenic composition of embodiment 84, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    86.  The  immunogenic  composition  of  embodiment  85,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    87. The  immunogenic composition of any one of embodiments 81  to 86, wherein  the RNA  comprises SEQ ID NO: 57.    88. An immunogenic composition comprising a a lipid nanoparticle (LNP) comprising an RNA,  wherein the RNA encodes  the polypeptide of SEQ  ID NO: 58 and comprises  the nucleotide  sequence of SEQ ID NO: 59 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 59, and wherein the RNA comprises:  (a) modified uridines;   (b) a 5’ cap; and  wherein  the  LNP  comprises  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    89.  The  immunogenic  composition  of  embodiment  88, wherein  the  nucleotide  sequence  includes modified uridines in place of all uridines.    90. The immunogenic of embodiment 88 or 89, wherein the modified uridines are each N1‐ methyl‐pseudouridine.    91. The  immunogenic composition of any one of embodiments 88  to 90, wherein  the RNA  further comprises at least one, at least two, or all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     92. The immunogenic composition of embodiment 91, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    93.  The  immunogenic  composition  of  embodiment  92,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    94. The  immunogenic composition of any one of embodiments 88  to 93, wherein  the RNA  comprises SEQ ID NO: 60.    95. An  immunogenic composition comprising a  lipid nanoparticle (LNP) comprising an RNA,  wherein the RNA encodes  the polypeptide of SEQ  ID NO: 61 and comprises  the nucleotide  sequence of SEQ ID NO: 62 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 62, and wherein the RNA comprises:  (a) modified uridines;   (b) a 5’ cap; and  wherein  the  LNP  comprises  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    96.  The  immunogenic  composition  of  embodiment  95, wherein  the  nucleotide  sequence  includes modified uridines in place of all uridines.    97. The immunogenic of embodiment 95 or 96, wherein the modified uridines are each N1‐ methyl‐pseudouridine.    98. The  immunogenic composition of any one of embodiments 95  to 97, wherein  the RNA  further comprises at least one, at least two, or all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     99. The immunogenic composition of embodiment 98, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    100.  The  immunogenic  composition  of  embodiment  99,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    101. The immunogenic composition of any one of embodiments 95 to 100, wherein the RNA  comprises SEQ ID NO: 63.    102. An immunogenic composition comprising a first RNA and a second RNA, wherein:  the first RNA encodes the polypeptide of SEQ ID NO: 7 and comprises the nucleotide  sequence of SEQ ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 9, and  the  second  RNA  encodes  the  polypeptide  of  SEQ  ID  NO:  49  and  comprises  the  nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at  85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least  97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 50, and   wherein each of the first RNA and the second RNA comprise:  (a) modified uridines; and   (b) a 5’ cap, and  wherein the first RNA and the second RNA are formulated in lipid nanoparticles (LNPs),  wherein  the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    103. The immunogenic composition of embodiment 102, wherein the first RNA and the second  RNA are formulated in the same lipid nanoparticles.    104. The immunogenic composition of embodiment 102, wherein the first RNA and the second  RNA are formulated in seperate lipid nanoparticles.    105. The immunogenic composition of any one of embodiments 102 to 104, wherein each of  the first RNA and the second RNA include modified uridines in place of all uridines.    106. The immunogenic of any one of embodiments 102 to 105, wherein the modified uridines  are each N1‐methyl‐pseudouridine.    107. The immunogenic composition of any one of embodiments 102 to 106, wherein the first  RNA and the second RNA each indepedently comprise at least one, at least two, or all of the  following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     108. The immunogenic composition of embodiment 107, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    109.  The  immunogenic  composition  of  embodiment  107,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    110. The immunogenic composition of any one of embodiments 102 to 109, wherein the first  RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 51.    111. An immunogenic composition comprising a first RNA and a second RNA, wherein:  the first RNA encodes the polypeptide of SEQ ID NO: 7 and comprises the nucleotide  sequence of SEQ ID NO: 9 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 9, and  the second RNA encodes the polypeptide of SEQ ID NO: 55, 58, or 61 and comprises  the nucleotide sequence of SEQ ID NO: 56, 59, or 62, or a nucleotide sequence that is at least  80% (e.g., at 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least  95%, at least 97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 56, 59, or 62, and   wherein each of the first RNA and the second RNA comprise:  (a) modified uridines; and   (b) a 5’ cap, and  wherein the first RNA and the second RNA are formulated in lipid nanoparticles (LNPs),  wherein  the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    112. The immunogenic composition of embodiment 111, wherein the first RNA and the second  RNA are formulated in separate lipid nanoparticles.    113. The immunogenic composition of embodiment 111, wherein the first RNA and the second  RNA are formulated in the same lipid nanoparticles.    114. The immunogenic composition of any one of embodiments 111 to 113, wherein the first  RNA and the second RNA each include modified uridines in place of all uridines.    115. The  immunogenic  composition of  any one of  embodiments 111  to 114, wherein  the  modified uridines are each N1‐methyl‐pseudouridine.    116. The immunogenic composition of any one of embodiments 111 to 115, wherein the first  RNA and the second RNA each independently further comprise at least one, at least two, or  all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     117. The immunogenic composition of any one of embodiments 111 to 116, wherein the  poly‐A sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides,  wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker  sequence.    118.  The  immunogenic  composition  of  embodiment  117,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.      119. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 56.    120. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 59.    121. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 9 and the second RNA comprises SEQ ID NO: 62.    122. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 57.    123. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 60.    124. The immunogenic composition of any one of embodiments 111 to 118, wherein the first  RNA comprises SEQ ID NO: 20 and the second RNA comprises SEQ ID NO: 63.    125. An immunogenic composition comprising a first RNA and a second RNA, wherein:  the first RNA encodes the polypeptide of SEQ ID NO: 58 and comprises the nucleotide  sequence of SEQ ID NO: 59 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 59, and  the second RNA encodes the polypeptide of SEQ ID NO: 49, 55, or 61 and comprises  the nucleotide sequence of SEQ ID NO: 50, 56, or 62, or a nucleotide sequence that is at least  80% (e.g., at 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least  95%, at least 97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 50, 56, or 62, and   wherein each of the first RNA and the second RNA comprise:  (a) modified uridines; and   (b) a 5’ cap, and  wherein the first RNA and the second RNA are formulated in lipid nanoparticles (LNPs),  wherein  the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    126. The immunogenic composition of embodiment 125, wherein the first RNA and the second  RNA are formulated in separate lipid nanoparticles.    127. The immunogenic composition of embodiment 125, wherein the first RNA and the second  RNA are formulated in the same lipid nanoparticles.    128. The immunogenic composition of any one of embodiments 125 to 127, wherein the first  RNA and the second RNA each include modified uridines in place of all uridines.    129. The immunogenic of any one of embodiments 125 to 128, wherein the modified uridines  are each N1‐methyl‐pseudouridine.    130. The immunogenic composition of any one of embodiments 125 to 129, wherein the first  RNA and the second RNA each independently further comprise at least one, at least two, or  all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     131. The immunogenic composition of embodiment 130, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    132.  The  immunogenic  composition  of  embodiment  130,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    133. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 59 and the second RNA comprises SEQ ID NO: 50.    134. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 59 and the second RNA comprises SEQ ID NO: 56.    135. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 59 and the second RNA comprises SEQ ID NO: 62.    136. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 51.    137. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 57.    138. The immunogenic composition of any one of embodiments 125 to 132, wherein the first  RNA comprises SEQ ID NO: 60 and the second RNA comprises SEQ ID NO: 63.    139. An immunogenic composition comprising a first RNA and a second RNA, wherein:  the first RNA encodes the polypeptide of SEQ ID NO: 49 and comprises the nucleotide  sequence of SEQ ID NO: 50 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 50, and  the second RNA encodes the polypeptide of SEQ ID NO: 55 or 61 and comprises the  nucleotide sequence of SEQ  ID NO: 56 or 62, or a nucleotide sequence that  is at  least 80%  (e.g., at 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,  at least 97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 56 or 62, and   wherein each of the first RNA and the second RNA comprise:  (a) modified uridines; and   (b) a 5’ cap, and  wherein the first RNA and the second RNA are formulated in lipid nanoparticles (LNPs),  wherein  the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    140. The immunogenic composition of embodiment 139, wherein the first RNA and the second  RNA are formulated in separate lipid nanoparticles.    141. The immunogenic composition of embodiment 139, wherein the first RNA and the second  RNA are formulated in the same lipid nanoparticles.    142. The immunogenic composition of any one of embodiments 139 to 141, wherein the first  RNA and the second RNA each include modified uridines in place of all uridines.    143. The immunogenic of any one of embodiments 139 to 142, wherein the modified uridines  are each N1‐methyl‐pseudouridine.    144. The immunogenic composition of any one of embodiments 139 to 143, wherein the first  RNA and the second RNA further each independently further comprise at least one, at least  two, or all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     145. The immunogenic composition of embodiment 144, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    146.  The  immunogenic  composition  of  embodiment  144,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    147. The immunogenic composition of any one of embodiments 139 to 146, wherein the first  RNA comprises SEQ ID NO: 50 and the second RNA comprises SEQ ID NO: 56.    148. The immunogenic composition of any one of embodiments 139 to 146, wherein the first  RNA comprises SEQ ID NO: 50 and the second RNA comprises SEQ ID NO: 62.    149. The immunogenic composition of any one of embodiments 139 to 146, wherein the first  RNA comprises SEQ ID NO: 51 and the second RNA comprises SEQ ID NO: 57.    150. The immunogenic composition of any one of embodiments 139 to 146, wherein the first  RNA comprises SEQ ID NO: 51 and the second RNA comprises SEQ ID NO: 63.    151. An immunogenic composition comprising a first RNA and a second RNA, wherein:  the first RNA encodes the polypeptide of SEQ ID NO: 55 and comprises the nucleotide  sequence of SEQ ID NO: 56 or a nucleotide sequence that is at least 80% (e.g., at 85%, at least  90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97%, at least  98%, or 99% or higher) identical to SEQ ID NO: 56, and  the  second  RNA  encodes  the  polypeptide  of  SEQ  ID  NO:  61  and  comprises  the  nucleotide sequence of SEQ ID NO: 62, or a nucleotide sequence that is at least 80% (e.g., at  85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least  97%, at least 98%, or 99% or higher) identical to SEQ ID NO: 62, and   wherein each of the first RNA and the second RNA comprise:  (a) modified uridines; and   (b) a 5’ cap, and  wherein the first RNA and the second RNA are formulated in lipid nanoparticles (LNPs),  wherein  the  LNPs  comprise  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐diyl)bis(2‐ hexyldecanoate),  2‐[(polyethylene  glycol)‐2000]‐N,N‐ditetradecylacetamide,  1,2‐Distearoyl‐ sn‐glycero‐3‐phosphocholine, and cholesterol.    152. The immunogenic composition of embodiment 151, wherein the first RNA and the second  RNA are formulated in separate lipid nanoparticles.    153. The immunogenic composition of embodiment 151, wherein the first RNA and the second  RNA are formulated in the same lipid nanoparticles.    154. The immunogenic composition of any one of embodiments 151 to 153, wherein the first  RNA and the second RNA each include modified uridines in place of all uridines.    155. The immunogenic of any one of embodiments 151 to 154, wherein the modified uridines  are each N1‐methyl‐pseudouridine.    156. The immunogenic composition of any one of embodiments 151 to 155, wherein the first  RNA and the second RNA each independently further comprise at least one, at least two, or  all of the following features:  a 5‘ untranslated region (UTR) comprising SEQ ID NO: 12;  a 3’ untranslated region (UTR) comprising SEQ ID NO: 13; and  a poly‐A sequence of at least 100 A nucleotides.     157. The immunogenic composition of embodiment 156, wherein the poly‐A sequence  comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30  adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.    158.  The  immunogenic  composition  of  embodiment  156,  wherein  the  poly‐A  sequence  comprises SEQ ID NO: 14.    159. The immunogenic composition of any one of embodiments 151 to 158, wherein the first  RNA comprises SEQ ID NO: 57 and the second RNA comprises SEQ ID NO: 63.    160. The immunogenic composition of any one of embodiments 74 to 159, wherein the 5’‐cap  is or comprises m2 7,3’‐OGppp(m1 2’‐O)ApG.    161. The immunogenic composition of any one of embodiments 74 to 160, wherein the LNP  comprises  about  40  to  about  50 mole  percent  ((4‐hydroxybutyl)azanediyl)bis(hexane‐6,1‐ diyl)bis(2‐hexyldecanoate), about 35 to about 45 mole percent cholesterol, about 5 to about  15 mole percent 1,2‐Distearoyl‐sn‐glycero‐3‐phosphocholine, and about 1 to about 10 mole  percent 2‐[(polyethylene glycol)‐2000]‐N,N‐ditetradecylacetamide.    162.  The  immunogenic  composition  of  any  one  of  embodiments  74  to  161, wherein  the  composition comprises a plurality of LNPs, wherein the average diameter of the plurality of  LNPs is about 30 nm to about 200 nm or about 60 nm to about 120 nm (e.g., as determined  by dynamic light scattering measurements).    163. A method of eliciting an immune response against SARS‐CoV‐2 comprising administering  the immunogenic composition of any one of embodiments 74 to 162.    164. The method of embodiment 163, wherein  the  immune response  is elicited against an  Omicron variant of SARS‐CoV‐2.     165. The method of embodiment 163, wherein the immune response is elicited against a Beta  variant of SARS‐CoV‐2.     166. The method of embodiment 163, wherein  the  immune response  is elicited against an  Alpha variant of SARS‐CoV‐2.     167. The method of embodiment 163, wherein the immune response is elicited against a Delta  variant of SARS‐CoV‐2.     168. The method of embodiment 163, wherein  the  immune  response  is elicited  against  a  Wuhan strain, an Omicron variant, a Beta variant, an Alpha variant, and a Delta variant of  SARS‐CoV‐2.       Citation of documents and studies referenced herein is not intended as an admission that any  of the foregoing is pertinent prior art. All statements as to the contents of these documents  are based on the information available to the applicants and do not constitute any admission  as to the correctness of the contents of these documents.  The following description is presented to enable a person of ordinary skill in the art to make  and  use  the  various  embodiments.  Descriptions  of  specific  devices,  techniques,  and  applications are provided only as examples. Various modifications to the examples described  herein will be readily apparent to those of ordinary skill in the art, and the general principles  defined herein may be applied to other examples and applications without departing from the  spirit and scope of the various embodiments. Thus, the various embodiments are not intended  to be limited to the examples described herein and shown, but are to be accorded the scope  consistent with the claims.          
Examples    Example 1: Immunogenicity study of BNT162b3 variants BNT162b3c and BNT162b3d  To get an idea about the potential potency of transmembrane‐anchored RBD‐based vaccine  antigens  (Schematic  in  Figure  6;  BNT162b3c  (1)  and  BNT162b3d  (2)),  BALB/c mice  were  immunized IM once with 4 µg LNP‐C12 formulated mRNA or with buffer as control. The non‐ clinical  LNP‐C12  formulated  mRNAs  were  used  as  surrogate  for  the  BNT162b3  variants  BNT162b3c  and BNT162b3d.  The  immunogenicity of  the RNA  vaccine was  investigated by  focusing on the antibody immune response.  ELISA data 6, 14 and 21 d after the first immunization show an early, dose‐dependent immune  activation against the S1 protein and the receptor binding domain (Figure 7). Sera obtained 6,  14 and 21 d after immunization show high SARS‐CoV‐2 pseudovirus neutralization, correlating  with the increase of IgG antibody titers (Figure 8).    Example 2: Neutralization of SARS‐CoV‐2 Omicron lineage (a.k.a. B.1.1.529) pseudovirus by  BNT162b2 vaccine‐elicited human sera  Materials and Methods:  A recombinant replication‐deficient VSV vector that encodes green fluorescent protein (GFP)  and luciferase (Luc) instead of the VSV‐glycoprotein (VSV‐G) was pseudotyped with Wuhan‐ Hu‐1 isolate SARS‐CoV‐2 spike (S) (GenBank: QHD43416.1), and a variant spike harbouring the  mutations  found  in  the  S protein of  the Omicron  (B.1.1.529)  lineage  (A67V,  ∆69‐70, T95I,  G142D, ∆143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S,  S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K,  P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) according to published pseudotyping  protocols. In brief, HEK293T/17 monolayers transfected to express the respective SARS‐CoV‐ 2  S  truncated  of  the  C‐terminal  cytoplasmic  19  amino  acids  (SARS‐CoV‐2‐S(CΔ19))  were  inoculated with VSVΔG‐GFP/Luc vector. After incubation for 2 h at 37 °C, the  inoculum was  removed, and  cells were washed with PBS before medium  supplemented with anti‐VSV‐G  antibody (clone 8G5F11, Kerafast) was added to neutralise residual input virus. VSV‐SARS‐CoV‐ 2 pseudovirus‐containing medium was collected 20 h after  inoculation, 0.2‐μm‐filtered and  stored at −80 °C.  For pseudovirus neutralisation assays, 40,000 Vero 76  cells were  seeded per 96‐well. Sera  were serially diluted 1:2 in culture medium starting with a 1:10 dilution (dilution range of 1:10  to  1:10,240).  VSV‐SARS‐CoV‐2‐S  pseudoparticles  were  diluted  in  culture  medium  for  a  fluorescent focus unit (ffu) count in the assay of ~200 TU in the assay. Serum dilutions were  mixed 1:1 with pseudovirus for 30 minutes at room temperature prior to addition to Vero 76  cell monolayers in 96‐well plates and incubation at 37 °C for 16‐24 hours. Supernatants were  removed,  and  the  cells were  lysed with  luciferase  reagent  (Promega).  Luminescence was  recorded, and neutralisation  titers were  calculated as  the  reciprocal of  the highest  serum  dilution that still resulted in 50% reduction in luminescence. Results were reported as GMT of  duplicates. If no neutralization was observed, an arbitrary titer value of 5 (half of the limit of  detection [LOD]) was reported.  Sera (N=19‐20) were collected from subjects 21 days after receiving the second 30 µg dose or  one month after receiving the third 30 µg dose of BNT162b2. Each serum was tested for its  neutralizing antibody  titer against wild‐type SARS‐CoV‐2 Wuhan Hu‐1 and Omicron  lineage  (B.1.1.529)  spike  protein  pseudotyped  VSV  by  a  50%  neutralization  assay  (pVNT50).  The  Omicron‐strain spike protein used in the neutralization assay carried the following amino acid  changes  compared  to  the Wuhan  reference: A67V,  ∆69‐70,  T95I, G142D,  ∆143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A,  Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y,  N856K, Q954H, N969K, L981F.  BNT162b2‐immune  sera  generated  at  21  days  after  the  second  dose  displayed  effective  neutralization of the SARS‐CoV‐2 Wuhan Hu‐1 pseudotyped reference. However, more than a  25‐fold  reduction  in  neutralization  titers  against  the Omicron  variant was observed when  compared to the Wuhan reference (geometric mean titer [GMT] of 6 vs. 155). Importantly, the  third dose significantly  increased the neutralizing antibody titers against the Omicron strain  pseudovirus by 25‐fold. Hence, neutralization titers against the Omicron variant pseudovirus  after three doses of BNT162b2 were comparable to the neutralization titers against the wild‐ type strain observed in sera from individuals who received two doses of BNT162b2 (GMT of  154 vs. 155).  Example 3: Additional data for neutralization of SARS‐CoV‐2 Omicron lineage (a.k.a.  B.1.1.529) pseudovirus by BNT162b2 vaccine‐elicited human sera  Further  to  the  study and data as described  in Example 2, a  longitudinal analysis of  neutralizing  titers was  also performed  in  an  independent  smaller  subset of  subjects.  Sera  drawn at 21 days after dose 2 exhibited a 19.6‐fold reduction  in GMT against the Omicron  variant compared  to  the Wuhan  reference pseudovirus  (Fig. 12; GMT of 6 vs. 118). Serum  obtained  from  study participants  just prior  to  receiving  the  third dose of BNT162b2  (at  a  median 251 days following dose 2) had considerably reduced neutralizing titers against the  Wuhan pseudovirus  (GMT of 14) while  the Omicron‐specific  titers were below  the  limit of  detection. The third dose of BNT162b2 resulted in a significant increase in neutralizing titers  against the Wuhan pseudovirus (GMT of 254) and a >26.6‐fold increase in neutralizing titers  against Omicron at 1 month after dose 3 compared to titers at 21 days after dose 2 (GMT of  160 vs. 6). In all 9 subjects reduced but effective neutralization of Omicron was observed up  to 3 months after the third dose (3.2‐fold reduction compared to 1 month after dose 3; GMT  of 50 vs. 160), whereas Wuhan‐specific neutralizing GMTs remained stable.   In summary, a third dose of BNT162b2 boosts Omicron neutralization capability to a  level similar to the one observed after two doses against the Wuhan pseudovirus. Thus, the  data indicate that providing a third dose of BNT162b2 can improve protection against infection  with the Omicron variant.  Example 4: Neutralization of other SARS‐CoV‐2 lineage pseudovirus by BNT162b2 vaccine‐ elicited human sera  As described  in Example 2 and Example 3, each  serum was also  tested  for  its neutralizing  antibody  titer  against  Beta  and  Delta  lineage  spike  protein  pseudotyped  VSV  by  a  50%  neutralization assay (pVNT50) (data not shown).    A recombinant replication‐deficient VSV vector that encodes green fluorescent protein (GFP)  and luciferase (Luc) instead of the VSV‐glycoprotein (VSV‐G) was pseudotyped with Wuhan‐ Hu‐1 isolate SARS‐CoV‐2 spike (S) (GenBank: QHD43416.1), and a variant spike harbouring the  mutations found in the S protein of the Beta lineage (mutations: L18F, D80A, D215G, R246I,  Δ242–244, K417N, E484K, N501Y, D614G, A701V), and  the Delta  lineage  (mutations: T19R,  G142D, Δ157/158, K417N, L452R, T478K, D614G, P681R, D950N, K986P, V987P), according to  published pseudotyping protocols.   For  sera  collected  21  days  after  a  second  dose  of  BNT162b2,  PVNT50  was  reduced  by  approximately 6.7‐fold (GMT of 24 vs 155) for the Beta variant and approximately 2.2‐fold for  the Delta variant (GMT of 73 vs 155) as compared to the Wuhan variant, but were significantly  higher than the neutralization response against the delta variant. The third dose of BNT162b2  also  increased neutralizing activity against Beta and Delta pseudoviruses, with GMTs of 279  and 413, respectively.  Example 5: T cell epitope conservation in the Omicron Spike variant In addition to humoral immunity, T‐cell mediated immunity is another layer of defense, in  particular for preventing severe COVID‐19. Previous observations that efficacy against  disease is already established about 12 days after the first dose of BNT162b2 before the  second dose has been administered and prior to the onset of high neutralizing titers further  highlights the potential protective role of the T cell response. Prior reports have shown that  CD8 T cell responses in individuals vaccinated with BNT162b2 are polyepitopic.   To assess the risk of immune evasion of CD8+  T cell responses by Omicron, a set of HLA class  I restricted T cell epitopes from the Wuhan spike protein sequence that were reported in the  Immune Epitope Database to be immunogenic (IEDB, n=244) were investigated (the  procedure used to identify these epitopes is described in the below paragraph). Despite the  multitude of mutations in the Omicron spike protein, 85.25% (n=208) of the described  epitopes were not impacted on the amino acid sequence level, indicating that the targets of  the vast majority of T cell responses elicited by BNT162b2 may still be conserved in the  Omicron variant (Fig. 13). Early laboratory studies confirm that CD8+ T cell recognition of  Omicron epitopes are preserved in COVID‐19 recovered individuals exposed early in the  pandemic and that the Omicron VOC has not evolved extensive T‐cell escape mutations at  this time.    To estimate the rate of nonsynonymous mutation in T cell epitopes in the spike glycoprotein,  the Immune Epitope Database ( was used to obtain epitopes 
Figure imgf000610_0001
confirmed for T cell reactivity in experimental assays. The database was filtered using the  following criteria: Organism: SARS‐COV2; Antigen: Spike glycoprotein; Positive Assay; No B  cell assays; No MHC assays; MHC Restriction Type: Class I; Host: Homo sapiens (human). The  resulting table was filtered by removing epitopes that were “deduced from a reactive  overlapping peptide pool”, as well as epitopes longer than 14 amino acids in order to restrict  the dataset to confirmed minimal epitopes only. Of the 251 unique epitope sequences  obtained in this approach, 244 were found in the Wuhan strain Spike glycoprotein. Of these,  36 epitopes (14.75%) included a position reported to be mutated in Omicron by the  sequence analysis disclosed herein. Results are summarized in Figure 10.  Also shown are the  numbers of predicted MHC‐I epitopes mutated in each of the Alpha, Beta, Gamma, Delta  SARS‐CoV‐2 variants.  Figure 13 depicts the locations of the T cell epitopes within the Spike  Protein, and indicates which epitopes are conserved or mutated in the Spike protein from  the Omicron variant.  Example 6: Exemplary Dosing Regimens  In some embodiments, compositions and methods disclosed herein can be used in accordance  with an exemplary vaccination regimen as illustrated in Figure 14.      Primary Dosing Regimens  In some embodiments, subjects are administered a primary dosing regimen.  A primary dosing  regimen can comprise one or more doses. For example,  in  some embodiments, a primary  dosing regimen comprises a single dose (PD1). In some embodiments a primary dosing regimen  comprises a first dose (PD1) and a second dose (PD2). In some embodiments, a primary dosing  regimen comprises a first dose, a second dose, and a third dose (PD3). In some embodiments,  a primary dosing regimen comprises a first dose, a second dose, a third dose, and one or more  additional doses (PDn) of any one of the pharmaceutical compositions described herein.    In  some  embodiments,  PD1  comprises  administering  1  to  100  ug  of  RNA.  In  some  embodiments, PD1 comprises administering 1 to 60 ug of RNA    In some embodiments, PD1  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD1  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD1 comprises administering about  3 ug of RNA.  In some embodiments, PD1 comprises administering about 5 ug of RNA.  In some  embodiments, PD1 comprises administering about 10 ug of RNA.  In some embodiments, PD1  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD1  comprises  administering about 20 ug of RNA.  In some embodiments, PD1 comprises administering about  30 ug of RNA.  In some embodiments, PD1 comprises administering about 50 ug of RNA.  In  some embodiments, PD1 comprises administering about 60 ug of RNA.    In  some  embodiments,  PD2  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PD2 comprises administering 1 to 60 ug of RNA.    In some embodiments, PD2  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD2  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD2 comprises administering about  3  ug.    In  some  embodiments,  PD2  comprises  administering  about  5  ug  of  RNA.    In  some  embodiments, PD2 comprises administering about 10 ug of RNA.  In some embodiments, PD2  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD2  comprises  administering about 20 ug RNA.  In some embodiments, PD2 comprises administering about  30 ug of RNA.  In some embodiments, PD2 comprises administering about 50 ug of RNA.  In  some embodiments, PD2 comprises administering about 60 ug of RNA.    In  some  embodiments,  PD3  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PD3 comprises administering 1 to 60 ug of RNA.    In some embodiments, PD3  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PD 3  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PD3 comprises administering about  3 ug of RNA.  In some embodiments, PD3 comprises administering about 5 ug of RNA.  In some  embodiments, PD3 comprises administering about 10 ug of RNA.  In some embodiments, PD3  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PD3  comprises  administering about 20 ug of RNA.  In some embodiments, PD3 comprises administering about  30 ug of RNA.  In some embodiments, PD3 comprises administering about 50 ug of RNA.  In  some embodiments, PD3 comprises administering about 60 ug of RNA.    In  some  embodiments,  PDn  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, PDn comprises administering 1 to 60 ug of RNA.    In some embodiments, PDn  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  PDn  comprises  administering 1 to 30 ug of RNA.  In some embodiments, PDn comprises administering about  3 ug of RNA.  In some embodiments, PDn comprises administering about 5 ug of RNA.  In some  embodiments, PDn comprises administering about 10 ug of RNA.  In some embodiments, PDn  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  PDn  comprises  administering about 20 ug of RNA.  In some embodiments, PDn comprises administering about  30 ug of RNA.  In some embodiments, PDn comprises administering about 50 ug of RNA.  In  some embodiments, PDn comprises administering about 60 ug of RNA.    In  some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD1 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In some embodiments, PD1 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD1 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In  some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD2 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PD2 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD2 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.      In  some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PD3 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PD3 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PD3 comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In  some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, PDn comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic  fragment  thereof  from  the Wuhan  strain  and one or more  additional RNAs  encoding a Spike protein or an immunogenic fragment thereof from a SARS‐CoV‐2 strain that  is prevalent and/or spreading rapidly  in a relevant  jurisdiction.    In some embodiments, PDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  an  alpha  variant.    In  some  embodiments,  PDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, PDn comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations  from a delta variant.    In some embodiments, PDn comprises an RNA encoding a  SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof from the Wuhan strain and an  RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof comprising  one or more mutations from an Omicron variant.    In some embodiments, PD1, PD2, PD3, and PDn can each independently comprise a plurality of  (e.g., at least two) RNA (e.g., mRNA) compositions described herein.  In some embodiments  PD1, PD2, PD3, and PDn can each independently comprise a first and a second RNA (e.g., mRNA)  composition.    In  some  embodiments,  at  least  one  of  a  plurality  of  RNA  (e.g.,  mRNA)  compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least  one of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding  a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  from a different SARS‐CoV‐2  variant.  In some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions  comprises an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof from a Wuhan strain of SARS‐CoV‐2.  In some embodiments, at least one of a plurarity  of RNA (e.g., mRNA) compositions comprises an RNA encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising  one  or more mutations  from  a  variant  that  is  prevalent and/or spreading rapidly in a relevant jurisdiction.   In some embodiments, at least  one of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations from an alpha variant.    In some embodiments, at  least one of a plurality of RNA  (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof comprising one or more mutations from a delta variant.    In some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  comprising one or more mutations from an Omicron variant.     In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or  PDn  can  each  independently  comprise  at  least  two  different  RNA  (e.g.,  mRNA)  constructs  (e.g.,  differing  in  at  protein‐encoding  sequences).  For  example,  in  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2, PD3, and/or PDn  can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof  from a Wuhan strain of SARS‐CoV‐2 and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations  from  a  variant  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.  