WO2023147092A2

WO2023147092A2 - Coronavirus vaccine

Info

Publication number: WO2023147092A2
Application number: PCT/US2023/011791
Authority: WO
Inventors: Ye Che; Kena Anne SWANSON
Original assignee: BioNTech SE; Pfizer Inc.
Priority date: 2022-01-28
Filing date: 2023-01-27
Publication date: 2023-08-03
Also published as: WO2023147091A1; WO2023147092A9; WO2023147092A3

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: SI 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-naive 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 μ olf 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 Nl- 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 naive 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.l.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.ell (2022)) rendered BA.1 capable of partially escaping previously established SARS-CoV- 2 wild-type strain (Wuhan-Hu-l)-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 A143-145, 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 ay μ aln 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.l.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 capl 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 'Kozak sequence' to increase translational efficiency (e.g., SEQ ID NO: 12) may be used. As 3’-UTR sequence, a combination of two sequence elements (Fl 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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ: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 NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:104 or SEQ ID NQ: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 NQ: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 NQ:104 or SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ:70, and comprises the following substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:70, and comprises the following substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 NQ: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 NQ:70, and comprises the following substitutions relative to SEQ ID NQ: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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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'-O}Gppp(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 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 NQ:70, or does not comprise a D980P substitution relative to SEQ ID NQ:104 or SEQ ID NQ: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 NQ:70, or does not comprise a D980P substitution relative to SEQ ID NQ:104 or SEQ ID NQ: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) d So1main 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-Sl (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-naive 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- naive 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 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 (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 VNso 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) VNso 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. B_MEM 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 B_MEM cell (B_MEM - CD3-CD19+CD20+lgD- CD38^int/low) frequencies via B cell bait staining. (A) Schematic of one-dimensional staining of B_MEM 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 B_MEM cells for the four groups of individuals were analyzed. Variant- specific B_MEM cell frequencies were normalized to Wuhan frequencies for S protein (D) and RBD- (E) binding. (F) Depicts the frequency ratios of RBD protein specific B_MEM cells over full- length S protein-specific B_MEM cells.

Fig. 18. Omicron BA.1 breakthrough infection of BNT162b2 double- and triple-vaccinated individuals primarily boosts B_MEM 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+lgD-CD38^int/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 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 naive 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. Frequencies for these three

B_MEM 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 B_MEM 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 B_MEM 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 4^th (booster) dose of BNT162b2. "OMI" refers to sera from subjects administered an Omicron BA.1-specific 4^th (booster) dose. Also shown is the fold-change in titer from before administration of the 4^th dose to after administration of the 4^th 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 4^th dose of an Omicron BA.1-specific RNA vaccine as the 4^th dose, as compared to subjects administered BNT162b2 as the 4^th 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+0mi), 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 (Dl-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 4^th dose of BNT162b2. "OMI" refers to sera from subjects administered an Omicron BA.1-specific 4^th 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 4^th dose. Also shown is the fold-rise in titer from before administration of a 4^th dose to 7 days after administration of a 4^th 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 4^th 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) pVN so 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, 4^th sequence corresponds to Omicron BA.1 variant, and 5^th 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 naive, (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-naive 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-naive 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-naive 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-naive 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, A, 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; A, 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 otheramino 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. Koi bl, 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.n Im. 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 (a, (3, y, and 6), 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:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDI PIGAGICASYQ TQTNSPRRARSVASQSI IAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLSSNFGAI SS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGN CDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

( SEQ ID NO : 1 )

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

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.

MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAISGTNGTKRFD NPVLPFNDGVYFASTEKSNIIRGWI FGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLDVYYHKNNKSWMES EFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLGRDLPQGFSALEPLVDLP IGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKS FTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCY GVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRL FRKSNLKPFERDI STEP YQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVWLSFELLHAPATVCGPKKS TNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS NQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKS HRRARSVASQSI IAYTMSLGAENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQ YGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLAD AGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQI PFAMQMAYR FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAI SSVLNDI LSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQS APHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWI GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGK YEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT ( SEQ ID NO : 105 )

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 Al, 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:

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNG TKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIVNNATNWIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEP LVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFS TFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVC GPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDI PIGAGICASYQ TQTNSPRRARSVASQSI IAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNK VTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLSSNFGAI SS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGN CDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASWNIQKEIDRLNEVAKNLNESLIDL

QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT ( SEQ ID NO : 7 )

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

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).

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-lnclusive Coronavirus Spike Protein Modification Combinations

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.l.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.l.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 S s1ubunit 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.l.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.l.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 S s1ubunit 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.

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.l.1.7 ("Variant of Concern 202012/01" (VOC-202012/01)

B.l.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.l.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.l.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.l.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, 1692V, M1229I, and optionally S1147L.

P.l (B.l.1.248)

Lineage B.l.1.248, known as the Brazil(ian) variant, is one of the variants of SARS-CoV-2 which has been named P.l lineage. P.l 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.l, and B.1.351 variants, and also carries the same AH69/AV70 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.

Table 6: Overview of circulating SARS-CoV-2 strains which are VOI/VOC

Lineage Amino acid substitution

B.l.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

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.l, 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, A24-26, A27S, AO24-26, A27S, A69/70, 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, A24-26, A27S, G142D, K147E, W152R, F157L, 1210V, 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, A24-26, A27S, G142D, K147E, W152R, F157L, 1210V, 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, A24-26, A27S, G142D, K147E, W152R, F157L, 1210V, 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, A24-26, A27S, A69/70, 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, A24-26, A27S, V83A, G142D, A144, 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.l variant. In some embodiments, the one or more mutations characteristic of an Omicron XBB.l variant include G252V. In some embodiments, the one or more mutations characteristic of an Omicron XBB.l variant include T19I, A24-26, A27S, V83A, G142D, A144, 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.l 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, A24-26, A27S, V83A, G142D, A144, 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.l.3 variant include G252V and A484T. In some embodiments, the one or more mutations characteristic of an Omicron XBB.1.3 variant include T19I, A24-26, A27S, V83A, G142D, A144, 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, A24-26, A27S, A69/70, 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: l 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 S f1ragment (SI) (the s Su1bunit 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 S f1ragment (SI) (the S s1ubunit 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 su Sb1unit 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 W02017/0810822220 (e.g., SEQ ID NOs: 1-1115 and 1728, or fragments variants thereof) and WQ2019008001. 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

Table B: Exemplary nucleotide sequences encoding signal sequences

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 W02017/081082 (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-fi briti n- 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 BNT162bl (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 (CDllc+) 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 (CDllc+), 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 at https: ww.who.int/activities/tracking-sars-cov-2- 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 ?

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 'Kozak sequence' to increase translational efficiency. In one embodiment, a combination of two sequence elements (Fl 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.

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 Pl 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 cDN A, 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 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. 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:

The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA.

The structure of uridine is:

UTP (uridine 5'-triphosphate) has the following structure:

Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:

"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:

N1-methyl-pseudo-UTP has the following structure:

Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:

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 (m1.ψ ). 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 Nl- 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), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2- thio-uridine (m⁵s²U), l-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), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m⁵Um), 2'-O-methyl-pseudouridine (ψ m), 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 Nl- 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 capl 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 "capl" 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' -O}Gppp(m₁ ^{2' -O})ApG (also sometimes referred to as m₂ ⁷'^{3' O}G(5')ppp(5')m^{2' -O}ApG), which has the following structure:

Below is an exemplary Capl RNA, which comprises RNA and m2⁷'^{3 O}G(5')ppp(5')m^{2 -O}ApG:

Below is another exemplary Capl RNA (no cap analog):

In some embodiments, the RNA is modified with "CapO" structures using, in one embodiment, the cap analog anti-reverse cap (ARCA Cap ( m₂ ⁷'^{3' O}G(5')ppp(5')G)) with the structure:

Below is an exemplary CapO RNA comprising RNA and m₂ ⁷'^{3' O}G(5')ppp(5')G:

In some embodiments, the "CapO" structures are generated using the cap analog Beta-S-ARCA

( m₂ ⁷'^{2' O}G(5')ppSp(5')G) with the structure:

Below is an exemplary CapO RNA comprising Beta-S-ARCA ( m₂ ⁷'^{2' O}G(5')ppSp(5')G) and RNA:

The "DI" 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' O}GppSpG) or m₂ ⁷'^{3' O}Gppp(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 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al 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 WQ2016/005324, 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 'Kozak sequence' to increase translational efficiency. In one embodiment, a combination of two sequence elements (Fl 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 fortranscription 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- carboxy methy la minomethyl-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), l-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), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m⁵Um), 2'-O-methyl-pseudouridine (ipm), 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 RNaselll 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 'Kozak sequence' to increase translational 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.

Fl 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 Capl 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'-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. 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

BNT162al; 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)-tai I ). Compared to the uRNA, modRNA contains 1-methyl- pseudouridine instead of uridine. The preferred 5' cap structure is m₂ ^{7, 3'-O}Gppp(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'-O}Gppp(m1^{2' -O})ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

BNT162b2; RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7)

Structure m₂ ^{7, 3'-O}Gppp(m1^{2' -O})ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

BNT162bl; RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5)

Structure m₂ ^{7, 3'-O}Gppp(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'-O}Gppp(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'-O}Gppp(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.il (SEQ ID NO: 57; SEQ ID NO: 55)

Structure m₂ ^{7, 3'-O}Gppp(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'-O}Gppp(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 m₂ ^{7, 3'-O}Gppp(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 Delta variant of SARS-CoV-2

Nucleotide Sequence of RBP020.il (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.

Sequences of RBP020.il 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'-O}Gppp(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.

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.