In some embodiments a plurality of RNA (e.g., mRNA) compositions given in PD1,  PD2, PD3, and/or PDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof  derived from a Wuhan strain of SARS‐ CoV‐2 and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof comprising one or more mutations from a variant that is prevalent and/or spreading  rapidly in a relevant jurisdiction.  In some such embodiments, a variant can be an alpha variant.  In some such embodiments, a variant can be a delta variant.  In some such embodiments a  variant can be an Omicron variant.   In some embodiments, each of a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2,  PD3, and/or PDn can independently comprise at least two RNA (e.g., mRNA)s, each encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction.  In  some embodiments, each of a plurality of RNA (e.g., mRNA) compositions given in PD1, PD2,  PD3, and/or PDn can  independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S  protein or an immunogenic fragment thereof  from an alpha variant and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations from a delta variant.  In some embodiments, each of a plurality of RNA (e.g.,  mRNA) compositions given  in PD1, PD2, PD3, and/or PDn can  independently comprise an RNA  (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  from an  alpha variant and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic  fragment  thereof  comprising  one  or more mutations  from  an Omicron  variant.    In  some  embodiments, each of a plurality of RNA  (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or PDn can independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof  from a delta variant and an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations from an Omicron variant.    In some embodiments, PD1, PD2, PD3, and/or PDn each comprise a plurality of RNA (e.g., mRNA)  compositions, wherein each RNA  (e.g., mRNA) composition  is separately administered  to a  subject.  For  example,  in  some  embodiments  each  RNA  (e.g.,  mRNA)  composition  is  administered  via  intramuscular  injection  at different  injection  sites.  For example,  in  some  embodiments, a first and second RNA (e.g., mRNA) composition given in PD1, PD2, PD3, and/or  PDn are separately administered to different arms of a subject via intramuscular injection.   In some embodiments, PD1, PD2, PD3, and/or PDn comprise administering a plurality of RNA  molecules, wherein each RNA molecule encodes a Spike protein comprising mutations from a  different SARS‐CoV‐2 variant, and wherein the plurality of RNA molecules are administered to  the subject in a single formulation.  In some embodiments, the single formulation comprises  an RNA encoding a Spike protein or an immunogenic variant thereof from the Wuhan strain  and  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising one or more mutations from an alpha variant.  In some embodiments, the single  formulation  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or  an immunogenic fragment thereof comprising one or more mutations from a beta variant.  In  some embodiments, the single formulation comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from a delta variant.  In some embodiments, the single formulation comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from an Omicron variant.    In some embodiments, the length of time between PD1 and PD2 (PI1) is at least about 1 week,  at  least  about  2  weeks,  at  least  about  3  weeks,  or  at  least  about  4  weeks.    In  some  embodiments, PI1 is about 1 week to about 12 weeks. In some embodiments, PI1 is about 1  week to about 10 weeks.  In some embodiments, PI1 is about 2 weeks to about 10 weeks.  In  some embodiments, PI 1 is about 2 weeks to about 8 weeks.  In some embodiments, PI1 is about  3 weeks to about 8 weeks.  In some embodiments, PI1 is about 4 weeks to about 8 weeks.  In  some embodiments, PI1 is about 6 weeks to about 8 weeks.  In some embodiments PI1 is about  3 to about 4 weeks.  In some embodiments, PI1 is about 1 week.  In some embodiments, PI1 is  about 2 weeks.  In some embodiments, PI1 is about 3 weeks. In some embodiments, PI1 is about  4 weeks.  In some embodiments, PI1 is about 5 weeks.  In some embodiments, PI1 is about 6  weeks.    In some embodiments, PI1 is about 7 weeks.    In some embodiments, PI1 is about 8  weeks.  In some embodiments, PI1 is about 9 weeks.  In some embodiments, PI1 is about 10  weeks.  In some embodiments, PI1 is about 11 weeks.  In some embodiments, PI1 is about 12  weeks.    In some embodiments, the length of time between PD2 and PD3 (PI2) is at least about 1 week,  at least about 2 weeks, or at least about 3 weeks.  In some embodiments, PI2 is about 1 week  to about 12 weeks. In some embodiments, PI2 is about 1 week to about 10 weeks.  In some  embodiments, PI2 is about 2 weeks to about 10 weeks.  In some embodiments, PI2 is about 2  weeks to about 8 weeks.  In some embodiments, PI2 is about 3 weeks to about 8 weeks.  In  some embodiments, PI2 is about 4 weeks to about 8 weeks.  In some embodiments, PI2 is about  6 weeks to about 8 weeks.  In some embodiments PI2 is about 3 to about 4 weeks.  In some  embodiments, PI2  is about 1 week.    In some embodiments, PI2  is about 2 weeks.    In some  embodiments, PI2  is about 3 weeks.  In some embodiments, PI2  is about 4 weeks.    In  some  embodiments, PI2 is about 5 weeks.    In some embodiments, PI2 is about 6 weeks.    In some  embodiments, PI2 is about 7 weeks.    In some embodiments, PI2 is about 8 weeks.    In some  embodiments, PI2 is about 9 weeks.  In some embodiments, PI2 is about 10 weeks.  In some  embodiments, PI2 is about 11 weeks.  In some embodiments, PI2 is about 12 weeks.    In some embodiments, the length of time between PD3 and a subsequent dose that is part of  the  Primary  Dosing  Regimen,  or  between  doses  for  any  dose  beyond  PD3  (PIn)  is  each  separately and independently selected from: about 1 week or more, about 2 weeks or more,  or about 3 weeks or more.  In some embodiments, PIn is about 1 week to about 12 weeks. In  some embodiments, PIn is about 1 week to about 10 weeks.  In some embodiments, PIn is about  2 weeks to about 10 weeks.  In some embodiments, PIn is about 2 weeks to about 8 weeks.  In  some embodiments, PIn is about 3 weeks to about 8 weeks.  In some embodiments, PIn is about  4 weeks to about 8 weeks.  In some embodiments, PIn is about 6 weeks to about 8 weeks.  In  some embodiments PI n is about 3 to about 4 weeks.    In some embodiments, PI2 is about 1  week.    In some embodiments, PIn  is about 2 weeks.    In  some embodiments, PIn  is about 3  weeks.  In some embodiments, PIn  is about 4 weeks.    In some embodiments, PIn  is about 5  weeks.    In some embodiments, PInis about 6 weeks.    In some embodiments, PIn is about 7  weeks.    In some embodiments, PInis about 8 weeks.    In some embodiments, PIn is about 9  weeks.  In some embodiments, PIn is about 10 weeks.  In some embodiments, PIn is about 11  weeks.  In some embodiments, PIn is about 12 weeks.    In some embodiments, one or more compositions adminstered in PD1 are formulated in a Tris  buffer.  In some embodiments, one or more compositions administered in PD2 are formulated  in a Tris buffer.  In some embodiments, one or more compositions administering in PD3 are  formulated in a Tris buffer.  In some embodiments, one or more compositions adminsitered  in PDn are formulated in a Tris buffer.    In some embodiments, the primary dosing regimen comprises administering two or more RNA  (e.g., mRNA)  compositions  described  herein,  and  at  least  two  of  the  RNA  (e.g.,  mRNA)  compositions have different formulations.  In some embodiments, the primary dosing regimen  comprises PD1 and PD2, where PD1 comprises administering an RNA (e.g., mRNA) formulated  in a Tris buffer and PD2 comprises administering an RNA  (e.g., mRNA)  formulated  in a PBS  buffer.  In some embodiments, the primary dosing regimen comprises PD1 and PD2, where PD1  comprises administering an RNA (e.g., mRNA) formulated in a PBS buffer and PD2 comprises  administering an RNA (e.g., mRNA) formulated in a Tris buffer.    In some embodiments, one or more RNA  (e.g., mRNA) compositions given  in PD1, PD2, PD3,  and/or PDn can be administered in combination with another vaccine. In some embodiments,  another vaccine is for a disease that is not COVID‐19.  In some embodiments, the disease is  one that  increases deleterious effects of SARS‐CoV‐2 when a subject  is coinfected with the  disease  and  SARS‐CoV‐2.    In  some  embodiments,  the  disease  is  one  that  increases  the  transmission rate of SARS‐CoV‐2 when a subject is coinfected with the disease and SARS‐CoV‐ 2.    In some embodiments, another vaccine  is a different commerically available vaccine.  In  some embodiments, the different commercially available vaccine is an RNA vaccine.  In some  embodiments, the different commercially available vaccine is a polypeptide‐based vaccine. In  some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g.,  mRNA)  compositions  given  in  PD1,  PD2,  PD3,  and/or  PDn  are  separately  administered,  for  example,  in some embodiments via  intramuscular  injection, at different  injection sites. For  example, in some embodiments, an influenza vaccine and one or more SARS‐CoV‐2 RNA (e.g.,  mRNA)  compositions  described  herein  given  in  PD1,  PD2,  PD3,  and/or  PDn  are  separately  administered to different arms of a subject via intramuscular injection.    Booster Dosing Regimens In  some  embodiments,  methods  of  vaccination  disclosed  herein  comprise  one  or  more  Booster Dosing Regimens.   The Booster Dosing Regimens disclosed herein comprise one or  more doses.    In some embodiments, a Booster Dosing Regimen  is administered to patients  who have been administered a Primary Dosing Regimen (e.g., as described herein). In some  embodiments a Booster Dosing Regimen  is administed to patients who have not received a  pharmaceutical  composition  disclosed  herein.  In  some  embodiments  a  Booster  Dosing  Regimen  is administered to patients who have been previously vaccinated with a COVID‐19  vaccine that is different from the vaccine administered in a Primary Dosing Regimen.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks,  at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least  10 weeks, at least 11 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4  months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9  months, at  least 10 months, at  least 11 months, or at  least 12 months or  longer.  In  some  embodiments,  the  length  of  time  between  the  Primary Dosing  Regimen  and  the  Booster  Dosing Regimen  is about 1 month.    In some embodiments, the  length of time between the  Primary Dosing Regimen and the Booster Dosing Regimen is at least about 2 months.  In some  embodiments,  the  length  of  time  between  the  Primary Dosing  Regimen  and  the  Booster  Dosing Regimen is at least about 3 months.  In some embodiments, the length of time between  the Primary Dosing Regimen and the Booster Dosing Regimen is at least about 4 months.  In  some embodiments, the length of time between the Primary Dosing Regimen and the Booster  Dosing Regimen is at least about 5 months.  In some embodiments, the length of time between  the Primary Dosing Regimen and the Booster Dosing Regimen is at least about 6 months.  In  some embodiments, the length of time between the Primary Dosing Regimen and the Booster  Dosing Regimen is from about 1 month to about 48 months.  In some embodiments, the length  of time between the Primary Dosing Regimen and the Booster Dosing Regimen is from about  1 month to about 36 months.  In some embodiments, the length of time between the primary  dosing regimen and the Booster Dosing Regimen is from about 1 month to about 24 months.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is from about 2 months to about 24 months.  In some embodiments,  the length of time between the Primary Dosing Regimen and the Booster Dosing Regimen is  from about 3 months to about 24 months.  In some embodiments, the length of time between  the primary dosing regimen and the Booster Dosing Regimen is from about 3 months to about  18 months.  In some embodiments, the length of time between the primary dosing regimen  and  the  Booster Dosing  Regimen  is  from  about  3 months  to  about  12 months.    In  some  embodiments, the length of time between the primary dosing regimen and the Booster Dosing  Regimen is from about 6 months to about 12 months.  In some embodiments, the length of  time between the Primary Dosing Regimen and the Booster Dosing Regimen is from about 3  months to about 9 months.  In some embodiments, the length of time between the Primary  Dosing Regimen and the Booster Dosing Regimen is from about 5 months to about 7 months.   In  some  embodiments,  the  length  of  time  between  the  Primary Dosing Regimen  and  the  Booster Dosing Regimen is about 6 months.    In some embodiments, subjects are administered a Booster Dosing Regimen.  A Booster dosing  regimen can comprise one or more doses. For example,  in  some embodiments, a Booster  Dosing  Regimen  comprises  a  single  dose  (BD1).  In  some  embodiments  a  Booster  Dosing  Regimen  comprises  a  first  dose  (BD1)  and  a  second  dose  (BD2).  In  some  embodiments,  a  Booster Dosing Regimen comprises a first dose, a second dose, and a third dose (BD3). In some  embodiments, a Booster Dosing Regimen comprises a first dose, a second dose, a third dose,  and  one  or more  additional  doses  (BDn)  of  any  one  of  the  pharmaceutical  compositions  described herein.    In  some  embodiments,  BD1  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD1 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD1  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD1  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD1 comprises administering about  3 ug of RNA.  In some embodiments, BD1 comprises administering about 5 ug of RNA.  In some  embodiments, BD1 comprises administering about 10 ug of RNA.  In some embodiments, BD1  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD1  comprises  administering about 20 ug of RNA.  In some embodiments, BD1 comprises administering about  30 ug of RNA.  In some embodiments, BD1 comprises administering about 50 ug of RNA.  In  some embodiments, BD1 comprises administering about 60 ug of RNA.    In  some  embodiments,  BD2  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD2 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD2  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD2  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD2 comprises administering about  3  ug.    In  some  embodiments,  BD2  comprises  administering  about  5  ug  of  RNA.    In  some  embodiments, BD2 comprises administering about 10 ug of RNA.  In some embodiments, BD2  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD2  comprises  administering about 20 ug RNA.  In some embodiments, BD2 comprises administering about  30 ug of RNA.  In some embodiments, BD2 comprises administering about 50 ug of RNA.  In  some embodiments, BD2 comprises administering about 60 ug of RNA.    In  some  embodiments,  BD3  comprises  administering  1  to  100  ug  of  RNA.    In  some  embodiments, BD3 comprises administering 1 to 60 ug of RNA.   In some embodiments, BD3  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BD3  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BD3 comprises administering about  3 ug of RNA.  In some embodiments, BD3 comprises administering about 5 ug of RNA.  In some  embodiments, BD3 comprises administering about 10 ug of RNA.  In some embodiments, BD3  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD3  comprises  administering about 20 ug of RNA.  In some embodiments, BD3 comprises administering about  30 ug of RNA.  In some embodiments, BD3 comprises administering about 50 ug of RNA.  In  some embodiments, BD3 comprises administering about 60 ug of RNA.    In  some  embodiments,  BDn  comprises  administering  1  to  100  ug  of  RNA.  In  some  embodiments, BDn comprises administering 1 to 60 ug of RNA.   In some embodiments, BDn  comprises  administering  1  to  50  ug  of  RNA.    In  some  embodiments,  BDn  comprises  administering 1 to 30 ug of RNA.  In some embodiments, BDn comprises administering about  3 ug of RNA.  In some embodiments, BDn comprises administering about 5 ug of RNA.  In some  embodiments, BDn comprises administering about 10 ug of RNA.  In some embodiments, BDn  comprises  administering  about  15  ug  of  RNA.    In  some  embodiments,  BD n  comprises  administering about 20 ug of RNA.  In some embodiments, BDn comprises administering about  30 ug of RNA.  In some embodiments, BDn comprises administering about 60 ug of RNA.  In  some embodiments, BDn comprises administering about 50 ug of RNA.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD1 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD1 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD1  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD1  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD1 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.    In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD2 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.   In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD2 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD2  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD2  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD2 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.      In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BD3 comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.  In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BD3 comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BD3  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BD3  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In  some embodiments, BD3 comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.    In  some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain.  In some embodiments, BDn comprises  an RNA encoding a Spike protein or an  immunogenic  fragment  thereof  from a SARS‐CoV‐2  strain  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from a delta variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment thereof comprising one or more mutations  from a beta variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an Omicron variant.    In  some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an  immunogenic fragment thereof from the Wuhan strain and one or more RNA encoding a Spike  protein  or  an  immunogenic  fragment  thereof  from  a  SARS‐CoV‐2  strain  that  is  prevalent  and/or spreading rapidly in a relevant jurisdiction.  In some embodiments, BDn comprises an  RNA encoding  a  SARS‐CoV‐2  Spike protein or an  immunogenic  fragment  thereof  from  the  Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment  thereof comprising one or more mutations from a alpha variant.  In some embodiments, BDn  comprises an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic fragment thereof  from the Wuhan strain and an RNA encoding a SARS‐CoV‐2 Spike protein or an immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments,  BDn  comprises  an  RNA  encoding  a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic fragment thereof from the Wuhan strain and an RNA encoding a SARS‐CoV‐2  Spike protein or an immunogenic fragment thereof comprising one or more mutations from a  beta variant.    In some embodiments, BDn comprises an RNA encoding a SARS‐CoV‐2 Spike  protein or an immunogenic fragment thereof from the Wuhan strain and an RNA encoding a  SARS‐CoV‐2  Spike  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more  mutations from an Omicron variant.      In some embodiments, BD1, BD2, BD3, and BDn can each independently comprise a plurality of  (e.g., at least two) RNA (e.g., mRNA) compositions described herein.  In some embodiments  BD1, BD2, BD3, and BDn can each independently comprise a first and a second RNA (e.g., mRNA)  composition.  In some embodiments, BD1, BD2, BD3, and BDn can each independently comprise  a plurality of (e.g., at least two) RNA (e.g., mRNA) compositions, wherein , at least one of the  plurality of RNA (e.g., mRNA) compositions comprises BNT162b2 (e.g., as described herein). In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  from a different SARS‐CoV‐2 variant (e.g., a variant that is prevalent or rapidly spreading in a  relevant jurisdiction, e.g., a variant disclosed herein).  In some embodiments, at least one of a  plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof from a Wuhan strain of SARS‐CoV‐2.  In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one  or more mutations  from a variant  that  is prevalent and/or  spreading  rapidly  in a  relevant  jurisdiction.    In  some  embodiments,  at  least  one  of  a  plurality  of  RNA  (e.g.,  mRNA)  compositions  comprises  an  RNA  (e.g.,  mRNA)  encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic fragment thereof comprising one or more mutations from an alpha variant.  In  some embodiments, at least one of a plurality of RNA (e.g., mRNA) compositions comprises  an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof  comprising one or more mutations from a delta variant.  In some embodiments, at least one  of a plurality of RNA (e.g., mRNA) compositions comprises an RNA (e.g., mRNA) encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from an Omicron variant.     In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can each indendently comprise at least two different RNA (e.g., mRNA) constructs  (e.g., RNA  constructs  having  differing  protein‐encoding  sequences).  For  example,  in  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or BDn  can each indendently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof from a Wuhan strain of SARS‐CoV‐2 and an RNA (e.g., mRNA)  encoding a SARS‐CoV‐2 S protein or an  immunogenic  fragment  thereof  comprising one or  more mutations  from  a  variant  that  is  prevalent  and/or  spreading  rapidly  in  a  relevant  jurisdiction.  In some embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1,  BD2, BD3, and/or BDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof derived from a Wuhan strain of SARS‐ CoV‐2 and an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment  thereof comprising one or more mutations from a variant that is prevalent and/or spreading  rapidly in a relevant jurisdiction.  In some such embodiments, a variant can be an alpha variant.  In some such embodiments, a variant can be a delta variant.  In some such embodiments a  variant can be an Omicron variant.   In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can each independently comprise at least two RNA (e.g., mRNA)s each encoding a  SARS‐CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations  from a distinct variant that is prevalent and/or spreading rapidly in a relevant jurisdiction.  In  some embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or  BDn can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein  or an immunogenic fragment thereof from an alpha variant and an RNA (e.g., mRNA) encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or  more  mutations  from  a  delta  variant.    In  some  embodiments  a  plurality  of  RNA  (e.g., mRNA)  compositions given in BD1, BD2, BD3, and/or BDn can each independently comprise an RNA (e.g.,  mRNA) encoding a SARS‐CoV‐2 S protein or an immunogenic fragment thereof from an alpha  variant  and  an  RNA  (e.g., mRNA)  encoding  a  SARS‐CoV‐2  S  protein  or  an  immunogenic  fragment  thereof  comprising  one  or more mutations  from  an Omicron  variant.    In  some  embodiments a plurality of RNA (e.g., mRNA) compositions given in BD1, BD2, BD3, and/or BDn  can each independently comprise an RNA (e.g., mRNA) encoding a SARS‐CoV‐2 S protein or an  immunogenic fragment thereof from a delta variant and an RNA (e.g., mRNA) encoding a SARS‐ CoV‐2 S protein or an immunogenic fragment thereof comprising one or more mutations from  an Omicron variant.    In some embodiments, a plurality of RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn are separately administered to a subject, for example, in some embodiments via  intramuscular injection, at different injection sites. For example, in some embodiments, a first  and second RNA (e.g., mRNA) composition given  in BD1, BD2, BD3, and/or BDn are separately  administered to different arms of a subject via intramuscular injection.   In some embodiments, the length of time between BD1 and BD2 (BI1) is at least about 1 week,  at  least  about  2  weeks,  at  least  about  3  weeks,  or  at  least  about  4  weeks.    In  some  embodiments, BI1 is about 1 week to about 12 weeks.  In some embodiments, BI1 is about 1  week to about 10 weeks.  In some embodiments, BI1 is about 2 weeks to about 10 weeks.  In  some embodiments, BI1 is about 2 weeks to about 8 weeks.  In some embodiments, BI1 is about  3 weeks to about 8 weeks.  In some embodiments, BI1 is about 4 weeks to about 8 weeks.  In  some embodiments, BI1 is about 6 weeks to about 8 weeks.  In some embodiments BI1 is about  3 to about 4 weeks.  In some embodiments, BI1 is about 1 week.  In some embodiments, BI1 is  about 2 weeks.    In some embodiments, BI1  is about 3 weeks.  In some embodiments, BI1  is  about 4 weeks. In some embodiments, BI1 is about 5 weeks. In some embodiments, BI1 is about  6 weeks.  In some embodiments, BI1 is about 7 weeks.  In some embodiments, BI1 is about 8  weeks.  In some embodiments, BI1 is about 9 weeks.  In some embodiments, BI1 is about 10  weeks.  In some embodiments, the length of time between BD2 and BD3 (BI2) is at least about 1 week,  at least about 2 weeks, or at least about 3 weeks.  In some embodiments, BI2 is about 1 week  to about 12 weeks. In some embodiments, BI2 is about 1 week to about 10 weeks.  In some  embodiments, BI2 is about 2 weeks to about 10 weeks.  In some embodiments, BI2 is about 2  weeks to about 8 weeks.  In some embodiments, BI2 is about 3 weeks to about 8 weeks.  In  some embodiments, BI2 is about 4 weeks to about 8 weeks.  In some embodiments, BI2 is about  6 weeks to about 8 weeks.  In some embodiments BI2 is about 3 to about 4 weeks.  In some  embodiments, BI2  is about 1 week.    In some embodiments, BI2  is about 2 weeks.    In some  embodiments, BI2  is about 3 weeks.  In  some embodiments, BI2  is about 4 weeks.  In  some  embodiments, BI2  is about 5 weeks.  In  some embodiments, BI2  is about 6 weeks.  In  some  embodiments, BI2  is about 7 weeks.  In  some embodiments, BI2  is about 8 weeks.  In  some  embodiments, BI2 is about 9 weeks. In some embodiments, BI2 is about 10 weeks.  In some embodiments, the length of time between BD3 and a subsequent dose that is part of  the  Booster  Dosing  Regimen,  or  between  doses  for  any  dose  beyond  BD3  (BIn)  is  each  separately and independently selected from: about 1 week or more, about 2 weeks or more,  or about 3 weeks or more.  In some embodiments, BIn is about 1 week to about 12 weeks. In  some embodiments, BI n  is about 1 week  to about 10 weeks.    In some embodiments, BIn  is  about 2 weeks to about 10 weeks.    In some embodiments, BIn  is about 2 weeks to about 8  weeks.  In some embodiments, BIn is about 3 weeks to about 8 weeks.  In some embodiments,  BIn is about 4 weeks to about 8 weeks.  In some embodiments, BIn is about 6 weeks to about  8 weeks.  In some embodiments BIn is about 3 to about 4 weeks.  In some embodiments, BIn is  about 1 week.  In some embodiments, BIn is about 2 weeks.  In some embodiments, BIn is about  3 weeks. In some embodiments, BIn is about 4 weeks. In some embodiments, BIn is about 5  weeks.  In  some embodiments, BIn  is about 6 weeks.  In some embodiments, BIn  is about 7  weeks.  In  some embodiments, BIn  is about 8 weeks.  In some embodiments, BIn  is about 9  weeks. In some embodiments, BIn is about 10 weeks.  In some embodiments, one or more compositions adminstered in BD1 are formulated in a Tris  buffer.  In some embodiments, one or more compositions administered in BD2 are formulated  in a Tris buffer.  In some embodiments, one or more compositions administering in BD3 are  formulated in a Tris buffer.  In some embodiments, one or more compositions adminsitered  in BD3 are formulated in a Tris buffer.    In some embodiments, the Booster dosing regimen comprises administering two or more RNA  (e.g., mRNA)  compositions  described  herein,  and  at  least  two  of  the  RNA  (e.g.,  mRNA)  compositions  have  differnent  formulations.    In  some  embodiments,  the  Booster  dosing  regimen comprises BD1 and BD2, where BD1 comprises administering an RNA  (e.g., mRNA)  formulated in a Tris buffer and BD2 comprises administering an RNA (e.g., mRNA) formulated  in a PBS buffer.  In some embodiments, the Booster dosing regimen comprises BD1 and BD2,  where BD1 comprises administering an RNA (e.g., mRNA) formulated in a PBS buffer and BD2  comprises administering an RNA (e.g., mRNA) formulated in a Tris buffer.    In some embodiments, one or more RNA (e.g., mRNA) compositions given  in BD1, BD2, BD3,  and/or BDn can be administered in combination with another vaccine. In some embodiments,  another vaccine is for a disease that is not COVID‐19.  In some embodiments, the disease is  one that  increases deleterious effects of SARS‐CoV‐2 when a subject  is coinfected with the  disease  and  SARS‐CoV‐2.    In  some  embodiments,  the  disease  is  one  that  increases  the  transmission rate of SARS‐CoV‐2 when a subject is coinfected with the disease and SARS‐CoV‐ 2.    In some embodiments, another vaccine  is a different commerically available vaccine.  In  some embodiments, the different commercially available vaccine is an RNA vaccine.  In some  embodiments, the different commercially available vaccine is a polypeptide‐based vaccine. In  some embodiments, another vaccine (e.g., as described herein) and one or more RNA (e.g.,  mRNA)  compositions  given  in  BD1,  BD2,  BD3,  and/or  BDn  are  separately  administered,  for  example,  in some embodiments via  intramuscular  injection, at different  injection sites. For  example, in some embodiments, an influenza vaccine and one or more SARS‐CoV‐2 RNA (e.g.,  mRNA)  compositions  described  herein  given  in  BD1,  BD2,  BD3,  and/or  BDn  are  separately  administered to different arms of a subject via intramuscular injection.    Additional Booster Regimens  In some embodiments, methods of vaccination disclosed herein comprise administering more  than one Booster Dosing Regimen.    In some embodiments, more  than one Booster Dosing  Regimen may need to be administered to increase neutralizing antibody response.  In some  embodiments, more than one booster dosing regimen may be needed to counteract a SARS‐ CoV‐2  strain  that has been  shown  to have  a high  likelihood of  evading  immune  response  elicited  by  vaccines  that  a  patient  has  previously  received.    In  some  embodiments,  an  additional Booster Dosing Regimen is administered to a patient who has been determined to  produce low concentrations of neutralizing antibodies.  In some embodiments, an additional  booster dosing regimen is administered to a patient who has been determined to have a high  likelihood of being susceptible to SARS‐CoV‐2 infection, despite previous vaccination (e.g., an  immunocompromised patient, a cancer patient, and/or an organ transplant patient).  The description provided above for the first Booster Dosing Regimen also describes the one or  more additional Booster Dosing Regimens.   The  interval of  time between  the  first Booster  Dosing  Regimen  and  a  second  Booster  Dosing  Regimen,  or  between  subsequent  Booster  Dosing Regimens can be any of the acceptable intervals of time described above between the  Primary Dosing Regimen and the First Booster Dosing Regimen.    In some embodiments, a dosing regimen comprises a primary regimen and a booster regimen,  wherein at least one dose given in the primary regimen and/or the booster regimen comprises  a composition comprising an RNA that encodes a S protein or immungenic fragment thereof  from a variant that is prevalent or is spreading rapidly in a relevant jurisdiction (e.g., Omicron  variant  as  described  herein).  For  example,  in  some  embodiments,  a  primary  regimen  comprises at least 2 doses of BNT162b2 (e.g., encoding a Wuhan strain), for example, given at  least  3 weeks  apart,  and  a  booster  regimen  comprises  at  least  1  dose  of  a  composition  comprising RNA that encodes a S protein or immungenic fragment thereof from a variant that  is prevalent or is spreading rapidly in a relevant jurisdiction (e.g., Omicron variant as described  herein). In some such embodiments, such a dose of a booster regimen may further comprise  an RNA  that encodes a S protein or  immungenic  fragement  thereof  from a Wuhan  strain,  which can be administered with an RNA that encodes a S protein or  immungenic fragment  thereof from a variant that is prevalent or is spreading rapidly in a relevant jurisdiction (e.g.,  Omicron variant as described herein), as a single mixture, or as two separate compositions,  for example, in 1:1 weight ratio. In some embodiments, a booster regimen can also comprise  at least 1 dose of BNT162b2, which can be administered as a first booster dose or a subsequent  booster dose.   In some embodiments, an RNA composition described herein is given as a booster at a dose  that is higher than the doses given during a primary regimen (primary doses) and/or the dose  given for a first booster, if any. For example, in some embodiments, such a dose may be 60  ug; or in some embodiments such a dose may be higher than 30 ug and lower than 60 ug (e.g.,  55 ug, 50 ug, or lower). In some embodiments, an RNA composition described herein is given  as a booster at least 3‐12 months or 4‐12 months, or 5‐12 months, or 6‐12 months after the  last dose (e.g., the  last dose of a primary regimen or a first dose of a booster regimen).    In  some embodiments, the primary doses and/or the first booster dose (if any) may comprise  BNT162b2, for example at 30 ug per dose.  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 49 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  49). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 50 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 50).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 51 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 51).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 55 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  55. In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 56 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 56).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 57).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 58 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  58). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 59 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 59).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 60).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 61 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  61). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 62 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 62).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 63 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 63).  In some embodiments, the formulations disclosed herein can be used to carry out any of the  dosing regimens described in Table 28 (below).     
Figure imgf000636_0001
Figure imgf000637_0001
Figure imgf000638_0001
In some embodiments of certain exemplary dosing regimens as described in Table 28 above,  an RNA  composition  described herein  (e.g.,  comprising RNA  encoding  a  variant described  herein)  is  given  in  a  first  dose  of  a  primary  regimen.    In  some  embodiments  of  certain  exemplary dosing regimens as described  in Table 28 above, an RNA composition described  herein (e.g., comprising RNA encoding a variant described herein) is given in a second dose of  a primary regimen.  In some embodiments of certain exemplary dosing regimens as described  in Table 28 above, an RNA composition described herein  (e.g., comprising RNA encoding a  variant described herein) is given in a first dose and a second dose of a primary regimen.  In  some embodiments of certain exemplary dosing regimens as described in Table 28 above, an  RNA composition described herein (e.g., comprising RNA encoding a variant described herein)  is given in a first dose of a booster regimen.  In some embodiments of certain exemplary dosing  regimens  as  described  in  Table  28  above,  an  RNA  composition  described  herein  (e.g.,  comprising RNA encoding a variant described herein)  is given in a second dose of a booster  regimen.  In some embodiments of certain exemplary dosing regimens as described in Table  28 above, an RNA  composition described herein  (e.g.,  comprising RNA encoding a  variant  described herein)  is given  in a first dose and a second dose of a booster regimen.    In some  embodiments of certain exemplary dosing regimens as described in Table 28 above, an RNA  composition described herein (e.g., comprising RNA encoding a variant described herein)  is  given in a first dose and a second dose of a primary regimen and also in at least one dose of a  booster regimen.  In some embodiments of certain exemplary dosing regimens as described  in Table 28 above, an RNA composition described herein  (e.g., comprising RNA encoding a  variant described herein) is given in at least one dose (including, e.g., at least two doses) of a  booster regimen and BNT162b2 is given in a primary regimen. In some embodiments of certain  exemplary dosing regimens as described  in Table 28 above, an RNA composition described  herein (e.g., comprising RNA encoding a variant described herein) is given in a second dose of  a booster regimen and BNT162b2 is given in a primary regimen and in a first dose of a booster  regimen. In some embodiments, an RNA composition described herein (e.g., comprising RNA  encoding a variant described herein) comprises an RNA encoding a polypeptide as set forth in  SEQ ID NO: 49 or an immunogenic fragment thereof, or a variant thereof (e.g., having at least  70% or more, including, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,  at least 97%, at least 98%, or higher, identity to SEQ ID NO: 49). In some embodiments, an RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described  herein)  comprises an RNA  that  includes  the  sequence of SEQ  ID NO: 50 or a variant  thereof  (e.g.,  having at least 70% or more, including, e.g., at least 80%, at least 85%, at least 90%, at least  95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO: 50).  In some  embodiments, an RNA composition described herein (e.g., comprising RNA encoding a variant  described herein) comprises an RNA that includes the sequence of SEQ ID NO: 51 or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  51).    In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 55 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  55). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 56 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 56).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 57 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 57).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 58 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  58). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 59 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 59).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 60 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 60).  In some embodiments, an RNA composition described herein comprises an RNA encoding a  polypeptide as set forth in SEQ ID NO: 61 or an immunogenic fragment thereof, or a variant  thereof (e.g., having at least 70% or more, including, e.g., at least 80%, at least 85%, at least  90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher, identity to SEQ ID NO:  61). In some embodiments, an RNA composition comprises an RNA that includes the sequence  of SEQ ID NO: 62 or a variant thereof (e.g., having at least 70% or more, including, e.g., at least  80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or higher,  identity to SEQ  ID NO: 62).    In some embodiments, an RNA composition comprises an RNA  that includes the sequence of SEQ ID NO: 63 or a variant thereof (e.g., having at least 70% or  more,  including, e.g., at  least 80%, at  least 85%, at  least 90%, at  least 95%, at  least 96%, at  least 97%, at least 98%, or higher, identity to SEQ ID NO: 63).  In  some  embodiments,  such  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding a variant described herein) can  further comprise RNA encoding a S protein or an  immungenic fragment thereof of a different strain (e.g., a Wuhan strain). By way of example,  in some embodiments, a second dose of a booster regimen of Regimens #9‐11 as described in  Table 28 above  can  comprise an RNA  composition described herein  (e.g.,  comprising RNA  encoding a variant described herein such as Omicron, for example, in one embodiment RNA  as described in this Example) and a BNT162b2 construct, for example, in 1: 1 weight ratio.    In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary  regimen and a  first dose and a  second dose of a booster  regimen each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described herein such as Omicron, for example, in one embodiment RNA as described in this  Example).  In  some  such  embodiments,  a  second  dose  of  a  booster  regimen may  not  be  necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary  regimen and a  first dose and a  second dose of a booster  regimen each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described herein such as Omicron, for example, in one embodiment RNA as described in this  Example).  In  some  such  embodiments,  a  second  dose  of  a  booster  regimen may  not  be  necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary regimen each comprise a BNT162b2 construct, and a first dose and a second  dose  of  a  booster  regimen  each  comprise  an  RNA  composition  described  herein  (e.g.,  comprising RNA encoding a variant described herein such as Omicron,  for example,  in one  embodiment RNA as described in this Example). In some such embodiments, a second dose of  a booster regimen may not be necessary.   In some embodiments of Regimen #6 as described in Table 28 above, a first dose and a second  dose of a primary regimen and a first dose of a booster regimen each comprise a BNT162b2  construct, and a second dose of a booster regimen comprises an RNA composition described  herein  (e.g.,  comprising  RNA  encoding  a  variant  described  herein  such  as  Omicron,  for  example, in one embodiment RNA as described in this Example).     Example 7: Omicron breakthrough infection drives cross‐variant neutralization and memory  B cell formation  The present Example shows that an Omicron breakthrough infection in individuals double‐ and  triple‐vaccinated  with  BNT162b2  drives  cross  variant  neutralization  and  memory  B  cell  formation,  including production of neutralizing antibodies and B cell  responses  toward   an  Omicron variant. One of ordinary skill in the art reading the present Example will understand  that  such  findings  can  be  extended  to  administration  of  an  RNA  (e.g.,  mRNA)  vaccine  comprising an RNA encoding a SARS‐CoV‐2 S protein having mutations characteristic of an  Omicron variant (e.g., ones as described herein) to subjects who were previously administered  two or three doses of SARS‐CoV‐2 vaccines (e.g., in some embodiments developed based on a  S protein from a Wuhan‐Hu‐1 strain).  Omicron is the evolutionarily most distinct SARS‐CoV‐2 variant of concern (VOC) to date. To  address how Omicron breakthrough infection can potentially reshape SARS‐CoV‐2 recognition  in vaccinated individuals, the effects of Omicron breakthrough infection were investigated on  serum  neutralization  and  BMEM  cell  antigen  recognition  in  BNT162b2  double‐  and  triple‐ vaccinated individuals. Omicron breakthrough infection induced broad neutralization of VOCs  including  Omicron, with  substantially  stronger  neutralization  compared  to  Omicron‐naïve  double‐ and triple‐vaccinees. Broad recognition of VOCs by BMEM cells from BNT162b2 double‐  and  triple‐vaccinated  individuals  was  boosted  by  Omicron  breakthrough  infection,  with  recognition primarily against conserved epitopes shared broadly between variants rather than  Omicron‐specific  epitopes.  The  data  presented  herein  demonstrate  that  an  Omicron  breakthrough  infection  efficiently  broadens  neutralizing  antibody  and/or  B  cell  responses  towards multiple variants and suggest that a vaccine adapted to the Omicron S protein may  be able to reshape the immune repertoire.      Introduction  Containment  of  the  current  COVID‐19  pandemic  requires  the  generation  of  durable  and  sufficiently broad immunity that provides protection against circulating and future variants of  SARS‐CoV‐2. The titer of neutralizing antibodies to SARS‐CoV‐2, and the binding of antibodies  to the spike (S) glycoprotein and its receptor‐binding domain (RBD) are considered correlates  of protection against  infection  (D. S. Khoury et al., “Neutralizing antibody  levels are highly  predictive of immune protection from symptomatic SARS‐CoV‐2 infection,“ Nature medicine.  27, 1205–1211  (2021), doi:10.1038/s41591‐021‐01377‐8; and P. B. Gilbert et al., “Immune  correlates analysis of the mRNA‐1273 COVID‐19 vaccine efficacy clinical trial,“ Science (New  York, N.Y.). 375, 43–50  (2022), doi:10.1126/science.abm3425). Currently available vaccines  are  based  on  the  ancestral Wuhan‐Hu‐1  strain  and  induce  antibodies with  a  neutralizing  capacity that exceeds the breadth elicited by infection with the Wuhan strain, or with variants  of concern (VOCs) (K. Röltgen et al., “Immune imprinting, breadth of variant recognition, and  germinal  center  response  in  human  SARS‐CoV‐2  infection  and  vaccination,“  Cell  (2022),  doi:10.1016/j.cell.2022.01.018). However, protective titers wane over time (J. P. Evans et al.,  “Neutralizing antibody responses elicited by SARS‐CoV‐2 mRNA vaccination wane over time  and are boosted by breakthrough infection,“ Science translational medicine, eabn8057 (2022),  doi:10.1126/scitranslmed.abn8057; S. Yamayoshi et al., “Antibody titers against SARS‐CoV‐2  decline,  but  do  not  disappear  for  several months,“  EclinicalMedicine,  32,  100734  (2021),  doi:10.1016/j.eclinm.2021.100734; W. N. Chia et al., “Dynamics of SARS‐CoV‐2 neutralising  antibody  responses and duration of  immunity,“ The  Lancet Microbe, 2, e240‐e249  (2021),  doi:10.1016/S2666‐5247(21)00025‐2;  Y.  Goldberg  et  al.,  “Waning  Immunity  after  the  BNT162b2  Vaccine  in  Israel,“  The  New  England  journal  of  medicine.  385,  e85  (2021),  doi:10.1056/NEJMoa2114228. and routine booster vaccinations are thought to be needed to  trigger recall immunity and maintain efficacy against new VOCs (A. R. Falsey et al., “SARS‐CoV‐ 2 Neutralization with BNT162b2 Vaccine Dose 3,“ The New England journal of medicine. 385,  1627–1629 (2021), doi:10.1056/NEJMc2113468; A. Choi et al., “Safety and immunogenicity of  SARS‐CoV‐2 variant mRNA vaccine boosters  in healthy adults,“ Nature medicine. 27, 2025– 2031  (2021),  doi:10.1038/s41591‐021‐01527‐y;  and  N.  Andrews  et  al.,  “Effectiveness  of  COVID‐19 booster vaccines against covid‐19 related symptoms, hospitalisation and death  in  England,“ Nature medicine (2022), doi:10.1038/s41591‐022‐01699‐1 .  Long‐lived  memory  B  (BMEM)  cells  are  the  basis  for  the  recall  response  upon  antigen  reencounter either by  infection or booster vaccination. They play an  important  role  in  the  maintenance  and  evolution of  the  antiviral  antibody  response  against  variants,  since  low‐ affinity  selection  mechanisms  during  the  germinal  center  reaction  and  continued  hypermutation of BMEM cells expand the breadth of viral variant recognition over time ( W.  E.  Purtha, et al., “Memory B cells, but not long‐lived plasma cells, possess antigen specificities  for  viral  escape mutants,“  The  Journal  of  experimental medicine,  208,  2599–2606  (2011)  doi:10.1084/jem.20110740; and Y. Adachi et al., “Distinct germinal center selection at  local  sites shapes memory B cell response to viral escape,“ The Journal of experimental medicine.  212, 1709–1723 (2015), doi:10.1084/jem.20142284).  How  vaccine‐mediated protective  immunity will evolve over  time and will be modified by  iterations of exposure to COVID‐19 vaccines and  infections with  increasingly divergent viral  variants, is of particular relevance with the emergence of antigenically distinct VOCs. Omicron  is  the evolutionarily most distant  reported VOC with a hitherto unprecedented number of  amino acid alterations in its S glycoprotein, including at least 15 amino acid changes in the RBD  and extensive changes  in  the N‐terminal domain  (NTD). These alterations are predicted  to  affect most neutralizing antibody epitopes. In addition, Omicron is highly transmissible, and  its sublineages BA.1 and BA.2 have spread rapidly across the globe, outcompeting Delta within  weeks to become the dominant circulating VOC (W. Dejnirattisai et al., “SARS‐CoV‐2 Omicron‐ B.1.1.529 leads to widespread escape from neutralizing antibody responses,“ Cell. 185, 467‐ 484.e15 (2022), doi:10.1016/j.cell.2021.12.046; and   M. Hoffmann et al., “The Omicron  variant  is highly resistant against antibody‐mediated neutralization,“ Cell. 185, 447‐456.e11  (2022), doi:10.1016/j.cell.2021.12.032).   To date, over 1 billion people worldwide have been vaccinated with the mRNA‐based COVID‐ 19 vaccine BNT162b2 and have received the primary 2‐dose series or further boosters. This  vaccine is contributing substantially to the pattern of population immunity in many regions on  which further immune editing and effects of currently spreading variants will build upon.  To characterize the effect of Omicron breakthrough infection on the magnitude and breadth  of serum neutralizing activity and BMEM cells, blood samples from individuals that were double‐  or triple‐vaccinated with BNT162b2 were studied.  As understanding of the antigen‐specific B cell memory pool  is a critical determinant of an  individual’s ability to respond to newly emerging variants, this data can help to guide vaccine  development.      Results and discussion  Cohorts and sampling  Blood samples have been sourced from the biosample collection of BNT162b2 vaccine trials,  and a biobank of prospectively collected samples from vaccinated individuals with subsequent  SARS‐CoV‐2  Omicron  breakthrough  infection.  Samples  were  selected  to  investigate  biomarkers in four independent groups, namely individuals who were (i) double‐ or (ii) triple‐ vaccinated with BNT162b2 without a prior or breakthrough  infection at the time of sample  collection  (BNT162b22,  BNT162b23)  and  individuals  who  were  (iii)  double‐  or  (iv)  triple‐ vaccinated with BNT162b2 and who experienced breakthrough infection with the SARS‐CoV‐ 2  Omicron  variant  after  a  median  of  approximately  5  months  or  4  weeks,  respectively  (BNT162b22 + Omi, BNT162b23 + Omi) (see materials and methods below). Immune sera were  used to characterize Omicron infection‐associated changes to the magnitude and the breadth  of  serum  neutralizing  activity.  PBMCs  were  used  to  characterize  the  VOC‐specificity  of  peripheral BMEM cells recognizing the respective full‐length SARS‐CoV‐2 S protein or  its RBD  (Fig. 15).     Omicron  breakthrough  infection  of  BNT162b2  double‐  and  triple‐vaccinated  individuals  induces broad neutralization of Omicron BA.1, BA.2 and other VOCs  To evaluate the neutralizing activity of immune sera, two orthogonal test systems were used:  a well‐characterized  pseudovirus  neutralization  test  (pVNT)  to  investigate  the  breadth  of  inhibition  of  virus  entry  in  a  propagation‐deficient  set‐up,  as  well  as  a  live  SARS‐CoV‐2  neutralization test (VNT) designed to evaluate neutralization during multicycle replication of  authentic  virus with  the antibodies maintained  throughout  the entire  test period.  For  the  former,  pseudoviruses  bearing  S  proteins  comprising mutations  characteristic  of Omicron  sublineages BA.1 or BA.2, other SARS‐CoV‐2 VOCs (Wuhan, Alpha, Beta, Delta) were used to  assess  breadth  while    pseudoviruses  bearing  the  S  proteins  of  SARS‐CoV‐1  (T.  Li  et  al.,  “Phylogenetic  supertree  reveals  detailed  evolution  of  SARS‐CoV‐2,“  Scientific  reports,  10,  22366  (2020),  doi:10.1038/s41598‐020‐79484‐8)  was  used  to  detect  potential  pan‐ Sarbecovirus neutralizing activity (C.‐W. Tan et al., “Pan‐Sarbecovirus Neutralizing Antibodies  in BNT162b2‐Immunized SARS‐CoV‐1 Survivors,“ The New England journal of medicine, 385,  1401–1406 (2021), doi:10.1056/NEJMoa2108453).   As reported previously (A. R. Falsey et al., “SARS‐CoV‐2 Neutralization with BNT162b2 Vaccine  Dose  3,”  The  New  England  journal  of  medicine,  385,  1627–1629  (2021),  doi:10.1056/NEJMc2113468; and C.‐W. Tan et al., “Pan‐Sarbecovirus Neutralizing Antibodies  in BNT162b2‐Immunized SARS‐CoV‐1 Survivors,“ The New England journal of medicine. 385,  1401–1406  (2021),  doi:10.1056/NEJMoa2108453),  in  Omicron‐naïve  double‐vaccinated  individuals 50% pseudovirus neutralization (pVN50) geometric mean titers (GMTs) of Beta and  Delta  VOCs  were  reduced,  and  neutralization  of  both  Omicron  sublineages  was  virtually  undetectable.  In Omicron‐naïve triple‐vaccinated  individuals, pVN50 GMTs against all tested  VOCs were substantially higher with robust neutralization of Alpha, Beta and Delta variants.  While GMTs against Omicron BA.1 were significantly lower compared to Wuhan (GMT 160 vs  398),  titers  against  Omicron  BA.2  were  also  considerably  reduced  at  211.  Thus,  triple  vaccination induced a similar level of neutralization against the two Omicron sublineages  (Fig.  16, A) (A. Muik et al., “Neutralization of SARS‐CoV‐2 Omicron by BNT162b2 mRNA vaccine‐ elicited  human  sera,“  Science  (New  York,  N.Y.),  375,  678–680  (2022),  doi:10.1126/science.abn7591; C.‐W. Tan et al., “Pan‐Sarbecovirus Neutralizing Antibodies in  BNT162b2‐Immunized  SARS‐CoV‐1  Survivors,“  The New  England  journal  of medicine,  385,  1401–1406  (2021),  doi:10.1056/NEJMoa2108453;  J.  Liu  et  al.,  “BNT162b2‐elicited  neutralization  of  B.1.617  and  other  SARS‐CoV‐2  variants,“  Nature,  596,  273–275  (2021),  doi:10.1038/s41586‐021‐03693‐y;  A.  Muik  et  al.,  “Neutralization  of  SARS‐CoV‐2  lineage  B.1.1.7 pseudovirus by BNT162b2 vaccine‐elicited human sera,“ Science (New York, N.Y.), 371,  1152–1153  (2021), doi:10.1126/science.abg6105; and Y.  Liu et al.,  “Neutralizing Activity of  BNT162b2‐Elicited Serum,“ The New England  journal of medicine, 384, 1466–1468  (2021),  doi:10.1056/NEJMc2102017).  Omicron  breakthrough  infection  had  a marked  effect  on magnitude  and  breadth  of  the  neutralizing antibody response of both double‐ and triple‐vaccinated individuals, with slightly  higher pVN50 GMTs observed in the triple‐vaccinated individuals (Fig. 16, A). The pVN50 GMT  of double‐vaccinated individuals with breakthrough infection against Omicron BA.1 and BA.2  was more  than 100‐fold and 35‐fold above  the GMTs of Omicron‐naïve double‐vaccinated  individuals. Immune sera from double‐vaccinated individuals with breakthrough infection had  broad neutralizing activity, with higher pVN50 GMTs against Beta and Delta than observed in  Omicron‐naïve triple‐vaccinated individuals (GMT 740 vs. 222 and 571 vs. 370).  The effect of Omicron breakthrough infection on the neutralization of Omicron BA.1 and BA.2  pseudovirus  was  less  pronounced  when  looking  at  triple‐vaccinated  individuals  (approximately 7‐fold and 4‐fold increased neutralization compared to Omicron‐naïve triple‐ vaccinated  individuals). pVN50 GMTs against Omicron BA.1, BA.2 and Delta were 1029, 836  and 1103 in triple‐vaccinated Omicron breakthrough individuals as compared to 160, 211 and  370 in the Omicron‐naïve triple‐vaccinated. GMTs against all SARS‐CoV‐2 VOCs, including Beta  and Omicron, were close to titers against the Wuhan reference, while noticeably reduced in  triple‐vaccinated Omicron‐naïve individuals.   Likewise, while sera from vaccinated Omicron‐naïve individuals had no detectable or only poor  pVN50  titers  against  the  phylogenetically more  distant  SARS‐CoV‐1,  convalescent  sera  of  double‐ and even more markedly of triple‐vaccinated Omicron  infected  individuals robustly  neutralized SARS‐CoV‐1 pseudovirus (Fig. 16, A and B). Nine out of 18 breakthrough infected  individuals (four double‐vaccinated and five triple‐vaccinated) had SARS‐CoV‐1 pVN50 GMTs  comparable  to  or  above  those  against  the  Wuhan  reference  in  Omicron‐naïve  double‐ vaccinated individuals (GMT≥120).  Authentic live SARS‐CoV‐2 virus neutralization assays conducted with Wuhan, Beta, Delta and  Omicron BA.1 pseudoviruses also showed similar findings (Fig. 16, B).  In BNT162b2 double‐  and triple‐vaccinated individuals, Omicron infection was associated with a strongly increased  neutralizing activity against Omicron BA.1 with 50% virus neutralization (VN50) GMTs  in the  same range as against the Wuhan strain (Fig. 16, B; GMT 493 vs. 381 and GMT 538 vs. 613).  Similarly, Omicron convalescent double‐ and triple‐vaccinated individuals showed comparable  levels of neutralization against other variants as well (e.g., GMT 493 and 729 against Beta),  indicating a wide breadth of neutralizing activity.  In  aggregate,  these  data  demonstrate  that  SARS‐CoV‐2  Omicron  breakthrough  infection  induces  neutralization  activity  of  profound  breadth  in  vaccine‐experienced  individuals,  a  finding further supported by the calculated ratios of VN50 GMTs against the Wuhan strain and  SARS‐CoV‐2  VOCs  (Fig.  16,  C). While  double‐  and  to  a  lesser  extent  also  triple‐BNT162b2  vaccinated  Omicron‐naïve  individuals  displayed  marked  differences  in  neutralization  proficiency against VOCs, neutralization activity of Omicron convalescent subjects was leveled  to almost the same range of high performance against all variant strains tested.  Likewise, Omicron breakthrough  infection had a  similarly broad neutralization augmenting  effect  in  individuals  vaccinated  with  other  approved  COVID‐19  vaccines  or  heterologous  regimens (Fig. 19; Table 29).  Table 29. Individuals vaccinated with other approved COVID‐19 vaccines or mixed regimens  after subsequent Omicron breakthrough infection 
Figure imgf000648_0001
Figure imgf000649_0001
n/a, not available; N/A, not applicable; AZ, AstraZeneca AZD1222; BNT, BioNTech/Pfizer  BNT162b2; J&J, Johnson & Johnson Ad26.COV2.S; MOD, Moderna mRNA‐1273; BNT4,  BNT162b2 four‐dose series; MOD2, mRNA‐1273 two‐dose series; MOD3, mRNA‐1273 three‐ dose series    BMEM cells of BNT162b2 double‐ and of triple‐vaccinated  individuals broadly recognize VOCs  and are further boosted by Omicron breakthrough infection  Next, the phenotype and quantity of SARS‐CoV‐2 S protein specific B cells were investigated.  Flow  cytometry‐based  B  cell  phenotyping  assays  were  used  for  differential  detection  of  variant‐specific S protein‐binding B cells in bulk PBMCs. All S protein‐ and RBD‐specific B cells  in the peripheral blood were found to be of a BMEM phenotype (BMEM; CD20highCD38int/neg), as  antigen‐specific plasmablasts or naïve B cells were not detected (data not shown). The assays  therefore allowed the differentiation for each of the SARS‐CoV‐2 variants between BMEM cells  recognizing the full S protein or its RBD that is a hotspot for amino acid alterations, and variant‐ specific antigenic epitopes (Fig. 17, A).   The overall  frequency of antigen‐specific BMEM cells varied across  the different groups. The  frequency of BMEM cells  in Omicron‐naïve double‐vaccinated  individuals was  low at an early  time point after vaccination and  increased over time: At 5 months as compared to 3 weeks  after the second BNT162b2 dose, S protein‐specific BMEM cells almost quadrupled, RBD‐specific  ones tripled across all VOCs thereby reaching quantities similar to those observed in Omicron‐ naïve triple‐vaccinated individuals (Fig. 17, B and C).  Double or triple BNT162b2‐vaccinated individuals with a SARS‐CoV‐2 Omicron breakthrough  infection exhibited a strongly increased frequency of BMEM cells, which was higher than those  of Omicron‐naïve triple‐vaccinated individuals (Fig. 17, B and D).   In all groups,  including Omicron‐naïve and Omicron  infected  individuals, BMEM cells against  Omicron BA.1 S protein were detectable at frequencies comparable to those against Wuhan  and other tested VOCs (Fig 17, B and D), whereas the frequency of BMEM cells against Omicron  BA.1 RBD was slightly lower compared to the other variants (Fig. 17, C and E).   The ratios of RBD protein to S protein binding within the different groups was then compared  and found to be biased towards S protein recognition for the Omicron BA.1 VOC, particularly  in  the Omicron‐naïve  groups  (Fig.  17,  F).  In  the Omicron‐experienced  groups  this  ratio  is  higher,  indicating  that  an  Omicron  breakthrough  infection  improved  Omicron  BA.1  RBD  recognition.     Omicron  breakthrough  infection  in  BNT162b2  double‐  and  triple‐vaccinated  individuals  primarily boosts BMEM cells against conserved epitopes shared broadly between S proteins of  Wuhan and other VOCs rather than strictly Omicron S‐specific epitopes.   These  findings  indicate  that  Omicron  infection  in  vaccinated  individuals  boosts  not  only  neutralizing activity and BMEM  cells against Omicron BA.1, but broadly augments  immunity  against various VOCs. To  investigate the specificity of antibody responses at a cellular  level,  multi‐parameter analyses of BMEM cells stained with fluorescently labeled variant‐specific S or  RBD proteins were performed.   A  combinatorial gating  strategy was applied  to distinguish between BMEM cell  subsets  that  could identify only single variant‐specific epitopes of Wuhan, Alpha, Delta or Omicron BA.1,  versus those that could identify any given combination thereof (Fig 18, A).   In a first analysis, BMEM cell recognition of Wuhan and Omicron BA.1 S and RBD proteins was  evaluated (Fig. 18, B, C, and D). The SARS‐CoV‐2 Omicron variant has 37 amino acid alterations  in the S protein compared to the Wuhan parental strain, of which 15 alterations are  in the  RBD, an immunodominant target of neutralizing antibodies induced by COVID‐19 vaccines or  by SARS‐CoV‐2 infections.   Staining with  full  length  S proteins  showed  that  the  largest proportion of BMEM  cells  from  Omicron‐naïve  double‐vaccinated  individuals,  and  even more  predominantly  from  triple‐ vaccinated  individuals were directed against epitopes shared by both Wuhan and Omicron  BA.1 SARS‐CoV‐2 variants. Consistent with the observation that vaccination with BNT162b2  can  elicit  immune  responses  against  wild‐type  epitopes  that  do  not  recognize  the  corresponding  altered  epitopes  in  the Omicron BA.1  S protein  (Fig. 18, B  and C),  in most  individuals a smaller but clearly detectable proportion of BMEM cells was found that recognized  only Wuhan S protein or RBD. Consistent with  the  lack of exposure, no BMEM cells binding  exclusively  to  Omicron  BA.1  S  or  RBD  protein  were  detected  in  these  Omicron‐naïve  individuals.  In Omicron convalescent individuals, frequencies of BMEM cells recognizing S protein epitopes  shared between Wuhan and Omicron BA.1 were significantly higher than in the Omicron‐naïve  ones (Fig. 18, B and C). In most of these subjects, a small proportion of exclusively Wuhan S  protein‐specific  BMEM  cells was  found,  as well  as  a  slightly  lower  frequency  of  exclusively  Omicron BA.1 variant S protein‐specific ones.  A  similar  but  slightly  different  pattern was  observed  by  B  cell  staining with  labeled  RBD  proteins  (Fig.  18,  B  and  D).  Again,  Omicron  breakthrough  infection  of  double‐/triple‐ vaccinated  individuals  was  found  to  primarily  boost  BMEM  cells  reactive  with  conserved  epitopes. A moderate boost of Wuhan‐specific reactivities was observed; however, only small  populations of Omicron‐RBD‐specific BMEM cells were detected in the tested individuals (Fig.  18, D).  Next, the combinatorial gating approach was used to identify the subsets of S protein or RBD  binding BMEM  cells  that either bind exclusively  to Wuhan or Omicron BA.1, or  to  common  epitopes conserved broadly across all four variants, Wuhan, Alpha, Delta and Omicron BA.1  (Fig 18, E). Across all  four  study groups,  the  frequency of BMEM  cells  recognizing S protein  epitopes was  found  to be  conserved  across  all  tested  variants,  accounting  for  the  largest  fraction of the pool of S protein‐binding BMEM cells (Fig. 18, F, all 4+ve). The S protein of the  Wuhan strain does not have an exclusive amino acid change  that distinguishes  it  from  the  spike proteins of the Alpha, Delta, or Omicron BA.1 VOCs. Accordingly, BMEM cells exclusively  recognizing the Wuhan S protein were hardly detected in any individual (Fig. 18, F). In several  individuals  with  Omicron  breakthrough  infection,  a  small  proportion  of  BMEM  cells  was  detected that bound exclusively to Omicron BA.1 S protein (Fig. 18, F), whereas almost none  of the individuals displayed a strictly Omicron BA.1 RBD‐specific response (Fig. 18, G).   These  findings  indicate  that  SARS‐CoV‐2  Omicron  breakthrough  infection  in  vaccinated  individuals primarily expands a broad BMEM cell repertoire against conserved S protein and RBD  epitopes, rather than inducing large numbers of Omicron‐specific BMEM cells.   