10 20 30 40 50 53

AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCCGCC ACC hAg-Kozak

63 73 83 93 103 113

AUGUUCGUGU UCCUGGUGCU GCUGCCUCUG GUGUCCAGCC AGUGUGUGAA CCUGACCACC

M F V F L V L L P L V S S Q C V N L T T

S protein mut4

123 133 143 153 163 173

AGAACACAGC UGCCUCCAGC CUACACCAAC AGCUUUACCA GAGGCGUGUA CUACCCCGAC

R T Q L P P A Y T N S F T R G V Y Y P D

S protein mut3

183 193 203 213 223 233

AAGGUGUUCA GAUCCAGCGU GCUGCACUCU ACCCAGGACC UGUUCCUGCC UUUCUUCAGC

K V F R S S V L H S T Q D L F L P F F S

S protein mut3

243 253 263 273 283 293

AACGUGACCU GGUUCCACGC CAUCUCCGGC ACCAAUGGCA CCAAGAGAUU CGACAACCCC N V T W F H A I S G T N G T K R F D N P

S protein mut3

303 313 323 333 343 353

GUGCUGCCCU UCAACGACGG GGUGUACUUU GCCAGCACCG AGAAGUCCAA CAUCAUCAGA

V L P F N D G V Y F A S T E K S N I I R

S protein mut3

363 373 383 393 403 413

GGCUGGAUCU UCGGCACCAC ACUGGACAGC AAGACCCAGA GCCUGCUGAU CGUGAACAAC

G W I F G T T L D S K T Q S L L I V N N

S protein mut3

423 433 443 453 463 473

GCCACCAACG UGGUCAUCAA AGUGUGCGAG UUCCAGUUCU GCAACGACCC CUUCCUGGGC

A T N V V I K V C E F Q F C N D P F L G

S protein mut3

483 493 503 513 523 533

GUCUACCACA AGAACAACAA GAGCUGGAUG GAAAGCGAGU UCCGGGUGUA CAGCAGCGCC

V Y H K N N K S W M E S E F R V Y S S A

S protein mut3 543 553 563 573 583 593

AACAACUGCA CCUUCGAGUA CGUGUCCCAG CCUUUCCUGA UGGACCUGGA AGGCAAGCAG N N C T F E Y V S Q P F L M D L E G K Q

S protein mut3

603 613 623 633 643 653

GGCAACUUCA AGAACCUGCG CGAGUUCGUG UUUAAGAACA UCGACGGCUA CUUCAAGAUC

G N F K N L R E F V F K N I D G Y F K I

S protein mut3

663 673 683 693 703 713

UACAGCAAGC ACACCCCUAU CAACCUCGUG CGGGAUCUGC CUCAGGGCUU CUCUGCUCUG

Y S K H T P I N L V R D L P Q G F S A L

S protein mut3

723 733 743 753 763 773

GAACCCCUGG UGGAUCUGCC CAUCGGCAUC AACAUCACCC GGUUUCAGAC ACUGCUGGCC E P L V D L P I G I N I T R F Q T L L A

S protein mut3

783 793 803 813 823 833

CUGCACAGAA GCUACCUGAC ACCUGGCGAU AGCAGCAGCG GAUGGACAGC UGGUGCCGCC

L H R S Y L T P G D S S S G W T A G A A

S protein mut3

843 853 863 873 883 893

GCUUACUAUG UGGGCUACCU GCAGCCUAGA ACCUUCCUGC UGAAGUACAA CGAGAACGGC

A Y Y V G Y L Q P R T F L L K Y N E N G

S protein mut3

903 913 923 933 943 953

ACCAUCACCG ACGCCGUGGA UUGUGCUCUG GAUCCUCUGA GCGAGACAAA GUGCACCCUG

T I T D A V D C A L D P I S E T K C T L

S protein mut3

963 973 983 993 1003 1013

AAGUCCUUCA CCGUGGAAAA GGGCAUCUAC CAGACCAGCA ACUUCCGGGU GCAGCCCACC K S F T V E K G I Y Q T S N F R V Q P T

S protein mut3

1023 1033 1043 1053 1063 1073

GAAUCCAUCG UGCGGUUCCC CAAUAUCACC AAUCUGUGCC CCUUCGGCGA GGUGUUCAAU

E S I V R F P N I T N I C P F G E V E N

S protein mut3

1083 1093 1103 1113 1123 1133

GCCACCAGAU UCGCCUCUGU GUACGCCUGG AACCGGAAGC GGAUCAGCAA UUGCGUGGCC

A T R F A S V Y A W N R K R I S N C V A

S protein mut3

1143 1153 1163 1173 1183 1193

GACUACUCCG UGCUGUACAA CUCCGCCAGC UUCAGCACCU UCAAGUGCUA CGGCGUGUCC

D Y S V L Y N S A S F S T F K C Y G V S

S protein mut3

1203 1213 1223 1233 1243 1253

CCUACCAAGC UGAACGACCU GUGCUUCACA AACGUGUACG CCGACAGCUU CGUGAUCCGG

P T K L N D L C F T N V Y A D S F V I R

S protein mut3 1263 1273 1283 1293 1303 1313

GGAGAUGAAG UGCGGCAGAU UGCCCCUGGA CAGACAGGCA AGAUCGCCGA CUACAACUAC

G D E V R Q I A P G Q T G K I A D Y N Y

S protein mut3

1323 1333 1343 1353 1363 1373

AAGCUGCCCG ACGACUUCAC CGGCUGUGUG AUUGCCUGGA ACAGCAACAA CCUGGACUCC

K L P D D F T G C V I A W N S N N L D S

S protein mut3

1383 1393 1403 1413 1423 1433

AAAGUCGGCG GCAACUACAA UUACCUGUAC CGGCUGUUCC GGAAGUCCAA UCUGAAGCCC

K V G G N Y N Y L Y R L F R K S N L K P

S protein mut3

1443 1453 1463 1473 1483 1493

UUCGAGCGGG ACAUCUCCAC CGAGAUCUAU CAGGCCGGCA GCACCCCUUG UAACGGCGUG

P E R D I S T E I Y Q A G S T P C N G V

S protein mut3

1503 1513 1523 1533 1543 1553

GAAGGCUUCA ACUGCUACUU CCCACUGCAG UCCUACGGCU UUCAGCCCAC AGACGGCGUG

E G F N C Y F P L Q S Y G F Q P T Y G V

S protein mut3

1563 1573 1583 1593 1603 1613

GGCUAUCAGC CCUACAGAGU GGUGGUGCUG AGCUUCGAAC UGCUGCAUGC CCCUGCCACA

G Y Q P Y R V V V L S F E L L H A P A T

S protein mut3

1623 1633 1643 1653 1663 1673

GUGUGCGGCC CUAAGAAAAG CACCAAUCUC GUGAAGAACA AAUGCGUGAA CUUCAACUUC

V C G P K K S T N L V K N K C V N F N F

S protein mut3

1683 1693 1703 1713 1723 1733

AACGGCCUGA CCGGCACCGG CGUGCUGACA GAGAGCAACA AGAAGUUCCU GCCAUUCCAG

N G L T G T G V L T E S N K K F L P F Q

S protein mut3

1743 1753 1763 1773 1783 1793

CAGUUUGGCC GGGAUAUCGA CGAUACCACA GACGCCGUUA GAGAUCCCCA GACACUGGAA

Q F G R D I D D T T D A V R D P Q T L E

S protein mut3

1803 1813 1823 1833 1843 1853

AUCCUGGACA UCACCCCUUG CAGCUUCGGC GGAGUGUCUG UGAUCACCCC UGGCACCAAC

I L D I I P C S F G G V S V I T P G T N

S protein mut3

1863 1873 1883 1893 1903 1913

ACCAGCAAUC AGGUGGCAGU GCUGUACCAG GGCGUGAACU GUACCGAAGU GCCCGUGGCC

T S N Q V A V L Y Q G V N G T E V P V A

S protein mut3

1923 1933 1943 1953 1963 1973

AUUCACGCCG AUCAGCUGAC ACCUACAUGG CGGGUGUACU CCACCGGCAG CAAUGUGUUU

I H A D Q L T P T W R V Y S T G S N V F

S protein mut3 1983 1993 2003 2013 2023 2033

CAGACCAGAG CCGGCUGUCU GAUCGGAGCC GAGCACGUGA ACAAUAGCUA CGAGUGCGAC

Q T R A G C L I G A E H V N N S Y E C D

S protein mut3

2043 2053 2063 2073 2083 2093

AUCCCCAUCG GCGCUGGAAU CUGCGCCAGC UACCAGACAC AGACAAACAG CGACCGGAGA

I P I G A G I C A S Y Q T Q T N S H R R

S protein mut3

2103 2113 2123 2133 2143 2153

GCCAGAAGCG UGGCCAGCCA GAGCAUCAUU GCCUACACAA UGUCUCUGGG CGCCGAGAAC A R S V A S Q S I I A Y T M S L G A E N

S protein mut3

2163 2173 2183 2193 2203 2213

AGCGUGGCCU ACUCCAACAA CUCUAUCGCU AUCCCCAJCA ACUUCACCAU CAGCGUGACC S V A Y S N N S I A I P ? N E T I S V T

S protein mut3

2223 2233 2243 2253 2263 2273

ACAGAGAUCC UGCCUGUGUC CAUGACCAAG ACCAGCGUGG ACUGCACCAU GUACAUCUGC

T E I L P V S M T K T S V D C T M Y I C

S protein mut3

2283 2293 2303 2313 2323 2333

GGCGAUUCCA CCGAGUGCUC CAACCUGCUG CUGCAGUACG GCAGCUUCUG CACCCAGCUG

G D S T E C S N L L L Q Y G S F C T Q L

S protein mut3

2343 2353 2363 2373 2383 2393

AAUAGAGCCC UGACAGGGAU CGCCGUGGAA CAGGACAAGA ACACCCAAGA GGUGUUCGCC N R A L T G I A V E Q D K N T Q E V F A

S protein mut3

2403 2413 2423 2433 2443 2453

CAAGUGAAGC AGAUCUACAA GACCCCUCCU AUCAAGGACU UCGGCGGCUU CAAUUUCAGC Q V K Q I Y K T P P I K D F G G F N F S

S protein mut3

2463 2473 2483 2493 2503 2513

CAGAUUCUGC CCGAUCCUAG CAAGCCCAGC AAGCGGAGCU UCAUCGAGGA CCUGCUGUUC

Q I L P D F S K P S K R S F I E D L L F

S protein mut3

2523 2533 2543 2553 2563 2573

AACAAAGUGA CACUGGCCGA CGCCGGCUUC AUCAAGCAGU AUGGCGAUUG UCUGGGCGAC

N K V T L A D A G F I K Q Y G D C L G D

S protein mut3

2583 2593 2603 2613 2623 2633

AUUGCCGCCA GGGAUCUGAU UUGCGCCCAG AAGUUUAACG GACUGACAGU GCUGCCUCCU

I A A R D L I C A Q K F N G L T V L P P

S protein mut3

2643 2653 2663 2673 2683 2693

CUGCUGACCG AUGAGAUGAU CGCCCAGUAC ACAUCUGCCC UGCUGGCCGG CACAAUCACA L L T D E M I A Q Y T S A L L A G T I T

S protein mut3 2703 2713 2723 2733 2743 2753

AGCGGCUGGA CAUUUGGAGC AGGCGCCGCU CUGCAGAUCC CCUUUGCUAU GCAGAUGGCC

S G W T F G A G A A L Q I P F A M Q M A

S protein mut3

2763 2773 2783 2793 2803 2813

UACCGGUUCA ACGGCAUCGG AGUGACCCAG AAUGUGCUGU ACGAGAACCA GAAGCUGAUC

Y R F N G I G V T Q N V L Y E N Q K L I

S protein mut3

2823 2833 2843 2853 2863 2873

GCCAACCAGU UCAACAGCGC CAUCGGCAAG AUCCAGGACA GCCUGAGCAG CACAGCAAGC

A N Q F N S A I G K I Q D S L S S T A S

S protein mut3

2883 2893 2903 2913 2923 2933

GCCCUGGGAA AGCUGCAGGA CGUGGUCAAC CAGAAUGCCC AGGCACUGAA CACCCUGGUC A L G K L Q D V V N Q N A Q A L N T L V

S protein mut3

2943 2953 2963 2973 2983 2993

AAGCAGCUGU CCUCCAACUU CGGCGCCAUC AGCUCUGUGC UGAACGAUAU CCUGGCCAGA

K Q L S S N F G A I S S V L N D T E A R

S protein mut3

3003 3013 3023 3033 3043 3053

CUGGAC GAGAGGCCGA GGUGCAGAUC GACAGACUGA UCACAGGCAG ACUGCAGAGC L D P F E A E V Q I D R L I T G R L Q S

S protein mut3

3063 3073 3083 3093 3103 3113

CUCCAGACAU ACGUGACCCA GCAGCUGAUC AGAGCCGCCG AGAUUAGAGC CUCUGCCAAU L Q T Y V T Q Q L I R A A E I R A S A N

S protein mut3

3123 3133 3143 3153 3163 3173

CUGGCCGCCA CCAAGAUGUC UGAGUGUGUG CUGGGCCAGA GCAAGAGAGU GGACUUUUGC

L A A T K M S E C V L G Q S K R V D F C

S protein mut3

3183 3193 3203 3213 3223 3233

GGCAAGGGCU ACCACCUGAU GAGCUUCCCU CAGUCUGCCC CUCACGGCGU GGUGUUUCUG G K G Y H L M S F P Q S A P H G V V F L

S protein mut3

3243 3253 3263 3273 3283 3293

CACGUGACAU AUGUGCCCGC UCAAGAGAAG AAUUUCACCA CCGCUCCAGC CAUCUGCCAC H V T Y V P A Q E K N F T T A P A I C H

S protein mut3

3303 3313 3323 3333 3343 3353

GACGGCAAAG CCCACUUUCC UAGAGAAGGC GUGUUCGUGU CCAACGGCAC CCAUUGGUUC

D G K A H F P R E G V F V S N G T H W F

S protein mut3

3363 3373 3383 3393 3403 3413

GUGACACAGC GGAACUUCUA CGAGCCCCAG AUCAUCACCA CCCACAACAC CUUCGUGUCU

V T Q R N F Y E P Q I T T T E N T F V S

S protein mut3 3423 3433 3443 3453 3463 3473

GGCAACUGCG ACGUCGUGAU CGGCAUUGUG AACAAUACCG UGUACGACCC UCUGCAGCCC

G N C D V V I G I V N N T V Y D P L Q P

S protein mut3

3483 3493 3503 3513 3523 3533

GAGCUGGACA GCUUCAAAGA GGAACUGGAC AAGUACUUUA AGAACCACAC AAGCCCCGAC

E L D S F K E E L D K Y F K N H T S P D

S protein mut3

3543 3553 3563 3573 3583 3593

GUGGACCUGG GCGAUAUCAG CGGAAUCAAU GCCAGCGUCG UGAACAUCCA GAAAGAGAUC

V D L G D I S G I N A S V V N I Q K E I

S protein mut3

3603 3613 3623 3633 3643 3653

GACCGGCUGA ACGAGGUGGC CAAGAAUCUG AACGAGAGCC UGAUCGACCU GCAAGAACUG

D R L N E V A K N L N E S L I D L Q E L

S protein mut3

3663 3673 3683 3693 3703 3713

GGGAAGUACG AGCAGUACAU CAAGUGGCCC UGGUACAUCU GGCUGGGCUU UAUCGCCGGA

G K Y E Q Y I K W P W Y I W L G F I A G

S protein mut3

3723 3733 3743 3753 3763 3773

CUGAUUGCCA UCGUGAUGGU CACAAUCAUG CUGUGUUGCA UGACCAGCUG CUGUAGCUGC

L I A I V M V T I M L C C M T S C C S C

S protein mut3

3783 3793 3803 3813 3823 3833

CUGAAGGGCU GUUGUAGCUG UGGCAGCUGC UGCAAGUUCG ACGAGGACGA UUCUGAGCCC

L K G C C S C G S C C K F D E D D S E P

S protein mut3

3843 3853 3863 3869

GUGCUGAAGG GCGUGAAACU GCACUACACA UGAUGA

V L K G V K L H Y T * *

S protein mut3

3879 3889 3899 3909 3919 3929

GAUCUGCUGG UACUGCAUGC ACGCAAUGCU AGCUGCCCCU UUCCCGUCCU GGGUACCCCG

Fl element

3939 3949 3959 3969 3979 3989

AGUCUCCCCC GACCUCGGGU CCCAGGUAUG CUCCCACCUC CACCUGCCCC ACUCACCACC

Fl element

3999 4009 4019 4029 4039 4049

UCUGCUAGUU CCAGACACCU CCCAAGCACG CAGCAAUGCA GCUCAAAACG CUUAGCCUAG

Fl element

4059 4069 4079 4089 4099 4109

CCACACCCCC ACGGGAAACA GCAGUGAUUA ACCUUUAGCA AUAAACGAAA GUUUAACUAA

Fl element 4119 4129 4139 4149 4159 4164

GCUAUACUAA CCCCAGGGUU GGUCAAUUUC GUGCCAGCCA CACCCUGGAG CUAGC

Fl element

4174 4184 4194 4204 4214 4224

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCAUAUGACU AAAAAAAAAA AAAAAAAAAA

Poly(A)

4234 4244 4254 4264 4274

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

Poly(A)

Sequences of RBP020.14 are also shown in Table 10.

Nucleotide Sequence of RBP020.16 (Delta-specific vaccine)

10 20 30 40 50 53

AGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCCGCC ACC hAg-Kozak

63 73 83 93 103 113

ATGTTCGTGT TCCTGGTGCT GCTGCCTCTG GTGTCCAGCC AGTGTGTGAA CCTGAGAACC

M F V F L V L L P L V S S Q C V N L J: T

S protein mut4

123 133 143 153 163 173

AGAACACAGC TGCCTCCAGC CTACACCAAC AGCTTTACCA GAGGCGTGTA CTACCCCGAC

R T Q L P P A Y T N S F T R G V Y Y P D

S protein mut4

183 193 203 213 223 233

AAGGTGTTCA GATCCAGCGT GCTGCACTCT ACCCAGGACC TGTTCCTGCC TTTCTTCAGC

K V F R S S V L H S T Q D L F L P F F S

S protein mut4

243 253 263 273 283 293

AACGTGACCT GGTTCCACGC CATCCACGTG TCCGGCACCA ATGGCACCAA GAGATTCGAC N V T W F H A I H V S G T N G T K R F D

S protein mut4

303 313 323 333 343 353

AACCCCGTGC TGCCCTTCAA CGACGGGGTG TACTTTGCCA GCACCGAGAA GTCCAACATC

N P V L P F N D G V Y F A S T E K S N I

S protein mut4

363 373 383 393 403 413

ATCAGAGGCT GGATCTTCGG CACCACACTG GACAGCAAGA CCCAGAGCCT GCTGATCGTG

I R G W I F G T T L D S K T Q S L L I V

S protein mut4

423 433 443 453 463 473

AACAACGCCA CCAACGTGGT CATCAAAGTG TGCGAGTTCC AGTTCTGCAA CGACCCCTTC

N N A T N V V I K V C E F Q F C N D P F

S protein mut4

483 493 503 513 523 533

CTGGACGTCT ACTACCACAA GAACAACAAG AGCTGGATGG AAAGCGGCGT GTACAGCAGC L E V Y Y H K N N K S W M E S G V Y S S

S protein mut4 543 553 563 573 583 593

GCCAACAACT GCACCTTCGA GTACGTGTCC CAGCCTTTCC TGATGGACCT GGAAGGCAAG

A N N C T F E Y V S Q P F L M D L E G K

S protein mut4

603 613 623 633 643 653

CAGGGCAACT TCAAGAACCT GCGCGAGTTC GTGTTTAAGA ACATCGACGG CTACTTCAAG

Q G N F K N L R E F V F K N I D G Y F K

S protein mut4

663 673 683 693 703 713

ATCTACAGCA AGCACACCCC TATCAACCTC GTGCGGGATC TGCCTCAGGG CTTCTCTGCT N L V R D L P Q G F S A

protein mut4

723 733 743 753 763 773

CTGGAACCCC TGGTGGATCT GCCCATCGGC ATCAACATCA CCCGGTTTCA GACACTGCTG L E P L V D L P I G I N I T R F Q T L L

S protein mut4

783 793 803 813 823 833

GCCCTGCACA GAAGCTACCT GACACCTGGC GATAGCAGCA GCGGATGGAC AGCTGGTGCC

A L H R S Y L T P G D S S S G W T A G A

S protein mut4

843 853 863 873 883 893

GCCGCTTACT ATGTGGGCTA CCTGCAGCCT AGAACCTTCC TGCTGAAGTA CAACGAGAAC

A A Y Y V G Y L Q P R T F I L K Y N E N

S protein mut4

903 913 923 933 943 953

GGCACCATCA CCGACGCCGT GGATTGTGCT CTGGATCCTC TGAGCGAGAC AAAGTGCACC

G T I T D A V D C A L D P L S E T K C T

S protein mut4

963 973 983 993 1003 1013

CTGAAGTCCT TCACCGTGGA AAAGGGCATC TACCAGACCA GCAACTTCCG GGTGCAGCCC L K S F T V E K G I Y Q T S N F R V Q P

S protein mut4

1023 1033 1043 1053 1063 1073

ACCGAATCCA TCGTGCGGTT CCCCAATATC ACCAATCTGT GCCCCTTCGG CGAGGTGTTC

T E S I V R F P N I T N L C P F G E V F

S protein mut4

1083 1093 1103 1113 1123 1133

AATGCCACCA GATTCGCCTC TGTGTACGCC TGGAACCGGA AGCGGATCAG CAATTGCGTG

N A T R F A S V Y A W N R K R I S N C V

S protein mut4

1143 1153 1163 1173 1183 1193

GCCGACTACT CCGTGCTGTA CAACTCCGCC AGCTTCAGCA CCTTCAAGTG CTACGGCGTG

A D Y S V L Y N S A S F S T F K C Y G V

S protein mut4

1203 1213 1223 1233 1243 1253

TCCCCTACCA AGCTGAACGA CCTGTGCTTC ACAAACGTGT ACGCCGACAG CTTCGTGATC

S P T K L N D L C F T N V Y A D S F V I

S protein mut4 1263 1273 1283 1293 1303 1313

CGGGGAGATG AAGTGCGGCA GATTGCCCCT GGACAGACAG GCAAGATCGC CGACTACAAC R G D E V R Q I A P G Q T G K I A D Y N

S protein mut4

1323 1333 1343 1353 1363 1373

TACAAGCTGC CCGACGACTT CACCGGCTGT GTGATTGCCT GGAACAGCAA CAACCTGGAC

Y K L F D D F T G C V I A W N S N N L D

S protein mut4

1383 1393 1403 1413 1423 1433

TCCAAAGTCG GCGGCAACTA CAATTACAGG TACCGGCTGT TCCGGAAGTC CAATCTGAAG

S K V G G N Y N Y R: Y R L F R K S N L K

S protein mut4

1443 1453 1463 1473 1483 1493

CCCTTCGAGC GGGACATCTC CACCGAGATC TATCAGGCCG GCAGCGAGCC TTGTAACGGC

P F E R D I S T E I Y Q A G S K P C N G

S protein mut4

1503 1513 1523 1533 1543 1553

GTGGAAGGCT TCAACTGCTA CTTCCCACTG CAGTCCTACG GCTTTCAGCC CACAAATGGC

V E G F N C Y F P L Q S Y G F Q P I N G

S protein mut4

1563 1573 1583 1593 1603 1613

GTGGGCTATC AGCCCTACAG AGTGGTGGTG CTGAGCTTCG AACTGCTGCA TGCCCCTGCC

V G Y Q P Y R V V V L S F E L L H A P A

S protein mut4

1623 1633 1643 1653 1663 1673

ACAGTGTGCG GCCCTAAGAA AAGCACCAAT CTCGTGAAGA ACAAATGCGT GAACTTCAAC

T V C G P K K S T N L V K N K C V N F N

S protein mut4

1683 1693 1703 1713 1723 1733

TTCAACGGCC TGACCGGCAC CGGCGTGCTG ACAGAGAGCA ACAAGAAGTT CCTGCCATTC

F N G L T G T G V L T E S N K K F L P F

S protein mut4

1743 1753 1763 1773 1783 1793

CAGCAGTTTG GCCGGGATAT CGCCGATACC ACAGACGCCG TTAGAGATCC CCAGACACTG

Q Q F G R D I A D T I D A V R D P Q T L

S protein mut4

1803 1813 1823 1833 1843 1853

GAAATCCTGG ACATCACCCC TTGCAGCTTC GGCGGAGTGT CTGTGATCAC CCCTGGCACC E I L D I T P C S F G G V S V I T P G T

S protein mut4

1863 1873 1883 1893 1903 1913

AACACCAGCA ATCAGGTGGC AGTGCTGTAC CAGGGGGTGA ACTGTACCGA AGTGCCCGTG N T S N Q V A V L Y Q G V N C I E V P V

S protein mut4

1923 1933 1943 1953 1963 1973

GCCATTCACG CCGATCAGCT GACACCTACA TGGCGGGTGT ACTCCACCGG CAGCAATGTG

A F T W R V Y S T G S N V

protein mut4 1983 1993 2003 2013 2023 2033

TTTCAGACCA GAGCCGGCTG TCTGATCGGA GCCGAGCACG TGAACAATAG CTACGAGTGC

F Q T R A G C L I G A E H V N N S Y E C

S protein mut4

2043 2053 2063 2073 2083 2093

GACATCCCCA TCGGCGCTGG AATCTGCGCC AGCTACCAGA CACAGACAAA CAGCAGGCGG

D I P I G A G I C A S Y Q T Q T N S A R

S protein mut4

2103 2113 2123 2133 2143 2153

AGAGCCAGAA GCGTGGCCAG CCAGAGCATC ATTGCCTACA CAATGTCTCT GGGCGCCGAG

R A R S V A S Q S I I A Y I M S L G A E

S protein mut4

2163 2173 2183 2193 2203 2213

AACAGCGTGG CCTACTCCAA CAACTCTATC GCTATCCCCA CCAACTTCAC CATCAGCGTG

N S V A Y S N N S I A I R I N F T I S V

S protein mut4

2223 2233 2243 2253 2263 2273

ACCACAGAGA TCCTGCCTGT GTCCATGACC AAGACCAGCG TGGACTGCAC CATGTACATC

T I E I I P V S M T K T S V D C T M Y I

S protein mut4

2283 2293 2303 2313 2323 2333

TGCGGCGATT CCACCGAGTG CTCCAACCTG CTGCTGCAGT ACGGCAGCTT CTGCACCCAG

C G D S T E C S N L L L Q Y G S F C T Q

S protein mut4

2343 2353 2363 2373 2383 2393

CTGAATAGAG CCCTGACAGG GATCGCCGTG GAACAGGACA AGAACACCCA AGAGGTGTTC

L N R A L T G I A V E Q D K N T Q E V F

S protein mut4

2403 2413 2423 2433 2443 2453

GCCCAAGTGA AGCAGATCTA CAAGACCCCT CCTATCAAGG ACTTCGGCGG CTTCAATTTC A Q V K Q I Y K T P P I K D F G G F N F

S protein mut4

2463 2473 2483 2493 2503 2513

AGCCAGATTC TGCCCGATCC TAGCAAGCCC AGCAAGCGGA GCTTCATCGA GGACCTGCTG

S Q I L P D P S K P S K R S F I E D L L

S protein mut4

2523 2533 2543 2553 2563 2573

TTCAACAAAG TGACACTGGC CGACGCCGGC TTCATCAAGC AGTATGGCGA TTGTCTGGGC

F N K V T L A D A G F I K Q Y G D C L G

S protein mut4

2583 2593 2603 2613 2623 2633

GACATTGCCG CCAGGGATCT GATTTGCGCC CAGAAGTTTA ACGGACTGAC AGTGCTGCCT

D I A A R D L I C A Q K F N G L T V L P

S protein mut4

2643 2653 2663 2673 2683 2693

CCTCTGCTGA CCGATGAGAT GATCGCCCAG TACACATCTG CCCTGCTGGC CGGCACAATC

P L L T D E M I A Q Y T S A L L A G T I

S protein mut4 2703 2713 2723 2733 2743 2753

ACAAGCGGCT GGACATTTGG AGCAGGCGCC GCTCTGCAGA TCCCCTTTGC TATGCAGATG

T S G W T F G A G A A L Q I F F A M Q M

S protein mut4

2763 2773 2783 2793 2803 2813

GCCTACCGGT TCAACGGCAT CGGAGTGACC CAGAATGTGC TGTACGAGAA CCAGAAGCTG

A Y R F N G I G V T Q N V L Y E N Q K L

S protein mut4

2823 2833 2843 2853 2863 2873

ATCGCCAACC AGTTCAACAG CGCCATCGGC AAGATCCAGG ACAGCCTGAG CAGCACAGCA I A N Q F N S A I G K I Q D S L S S T A

S protein mut4

2883 2893 2903 2913 2923 2933

AGCGCCCTGG GAAAGCTGCA GAAGGTGGTC AACCAGAATG CCCAGGCACT GAACACCCTG

S A L G K L Q N V V N Q N A Q A L N T L

S protein mut4

2943 2953 2963 2973 2983 2993

GTCAAGCAGC TGTCCTCCAA CTTCGGCGCC ATCAGCTCTG TGCTGAACGA TATCCTGAGC

V K Q L S S N F G A L S

S protein

3003 3013 3023 3033 3043 3053

AGACTGGACC CTCCTGAGGC CGAGGTGCAG ATCGACAGAC TGATCACAGG CAGACTGCAG

R L D P R E A E V Q I D R L I T G R L Q

S protein mut4

3063 3073 3083 3093 3103 3113

AGCCTCCAGA CATACGTGAC CCAGCAGCTG ATCAGAGCCG CCGAGATTAG AGCCTCTGCC

S L Q T Y V T Q Q L R A S A

S protein

3123 3133 3143 3153 3163 3173

AATCTGGCCG CCACCAAGAT GTCTGAGTGT GTGCTGGGCC AGAGCAAGAG AGTGGACTTT

N L A A T K M S E C V L G Q S K R V D F

S protein mut4

3183 3193 3203 3213 3223 3233

TGCGGCAAGG GCTACCACCT GATGAGCTTC CCTCAGTCTG CCCCTCACGG CGTGGTGTTT

C G K G Y H L M S F P Q S A P H G V V F

S protein mut4

3243 3253 3263 3273 3283 3293

CTGCACGTGA CATATGTGCC CGCTCAAGAG AAGAATTTCA CCACCGCTCC AGCCATCTGC

L H V T Y V P A Q E K N F T T A P A I C

S protein mut4

3303 3313 3323 3333 3343 3353

CACGACGGCA AAGCCCACTT TCCTAGAGAA GGCGTGTTCG TGTCCAACGG CACCCATTGG H D G K A H F P R E G V F V S N G T H W

S protein mut4

3363 3373 3383 3393 3403 3413

TTCGTGACAC AGCGGAACTT CTACGAGCCC CAGATCATCA CCACCGACAA CACCTTCGTG F V T Q R N F Y E P Q I I T T D N T F V

S protein mut4 3423 3433 3443 3453 3463 3473

TCTGGCAACT GCGACGTCGT GATCGGCATT GTGAACAATA CCGTGTACGA CCCTCTGCAG

S G N C D V V I G I V N N T V Y D P L Q

S protein mut4

3483 3493 3503 3513 3523 3533

CCCGAGCTGG ACAGCTTCAA AGAGGAACTG GACAAGTACT TTAAGAACCA CACAAGCCCC

P E L D S F K E E L D K Y F K N H T S P

S protein mut4

3543 3553 3563 3573 3583 3593

GACGTGGACC TGGGCGATAT CAGCGGAATC AATGCCAGCG TCGTGAACAT CCAGAAAGAG

D V D L G D L S G T N A S V V N L Q K E

S protein mut4

3603 3613 3623 3633 3643 3653

ATCGACCGGC TGAACGAGGT GGCCAAGAAT CTGAACGAGA GCCTGATCGA CCTGCAAGAA

I D R L N E V A K N L N E S L T D L Q E

S protein mut4

3663 3673 3683 3693 3703 3713

CTGGGGAAGT ACGAGCAGTA CATCAAGTGG CCCTGGTACA TCTGGCTGGG CTTTATCGCC

L G K Y E Q Y L K W P W Y L W L G F L A

S protein mut4

3723 3733 3743 3753 3763 3773

GGACTGATTG CCATCGTGAT GGTCACAATC ATGCTGTGTT GCATGACCAG CTGCTGTAGC

G L I A L V M V T T M L C C M T S C C S

S protein mut4

3783 3793 3803 3813 3823 3833

TGCCTGAAGG GCTGTTGTAG CTGTGGCAGC TGCTGCAAGT TCGACGAGGA CGATTCTGAG

C L K G C C S C G S C C K F D E D D S E

S protein mut4

3843 3853 3863 3872

CCCGTGCTGA AGGGCGTGAA ACTGCACTAC ACATGATGA

P V L K G V K L H Y T * *

S protein mut4

3882 3892 3902 3912 3922 3932

TTTCACCTGG TACTGCATGC ACGCAATGCT AGCTGCCCCT TTCCCGTCCT GGGTACCCCG

Fl element

3942 3952 3962 3972 3982 3992

AGTCTCCCCC GACCTCGGGT CCCAGGTATG CTCCCACCTC CACCTGCCCC ACTCACCACC

Fl element

4002 4012 4022 4032 4042 4052

TCTGCTAGTT CCAGACACCT CCCAAGCACG CAGCAATGCA GCTCAAAACG CTTAGCCTAG

Fl element

4062 4072 4082 4092 4102 4112

CCACACCCCC ACGGGAAACA GCAGTGATTA ACCTTTAGCA ATAAACGAAA GTTTAACTAA

Fl element 4122 4132 4142 4152 4162 4167

GCTATACTAA CCCCAGGGTT GGTCAATTTC GTGCCAGCCA CACCCTGGAG CTAGC

Fl element

4177 4187 4197 4207 4217 4227

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA GCATATGACT AAAAAAAAAA AAAAAAAAAA

Poly(A)

4237 4247 4257 4267 4277

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

Poly(A)

Sequences of RBP020.14 are also shown in Table 11.

Table 11: Sequences of RBP020.16 (Delta-specific RNA vaccine)

RBP020.17 (Omicron BA.1-specific RNA vaccine) as described in Table 20 above contains the following mutations with respect to Wuhan (WT SEQ ID NO: 1):

A67V, AHV69-70, T95I, G142D, AVYY143-145, AN211, L212I, R214_D215insEPE, 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,

KV986-987PP.