To  further  dissect  this  response,  the  BMEM  subsets  directed  against  the  RBD  were  characterized. The combinatorial Boolean gating approach was used to discern BMEM cells with  distinct binding patterns  in  the  spectrum of  strictly  variant‐specific  and  common epitopes  shared by several variants. Multiple sequence alignments revealed that the Omicron BA.1 RBD  diverges from the RBD sequence regions conserved in Wuhan, Alpha and Delta by 13 single  amino  acid  alterations.  All  Omicron  convalescent  individuals  were  found  to  have  robust  frequencies of BMEM cells that recognized Wuhan, Alpha as well as the Delta VOC RBDs, but  not Omicron BA.1 RBD, while BMEM cells exclusively  reactive with Omicron BA.1 RBD were  almost absent in most of those individuals (Fig. 18, H). BMEM cells that exclusively recognized  the  Omicron  BA.1  and  Alpha  RBDs,  or  the  Omicron  BA.1  and  Delta  RBDs were  also  not  detected.  Furthermore,  in  all  individuals  two  additional  subsets  of  RBD‐specific  BMEM  cells  were  identified. One subset was characterized by binding to Wuhan, Alpha and Omicron BA.1, but  not Delta, RBD. The other population exhibited binding to Wuhan and Alpha but not Omicron  BA.1 or Delta RBD (Fig. 18, H). Sequence alignment identified L452R as the only RBD mutation  unique for Delta that is not shared by the other 3 variant RBDs (Fig. 18, I top). Similarly, the  only RBD site conserved in Wuhan and Alpha but altered in Delta and Omicron BA.1 was found  to be T478K (Fig 18, I bottom). Both L452R and T478K alterations are known to be associated  with the evasion of vaccine induced neutralizing antibody responses. Of note, no BMEM cells  were detected in all combinatorial subgroups in which multiple sequence alignment failed to  identify unique epitopes in the RBD sequence that satisfied the Boolean selection criteria (e.g.,  Wuhan only or Wuhan and Omicron BA.1, but not Alpha, Delta). These findings indicate that  the BMEM cell response against RBD is driven by specificities induced through prior vaccination  with BNT162b2 and not substantially  redirected against new RBD epitopes mutated  in  the  Omicron variant after infection.    Summary   SARS‐CoV‐2 Omicron  is a partial  immune escape variant with an unprecedented number of  amino acid alterations in the S protein at sites of neutralizing antibody binding, distinguishing  it  from previously  reported  variants. Recent neutralizing antibody mapping  and molecular  modeling  studies  strongly  support  the  functional  relevance of  these alterations,  and  their  importance  is  confirmed  by  the  observation  that  double‐vaccinated  individuals  have  no  detectable neutralizing activity against SARS‐CoV‐2 Omicron .   The  findings  presented  herein  show  that  Omicron  breakthrough  infection  of  vaccinated  individuals boosts not only neutralizing activity and BMEM cells against Omicron but broadly  augments immunity against various VOCs, and also provide insights into how broad immunity  is acheived  The data presented herein  indicate that  initial exposure to the Wuhan strain S protein may  have shaped the formation of BMEM cells and imprinted against the formation of novel BMEM  cell  responses  against  the  more  distinctive  epitopes  of  the  Omicron  variant.  Similar  observations have been reported from vaccinated individuals who experienced breakthrough  infections with  the delta variant  (K. Röltgen et al., “Immune  imprinting, breadth of variant  recognition, and germinal center response in human SARS‐CoV‐2 infection and vaccination,“  Cell  (2022),  doi:10.1016/j.cell.2022.01.018.).    As  demonstrated  in  the  present  Example,  Omicron  breakthrough  infection  primarily  expands  a  broad  BMEM  cell  repertoire  against  conserved S protein and RBD epitopes, rather than inducing considerable numbers of strictly  Omicron‐specific BMEM cells.   Thus, Omicron breakthrough infection in double‐vaccinated individuals leads to expansion of  the pre‐existing BMEM cell pool, similar to a third dose of booster vaccination. However, there  are  clear  differences  in  the  immune  response  pattern  induced  by  a  homologous  vaccine  booster as compared to an Omicron breakthrough  infection. Despite the focus of the B cell  memory response on conserved epitopes, Omicron breakthrough  infection  leads to a more  substantial increase in antibody neutralization titers against Omicron, as well as pronounced  cross‐neutralization of both the ancestral and the novel SARS CoV‐2 variants. These effects are  particularly striking in double‐vaccinated individuals.  Without  wishing  to  be  bound  by  theory,  three  findings  may  point  to  potentially  complementary and synergistic mechanisms responsible for these results:    First,  an  overall  increase  of  S  protein‐specific  BMEM  cells.  Omicron‐convalescent  double‐ vaccinated individuals have a higher frequency of BMEM cells and higher neutralizing antibody  titers  against  all  VOCs  as  compared  to  triple‐vaccinated  individuals.  That  breakthrough  infection elicits a stronger neutralizing antibody response than the 3rd vaccine dose in double‐ vaccinated  individuals  is  not  apparent  from  previous  studies  describing  breakthrough  infections  with  other  variants  (Evans  et  al.,  Science  Translational  Medicine  (2022)  14,  eabn8057) and may be explained by poor neutralization of the Omicron variant in the initial  phase of infection, potentially causing a greater or prolonged antigen exposure of the immune  system to the altered S protein.  Second, a stronger bias on RBD‐specific BMEM cell responses. Omicron breakthrough infection  promotes proportionally more pronounced boosting of RBD‐specific BMEM cells than of BMEM  cells that recognize S protein‐specific epitopes outside the RBD. Therefore, Omicron‐infected  individuals have a significantly higher ratio of RBD/S protein‐specific BMEM cells compared to  vaccinated Omicron‐naïve individuals. The RBD is a key domain of the S protein that binds to  the SARS‐CoV‐2 receptor ACE2 and has multiple neutralizing antibody binding sites in regions  that are not affected by Omicron alterations, e.g.,  position  L452. An increased focus of the  immune response on this domain could promote BMEM cells producing neutralizing antibodies  against RBD epitopes that are not altered in Omicron.    Third, the  induction of broadly neutralizing antibodies. The majority of sera  from Omicron‐ convalescent  but  not  from  Omicron‐naïve  vaccinated  individuals  was  found  to  robustly  neutralize  SARS‐CoV‐1.  This may  indicate  that Omicron  infection  in  vaccinated  individuals  stimulates  BMEM  cells  that  form  neutralizing  antibodies  against  spike  protein  epitopes  conserved  in  the  SARS‐CoV‐1  and  SARS‐CoV‐2  families.  It  was  reported  that  broadly  neutralizing  antibodies  are  present  in  SARS‐CoV‐1  infected  individuals  vaccinated  with  BNT162b2.    Such  pan‐Serbecovirus  immune  responses  are  thought  to  be  triggered  by  neutralizing antibodies to highly conserved S protein domains. The greater antigenic distance  of  the Omicron spike protein  from  the other SARS‐Cov‐2 strains may promote  targeting of  conserved subdominant neutralizing epitopes as  recently described  to be  located  in  the C‐ terminal portion of the spike protein.   In aggregate, these results indicate that despite possible imprinting of the immune response  by  previous  vaccination,  the  preformed  B‐cell  memory  pool  can  be  refocused  and  quantitatively remodeled by exposure to heterologous S proteins to allow neutralization of  variants that evade a previously established neutralizing antibody response.     In conclusion, while the data are based on samples from individuals exposed to the Omicron  S protein as a result of infection, the findings presented herein support that a vaccine adapted  to the Omicron S protein can similarly reshape the B‐cell memory repertoire and therefore  can be more beneficial  than an extended series of boosters with  the existing Wuhan‐Hu‐1  spike based vaccines.    Materials and Methods  Recruitment of participants and sample collection  Individuals  from  the SARS‐CoV‐2 Omicron‐naïve BNT162b2 double‐vaccinated  (BNT162b22)  and  triple‐vaccinated  (BNT162b23)  cohorts  provided  informed  consent  as  part  of  their  participation  in a clinical  trial  (the Phase 1/2  trial BNT162‐01  [NCT04380701],  the Phase 2  rollover trial BNT162‐14 [NCT04949490], or as part of the BNT162‐17 [NCT05004181] trial).  Participants from the SARS‐CoV‐2 Omicron convalescent double‐ and triple vaccinated cohorts  (BNT162b22 + Omi and BNT162b23 + Omi cohorts,  respectively) and  individuals vaccinated  with  other  approved  COVID‐19  vaccines  or  mixed  regimens  with  subsequent  Omicron  breakthrough infection were recruited from University Hospital, Goethe University Frankfurt  as  part  of  a  research  program  that  recruited  patients  that  had  experienced  Omicron  breakthrough  infection  following  vaccination  for COVID‐19,  to provide blood  samples  and  clinical  data  for  research.  Infection with  the Omicron  strain was  confirmed with  variant‐ specific PCR or  sequencing,  and participants were  free of  symptoms  at  the  time of blood  collection.   Sampling timepoints are provided in Fig. 15.   Serum was isolated by centrifugation 2000 x g for 10 minutes and cryopreserved until use. Li‐ Heparin blood  samples were  isolated by density gradient  centrifugation using Ficoll‐Paque  PLUS (Cytiva) and were subsequently cryopreserved until use.     VSV‐SARS‐CoV‐2 S variant pseudovirus generation  A recombinant replication‐deficient vesicular stomatitis virus (VSV) vector that encodes green  fluorescent  protein  (GFP)  and  luciferase  instead  of  the  VSV‐glycoprotein  (VSV‐G)  was  pseudotyped with SARS‐CoV‐1 spike (S) (UniProt Ref: P59594) and with SARS‐CoV‐2 S derived  from either the Wuhan reference strain (NCBI Ref: 43740568), the Alpha variant (mutations:  Δ69/70,  Δ144,  N501Y,  A570D,  D614G,  P681H,  T716I,  S982A,  D1118H),  the  Beta  variant  (mutations: L18F, D80A, D215G, Δ242–244, R246I, K417N, E484K, N501Y, D614G, A701V), the  Delta  variant  (mutations:  T19R,  G142D,  E156G,  Δ157/158,  K417N,  L452R,  T478K,  D614G,  P681R, D950N) the Omicron BA.1 variant (mutations: A67V, Δ69/70, T95I, G142D, Δ143‐145,  Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K,  E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K,  D796Y, N856K, Q954H, N969K, L981F) or the Omicron BA.2 variant (mutations: T19I, Δ24‐26,  A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K,  S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K,  D796Y, Q954H, N969K) according to published pseudotyping protocols (M. Berger Rentsch, G.  Zimmer,  A  vesicular  stomatitis  virus  replicon‐based  bioassay  for  the  rapid  and  sensitive  determination  of  multi‐species  type  I  interferon.  PloS  one.  6,  e25858  (2011),  doi:10.1371/journal.pone.0025858).   In brief, HEK293T/17 monolayers (ATCC® CRL‐11268™) cultured in Dulbecco’s modified Eagle’s  medium  (DMEM) with GlutaMAX™  (Gibco)  supplemented with  10%  heat‐inactivated  fetal  bovine  serum  (FBS  [Sigma‐Aldrich])  (referred  to as medium) were  transfected with Sanger  sequencing‐verified  SARS‐CoV‐1  or  variant‐specific  SARS‐CoV‐2  S  expression  plasmid with  Lipofectamine LTX (Life Technologies) following the manufacturer’s instructions. At 24 hours  VSV‐G complemented VSVΔG vector. After incubation for 2 hours at 37 °C with 7.5% CO2, cells  were washed twice with phosphate buffered saline (PBS) before medium supplemented with  anti‐VSV‐G antibody  (clone 8G5F11, Kerafast  Inc.) was added  to neutralize  residual VSV‐G‐ complemented input virus. VSV‐SARS‐CoV‐2‐S pseudotype‐containing medium was harvested  20 hours after inoculation, passed through a 0.2 µm filter (Nalgene) and stored at ‐80 °C. The  pseudovirus batches were titrated on Vero 76 cells (ATCC® CRL‐1587™) cultured in medium.  The  relative  luciferase  units  induced  by  a  defined  volume  of  a Wuhan  spike  pseudovirus  reference batch previously described in Muik et al. (Muik et al., “Neutralization of SARS‐CoV‐ 2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine‐elicited human sera. Science (New York,  N.Y.).  371,  1152–1153  (2021),  doi:10.1126/science.abg6105“)  that  corresponds  to  an  infectious titer of 200 transducing units (TU) per mL, was used as a comparator. Input volumes  for the SARS‐CoV‐2 variant pseudovirus batches were calculated to normalize the infectious  titer based on the relative luciferase units relative to the reference.     Pseudovirus neutralization assay  Vero 76 cells were seeded in 96‐well white, flat‐bottom plates (Thermo Scientific) at 40,000  cells/well  in medium 4 hours prior to  the assay and cultured at 37  °C with 7.5% CO2. Each  serum was serially diluted 2‐fold in medium with the first dilution being 1:5 (Omicron naïve  double‐ and triple BNT162b2 vaccinated; dilution range of 1:5 to 1:5,120) or 1:30 (double‐ and  triple BNT162b2 vaccinated after subsequent Omicron breakthrough infection; dilution range  of 1:30 to 1:30,720). VSV‐SARS‐CoV‐2‐S/VSV‐SARS‐CoV‐1‐S particles were diluted in medium  to obtain 200 TU in the assay. Serum dilutions were mixed 1:1 with pseudovirus (n=2 technical  replicates  per  serum  per  pseudovirus)  for  30 minutes  at  room  temperature  before  being  added  to  Vero  76  cell monolayers  and  incubated  at  37  °C  with  7.5%  CO2  for  24  hours.  Supernatants were  removed  and  the  cells were  lysed with  luciferase  reagent  (Promega).  Luminescence was  recorded on a CLARIOstar® Plus microplate  reader  (BMG Labtech), and  neutralization titers were calculated as the reciprocal of the highest serum dilution that still  resulted in 50% reduction in luminescence. Results were expressed as geometric mean titers  (GMT) of duplicates. If no neutralization was observed, an arbitrary titer value of half of the  limit of detection [LOD] was reported.     Live SARS‐CoV‐2 neutralization assay  SARS‐CoV‐2 virus neutralization titers were determined by a microneutralization assay based  on  cytopathic effect  (CPE)  at VisMederi  S.r.l.,  Siena,  Italy.  In brief, heat‐inactivated  serum  samples from participants were serially diluted 1:2 (starting at 1:10) and incubated for 1 hour  at 37 °C with 100 TCID50 of live Wuhan‐like SARS‐CoV‐2 virus strain 2019‐nCOV/ITALY‐INMI1  (GenBank: MT066156), Beta virus strain Human nCoV19 isolate/England ex‐SA/HCM002/2021  (mutations:  D80A,  D215G,  Δ242–244,  K417N,  E484K,  N501Y,  D614G,  A701V),  sequence‐ verified Delta strain isolated from a nasopharyngeal swab (mutations: T19R, G142D, E156G,  Δ157/158,  L452R,  T478K,  D614G,  P681R,  R682Q,  D950N)  or  Omicron  BA.1  strain  hCoV‐ 19/Belgium/rega‐20174/2021 (mutations: A67V, Δ69/70, T95I, G142D, Δ143‐145, Δ211, L212I,  ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R,  G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K,  Q954H, N969K, L981F) to allow any antigen‐specific antibodies to bind to the virus. The 2019‐ nCOV/ITALY‐INMI1  strain  S protein  is  identical  in  sequence  to  the wild‐type  SARS‐CoV‐2  S  (Wuhan‐Hu‐1 isolate). Vero E6 (ATCC® CRL‐1586™) cell monolayers were inoculated with the  serum/virus mix in 96‐well plates and incubated for 3 days (2019‐nCOV/ITALY‐INMI1 strain)  or 4 days (Beta, Delta and Omicron BA.1 variant strain) to allow infection by non‐neutralized  virus. The plates were observed under an inverted light microscope and the wells were scored  as positive for SARS‐CoV‐2 infection (i.e., showing CPE) or negative for SARS‐CoV‐2 infection  (i.e., cells were alive without CPE). The neutralization titer was determined as the reciprocal  of the highest serum dilution that protected more than 50% of cells from CPE and reported as  GMT of duplicates. If no neutralization was observed, an arbitrary titer value of 5 (half of the  limit of detection [LOD]) was reported.     Detection and characterization of SARS‐CoV‐2‐specific B cells with flow cytometry  Spike/RBD‐specific B cells were detected using recombinant, biotinylated SARS‐CoV‐2 Spike  (Acro Biosystems: Wuhan – SPN‐C82E9, Alpha – SPN‐C82E5, Delta – SPN‐C82Ec, Omicron –  SPN‐C82Ee) and RBD (Acro Biosystems: Wuhan – SPD‐B28E9, Alpha – SPD‐C82E6, Delta – SPD‐ C82Ed,  Omicron  –  SPD‐C82E4)  proteins.  Recombinant  Spike  and  RBD  proteins  were  tetramerized with  fluorescently  labeled  Streptavidin  (BioLegend,  BD  Biosciences)  in  a  4:1  molar ratio for 1 h at 4 °C in the dark. Afterwards samples were spun down for 10 min at 4°C  to remove eventual precipitates.   For  flow cytometric analysis, PBMCs were thawed and 5x106 cells per sample were seeded  into 96 U‐bottom plates. Cells were blocked for Fc‐receptor‐binding (Human BD Fc Block™, BD  Biosciences) and statured with  free biotin  (D‐Biotin,  Invitrogen, 1 µM)  in  flow buffer  (DPBS  (Gibco) supplemented with 2% FBS (Sigma), 2 mM EDTA (Sigma‐Aldrich)) for 20 min at 4 °C.  Cells were washed and labeled with BCR bait tetramers supplemented with free Biotin in flow  buffer (D‐Biotin, Invitrogen, 2 µg/ml) for 1 h at 4 °C  in the dark (2 µg/ml for Spike and 0,25  µg/ml for RBD proteins). Cells were washed with flow buffer and stained for viability (Fixable  Viability  Dye  eFluor™  780,  eBioscience)  and  surface  markers  (CD3  –  clone:  UCHT1(BD  Biosciences), CD4 – clone: SK3 (BD Biosciences), CD185 (CXCR5) – clone: RF8B2 (BioLegend),  CD279 (PD‐1) – clone: EH12.1(BD Biosciences), CD278 (ICOS) – clone: C398.4A (BioLegend) ,  CD19‐ clone: SJ25C1(BD Biosciences), CD20 – clone: 2H7(BD Biosciences), CD21 – clone: B‐ ly4(BD Biosciences),  CD27 – clone: L128(BD Biosciences), CD38 – clone: HIT2(BD Biosciences),  CD11c – clone: S‐HCL‐3(BD Biosciences), CD138 – clone: MI15(BD Biosciences),  IgG  ‐ clone:  G18‐145(BD  Biosciences),  IgM  –  clone:  G20‐127(BD  Biosciences),  IgD  –  clone:  IA6‐2(BD  Biosciences), CD14 – clone: MφP9  (BD Biosciences, dump channel), CD16 – clone: 3G8  (BD  Biosciences, dump channel)) in flow buffer supplemented with Brilliant Stain Buffer Plus (BD  Biosciences, according to the manufacturer’s  instructions) for 20 min at 4 °C. Samples were  washed  and  fixed  with  BDTM  Stabilizing  Fixative  (BD  Biosciences,  according  to  the  manufacturer’s instructions) prior to data acquisition on a BD Symphony A3 flow cytometer.  FCS  3.0  files were  exported  from  BD  Diva  Software  and  analyzed  using  FlowJo  software  (Version 10.7.1.).  Debris and doublets were discriminated via FSC/SSC. Then dead cells and monocytes (CD14,  CD16 – Viability/Dump channel) were excluded. CD19 positive B cells were analyzed for IgD  and CD27 expression, thereby naïve B cells were discriminated as IgD+ cells with the Boolean  ‘make  non‐gate’  function.  Within  non‐naïve  B  cells  Plasmablasts  (CD38high  CD20low)  and  memory B cells (BMEMs CD38int/lowCD20high) were distinguished. BMEM cells were analyzed for B  cell bait binding. SARS‐CoV‐2 Spike reactivities were assessed by gating on each Spike/RBD  variant tested by plotting against the CD20 signal. Bait gates were overlayed onto total BMEM  cells and displayed as NxN‐Plots for the four bait channels.    Statistical analysis  The statistical method of aggregation used for the analysis of antibody titers is the geometric  mean and for the ratio of SARS‐CoV‐2 VOC titer and Wuhan titer the geometric mean and the  corresponding 95% confidence interval. The use of the geometric mean accounts for the non‐ normal distribution of antibody titers, which span several orders of magnitude. The Friedman  test with Dunn’s correction for multiple comparisons was used to conduct pairwise signed‐ rank tests of group geometric mean neutralizing antibody titers with a common control group.  Flow  cytometric  frequencies  were  analyzed  with  and  tables  were  exported  from  FlowJo  software (Version 10.7.1.). Statistical analysis of cumulative memory B cell frequencies was  the mean and standard errors of the mean (SEM). All statistical analyses were performed using  GraphPad Prism software version 9.  Example 8: Induced antibody response of vaccines encoding a SARS‐CoV‐2 S protein from an  Omicron variant  To  test the efficacy of an RNA vaccine encoding a SARS‐CoV‐2 S protein comprising one or  more mutations  characteristic  of  an  Omicron  variant,  subjects  previously  administered  a  primary regimen comprising two doses of 30 ug of RNA encoding a SARS‐CoV‐2 S protein from  a Wuhan strain (e.g., BNT162b2), and a booster regimen comprising a dose of 30 ug of RNA  encoding a SARS‐CoV‐2 S protein from a Wuhan strain (i.e., a Wuhan specific booster, e.g.,   BNT162b2) were  administered  a  further  booster  dose  comprising  either  (i)  30  ug  of  RNA  encoding an SARS‐CoV‐2 S protein from a Wuhan strain (e.g., BNT162b2), or (ii) 30 ug of RNA  encoding a SARS‐CoV‐2 S protein comprising mutations that are characteristic of an Omicron  variant  (i.e.,  an  Omicron  specific  booster,  e.g.,  RNA  encoding  a  SARS‐CoV‐2  S  protein  comprising  an  amino  acid  sequence  of  SEQ  ID  NO:  49,  and/or  comprising  a  nucleotide  sequence of SEQ ID NOs: 50 and/or 51) (the dose administered as part of the second booster  regimen is referred to as a “4th dose“ in the figures).  Sera was collected from subjects at the  time of administering the second booster regimen and one month afterwards.  Neutralization  antibody  titers  were  determined  using  a  Fluorescent  Focus  Reduction  Neutralization Test (“FFRNT“).  Suitable FFRNT assays are known in the art, and include, e.g.,  the assays described in Zou J, Xia H, Xie X, et al. “Neutralization against Omicron SARS‐CoV‐2  from previous non‐Omicron  infection," Nat Commun 2022;13:852, the contents of which  is  incorporated by reference herein in its entirety.  Additional exemplary neutralization assays  include those described in the previous examples, as well as those described in Bewley, Kevin  R., et al. "Quantification of SARS‐CoV‐2 neutralizing antibody by wild‐type plaque reduction  neutralization,  microneutralization  and  pseudotyped  virus  neutralization  assays."  Nature  Protocols 16.6  (2021): 3114‐3140.   As shown  in Fig. 20, A, subjects administered a second  booster  regimen  comprising  a  dose  of  RNA  encoding  a  SARS‐CoV‐2  S  protein  comprising  mutations  characteristic  of  an  Omicron  variant  exhibited  significant  increases  in  concentrations  of  neutralization  antibodies  against  an  Omicron  variant,  as  compared  to  subjects administered a second booster regimen comprising a dose of RNA encoding a SARS‐ CoV‐2 S protein of a Wuhan strain.   Specifically, subjects administered an Omicron specific  booster exhibited a GMR that was 1.79‐fold higher and a GMFR that was 2.31 fold higher than  that observed in subjects administered a fourth dose of RNA encoding a SARS‐CoV‐2 S protein  from a Wuhan strain.  The superior immune response induced by an Omicron‐specific booster  against an Omicron variant was further increased in subjects previously infected with SARS‐ CoV‐2  (as  determined  by  an  antigen  test)  or  currently  infected  with  SARS‐CoV‐2  (as  determined by PCR).  See Fig. 20, B, which shows that a subject population including previously  and/or currently infected subjects exhibited a GMR that is 2.94 fold higher, and a GMFR ratio  that  is 1.97  fold higher that observed  in a subject population comprising previously and/or  currently  infected  subjects administered an RNA vaccine encoding a SARS‐CoV‐2 S protein  froma Wuhan strain.    Pseudovirus  neutralization  assays were  also  performed  using  a  pseudovirus  comprising  a  SARS‐CoV‐2  S  protein  of  a Wuhan  strain,  using  the  same  sera  samples  discussed  above.   Subjects  administered  RNA  encoding  a  SARS‐CoV‐2  S  protein  from  a Wuhan  strain  (e.g.,  BNT162b2) exhibited titers of neutralization antibodies that were similar to those observed in  subjects administered an Omicron‐specific booster, demonstrating that the two vaccines are  at  least similarly effective  in  their ability  to  induce an antibody  response against a Wuhan  strain.  See Fig., 20C, which shows that the GMR and GMFR observed in subjects administered  a Wuhan specific booster (e.g., BNT162b2) is similar to that observed in subjects administered  an Omicron specific booster (OMI).  In subjects previously infected with SARS‐CoV‐2 (e.g., as  determined by an antigen assay) or currently infected with SARS‐CoV‐2 (e.g., as determined  by  a  PCR  assay),  subjects  administered  an  Omicron  specific  booster  demonstrated  an  improved  immune  response as compared  to subjects administered a booster specific  for a  Wuhan strain.  See Fig., 20D, which shows that the GMR for subjects administered an Omicron  specific booster is about 1.4 fold that of subjects administered a Wuhan specific booster.  Subjects  administered  an Omicron  specific booster  also demonstrated  a  superior  immune  response against a delta variant in pseudovirus neutralization assays.  See Fig., 20E, showing  that the GMFR for subjects administered an Omicron‐specific booster is about 1.20 fold higher  than that observed in subjects administered a Wuhan specific booster.  The superior immune  response induced by an Omicron specific booster against a delta variant was further increased  in sera from subjects previously and/or currently infected with SARS‐CoV‐2.  See Fig., 20F.    Example 9: Immunogenicity study of Vaccines Encoding S proteins of SARS‐CoV‐2 Variants  in Vaccine‐Naïve Subjects   To  test  the  immunogenicity  of  various  variant  specific  vaccines  in  vaccine  naïve  subjects,  vaccine naïve mice were immunized twice with (a) saline (negative control), (b) an RNA vaccine  encoding a SARS‐CoV‐2 S protein from a Wuhan strain, (c) an RNA vaccine encoding a SARS‐ CoV‐2  S  protein  having mutations  characteristic  of  an Omicron  variant  (Omi),  (d)  an RNA  vaccine encoding a SARS‐CoV‐2 S protein having mutations characteristic of a delta variant  (Delta), (e) a bivalent vaccine comprising RNA encoding a SARS‐CoV‐2 S protein from a Wuhan  strain and a SARS‐CoV‐2 S protein comprising mutations characteristic of an Omicron variant  (b2+Omi), and (f) a bivalent vaccine comprising RNA encoding a SARS‐CoV‐2 S protein having  mutations characteristic of a delta variant and RNA encoding a SARS‐CoV‐2 S protein having  mutations characteristics of an Omicron variant (Delta+Omi). The immunogenicity of the RNA  vaccines was investigated by focusing on the antibody immune response.  Sera was obtained 7 days after immunization, and analyzed using a pseudovirus neutralization  assay (e.g., the assay described in Example 2), using pseudoviruses comprising a SARS‐CoV‐2  S protein from a Wuhan strain, a SARS‐CoV‐2 S protein comprising mutations characteristic of  a beta variant, a SARS‐CoV‐2 S protein comprising mutations characteristic of a delta variant,  or  a  SARS‐CoV‐2  S protein  comprising mutations  characteristic of  an Omicron  variant.   As  shown  in Fig., 21, bivalent vaccines were  found  to elicit  the broadest  immune  response  in  vaccine naïve mice.  Example  10:  Induced  antibody  response  of  vaccines  encoding  a  SARS‐CoV‐2  S  protein  comprising one or more mutations characteristic of a Beta variant  in  subjects previously  administered an RNA vaccine encoding a SARS‐CoV‐2 S protein from a Wuhan strain  To  test the efficacy of an RNA vaccine encoding a SARS‐CoV‐2 S protein comprising one or  more mutations characteristic of a Beta variant, subjects previously administered a primary  regimen comprising two doses each of 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a  Wuhan strain (in the present example, BNT162b2 (SEQ  ID NO: 20)), were administered two  booster doses, each comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein comprising one  or more mutations characteristic of a Beta variant  (referred  to hereafter as a Beta‐specific  vaccine).  In the present Example, construct RBP020.11 was administered as the Beta‐specific  vaccine.  While  in  the  present  Example,  the  two  booster  doses  were  administered  approximately  one  month  apart,  in  some  embodiments,  the  two  booster  doses  can  be  administered at least 3 weeks apart, at least 4 weeks apart, at least 5 weeks apart, at least 6  weeks apart, at least 7 weeks apart, at least 8 weeks apart, or longer (e.g., in accordance with  exemplary dosing regimens as described herein).  Sera were  collected  from  subjects  before  administeration  of  BNT162b2,  one month  after  administering two primary doses of BNT162b2, one month after administering a first dose of  a Beta‐specific vaccine, and one month after administering a second dose of a Beta‐specific  vaccine.   Neutralization  antibody  titers  against  a  pseudovirus  comprising  a  SARS‐CoV‐2  S  protein  of  a Wuhan  strain  or  a  SARS‐CoV‐2  S  protein  comprising  one  or more mutations  characteristic  of  a  Beta  variant  were measured  using  a  pseudovirus  neutralization  assay  (results  shown  in Fig. 22).   Subjects exhibited an  increase  in neutralization antibody  titers  against both a Wuhan strain of SARS‐CoV‐2 and a Beta varaint following adminstration of the  third and fourth doses of a Beta‐specific vaccine.    Example  11:  Induced  antibody  response  of  vaccines  encoding  a  SARS‐CoV‐2  S  protein  comprising one or more mutations characteristic of a Beta variant in vaccine naïve subjects  To test the efficacy of an RNA vaccine encoding a SARS‐CoV‐2 S protein comprising one or  more mutations characteristic of a Beta variant in vaccine naïve subjects, subjects who had  not previouly been administered a SARS‐CoV‐2 vaccine, and did not show evidence of prior  or current infection with SARS‐CoV‐2 (e.g., as assessed by an antibody test and/or a PCR test)  were administered two doses each of 30 ug of RNA encoding a SARS‐CoV‐2 S protein  comprising one or more mutations characteristic of a Beta variant (in the present example,  RBP020.11).  Sera was collected one month after administration of a second dose, and  neutralization antibody titers were measured using a viral neutralization assay, using viral  particles comprising either a SARS‐CoV‐2 S protein from a Wuhan strain or a SARS‐CoV‐2 S  protein having one or more mutations characteristic of a Beta variant.  Tables 22 and 23,  below, show the results for the neutralization assay against Beta variant (results for the  neutralization assay against a Wuhan strain are not shown).  As shown in the tables,  compared to vaccine‐naïve subjects administered two doses of an RNA vaccine encoding a  SARS‐CoV‐2 S protein from a Wuhan strain (in the present Example, BNT162b2), an RNA  vaccine encoding a SARS‐CoV‐2 S protein having mutations characteristic of a Beta variant  was found to induce a significantly stronger antibody response against a Beta variant.       
Figure imgf000665_0001
Figure imgf000666_0001
Example  12:  Induced  antibody  response  and  reactogenecity  of  BNT162b2  or  Omicron‐ specific vaccine as monovalent, bivalent and high dose in participants 55+ years of age  To test the efficacy and safety of (i) higher doses of RNA vaccines (e.g., as described herein),  (ii) RNA vaccines encoding a SARS‐CoV‐2 S protein having one or more mutations characteristic of an Omicron variant (an Omicron specific vaccine), and (iii) a bivalent vaccine comprising an RNA encoding a SARS‐CoV‐2 S protein from a Wuhan variant and RNA encoding a SARS‐CoV‐2 S  protein  having  one  or  more  mutations  characteristic  of  an  Omicron  variant,  subjects previously administered at least one dose of an RNA vaccine encoding a SARS‐CoV‐2 S protein of a Wuhan strain were administered one of several booster doses (e.g., as described herein). Specifically, subjects who had previously been administered two doses of 30 ug of an RNA vaccine  encoding  a  SARS‐CoV‐2  S  protein  from  a Wuhan  strain  (in  the  present  example, BNT162b2), and a third dose of 30 ug of RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain (also BNT162b2 in the present example), were administered a fourth dose comprising: (a) 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, (b) 60 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain, (c) 30 ug of an Omicron‐specific vaccine, (d) 60 ug of an Omicron‐specific vaccine, e) 30 ug of a bivalent RNA vaccine (Omicron‐adapted bivalent vaccine), comprising 15 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 15 ug of RNA encoding a SARS‐CoV‐2 S protein comprising mutations characteristic of an Omicron variant, or (f) 60 ug of a bivalent RNA vaccine (Omicron‐adapted bivalent vaccine), comprising 30 ug of RNA encoding a SARS‐CoV‐2 S protein from a Wuhan strain and 30 ug of RNA encoding a SARS‐CoV‐2 S protein comprising mutations characteristic of an Omicron variant. In the present example, for the fourth dose, the RNA encoding a SARS‐CoV‐2 S protein from a  Wuhan  variant  was  BNT162b2,  and  the  RNA  encoding  a  SARS‐CoV‐2  S  protein  having  mutations characteristic of an Omicron variant comprised the nucleotide sequence of SEQ ID  NO: 51.    Sera samples were collected at the time of administering the 4th dose and 7 days afterward,  and tested for neutralization antibody titers against a viral particle comprising a SARS‐CoV‐2 S  protein from a Wuhan strain, or a SARS‐CoV‐2 S protein comprising mutations characteristic  of a Delta variant or an Omicron variant.    Neutralization  antibody  titers  were  determined  using  a  Fluorescent  Focus  Reduction  Neutralization Test  (“FFRNT“).   Suitable FFRNT assays are known  in the art, as discussed  in  Example 8.  The neutralization responses are shown in Fig. 23.  As shown in Fig. 23 (A) subjects administered a fourth dose of 30 ug of an Omciron‐specific  vaccine  exhibited  an  increase  in  neutralization  antibodies  against  an  Omicron  variant  as  compared to subjects administered a fourth dose of 30 ug of BNT162b2.  Administering 60 ug  of  RNA  increased  neutralization  responses  both  for  BNT162b2  and  an  Omicron‐specific  vaccine, with  60  ug  of  an Omicron‐specific  vaccine  showing  a  stronger  immune  response  against  an  Omicron  variant.  As  shown  in  Fig.  23  (B),  similar  effects  were  observed  in  a  population that  included subjects previously or currently  infected with SARS‐CoV‐2 (e.g., as  determined by an antibody test and a PCR test, respectively).  Fig. 23 (C‐D) provides data for neutalization responses against a Wuhan strain of SARS‐CoV‐2  in a population of subjects excluding subjects previously or currently infected with SARS‐CoV‐ 2 (Fig. 23(C)) and a population of subjects including these subjects (Fig. 23(D)).  Fig. 23 (E‐F) provides data for neutralization responses against a Delta variant in a population  of subjects excluding subjects previously or currently infected with SARS‐CoV‐2 (Fig. 23(E)) and  a population of subjects including these subjects (Fig. 23(F)).    Fig. 23 (G) shows neutralization responses as compared to subjects administered a 4th dose  of 30 ug of BNT162b2.   As can be seen  in the table, an Omicron‐specific vaccine  induced a  strong response against an Omicron variant, and responses that were at least comparable to  that of BNT162b2 for other variants.  A bivalent vaccine (Omicron‐adapted bivalent vaccine)  produced a strong immue response against each SARS‐CoV‐2 variant tested, both at 30 ug and  60 ug doses.  Reactogenicity of the tested  4th doses was also monitored in patients for 7 days following  administration of the 4th dose.  Fig. 24 (A) shows local immune responses observed in  subjects of different groups as indicated.  As can be seen in the figure, 60 ug doses of an  Omicron specific vaccine and a bivalent vaccine were found to be more likely to produce  pain at the injection site, as compared to that observed with other tested booster doses;  however, the pain was rated as mild or moderate for both doses.  Redness and swelling  responses were low and comparable at each dose tested.  Fig. 24 (B) shows systemic immune responses observed in subjects of different groups as  indicated.  Systemic responses (as characterized by fever, fatigue, headache, chills, vomiting,  diarrhea, muscle pain, or joint pain) were similar for each dose, while fatigue trended higher  with the 60 ug doses.   The immune responses and reactogenicity of Omicron‐adapted vaccines (monovalent and  bivalent vaccines as described in this Example) as a booster dose are also confirmed in a  Phase 2/3 trial in over 1,000 participants 56 years of age and older. The Omicron‐adapted  vaccines (monovalent or bivalent; and 30 ug or 60 ug) given as a booster dose elicited  substantially higher neutralizing antibody responses against Omicron BA.1 when compared  to that induced by BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan strain). The  pre‐specified criterion for superiority was measured by the ratio of neutralizing geometric  mean titers (GMR) with the lower bound of the 95% confidence interval >1. The geometric  mean ratios (GMRs) for the monovalent Omicron‐specific vaccine (30 µg and 60 µg)  compared to that induced by BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan  strain) were 2.23 (95% CI: 1.65, 3.00) and 3.15 (95% CI: 2.38, 4.16), respectively. The GMRs  for the Omicron‐adapted bivalent 30 µg and 60 µg vaccines (as described in this Example)  compared to that induced by BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan  strain) were 1.56 (95% CI: 1.17, 2.08) and 1.97 (95% CI: 1.45, 2.68), respectively. The  monovalent Omicron‐specific vaccine 30 µg and 60 µg achieved a lower bound 95%  confidence interval for GMR of >1.5, demonstrating superiorty against Omicron and  satisfying the regulatory requirement of super superiority.  One month after administration, a booster dose of the Omicron‐adapted monovalent  vaccine (30 µg and 60 µg) increased neutralizing geometric mean titers (GMT) against  Omicron BA.1 13.5 and 19.6‐fold above pre‐booster dose levels, while a booster dose of the  Omicron‐adapted bivalent vaccine (30 µg and 60 µg) conferred a 9.1 and 10.9‐fold increase  in neutralizing GMTs against Omicron BA.1. Both Omicron‐adapted vaccines (e.g.,  monovalent and bivalent vaccines) were well‐tolerated in participants who received one or  the other Omicron‐adapted vaccine, and demonstrated a favorable safety and tolerability  profile similar to that of BNT162b2 (encoding a SARS‐CoV‐2 S protein from a Wuhan strain). Additionally, in a SARS‐CoV‐2 live virus neutralization assay tested on sera from participants  over 56 years of age and older receiving an Omicron‐adapted vaccine (e.g., monovalent or  bivalent vaccine as described in this Example), sera also neutralized Omicron BA.4/BA.5 with  titers lower than Omicron BA.1.   Example  13:  Omicron  breakthrough  infection  drives  cross‐variant  neutralization  and  memory B cell formation, but to a lesser extent against Omicron BA.4 and BA.5  New Omicron sublineages that harbor further alterations in the SARS‐CoV‐2 S protein  continue to arise, with BA.4 and BA.5 deemed VOCs by the European Centre for Disease  Prevention and Control (ECDC) on the May 12, 2022 (Euopean Centre for Disease Prevention  and Control, Epidemiological update: SARS‐CoV‐2 Omicron sub‐lineages BA.4 and BA.5  (2022) (available at https://www.ecdc.europa.eu/en/news‐events/epidemiological‐update‐ sarscov‐2‐omicron‐sub‐lineages‐ba4‐and‐ba5)).    The present Example 13 is an extension of Example 7, in which the serum samples collected  from BA.1‐breathrough cases as described in Example 7 were further analyzed for their  neutralization activity against Omicron BA.4 and BA.5 variants.   As described in Example 7, in Omicron‐naïve double‐vaccinated individuals, 50% pseudovirus  neutralization (pVN50) geometric mean titers (GMTs) of Beta and Delta VOCs were found to  be reduced as compared to the Wuhan strain, while neutralization of Omicron sublineages  BA.1 and BA.2 was virtually undectable.  In this present Example, Fig. 25(a) shows that  neutralization titers of BA.4/5 was also virtually undetectable in double‐vaccinated, BA.1‐ breakthrough patients. As described in Example 7, Omicron‐naïve triple‐vaccinated individuals exhibited pVN50  GMTs against all tested VOCs that were substantially higher as compared to double‐ vaccinated individuals.  Robust neutralization of Alpha, Beta and Delta variants was  observed, while neutralization of Omicron BA.1 and BA.2 was reduced as compared to  Wuhan (GMT 160 and 211 vs 398).   As shown in Fig. 25(A) of the present Example,  neutralization of Omicron BA.4/5 was further reduced (GMT 74) in Omicron‐naïve triple‐ vaccinating patients, corresponding to a 5‐fold lower titer as compared to the Wuhan strain.  As shown in Fig. 25(b), Omicron BA.1 breakthrough infection was found to have only a minor  boosting effect on neutralization of BA.4/5.  In double‐vaccinated patients, pVN50 GMTs  against Omicron BA.4/5 were significantly below those against Wuhan (GMT 135 vs. 740).  A  similar pattern was observed with BA.1 convalescent and control sera from triple‐vaccinated  individuals.  As noted in Example 7, BA.1 convalescent sera exhibited high pVN50 GMTs  against previous SARS‐CoV‐2 VOCs, including Beta (1182), Omicron BA.1 (1029), and Omicron  BA.2 (836), which were close to titers against the Wuhan reference (1182).  In contrast, as  shown in Fig. 25(b), neutralization of BA.4/5 in triple‐vaccinated individuals with a  breakthrough infection of BA.1 was significantly reduced as compared to the Wuhan strain,  with pVN50 GMTs of 197, 6‐fold lower than against the Wuhan strain.  Of note, in all cohorts, neutralizing titers against BA.4/5 were closer to the low level  observed against the phylogenetically more distant SARS‐CoV‐1 pseudovirus than that seen  against Wuhan.  Comparing the ratios of SARS‐CoV‐2 VOC and SARS‐CoV‐1 pVN50 GMTs  normalized against Wuhan (Fig. 25(c)), it is remarkable that breakthrough infection with  Omicron BA.1 does not lead to more efficient cross‐neutralization of Omicron BA.4/5 in  double‐vaccinated and triple‐vaccinated individuals. In aggregate, these data demonstrate  that Omicron BA.1 breakthrough infection of vaccine‐experienced individuals mediates  broadly neutralizing activity against BA.1, BA.2 and several previous SARS‐CoV‐2 variants, but  not for BA.4/5.  As shown in Fig. 26, similar results were found for patients previously administered a non‐ BNT162b2 vaccine and who had a BA.1 breakthrough infection.  As described  in  Example  7, Omicron BA.1 breakthrough  infection  in BNT162b2‐vaccinated  individuals was found to produce strong neutralizing activity against Omicron BA.1, BA.2 and  previous SARS‐CoV‐2 VOCs, primarily by expanding BMEM cells against epitopes shared broadly  across the different SARS‐CoV‐2 strains.  These data demonstrate that a vaccination‐imprinted  BMEM cell pool has sufficient plasticity to be remodeled by exposure to a heterologous SARS‐ CoV‐2 S protein. While selective amplification of BMEM cells recognizing shared epitopes allows  for  effective  neutralization  of most  variants  that  evade  previously  established  immunity,  susceptibility to escape by variants that acquire alterations at hitherto conserved sites may be  heightened. The  significantly  reduced  neutralizing  activity  against  the  Omicron  BA.4/5  pseudovirus, which harbors the additional alterations L452R and F486V in the RBD, supports  a mechanism of immune evasion by loss of the few remaining conserved epitopes.  Discussion  Surprisingly, and contrary to the results observed in Example 7, neutralization of Omicron  sublineages BA.4 and BA.5 was not enhanced in BA.1‐breakthrough patients, with titers  instead comparable to those against the phylogenetically more distant SARS‐CoV‐1. While  the present Example focused on individuals vaccinated with the BNT162b2 mRNA vaccine, in  individuals vaccinated with CoronaVac (a whole, inactivated virus vaccine developed by  Sinovac Biotech), similar observations have recently been reported, suggesting that Omicron  BA.4/5 can bypass BA.1 infection‐mediated boosting of humoral immunity (Y. Cao et al.,  BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection, bioRxiv: the  preprint server for biology (2022)).  The present disclosure provides insights into how immunity against multiple variants is  achieved in BA.1 breakthrough cases, why Omicron BA.4 and BA.5 sublineages can partially  escape neutralization, and provides vaccination protocols and technologies to enhance  protection across coronavirus strains and lineages, specifically including across Omicron  lineages (e.g., including BA.4 and/or BA.5). Without wishing to be bound by any particular  theory, the present disclosure proposes that initial exposure to the Wuhan strain S protein  may shape formation of BMEM cells and imprint against novel BMEM cell responses recognizing  epitopes distinctive for the Omicron BA.1 variant.  Omicron BA.1 breakthrough infection in BNT162b2‐vaccinated individuals primarily expands  a broad BMEM cell repertoire against conserved SARS‐CoV‐2 S protein and RBD epitopes,  rather than inducing strictly Omicron BA.1‐specific BMEM cells.  As compared to the immune  response induced by a homologous vaccine booster, an Omicron BA.1 breakthrough  infection leads to a more substantial increase in antibody neutralization titers against  Omicron and a robust cross‐neutralization of many SARS CoV‐2 variants.   As noted in Example 7, one potential explanation for the broad neutralization elicted by a  BA.1 breakthrough infection is the induction of broadly neutralizing antibodies. Sera from  Omicron BA.1‐convalescent vaccinated individuals was found to neutralize SARS‐CoV‐2  Omicron BA.4/5 and SARS‐CoV‐1 to a far lesser extent than previous SARS‐CoV‐2 VOCs  including BA.1 and BA.2. This finding indicates that Omicron BA.1 infection in vaccinated  individuals stimulates BMEM cells that produce neutralizing antibodies against S protein  epitopes conserved in the SARS‐CoV‐2 variants up to and including Omicron BA.2, but that  have mostly been lost in BA.4/5 and are for the most part not shared by SARS‐CoV‐1.  The greater antigenic distance of the Omicron BA.1 S protein from earlier SARS‐CoV‐2 strains  may promote targeting of conserved subdominant neutralizing epitopes as recently  described to be located, e.g., in cryptic sites within a portion of the RBD distinct from the  receptor‐binding motif (Li, Tingting, et al. "Cross‐neutralizing antibodies bind a SARS‐CoV‐2  cryptic site and resist circulating variants," Nature communications 12.1 (2021): 1‐12, and  Yuan, Meng, et al. "A highly conserved cryptic epitope in the receptor binding domains of  SARS‐CoV‐2 and SARS‐CoV" Science 368.6491 (2020): 630‐633) or in the membrane proximal  S glycoprotein subunit designated S2 (Pinto, Dora, et al. "Broad betacoronavirus  neutralization by a stem helix–specific human antibody." Science 373.6559 (2021): 1109‐ 1116. Li, Wenwei, et al. "Structural basis and mode of action for two broadly neutralizing  antibodies against SARS‐CoV‐2 emerging variants of concern." Cell reports 38.2 (2022):  110210; Hurlburt, Nicholas K., et al. "Structural definition of a pan‐sarbecovirus neutralizing  epitope on the spike S2 subunit." Communications biology 5.1 (2022): 1‐13).  As noted in Example 7, Omicron BA.1‐infected individuals appear to have a significantly  higher RBD/S protein‐specific BMEM cell ratio as compared to vaccinated Omicron‐naïve  individuals. Omicron BA.1 carries multiple S protein alterations in key neutralizing antibody  binding sites of the NTD (such as del69/70 and del143‐145) that dramatically reduce the  targeting surface for memory B cell responses in this region. Although the Omicron BA.1 RBD  harbors multiple alterations, some neutralizing antibody binding sites are unaffected (20). An  expansion of BMEM cells that produce neutralizing antibodies against RBD epitopes that are  not altered in Omicron BA.1, such as those at position L452 as indicated in the present  Example, could help to rapidly restore neutralization of the BA.1 and BA.2 variants.  Importantly, the strong neutralization of Omicron BA.1 and BA.2 should not mask the fact  that the neutralizing BMEM immune response in Omicron BA.1 convalescent vaccinated  individuals is driven by a smaller number of epitopes. The significantly reduced neutralizing  activity against the Omicron BA.4/5 pseudovirus, which harbors the additional alterations  L452R and F486V in the RBD, demonstrates the mechanism of immune evasion by loss of the  few remaining conserved epitopes. Meanwhile, further sublineages with L452 alterations  (e.g., BA.2.12.1) are being reported to evade humoral immunity elicited by BA.1  breakthrough infection (Y. Cao et al., cited above).  The present disclosure proposes that immunity in the early stages of Omicron BA.1 infection  in vaccinated individuals may be based on recognition of conserved epitopes, and narrowly  focused on a small number of neutralizing sites that are not altered in Omicron BA.1 and  BA.2. Such a narrow immune response bears a high risk that those few epitopes may be lost  by acquisition of further alterations in the course of the on‐going evolution of Omicron and  may result in immune escape, as experienced with sublineages BA.2.12.1, BA.4 and BA.5 (Y.  Cao et al., cited above, and K. Khan et al., Omicron sub‐lineages BA.4/BA.5 escape BA.1  infection elicited neutralizing immunity (2022)). Importantly, Omicron BA.1 breakthrough  infection does not appear to reduce the overall spectrum of (Wuhan) S glycoprotein‐specific  memory B cells, as memory B cells that do not recognize Omicron BA.1 S remain detectable  in blood at similar frequencies. Wuhan‐specific (non‐Omicron BA.1 reactive) BMEM cells were  consistently detected in Omicron BA.1 breakthrough infected individuals at levels similar to  those in Omicron‐naïve double‐/triple‐vaccinated individuals. Withouth wishing to be bound  by any particular theory, the present disclosure notes that these findings may reflect an  increase of the total BMEM cell repertoire by selective amplification of BMEM cells that  recognize shared epitopes.  The present Example, among other things, provides insights that it may be more beneficial  for a subject who has been infected or administered at least one dose (including, e.g., at  least two , at least three doses) of vaccine(s) adapted to a Wuhan strain (e.g., but not limited  to a protein based vaccine or RNA‐based vaccines such as BNT162b2, Moderna mRNA‐1273)  to receive at least one dose of a vaccine (e.g., a protein or RNA‐based vaccine) adapted to a  strain that is not an Omicron BA.1. In some embodiments, a vaccine that is adapted to a  strain that is not an Omicron BA.1 can be or comprise a vaccine that is adapted to Omicron  BA.4 and/or Omicron BA.5. The present Example, among other things, also provides insights  that vaccine‐naïve subjects without prior SARS‐CoV‐2 infection may be desirable to be  administered a combination of vaccines, which comprises at least one dose of a vaccine  adapted to a WuHan strain (e.g., RNA vaccine such as in some embodiment BNT162b2) and  at least one dose of a vaccine adapted to a strain that is not an Omicron BA.1.  In some  embodiments, such vaccines in a combination may be administered at different times, for  example, in some embodiments as primary doses and/or booster doses administered apart  by a pre‐determined period of time (e.g., according to certain dosing regimens as described  herein).  In some embodiments, such vaccines in a combination may be administered as a  single multivalent vaccine.    MATERIALS AND METHODS  Serum samples, neutralization assays, and all other experiments described in the present  Example were performed as in Example 7.  The BA.4/5 VSV‐SARS‐CoV‐2 S variant  pseudovirus generation A recombinant replication‐deficient vesicular stomatitis virus (VSV)  vector that encodes green fluorescent protein (GFP) and luciferase instead of the VSV‐ glycoprotein (VSV‐G) was pseudotyped comprised a SARS‐CoV‐2 S protein comprising the  following mutations relative to the Wuhan strain: T19I, Δ24‐26, A27S, Δ69/70, G142D,  V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N,  T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y,  Q954H, N969K).    Example 14. Omicron BA.2 breakthrough infection of vaccinated individuals induces broad  cross neutralization against Omicron BA.1, BA.2 and other VOCs, including BA.4 and BA.5.   The present Example shows that a BA.2 Omicron breakthrough infection in individuals triple‐ vaccinated  with  BNT162b2  surprisingly  drives  superior  cross  variant  neutralization  as  compared  to  a BA.1‐breakthrough  infection  in  individuals  triple‐vaccinated with BN162b2,  including improved production of neutralizing antibodies against  a BA.4/5 Omicron variant.  Thus,  among  other  things,  the  present  disclosure  demonstrates  feasibility  of  defining  immunologically  synergistic categories of coronavirus  strains and/or  sequences  (e.g.,  spike  protein sequences).  In some embodiments of improved coronavirus vaccination strategies provided by the present  disclosure, a subject is exposed to each of at least two different such synergistic categories.  In  some embodiments, a subject is, has been or becomes infected with a virus of a first category,  and receives at least one dose of a vaccine of a second category, characterized by immunologic  synergy with the first category.  Alternatively or additionally, in some embodiments, a subject  receives or has received doses of first and second vaccines of such first and second categories.   In some embodiments, vaccines of different categories may be separately administered (e.g.,  at different points  in  time and/or  to different  sites on a  subject).    In  some embodiments,  vaccines of different categories may be administered together (e.g., at substantially the same  time and/or to approximately or exactly the same site and/or in a single composition).  As compared to Omicron BA.1 breakthrough infection of vaccinated individuals, which induces  lower neutralization against Omicron BA.4/BA.5 relative to neutralization against other SARS‐ CoV‐2 variants (including, e.g., Wuhan‐Hu‐1 strain, alpha variant, beta variant, delta variant,  Omicron BA.1, Omicron BA.2, and Omicron BA.2.11.2), the present Example shows that a BA.2  Omicron breakthrough infection in individuals vaccinated with BNT162b2 surprisingly drives  superior cross variant neutralization, including improved production of neutralizing antibodies  against  a BA.4/5 Omicron variant. Thus, in some embodiments, the present disclosure, among  other things, demonstrates that SARS‐CoV‐2 strains and/or variants can be grouped  into at  least two different categories such that a subject who is exposed to a SARS‐CoV‐2 strain and/or  variant  from  each  of  such  two  different  categories  can  benefit  from  immunologically  syngergistic protection conferred by such two different catergories. In some embodiments, a  first  category of  SARS‐CoV‐2  strains/variants  comprises: Wuhan‐Hu‐1  strain, alpha variant,  beta  variant,  delta  variant,  Omicron  BA.1,  and  subvariants  derived  from  aforementioned  strains and/or variants; while a second category comprises Omicron BA.2, Omicron BA.2.12.1,  Omicron BA.4/BA.5, and subvariants derived  from aforementioned strains and/or variants.  Thus, in some embodiments, the present disclosure, among other things, provide insights that  a combination of at least one dose (including, e.g., at least 1, at least 2, at least 3, at least 4,  or more) of a first vaccine (e.g., an mRNA vaccine as described herein that encodes a spike  protein polypeptide) that comprises or delivers a SARS‐CoV‐2 spike protein polypeptide with  a  sequence  characteristic  of  a  first  category  as  described  above,  and  at  least  one  dose  (including, e.g., at least 1, at least 2, at least 3, at least 4, or more) of a second vaccine (e.g.,  an mRNA vaccine described herein that encodes a spike protein polypeptide) that comprises  or delivers a SARS‐CoV‐2 spike protein polypeptide with a sequence characteristic of a second  category as described above can synergistically provide superior cross variant neutralization,  including enhanced production of neutralizing antibodies toward a BA.4/5 Omicron variant.  In  some  embodiments,  the  present  disclosure  specifically  teaches  surprising  efficacy  of  administering at  least one dose of a  vaccine  (e.g., an mRNA  vaccine  that encodes a  spike  protein  polypeptide  as  described  herein)  that  comprises  or  delivers  a  SARS‐CoV‐2  spike  protein polypeptide with sequences characteristic of a BA.2 Omicron variant  to subjects who  have received at least one (e.g., 2, 3, or more) doses of a vaccine (e.g., vaccine that encodes a  spike protein polypeptide as described herein) that comprises or delivers a SARS‐CoV‐2 spike  protein polypeptide with sequences characteristic of a Wuhan‐Hu‐1 strain).     Background  Emergence of the SARS‐CoV‐2 Omicron variant of concern (VOC) in November 2021 (Ref. 1)  can  be  considered  a  turning  point  in  the  COVID‐19  pandemic,  owing  to  its  ability  to  substantially escape previously established  immunity. Omicron BA.1, which displaced Delta  within weeks as the predominant circulating VOC, had acquired significant alterations in the  receptor binding domain (RBD) and N‐terminal domain (NTD) (Ref. 2). These changes resulted  in a  loss of many epitopes  recognized by neutralizing antibodies  (Refs. 3‐4) and drastically  impaired  humoral  immunity  induced by  vaccines based on  the  ancestral Wuhan  strain  or  exposure  to  the  ancestral  strain  or  previous  variants  (Refs.  5‐7).  BA.1 was  subsequently  displaced by the BA.2 variant, which in turn gave rise to further sub‐lineages. BA.4 and BA.5,  which are derived from BA.2, are currently becoming the dominant variants in many countries  across the globe with multiple studies suggesting a significant change in antigenic properties  compared to BA.2, and especially compared to BA.1 (Refs. 8‐9). As BA.4 and BA.5 share an  identical  S  glycoprotein  sequence,  they  are  referred herein  as BA.4/5. While many of  the  amino acid changes in the RBD are shared between Omicron sub‐lineages, alterations within  the NTD of BA.2‐derived sub‐lineages including BA.4/5 are mostly distinct from those found in  BA.1 (Figure 32).  A vast majority worldwide have been  immunized with  the vaccines adapted Wuhan strain,  including, e.g., mRNA vaccines such as BNT162b2 and mRNA‐1273 (Ref. 10), which have thus  substantially shaped SARS‐CoV‐2 population immunity. However, emergence of the immune  escape  variant  Omicron  BA.1  led  to  a  steep  increase  in  the  occurrence  of  breakthrough  infections  in  vaccinated  individuals.  It  has  been  reported  that  SARS‐CoV‐2  variant  breakthrough  infection  can  reshape  humoral  immunity,  thereby  modulating  neutralizing  antibody titers against other variants (Refs. 8, 11, 12). However, as previously reported, BA.1  breakthrough infection may not provide strong immunity against Omicron BA.4/5.      Certain findings  In order to determine if BA.2 breakthrough infection would refocus immunity against Omicron  BA.2  and  BA.2‐derived  sub‐lineages  such  as  BA.4/5,  the  magnitude  and  breadth  of  the  neutralizing antibody response was studied in samples from  individuals who had received a  triple  vaccination  scheme with mRNA  vaccines  (BNT162b2/mRNA‐1273)  and  subsequently  experienced  SARS‐CoV‐2  breakthrough  infections  between March  and May  2022,  during  which period the BA.2 lineage was dominant in Germany (All Vax + Omi BA.2).  Such findings  have  important  implications  for  ongoing  efforts  of  vaccine  design,  as  containment  of  the  COVID‐19 pandemic  requires  the generation of durable and sufficiently broad  immunity  to  provide protection against current and future variants of SARS‐CoV‐2.  Two reference cohorts were generated from data previously published in Quandt et al. (Ref.  12),  comprising  (i)  individuals  triple‐vaccinated  with  BNT162b2  without  a  prior  or  breakthrough  SARS‐CoV‐2  infection  at  the  time  of  sample  collection  (BNT162b23)  and  (ii)  individuals who were triple‐vaccinated with mRNA vaccines with subsequent breakthrough‐ infection during a period of Omicron BA.1 dominance (All Vax + Omi BA.1).  Breakthrough infection with the SARS‐CoV‐2 Omicron BA.1 and BA.2 occurred at a median of  approximately 4 months or 3 weeks, respectively, after triple‐vaccination with an mRNA‐based  COVID‐19  vaccine  (BNT162b2,  mRNA‐1273,  or  heterologous  regimens  comprising  both  vaccines; all Vax + Omi BA.1, all Vax + Omi BA.2) (Fig. 28). Immune sera used to characterize  serum  neutralizing  activity  were  collected  at  a median  28  days  post‐vaccination  for  the  BNT162b23 cohort, 43 days post‐BA.1 breakthrough for the All Vax + Omi BA.1 cohort, and 39  days post BA.2 breakthrough infection for the All Vax + Omi BA.2 cohort. Median ages of the  cohorts were  similar  (32‐38  years). The BA.2.12.1 neutralization data was generated  from  serum samples from cohorts BNT162b3 and All Vax+Om BA. 1 for this study.  To evaluate the neutralizing activity of immune sera, a pseudovirus neutralization test (pVNT),  for example, as described in Refs. 13, 14, were used. Pseudoviruses bearing the S glycoproteins  of  SARS‐CoV‐2 Wuhan,  Alpha,  Beta, Delta, Omicron  BA.1,  BA.2,  BA.2.12.1,  as well  as  the  recently emerged Omicron sub‐lineages BA.4 and BA.5 were applied to assess neutralization  breadth. As BA.4 and BA.5 share an identical S glycoprotein sequence, including key alterations  L452R and F486V, they are referred herein as BA.4/5. In addition, SARS‐CoV (herein referred  to  as  SARS‐CoV‐1;  Ref.  15) was  assayed  to  detect  potential  pan‐Sarbecovirus  neutralizing  activity.   As  reported previously  in Ref. 12, 50% pseudovirus neutralization  (pVN50) geometric mean  titers (GMTs) against Omicron BA.1 and BA.2 of immune sera from SARS‐CoV‐2 naïve triple‐ vaccinated individuals were considerably reduced compared to the Wuhan strain (GMT 160  and  221  versus  398). Neutralizing  activity  against  BA.2.12.1  and  BA.4/5 was  even  further  reduced (GMTs 111 and 74), corresponding to a 5.4‐fold lower titer for BA.4/5 as compared  to the Wuhan strain (Fig. 29(A)).  Omicron  BA.2  breakthrough  infection markedly  increased  pVN50  GMTs  against  BA.2  and  BA.2.12.1  compared  to  SARS‐CoV‐2‐naïve  triple‐vaccinated  immune  sera,  such  that  neutralization of BA.2 after breakthrough infection was comparable to the Wuhan strain (Fig.  30 (B‐C)). Similarly, BA.1 breakthrough infection conferred robust neutralizing activity against  BA.1 (Fig. 29(B), Fig. 30(A). Importantly, while pVN50 GMTs against BA.4/5 in BA.2 convalescent  sera were lower than against the Wuhan strain (GMTs 391 versus 922, i.e., 2.4‐fold reduction),  this reduction was still less than that observed in the Omicron‐naïve BNT162b23 cohort, whose  sera showed a 5.4‐fold reduction of BA.4/5 neutralizing activity (Fig. 30(C)). By contrast, pVN50  GMTs  against  BA.4/5  and Wuhan  after  BA.1  breakthrough  infection were  266  and  1327,  respectively (i.e., 5‐fold reduction; Fig. 29(B)). Hence, Omicron BA.1 breakthrough infection of  triple‐vaccinated  individuals did not  lead  to more efficient cross‐neutralization of Omicron  BA.4/5  as  compared  with  triple‐vaccinated  Omicron‐naïve  individuals.  