RBP020.20 (Omicron BA.1-specific RNA vaccine) as described in Table 21 contains the following mutations compared to Wuhan (WT SEQ ID NO: 1): A67V, AHV69-70, T95I, G142D, AVYY143-145, AN211, L212I, R214_D215insEPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, PRR681-683HGS, R685S, N764K, D796Y, F817P, N856K, A892P, A899P, A942P, Q954H, N969K, L981F, D985P, V97P (furin site, superspike/P6').

Table 23: Description of sequences of RBP020.21 (Omicron BA.2-specific RNA vaccine) as described in Table 22 above

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)-re licase-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

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

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:

Component Function Proportion (mol%)

ALC-0315 [i] Functional lipid 47.5

ALC-0159 [2] Functional lipid 1.8

DSPC [3] Structural lipid 10.0

Cholesterol, synthetic Structural lipid 40.7

Component Function Concentration (mg/mL)

Drug Substance Active 0.5

ALC-0315 [1] Functional lipid 7.17

ALC-0159 [2] Functional lipid 0.89

DSPC [3] Structural lipid 1.56

Cholesterol, synthetic Structural lipid 3.1

Sucrose Cryoprotectant 102.69

NaCI Buffer 6.0

KCI Buffer 0.15

Na₂HPO₄ Buffer 1.08

KH₂PO₄ Buffer 0.18

Water for injection Solvent/Vehicle q.s.

Component Function Concentration (mg/mL)

Drug Substance Active 1.0

ALC-0315 [i] Functional lipid 13.56

ALC-0159 [₂] Functional lipid 1.77

DSPC [3] Structural lipid 3.11

Cholesterol, synthetic Structural lipid 6.20

Sucrose Cryoprotectant 102.69 NaCI Buffer 6.0

KCI Buffer 0.15

Na₂HPO₄ Buffer 1.08

KH₂PO₄ Buffer 0.15

Water for injection Solvent/Vehicle q.s.

[1] ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2- hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino] hexyl 2-hexyldecanoate

[2] ALC-0159 = 2-[(polyethyleneglycol)-2000]-N,Nditetradecylacetamide/2-[2-(ω-methoxy

(polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide

[3] DSPC = l,2-Distearoyl-sn-glycero-3-phosphocholine q.s. = quantum satis (as much as may suffice)

ALC-0315:

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 HCI), 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 cationica I ly 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 cationica I ly 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., Lachelt, 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 nanoool5.ll (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 diameterthat 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 cation ica I ly 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 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). Here "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". 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 orcationicaIly 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((3-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 apolargroups 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), 1,2-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-[l,3]-dioxolane (DLin-K-XTC2-DMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l,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-propana minium 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-[(3(3)-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), Nl-[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), dimyristoyl phosphatidylcholine (DM PC), dipentadecanoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC), diarachidoyl phosphatidylcholine (DAPC), dibehenoyl phosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-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 (Choi) 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:

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:

In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):

(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)-, -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=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)-, -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=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;

R^a 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 (111 B) :

(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 ( I II B).

In other embodiments of Formula (III), the lipid has one of the following structures (I I IC) or (IIID):

(inc) (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 (IIIIF):

(IIIIE) (IIIIF)

In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIIH), (Illi), or (IIIJ):

(IIIG) (IIIIH)

(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:

R^7a and R^7b are, at each occurrence, independently H or C₁-C₁₂ alkyl; and a is an integer from 2 to 12, wherein R^7a, R^7b 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 R^7a is H. For example, in some embodiments, R^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R^7b 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:

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).

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.

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:

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 naive 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 administeringto 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-a, or IFN-|3, 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-a) or interferon-gamma (IFN-y), interleukins, such as IL2, IL7, 1 L12, 1 L15 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-a.

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, IFNe, 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 IFN^l have antiviral, antibacterial, and anticancer properties.

Type II interferon (IFNy 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 I L10R2 (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 cel I, 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 CHI, 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, lgG2, lgG3, lgG4, 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., FcyR 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 lgG3 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 lgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an lgG4 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, lgG2, lgG3, and lgG4.

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, U82007/0269422, U82010/0113339, W02009/083804, and W02009/133208, 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 (3-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), |3-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, 1 L12, IFNα, IFNy, 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% NaCI, saline), e.g., by a one-step dilution process. In some embodiments, bacteriostatic sodium chloride solution (e.g., 0.9% NaCI, 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 4-5°C or below, about -20°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. 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.ln 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

I 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

II 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 Pa02/Fi02<300mm Hg); respiratoryfailure (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; Sl-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 l-10x that of a previous dose (e.g., lx-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 lOx 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 ChAdxOxl vaccine, an NVX-CoV2373 vaccine, a CvnCoV vaccine, a GAM-COVIDOVac vaccine, a CoronaVac vaccine, a BBlBP-CorV vaccine, an Ad5-nCoV vaccine, a zf2OOl 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).

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.l.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.l.1.248, B.l.1.7, B.l.617.2, and B.l.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 BNT162bl (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 (IFNy) 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 (IFNy) 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 correspondingto 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 VQC-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 "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, 1692V, 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 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 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 "B.l.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 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 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 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 the Omicron (B.l.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, A69-70, T95I, G142D, A143-145, A211, 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, A69-70, T95I, G142D, A143-145, A211, 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, A69-70, T95I, G142D, A143-145, A211, 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:

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:

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, 1472V, 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, 1472V, 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, 1472V, 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, 1472V, 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 VQC-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, A69-70, T95I, G142D, A143-145, A211, 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:

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.

A67V, A69-70, T95I, G142D, A143-145, A211, 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.

A67V, A69-70, T95I, G142D, A143-145, A211, 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.

A67V, A69-70, T95I, G142D, A143-145, A211, 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 spike changes in Omicron BA.2 variant include T19I, A24-26, 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.

T19I, A24-26, A27S, A69/70, 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 l-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.l.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:

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, A24-26, 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.

T19I, A24-26, A27S, A69/70, 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, A69-70, T95I, G142D, A143-145, A211, 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, A24-26, 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:

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, A69-70, T95I, G142D, A143-145, A211, 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.l.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.l.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, A69-70, T95I, G142D, A143-145, A211, 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.

A67V, A69-70, T95I, G142D, A143-145, A211, 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, A24-26, 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, A24-26, A27S, A69/70, 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, A69-70, T95I, G142D, A143-145, A211, 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, A24-26, 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.

A67V, A69-70, T95I, G142D, A143-145, A211, 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, A24-26, A27S, A69/70, 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.

T19I, A24-26, 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:

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 ORFlab 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 C7p2fu 11 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 ll;jiacl89, 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 (Ml), 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 I FN - and TNF-a, 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 y-chain and one 6-chain. This group of T cells is much less common (2% of total T cells) than the a (3 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 helperT 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, K and A 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 lowT 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 ( PD_n) 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, 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, PD_n comprises administering 1 to 100 ug of RNA. In some embodiments, PD_n comprises administering 1 to 60 ug of RNA. In some embodiments, PD_n comprises administering 1 to 50 ug of RNA. In some embodiments, PD_n comprises administering 1 to 30 ug of RNA. In some embodiments, PD_n comprises administering about 3 ug of RNA. In some embodiments, PD_n comprises administering about 5 ug of RNA. In some embodiments, PD_n comprises administering about 10 ug of RNA. In some embodiments, PD_n comprises administering about 15 ug of RNA. In some embodiments, PD_n comprises administering about 20 ug of RNA. In some embodiments, PD_n comprises administering about 30 ug of RNA. In some embodiments, PD_n comprises administering about 50 ug of RNA. In some embodiments, PD_n 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, PD_n comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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 PD_n 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 PD_n 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 PD_n 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 PD_n 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 PD_n 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 PD_n can independently comprise at least two RNA (e.g., mRNA)s, each encoding a SARS-CoV-2 S protein or an immunogenic fragment thereof com prising 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 PD_n 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 PD_n 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 PD_n 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 PD_n 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 PD_n are separately administered to different arms of a subject via intramuscular injection.

In some embodiments, PD₁, PD₂, PD₃, and/or PD_n 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 ( Ph) 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, PI₁is about 6 weeks to about 8 weeks. In some embodiments PI₁is 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, PI₁is about 3 weeks. In some embodiments, PI₁is about

4 weeks. In some embodiments, PI₁ is about 5 weeks. In some embodiments, PI₁ is about 6 weeks. In some embodiments, Pl₁ is about 7 weeks. In some embodiments, Pl₁ is about 8 weeks. In some embodiments, Pl₁ is about 9 weeks. In some embodiments, Pl₁ is about 10 weeks. In some embodiments, Pl₁ is about 11 weeks. In some embodiments, Pl₁ 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, Pl₂ is about 1 week to about 12 weeks. In some embodiments, Pl₂ is about 1 week to about 10 weeks. In some embodiments, Pl₂ is about 2 weeks to about 10 weeks. In some embodiments, Pl₂ is about 2 weeks to about 8 weeks. In some embodiments, Pl₂ is about 3 weeks to about 8 weeks. In some embodiments, Pl₂ is about 4 weeks to about 8 weeks. In some embodiments, Pl₂ is about

6 weeks to about 8 weeks. In some embodiments Pl₂ is about 3 to about 4 weeks. In some embodiments, Pl₂ is about 1 week. In some embodiments, Pl₂ is about 2 weeks. In some embodiments, Pl₂ is about 3 weeks. In some embodiments, Pl₂ is about 4 weeks. In some embodiments, Pl₂ is about 5 weeks. In some embodiments, Pl₂ is about 6 weeks. In some embodiments, Pl₂ is about 7 weeks. In some embodiments, Pl₂ is about 8 weeks. In some embodiments, Pl₂ is about 9 weeks. In some embodiments, Pl₂ is about 10 weeks. In some embodiments, Pl₂ is about 11 weeks. In some embodiments, Pl₂ 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 (Pl_n) 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, Pl_n is about 1 week to about 12 weeks. In some embodiments, Pl_n is about 1 week to about 10 weeks. In some embodiments, Pin is about 2 weeks to about 10 weeks. In some embodiments, Pl_n is about 2 weeks to about 8 weeks. In some embodiments, Pl_n is about 3 weeks to about 8 weeks. In some embodiments, Pl_n is about 4 weeks to about 8 weeks. In some embodiments, Pl_n is about 6 weeks to about 8 weeks. In some embodiments Pl_n is about 3 to about 4 weeks. In some embodiments, Pl₂ is about 1

In some embodiments, Pl_n is about 2 weeks. In some embodiments, Pl_n is about 3 weeks. In some embodiments, Pl_n is about 4 weeks. In some embodiments, Pl_n is about 5 weeks. In some embodiments, Pl_nis about 6 weeks. In some embodiments, Pl_n is about 7 weeks. In some embodiments, Pl_nis about 8 weeks. In some embodiments, Pl_n is about 9 weeks. In some embodiments, Pl_n is about 10 weeks. In some embodiments, Pl_n is about 11 weeks. In some embodiments, Pl_n 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 PD_n 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 PD_n 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 PD_n 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 PD_n 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 (BD_n) 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, BD_n comprises administering 1 to 100 ug of RNA. In some embodiments, BD_n comprises administering 1 to 60 ug of RNA. In some embodiments, BD_n comprises administering 1 to 50 ug of RNA. In some embodiments, BD_n comprises administering 1 to 30 ug of RNA. In some embodiments, BD_n comprises administering about 3 ug of RNA. In some embodiments, BD_n comprises administering about 5 ug of RNA. In some embodiments, BD_n comprises administering about 10 ug of RNA. In some embodiments, BD_n comprises administering about 15 ug of RNA. In some embodiments, BD_n comprises administering about 20 ug of RNA. In some embodiments, BD_n comprises administering about 30 ug of RNA. In some embodiments, BD_n comprises administering about 60 ug of RNA. In some embodiments, BD_n 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, BD_n comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereof from the Wuhan strain. In some embodiments, BD_n 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, BD_n 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, BD_n 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, BD_n 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, BD_n 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, BD_n 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, BD_n 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, BD_n 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_n 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, BD_n 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 BD_n 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 BD_n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, BD₁, BD₂, BD₃, and BD_n 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 BD_n 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 BD_n 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 BD_n 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 BD_n 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 com prising 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 BD_n 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 BD_n 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 BD_n 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 BD_n 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, BI₂is 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 (Bl_n) 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, Bl_n is about 1 week to about 12 weeks. In some embodiments, Bl_n is about 1 week to about 10 weeks. In some embodiments, Bl_n is about 2 weeks to about 10 weeks. In some embodiments, Bl_n is about 2 weeks to about 8 weeks. In some embodiments, Bl_n is about 3 weeks to about 8 weeks. In some embodiments, BI_n is about 4 weeks to about 8 weeks. In some embodiments, Bl_n is about 6 weeks to about 8 weeks. In some embodiments Bl_n is about 3 to about 4 weeks. In some embodiments, Bl_n is about 1 week. In some embodiments, Bl_n is about 2 weeks. In some embodiments, Bl_n is about 3 weeks. In some embodiments, Bl_n is about 4 weeks. In some embodiments, Bl_n is about 5 weeks. In some embodiments, Bl_n is about 6 weeks. In some embodiments, Bl_n is about 7 weeks. In some embodiments, Bl_n is about 8 weeks. In some embodiments, Bl_n is about 9 weeks. In some embodiments, Bl_n 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 BD_n 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 BD_n 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 BD_n 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).

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 su Sb1unit of the SARS-CoV-2 S protein, or the receptor binding domain (RBD) of the su S1bunit 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-hexyldeca noate), 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

(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

(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

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

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;

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 Nl- 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;

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 Nl- 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;

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 Nl- 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

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

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

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.l.1.529) lineage (A67V, A69-70, T95I, G142D, A143-145, A211 , 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(CA19)) were inoculated with VSVAG-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, A69-70, T95I, G142D, A143-145, A211 , 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, A242-244, K417N, E484K, N501Y, D614G, A701V), and the Delta lineage (mutations: T19R, G142D, A157/158, 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 (https:// w.iedb .org/) was used to obtain epitopes 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

Primary Dosing Regimens

In some embodiments, PD_n comprises administering 1 to 100 ug of RNA. In some embodiments, PD_n comprises administering 1 to 60 ug of RNA. In some embodiments, PD_n comprises administering 1 to 50 ug of RNA. In some embodiments, PD_n comprises administering 1 to 30 ug of RNA. In some embodiments, PD_n comprises administering about 3 ug of RNA. In some embodiments, PD_n comprises administering about 5 ug of RNA. In some embodiments, PD_n comprises administering about 10 ug of RNA. In some embodiments, PD_n comprises administering about 15 ug of RNA. In some embodiments, PD_n comprises administering about 20 ug of RNA. In some embodiments, PD_n comprises administering about 30 ug of RNA. In some embodiments, PDn comprises administering about 50 ug of RNA. In some embodiments, PD_n comprises administering about 60 ug of RNA.

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, PD_n comprises an RNA encoding a SARS-CoV-2 Spike protein or an immunogenic fragment thereof from the Wuhan strain. In some embodiments, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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, PD_n 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/or PD_n 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, 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, Pl₂ 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 PI₁is 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

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 (Pl_n) 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, Pl_n is about 1 week to about 12 weeks. In some embodiments, Pl_n is about 1 week to about 10 weeks. In some embodiments, Plnis about

2 weeks to about 10 weeks. In some embodiments, Pl_n is about 2 weeks to about 8 weeks. In some embodiments, Pl_n is about 3 weeks to about 8 weeks. In some embodiments, Pl_n is about

4 weeks to about 8 weeks. In some embodiments, Pl_n is about 6 weeks to about 8 weeks. In some embodiments Pl_n is about 3 to about 4 weeks. In some embodiments, Pl₂ is about 1 week. In some embodiments, Pl_n is about 2 weeks. In some embodiments, Pl_n is about 3 weeks. In some embodiments, Pl_n is about 4 weeks. In some embodiments, Pl_n is about 5 weeks. In some embodiments, Pl_nis about 6 weeks. In some embodiments, Pl_n is about 7 weeks. In some embodiments, Plnis about 8 weeks. In some embodiments, Pl_n is about 9 weeks. In some embodiments, Pl_n is about 10 weeks. In some embodiments, Pl_n is about 11 weeks. In some embodiments, Pl_n is about 12 weeks.

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, 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, BD₁, BD₂, BD₃, and BD_n 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 BD_n can each independently comprise a first and a second RNA (e.g., mRNA) composition. In some embodiments, BD₁, BD₂, BD₃, and BD_n 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 com prising 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 BD_n 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 BD_n 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 BD_n 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 BD_n 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 com prising 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 BD_n 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 BD_n 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, 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, Bl₂ 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. I n some embodiments, BI₂ is about 5 weeks. In some embodiments, BI₂ is about 6 weeks. I n some embodiments, BI₂ is about 7 weeks. In some embodiments, BI₂ is about 8 weeks. I n 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 (Bl_n) 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, Bl_n is about 1 week to about 12 weeks. In some embodiments, Bl_n is about 1 week to about 10 weeks. In some embodiments, Bl_n is about 2 weeks to about 10 weeks. In some embodiments, Bl_n is about 2 weeks to about 8 weeks. In some embodiments, Bl_n is about 3 weeks to about 8 weeks. In some embodiments, Bln is about 4 weeks to about 8 weeks. In some embodiments, Bl_n is about 6 weeks to about 8 weeks. In some embodiments Bl_n is about 3 to about 4 weeks. In some embodiments, Bl_n is about 1 week. In some embodiments, Bl_n is about 2 weeks. In some embodiments, Bl_n is about 3 weeks. In some embodiments, Bl_n is about 4 weeks. In some embodiments, Bl_n is about 5 weeks. In some embodiments, Bl_n is about 6 weeks. In some embodiments, Bl_n is about 7 weeks. In some embodiments, Bl_n is about 8 weeks. In some embodiments, Bl_n is about 9 weeks. In some embodiments, Bl_n 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.

Additional Booster Regimens

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.

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 B_MEM 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-naive double- and triple-vaccinees. Broad recognition of VOCs by B_MEM 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. Rbltgen 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:10.1016/52666-5247(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-l .

Long-lived memory B (B_MEM) 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 B_MEM 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.l.1.529 leads to widespread escape from neutralizing antibody responses," Cell. 185, 467- 484.el5 (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. ell (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 B_MEM 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 B_MEM 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.l, 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-lmmunized 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-lmmunized SARS-CoV-1 Survivors," The New England journal of medicine. 385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453), in Omicron-naive 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-naive 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-lmmunized 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.l.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-naive 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-naive 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-naive 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-naive 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-naive individuals.

Likewise, while sera from vaccinated Omicron-naive 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-naive 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 VNso 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-naive 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

n/a, not available; N/A, not app icable; 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

B_MEM 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 B_MEM phenotype (B_MEM; CD^20highCD^38int/neg), as antigen-specific plasmablasts or naive B cells were not detected (data not shown). The assays therefore allowed the differentiation for each of the SARS-CoV-2 variants between B_MEM 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 B_MEM cells varied across the different groups. The frequency of B_MEM cells in Omicron-naive 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 B_MEM cells almost quadrupled, RBD-specific ones tripled across all VOCs thereby reaching quantities similar to those observed in Omicron- naive 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 B_MEM cells, which was higher than those of Omicron-naive triple-vaccinated individuals (Fig. 17, B and D).

In all groups, including Omicron-naive and Omicron infected individuals, B_MEM 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 B_MEM 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-naive 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 B_MEM 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 B_MEM cells stained with fluorescently labeled variant-specific S or RBD proteins were performed.

A combinatorial gating strategy was applied to distinguish between B_MEM 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, B_MEM 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 B_MEM cells from Omicron-naive 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 B_MEM cells was found that recognized only Wuhan S protein or RBD. Consistent with the lack of exposure, no B_MEM cells binding exclusively to Omicron BA.1 S or RBD protein were detected in these Omicron-naive individuals.

In Omicron convalescent individuals, frequencies of B_MEM cells recognizing S protein epitopes shared between Wuhan and Omicron BA.1 were significantly higherthan in the Omicron-naive ones (Fig. 18, B and C). In most of these subjects, a small proportion of exclusively Wuhan S protein-specific B_MEM 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 B_MEM cells reactive with conserved epitopes. A moderate boost of Wuhan-specific reactivities was observed; however, only small populations of Omicron-RBD-specific B_MEM 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 B_MEM 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 B_MEM 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 B_MEM 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, B_MEM 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 B_MEM 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 B_MEM cell repertoire against conserved S protein and RBD epitopes, rather than inducing large numbers of Omicron-specific B_MEM cells.

To further dissect this response, the B_MEM subsets directed against the RBD were characterized. The combinatorial Boolean gating approach was used to discern B_MEM 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 B_MEM cells that recognized Wuhan, Alpha as well as the Delta VOC RBDs, but not Omicron BA.1 RBD, while B_MEM cells exclusively reactive with Omicron BA.1 RBD were almost absent in most of those individuals (Fig. 18, H). B_MEM 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 B_MEM 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 B_MEM 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 B_MEM 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 B_MEM 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 B_MEM cells and imprinted against the formation of novel B_MEM 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. Rbltgen 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 B_MEM cell repertoire against conserved S protein and RBD epitopes, rather than inducing considerable numbers of strictly Omicron-specific B_MEM cells.