In  both  cohorts,  neutralizing  titers  against  BA.4/5  were  closer  to  the  low  level  observed  against  the  phylogenetically more distant SARS‐CoV‐1 than that seen against Wuhan (Fig. 29). Of note, the  pVN50 GMTs against the Wuhan strain after BA.1 breakthrough infection were slightly higher  than those observed for BA.2 breakthrough infection (GMTs 1327 versus 922), which, without  wishing to be bound by a particular theory, may relate to the longer interval between the third  vaccination and the infection (median 22 days for BA.1 versus 127.5 days for BA.2) (Fig. 30).  A separate analysis was conducted including only individuals triple‐vaccinated with BNT162b2  (with  BA.2  or  BA.1  breakthrough  infections,  or  Omicron‐naïve).  In  these  analyses  similar  observations regarding BA.4/5 neutralizing activities were made: pVN50 GMTs against BA.4/5  in BA.2 convalescent sera were 2.4‐fold  lower than against the Wuhan strain, whereas the  reduction was 6‐fold after BA.1 breakthrough infections (Fig. 30). While relative neutralization  of BA.2 and BA.2.12.1 was comparable in BA.2 and BA.1 convalescent sera, neutralizing activity  against these variants remained slightly above that seen in Omicron‐naïve sera.  Immune sera from triple‐vaccinated Omicron naïve individuals had broad neutralizing activity  against ancestral SARS‐CoV‐2 VOCs. Neutralizing activity against Beta was slightly higher  in  BA.1 convalescent sera, whereas neutralization of Alpha and Delta was not affected by BA.1  or BA.2 breakthrough infections (Fig. 30(C)).  In aggregate, these data demonstrate that Omicron BA.2 breakthrough infections of vaccine‐ experienced individuals mediate broadly neutralizing activity against BA.1, BA.2, BA.2.12.1 and  several ancestral SARS‐CoV‐2 variants. Moreover, neutralizing activity against BA.4/5, while  lower  than  against  the  Wuhan  reference,  is  provided  to  a  larger  extent  than  in  BA.1  convalescent sera.   Recent studies have demonstrated that Omicron BA.1 breakthrough  infection  in  individuals  vaccinated with an mRNA vaccine (BNT162b2 or mRNA‐1273) boosts serum neutralizing titers  not only against the ancestral Wuhan strain, but also against VOCs including BA.2 (Refs. 8, 11,  12). This effect was seen in triple‐vaccinated individuals but was particularly evident in double‐ vaccinated  individuals,  whose  sera  contain  little  to  no  neutralizing  activity  against  BA.2.  However,  BA.1  breakthrough  infection  did  not  induce  strong  neutralizing  activity  against  BA.4/5, VOCs  that are currently establishing dominance worldwide. Without wishing  to be  bound by a particular theory, this  immune escape has been attributed to the amplification  and/or recall of pre‐existing neutralizing antibody responses that recognize epitopes absent  in the Omicron sub‐lineages BA.2.12.1, BA.4, and BA.5.  The present Example, among other things, provide insights that BA.2 breakthrough infections  trigger  recall  responses which mediate  enhanced  neutralization  of  the  BA.2‐derived  sub‐ lineages,  including BA.4/5,  indicating that higher S protein sequence similarity among BA.2,  BA.2.12.1, and BA.4/5 drives more efficient cross‐neutralization compared to breakthrough  infections with the more distant BA.1 variant. Notwithstanding the importance of vaccination  with  currently  approved Wuhan‐derived  vaccines  such  as  BNT162b2  that  offer  effective  protection from severe disease by current VOCs including Omicron BA.1 and BA.2, the present  findings of broadly cross‐neutralizing activity against current VOCs including BA.4/5 after BA.2  breakthrough infection provides insights, among other things, that a combination of a vaccine  adapted  to Wuhan strain sequence or a variant sequence  from  the same  immunologically‐ related category as discussed above (e.g., alpha strain, beta strain, delta strain,  Omicron BA.1)  and a vaccine adapted  to  the BA.2 variant sequence or a variant sequence  from  the same  immunologically‐related  category  as  discussed  above  (e.g.,  Omicron  BA.2.12.1,  Omicron  BA.4/BA.5)  can  provide  enhanced  cross‐neutralization  activity  against  variants  from  two  different  categories.  In  some  embodiments,  the  present  Example  provides  evidence  that  supports  implementation of  licensure procedures modelled on that of seasonal flu vaccines  that  use  the  latest  epidemiological  data  to  select  for  COVID‐19  vaccine  strains.  In  some  embodiments, the present Example further provides evidence that supports establishment of  rapid  strain  selection  for  seasonal  updates  of  COVID‐19  vaccines,  similar  to  the  selection  process practiced by the World Health Organization (WHO) Global Influenza Surveillance and  Response  System  (GISRS),  and/or  agreement  on  accelerated  approval  pathways based  on  surrogate immunogenicity endpoints.  Neutralization titers from subjects vaccinated against SARS‐CoV‐2 and who have had a BA.1  or a BA.2 breakthrough infection are shown in Fig. 30(A) and (B), respectively, and GMRs for  both groups of subjects are shown in Fig. 30(C).   As shown in Figs. 30(A) and (B), sera from  subjects previously vaccinated against SARS‐CoV‐2, and who had a breakthrough infection  with either BA.1 or BA.2, were found to have significant neutralization titers against  pseudovirus comprising a SARS‐CoV‐2 S protein of a Wuhan strain, an Alpha variant, a Beta  variant, a Delta Variant, and an Omicron BA.1 variant.  As noted previously, neutralization  titers against BA.2 are somewhat lower in sera from BA.1 breakthrough patients (GMT of 875  for BA.2 vs 1327 for Wuhan strain) and are lower still against BA.2.12.1 and BA.4/5 (GMTs of  584 and 266, respectively as compared to 1327 for Wuhan).  BA.2 breakthrough patients  show similar neutralization responses as BA.1 breakthrough patients against a SARS‐CoV‐2  Wuhan strain, Alpha variant, Beta variant, and Delta variant.  The neutralization response  against Omicron BA.1 is somewhat higher in BA.1‐breakthrough patients than in BA.2‐ breakthrough patients (GMR of 0.76 as compared to 0.60) , while neutralization titers against  Omicron BA.2 are higher in BA.2‐breakthrough patients than in BA.1‐breakthrough patients  (GMR of 0.94 vs 0.66).  Surprisingly, however, neutralization responses against BA.4/5 are  significantly higher in BA.2‐breakthrough patients (GMR of 0.39 in BA.2 breakthrough  patients, as compared to a GMR of 0.2 in BA.1 breakthrough subjects).  The present  disclosure therefore documents that a broader immune response can be elicited by a BA.2‐ breakthrough infection as compared to a BA.1 breakthrough infection in subject vaccinated  against SARS‐CoV‐2, and teaches that  administering a booster vaccine comprising RNA  encoding an S protein comprising mutations characteristic of a BA.2 Omicron variant can  achieve surprising and unexpected benefits.     Furthermore, the present disclosure provides an insight that, given similarities among  S  protein sequences of BA.2 and BA.4/5 variants, combining vaccination doses that comprise  or deliver BA.4 and/or BA.5 variant spike sequences with those of that comprise or deliver  Wuhan spike sequences may also achieve particularly broad immunization (i.e., synergistic  immunization as described herein).  In some embodiments,  these findings suggest that syngergistic categories of coronavirus  strain and/or variant sequences (e.g., SARS‐CoV‐2 strain and/or variant sequences) can be  defined, for example, in some embodiments based on shared amino acid alterations in S  glycoprotein of coronavirus strain and/or variant sequences. For example, while many of the  amino acid changes in the RBD of S protein are shared between Omicron sub‐lineages (e.g.,  BA.1, BA.2, BA.2.12.1, and BA.4/5), alterations within the NTD of BA.2 and BA.2‐derived sub‐ lineages including BA.4/5 are mostly distinct from those found in BA.1. Therefore, in some  embodiments, synergistic categories of coronavirus strain and/or variant sequences (e.g.,  SARS‐CoV‐2 strain and/or variant sequences) can be defined based on the degree of shared  amino acid mutations present with the NTD of a S protein. For example, in some  embodiments where two SARS‐CoV‐2 strain and/or variant sequences share at least 50%  (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more) of the amino  acid mutations present in the NTD of a S protein, both SARS‐CoV‐2 strain and variant  sequences can be grouped into the same category. In some embodiments where two SARS‐ CoV‐2 strain and/or variant sequences share no more 50% (including, e.g., no more than  45%, no more than 40%, no more than 30%, or lower) of the amino acid mutations present  in the NTD of a S protein, both SARS‐CoV‐2 strain and variant sequences can be grouped into  different categories. Among other things, the present findings provide insights that exposing  subjects (e.g., via infection and/or vaccination) to at least two antigens that are of different  synergistic categories (e.g., as shown in the table below) can produce a more robust immune  response (e.g., broadening the spectrum of cross‐neutralization against different variants  and/or producing an immune response that is less prone to immune escape).  
Figure imgf000683_0001
Figure imgf000684_0001
  For example, in some embodiments, vaccine‐naïve subjects without prior infection may be  administered a combination of vaccines, at least two of which are each adapted to a SARS‐ CoV‐2 strain of different synergistic caterogies (e.g., as described herein). In some  embodiments, such vaccines in a combination may be administered at different times, for  example, in some embodiments as a first dose and a second dose administered apart by a  pre‐determined period of time (e.g., according to certain dosing regimens as described  herein).  In some embodiments, such vaccines in a combination may be administered as a  multivalent vaccine. In some embodiments, subject infected or vaccinated with a SARS‐CoV‐ 2 strain of one category may be administered with a vaccine adapted to a SARS‐CoV‐2 strain  of a different category (e.g., as described herein). In some embodiments, such a vaccine may  be a polypeptide‐based or RNA‐based vaccine.  While  the  present  findings  are  based  on  retrospective  analyses  of  samples  derived  from  different studies, using relatively small samples sizes and cohorts that are not  fully aligned  regarding  immunization  intervals  and  demographic  characteristics  such  as  age  and  sex  of  individuals,  the present  findings provide useful  insights  for vaccine design and vaccination  strategies  for  improving  cross‐neutralization  against  a  broader  spectrum  of  SARS‐COV‐2  variants.     Materials and Methods  Recruitment of participants and sample collection  Individuals  from  the  SARS‐CoV‐2‐naïve  BNT162b2  triple‐vaccinated  (BNT162b23)  cohort  provided  informed  consent  as  part  of  their  participation  in  the  Phase  2  trial  BNT162‐17  (NCT05004181). Individuals with Omicron BA.1 or BA.2 breakthrough infection (All Vax + Omi  BA.1 and All Vax + Omi BA.2 cohorts) were triple‐vaccinated, e.g., with one or more doses of  BNT162b2,  Moderna  mRNA‐1273,  AstraZeneca  ChAdOx1‐S  recombinant  vaccine,  or  a  combination  thereof,  and were  recruited  to  provide  blood  samples  and  clinical  data  for  research.  Omicron  infections  were  confirmed  with  variant‐specific  PCR  either  between  November 2021 and mid‐January 2022 (All Vax + Omi BA.1) or between March 2022 and May  2022, at  times were sub‐lineages BA.1 or BA.2,  respectively, were dominant  (Ref. 24). The  infections  of  certain  participants  (e.g.,  at  least  7  participants)  in  this  study were  further  characterized by genome sequencing, and genome sequencing confirmed Omicron BA.1 or  BA.2 infection.  Participants were free of symptoms at the time of blood collection. Table 32 is a summary of  characteristics  of  vaccinated  individuals  analyzed  for  neutralizing  antibody  responses.  All  participants had no documented history of SARS‐CoV‐2 infection prior to vaccination.  Table 32 
Figure imgf000685_0001
N/A: not applicable; n/a, not available; D, Dose; Yrs, Years; n, Number.  *, Negative SARS‐CoV‐2 PCR test at the time of enrollment  #, No evidence of prior SARS‐CoV‐2 infection (based on COVID‐19 symptoms/signs and SARS‐ CoV‐2 PCR test)  Participants received the primary 2‐dose series of BNT162b2 vaccine as part of a  governmental vaccination program and the interval between doses was not recorded  †, Omicron BA.1 infection confirmed at time of recruitment to the research study.  Serum  was  isolated  by  centrifugation  of  drawn  blood  at  2000  x  g  for  10  minutes  and  cryopreserved until use.     VSV‐SARS‐CoV‐2 S variant pseudovirus generation   A recombinant replication‐deficient vesicular stomatitis virus (VSV) vector that encodes  green fluorescent protein (GFP) and luciferase instead of the VSV‐glycoprotein (VSV‐G) was  pseudotyped with SARS‐CoV‐1 S glycoprotein (UniProt Ref: P59594) and with SARS‐CoV‐2 S  glycoprotein derived from either the Wuhan reference strain (NCBI Ref: 43740568), the  Alpha variant (alterations: Δ69/70, Δ144, N501Y, A570D, D614G, P681H, T716I, S982A,  D1118H), the Beta variant (alterations: L18F, D80A, D215G, Δ242–244, R246I, K417N, E484K,  N501Y, D614G, A701V), the Delta variant (alterations: T19R, G142D, E156G, Δ157/158,  K417N, L452R, T478K, D614G, P681R, D950N) the Omicron BA.1 variant (alterations: A67V,  Δ69/70, T95I, G142D, Δ143‐145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F,  K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K,  D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), the Omicron  BA.2 variant (alterations: T19I, Δ24‐26, A27S, G142D, V213G, G339D, S371F, S373P, S375F,  T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H,  D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), the Omicron BA.2.12.1  variant (alterations: T19I, Δ24‐26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A,  D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H,  D614G, H655Y, N679K, P681H, S704L, N764K, D796Y, Q954H, N969K), or the Omicron BA.4/5  variant (alterations: T19I, Δ24‐26, A27S, Δ69/70, G142D, V213G, G339D, S371F, S373P,  S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R,  N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) according to  published pseudotyping protocols (Ref. 49).  A  diagram  of  SARS‐CoV‐2  S  glycoprotein  alterations  is  shown  in  Fig.  31  and  a  separate  alignment of S glycoprotein alterations in Omicron sub‐lineages is displayed in Fig. 32. In brief,  HEK293T/17  monolayers  (ATCC®  CRL‐11268™)  cultured  in  Dulbecco’s  modified  Eagle’s  medium  (DMEM) with GlutaMAX™  (Gibco)  supplemented with  10%  heat‐inactivated  fetal  bovine  serum  (FBS  [Sigma‐Aldrich])  (referred  to as medium) were  transfected with Sanger  sequencing‐verified  SARS‐CoV‐1  or  variant‐specific  SARS‐CoV‐2  S  expression  plasmid with  Lipofectamine LTX (Life Technologies) following the manufacturer’s instructions. At 24 hours  VSV‐G complemented VSVΔG vector. After incubation for 2 hours at 37 °C with 7.5% CO2, cells  were washed twice with phosphate buffered saline (PBS) before medium supplemented with  anti‐VSV‐G antibody  (clone 8G5F11, Kerafast  Inc.) was added  to neutralize  residual VSV‐G‐ complemented input virus. VSV‐SARS‐CoV‐2‐S pseudotype‐containing medium was harvested  20 hours after inoculation, passed through a 0.2 µm filter (Nalgene) and stored at ‐80 °C. The  pseudovirus batches were titrated on Vero 76 cells (ATCC® CRL‐1587™) cultured in medium.  The  relative  luciferase  units  induced  by  a  defined  volume  of  a  Wuhan  S  glycoprotein  pseudovirus reference batch previously described in Muik et al., 2021, that corresponds to an  infectious titer of 200 transducing units (TU) per mL, was used as a comparator. Input volumes  for the SARS‐CoV‐2 variant pseudovirus batches were calculated to normalize the infectious  titer based on the relative luciferase units relative to the reference.    Pseudovirus neutralization assay   Vero 76 cells were seeded in 96‐well white, flat‐bottom plates (Thermo Scientific) at 40,000  cells/well in medium 4 hours prior to the assay and cultured at 37 °C with 7.5% CO2. Each  individual serum was serially diluted 2‐fold in medium with the first dilution being 1:5  (Omicron‐naïve triple BNT162b2 vaccinated; dilution range of 1:5 to 1:5,120) or 1:30 (triple  vaccinated after subsequent Omicron BA.1 or BA.2 breakthrough infection; dilution range of  1:30 to 1:30,720). In the case of the SARS‐CoV‐1 pseudovirus assay, the serum of all  individuals was initially diluted 1:5 (dilution range of 1:5 to 1:5,120). VSV‐SARS‐CoV‐2‐S/VSV‐ SARS‐CoV‐1‐S particles were diluted in medium to obtain 200 TU in the assay. Serum  dilutions were mixed 1:1 with pseudovirus (n=2 technical replicates per serum per  pseudovirus) for 30 minutes at room temperature before being added to Vero 76 cell  monolayers and incubated at 37 °C with 7.5% CO2 for 24 hours. Supernatants were removed  and the cells were lysed with luciferase reagent (Promega). Luminescence was recorded on a  CLARIOstar® Plus microplate reader (BMG Labtech), and neutralization titers were calculated  as the reciprocal of the highest serum dilution that still resulted in 50% reduction in  luminescence. Results were expressed as geometric mean titers (GMT) of duplicates. If no  neutralization was observed, an arbitrary titer value of half of the limit of detection [LOD]  was reported.     Statistical analysis  The statistical method of aggregation used for the analysis of antibody titers is the geometric  mean and for the ratio of SARS‐CoV‐2 VOC titer and Wuhan titer the geometric mean and  the corresponding 95% confidence interval. The use of the geometric mean accounts for the  non‐normal distribution of antibody titers, which span several orders of magnitude. The  Friedman test with Dunn’s correction for multiple comparisons was used to conduct pairwise  signed‐rank tests of group geometric mean neutralizing antibody titers with a common  control group. All statistical analyses were performed using GraphPad Prism software  version 9.  References cited in Example 14  1.  WHO Technical Advisory Group on SARS‐CoV‐2 Virus Evolution (TAG‐VE),  Classification of Omicron (B.1.1.529): SARS‐CoV‐2 Variant of Concern (2021).    2.  WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing response  to Omicron SARS‐CoV‐2 variant: Technical brief and priority actions for Member States  (2022).    3.  M. Hoffmann et al., The Omicron variant is highly resistant against antibody‐mediated  neutralization. Cell. 185, 447‐456.e11 (2022), doi:10.1016/j.cell.2021.12.032.    4.  W. Dejnirattisai et al., SARS‐CoV‐2 Omicron‐B.1.1.529 leads to widespread escape  from neutralizing antibody responses. Cell. 185, 467‐484.e15 (2022),  doi:10.1016/j.cell.2021.12.046.    5.  V. Servellita et al., Neutralizing immunity in vaccine breakthrough infections from the  SARS‐CoV‐2 Omicron and Delta variants. Cell. 185, 1539‐1548.e5 (2022),  doi:10.1016/j.cell.2022.03.019.    6.  C. Kurhade et al., Neutralization of Omicron BA.1, BA.2, and BA.3 SARS‐CoV‐2 by 3  doses of BNT162b2 vaccine. Nature communications. 13, 255 (2022), doi:10.1038/s41467‐ 022‐30681‐1.    7.  Y. Cao et al., Omicron escapes the majority of existing SARS‐CoV‐2 neutralizing  antibodies. Nature. 602, 657–663 (2022), doi:10.1038/s41586‐021‐04385‐3.    8.  Y. Cao et al., BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron  infection. Nature (2022), doi:10.1038/s41586‐022‐04980‐y.    9.  N. P. Hachmann et al., Neutralization Escape by SARS‐CoV‐2 Omicron Subvariants  BA.2.12.1, BA.4, and BA.5. The New England journal of medicine (2022),  doi:10.1056/NEJMc2206576.    10.  E. Mathieu et al., A global database of COVID‐19 vaccinations. Nature human  behaviour. 5, 947–953 (2021), doi:10.1038/s41562‐021‐01122‐8.    11.  C. I. Kaku et al., Recall of pre‐existing cross‐reactive B cell memory following Omicron  BA.1 breakthrough infection. Science immunology, eabq3511 (2022),  doi:10.1126/sciimmunol.abq3511.    12.  J. Quandt et al., Omicron BA.1 breakthrough infection drives cross‐variant  neutralization and memory B cell formation against conserved epitopes. Science  immunology, eabq2427 (2022), doi:10.1126/sciimmunol.abq2427.    13.  A. Muik et al., Neutralization of SARS‐CoV‐2 Omicron by BNT162b2 mRNA vaccine‐ elicited human sera. Science (New York, N.Y.). 375, 678–680 (2022),  doi:10.1126/science.abn7591.    14.  A. Muik et al., Neutralization of SARS‐CoV‐2 lineage B.1.1.7 pseudovirus by BNT162b2  vaccine‐elicited human sera. Science (New York, N.Y.). 371, 1152–1153 (2021),  doi:10.1126/science.abg6105.    15.  C.‐W. Tan et al., Pan‐Sarbecovirus Neutralizing Antibodies in BNT162b2‐Immunized  SARS‐CoV‐1 Survivors. The New England journal of medicine. 385, 1401–1406 (2021),  doi:10.1056/NEJMoa2108453.  Example 15: Further updates on immune responses elicited by  vaccines encoding a SARS‐ CoV‐2 S protein from an Omicron variant  Following the experiment described in Example 8, further subjects were enrolled in a clinical  trial investigating RNA vaccines encoding a SARS‐CoV‐2 S protein comprising one or more  mutations characteristic of a BA.1 Omicron variant.  In the present Example, subjects (18 to  55 years of age with or without evidence of prior infection) were administered a 2nd booster  (4th dose) of either 30 ug of RNA encoding a SARS‐CoV‐2 S protein of a Wuhan strain (in the  present Example, BNT162b2) or 30 µg of RNA encoding an SARS‐CoV‐2 S protein having one  or more mutations characteristic of an Omicron variant (in the present example, BNT162b2  OMI, which encodes a SARS‐CoV‐2 S protein having mutations characteristic of a BA.1  Omicron variant, comprises SEQ ID NOs: 50 and 51, and encodes an amino acid of SEQ ID  NO: 49).  In a primary immunogenicity analysis of participants without evidence of prior infection,  BNT162b2 OMI (N=132) elicited a superior neutralizing antibody response to the BA.1  Omicron SARS‐CoV‐2 virus compared to BNT162b2 (N=141). The BNT162b2 OMI GMT  against BA.1 Omicron was 1929 (CI: 1632, 2281) compared to a BNT162b2 GMT of 1100 (CI:  932, 1297); GMT ratio 1.75 (95% CI: 1.39, 2.22).  Compared to BNT162b2, BNT162b2 OMI elicited a similar neutralizing antibody response to  a Wuhan strain of SARS‐CoV‐2. BNT162b2 OMI GMT was 11997 (CI: 10554, 13638) compared  to a BNT162b2 GMT of 12009 (CI: 10744, 13425).  The data suggests that an Omicron monovalent vaccine as the 2nd booster vaccination (4th  dose) improves a neutralizing antibody response to BA.1 Omicron compared to an RNA  vaccine encoding an S protein of a Wuhan strain, and does not negatively affect a  neutralizing antibody response to a Wuhan strain of SARS‐CoV‐2.     Example 16.  Immunological impact of VOC vaccination  The present Example describes immunological impacts of administration of BNT162b2  vaccines encoding spike proteins from certain variants of concern (“VOC”).  In particular, the  present Example describes immunological impacts of administration of a “booster dose” to  subjects (in this Example, mice) who have received two doses (i.e., according to an  established model immunization protocol) of the “original” BNT162b2 vaccine (i.e., encoding  the Wuhan spike protein, as described herein).  Figure 33 presents the  immunization protocol utilized  in the present Example.   Specifically,  BALB/c mice were immunized twice (1 ug each dose) with BNT 162b2, and then at a later time  point with a BNT162b2/VOC  (1 ug each dose).    Immunization occurred up  to 3 or 4  times.   Animals were bled regularly to analyze antibody immune response by ELISA and pseudovirus  neutralization assay.  At the end of the trial, animals were euthanized and T cell response in  the spleen was analyzed.    Boosting was performed with:  (a) the original BNT162b2  (“BNT162b2”);  (b) BNT162b2 OMI  BA.1 (“OMI BA.1”); (c) BNT162b2 OMI BA.4/5 (“OMI BA.4/5”); (d) BNT162b2 + OMI   BA.4/5  (0.5 ug each); (e) OMI BA.1 + OMI BA.4/5 (0.5 ug each); and (f) BNT162b2 + OMI BA.1 + OMI  BA.4/5 (0.33 ug each).    Omicron variants BA.4 and BA.5 were first reported in circulation in January 2022, and were  becoming dominant variants by  June 2022.   Both of  these  lineages contain  the amino‐acid  substitutions F486V, and R493Q. Preliminary studies suggest a significant change in antigenic  properties  of  BA.4  and  BA.5  compared  to  BA.1  and  BA.2,  especially  compared  to  BA.1.  Additionally,  as  increasing  trends  in  BA.5  variant  proportions  are  observed  in  particular  locations  (e.g.,  in  Portugal),  COVID‐19  case  numbers  and  test  positivity  rate  have  also  increased.   The present disclosure proposes that   BA.4/5 (which, given their common spike  protein mutations, are considered together in the present Example) could represent escape  VOC.   The present disclosure demonstrates particular benefits of dosing  regimens  (e.g., as  described herein and specifically as exemplified in the present Example) that include one or  more doses of a vaccine that comprises or delivers (e.g., via expression of an administered  RNA) a spike protein that includes relevant BA.4/5 sequences (e.g., amino acid substitutions).   Figures 34 and 35 present baseline (determined at day 104, pre‐boost) geometric mean  titers (“GMT”s) relative to various SARS‐CoV‐2 strains, as indicated.  As can be seen, baseline  immunization of the different mouse cohorts was comparable.  Specifically, group GMTs per  pseudovirus were consistently in the same ballpark between cohorts; no difference greater  than about 2‐fold was observed.  Consistent with observations made in human populations,  as noted above, neutralizing GMTs against the Wuhan strain were considerably higher (GMT  of up to 3,044) as compared to those against VOCs.  Overall, the order of GMTs was Wuhan >  BA.1 ≅ BA.2 > BA.2.12.1 > BA.4/5.  Figure 36 shows baseline (determined at day 104, pre‐boost) cross‐neutralization analysis  and demonstrates that baseline immunization of cohorts with respect to cross‐neutralization  capacity is comparable.  Specifically, at baseline, calculated variant/Wuhan ref GMT ratios  indicated that cross‐neutralization capacity was quite comparable between cohorts (only  one outlier in the BNT162b2 monovalent group re. BA.1 neutralization was observed).  Again  consistent with observations of human populations, BA.1 = BA.2 > BA.2.12.1 > BA.4/5  Figures 37 ‐ 39 present data obtained seven days post‐boost and document remarkable  effectiveness of BA.4/5, and in particular of monovalent BA.4/5, in achieving significant  geometric mean fold increase of GMTs (Figures 37 and 38) and effective cross‐neutralization  (Figure 39).  As can be seen, BNT162b2 booster immunization resulted in a comparable titer  increase against all VOCs (3.9‐7.1 fold), whereas monovalent BA.1 and BA.4/5 boosters  resulted in a considerably stronger increase in the homologous VOC titer (16.8‐fold for BA.1,  67.3‐fold for BA.4/5).   The monovalent BA.4/5 booster was the most effective in driving titer increases across the  pseudovirus panel tested.  Bivalent boosters showed a similar but attenuated trend  compared to the monovalent VOC boosters; amongst bivalent boosters the b2 + BA.4/5  combination was most effective in driving broad cross‐neutralization. The trivalent booster  (b2 + BA.1 + BA.4/5) was superior to the bivalent boosters and gave intermediate  immunization between the bivalent b2 + BA.4/5 and monovalent BA.4/5 booster.  Figure 39, among other things, presents calculated variant/Wuhan ref GMT ratios, which  indicate that:   (i) BNT162b2 booster results in relatively poor cross‐neutralization, especially of BA.2  and descendants (BA.2.12.1, BA.4/5)  (ii) BA.1 booster results in superior cross‐neutralization of BA.1, but still relatively  poor neutralization of BA.2.12.1, BA.4/5  (iii) BA.4/5 booster results in balanced pan‐Omicron neutralization with very  encouraging neutralization against BA.2, BA.2.12.1 and BA.4/5  Bivalent boosters showed a similar but attenuated trend compared to the monovalent VOC  boosters; among bivalent boosters the b2 + BA.4/5 combination was most effective in  driving broad cross‐neutralization; the trivalent booster (b2 + BA.1 + BA.4/5) elicited  comparable cross‐neutralization to the BA.1/BA.4/5 booster.    The present specification demonstrates remarkable efficacy of BA.4/5 immunization  (and specifically of BNT162b2 + BA.4/5 immnunization, e.g., with sequences provided  herein).  Futhermore, the present specification demonstrates efficacy of BA.4/5  immunization in monovalent, bivalent, and trivalent formats, and documents surprising  efficacy of monovalent BA.4/5.    The present disclosure specifically demonstrates remarkable usefulness of one or  more BA.4/5 doses administered to subjects who have previously been immunized (e.g.,  with a Wuhan vaccine, such as with at least (or exactly) two doses of a Wuhan vaccine.   Without wishing to be bound by any particular theory, the present disclosure teahces that  immunological characteristics of the omicron BA.4/5 spike may render it particularly useful  or effective for immunization of subjects, including those who have been immunized (e.g.,  via prior administration of one or more vaccine doses and/or by prior infection) with the  Wuhan strain (and/or with one or more strains immnuologically related to the Wuhan  strain), including specifically by vaccination with one or more (e.g., 1, 2, 3, 4 or more) doses  of original BNT162b2.      Example 17.  Selection and characterization of exemplary spike protein variants.   The present Example describes design and characterization of various spike variant  sequences for use in an RNA vaccine (e.g., a vaccine comprising an RNA, for example, in  some embodiments mRNA, encoding a spike protein sequence from a coronarvirus, e.g.,  SARS‐CoV‐2).    As described herein, in some embodiments, certain mutations may be introduced in a SARS‐ CoV‐2 spike protein‐encoding sequence that contribute to an increase in the immunogenicity  of spike protein antigens, e.g., by improving expression of the spike protein, improving the  stability of the spike protein, improving the stability of a particular confirmation of the spike  protein (e.g., improving stabilization of a prefusion conformation), and/or increasing the  number of neutralization‐sensitive epitopes on the spike protein.  Certain mutations are  known in the art, e.g., as disclosed in WO 2021243122 A2 and Hsieh, Ching‐Lin, et al.  ("Structure‐based design of prefusion‐stabilized SARS‐CoV‐2 spikes," Science 369.6510  (2020): 1501‐1505), the contents of each which are incorporated by reference herein in their  entirety.     In this Example, various combinations of proline substitutions were introduced into the  amino acid sequence of a SARS‐CoV‐2 S protein.  Exemplary combinations of such  combinations of proline mutations are shown in Fig. 40.  The positions of the mutations as  listed in Fig. 40 are shown with respect to the spike protein sequence according to SEQ ID  NO: 1 (SARS‐CoV‐2 Wuhan, i.e., wildtype strain). A spike protein sequence containing proline  mutations corresponding to K986P and V987P in the Wuhan strain is desginated as „P2“.   A  spike protein sequence containing proline mutations corresponding to K986P, V987P, F817P,  A892P, A899P, and A942P in the Wuhan strain is designated as “P6“.  A spike protein  sequence containing proline mutations corresponding to D985P and V987P in the Wuhan  strain is designated as “P2‘ “.  A spike protein sequence containing proline mutations  corresponding to V987P, F817P, A892P, A899P, and A942P is designated as “P5‘ “.  A spike  protein sequence containing proline mutations corresponding to D985P, V987P, F817P,  A892P, A899P, and A942P is designated as “P6‘ “.  A spike protein sequence containing  proline mutations corresponding to D985P, K986P, F817P, A892P, A899P, A942P is  designated as “P6‘‘ “.  