Thus, Omicron breakthrough infection in double-vaccinated individuals leads to expansion of the pre-existing B_MEM 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 B_MEM cells. Omicron-convalescent double- vaccinated individuals have a higher frequency of B_MEM 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 B_MEM cell responses. Omicron breakthrough infection promotes proportionally more pronounced boosting of RBD-specific B_MEM cells than of B_MEM cells that recognize S protein-specific epitopes outside the RBD. Therefore, Omicron-infected individuals have a significantly higher ratio of RBD/S protein-specific B_MEM cells compared to vaccinated Omicron-naive 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 B_MEM 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-naive vaccinated individuals was found to robustly neutralize SARS-CoV-1. This may indicate that Omicron infection in vaccinated individuals stimulates B_MEM 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-naive 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: A69/70, A144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), the Beta variant (mutations: L18F, D80A, D215G, A242-244, R246I, K417N, E484K, N501Y, D614G, A701V), the Delta variant (mutations: T19R, G142D, E156G, A157/158, K417N, L452R, T478K, D614G, P681R, D950N) the Omicron BA.1 variant (mutations: A67V, A69/70, T95I, G142D, A143-145, A211, 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, A24-26, 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 VSVAG 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 pm 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 a I., "Neutralization of SARS-CoV- 2 lineage B.l.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 naive 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-INMIl (GenBank: MT066156), Beta virus strain Human nCoV19 isolate/England ex-SA/HCM002/2021 (mutations: D80A, D215G, A242-244, K417N, E484K, N501Y, D614G, A701V), sequence- verified Delta strain isolated from a nasopharyngeal swab (mutations: T19R, G142D, E156G, A157/158, L452R, T478K, D614G, P681R, R682Q, D950N) or Omicron BA.1 strain hCoV- 19/Belgium/rega-20174/2021 (mutations: A67V, A69/70, T95I, G142D, A143-145, A211, 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-INMIl 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-INMIl 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 (Aero Biosystems: Wuhan - SPN-C82E9, Alpha - SPN-C82E5, Delta - SPN-C82Ec, Omicron - SPN-C82Ee) and RBD (Aero 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), CDllc - 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: MΦ P9 (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 naive B cells were discriminated as lgD⁺ cells with the Boolean 'make non-gate' function. Within non-naive B cells Plasmablasts (CD38^high CD20^low) and memory B cells (B_MEMS CD38^int/lowCD20^hlgh) were distinguished. B_MEM 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 B_MEM 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-Naive Subjects

To test the immunogenicity of various variant specific vaccines in vaccine naive subjects, vaccine naive 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+0mi), 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 naive 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.il 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 naive 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 naive 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.il). 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-naive 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.

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 4^th doses was also monitored in patients for 7 days following administration of the 4^th 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% Cl: 1.65, 3.00) and 3.15 (95% Cl: 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% Cl: 1.17, 2.08) and 1.97 (95% Cl: 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 superiorly 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-naive 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-naive 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-naive 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 B_MEM cells against epitopes shared broadly across the different SARS-CoV-2 strains. These data demonstrate that a vaccination-imprinted

B_MEM cell pool has sufficient plasticity to be remodeled by exposure to a heterologous SARS- CoV-2 S protein. While selective amplification of B_MEM 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 B_MEM cells and imprint against novel B_MEM cell responses recognizing epitopes distinctive for the Omicron BA.1 variant. Omicron BA.1 breakthrough infection in BNT162b2-vaccinated individuals primarily expands a broad B_MEM cell repertoire against conserved SARS-CoV-2 S protein and RBD epitopes, rather than inducing strictly Omicron BA.1-specific B_MEM 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 B_MEM 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 B_MEM cell ratio as compared to vaccinated Omicron-naive individuals. Omicron BA.1 carries multiple S protein alterations in key neutralizing antibody binding sites of the NTD (such as del69/70 and dell43-145) 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 B_MEM 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 B_MEM 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) B_MEM cells were consistently detected in Omicron BA.1 breakthrough infected individuals at levels similar to those in Omicron-naive 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 B_MEM cell repertoire by selective amplification of B_MEM 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-naive 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, A 24-26, A27S, A69/70, 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 orderto 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 naive 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-naive 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-naive 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-naive 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 pVN so 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-naive). 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-naive sera.

Immune sera from triple-vaccinated Omicron naive 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).

For example, in some embodiments, vaccine-naive 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-naive 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 ChAdOxl-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

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)

4, 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: A69/70, A144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), the Beta variant (alterations: L18F, D80A, D215G, A242-244, R246I, K417N, E484K, N501Y, D614G, A701V), the Delta variant (alterations: T19R, G142D, E156G, A157/158, K417N, L452R, T478K, D614G, P681R, D950N) the Omicron BA.1 variant (alterations: A67V, A69/70, T95I, G142D, A143-145, A211, 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, A24-26, 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, A24-26, 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, A24-26, A27S, A69/70, 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 pm 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/we 11 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-naive 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.l.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.ell (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.el5 (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-lmmunized 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 (Cl: 1632, 2281) compared to a BNT162b2 GMT of 1100 (Cl: 932, 1297); GMT ratio 1.75 (95% Cl: 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 (Cl: 10554, 13638) compared to a BNT162b2 GMT of 12009 (Cl: 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 S s1ubunit 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.

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/lmmunogenicity 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 C7p2fu II of WO2021/188969), e.g., a dose of a combination of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2fu 11 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 C7p2f ul I of WO2021/188969), e.g., each dose of a combination of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2f ul I 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 C7p2fu 11 of WO2021/188969), e.g., each dose of a combination of BNT162b5 and an RNA encoding SEQ ID NO: RS C7p2f ul I 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 evaluated 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-Barre 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- y 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. Specificia I ly, 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

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

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.

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

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

Table 38: 1-month PD2: GMT across vaccines against SARS-CoV-2 variants

SEQUENCE LISTING

<110> BioNTech SE

<120> CORONAVIRUS VACCINE

<130> 2013237-0549

<160> 54

<170> Patentin version 3.5

<210> 1

<211> 1273

<212> PRT

<213> Artificial Sequence

<220>

<223> S protein

<400> 1

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu 35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Ala lie His Vai Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80

Asn Pro Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn lie lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser

100 105 110 Lys Thr Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie

115 120 125

Lys Vai Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Vai Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Vai Phe Lys Asn lie Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr

195 200 205

Pro lie Asn Leu Vai Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Vai Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr 225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala

275 280 285

Vai Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai

305 310 315 320 Gin Pro Thr Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

340 345 350

Trp Asn Arg Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe 385 390 395 400

Vai lie Arg Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly

405 410 415

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

485 490 495

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai cys Gly Pro Lys 515 520 525

Lys Ser Thr Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560

Pro Phe Gin Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai

565 570 575

Arg Asp Pro Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe

580 585 590

Gly Gly Vai Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai

595 600 605

Ala Vai Leu Tyr Gin Asp Vai Asn Cys Thr Glu Vai Pro Vai Ala lie

610 615 620

His Ala Asp Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser

625 630 635 640

Asn Vai Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Vai

645 650 655

Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala

660 665 670

Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Vai Ala

675 680 685

Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Vai Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie

705 710 715 720 Ser Vai Thr Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai

725 730 735

Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr

755 760 765

Gly lie Ala Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin

770 775 780

Vai Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe 785 790 795 800

Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe lie Glu Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly

820 825 830

Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp

835 840 845

Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Vai Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880

Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie

885 890 895

Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr

900 905 910 Gin Asn Vai Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn

915 920 925

Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gin Asp Vai Vai Asn Gin Asn Ala Gin Ala Leu Asn 945 950 955 960

Thr Leu Vai Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Vai

965 970 975

Leu Asn Asp lie Leu Ser Arg Leu Asp Lys Vai Glu Ala Glu Vai Gin

980 985 990 lie Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai

995 1000 1005

Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys 1025 1030 1035

Arg Vai Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gin Ser Ala Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai 1055 1060 1065

Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His 1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn 1085 1090 1095

Gly Thr His Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin

1100 1105 1110 lie lie Thr Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai

1115 1120 1125

Vai lie Gly lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn

1160 1165 1170

Ala Ser Vai Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu

1175 1180 1185

Vai Ala Lys Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu

1205 1210 1215

Gly Phe lie Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met 1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260

Vai Leu Lys Gly Vai Lys Leu His Tyr Thr 1265 1270

<210> 2

<211> 3819

<212> RNA

<213> Artificial Sequence <220>

<223> Coding Sequence

<400> 2 auguuugugu uucuugugcu gcugccucuu gugucuucuc agugugugaa uuugacaaca 60 agaacacagc ugccaccagc uuauacaaau ucuuuuacca gaggagugua uuauccugau 120 aaaguguuua gaucuucugu gcugcacagc acacaggacc uguuucugcc auuuuuuagc 180 aaugugacau gguuucaugc aauucaugug ucuggaacaa auggaacaaa aagauuugau 240 aauccugugc ugccuuuuaa ugauggagug uauuuugcuu caacagaaaa gucaaauauu 300 auuagaggau ggauuuuugg aacaacacug gauucuaaaa cacagucucu gcugauugug 360 aauaaugcaa caaauguggu gauuaaagug ugugaauuuc aguuuuguaa ugauccuuuu 420 cugggagugu auuaucacaa aaauaauaaa ucuuggaugg aaucugaauu uagaguguau 480 uccucugcaa auaauuguac auuugaauau gugucucagc cuuuucugau ggaucuggaa 540 ggaaaacagg gcaauuuuaa aaaucugaga gaauuugugu uuaaaaauau ugauggauau 600 uuuaaaauuu auucuaaaca cacaccaauu aauuuaguga gagaucugcc ucagggauuu 660 ucugcucugg aaccucuggu ggaucugcca auuggcauua auauuacaag auuucagaca 720 cugcuggcuc ugcacagauc uuaucugaca ccuggagauu cuucuucugg auggacagcc 780 ggagcugcag cuuauuaugu gggcuaucug cagccaagaa cauuucugcu gaaauauaau 840 gaaaauggaa caauuacaga ugcuguggau ugugcucugg auccucuguc ugaaacaaaa 900 uguacauuaa aaucuuuuac aguggaaaaa ggcauuuauc agacaucuaa uuuuagagug 960 cagccaacag aaucuauugu gagauuucca aauauuacaa aucugugucc auuuggagaa 1020 guguuuaaug caacaagauu ugcaucugug uaugcaugga auagaaaaag aauuucuaau 1080 uguguggcug auuauucugu gcuguauaau agugcuucuu uuuccacauu uaaauguuau 1140 ggagugucuc caacaaaauu aaaugauuua uguuuuacaa auguguaugc ugauucuuuu 1200 gugaucagag gugaugaagu gagacagauu gcccccggac agacaggaaa aauugcugau 1260 uacaauuaca aacugccuga ugauuuuaca ggauguguga uugcuuggaa uucuaauaau 1320 uuagauucua aagugggagg aaauuacaau uaucuguaca gacuguuuag aaaaucaaau 1380 cugaaaccuu uugaaagaga uauuucaaca gaaauuuauc aggcuggauc aacaccuugu 1440 aauggagugg aaggauuuaa uuguuauuuu ccauuacaga gcuauggauu ucagccaacc 1500 aauggugugg gauaucagcc auauagagug guggugcugu cuuuugaacu gcugcaugca 1560 ccugcaacag uguguggacc uaaaaaaucu acaaauuuag ugaaaaauaa augugugaau 1620 uuuaauuuua auggauuaac aggaacagga gugcugacag aaucuaauaa aaaauuucug 1680 ccuuuucagc aguuuggcag agauauugca gauaccacag augcagugag agauccucag 1740 acauuagaaa uucuggauau uacaccuugu ucuuuugggg gugugucugu gauuacaccu 1800 ggaacaaaua caucuaauca gguggcugug cuguaucagg augugaauug uacagaagug 1860 ccaguggcaa uucaugcaga ucagcugaca ccaacaugga gaguguauuc uacaggaucu 1920 aauguguuuc agacaagagc aggaugucug auuggagcag aacaugugaa uaauucuuau 1980 gaaugugaua uuccaauugg agcaggcauu ugugcaucuu aucagacaca gacaaauucc 2040 ccaaggagag caagaucugu ggcaucucag ucuauuauug cauacaccau gucucuggga 2100 gcagaaaauu cuguggcaua uucuaauaau ucuauugcua uuccaacaaa uuuuaccauu 2160 ucugugacaa cagaaauuuu accugugucu augacaaaaa caucugugga uuguaccaug 2220 uacauuugug gagauucuac agaauguucu aaucugcugc ugcaguaugg aucuuuuugu 2280 acacagcuga auagagcuuu aacaggaauu gcuguggaac aggauaaaaa uacacaggaa 2340 guguuugcuc aggugaaaca gauuuacaaa acaccaccaa uuaaagauuu uggaggauuu 2400 aauuuuagcc agauucugcc ugauccuucu aaaccuucua aaagaucuuu uauugaagau 2460 cugcuguuua auaaagugac acuggcagau gcaggauuua uuaaacagua uggagauugc 2520 cugggugaua uugcugcaag agaucugauu ugugcucaga aauuuaaugg acugacagug 2580 cugccuccuc ugcugacaga ugaaaugauu gcucaguaca caucugcuuu acuggcugga 2640 acaauuacaa gcggauggac auuuggagcu ggagcugcuc ugcagauucc uuuugcaaug 2700 cagauggcuu acagauuuaa uggaauugga gugacacaga auguguuaua ugaaaaucag 2760 aaacugauug caaaucaguu uaauucugca auuggcaaaa uucaggauuc ucugucuucu 2820 acagcuucug cucugggaaa acugcaggau guggugaauc agaaugcaca ggcacugaau 2880 acucugguga aacagcuguc uagcaauuuu ggggcaauuu cuucugugcu gaaugauauu 2940 cugucuagac uggauaaagu ggaagcugaa gugcagauug auagacugau cacaggaaga 3000 cugcagucuc ugcagacuua ugugacacag cagcugauua gagcugcuga aauuagagcu 3060 ucugcuaauc uggcugcuac aaaaaugucu gaaugugugc ugggacaguc aaaaagagug 3120 gauuuuugug gaaaaggaua ucaucugaug ucuuuuccac agucugcucc acauggagug 3180 guguuuuuac augugacaua ugugccagca caggaaaaga auuuuaccac agcaccagca 3240 auuugucaug auggaaaagc acauuuucca agagaaggag uguuuguguc uaauggaaca 3300 cauugguuug ugacacagag aaauuuuuau gaaccucaga uuauuacaac agauaauaca 3360 uuugugucag gaaauuguga uguggugauu ggaauuguga auaauacagu guaugaucca 3420 cugcagccag aacuggauuc uuuuaaagaa gaacuggaua aauauuuuaa aaaucacaca 3480 ucuccugaug uggauuuagg agauauuucu ggaaucaaug caucuguggu gaauauucag 3540 aaagaaauug auagacugaa ugaaguggcc aaaaaucuga augaaucucu gauugaucug 3600 caggaacuug gaaaauauga acaguacauu aaauggccuu gguacauuug gcuuggauuu 3660 auugcaggau uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu 3720 uguucuuguu uaaaaggaug uuguucuugu ggaagcuguu guaaauuuga ugaagaugau 3780 ucugaaccug uguuaaaagg agugaaauug cauuacaca 3819

<210> 3

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> S Protein RBD Fusion

<400> 3

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Gly Glu Vai Phe

20 25 30 Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg Lys Arg lie

35 40 45

Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu Asn Asp Leu 65 70 75 80

Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg Gly Asp Glu

85 90 95

Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp lie Ser Thr

145 150 155 160

Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly

180 185 190

Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Vai Cys Gly Pro Lys

210 215 <210> 4

<211> 654

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding Sequence

<400> 4 auguuugugu uucuugugcu gcugccucuu gugucuucuc aguguguggu gagauuucca 60 aauauuacaa aucugugucc auuuggagaa guguuuaaug caacaagauu ugcaucugug 120 uaugcaugga auagaaaaag aauuucuaau uguguggcug auuauucugu gcuguauaau 180 agugcuucuu uuuccacauu uaaauguuau ggagugucuc caacaaaauu aaaugauuua 240 uguuuuacaa auguguaugc ugauucuuuu gugaucagag gugaugaagu gagacagauu 300 gcccccggac agacaggaaa aauugcugau uacaauuaca aacugccuga ugauuuuaca 360 ggauguguga uugcuuggaa uucuaauaau uuagauucua aagugggagg aaauuacaau 420 uaucuguaca gacuguuuag aaaaucaaau cugaaaccuu uugaaagaga uauuucaaca 480 gaaauuuauc aggcuggauc aacaccuugu aauggagugg aaggauuuaa uuguuauuuu 540 ccauuacaga gcuauggauu ucagccaacc aauggugugg gauaucagcc auauagagug 600 guggugcugu cuuuugaacu gcugcaugca ccugcaacag uguguggacc uaaa 654

<210> 5

<211> 266

<212> PRT

<213> Artificial Sequence

<220>

<223> S Protein RBD Fusion

<400> 5

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Gly Glu Vai Phe

20 25 30 Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg Lys Arg lie

35 40 45

Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu Asn Asp Leu 65 70 75 80

Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg Gly Asp Glu

85 90 95

Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp lie Ser Thr 145 150 155 160

Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly

180 185 190

Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Vai Cys Gly Pro Lys Gly Ser Pro Gly Ser Gly

210 215 220

Ser Gly Ser Gly Tyr lie Pro Glu Ala Pro Arg Asp Gly Gin Ala Tyr

225 230 235 240 Vai Arg Lys Asp Gly Glu Trp Vai Leu Leu Ser Thr Phe Leu Gly Arg 245 250 255

Ser Leu Glu Vai Leu Phe Gin Gly Pro Gly

260 265

<210> 6 <211> 798 <212> RNA <213> Artificial Sequence <220> <223> Coding Sequence

<400> 6 auguuugugu uucuugugcu gcugccucuu gugucuucuc aguguguggu gagauuucca 60 aauauuacaa aucugugucc auuuggagaa guguuuaaug caacaagauu ugcaucugug 120 uaugcaugga auagaaaaag aauuucuaau uguguggcug auuauucugu gcuguauaau 180 agugcuucuu uuuccacauu uaaauguuau ggagugucuc caacaaaauu aaaugauuua 240 uguuuuacaa auguguaugc ugauucuuuu gugaucagag gugaugaagu gagacagauu 300 gcccccggac agacaggaaa aauugcugau uacaauuaca aacugccuga ugauuuuaca 360 ggauguguga uugcuuggaa uucuaauaau uuagauucua aagugggagg aaauuacaau 420 uaucuguaca gacuguuuag aaaaucaaau cugaaaccuu uugaaagaga uauuucaaca 480 gaaauuuauc aggcuggauc aacaccuugu aauggagugg aaggauuuaa uuguuauuuu 540 ccauuacaga gcuauggauu ucagccaacc aauggugugg gauaucagcc auauagagug 600 guggugcugu cuuuugaacu gcugcaugca ccugcaacag uguguggacc uaaaggcucc 660 cccggcuccg gcuccggauc ugguuauauu ccugaagcuc caagagaugg gcaagcuuac 720 guucguaaag auggcgaaug gguauuacuu ucuaccuuuu uaggccgguc ccuggaggug 780 cuguuccagg gccccggc 798

<210> 7

<211> 1273 <212> PRT

<213> Artificial Sequence

<220>

<223> S Protein Variant

<400> 7

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu 35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Ala lie His Vai Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80

Asn Pro Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn lie lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie

115 120 125

Lys Vai Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Vai Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu

165 170 175 Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Vai Phe Lys Asn lie Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr

195 200 205

Pro lie Asn Leu Vai Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Vai Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala

275 280 285

Vai Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai

305 310 315 320

Gin Pro Thr Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

340 345 350

Trp Asn Arg Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe 385 390 395 400

Vai lie Arg Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly

405 410 415

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

485 490 495

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560

Pro Phe Gin Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai

565 570 575 Arg Asp Pro Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe

580 585 590

Gly Gly Vai Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai

595 600 605

Ala Vai Leu Tyr Gin Asp Vai Asn Cys Thr Glu Vai Pro Vai Ala lie

610 615 620

His Ala Asp Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser

625 630 635 640

Asn Vai Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Vai

645 650 655

Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala

660 665 670

Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Vai Ala

675 680 685

Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Vai Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie

705 710 715 720

Ser Vai Thr Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai

725 730 735

Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr

755 760 765 Gly lie Ala Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin

770 775 780

Vai Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe 785 790 795 800

Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe lie Glu Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly

820 825 830

Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp

835 840 845

Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Vai Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie

885 890 895

Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr

900 905 910

Gin Asn Vai Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn

915 920 925

Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gin Asp Vai Vai Asn Gin Asn Ala Gin Ala Leu Asn

945 950 955 960

Thr Leu Vai Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Vai

965 970 975 Leu Asn Asp lie Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Vai Gin

980 985 990 lie Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai

995 1000 1005

Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys 1025 1030 1035

Arg Vai Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gin Ser Ala Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai 1055 1060 1065

Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His 1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn 1085 1090 1095

Gly Thr His Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin

1100 1105 1110 lie lie Thr Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai

1115 1120 1125

Vai lie Gly lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155 His Thr Ser Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn

1160 1165 1170

Ala Ser Vai Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu

1175 1180 1185

Vai Ala Lys Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu 1190 1195 1200

Gly Lys Tyr Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu

1205 1210 1215

Gly Phe lie Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met

1220 1225 1230

Leu cys cys Met Thr Ser cys Cys Ser Cys Leu Lys Gly cys cys

1235 1240 1245

Ser Cys Gly Ser cys cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260

Vai Leu Lys Gly Vai Lys Leu His Tyr Thr

1265 1270

<210> 8

<211> 3819

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding Sequence

<400> 8 auguuugugu uucuugugcu gcugccucuu gugucuucuc agugugugaa uuugacaaca 60 agaacacagc ugccaccagc uuauacaaau ucuuuuacca gaggagugua uuauccugau 120 aaaguguuua gaucuucugu gcugcacagc acacaggacc uguuucugcc auuuuuuagc 180 aaugugacau gguuucaugc aauucaugug ucuggaacaa auggaacaaa aagauuugau 240 aauccugugc ugccuuuuaa ugauggagug uauuuugcuu caacagaaaa gucaaauauu 300 auuagaggau ggauuuuugg aacaacacug gauucuaaaa cacagucucu gcugauugug 360 aauaaugcaa caaauguggu gauuaaagug ugugaauuuc aguuuuguaa ugauccuuuu 420 cugggagugu auuaucacaa aaauaauaaa ucuuggaugg aaucugaauu uagaguguau 480 uccucugcaa auaauuguac auuugaauau gugucucagc cuuuucugau ggaucuggaa 540 ggaaaacagg gcaauuuuaa aaaucugaga gaauuugugu uuaaaaauau ugauggauau 600 uuuaaaauuu auucuaaaca cacaccaauu aauuuaguga gagaucugcc ucagggauuu 660 ucugcucugg aaccucuggu ggaucugcca auuggcauua auauuacaag auuucagaca 720 cugcuggcuc ugcacagauc uuaucugaca ccuggagauu cuucuucugg auggacagcc 780 ggagcugcag cuuauuaugu gggcuaucug cagccaagaa cauuucugcu gaaauauaau 840 gaaaauggaa caauuacaga ugcuguggau ugugcucugg auccucuguc ugaaacaaaa 900 uguacauuaa aaucuuuuac aguggaaaaa ggcauuuauc agacaucuaa uuuuagagug 960 cagccaacag aaucuauugu gagauuucca aauauuacaa aucugugucc auuuggagaa 1020 guguuuaaug caacaagauu ugcaucugug uaugcaugga auagaaaaag aauuucuaau 1080 uguguggcug auuauucugu gcuguauaau agugcuucuu uuuccacauu uaaauguuau 1140 ggagugucuc caacaaaauu aaaugauuua uguuuuacaa auguguaugc ugauucuuuu 1200 gugaucagag gugaugaagu gagacagauu gcccccggac agacaggaaa aauugcugau 1260 uacaauuaca aacugccuga ugauuuuaca ggauguguga uugcuuggaa uucuaauaau 1320 uuagauucua aagugggagg aaauuacaau uaucuguaca gacuguuuag aaaaucaaau 1380 cugaaaccuu uugaaagaga uauuucaaca gaaauuuauc aggcuggauc aacaccuugu 1440 aauggagugg aaggauuuaa uuguuauuuu ccauuacaga gcuauggauu ucagccaacc 1500 aauggugugg gauaucagcc auauagagug guggugcugu cuuuugaacu gcugcaugca 1560 ccugcaacag uguguggacc uaaaaaaucu acaaauuuag ugaaaaauaa augugugaau 1620 uuuaauuuua auggauuaac aggaacagga gugcugacag aaucuaauaa aaaauuucug 1680 ccuuuucagc aguuuggcag agauauugca gauaccacag augcagugag agauccucag 1740 acauuagaaa uucuggauau uacaccuugu ucuuuugggg gugugucugu gauuacaccu 1800 ggaacaaaua caucuaauca gguggcugug cuguaucagg augugaauug uacagaagug 1860 ccaguggcaa uucaugcaga ucagcugaca ccaacaugga gaguguauuc uacaggaucu 1920 aauguguuuc agacaagagc aggaugucug auuggagcag aacaugugaa uaauucuuau 1980 gaaugugaua uuccaauugg agcaggcauu ugugcaucuu aucagacaca gacaaauucc 2040 ccaaggagag caagaucugu ggcaucucag ucuauuauug cauacaccau gucucuggga 2100 gcagaaaauu cuguggcaua uucuaauaau ucuauugcua uuccaacaaa uuuuaccauu 2160 ucugugacaa cagaaauuuu accugugucu augacaaaaa caucugugga uuguaccaug 2220 uacauuugug gagauucuac agaauguucu aaucugcugc ugcaguaugg aucuuuuugu 2280 acacagcuga auagagcuuu aacaggaauu gcuguggaac aggauaaaaa uacacaggaa 2340 guguuugcuc aggugaaaca gauuuacaaa acaccaccaa uuaaagauuu uggaggauuu 2400 aauuuuagcc agauucugcc ugauccuucu aaaccuucua aaagaucuuu uauugaagau 2460 cugcuguuua auaaagugac acuggcagau gcaggauuua uuaaacagua uggagauugc 2520 cugggugaua uugcugcaag agaucugauu ugugcucaga aauuuaaugg acugacagug 2580 cugccuccuc ugcugacaga ugaaaugauu gcucaguaca caucugcuuu acuggcugga 2640 acaauuacaa gcggauggac auuuggagcu ggagcugcuc ugcagauucc uuuugcaaug 2700 cagauggcuu acagauuuaa uggaauugga gugacacaga auguguuaua ugaaaaucag 2760 aaacugauug caaaucaguu uaauucugca auuggcaaaa uucaggauuc ucugucuucu 2820 acagcuucug cucugggaaa acugcaggau guggugaauc agaaugcaca ggcacugaau 2880 acucugguga aacagcuguc uagcaauuuu ggggcaauuu cuucugugcu gaaugauauu 2940 cugucuagac uggauccucc ugaagcugaa gugcagauug auagacugau cacaggaaga 3000 cugcagucuc ugcagacuua ugugacacag cagcugauua gagcugcuga aauuagagcu 3060 ucugcuaauc uggcugcuac aaaaaugucu gaaugugugc ugggacaguc aaaaagagug 3120 gauuuuugug gaaaaggaua ucaucugaug ucuuuuccac agucugcucc acauggagug 3180 guguuuuuac augugacaua ugugccagca caggaaaaga auuuuaccac agcaccagca 3240 auuugucaug auggaaaagc acauuuucca agagaaggag uguuuguguc uaauggaaca 3300 cauugguuug ugacacagag aaauuuuuau gaaccucaga uuauuacaac agauaauaca 3360 uuugugucag gaaauuguga uguggugauu ggaauuguga auaauacagu guaugaucca 3420 cugcagccag aacuggauuc uuuuaaagaa gaacuggaua aauauuuuaa aaaucacaca 3480 ucuccugaug uggauuuagg agauauuucu ggaaucaaug caucuguggu gaauauucag 3540 aaagaaauug auagacugaa ugaaguggcc aaaaaucuga augaaucucu gauugaucug 3600 caggaacuug gaaaauauga acaguacauu aaauggccuu gguacauuug gcuuggauuu 3660 auugcaggau uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu 3720 uguucuuguu uaaaaggaug uuguucuugu ggaagcuguu guaaauuuga ugaagaugau 3780 ucugaaccug uguuaaaagg agugaaauug cauuacaca 3819

<210> 9

<211> 3819

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding Sequence

<400> 9 auguucgugu uccuggugcu gcugccucug guguccagcc agugugugaa ccugaccacc 60 agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgac 120 aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180 aacgugaccu gguuccacgc cauccacgug uccggcacca auggcaccaa gagauucgac 240 aaccccgugc ugcccuucaa cgacggggug uacuuugcca gcaccgagaa guccaacauc 300 aucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgug 360 aacaacgcca ccaacguggu caucaaagug ugcgaguucc aguucugcaa cgaccccuuc 420 cugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguac 480 agcagcgcca acaacugcac cuucgaguac gugucccagc cuuuccugau ggaccuggaa 540 ggcaagcagg gcaacuucaa gaaccugcgc gaguucgugu uuaagaacau cgacggcuac 600 uucaagaucu acagcaagca caccccuauc aaccucgugc gggaucugcc ucagggcuuc 660 ucugcucugg aaccccuggu ggaucugccc aucggcauca acaucacccg guuucagaca 720 cugcuggccc ugcacagaag cuaccugaca ccuggcgaua gcagcagcgg auggacagcu 780 ggugccgccg cuuacuaugu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840 gagaacggca ccaucaccga cgccguggau ugugcucugg auccucugag cgagacaaag 900 ugcacccuga aguccuucac cguggaaaag ggcaucuacc agaccagcaa cuuccgggug 960 cagcccaccg aauccaucgu gcgguucccc aauaucacca aucugugccc cuucggcgag 1020 guguucaaug ccaccagauu cgccucugug uacgccugga accggaagcg gaucagcaau 1080 ugcguggccg acuacuccgu gcuguacaac uccgccagcu ucagcaccuu caagugcuac 1140 ggcguguccc cuaccaagcu gaacgaccug ugcuucacaa acguguacgc cgacagcuuc 1200 gugauccggg gagaugaagu gcggcagauu gccccuggac agacaggcaa gaucgccgac 1260 uacaacuaca agcugcccga cgacuucacc ggcuguguga uugccuggaa cagcaacaac 1320 cuggacucca aagucggcgg caacuacaau uaccuguacc ggcuguuccg gaaguccaau 1380 cugaagcccu ucgagcggga caucuccacc gagaucuauc aggccggcag caccccuugu 1440 aacggcgugg aaggcuucaa cugcuacuuc ccacugcagu ccuacggcuu ucagcccaca 1500 aauggcgugg gcuaucagcc cuacagagug guggugcuga gcuucgaacu gcugcaugcc 1560 ccugccacag ugugcggccc uaagaaaagc accaaucucg ugaagaacaa augcgugaac 1620 uucaacuuca acggccugac cggcaccggc gugcugacag agagcaacaa gaaguuccug 1680 ccauuccagc aguuuggccg ggauaucgcc gauaccacag acgccguuag agauccccag 1740 acacuggaaa uccuggacau caccccuugc agcuucggcg gagugucugu gaucaccccu 1800 ggcaccaaca ccagcaauca gguggcagug cuguaccagg acgugaacug uaccgaagug 1860 cccguggcca uucacgccga ucagcugaca ccuacauggc ggguguacuc caccggcagc 1920 aauguguuuc agaccagagc cggcugucug aucggagccg agcacgugaa caauagcuac 1980 gagugcgaca uccccaucgg cgcuggaauc ugcgccagcu accagacaca gacaaacagc 2040 ccucggagag ccagaagcgu ggccagccag agcaucauug ccuacacaau gucucugggc 2100 gccgagaaca gcguggccua cuccaacaac ucuaucgcua uccccaccaa cuucaccauc 2160 agcgugacca cagagauccu gccugugucc augaccaaga ccagcgugga cugcaccaug 2220 uacaucugcg gcgauuccac cgagugcucc aaccugcugc ugcaguacgg cagcuucugc 2280 acccagcuga auagagcccu gacagggauc gccguggaac aggacaagaa cacccaagag 2340 guguucgccc aagugaagca gaucuacaag accccuccua ucaaggacuu cggcggcuuc 2400 aauuucagcc agauucugcc cgauccuagc aagcccagca agcggagcuu caucgaggac 2460 cugcuguuca acaaagugac acuggccgac gccggcuuca ucaagcagua uggcgauugu 2520 cugggcgaca uugccgccag ggaucugauu ugcgcccaga aguuuaacgg acugacagug 2580 cugccuccuc ugcugaccga ugagaugauc gcccaguaca caucugcccu gcuggccggc 2640 acaaucacaa gcggcuggac auuuggagca ggcgccgcuc ugcagauccc cuuugcuaug 2700 cagauggccu accgguucaa cggcaucgga gugacccaga augugcugua cgagaaccag 2760 aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggacag ccugagcagc 2820 acagcaagcg cccugggaaa gcugcaggac guggucaacc agaaugccca ggcacugaac 2880 acccugguca agcagcuguc cuccaacuuc ggcgccauca gcucugugcu gaacgauauc 2940 cugagcagac uggacccucc ugaggccgag gugcagaucg acagacugau cacaggcaga 3000 cugcagagcc uccagacaua cgugacccag cagcugauca gagccgccga gauuagagcc 3060 ucugccaauc uggccgccac caagaugucu gagugugugc ugggccagag caagagagug 3120 gacuuuugcg gcaagggcua ccaccugaug agcuucccuc agucugcccc ucacggcgug 3180 guguuucugc acgugacaua ugugcccgcu caagagaaga auuucaccac cgcuccagcc 3240 aucugccacg acggcaaagc ccacuuuccu agagaaggcg uguucguguc caacggcacc 3300 cauugguucg ugacacagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360 uucgugucug gcaacugcga cgucgugauc ggcauuguga acaauaccgu guacgacccu 3420 cugcagcccg agcuggacag cuucaaagag gaacuggaca aguacuuuaa gaaccacaca 3480 agccccgacg uggaccuggg cgauaucagc ggaaucaaug ccagcgucgu gaacauccag 3540 aaagagaucg accggcugaa cgagguggcc aagaaucuga acgagagccu gaucgaccug 3600 caagaacugg ggaaguacga gcaguacauc aaguggcccu gguacaucug gcugggcuuu 3660 aucgccggac ugauugccau cgugaugguc acaaucaugc uguguugcau gaccagcugc 3720 uguagcugcc ugaagggcug uuguagcugu ggcagcugcu gcaaguucga cgaggacgau 3780 ucugagcccg ugcugaaggg cgugaaacug cacuacaca 3819

<210> 10

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> Foldon Sequence

<400> 10

Gly Ser Gly Tyr lie Pro Glu Ala Pro Arg Asp Gly Gin Ala Tyr Vai

1 5 10 15

Arg Lys Asp Gly Glu Trp Vai Leu Leu Ser Thr Phe Leu Gly Arg Ser

20 25 30

Leu Glu Vai Leu Phe Gin Gly Pro Gly 35 40

<210> 11

<211> 123

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding Sequence

<400> 11 ggaucugguu auauuccuga agcuccaaga gaugggcaag cuuacguucg uaaagauggc 60 gaauggguau uacuuucuac cuuuuuaggc cggucccugg aggugcuguu ccagggcccc 120 ggc 123

<210> 12

<211> 47

<212> RNA

<213> Artificial Sequence <220>

<223> 5 ' -UTR

<400> 12 aacuaguauu cuucuggucc ccacagacuc agagagaacc cgccacc 47

<210> 13

<211> 278

<212> RNA

<213> Artificial Sequence

<220>

<223> 3 ' -UTR

<400> 13 cugguacugc augcacgcaa ugcuagcugc cccuuucccg uccuggguac cccgagucuc 60 ccccgaccuc gggucccagg uaugcuccca ccuccaccug ccccacucac caccucugcu 120 aguuccagac accucccaag cacgcagcaa ugcagcucaa aacgcuuagc cuagccacac 180 ccccacggga aacagcagug auuaaccuuu agcaauaaac gaaaguuuaa cuaagcuaua 240 cuaaccccag gguuggucaa uuucgugcca gccacacc 278

<210> 14

<211> 110

<212> RNA

<213> Artificial Sequence

<220>

<223> A30L70

<400> 14 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gcauaugacu aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 110

<210> 15

<211> 4282

<212> RNA

<213> Artificial Sequence

<220>

<223> RBL063.1 <400> 15 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu 60 guuucuugug cugcugccuc uugugucuuc ucagugugug aauuugacaa caagaacaca 120 gcugccacca gcuuauacaa auucuuuuac cagaggagug uauuauccug auaaaguguu 180 uagaucuucu gugcugcaca gcacacagga ccuguuucug ccauuuuuua gcaaugugac 240 augguuucau gcaauucaug ugucuggaac aaauggaaca aaaagauuug auaauccugu 300 gcugccuuuu aaugauggag uguauuuugc uucaacagaa aagucaaaua uuauuagagg 360 auggauuuuu ggaacaacac uggauucuaa aacacagucu cugcugauug ugaauaaugc 420 aacaaaugug gugauuaaag ugugugaauu ucaguuuugu aaugauccuu uucugggagu 480 guauuaucac aaaaauaaua aaucuuggau ggaaucugaa uuuagagugu auuccucugc 540 aaauaauugu acauuugaau augugucuca gccuuuucug auggaucugg aaggaaaaca 600 gggcaauuuu aaaaaucuga gagaauuugu guuuaaaaau auugauggau auuuuaaaau 660 uuauucuaaa cacacaccaa uuaauuuagu gagagaucug ccucagggau uuucugcucu 720 ggaaccucug guggaucugc caauuggcau uaauauuaca agauuucaga cacugcuggc 780 ucugcacaga ucuuaucuga caccuggaga uucuucuucu ggauggacag ccggagcugc 840 agcuuauuau gugggcuauc ugcagccaag aacauuucug cugaaauaua augaaaaugg 900 aacaauuaca gaugcugugg auugugcucu ggauccucug ucugaaacaa aauguacauu 960 aaaaucuuuu acaguggaaa aaggcauuua ucagacaucu aauuuuagag ugcagccaac 1020 agaaucuauu gugagauuuc caaauauuac aaaucugugu ccauuuggag aaguguuuaa 1080 ugcaacaaga uuugcaucug uguaugcaug gaauagaaaa agaauuucua auuguguggc 1140 ugauuauucu gugcuguaua auagugcuuc uuuuuccaca uuuaaauguu auggaguguc 1200 uccaacaaaa uuaaaugauu uauguuuuac aaauguguau gcugauucuu uugugaucag 1260 aggugaugaa gugagacaga uugcccccgg acagacagga aaaauugcug auuacaauua 1320 caaacugccu gaugauuuua caggaugugu gauugcuugg aauucuaaua auuuagauuc 1380 uaaaguggga ggaaauuaca auuaucugua cagacuguuu agaaaaucaa aucugaaacc 1440 uuuugaaaga gauauuucaa cagaaauuua ucaggcugga ucaacaccuu guaauggagu 1500 ggaaggauuu aauuguuauu uuccauuaca gagcuaugga uuucagccaa ccaauggugu 1560 gggauaucag ccauauagag ugguggugcu gucuuuugaa cugcugcaug caccugcaac 1620 agugugugga ccuaaaaaau cuacaaauuu agugaaaaau aaauguguga auuuuaauuu 1680 uaauggauua acaggaacag gagugcugac agaaucuaau aaaaaauuuc ugccuuuuca 1740 gcaguuuggc agagauauug cagauaccac agaugcagug agagauccuc agacauuaga 1800 aauucuggau auuacaccuu guucuuuugg gggugugucu gugauuacac cuggaacaaa 1860 uacaucuaau cagguggcug ugcuguauca ggaugugaau uguacagaag ugccaguggc 1920 aauucaugca gaucagcuga caccaacaug gagaguguau ucuacaggau cuaauguguu 1980 ucagacaaga gcaggauguc ugauuggagc agaacaugug aauaauucuu augaauguga 2040 uauuccaauu ggagcaggca uuugugcauc uuaucagaca cagacaaauu ccccaaggag 2100 agcaagaucu guggcaucuc agucuauuau ugcauacacc augucucugg gagcagaaaa 2160 uucuguggca uauucuaaua auucuauugc uauuccaaca aauuuuacca uuucugugac 2220 aacagaaauu uuaccugugu cuaugacaaa aacaucugug gauuguacca uguacauuug 2280 uggagauucu acagaauguu cuaaucugcu gcugcaguau ggaucuuuuu guacacagcu 2340 gaauagagcu uuaacaggaa uugcugugga acaggauaaa aauacacagg aaguguuugc 2400 ucaggugaaa cagauuuaca aaacaccacc aauuaaagau uuuggaggau uuaauuuuag 2460 ccagauucug ccugauccuu cuaaaccuuc uaaaagaucu uuuauugaag aucugcuguu 2520 uaauaaagug acacuggcag augcaggauu uauuaaacag uauggagauu gccuggguga 2580 uauugcugca agagaucuga uuugugcuca gaaauuuaau ggacugacag ugcugccucc 2640 ucugcugaca gaugaaauga uugcucagua cacaucugcu uuacuggcug gaacaauuac 2700 aagcggaugg acauuuggag cuggagcugc ucugcagauu ccuuuugcaa ugcagauggc 2760 uuacagauuu aauggaauug gagugacaca gaauguguua uaugaaaauc agaaacugau 2820 ugcaaaucag uuuaauucug caauuggcaa aauucaggau ucucugucuu cuacagcuuc 2880 ugcucuggga aaacugcagg auguggugaa ucagaaugca caggcacuga auacucuggu 2940 gaaacagcug ucuagcaauu uuggggcaau uucuucugug cugaaugaua uucugucuag 3000 acuggauccu ccugaagcug aagugcagau ugauagacug aucacaggaa gacugcaguc 3060 ucugcagacu uaugugacac agcagcugau uagagcugcu gaaauuagag cuucugcuaa 3120 ucuggcugcu acaaaaaugu cugaaugugu gcugggacag ucaaaaagag uggauuuuug 3180 uggaaaagga uaucaucuga ugucuuuucc acagucugcu ccacauggag ugguguuuuu 3240 acaugugaca uaugugccag cacaggaaaa gaauuuuacc acagcaccag caauuuguca 3300 ugauggaaaa gcacauuuuc caagagaagg aguguuugug ucuaauggaa cacauugguu 3360 ugugacacag agaaauuuuu augaaccuca gauuauuaca acagauaaua cauuuguguc 3420 aggaaauugu gaugugguga uuggaauugu gaauaauaca guguaugauc cacugcagcc 3480 agaacuggau ucuuuuaaag aagaacugga uaaauauuuu aaaaaucaca caucuccuga 3540 uguggauuua ggagauauuu cuggaaucaa ugcaucugug gugaauauuc agaaagaaau 3600 ugauagacug aaugaagugg ccaaaaaucu gaaugaaucu cugauugauc ugcaggaacu 3660 uggaaaauau gaacaguaca uuaaauggcc uugguacauu uggcuuggau uuauugcagg 3720 auuaauugca auugugaugg ugacaauuau guuauguugu augacaucau guuguucuug 3780 uuuaaaagga uguuguucuu guggaagcug uuguaaauuu gaugaagaug auucugaacc 3840 uguguuaaaa ggagugaaau ugcauuacac augaugacuc gagcugguac ugcaugcacg 3900 caaugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc 3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc 4020 aagcacgcag caaugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca 4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu 4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aa 4282

<210> 16 <211> 4282 <212> RNA <213> Artificial Sequence <220>

<223> RBL063.2

<400> 16 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguucgu 60 guuccuggug cugcugccuc ugguguccag ccagugugug aaccugacca ccagaacaca 120 gcugccucca gccuacacca acagcuuuac cagaggcgug uacuaccccg acaagguguu 180 cagauccagc gugcugcacu cuacccagga ccuguuccug ccuuucuuca gcaacgugac 240 cugguuccac gccauccacg uguccggcac caauggcacc aagagauucg acaaccccgu 300 gcugcccuuc aacgacgggg uguacuuugc cagcaccgag aaguccaaca ucaucagagg 360 cuggaucuuc ggcaccacac uggacagcaa gacccagagc cugcugaucg ugaacaacgc 420 caccaacgug gucaucaaag ugugcgaguu ccaguucugc aacgaccccu uccugggcgu 480 cuacuaccac aagaacaaca agagcuggau ggaaagcgag uuccgggugu acagcagcgc 540 caacaacugc accuucgagu acguguccca gccuuuccug auggaccugg aaggcaagca 600 gggcaacuuc aagaaccugc gcgaguucgu guuuaagaac aucgacggcu acuucaagau 660 cuacagcaag cacaccccua ucaaccucgu gcgggaucug ccucagggcu ucucugcucu 720 ggaaccccug guggaucugc ccaucggcau caacaucacc cgguuucaga cacugcuggc 780 ccugcacaga agcuaccuga caccuggcga uagcagcagc ggauggacag cuggugccgc 840 cgcuuacuau gugggcuacc ugcagccuag aaccuuccug cugaaguaca acgagaacgg 900 caccaucacc gacgccgugg auugugcucu ggauccucug agcgagacaa agugcacccu 960 gaaguccuuc accguggaaa agggcaucua ccagaccagc aacuuccggg ugcagcccac 1020 cgaauccauc gugcgguucc ccaauaucac caaucugugc cccuucggcg agguguucaa 1080 ugccaccaga uucgccucug uguacgccug gaaccggaag cggaucagca auugcguggc 1140 cgacuacucc gugcuguaca acuccgccag cuucagcacc uucaagugcu acggcguguc 1200 cccuaccaag cugaacgacc ugugcuucac aaacguguac gccgacagcu ucgugauccg 1260 gggagaugaa gugcggcaga uugccccugg acagacaggc aagaucgccg acuacaacua 1320 caagcugccc gacgacuuca ccggcugugu gauugccugg aacagcaaca accuggacuc 1380 caaagucggc ggcaacuaca auuaccugua ccggcuguuc cggaagucca aucugaagcc 1440 cuucgagcgg gacaucucca ccgagaucua ucaggccggc agcaccccuu guaacggcgu 1500 ggaaggcuuc aacugcuacu ucccacugca guccuacggc uuucagccca caaauggcgu 1560 gggcuaucag cccuacagag ugguggugcu gagcuucgaa cugcugcaug ccccugccac 1620 agugugcggc ccuaagaaaa gcaccaaucu cgugaagaac aaaugcguga acuucaacuu 1680 caacggccug accggcaccg gcgugcugac agagagcaac aagaaguucc ugccauucca 1740 gcaguuuggc cgggauaucg ccgauaccac agacgccguu agagaucccc agacacugga 1800 aauccuggac aucaccccuu gcagcuucgg cggagugucu gugaucaccc cuggcaccaa 1860 caccagcaau cagguggcag ugcuguacca ggacgugaac uguaccgaag ugcccguggc 1920 cauucacgcc gaucagcuga caccuacaug gcggguguac uccaccggca gcaauguguu 1980 ucagaccaga gccggcuguc ugaucggagc cgagcacgug aacaauagcu acgagugcga 2040 cauccccauc ggcgcuggaa ucugcgccag cuaccagaca cagacaaaca gcccucggag 2100 agccagaagc guggccagcc agagcaucau ugccuacaca augucucugg gcgccgagaa 2160 cagcguggcc uacuccaaca acucuaucgc uauccccacc aacuucacca ucagcgugac 2220 cacagagauc cugccugugu ccaugaccaa gaccagcgug gacugcacca uguacaucug 2280 cggcgauucc accgagugcu ccaaccugcu gcugcaguac ggcagcuucu gcacccagcu 2340 gaauagagcc cugacaggga ucgccgugga acaggacaag aacacccaag agguguucgc 2400 ccaagugaag cagaucuaca agaccccucc uaucaaggac uucggcggcu ucaauuucag 2460 ccagauucug cccgauccua gcaagcccag caagcggagc uucaucgagg accugcuguu 2520 caacaaagug acacuggccg acgccggcuu caucaagcag uauggcgauu gucugggcga 2580 cauugccgcc agggaucuga uuugcgccca gaaguuuaac ggacugacag ugcugccucc 2640 ucugcugacc gaugagauga ucgcccagua cacaucugcc cugcuggccg gcacaaucac 2700 aagcggcugg acauuuggag caggcgccgc ucugcagauc cccuuugcua ugcagauggc 2760 cuaccgguuc aacggcaucg gagugaccca gaaugugcug uacgagaacc agaagcugau 2820 cgccaaccag uucaacagcg ccaucggcaa gauccaggac agccugagca gcacagcaag 2880 cgcccuggga aagcugcagg acguggucaa ccagaaugcc caggcacuga acacccuggu 2940 caagcagcug uccuccaacu ucggcgccau cagcucugug cugaacgaua uccugagcag 3000 acuggacccu ccugaggccg aggugcagau cgacagacug aucacaggca gacugcagag 3060 ccuccagaca uacgugaccc agcagcugau cagagccgcc gagauuagag ccucugccaa 3120 ucuggccgcc accaagaugu cugagugugu gcugggccag agcaagagag uggacuuuug 3180 cggcaagggc uaccaccuga ugagcuuccc ucagucugcc ccucacggcg ugguguuucu 3240 gcacgugaca uaugugcccg cucaagagaa gaauuucacc accgcuccag ccaucugcca 3300 cgacggcaaa gcccacuuuc cuagagaagg cguguucgug uccaacggca cccauugguu 3360 cgugacacag cggaacuucu acgagcccca gaucaucacc accgacaaca ccuucguguc 3420 uggcaacugc gacgucguga ucggcauugu gaacaauacc guguacgacc cucugcagcc 3480 cgagcuggac agcuucaaag aggaacugga caaguacuuu aagaaccaca caagccccga 3540 cguggaccug ggcgauauca gcggaaucaa ugccagcguc gugaacaucc agaaagagau 3600 cgaccggcug aacgaggugg ccaagaaucu gaacgagagc cugaucgacc ugcaagaacu 3660 ggggaaguac gagcaguaca ucaaguggcc cugguacauc uggcugggcu uuaucgccgg 3720 acugauugcc aucgugaugg ucacaaucau gcuguguugc augaccagcu gcuguagcug 3780 ccugaagggc uguuguagcu guggcagcug cugcaaguuc gacgaggacg auucugagcc 3840 cgugcugaag ggcgugaaac ugcacuacac augaugacuc gagcugguac ugcaugcacg 3900 caaugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc 3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc 4020 aagcacgcag caaugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca 4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu 4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aa 4282 <210> 17

<211> 1261

<212> RNA

<213> Artificial Sequence

<220>

<223> RBL063.3

<400> 17 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu 60 guuucuugug cugcugccuc uugugucuuc ucagugugug gugagauuuc caaauauuac 120 aaaucugugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug uguaugcaug 180 gaauagaaaa agaauuucua auuguguggc ugauuauucu gugcuguaua auagugcuuc 240 uuuuuccaca uuuaaauguu auggaguguc uccaacaaaa uuaaaugauu uauguuuuac 300 aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga uugcccccgg 360 acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua caggaugugu 420 gauugcuugg aauucuaaua auuuagauuc uaaaguggga ggaaauuaca auuaucugua 480 cagacuguuu agaaaaucaa aucugaaacc uuuugaaaga gauauuucaa cagaaauuua 540 ucaggcugga ucaacaccuu guaauggagu ggaaggauuu aauuguuauu uuccauuaca 600 gagcuaugga uuucagccaa ccaauggugu gggauaucag ccauauagag ugguggugcu 660 gucuuuugaa cugcugcaug caccugcaac agugugugga ccuaaaggcu cccccggcuc 720 cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu acguucguaa 780 agauggcgaa uggguauuac uuucuaccuu uuuaggccgg ucccuggagg ugcuguucca 840 gggccccggc ugaugacucg agcugguacu gcaugcacgc aaugcuagcu gccccuuucc 900 cguccugggu accccgaguc ucccccgacc ucggguccca gguaugcucc caccuccacc 960 ugccccacuc accaccucug cuaguuccag acaccuccca agcacgcagc aaugcagcuc 1020 aaaacgcuua gccuagccac acccccacgg gaaacagcag ugauuaaccu uuagcaauaa 1080 acgaaaguuu aacuaagcua uacuaacccc aggguugguc aauuucgugc cagccacacc 1140 cuggagcuag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260 1261

<210> 18

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> S Protein RBD Fusion

<400> 18

Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Gly Glu Vai Phe 1 5 10 15

Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg Lys Arg lie

20 25 30

Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser Ala Ser Phe

35 40 45

Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu Asn Asp Leu

50 55 60

Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg Gly Asp Glu 65 70 75 80

Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala Asp Tyr Asn

85 90 95

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala Trp Asn Ser

100 105 110

Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

115 120 125

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp lie Ser Thr

130 135 140 Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai Glu Gly Phe

145 150 155 160

Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly

165 170 175

Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe Glu Leu Leu

180 185 190

His Ala Pro Ala Thr Vai Cys Gly Pro Lys Gly Ser Pro Gly Ser Gly

195 200 205

Ser Gly Ser Gly Tyr lie Pro Glu Ala Pro Arg Asp Gly Gin Ala Tyr

210 215 220

Vai Arg Lys Asp Gly Glu Trp Vai Leu Leu Ser Thr Phe Leu Gly Arg

225 230 235 240

Ser Leu Glu Vai Leu Phe Gin Gly Pro Gly

245 250

<210> 19

<211> 4283

<212> RNA

<213> Artificial Sequence

<220>

<223> RBP020.1

<400> 19 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugugu gaauuugaca acaagaacac 120 agcugccacc agcuuauaca aauucuuuua ccagaggagu guauuauccu gauaaagugu 180 uuagaucuuc ugugcugcac agcacacagg accuguuucu gccauuuuuu agcaauguga 240 caugguuuca ugcaauucau gugucuggaa caaauggaac aaaaagauuu gauaauccug 300 ugcugccuuu uaaugaugga guguauuuug cuucaacaga aaagucaaau auuauuagag 360 gauggauuuu uggaacaaca cuggauucua aaacacaguc ucugcugauu gugaauaaug 420 caacaaaugu ggugauuaaa gugugugaau uucaguuuug uaaugauccu uuucugggag 480 uguauuauca caaaaauaau aaaucuugga uggaaucuga auuuagagug uauuccucug 540 caaauaauug uacauuugaa uaugugucuc agccuuuucu gauggaucug gaaggaaaac 600 agggcaauuu uaaaaaucug agagaauuug uguuuaaaaa uauugaugga uauuuuaaaa 660 uuuauucuaa acacacacca auuaauuuag ugagagaucu gccucaggga uuuucugcuc 720 uggaaccucu gguggaucug ccaauuggca uuaauauuac aagauuucag acacugcugg 780 cucugcacag aucuuaucug acaccuggag auucuucuuc uggauggaca gccggagcug 840 cagcuuauua ugugggcuau cugcagccaa gaacauuucu gcugaaauau aaugaaaaug 900 gaacaauuac agaugcugug gauugugcuc uggauccucu gucugaaaca aaauguacau 960 uaaaaucuuu uacaguggaa aaaggcauuu aucagacauc uaauuuuaga gugcagccaa 1020 cagaaucuau ugugagauuu ccaaauauua caaaucugug uccauuugga gaaguguuua 1080 augcaacaag auuugcaucu guguaugcau ggaauagaaa aagaauuucu aauugugugg 1140 cugauuauuc ugugcuguau aauagugcuu cuuuuuccac auuuaaaugu uauggagugu 1200 cuccaacaaa auuaaaugau uuauguuuua caaaugugua ugcugauucu uuugugauca 1260 gaggugauga agugagacag auugcccccg gacagacagg aaaaauugcu gauuacaauu 1320 acaaacugcc ugaugauuuu acaggaugug ugauugcuug gaauucuaau aauuuagauu 1380 cuaaaguggg aggaaauuac aauuaucugu acagacuguu uagaaaauca aaucugaaac 1440 cuuuugaaag agauauuuca acagaaauuu aucaggcugg aucaacaccu uguaauggag 1500 uggaaggauu uaauuguuau uuuccauuac agagcuaugg auuucagcca accaauggug 1560 ugggauauca gccauauaga gugguggugc ugucuuuuga acugcugcau gcaccugcaa 1620 cagugugugg accuaaaaaa ucuacaaauu uagugaaaaa uaaaugugug aauuuuaauu 1680 uuaauggauu aacaggaaca ggagugcuga cagaaucuaa uaaaaaauuu cugccuuuuc 1740 agcaguuugg cagagauauu gcagauacca cagaugcagu gagagauccu cagacauuag 1800 aaauucugga uauuacaccu uguucuuuug gggguguguc ugugauuaca ccuggaacaa 1860 auacaucuaa ucagguggcu gugcuguauc aggaugugaa uuguacagaa gugccagugg 1920 caauucaugc agaucagcug acaccaacau ggagagugua uucuacagga ucuaaugugu 1980 uucagacaag agcaggaugu cugauuggag cagaacaugu gaauaauucu uaugaaugug 2040 auauuccaau uggagcaggc auuugugcau cuuaucagac acagacaaau uccccaagga 2100 gagcaagauc uguggcaucu cagucuauua uugcauacac caugucucug ggagcagaaa 2160 auucuguggc auauucuaau aauucuauug cuauuccaac aaauuuuacc auuucuguga 2220 caacagaaau uuuaccugug ucuaugacaa aaacaucugu ggauuguacc auguacauuu 2280 guggagauuc uacagaaugu ucuaaucugc ugcugcagua uggaucuuuu uguacacagc 2340 ugaauagagc uuuaacagga auugcugugg aacaggauaa aaauacacag gaaguguuug 2400 cucaggugaa acagauuuac aaaacaccac caauuaaaga uuuuggagga uuuaauuuua 2460 gccagauucu gccugauccu ucuaaaccuu cuaaaagauc uuuuauugaa gaucugcugu 2520 uuaauaaagu gacacuggca gaugcaggau uuauuaaaca guauggagau ugccugggug 2580 auauugcugc aagagaucug auuugugcuc agaaauuuaa uggacugaca gugcugccuc 2640 cucugcugac agaugaaaug auugcucagu acacaucugc uuuacuggcu ggaacaauua 2700 caagcggaug gacauuugga gcuggagcug cucugcagau uccuuuugca augcagaugg 2760 cuuacagauu uaauggaauu ggagugacac agaauguguu auaugaaaau cagaaacuga 2820 uugcaaauca guuuaauucu gcaauuggca aaauucagga uucucugucu ucuacagcuu 2880 cugcucuggg aaaacugcag gaugugguga aucagaaugc acaggcacug aauacucugg 2940 ugaaacagcu gucuagcaau uuuggggcaa uuucuucugu gcugaaugau auucugucua 3000 gacuggaucc uccugaagcu gaagugcaga uugauagacu gaucacagga agacugcagu 3060 cucugcagac uuaugugaca cagcagcuga uuagagcugc ugaaauuaga gcuucugcua 3120 aucuggcugc uacaaaaaug ucugaaugug ugcugggaca gucaaaaaga guggauuuuu 3180 guggaaaagg auaucaucug augucuuuuc cacagucugc uccacaugga gugguguuuu 3240 uacaugugac auaugugcca gcacaggaaa agaauuuuac cacagcacca gcaauuuguc 3300 augauggaaa agcacauuuu ccaagagaag gaguguuugu gucuaaugga acacauuggu 3360 uugugacaca gagaaauuuu uaugaaccuc agauuauuac aacagauaau acauuugugu 3420 caggaaauug ugauguggug auuggaauug ugaauaauac aguguaugau ccacugcagc 3480 cagaacugga uucuuuuaaa gaagaacugg auaaauauuu uaaaaaucac acaucuccug 3540 auguggauuu aggagauauu ucuggaauca augcaucugu ggugaauauu cagaaagaaa 3600 uugauagacu gaaugaagug gccaaaaauc ugaaugaauc ucugauugau cugcaggaac 3660 uuggaaaaua ugaacaguac auuaaauggc cuugguacau uuggcuugga uuuauugcag 3720 gauuaauugc aauugugaug gugacaauua uguuauguug uaugacauca uguuguucuu 3780 guuuaaaagg auguuguucu uguggaagcu guuguaaauu ugaugaagau gauucugaac 3840 cuguguuaaa aggagugaaa uugcauuaca caugaugacu cgagcuggua cugcaugcac 3900 gcaaugcuag cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc 3960 cagguaugcu cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc 4020 caagcacgca gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc 4080 agugauuaac cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg 4140 ucaauuucgu gccagccaca cccuggagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aaagcauaug acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aaa 4283

<210> 20 <211> 4283 <212> RNA <213> Artificial Sequence <220> <223> RBP020.2 <400> 20 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguucg 60 uguuccuggu gcugcugccu cuggugucca gccagugugu gaaccugacc accagaacac 120 agcugccucc agccuacacc aacagcuuua ccagaggcgu guacuacccc gacaaggugu 180 ucagauccag cgugcugcac ucuacccagg accuguuccu gccuuucuuc agcaacguga 240 ccugguucca cgccauccac guguccggca ccaauggcac caagagauuc gacaaccccg 300 ugcugcccuu caacgacggg guguacuuug ccagcaccga gaaguccaac aucaucagag 360 gcuggaucuu cggcaccaca cuggacagca agacccagag ccugcugauc gugaacaacg 420 ccaccaacgu ggucaucaaa gugugcgagu uccaguucug caacgacccc uuccugggcg 480 ucuacuacca caagaacaac aagagcugga uggaaagcga guuccgggug uacagcagcg 540 ccaacaacug caccuucgag uacguguccc agccuuuccu gauggaccug gaaggcaagc 600 agggcaacuu caagaaccug cgcgaguucg uguuuaagaa caucgacggc uacuucaaga 660 ucuacagcaa gcacaccccu aucaaccucg ugcgggaucu gccucagggc uucucugcuc 720 uggaaccccu gguggaucug cccaucggca ucaacaucac ccgguuucag acacugcugg 780 cccugcacag aagcuaccug acaccuggcg auagcagcag cggauggaca gcuggugccg 840 ccgcuuacua ugugggcuac cugcagccua gaaccuuccu gcugaaguac aacgagaacg 900 gcaccaucac cgacgccgug gauugugcuc uggauccucu gagcgagaca aagugcaccc 960 ugaaguccuu caccguggaa aagggcaucu accagaccag caacuuccgg gugcagccca 1020 ccgaauccau cgugcgguuc cccaauauca ccaaucugug ccccuucggc gagguguuca 1080 augccaccag auucgccucu guguacgccu ggaaccggaa gcggaucagc aauugcgugg 1140 ccgacuacuc cgugcuguac aacuccgcca gcuucagcac cuucaagugc uacggcgugu 1200 ccccuaccaa gcugaacgac cugugcuuca caaacgugua cgccgacagc uucgugaucc 1260 ggggagauga agugcggcag auugccccug gacagacagg caagaucgcc gacuacaacu 1320 acaagcugcc cgacgacuuc accggcugug ugauugccug gaacagcaac aaccuggacu 1380 ccaaagucgg cggcaacuac aauuaccugu accggcuguu ccggaagucc aaucugaagc 1440 ccuucgagcg ggacaucucc accgagaucu aucaggccgg cagcaccccu uguaacggcg 1500 uggaaggcuu caacugcuac uucccacugc aguccuacgg cuuucagccc acaaauggcg 1560 ugggcuauca gcccuacaga gugguggugc ugagcuucga acugcugcau gccccugcca 1620 cagugugcgg cccuaagaaa agcaccaauc ucgugaagaa caaaugcgug aacuucaacu 1680 ucaacggccu gaccggcacc ggcgugcuga cagagagcaa caagaaguuc cugccauucc 1740 agcaguuugg ccgggauauc gccgauacca cagacgccgu uagagauccc cagacacugg 1800 aaauccugga caucaccccu ugcagcuucg gcggaguguc ugugaucacc ccuggcacca 1860 acaccagcaa ucagguggca gugcuguacc aggacgugaa cuguaccgaa gugcccgugg 1920 ccauucacgc cgaucagcug acaccuacau ggcgggugua cuccaccggc agcaaugugu 1980 uucagaccag agccggcugu cugaucggag ccgagcacgu gaacaauagc uacgagugcg 2040 acauccccau cggcgcugga aucugcgcca gcuaccagac acagacaaac agcccucgga 2100 gagccagaag cguggccagc cagagcauca uugccuacac aaugucucug ggcgccgaga 2160 acagcguggc cuacuccaac aacucuaucg cuauccccac caacuucacc aucagcguga 2220 ccacagagau ccugccugug uccaugacca agaccagcgu ggacugcacc auguacaucu 2280 gcggcgauuc caccgagugc uccaaccugc ugcugcagua cggcagcuuc ugcacccagc 2340 ugaauagagc ccugacaggg aucgccgugg aacaggacaa gaacacccaa gagguguucg 2400 cccaagugaa gcagaucuac aagaccccuc cuaucaagga cuucggcggc uucaauuuca 2460 gccagauucu gcccgauccu agcaagccca gcaagcggag cuucaucgag gaccugcugu 2520 ucaacaaagu gacacuggcc gacgccggcu ucaucaagca guauggcgau ugucugggcg 2580 acauugccgc cagggaucug auuugcgccc agaaguuuaa cggacugaca gugcugccuc 2640 cucugcugac cgaugagaug aucgcccagu acacaucugc ccugcuggcc ggcacaauca 2700 caagcggcug gacauuugga gcaggcgccg cucugcagau ccccuuugcu augcagaugg 2760 ccuaccgguu caacggcauc ggagugaccc agaaugugcu guacgagaac cagaagcuga 2820 ucgccaacca guucaacagc gccaucggca agauccagga cagccugagc agcacagcaa 2880 gcgcccuggg aaagcugcag gacgugguca accagaaugc ccaggcacug aacacccugg 2940 ucaagcagcu guccuccaac uucggcgcca ucagcucugu gcugaacgau auccugagca 3000 gacuggaccc uccugaggcc gaggugcaga ucgacagacu gaucacaggc agacugcaga 3060 gccuccagac auacgugacc cagcagcuga ucagagccgc cgagauuaga gccucugcca 3120 aucuggccgc caccaagaug ucugagugug ugcugggcca gagcaagaga guggacuuuu 3180 gcggcaaggg cuaccaccug augagcuucc cucagucugc cccucacggc gugguguuuc 3240 ugcacgugac auaugugccc gcucaagaga agaauuucac caccgcucca gccaucugcc 3300 acgacggcaa agcccacuuu ccuagagaag gcguguucgu guccaacggc acccauuggu 3360 ucgugacaca gcggaacuuc uacgagcccc agaucaucac caccgacaac accuucgugu 3420 cuggcaacug cgacgucgug aucggcauug ugaacaauac cguguacgac ccucugcagc 3480 ccgagcugga cagcuucaaa gaggaacugg acaaguacuu uaagaaccac acaagccccg 3540 acguggaccu gggcgauauc agcggaauca augccagcgu cgugaacauc cagaaagaga 3600 ucgaccggcu gaacgaggug gccaagaauc ugaacgagag ccugaucgac cugcaagaac 3660 uggggaagua cgagcaguac aucaaguggc ccugguacau cuggcugggc uuuaucgccg 3720 gacugauugc caucgugaug gucacaauca ugcuguguug caugaccagc ugcuguagcu 3780 gccugaaggg cuguuguagc uguggcagcu gcugcaaguu cgacgaggac gauucugagc 3840 ccgugcugaa gggcgugaaa cugcacuaca caugaugacu cgagcuggua cugcaugcac 3900 gcaaugcuag cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc 3960 cagguaugcu cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc 4020 caagcacgca gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc 4080 agugauuaac cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg 4140 ucaauuucgu gccagccaca cccuggagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa 4200 aaagcauaug acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaaaaaaaa aaa 4283