A spike protein sequence containing proline mutations corresponding  to D985P, K986P, V987P, F817P, A892P, A899P, A942P is designated as “P7“.  The various combinations of proline mutations as described herein (e.g., as described in Fig.  40) can be introduced into coronavirus spike protein or an immunogenic fragment thereof.  In this Example, the various combinations of proline mutations as described in Figure 40 can  be introduced into a S protein, or an immunogenic fragment thereof, of SARS‐CoV‐2 strains  and variants as shown in Table 1.   Alternatively or additionally, additional mutations may be introduced into a coronavirus  spike protein or an immunogenic fragment thereof, for example, in some embodiments a S  protein, or an immunogenic fragment thereof, of SARS‐CoV‐2 strains and variants. In some  embodiments, such additional mutations were selected, for example, to (a) increase  adoption of the RBD‐up conformation to expose more neutralization‐sensitive epitopes on  the spike protein, (b) decrease adoption of the RBD‐down conformation, (c) increase  expression of the variant coronavirus spike protein compared to the reference (e.g., native  or wild‐type) coronavirus spike protein, (d) increase adoption of a prefusion conformation,  (e) decrease shedding of a S1 subunit of the variant coronavirus spike protein, and/or (f)  improve localization of the variant coronavirus spike protein to a host cell membrane.  Exemplary such mutations are disclosed in Fig. 41 under the columns “Mutation” and  “Mutation Type”. In some embodiments, furin cleavage site in a coronavirus protein or an  immunogenic fragement thereof comprising the amino acid sequence of RRAR can be  optionally mutated to the amino acid sequence (GSAS).   Fig. 41 shows certain combinations of mutations that were introduced in a coronavirus S  protein, and mRNA constructing encoding such coronavirus S protein variant. S protein  expression, ACE2 binding, and CR3022* epitope binding (Yuan et al., „A highly conserved  cryptic epitope in the receptor‐binding domains of SARS‐CoV‐2 and SARS‐CoV (2020) 368:  630) was evaluated for each mRNA encoding a S protein mutant (see Fig. 41).  Surprisingly,  as shown in Fig. 41, incorporation of a D985P mutation rather than a K986P mutation was  found to improve each of protein expression, CR3022 response and ACE2 response (compare  data for Mutant E and Mutant I).    Example 18.  Neutralization of Various SARS‐CoV‐2 VOCs by RNA‐vaccines encoding  Exemplary Spike Protein Variants.   The present Example describes immunological impacts of adminstration of RNA vaccines  encoding exemplary spike protein variants of a SARS‐CoV‐2 strain or VOC. In particular, the  present Example describes immunological impacts of administration of at least one dose  (including, e.g., at least two doses or at least three doses) to subjects (in this Example, mice).   Specifically, mice were immunized with at least one dose of: (i) BNT162b2; (ii) an RNA  encoding an S protein of a Wuhan strain with at least mutations K986P, V987P, and D614G  mutations at the furin cleavage site (R682G,  R683S,  R685S) (denoted as „P2 with D614G +  furin mutant“ in Fig. 42); (iii) an RNA encoding an S protein of a Wuhan strain with at least  mutations D985P, V987P, F817P, A892P, A899P, A942P, and D614G (denoted as „P6‘ with  D614G + intact furin“ in Fig. 42); and (iv) an RNA encoding an S protein of a Wuhan strain  with at least mutations D985P, V987P, F817P, A892P, A899P, A942P, and D614G as well as  mutations at the furin cleavage site (R682G,  R683S  R685S) (denoted as „P6‘ with D614G +  furin mutant“ in Fig. 42).  Animals were bled about one month after receiving a second dose  of vaccine, and sera was analyzed to determine antibody immune response, for example,  using a pseudovirus neutralization assay as described herein. Specifically, neutralizing  antibody titers against Wuhan (wildtype), Beta variant, Delta variant, and Omicron BA.1  variant were evaluated for each tested RNA vaccine, as indicated in Fig. 42.  Fig. 42 shows that an exemplary RNA construct encoding a SARS‐CoV‐2 S protein with at  least mutations at positions corresponding to D985P, V987P, F817P, A892P, A899P, A942P,  and D614G and the furin cleavage site (R682G,  R683S  R685S) in the Wuhan strain (e.g., SEQ  ID NO: 1) stimulated higher neutralization titers across VOCs including Wuhan, beta variant,  delta variant, and Omicron BA.1 variant.  Such mutations, as described in Tables 2A‐2B, are  numbered with respect to the Wuhan spike protein sequence (SEQ ID NO: 1), however, the  position of the particular mutation may vary as described herein depending on the strain of  SARS‐CoV‐2 spike protein sequence (see, e.g., alignment in Table 5).  Surprisingly, as shown  in Fig. 42, RNA encoding a SARS‐CoV‐2 S protein comprising the P6‘ set of mutations, the  D614G mutation, and an intact furin cleavage site was found to produce an immune  response that was similar to or worse than that of BNT162b2, but incorporation of furin site  mutations in the same construct was found to greatly increase immune response relative to  BNT162b2.  Immune responses were especially improved for SARS‐CoV‐2 variants,  demonstrating that BNT162b5 can induce an immune response with broader cross‐ neutralization as compared to BNT162b2. Similar experiments performed with a BA.4/5‐ adapted bivalent vaccine in vaccine‐naive mice did not replicate these results, although  improved immune responses were still observed for BQ.1.1 and XBB Omicron variants (data  shown in Figure 47.     Example 19: Immunogenicity Study of Vaccines Encoding a SARS‐CoV‐2 S Protein variant in  vaccine‐experienced healthy subjects  This Example describes a study that evaluated the safety, tolerability and immunogenicity of  a BNT162b, RNA‐based SARS‐CoV‐2 vaccine candidate given as a booster dose in adults to  prevent COVID‐19.  The evaluation was performed in subjects who comprised the following:  · 18 through 55 years of age and healthy (who may have had preexisting disease if it  was stable);  · had received 1 booster dose of a US‐authorized COVID‐19 vaccine (e.g., BNT162b2,  Moderna COVID‐19 vaccine, etc.), with the last dose having been 90 or more days  before the first visit of the study.  In some embodiments, subjects with at least one or more of the following characteristics  were excluded from the present study.   · History of severe adverse reaction associated with a vaccine and/or severe allergic  reaction (eg, anaphylaxis) to any component of the study vaccines.  · Known or suspected immunodeficiency.  · Bleeding diathesis or condition associated with prolonged bleeding that would have,  in the opinion of the investigator, contraindicated intramuscular injection.  · Women who were pregnant or breastfeeding.  · Other medical or psychiatric condition, or laboratory abnormality that may have  increased the risk of study participation or in the investigator's judgment, made the  participant inapprropriate for the study.  · Receipt of chronic systemic treatment with known immunosuppressent medications  (including cytotoxic agents or systemic corticosteroids), or radiotherapy, within 60  days before study vaccination and through end of study.  · Receipt of blood/plasma products, immunoglobulin, or monoclonal antibodies, from  60 days before study vaccination or planned receipt throughout the study.  · Participation in other studies involving a study intervention within 28 days of  randomization.   · Anticipated participation in other studies within 28 days after receipt of study  intervention in this study.  Exemplary Dosing Regimens:  Subjects in the present study received 1 of the 2 study vaccines: BNT162b5 Bivalent or  BNT162b2 Bivalent. 
Figure imgf000698_0001
  As described herein, BNT162b2 bivalent (WT/OMI BA.1) refers to a composition comprising  (i) an RNA encoding an S protein from a Wuhan strain, and (ii) an RNA encoding an S protein  from an Omicron BA.1 variant, wherein both S protein sequences each comprise 2 proline  mutations at positions corresponding to K986P and V987P in the Wuhan sequence (SEQ ID  NO: 1). In some embodiments, such an RNA vaccine sequence encoding an S protein from a  Wuhan sequence with 2 proline mutations is set forth in SEQ ID NO: 20 (and the  corresponding nucleotide sequence that encodes the S protein with 2 proline mutations is  set forth in SEQ ID NO: 9; and the amino acid sequence of the S protein with 2 proline  mutations is set forth in SEQ ID NO: 7). In some embodiments, such an RNA encoding an S  protein from an Omicron BA.1 variant with 2 proline mutations is set forth in SEQ ID NO: 51  (and the corresponding nucleotide sequence that encodes the S protein from Omicron BA.1  with 2 proline mutations is set forth in SEQ ID NO: 50; and the amino acid sequence of the S  protein from Omicron BA.1 with 2 proline mutations is set forth in SEQ ID NO: 49). In some  embodiments, both types of RNA molecules may be formulated in lipid nanoparticles (LNPs)  to form a bivalent vaccine (e.g., two populations of RNAs are mixed prior to LNP formulation;  or each RNA is formulated in a separate LNP composition, and then mixed together).   As described herein, BNT162b5 bivalent (WT/OMI BA.2) refers to a composition comprising  (i) an RNA encoding an S protein from a Wuhan strain, and (ii) an RNA encoding an S protein  from an Omicron BA.2 variant, wherein both S protein sequences each comprise (A) 6  proline mutations at positions corresponding to F817P, A892P, A899P, A942P, D985P, and  V987P; (B) furin cleavage site mutations at positions corresponding to R682G,  R683S,  and  R685S; and (C) a D614G mutation (all mutation positions of which are numbered  relative to the Wuhan sequence (SEQ ID NO: 1)). In some embodiments, such an RNA  sequence encoding an S protein from a Wuhan sequence with the aforementioned  mutations (A), (B), and (C) is set forth in SEQ ID NO: 83 (and the corresponding nucleotide  sequence that encodes the S protein with the aforementioned mutations (A), (B), and (C) is  set forth in SEQ ID NO: 81; and the amino acid sequence of the S protein with the  aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 80). In some  embodiments, such an RNA sequence encoding an S protein from an Omicron BA.2 sequence  with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 98 (and the  corresponding nucleotide sequence that encodes the S protein from an Omicron BA.2  variant with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 96; and  the amino acid sequence of the S protein from an Omicron BA.2 variant with the  aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 95). In some  embodiments, both types of RNA molecules may be formulated in lipid nanoparticles (LNPs)  to form a bivalent vaccine (e.g., two populations of RNAs are mixed prior to LNP formulation;  or each RNA is formulated in a separate LNP composition, and then mixed together).  In the present study, subjects received a single 30 microgram dose of study vaccine as  described above and a blood sample was taken at the indicated time below after  administration to evaluate neturalizing antibody titers against various SARS‐CoV‐2 strains  and variants (including, e.g., Wuhan, Omicron BA.1, Omicron BA.2, etc.)   Exemplary Primary Outcome Measures:  · Percentage of participants reporting local reactions [Time Frame: For 7 days following  the study vaccination] Pain at the injection site, redness, and swelling, as self‐reported in  electronic diaries  · Percentage of participants reporting systemic events [Time Frame: For 7 days following  the study vaccination] Fever, fatigue, headache, chills, vomiting, diarrhea, new or  worsened muscle pain, and new or worsened joint pain, as self‐reported in electronic  diaries  · Percentage of participants reporting adverse events [Time Frame: For 1 month following  the study vaccination].  · Percentage of participants reporting serious adverse events [Time Frame: For 6 months  following the study vaccination]  · Geometric Mean Titers (GMT) of SARS‐CoV‐2 Omicron (BA.2), Omicron (BA.1), and  reference strain neutralizing antibody levels for BNT162b5 Bivalent (WT/OMI BA.2) 30 µg  and BNT162b2 Bivalent (WT/OMI BA.1) 30 µg [Time Frame: Before study vaccination and  1 week, 1 month, 3 months and 6 months after study vaccination] as measured at the  central laboratory.  · Geometric Mean Fold Rise (GMFR) of SARS‐CoV‐2 Omicron (BA.2), Omicron (BA.1), and  reference strain neutralizing antibody levels for BNT162b5 Bivalent (WT/OMI BA.2) 30 µg  and BNT162b2 Bivalent (WT/OMI BA.1) 30 µg. [Time Frame: From before study  vaccination to 1 week, 1 month, 3 months and 6 months after study vaccination.]  · Percentages of participants with seroresponse to BNT162b5 Bivalent (WT/OMI BA.2) 30  µg and BNT162b2 Bivalent (WT/OMI BA.1) 30 µg in terms of GMTs of SARS‐CoV‐2  Omicron (BA.2), Omicron (BA.1), and reference strain neutralizing antibody levels.  [Time Frame:  1 week, 1 month, 3 months and 6 months after study vaccination.]  As  shown  in  Figs.  44(A)‐(C),  neutralization  titers  against  the Wuhan  strain were  higher  in  subjects admininistered BNT162b5 as compared to BNT162b2, one month after administering  a SARS‐CoV‐2 vaccine (GMFR of 4.1 vs 3.0 for all participants, 8.2 vs 5.6 for subjects without  evidence of infection prior to receiving a vaccine, and 2.8 vs 2.1 for subjects with evidence of  previous  infection).  Similarly, neutralization titers  in subjects administered BNT162b5 were  increased against Omicron BA.1 (GMFR of 5.5 vs 4.2), and BA.2 (GMFR of 5.1 vs 3.2) variants.   This  effect was  observed  for  all  patients,  regardless  of  previous  infection  status,  and  the  benefits provided by BNT162b5 were especially pronounced in subjections without evidence  of previous infection (see Fig. 44 C).  Thus, this clinical trial data confirmed the effects observed  in the previously described mouse and in vitro studies; specifically that BNT162b5 can induce  an  improved  immune  response  and/or  broader  cross‐neutralization  as  compared  to  BNT162b2.  Example 20: Safety/Immunogenicity study of Vaccines Encoding S proteins of SARS‐CoV‐2  Variants in healthy subjects   Recent evolution of SARS‐CoV‐2 is resulting in an emergence of new virus variants with  multiple mutations in the S protein, which might be associated with the lower efficacy of  some of the current vaccines. Therefore, the present study provides new approaches to  overcome waning immunity and/or the development of modified vaccines.  This Example describes a study to evaluate the safety, tolerability and immune responses of  an exemplary COVID‐19 RNA vaccine as a booster in COVID‐19 vaccine experienced healthy  adults (e.g., ages ≥56).    The COVID‐19 RNA vaccines tested include a B162b5 bivalent vaccine alone or in  combination with a T‐string RNA construct (e.g., an RNA construct comprising a sequence  that encodes at least two or more T cell epitopes). An exemplary T‐string construct is  described in e.g., WO2021/188969; the contents of each of which are incorporated herein by  reference in their entireties.    In some embodiments, a B162b5 bivalent vaccine tested in the present study is a  composition comprising (i) an RNA encoding a S protein from a Wuhan sequence, and (ii) an  RNA encoding a S protein from an Omicron variant (e.g., BA.1, BA.2, or BA.4/5), wherein  both S protein sequences each comprise (A) 6 proline mutations corresponding to F817P,  A892P, A899P, A942P, D985P, and V987P; (B) furin mutations at R682G,  R683S  R685S; and  (C) D614G mutation, all mutation positions of which are numbered relative to the Wuhan  sequence (SEQ ID NO: 1).  In some embodiments, both types of RNA moelcules (i) and (ii)  may be formulated in the lipid nanoparticles (LNPs) to form a bivalent vaccine (e.g., two  populations of RNAs are mixed prior to LNP formulation; or each RNA is formulated in a  separate LNP composition, followed by mixing together).  In some embodiments, an RNA encoding a S protein from a Wuhan sequence used in the  present study is described in Example 19. In some embodiments, an RNA encoding a S  protein from an Omicron BA.2 sequence used in the present study is described in Example  19. In some embodiments, an RNA vaccine sequence encoding a S protein from an Omicron  BA.1 sequence with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:  93 (and the corresponding nucleotide sequence that encodes the S protein from Omicron  BA.1 sequence with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO:  91; and the amino acid sequence of the S protein from Omicron BA.1 sequence with the  aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 90). In some  embodiments, an RNA vaccine sequence encoding a S protein from an Omicron BA.4/5  sequence with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 103  (and the corresponding nucleotide sequence that encodes the S protein from Omicron  BA.4/5 sequence with the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID  NO: 101; and the amino acid sequence of the S protein from Omicron BA.4/5 sequence with  the aforementioned mutations (A), (B), and (C) is set forth in SEQ ID NO: 100).  In some embodiments, subjects to be vaccinated with one of the COVID‐19 RNA vaccines  candidates described herein include triple‐vaccinated patients with an authorized vaccine  (e.g., BNT162b2, Moderna COVID‐19 vaccine or other COVID mRNA or protein‐based  vaccines).  In some embodiments, subjects are administered with at least 1 dose of  BNT162b5 (e.g., in some embodiments at 30 ug each) alone or in combination with a T‐string  construct (e.g., as described herein, e.g., an RNA encoding SEQ ID NO: RS C7p2full of  WO2021/188969), e.g., a dose of a combination of BNT162b5 and an RNA encoding SEQ ID  NO: RS C7p2full of up to about 100 ug RNA total. In some embodiments, subjects are  administered with at least 2 doses of BNT162b5 (e.g., in some embodiments at 30 ug each)  alone or in combination with a T‐string construct (e.g., as described herein, e.g., an RNA  encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g., each dose of a combination of  BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2full of up to about 100 ug RNA total,  wherein the two doses are administered, for example, at least 4 weeks or longer (including,  e.g., at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at  least 10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from one another.  In  some embodiments, subjects are administered with at least 3 doses of BNT162b5 (e.g., in  some embodiments at 30 ug each) alone or in combination with a T‐string construct (e.g., as  described herein, e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO2021/188969), e.g.,  each dose of a combination of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2full of up  to about 100 ug RNA total, wherein the first and the second doses and the second and third  doses are each independently administered at least 4 weeks or longer (including, e.g., at  least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least  10 weeks, at least 11 weeks, or at least 12 weeks, or longer) apart from one another.  In some embodiments, doses are administered by intramuscular injection, e.g., in the deltoid  muscle of the arm of subjects. In some embodiments, a combination of BNT162b5 and a T‐ string construct may be administered concomitantly as a single injection.      Exemplary characterization assays for evaluation:  Subjects are evalulated at various times during and after receiving treatment for safety and  any adverse reactions.  Safety is evaluated by physical assessment: a (complete) physical  examination include, at a minimum, assessments of the skin, lymphatic nodes,  cardiovascular, respiratory, gastrointestinal, and neurological systems. A brief (symptom‐ directed) physical examination. The brief physical examination includes an overall health  judgment. In‐depth physical examinations are required if obvious pathological signs are  visible or in the case the subject states any signs or symptoms.  Vital signs (comprising  systolic/diastolic blood pressure, pulse rate, respiratory rate, and oral body temperature) are  assessed at various timepoints.  Electrocardiogram: Standard 12‐lead ECGs are recorded.  Assessments of intensity for local reactions: Pain (perceived) at the injection site are  assessed as absent, mild, moderate, or severe. Erythema/redness and induration/swelling  are measured and recorded and then categorized during analysis as absent, mild, moderate,  or severe.  Assessments of intensity for systemic reactions: Symptoms of systemic reactions are  assessed as absent, mild, moderate, or severe. Surveillance of “confirmed COVID‐19” cases is  also performed.  Serological testing for SARS‐CoV‐2 N‐binding antibodies: Blood are drawn for serological  testing for SARS‐CoV‐2 N‐binding antibodies by serum SARS‐CoV‐2 nucleocapsid protein‐ specific antibody immunoassay at screening, which results can be useful for stratification.   SARS‐CoV‐2 sequencing: Swabs for SARS‐CoV‐2 genomic sequencing storage is collected for  SARS‐CoV‐2 genomic sequencing. The outcome include SARS‐CoV‐2 S protein sequences  and/or whole genome sequences and assigned (where possible) to known viral variants.  An adverse event of special interest (AESI), serious or non‐serious, is one of scientific and  medical concern specific to the present study. Exemplary AESIs include:  • Myocarditis (all Levels of Certainty including “Possible cases” (1 to 3) as per Brighton  Collaboration Case Definition), https://brightoncollaboration.us/myocarditis‐case‐definition‐ update/)  • Pericarditis (all Levels of Certainty including “Possible cases” (1 to 3) as per Brighton  Collaboration Case Definition)), https://brightoncollaboration.us/myocarditis‐case‐ definition‐update/)  • Anaphylaxis  • Thromboembolic events (e.g., deep vein thrombosis, stroke, myocardial infarction)  • Immune thrombocytopenia  • Immune based neurologic events (e.g., optic neuropathy, Guillain‐Barré syndrome)  Genetics: A blood sample (blood and/or isolated peripheral blood mononuclear cells) are  drawn. In some embodiments, these biosamples are used for human leukocyte antigen  typing of a subject to allow additional analysis, e.g., the characterization of T cell receptor  and B cell receptor sequences, in order to characterize the fine specificity of the immune  response. These blood samples can also be used to analyze transcriptional responses to  administration of the COVID‐19 RNA vaccine candidates using RNA sequencing methods.  Analysis of immune responses can provide better understanding of the mechanisms  underlying reactogenicity and the induction of antibody and T cell responses.  Immune responses: Immune responses are assessed at various timepoints in this study.  Exemplary humoral immune response assessments may include:  • Characterization of kinetics of titers, binding and functionality of antigen‐specific  sera antibodies e.g., using ELISA and virus neutralization tests (VNTs).  The cell‐mediated immune response assessments include:  Evaluation of antigen‐specific CD4 and CD8 T cells, including transcriptomic  profiling and functional characteristics such as effector functions measured by the  expression of cytokines such as (but not limited to) IL‐2, IFN‐γ, TNF‐α and the  activation marker CD40L using peptide pools from the antigen encoded by the RNA  component of the vaccine.  • Enumeration of antigen‐specific IFN‐ γ secreting cells that are evaluated using  assays including intracellular cytokine staining and enzyme‐linked immunosorbent  spot (ELISpot) assays.  Additional Characterizations/assessments: Additional characterizations/assessments can  include, but not limited to, phenotypic or functional characterization of antigen‐specific T  cells or B cells (e.g., by flow cytometry‐based phenotyping including multimer staining),  transcriptomics activity and analysis of T cell and B cell receptor repertoire (e.g., by next  generation sequencing, single‐cell RNA sequencing), and multiplex‐cytokine analysis. Such  analyses can also include human leukocyte antigen typing. Phenotypic or functional  characterization of other immune cell populations that may be relevant to understanding  the vaccine‐induced immune responses may be included in the present study.  Profiling of antibody Fc mediated effector functions may be performed using system  serology approach. This analysis may include, but is not limited to, evaluation of  antigenspecific antibody affinity, isotype and subclass, antibody Fc receptor binding and  antibody functionality such as antibody‐dependent cellular cytotoxicity, antibody‐dependent  natural killer cell activation, antibody‐dependent cellular phagocytosis (ADCP).  The present study may also include the characterization of molecular and cellular networks  that influence innate and adaptive immunity to the antigen encoded by the RNA component  of  the vaccine. This analysis can include the characterization of the transcriptional  signature induced by the encoded by the RNA component of the vaccine by RNA  sequencing (transcriptome analysis), complemented by the evaluation of potential  changes in cell composition and levels of cytokines, chemokines or inflammatory markers  induced by administration of the COVID‐19 RNA vaccine candidates.    Example 21.  Neutralization of Various SARS‐CoV‐2 VOCs by RNA vaccines encoding  Exemplary Spike Protein Variants in Vaccine Experienced Subjects  The present Example describes immunological impacts of adminstration of RNA vaccines  encoding exemplary spike protein variants of a SARS‐CoV‐2 strain or VOC. In particular, the  present Example describes immunological impacts of administration of at least one dose  (including, e.g., at least two doses or at least three doses) to subjects (in this Example, mice)  that have previously been administered one or more doses of another SARS‐CoV‐2 vaccine  that delivers a SARS‐CoV‐2 Spike protein comprising K986P and V987P (in the present  example, BNT162b2).   Mice were first immunized with two doses of BNT162b2, and then administered (i) a bivalent  vaccine comprising two RNAs, each encoding a SARS‐CoV‐2 S protein comprising K986P and  V987P mutations, wherein one of the RNAs encodes a SARS‐CoV‐2 S protein of a Wuhan  strain and the other RNA encodes a SARS‐CoV‐2 S protein comprising mutations  characteristic of a Omicron BA.4/5 variant („BNT162b2 Bivalent BA.4/5“ in Fig. 43); or (iii) a  bivalent RNA vaccine, comprising a first RNA encoding a SARS‐CoV‐2 S protein of a Wuhan  strain and a second RNA encoding a SARS‐COV‐2 S protein comprising mutations  characteristic of an Omicron BA.4/5 variant, wherein each of the first and the second RNA  comprise the P6‘ set of mutations (D985P, V987P, F817P, A892P, A899P, A942P), D614G, and  mutations at the furin cleavage site (R682G,  R683S, and R685S)  (denoted as „BNT162b5  Bivalent BA.4/5 in Fig. 43)).  Mutations shown relative to Wuhan S protein (e.g., SEQ ID NO:  1).  Animals were bled after receiving a third dose of vaccine, and sera was analyzed to  determine antibody immune response, for example, using a pseudovirus neutralization assay  as described herein. Specifically, neutralizing antibody titers against Wuhan (WT), and  Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/5  were evaluated for each tested RNA  vaccine, as indicated in Fig. 43.  Similar to the results shown in Fig. 42, the results in Fig. 43 show that the P6‘, D614G, and  furin cleavage site mutations can induce a stronger immune response as compared to K986P  and V987P in the context of a variant adapted vaccine (e.g., an Omicron‐adapted bivalent  vaccine) administered as a booster dose to vaccine‐experienced subjects.  Again, a stronger  immune response was found to be induced by BNT162b5 as compared to BNT162b2 for each  of the variants tested.  The immune response was particularly improved for the Omicron  BA.4/5 variant, with the P6‘, D614G, and furin cleavage site combination of mutations  inducing an immune response that was ~2.8‐fold higher than that induced by BNT162b2.   Surprisingly, immune responses were higher against non‐matched variants (e.g., the immune  response induced against the Omicron BA.1 variant was higher than for any variant for the  BNT162b2 bivalent vaccine), demonstrating that the P6‘, D614G, and furin site mutations can  induce a broader cross‐neutralization response than variant adapted versions of BNT162b2.    The above experiment was repeated with further Spike Protein variants.  Specificially, in the  repeat of the experiemnt, mice were first immunized with two doses of BNT162b2, and then  administered one of the following vaccines as a third dose:  (i) BNT162b2  (ii) a bivalent vaccine comprising two RNAs, each encoding a SARS‐CoV‐2 S  protein comprising K986P and V987P mutations, wherein one of the RNAs  encodes a SARS‐CoV‐2 S protein of a Wuhan strain and the other RNA encodes  a SARS‐CoV‐2 S protein comprising mutations characteristic of a Omicron  BA.4/5 variant („BNT162b2 Bivalent BA.4/5“ in Fig. 43);   (iii) a bivalent RNA vaccine, comprising a first RNA encoding a SARS‐CoV‐2 S  protein of a Wuhan strain and a second RNA encoding a SARS‐COV‐2 S protein  comprising mutations characteristic of an Omicron BA.4/5 variant, wherein  each of the first and the second RNA comprise the P6‘ set of mutations  (D985P, V987P, F817P, A892P, A899P, A942P), D614G, and mutations at the  furin cleavage site (R682G,  R683S, and R685S)  (denoted as „BNT162b5  Bivalent BA.4/5 in Fig. 43)), or  (iv) a bivalent RNA vaccine, comprising a first RNA encoding a SARS‐CoV‐2 S  protein of a Wuhan strain and a second RNA encoding a SARS‐COV‐2 S protein  comprising mutations characteristic of an Omicron BA.4/5 variant, wherein  each of the first and the second RNA comprise the P6 set of mutations (K986P,  V987P, F817P, A892P, A899P, A942P), D614G, mutations at the furin cleavage  site (R682G,  R683S, and R685S), and mutations to introduce a disulphide bod  (T547C and N978C) (denoted as „BNT162b6 Bivalent BA.4/5 in Fig. 45)).   Mutations shown relative to Wuhan S protein (e.g., SEQ ID NO: 1).    Again, sera were collected 1 month after administering a third dose, and neutralizing  antibody titers against Wuhan (wildtype), Delta variant, and Omicron variants BA.1 BA.2,  BA.2.12.1, BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB were evaluated for each tested RNA  vaccine, as indicated in Figs. 45 (A) and (B).  Titers are also shown in Table 33 below.  Table 33: Neutralizing titers induced by variant adapted BNT162b2 and BNT162b5 bivalent  vaccines, administered as booster doses 
Figure imgf000708_0001
  Figs. 45(A) and (B), and the titers in the above Table 33 again demontrate that RNA encoding  a SARS‐CoV‐2 S protein comprising the P6‘ set of mutations (D985P, V987P, F817P, A892P,  A899P, A942P), D614G, and furin site mutations (R682G,  R683S, R685S) can induce an  improved immune response (e.g., higher neutralization titers) as compared to RNA encoding  a SARS‐CoV‐2 S protein comprising K986P and V987P when administered to vaccine  experienced subjects.    Example 22: Further Exemplary Spike Protein Variants  Further Spike proteins were expressed, purified, and characterized  in vitro using the assays  described in the previous Examples.  Table 34 (below) lists the further S protein variants that  were expressed and purified, along with certain characteristic mutations present in the same.  Table 34: Further exemplary Spike protein variants  
Figure imgf000708_0002
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   Construct numbers 1‐87 were expressed, purified, and characterized in vitro, using the assays  described in the previous Examples.  The results of these assays are shown in Fig. 46.  Variants were also designed in which two or more cysteine residues were introduced, so as to  allow  for  the  formation  of  a  disulfide  bond.    Specifically, mutants  having  any  one  of  the  following  five  interprotomer  disulfide  bonds were  prepared:  (i) A570C‐N960C;  (ii)  A570C‐ K964C;  (iii)  A570C‐S967C;  (iv)  T547C‐N978C;  and  (v)  T547C‐S982C.    Said  mutants  also  comprised a D614G mutation, proline mutations, and furin site mutations, and are  listed  in  below Table 35.  Table 35: Exemplary mutations for introducing an interprotomer disulphide bond. 