<210> 21

<211> 1262

<212> RNA

<213> Artificial Sequence

<220>

<223> RBP020.3

<400> 21 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugugu ggugagauuu ccaaauauua 120 caaaucugug uccauuugga gaaguguuua augcaacaag auuugcaucu guguaugcau 180 ggaauagaaa aagaauuucu aauugugugg cugauuauuc ugugcuguau aauagugcuu 240 cuuuuuccac auuuaaaugu uauggagugu cuccaacaaa auuaaaugau uuauguuuua 300 caaaugugua ugcugauucu uuugugauca gaggugauga agugagacag auugcccccg 360 gacagacagg aaaaauugcu gauuacaauu acaaacugcc ugaugauuuu acaggaugug 420 ugauugcuug gaauucuaau aauuuagauu cuaaaguggg aggaaauuac aauuaucugu 480 acagacuguu uagaaaauca aaucugaaac cuuuugaaag agauauuuca acagaaauuu 540 aucaggcugg aucaacaccu uguaauggag uggaaggauu uaauuguuau uuuccauuac 600 agagcuaugg auuucagcca accaauggug ugggauauca gccauauaga gugguggugc 660 ugucuuuuga acugcugcau gcaccugcaa cagugugugg accuaaaggc ucccccggcu 720 ccggcuccgg aucugguuau auuccugaag cuccaagaga ugggcaagcu uacguucgua 780 aagauggcga auggguauua cuuucuaccu uuuuaggccg gucccuggag gugcuguucc 840 agggccccgg cugaugacuc gagcugguac ugcaugcacg caaugcuagc ugccccuuuc 900 ccguccuggg uaccccgagu cucccccgac cucggguccc agguaugcuc ccaccuccac 960 cugccccacu caccaccucu gcuaguucca gacaccuccc aagcacgcag caaugcagcu 1020 caaaacgcuu agccuagcca cacccccacg ggaaacagca gugauuaacc uuuagcaaua 1080 aacgaaaguu uaacuaagcu auacuaaccc caggguuggu caauuucgug ccagccacac 1140 ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagcauauga cuaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1260 aa 1262

<210> 22

<211> 1879

<212> PRT <213> Artificial Sequence <220>

<223> Viral Protein

<400> 22 Met Glu Lys Vai His Vai Asp lie Glu Glu Asp Ser Pro Phe Leu Arg 1 5 10 15

Ala Leu Gin Arg Ser Phe Pro Gin Phe Glu Vai Glu Ala Lys Gin Vai

20 25 30

Thr Asp Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala Ser 35 40 45

Lys Leu lie Glu Thr Glu Vai Asp Pro Ser Asp Thr lie Leu Asp lie

50 55 60

Gly Ser Ala Pro Ala Arg Arg Met Tyr Ser Lys His Lys Tyr His Cys 65 70 75 80 lie Cys Pro Met Arg Cys Ala Glu Asp Pro Asp Arg Leu Tyr Lys Tyr

85 90 95

Ala Thr Lys Leu Lys Lys Asn Cys Lys Glu lie Thr Asp Lys Glu Leu

100 105 110

Asp Lys Lys Met Lys Glu Leu Ala Ala Vai Met Ser Asp Pro Asp Leu

115 120 125

Glu Thr Glu Thr Met Cys Leu His Asp Asp Glu Ser Cys Arg Tyr Glu

130 135 140

Gly Gin Vai Ala Vai Tyr Gin Asp Vai Tyr Ala Vai Asp Gly Pro Thr 145 150 155 160

Ser Leu Tyr His Gin Ala Asn Lys Gly Vai Arg Vai Ala Tyr Trp lie

165 170 175

Gly Phe Asp Thr Thr Pro Phe Met Phe Lys Asn Leu Ala Gly Ala Tyr

180 185 190

Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Thr Vai Leu Thr Ala Arg

195 200 205 Asn lie Gly Leu Cys Ser Ser Asp Vai Met Glu Arg Ser Arg Arg Gly

210 215 220

Met Ser lie Leu Arg Lys Lys Tyr Leu Lys Pro Ser Asn Asn Vai Leu

225 230 235 240

Phe Ser Vai Gly Ser Thr lie Tyr His Glu Lys Arg Asp Leu Leu Arg

245 250 255

Ser Trp His Leu Pro Ser Vai Phe His Leu Arg Gly Lys Gin Asn Tyr

260 265 270

Thr Cys Arg Cys Glu Thr lie Vai Ser Cys Asp Gly Tyr Vai Vai Lys

275 280 285

Arg lie Ala lie Ser Pro Gly Leu Tyr Gly Lys Pro Ser Gly Tyr Ala

290 295 300

Ala Thr Met His Arg Glu Gly Phe Leu Cys Cys Lys Vai Thr Asp Thr 305 310 315 320

Leu Asn Gly Glu Arg Vai Ser Phe Pro Vai Cys Thr Tyr Vai Pro Ala

325 330 335

Thr Leu Cys Asp Gin Met Thr Gly lie Leu Ala Thr Asp Vai Ser Ala

340 345 350

Asp Asp Ala Gin Lys Leu Leu Vai Gly Leu Asn Gin Arg lie Vai Vai

355 360 365

Asn Gly Arg Thr Gin Arg Asn Thr Asn Thr Met Lys Asn Tyr Leu Leu

370 375 380

Pro Vai Vai Ala Gin Ala Phe Ala Arg Trp Ala Lys Glu Tyr Lys Glu

385 390 395 400 Asp Gin Glu Asp Glu Arg Pro Leu Gly Leu Arg Asp Arg Gin Leu Vai

405 410 415

Met Gly Cys Cys Trp Ala Phe Arg Arg His Lys lie Thr Ser lie Tyr

420 425 430

Lys Arg Pro Asp Thr Gin Thr lie lie Lys Vai Asn Ser Asp Phe His

435 440 445

Ser Phe Vai Leu Pro Arg lie Gly Ser Asn Thr Leu Glu lie Gly Leu

450 455 460

Arg Thr Arg lie Arg Lys Met Leu Glu Glu His Lys Glu Pro Ser Pro

465 470 475 480

Leu lie Thr Ala Glu Asp Vai Gin Glu Ala Lys Cys Ala Ala Asp Glu

485 490 495

Ala Lys Glu Vai Arg Glu Ala Glu Glu Leu Arg Ala Ala Leu Pro Pro

500 505 510

Leu Ala Ala Asp Vai Glu Glu Pro Thr Leu Glu Ala Asp Vai Asp Leu

515 520 525

Met Leu Gin Glu Ala Gly Ala Gly Ser Vai Glu Thr Pro Arg Gly Leu

530 535 540 lie Lys Vai Thr Ser Tyr Ala Gly Glu Asp Lys lie Gly Ser Tyr Ala

545 550 555 560

Vai Leu Ser Pro Gin Ala Vai Leu Lys Ser Glu Lys Leu Ser Cys lie

565 570 575

His Pro Leu Ala Glu Gin Vai lie Vai lie Thr His Ser Gly Arg Lys

580 585 590

Gly Arg Tyr Ala Vai Glu Pro Tyr His Gly Lys Vai Vai Vai Pro Glu

595 600 605 Gly His Ala lie Pro Vai Gin Asp Phe Gin Ala Leu Ser Glu Ser Ala

610 615 620

Thr lie Vai Tyr Asn Glu Arg Glu Phe Vai Asn Arg Tyr Leu His His

625 630 635 640 lie Ala Thr His Gly Gly Ala Leu Asn Thr Asp Glu Glu Tyr Tyr Lys

645 650 655

Thr Vai Lys Pro Ser Glu His Asp Gly Glu Tyr Leu Tyr Asp lie Asp

660 665 670

Arg Lys Gin Cys Vai Lys Lys Glu Leu Vai Thr Gly Leu Gly Leu Thr

675 680 685

Gly Glu Leu Vai Asp Pro Pro Phe His Glu Phe Ala Tyr Glu Ser Leu

690 695 700

Arg Thr Arg Pro Ala Ala Pro Tyr Gin Vai Pro Thr lie Gly Vai Tyr 705 710 715 720

Gly Vai Pro Gly Ser Gly Lys Ser Gly lie lie Lys Ser Ala Vai Thr

725 730 735

Lys Lys Asp Leu Vai Vai Ser Ala Lys Lys Glu Asn Cys Ala Glu lie

740 745 750 lie Arg Asp Vai Lys Lys Met Lys Gly Leu Asp Vai Asn Ala Arg Thr

755 760 765

Vai Asp Ser Vai Leu Leu Asn Gly Cys Lys His Pro Vai Glu Thr Leu

770 775 780

Tyr lie Asp Glu Ala Phe Ala Cys His Ala Gly Thr Leu Arg Ala Leu

785 790 795 800 lie Ala lie lie Arg Pro Lys Lys Ala Vai Leu Cys Gly Asp Pro Lys

805 810 815

Gin Cys Gly Phe Phe Asn Met Met Cys Leu Lys Vai His Phe Asn His

820 825 830

Glu lie Cys Thr Gin Vai Phe His Lys Ser lie Ser Arg Arg Cys Thr

835 840 845

Lys Ser Vai Thr Ser Vai Vai Ser Thr Leu Phe Tyr Asp Lys Lys Met

850 855 860

Arg Thr Thr Asn Pro Lys Glu Thr Lys lie Vai lie Asp Thr Thr Gly 865 870 875 880

Ser Thr Lys Pro Lys Gin Asp Asp Leu lie Leu Thr Cys Phe Arg Gly

885 890 895

Trp Vai Lys Gin Leu Gin lie Asp Tyr Lys Gly Asn Glu lie Met Thr

900 905 910

Ala Ala Ala Ser Gin Gly Leu Thr Arg Lys Gly Vai Tyr Ala Vai Arg

915 920 925

Tyr Lys Vai Asn Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Vai

930 935 940

Asn Vai Leu Leu Thr Arg Thr Glu Asp Arg lie Vai Trp Lys Thr Leu

945 950 955 960

Ala Gly Asp Pro Trp lie Lys Thr Leu Thr Ala Lys Tyr Pro Gly Asn

965 970 975

Phe Thr Ala Thr lie Glu Glu Trp Gin Ala Glu His Asp Ala lie Met

980 985 990

Arg His lie Leu Glu Arg Pro Asp Pro Thr Asp Vai Phe Gin Asn Lys

995 1000 1005 Ala Asn Vai Cys Trp Ala Lys Ala Leu Vai Pro Vai Leu Lys Thr

1010 1015 1020

Ala Gly lie Asp Met Thr Thr Glu Gin Trp Asn Thr Vai Asp Tyr 1025 1030 1035

Phe Glu Thr Asp Lys Ala His Ser Ala Glu lie Vai Leu Asn Gin 1040 1045 1050

Leu Cys Vai Arg Phe Phe Gly Leu Asp Leu Asp Ser Gly Leu Phe 1055 1060 1065

Ser Ala Pro Thr Vai Pro Leu Ser lie Arg Asn Asn His Trp Asp

1070 1075 1080

Asn Ser Pro Ser Pro Asn Met Tyr Gly Leu Asn Lys Glu Vai Vai 1085 1090 1095

Arg Gin Leu Ser Arg Arg Tyr Pro Gin Leu Pro Arg Ala Vai Ala

1100 1105 1110

Thr Gly Arg Vai Tyr Asp Met Asn Thr Gly Thr Leu Arg Asn Tyr

1115 1120 1125

Asp Pro Arg lie Asn Leu Vai Pro Vai Asn Arg Arg Leu Pro His

1130 1135 1140

Ala Leu Vai Leu His His Asn Glu His Pro Gin Ser Asp Phe Ser

1145 1150 1155

Ser Phe Vai Ser Lys Leu Lys Gly Arg Thr Vai Leu Vai Vai Gly

1160 1165 1170

Glu Lys Leu Ser Vai Pro Gly Lys Met Vai Asp Trp Leu Ser Asp

1175 1180 1185 Arg Pro Glu Ala Thr Phe Arg Ala Arg Leu Asp Leu Gly lie Pro

1190 1195 1200

Gly Asp Vai Pro Lys Tyr Asp lie lie Phe Vai Asn Vai Arg Thr

1205 1210 1215

Pro Tyr Lys Tyr His His Tyr Gin Gin Cys Glu Asp His Ala lie 1220 1225 1230

Lys Leu Ser Met Leu Thr Lys Lys Ala Cys Leu His Leu Asn Pro 1235 1240 1245

Gly Gly Thr Cys Vai Ser lie Gly Tyr Gly Tyr Ala Asp Arg Ala 1250 1255 1260

Ser Glu Ser lie lie Gly Ala lie Ala Arg Gin Phe Lys Phe Ser 1265 1270 1275

Arg Vai Cys Lys Pro Lys Ser Ser Leu Glu Glu Thr Glu Vai Leu 1280 1285 1290

Phe Vai Phe lie Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro

1295 1300 1305

Tyr Lys Leu Ser Ser Thr Leu Thr Asn lie Tyr Thr Gly Ser Arg

1310 1315 1320

Leu His Glu Ala Gly Cys Ala Pro Ser Tyr His Vai Vai Arg Gly 1325 1330 1335

Asp lie Ala Thr Ala Thr Glu Gly Vai lie lie Asn Ala Ala Asn 1340 1345 1350

Ser Lys Gly Gin Pro Gly Gly Gly Vai Cys Gly Ala Leu Tyr Lys 1355 1360 1365

Lys Phe Pro Glu Ser Phe Asp Leu Gin Pro lie Glu Vai Gly Lys 1370 1375 1380 Ala Arg Leu Vai Lys Gly Ala Ala Lys His lie lie His Ala Vai 1385 1390 1395

Gly Pro Asn Phe Asn Lys Vai Ser Glu Vai Glu Gly Asp Lys Gin

1400 1405 1410

Leu Ala Glu Ala Tyr Glu Ser lie Ala Lys lie Vai Asn Asp Asn

1415 1420 1425

Asn Tyr Lys Ser Vai Ala lie Pro Leu Leu Ser Thr Gly lie Phe 1430 1435 1440

Ser Gly Asn Lys Asp Arg Leu Thr Gin Ser Leu Asn His Leu Leu 1445 1450 1455

Thr Ala Leu Asp Thr Thr Asp Ala Asp Vai Ala lie Tyr Cys Arg 1460 1465 1470

Asp Lys Lys Trp Glu Met Thr Leu Lys Glu Ala Vai Ala Arg Arg 1475 1480 1485

Glu Ala Vai Glu Glu lie Cys lie Ser Asp Asp Ser Ser Vai Thr 1490 1495 1500

Glu Pro Asp Ala Glu Leu Vai Arg Vai His Pro Lys Ser Ser Leu 1505 1510 1515

Ala Gly Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser 1520 1525 1530

Tyr Leu Glu Gly Thr Lys Phe His Gin Ala Ala Lys Asp lie Ala 1535 1540 1545

Glu lie Asn Ala Met Trp Pro Vai Ala Thr Glu Ala Asn Glu Gin 1550 1555 1560 Vai Cys Met Tyr lie Leu Gly Glu Ser Met Ser Ser lie Arg Ser

1565 1570 1575

Lys Cys Pro Vai Glu Glu Ser Glu Ala Ser Thr Pro Pro Ser Thr 1580 1585 1590

Leu Pro Cys Leu Cys lie His Ala Met Thr Pro Glu Arg Vai Gin 1595 1600 1605

Arg Leu Lys Ala Ser Arg Pro Glu Gin lie Thr Vai Cys Ser Ser

1610 1615 1620

Phe Pro Leu Pro Lys Tyr Arg lie Thr Gly Vai Gin Lys lie Gin

1625 1630 1635

Cys Ser Gin Pro lie Leu Phe Ser Pro Lys Vai Pro Ala Tyr lie

1640 1645 1650

His Pro Arg Lys Tyr Leu Vai Glu Thr Pro Pro Vai Asp Glu Thr 1655 1660 1665

Pro Glu Pro Ser Ala Glu Asn Gin Ser Thr Glu Gly Thr Pro Glu 1670 1675 1680

Gin Pro Pro Leu lie Thr Glu Asp Glu Thr Arg Thr Arg Thr Pro 1685 1690 1695

Glu Pro lie lie lie Glu Glu Glu Glu Glu Asp Ser lie Ser Leu 1700 1705 1710

Leu Ser Asp Gly Pro Thr His Gin Vai Leu Gin Vai Glu Ala Asp 1715 1720 1725 lie His Gly Pro Pro Ser Vai Ser Ser Ser Ser Trp Ser lie Pro

1730 1735 1740

His Ala Ser Asp Phe Asp Vai Asp Ser Leu Ser lie Leu Asp Thr 1745 1750 1755 Leu Glu Gly Ala Ser Vai Thr Ser Gly Ala Thr Ser Ala Glu Thr

1760 1765 1770

Asn Ser Tyr Phe Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Vai 1775 1780 1785

Pro Ala Pro Arg Thr Vai Phe Arg Asn Pro Pro His Pro Ala Pro 1790 1795 1800

Arg Thr Arg Thr Pro Ser Leu Ala Pro Ser Arg Ala Cys Ser Arg

1805 1810 1815

Thr Ser Leu Vai Ser Thr Pro Pro Gly Vai Asn Arg Vai lie Thr

1820 1825 1830

Arg Glu Glu Leu Glu Ala Leu Thr Pro Ser Arg Thr Pro Ser Arg 1835 1840 1845

Ser Vai Ser Arg Thr Ser Leu Vai Ser Asn Pro Pro Gly Vai Asn 1850 1855 1860

Arg Vai lie Thr Arg Glu Glu Phe Glu Ala Phe Vai Ala Gin Gin 1865 1870 1875

Gin

<210> 23

<211> 613

<212> PRT

<213> Artificial Sequence

<220>

<223> Viral Protein

<400> 23

Arg Phe Asp Ala Gly Ala Tyr lie Phe Ser Ser Asp Thr Gly Gin Gly

1 5 10 15 His Leu Gin Gin Lys Ser Vai Arg Gin Thr Vai Leu Ser Glu Vai Vai

20 25 30

Leu Glu Arg Thr Glu Leu Glu lie Ser Tyr Ala Pro Arg Leu Asp Gin 35 40 45

Glu Lys Glu Glu Leu Leu Arg Lys Lys Leu Gin Leu Asn Pro Thr Pro

50 55 60

Ala Asn Arg Ser Arg Tyr Gin Ser Arg Lys Vai Glu Asn Met Lys Ala 65 70 75 80 lie Thr Ala Arg Arg lie Leu Gin Gly Leu Gly His Tyr Leu Lys Ala