Figure imgf000716_0001
Figure imgf000717_0001
  Sequences for the constructs provided in the above Table 34 are shown in the below Table 36.   Sequences are shown both with and without certain modifications introduced for expression  and purification purposes (modification include, e.g., a C‐terminal truncation fold, Precession  protease cleavage site, etc).  Modifications for expression are included in the top sequences  in each row and omitted in the bottom sequence.  Expression modifications also indicated in  the description provided in each row.  Modifications introduced to improve immunogenicity  also indicated in each row, and are the same for both top and bottom sequences.   Table 36: Sequences of exemplary Spike Protein variants 
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  Based on the in vitro results shown in Figure 46, constructs 70, 41, 84, 57, 75, 50, and 85 of  Table 34 were selected for immunogenicity testing in vivo.  Construct 50 of Table 34 showed  the greatest immunogenicity of the tested variants.    Example 23.  Neutralization of Various SARS‐CoV‐2 VOCs by RNA‐vaccines encoding  Exemplary Spike Protein Variants.   The present Example provides data demonstrating the immunogenicity of further SARS‐CoV‐ 2 variants described herein  (in particular, certain exemplary constructs  listed  in  the above  Table 34).   The study described in the present Example was conducted to evaluate the immunogenicity  of  new modified mRNA  construct  designs  of  the  protein mutants  described  in  previous  Example 22. The modRNAs  tested  in  the present example  included N1‐methylpseudouracil  and were encapsulated into lipid nanoparticles (LNPs). Groups containing 10 vaccine‐naieve  mice each were immunized with LNP formulations as a two‐dose series. Sera were collected  at Day 29 and Day 49 for neutralizing antibody responses. As indicated in below Tables 37 and  38, interprotomer A570C‐N960C was shown to provide improved immunogenicity.    Table 37: 7‐Day PD2: GMT across against SARS‐CoV‐2 variants 
Figure imgf000816_0001
  Table 38: 1‐month PD2: GMT across vaccines against SARS‐CoV‐2 variants 
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                                           SEQUENCE LISTING
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l    
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Claims

Claims  1.  A composition or medical preparation comprising an RNA encoding a SARS‐CoV‐2 S  polypeptide or an immunogenic fragment thereof, wherein the SARS‐CoV‐2 S polypeptide or  fragment comprises:   (a) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,  97%, 98%, 99%, or 100% identity to SEQ ID NO:1, and comprises one of the following  sets of amino substitutions relative to SEQ ID NO:1:  (1) D985P, V987P, F817P, A892P, A899P, and A942P;   (2) K986P, V987P, F817P, A892P, A899P, and A942P;   (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S,  and R685S;   (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;   (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or   (8) D985P, K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and  R685S;  (b)  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,  97%, 98%, 99%, or 100% identity to SEQ ID NO:69, and comprises one of the following sets of  amino substitutions relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S;   (c)  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,  97%, 98%, 99%, or 100% identity to SEQ ID NO:70, and comprises one of the following sets of  amino substitutions relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S; or  (d)  an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,  97%, 98%, 99%, or 100% identity to SEQ ID NO:104 or 105, and comprises one of the following  sets of amino substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.     
2.  The composition or medical preparation of claim 1, wherein the RNA comprises a  modified nucleoside in place of uridine.   
3.  The composition or medical preparation of claim 1 or 2, wherein the RNA comprises  modified uridines in place of all uridines.   
4.  The composition or medical preparation of any one of claims 1 to 3, wherein the  RNA comprises N1‐methyl‐pseudouridine (m1ψ) in place of all uridines.   
5.  The composition or medical preparation of any one of claims 1 to 4, wherein the  RNA comprises a 5’ cap.   
6.    The  composition  or medical  preparation  of  claim  5, wherein  the  5’  cap  is  or  comprises m2 7,3’‐OGppp(m1 2’‐O)ApG.   
7.  The composition or medical preparation of any one of claims 1 to 6, wherein the  RNA comprises a 5’‐UTR that is or comprises a modified human alpha‐globin 5’‐UTR.   
8.  The composition or medical preparation of any one of claims 1 to 7, wherein the  RNA comprises a 5’ UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide  sequence  that  is  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identical  to  the  nucleotide sequence of SEQ ID NO: 12.   
9.  The composition or medical preparation of any one of claims 1 to 8, wherein the  RNA  comprises  a  3’‐UTR  that  is  or  comprises  a  first  sequence  from  the  amino  terminal  enhancer  of  split  (AES) messenger  RNA  and  a  second  sequence  from  the mitochondrial  encoded 12S ribosomal RNA.   
10.  The composition or medical preparation of any one of claims 1 to 9, wherein the  RNA comprises a 3’ UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide  sequence  that  is  at  least  99%,  98%,  97%,  96%,  95%,  90%,  85%,  or  80%  identical  to  the  nucleotide sequence of SEQ ID NO: 13.   
11.  The composition or medical preparation of any one of claims 1 to 10, wherein the  RNA comprises a poly‐A sequence.   
12.  The composition or medical preparation of claim 11, wherein the poly‐A sequence  comprises at least 100 nucleotides.   
13.   The composition or medical preparation of claim 11 or 12, wherein  the poly‐A  sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the  30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.   
14.  The composition or medical preparation of any one of claims 11 to 13, wherein the  poly‐A  sequence comprises or consists of  the nucleotide  sequence of SEQ  ID NO: 14, or a  nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to  the nucleotide sequence of SEQ ID NO: 14.   
15.  The composition or medical preparation of any one of claims 1 to 14, wherein the  RNA is formulated or is to be formulated for intramuscular administration.   
16.  The composition or medical preparation of any one of claims 1 to 15, wherein the  RNA is formulated or is to be formulated as particles.   
17.  The composition or medical preparation of claim 16, wherein the particles are lipid  nanoparticles (LNPs) or lipoplex (LPX) particles.   
18.  The composition or medical preparation of claim 17, wherein the LNPs comprise a  cationically ionizable lipid, a neutral lipid, a sterol and a polymer‐lipid conjugate.   
19.  The composition or medical preparation of claim 17, wherein the lipoplex particles  are obtainable by mixing the RNA with liposomes.   
20.  The composition or medical preparation of any one of claims 1 to 19, wherein the  RNA is mRNA or saRNA.   
21.  The composition or medical preparation of any one of claims 1 to 20, which is a  pharmaceutical composition.   
22.  The composition or medical preparation of any one of claims 1 to 21, which is a  vaccine.   
23.    The  composition  or  medical  preparation  of  claim  21  or  22,  wherein  the  pharmaceutical  composition  further  comprises  one  or more  pharmaceutically  acceptable  carriers, diluents and/or excipients.   
24.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following  sets  of  amino  substitutions  relative to SEQ ID NO:1:  (1) D985P, V987P, F817P, A892P, A899P, and A942P;   (2) K986P, V987P, F817P, A892P, A899P, and A942P;   (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S,  and R685S;   (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;   (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or   (8) D985P, K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and  R685S.   
25.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
26.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
27.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
28.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:69; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following  sets  of  amino  substitutions  relative to SEQ ID NO:1:  (1) D985P, V987P, F817P, A892P, A899P, and A942P;   (2) K986P, V987P, F817P, A892P, A899P, and A942P;   (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S,  and R685S;   (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;   (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or   (8) D985P, K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and  R685S.   
29.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:69; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
30.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:69; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
31.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:69; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
32.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:70; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following  sets  of  amino  substitutions  relative to SEQ ID NO:1:  (1) D985P, V987P, F817P, A892P, A899P, and A942P;   (2) K986P, V987P, F817P, A892P, A899P, and A942P;   (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S,  and R685S;   (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;   (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or   (8) D985P, K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and  R685S.   
33.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:70; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
34.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:70; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
35.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:70; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
36.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:104 or SEQ ID NO:105; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:1,  and  comprises  one  of  the  following  sets  of  amino  substitutions  relative to SEQ ID NO:1:  (1) D985P, V987P, F817P, A892P, A899P, and A942P;   (2) K986P, V987P, F817P, A892P, A899P, and A942P;   (3) D985P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (4) K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and  R685S;   (5) D985P, K986P, V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S,  and R685S;   (6) D985P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S;   (7) K986P, V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; or   (8) D985P, K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and  R685S.   
37.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:104 or SEQ ID NO:105; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
38.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:104 or SEQ ID NO:105; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
39.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:104 or SEQ ID NO:105; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S. 
40.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
41.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
42.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
43.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
44.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
45.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
46.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
47.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, R682G, R683S, R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:69,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:69:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;   (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
48.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
49.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
50.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
51.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
52.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
53.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S. 
54.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
55.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
56.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
57.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, and A942P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
58.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
59.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
60.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, D614G, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
61.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
62.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: K986P, V987P,  F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
63.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ ID NO:1, and comprises the following substitutions relative to SEQ ID NO:1: D985P, K986P,  V987P, F817P, A892P, A899P, A942P, R682G, R683S, and R685S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
64.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
65.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: K983P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
66.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
67.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: K983P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
68.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID NO:70,  and  comprises one of  the  following  sets of amino  substitutions  relative to SEQ ID NO:70:  (1) D982P, V984P, F814P, A889P, A896P, and A939P;  (2) K983P, V984P, F814P, A889P, A896P, and A939P;  (3) D982P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S;  (4) K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; or  (5) D982P, K983P, V984P,  F814P, A889P, A896P, A939P, R679G, R680S, and  R682S.   
69.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
70.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: K983P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
71.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
72.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: K983P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
73.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:69, and comprises  the  following substitutions  relative  to SEQ  ID NO:69: D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
74.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:70, and comprises  the  following substitutions  relative  to SEQ  ID NO:70: D982P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
75.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:70, and comprises  the  following substitutions  relative  to SEQ  ID NO:70: K983P,  V984P, F814P, A889P, A896P, and A939P; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
76.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:70, and comprises  the  following substitutions  relative  to SEQ  ID NO:70: D982P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
77.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:70, and comprises  the  following substitutions  relative  to SEQ  ID NO:70: K983P,  V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO: 104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
78.  A composition or medical preparation comprising a first RNA encoding a first SARS‐ CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof,  and a  second RNA encoding a  second SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof, wherein:   (a) the first SARS‐CoV‐2 S polypeptide or fragment comprises an amino acid sequence  having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to  SEQ  ID NO:70, and comprises  the  following substitutions  relative  to SEQ  ID NO:70: D982P,  K983P, V984P, F814P, A889P, A896P, A939P, R679G, R680S, and R682S; and  (b)  the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  comprises  an  amino  acid  sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%  identity  to  SEQ  ID  NO:104  or  105,  and  comprises  one  of  the  following  sets  of  amino  substitutions relative to SEQ ID NO:104 or 105:  (1) D980P, V982P, F812P, A887P, A894P, and A937P;  (2) K981P, V982P, F812P, A887P, A894P, and A937P;  (3) D980P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S;  (4) K981P, V982P, F812P, A887P, A894P, A937P, R677G, R678S, and R680S; or  (5) D980P, K981P, V982P,  F812P, A887P, A894P, A937P, R677G, R678S, and  R680S.   
79.  The composition or medical preparation of any one of claims 24 to 78, wherein the  first RNA and the second RNA each comprise a modified nucleoside in place of uridine.   
80.  The composition or medical preparation of any one of claims 24 to 79, wherein the  first RNA and the second RNA each comprise modified uridines in place of all uridines.   
81.  The composition or medical preparation of any one of claims 24 to 80, wherein the  first RNA and the second RNA each comprise N1‐methyl‐pseudouridine (m1ψ) in place of all  uridines.   
82.  The composition or medical preparation of any one of claims 24 to 81, wherein the  first RNA and the second RNA each comprise a 5’ cap.   
83.  The composition or medical preparation of claim 82, wherein the 5’ cap comprises  m27,3’‐OGppp(m12’‐O)ApG.   
84.  The composition or medical preparation of any one of claims 24 to 83, wherein the  first RNA and the second RNA each comprise a 5’‐UTR that is or comprises a modified human  alpha‐globin 5’‐UTR.    85.  The composition or medical preparation of any one of claims 24 to 84, wherein the  first RNA and the second RNA each comprise a 5’ UTR comprising the nucleotide sequence of  SEQ ID NO: 12, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 
85%,  or 80% identical to the nucleotide sequence of SEQ ID NO: 12.   
86.  The composition or medical preparation of any one of claims 24 to 85, wherein the  first RNA and the second RNA each comprise a 3’‐UTR that  is or comprises a first sequence  from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from  the mitochondrial encoded 12S ribosomal RNA.   
87.  The composition or medical preparation of any one of claims 24 to 86, wherein the  first RNA and the second RNA each comprise a 3’ UTR comprising the nucleotide sequence of  SEQ ID NO: 13, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,  or 80% identical to the nucleotide sequence of SEQ ID NO: 13.   
88.  The composition or medical preparation of any one of claims 24 to 87, wherein the  first RNA and the second RNA each comprise a poly‐A sequence.   
89.  The composition or medical preparation of claim 88, wherein the first RNA and the  second RNA each comprise a poly‐A sequence that comprises at least 100 nucleotides.   
90.  The composition or medical preparation of claim 88 or 89, wherein the first RNA  and the second RNA each comprise a poly‐A sequence that comprises 30 adenine nucleotides  followed  by  70  adenine nucleotides, wherein  the  30  adenine  nucleotides  and  70  adenine  nucleotides are separated by a linker sequence.   
91.  The composition or medical preparation of any one of claims 88 to 90, wherein the  first RNA and the second RNA each comprise a poly‐A sequence that comprises or consists of  the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence that is at least 99%, 98%,  97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO: 14.   
92.  The composition or medical preparation of any one of claims 24 to 91, wherein the  first RNA and  the  second RNA are each  formulated or  to be  formulated  for  intramuscular  administration.   
93.  The composition or medical preparation of any one of claims 24 to 92, wherein the  first RNA and the second RNA are each formulated or to be formulated as particles.   
94.  The composition or medical preparation of claim 93, wherein the first RNA and the  second RNA are each to be formulated as lipid nanoparticles (LNPs) or lipoplex (LPX) particles.   
95.  The composition or medical preparation of claim 94, wherein the LNPs comprise a  cationically ionizable lipid, a neutral lipid, a sterol and a polymer‐lipid conjugate.   
96.   The  immunogenic composition of claim 94 or 95, wherein the first RNA and the  second RNA are formulated in separate LNPs.   
97.   The  immunogenic composition of claim 94 or 95, wherein the first RNA and the  second RNA are formulated in the same LNP.   
98.  The composition or medical preparation of claim 94, wherein the lipoplex particles  are obtainable by mixing the RNA with liposomes.   
99.  The composition or medical preparation of any one of claims 24 to 98, wherein the  first RNA and the second RNA are each mRNA, or wherein the first RNA and the second RNA  are each saRNA.   
100.  The composition or medical preparation of any one of claims 24 to 99, which is a  pharmaceutical composition.   
101.  The composition or medical preparation of any one of claims 24 to 100, which is  a vaccine.   
102.    The  composition  or medical  preparation  of  claim  100  or  101,  wherein  the  pharmaceutical  composition  further  comprises  one  or more  pharmaceutically  acceptable  carriers, diluents and/or excipients.   
103.  A method of inducing an immune response in a subject, the method comprising  administering to the subject the composition or medical preparation of any one of claims 1‐ 23 thereby inducing an immune response in the subject.   
104.  The method of claim 103, wherein the SARS‐CoV‐2 S polypeptide comprises an  amino acid sequence that does not comprise a D985P substitution relative to SEQ ID NO:1;  does not comprise a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or does not  comprise a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.   
105.  The method of claim 103 or 104,  further comprising administering a second  RNA  encoding  a  second  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof,  wherein the second SARS‐CoV‐2 S polypeptide or  immunogenic fragment  is a SARS‐CoV‐2 S  polypeptide of an Omicron variant that is not a BA.1 Omicron variant.   
106.  The method of claim 103 or 104,  further comprising administering a second,  different RNA encoding  a  second  SARS‐CoV‐2  S polypeptide or  an  immunogenic  fragment  thereof, wherein the second SARC‐CoV‐2 S polypeptide or fragment is selected from an SARS‐ CoV‐2 S polypeptide or fragment recited in claim 1.   
107.  A method of inducing an immune response in a subject, the method comprising  administering to the subject the composition or medical preparation of any one of claims 24‐ 102, thereby inducing an immune response in the subject.   
108.  The method of claim 107, wherein the SARS‐CoV‐2 S polypeptide comprises an  amino acid sequence that does not comprise a D985P substitution relative to SEQ ID NO:1;  does not comprise a D982P substitution relative to SEQ ID NO:69 or SEQ ID NO:70, or does not  comprise a D980P substitution relative to SEQ ID NO:104 or SEQ ID NO:105.   
109.  The method of claim 107 or 108,  further comprising administering a second  composition or medical preparation, wherein the second composition or medical preparation  comprises an RNA encoding an SARS‐CoV‐2 S polypeptide or an immunogenic fragment of an  Omicron variant that is not a BA.1 Omicron variant.     
110.  The method of claim 107 or 108,  further comprising administering a second  composition or medical preparation, wherein the second composition or medical preparation  comprises  a  third  RNA  encoding  a  third  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic  fragment  thereof,  and  a  fourth  RNA  encoding  a  fourth  SARS‐CoV‐2  S  polypeptide  or  an  immunogenic fragment thereof.   
111.  The method of  claim 110, wherein  the  third RNA encodes an  SARS‐CoV‐2  S  polypeptide or an  immunogenic  fragment  thereof  that  is a  first or a  second SARS‐CoV‐2 S  polypeptide  or  immunogenic  fragment  thereof  recited  in  any  one  of  claims  24‐102,  and  wherein the third RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that is different from  the  first  SARS‐CoV‐2  S  polypeptide  or  fragment  encoded  by  the  first  RNA  and/or  that  is  different from the second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.   
112.  The method of claim 110, wherein the  fourth RNA encodes an SARS‐CoV‐2 S  polypeptide or an  immunogenic  fragment  thereof  that  is a  first or a  second SARS‐CoV‐2 S  polypeptide  or  immunogenic  fragment  thereof  recited  in  any  one  of  claims  24‐102,  and  wherein  the  fourth RNA encodes a SARS‐CoV‐2 S polypeptide or  fragment  that  is different  from the first SARS‐CoV‐2 S polypeptide or fragment encoded by the first RNA and/or that is  different from the second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.     
113.  The method of  claim 110, wherein  the  third RNA encodes an  SARS‐CoV‐2  S  polypeptide or an  immunogenic  fragment  thereof  that  is a  first or a  second SARS‐CoV‐2 S  polypeptide  or  immunogenic  fragment  thereof  recited  in  any  one  of  claims  24‐102,  and  wherein the third RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that is different from  the first SARS‐CoV‐2 S polypeptide or fragment encoded by the first RNA and that is different  from the second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.     
114.  The method of claim 110, wherein the  fourth RNA encodes an SARS‐CoV‐2 S  polypeptide or an  immunogenic  fragment  thereof  that  is a  first or a  second SARS‐CoV‐2 S  polypeptide  or  immunogenic  fragment  thereof  recited  in  any  one  of  claims  24‐102,  and  wherein  the  fourth RNA encodes a SARS‐CoV‐2 S polypeptide or  fragment  that  is different  from  the  first SARS‐CoV‐2 S polypeptide or  fragment encoded by  the  first RNA and  that  is  different from the second SARS‐CoV‐2 S polypeptide or fragment encoded by the second RNA.     
115.  The method of  claim 110, wherein  the  third RNA encodes an  SARS‐CoV‐2  S  polypeptide or an  immunogenic  fragment  thereof  that  is a  first or a  second SARS‐CoV‐2 S  polypeptide or immunogenic fragment thereof recited in any one of claims 24‐102, wherein  the third RNA encodes a SARS‐CoV‐2 S polypeptide or fragment that is different from the first  SARS‐CoV‐2 S polypeptide or fragment encoded by the first RNA and that is different from the  second  SARS‐CoV‐2  S  polypeptide  or  fragment  encoded  by  the  second  RNA, wherein  the  fourth RNA encodes an SARS‐CoV‐2 S polypeptide or an immunogenic fragment thereof that  is a first or a second SARS‐CoV‐2 S polypeptide or immunogenic fragment thereof recited in  any one of claims 24‐102, wherein the  fourth RNA encodes a SARS‐CoV‐2 S polypeptide or  fragment that is different from the first SARS‐CoV‐2 S polypeptide or fragment encoded by the  first RNA and that is different from the second SARS‐CoV‐2 S polypeptide or fragment encoded  by the second RNA.     
116.  The method of claim 115, wherein each of the first, second, third, and fourth  RNAs encodes a different SARS‐CoV‐2 S polypeptide or immunogenic fragment thereof.   
117.     A method of inducing an immune response in a subject, the method comprising  administering to the subject (i) the composition or medical preparation of any one of claims  1‐23 and  (ii)  the composition or medical preparation of any one of claims 24‐102,  thereby  inducing an immune response in the subject.   
118.         The method of claim 117, wherein the subject receiving the composition or  medical  preparation  of  (ii) was  previously  administered with  the  composition  or medical  preparation of (i).   
119.         The method of claim 117, wherein the subject receiving the composition or  medical  preparation  of  (i) was  previously  administered  with  the  composition  or medical  preparation of (ii).   
120. The method of claim 117, wherein the composition or medical preparation of (i)  and the composition or medical preparation of (ii) are administered together as a multivalent  vaccine.   
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