85 90 95

Glu Gly Lys Vai Glu Cys Tyr Arg Thr Leu His Pro Vai Pro Leu Tyr

100 105 110

Ser Ser Ser Vai Asn Arg Ala Phe Ser Ser Pro Lys Vai Ala Vai Glu

115 120 125

Ala Cys Asn Ala Met Leu Lys Glu Asn Phe Pro Thr Vai Ala Ser Tyr

130 135 140

Cys lie lie Pro Glu Tyr Asp Ala Tyr Leu Asp Met Vai Asp Gly Ala

145 150 155 160

Ser Cys Cys Leu Asp Thr Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser

165 170 175

Phe Pro Lys Lys His Ser Tyr Leu Glu Pro Thr lie Arg Ser Ala Vai

180 185 190

Pro Ser Ala lie Gin Asn Thr Leu Gin Asn Vai Leu Ala Ala Ala Thr

195 200 205 Lys Arg Asn Cys Asn Vai Thr Gin Met Arg Glu Leu Pro Vai Leu Asp

210 215 220

Ser Ala Ala Phe Asn Vai Glu Cys Phe Lys Lys Tyr Ala Cys Asn Asn

225 230 235 240

Glu Tyr Trp Glu Thr Phe Lys Glu Asn Pro lie Arg Leu Thr Glu Glu

245 250 255

Asn Vai Vai Asn Tyr lie Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala

260 265 270

Leu Phe Ala Lys Thr His Asn Leu Asn Met Leu Gin Asp lie Pro Met

275 280 285

Asp Arg Phe Vai Met Asp Leu Lys Arg Asp Vai Lys Vai Thr Pro Gly

290 295 300

Thr Lys His Thr Glu Glu Arg Pro Lys Vai Gin Vai lie Gin Ala Ala

305 310 315 320

Asp Pro Leu Ala Thr Ala Tyr Leu Cys Gly lie His Arg Glu Leu Vai

325 330 335

Arg Arg Leu Asn Ala Vai Leu Leu Pro Asn lie His Thr Leu Phe Asp

340 345 350

Met Ser Ala Glu Asp Phe Asp Ala lie lie Ala Glu His Phe Gin Pro

355 360 365

Gly Asp Cys Vai Leu Glu Thr Asp lie Ala Ser Phe Asp Lys Ser Glu

370 375 380

Asp Asp Ala Met Ala Leu Thr Ala Leu Met lie Leu Glu Asp Leu Gly 385 390 395 400

Vai Asp Ala Glu Leu Leu Thr Leu lie Glu Ala Ala Phe Gly Glu lie

405 410 415 Ser Ser lie His Leu Pro Thr Lys Thr Lys Phe Lys Phe Gly Ala Met

420 425 430

Met Lys Ser Gly Met Phe Leu Thr Leu Phe Vai Asn Thr Vai lie Asn

435 440 445 lie Vai lie Ala Ser Arg Vai Leu Arg Glu Arg Leu Thr Gly Ser Pro

450 455 460

Cys Ala Ala Phe lie Gly Asp Asp Asn lie Vai Lys Gly Vai Lys Ser 465 470 475 480

Asp Lys Leu Met Ala Asp Arg Cys Ala Thr Trp Leu Asn Met Glu Vai

485 490 495

Lys lie lie Asp Ala Vai Vai Gly Glu Lys Ala Pro Tyr Phe Cys Gly

500 505 510

Gly Phe lie Leu Cys Asp Ser Vai Thr Gly Thr Ala Cys Arg Vai Ala

515 520 525

Asp Pro Leu Lys Arg Leu Phe Lys Leu Gly Lys Pro Leu Ala Ala Asp

530 535 540

Asp Glu His Asp Asp Asp Arg Arg Arg Ala Leu His Glu Glu Ser Thr 545 550 555 560

Arg Trp Asn Arg Vai Gly lie Leu Ser Glu Leu Cys Lys Ala Vai Glu

565 570 575

Ser Arg Tyr Glu Thr Vai Gly Thr Ser lie lie Vai Met Ala Met Thr

580 585 590

Thr Leu Ala Ser Ser Vai Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro

595 600 605 lie Thr Leu Tyr Gly 610

<210> 24

<211> 11917

<212> RNA

<213> Artificial Sequence

<220>

<223> RBS004.1

<400> 24 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg 780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa 1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga 1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu 4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa 5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc 5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu 6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc 7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga 7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugaauu 7620 ugacaacaag aacacagcug ccaccagcuu auacaaauuc uuuuaccaga ggaguguauu 7680 auccugauaa aguguuuaga ucuucugugc ugcacagcac acaggaccug uuucugccau 7740 uuuuuagcaa ugugacaugg uuucaugcaa uucauguguc uggaacaaau ggaacaaaaa 7800 gauuugauaa uccugugcug ccuuuuaaug auggagugua uuuugcuuca acagaaaagu 7860 caaauauuau uagaggaugg auuuuuggaa caacacugga uucuaaaaca cagucucugc 7920 ugauugugaa uaaugcaaca aaugugguga uuaaagugug ugaauuucag uuuuguaaug 7980 auccuuuucu gggaguguau uaucacaaaa auaauaaauc uuggauggaa ucugaauuua 8040 gaguguauuc cucugcaaau aauuguacau uugaauaugu gucucagccu uuucugaugg 8100 aucuggaagg aaaacagggc aauuuuaaaa aucugagaga auuuguguuu aaaaauauug 8160 auggauauuu uaaaauuuau ucuaaacaca caccaauuaa uuuagugaga gaucugccuc 8220 agggauuuuc ugcucuggaa ccucuggugg aucugccaau uggcauuaau auuacaagau 8280 uucagacacu gcuggcucug cacagaucuu aucugacacc uggagauucu ucuucuggau 8340 ggacagccgg agcugcagcu uauuaugugg gcuaucugca gccaagaaca uuucugcuga 8400 aauauaauga aaauggaaca auuacagaug cuguggauug ugcucuggau ccucugucug 8460 aaacaaaaug uacauuaaaa ucuuuuacag uggaaaaagg cauuuaucag acaucuaauu 8520 uuagagugca gccaacagaa ucuauuguga gauuuccaaa uauuacaaau cuguguccau 8580 uuggagaagu guuuaaugca acaagauuug caucugugua ugcauggaau agaaaaagaa 8640 uuucuaauug uguggcugau uauucugugc uguauaauag ugcuucuuuu uccacauuua 8700 aauguuaugg agugucucca acaaaauuaa augauuuaug uuuuacaaau guguaugcug 8760 auucuuuugu gaucagaggu gaugaaguga gacagauugc ccccggacag acaggaaaaa 8820 uugcugauua caauuacaaa cugccugaug auuuuacagg augugugauu gcuuggaauu 8880 cuaauaauuu agauucuaaa gugggaggaa auuacaauua ucuguacaga cuguuuagaa 8940 aaucaaaucu gaaaccuuuu gaaagagaua uuucaacaga aauuuaucag gcuggaucaa 9000 caccuuguaa uggaguggaa ggauuuaauu guuauuuucc auuacagagc uauggauuuc 9060 agccaaccaa ugguguggga uaucagccau auagaguggu ggugcugucu uuugaacugc 9120 ugcaugcacc ugcaacagug uguggaccua aaaaaucuac aaauuuagug aaaaauaaau 9180 gugugaauuu uaauuuuaau ggauuaacag gaacaggagu gcugacagaa ucuaauaaaa 9240 aauuucugcc uuuucagcag uuuggcagag auauugcaga uaccacagau gcagugagag 9300 auccucagac auuagaaauu cuggauauua caccuuguuc uuuugggggu gugucuguga 9360 uuacaccugg aacaaauaca ucuaaucagg uggcugugcu guaucaggau gugaauugua 9420 cagaagugcc aguggcaauu caugcagauc agcugacacc aacauggaga guguauucua 9480 caggaucuaa uguguuucag acaagagcag gaugucugau uggagcagaa caugugaaua 9540 auucuuauga augugauauu ccaauuggag caggcauuug ugcaucuuau cagacacaga 9600 caaauucccc aaggagagca agaucugugg caucucaguc uauuauugca uacaccaugu 9660 cucugggagc agaaaauucu guggcauauu cuaauaauuc uauugcuauu ccaacaaauu 9720 uuaccauuuc ugugacaaca gaaauuuuac cugugucuau gacaaaaaca ucuguggauu 9780 guaccaugua cauuugugga gauucuacag aauguucuaa ucugcugcug caguauggau 9840 cuuuuuguac acagcugaau agagcuuuaa caggaauugc uguggaacag gauaaaaaua 9900 cacaggaagu guuugcucag gugaaacaga uuuacaaaac accaccaauu aaagauuuug 9960 gaggauuuaa uuuuagccag auucugccug auccuucuaa accuucuaaa agaucuuuua 10020 uugaagaucu gcuguuuaau aaagugacac uggcagaugc aggauuuauu aaacaguaug 10080 gagauugccu gggugauauu gcugcaagag aucugauuug ugcucagaaa uuuaauggac 10140 ugacagugcu gccuccucug cugacagaug aaaugauugc ucaguacaca ucugcuuuac 10200 uggcuggaac aauuacaagc ggauggacau uuggagcugg agcugcucug cagauuccuu 10260 uugcaaugca gauggcuuac agauuuaaug gaauuggagu gacacagaau guguuauaug 10320 aaaaucagaa acugauugca aaucaguuua auucugcaau uggcaaaauu caggauucuc 10380 ugucuucuac agcuucugcu cugggaaaac ugcaggaugu ggugaaucag aaugcacagg 10440 cacugaauac ucuggugaaa cagcugucua gcaauuuugg ggcaauuucu ucugugcuga 10500 augauauucu gucuagacug gauccuccug aagcugaagu gcagauugau agacugauca 10560 caggaagacu gcagucucug cagacuuaug ugacacagca gcugauuaga gcugcugaaa 10620 uuagagcuuc ugcuaaucug gcugcuacaa aaaugucuga augugugcug ggacagucaa 10680 aaagagugga uuuuugugga aaaggauauc aucugauguc uuuuccacag ucugcuccac 10740 auggaguggu guuuuuacau gugacauaug ugccagcaca ggaaaagaau uuuaccacag 10800 caccagcaau uugucaugau ggaaaagcac auuuuccaag agaaggagug uuugugucua 10860 auggaacaca uugguuugug acacagagaa auuuuuauga accucagauu auuacaacag 10920 auaauacauu ugugucagga aauugugaug uggugauugg aauugugaau aauacagugu 10980 augauccacu gcagccagaa cuggauucuu uuaaagaaga acuggauaaa uauuuuaaaa 11040 aucacacauc uccugaugug gauuuaggag auauuucugg aaucaaugca ucugugguga 11100 auauucagaa agaaauugau agacugaaug aaguggccaa aaaucugaau gaaucucuga 11160 uugaucugca ggaacuugga aaauaugaac aguacauuaa auggccuugg uacauuuggc 11220 uuggauuuau ugcaggauua auugcaauug ugauggugac aauuauguua uguuguauga 11280 caucauguug uucuuguuua aaaggauguu guucuugugg aagcuguugu aaauuugaug 11340 aagaugauuc ugaaccugug uuaaaaggag ugaaauugca uuacacauga ugacucgagc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccacucacc accucugcua 11520 guuccagaca ccucccaagc acgcagcaau gcagcucaaa acgcuuagcc uagccacacc 11580 cccacgggaa acagcaguga uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa aauuuuuauu 11760 uuauuuuuuc uuuucuuuuc cgaaucggau uuuguuuuua auauuucaaa aaaaaaaaaa 11820 aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 11917

<210> 25 <211> 11917 <212> RNA <213> Artificial Sequence <220> <223> RBS004.2 <400> 25 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg 780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa 1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga 1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu 4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa 5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc 5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu 6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc 7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga 7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca 7560 agacuaguau guucguguuc cuggugcugc ugccucuggu guccagccag ugugugaacc 7620 ugaccaccag aacacagcug ccuccagccu acaccaacag cuuuaccaga ggcguguacu 7680 accccgacaa gguguucaga uccagcgugc ugcacucuac ccaggaccug uuccugccuu 7740 ucuucagcaa cgugaccugg uuccacgcca uccacguguc cggcaccaau ggcaccaaga 7800 gauucgacaa ccccgugcug cccuucaacg acggggugua cuuugccagc accgagaagu 7860 ccaacaucau cagaggcugg aucuucggca ccacacugga cagcaagacc cagagccugc 7920 ugaucgugaa caacgccacc aacgugguca ucaaagugug cgaguuccag uucugcaacg 7980 accccuuccu gggcgucuac uaccacaaga acaacaagag cuggauggaa agcgaguucc 8040 ggguguacag cagcgccaac aacugcaccu ucgaguacgu gucccagccu uuccugaugg 8100 accuggaagg caagcagggc aacuucaaga accugcgcga guucguguuu aagaacaucg 8160 acggcuacuu caagaucuac agcaagcaca ccccuaucaa ccucgugcgg gaucugccuc 8220 agggcuucuc ugcucuggaa ccccuggugg aucugcccau cggcaucaac aucacccggu 8280 uucagacacu gcuggcccug cacagaagcu accugacacc uggcgauagc agcagcggau 8340 ggacagcugg ugccgccgcu uacuaugugg gcuaccugca gccuagaacc uuccugcuga 8400 aguacaacga gaacggcacc aucaccgacg ccguggauug ugcucuggau ccucugagcg 8460 agacaaagug cacccugaag uccuucaccg uggaaaaggg caucuaccag accagcaacu 8520 uccgggugca gcccaccgaa uccaucgugc gguuccccaa uaucaccaau cugugccccu 8580 ucggcgaggu guucaaugcc accagauucg ccucugugua cgccuggaac cggaagcgga 8640 ucagcaauug cguggccgac uacuccgugc uguacaacuc cgccagcuuc agcaccuuca 8700 agugcuacgg cguguccccu accaagcuga acgaccugug cuucacaaac guguacgccg 8760 acagcuucgu gauccgggga gaugaagugc ggcagauugc cccuggacag acaggcaaga 8820 ucgccgacua caacuacaag cugcccgacg acuucaccgg cugugugauu gccuggaaca 8880 gcaacaaccu ggacuccaaa gucggcggca acuacaauua ccuguaccgg cuguuccgga 8940 aguccaaucu gaagcccuuc gagcgggaca ucuccaccga gaucuaucag gccggcagca 9000 ccccuuguaa cggcguggaa ggcuucaacu gcuacuuccc acugcagucc uacggcuuuc 9060 agcccacaaa uggcgugggc uaucagcccu acagaguggu ggugcugagc uucgaacugc 9120 ugcaugcccc ugccacagug ugcggcccua agaaaagcac caaucucgug aagaacaaau 9180 gcgugaacuu caacuucaac ggccugaccg gcaccggcgu gcugacagag agcaacaaga 9240 aguuccugcc auuccagcag uuuggccggg auaucgccga uaccacagac gccguuagag 9300 auccccagac acuggaaauc cuggacauca ccccuugcag cuucggcgga gugucuguga 9360 ucaccccugg caccaacacc agcaaucagg uggcagugcu guaccaggac gugaacugua 9420 ccgaagugcc cguggccauu cacgccgauc agcugacacc uacauggcgg guguacucca 9480 ccggcagcaa uguguuucag accagagccg gcugucugau cggagccgag cacgugaaca 9540 auagcuacga gugcgacauc cccaucggcg cuggaaucug cgccagcuac cagacacaga 9600 caaacagccc ucggagagcc agaagcgugg ccagccagag caucauugcc uacacaaugu 9660 cucugggcgc cgagaacagc guggccuacu ccaacaacuc uaucgcuauc cccaccaacu 9720 ucaccaucag cgugaccaca gagauccugc cuguguccau gaccaagacc agcguggacu 9780 gcaccaugua caucugcggc gauuccaccg agugcuccaa ccugcugcug caguacggca 9840 gcuucugcac ccagcugaau agagcccuga cagggaucgc cguggaacag gacaagaaca 9900 cccaagaggu guucgcccaa gugaagcaga ucuacaagac cccuccuauc aaggacuucg 9960 gcggcuucaa uuucagccag auucugcccg auccuagcaa gcccagcaag cggagcuuca 10020 ucgaggaccu gcuguucaac aaagugacac uggccgacgc cggcuucauc aagcaguaug 10080 gcgauugucu gggcgacauu gccgccaggg aucugauuug cgcccagaag uuuaacggac 10140 ugacagugcu gccuccucug cugaccgaug agaugaucgc ccaguacaca ucugcccugc 10200 uggccggcac aaucacaagc ggcuggacau uuggagcagg cgccgcucug cagauccccu 10260 uugcuaugca gauggccuac cgguucaacg gcaucggagu gacccagaau gugcuguacg 10320 agaaccagaa gcugaucgcc aaccaguuca acagcgccau cggcaagauc caggacagcc 10380 ugagcagcac agcaagcgcc cugggaaagc ugcaggacgu ggucaaccag aaugcccagg 10440 cacugaacac ccuggucaag cagcuguccu ccaacuucgg cgccaucagc ucugugcuga 10500 acgauauccu gagcagacug gacccuccug aggccgaggu gcagaucgac agacugauca 10560 caggcagacu gcagagccuc cagacauacg ugacccagca gcugaucaga gccgccgaga 10620 uuagagccuc ugccaaucug gccgccacca agaugucuga gugugugcug ggccagagca 10680 agagagugga cuuuugcggc aagggcuacc accugaugag cuucccucag ucugccccuc 10740 acggcguggu guuucugcac gugacauaug ugcccgcuca agagaagaau uucaccaccg 10800 cuccagccau cugccacgac ggcaaagccc acuuuccuag agaaggcgug uucgugucca 10860 acggcaccca uugguucgug acacagcgga acuucuacga gccccagauc aucaccaccg 10920 acaacaccuu cgugucuggc aacugcgacg ucgugaucgg cauugugaac aauaccgugu 10980 acgacccucu gcagcccgag cuggacagcu ucaaagagga acuggacaag uacuuuaaga 11040 accacacaag ccccgacgug gaccugggcg auaucagcgg aaucaaugcc agcgucguga 11100 acauccagaa agagaucgac cggcugaacg agguggccaa gaaucugaac gagagccuga 11160 ucgaccugca agaacugggg aaguacgagc aguacaucaa guggcccugg uacaucuggc 11220 ugggcuuuau cgccggacug auugccaucg ugauggucac aaucaugcug uguugcauga 11280 ccagcugcug uagcugccug aagggcuguu guagcugugg cagcugcugc aaguucgacg 11340 aggacgauuc ugagcccgug cugaagggcg ugaaacugca cuacacauga ugacucgagc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccacucacc accucugcua 11520 guuccagaca ccucccaagc acgcagcaau gcagcucaaa acgcuuagcc uagccacacc 11580 cccacgggaa acagcaguga uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa aauuuuuauu 11760 uuauuuuuuc uuuucuuuuc cgaaucggau uuuguuuuua auauuucaaa aaaaaaaaaa 11820 aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 11917

<210> 26

<211> 8896

<212> RNA

<213> Artificial Sequence

<220>

<223> RBS004.3

<400> 26 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg 780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa 1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga 1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu 4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa 5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc 5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu 6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc 7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga 7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugguga 7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug 7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc 7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa 7800 augauuuaug uuuuacaaau guguaugcug auucuuuugu gaucagaggu gaugaaguga 7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug 7920 auuuuacagg augugugauu gcuuggaauu cuaauaauuu agauucuaaa gugggaggaa 7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaaagagaua 8040 uuucaacaga aauuuaucag gcuggaucaa caccuuguaa uggaguggaa ggauuuaauu 8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau 8160 auagaguggu ggugcugucu uuugaacugc ugcaugcacc ugcaacagug uguggaccua 8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agagaugggc 8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggccgguccc 8340 uggaggugcu guuccagggc cccggcugau gacucgagcu gguacugcau gcacgcaaug 8400 cuagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua 8460 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca 8520 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau 8580 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu 8640 ucgugccagc cacaccgcgg ccgcaugaau acagcagcaa uuggcaagcu gcuuacauag 8700 aacucgcggc gauuggcaug ccgccuuaaa auuuuuauuu uauuuuuucu uuucuuuucc 8760 gaaucggauu uuguuuuuaa uauuucaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcau 8820 augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 8880 aaaaaaaaaa aaaaaa 8896

<210> 27

<211> 9079

<212> RNA

<213> Artificial Sequence

<220>

<223> RBS004.4

<400> 27 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua 660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg 780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac 840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa 1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg 1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc 1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag 1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua 1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug 1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug 1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga 1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua 2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga 2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc 2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug 3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua 3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau 3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau 3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc 3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac 4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga 4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu 4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc 4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg 4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa 5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc 5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg 5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu 5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc 6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu 6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac 6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa 6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg 6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua 6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg 6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac 6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug 6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca 7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc 7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau 7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug 7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc 7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga 7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca 7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugguga 7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug 7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc 7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa 7800 augauuuaug uuuuacaaau guguaugcug auucuuuugu gaucagaggu gaugaaguga 7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug 7920 auuuuacagg augugugauu gcuuggaauu cuaauaauuu agauucuaaa gugggaggaa 7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaaagagaua 8040 uuucaacaga aauuuaucag gcuggaucaa caccuuguaa uggaguggaa ggauuuaauu 8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau 8160 auagaguggu ggugcugucu uuugaacugc ugcaugcacc ugcaacagug uguggaccua 8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agagaugggc 8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggaagcggca 8340 gcggaucuga acaguacauu aaauggccuu gguacauuug gcuuggauuu auugcaggau 8400 uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu uguucuuguu 8460 uaaaaggaug uuguucuugu ggaagcuguu guaaauuuga ugaagaugau ucugaaccug 8520 uguuaaaagg agugaaauug cauuacacau gaugacucga gcugguacug caugcacgca 8580 augcuagcug ccccuuuccc guccugggua ccccgagucu cccccgaccu cgggucccag 8640 guaugcuccc accuccaccu gccccacuca ccaccucugc uaguuccaga caccucccaa 8700 gcacgcagca augcagcuca aaacgcuuag ccuagccaca cccccacggg aaacagcagu 8760 gauuaaccuu uagcaauaaa cgaaaguuua acuaagcuau acuaacccca ggguugguca 8820 auuucgugcc agccacaccg cggccgcaug aauacagcag caauuggcaa gcugcuuaca 8880 uagaacucgc ggcgauuggc augccgccuu aaaauuuuua uuuuauuuuu ucuuuucuuu 8940 uccgaaucgg auuuuguuuu uaauauuuca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 9000 cauaugacua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 9060 aaaaaaaaaa aaaaaaaaa 9079

<210> 28 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Vaccine Antigen <400> 28

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai 1 5 10 15

Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Gly Glu Vai Phe

20 25 30

Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg Lys Arg lie 35 40 45

Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser Ala Ser Phe

50 55 60

Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu Asn Asp Leu 65 70 75 80

Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg Gly Asp Glu

85 90 95

Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala Asp Tyr Asn

100 105 110

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala Trp Asn Ser

115 120 125

Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

130 135 140

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp lie Ser Thr 145 150 155 160

Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai Glu Gly Phe

165 170 175

Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly

180 185 190 Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe Glu Leu Leu

195 200 205

His Ala Pro Ala Thr Vai Cys Gly Pro Lys Gly Ser Pro Gly Ser Gly

210 215 220

Ser Gly Ser Gly Tyr lie Pro Glu Ala Pro Arg Asp Gly Gin Ala Tyr

225 230 235 240

Vai Arg Lys Asp Gly Glu Trp Vai Leu Leu Ser Thr Phe Leu Gly Ser

245 250 255

Gly Ser Gly Ser Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu

260 265 270

Gly Phe lie Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met Leu

275 280 285

Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys

290 295 300

Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Vai Leu Lys 305 310 315 320

Gly Vai Lys Leu His Tyr Thr

325

<210> 29

<211> 311

<212> PRT

<213> Artificial Sequence

<220>

<223> BNT162b3c

<400> 29

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15 Asn Leu Thr Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Gly

20 25 30

Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg

35 40 45

Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser

50 55 60

Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu 65 70 75 80

Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg

85 90 95

Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala

100 105 110

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala

115 120 125

Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr

130 135 140

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

145 150 155 160 lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai

165 170 175

Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro

180 185 190

Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe

195 200 205

Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys Gly Ser Pro

210 215 220 Gly Ser Gly Ser Gly Ser Gly Tyr lie Pro Glu Ala Pro Arg Asp Gly 225 230 235 240

Gin Ala Tyr Vai Arg Lys Asp Gly Glu Trp Vai Leu Leu Ser Thr Phe

245 250 255

Leu Gly Ser Gly Ser Gly Ser Glu Gin Tyr lie Lys Trp Pro Trp Tyr

260 265 270 lie Trp Leu Gly Phe lie Ala Gly Leu lie Ala lie Vai Met Vai Thr

275 280 285 lie Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys

290 295 300

Cys Ser Cys Gly Ser Cys Cys

305 310

<210> 30

<211> 1397

<212> RNA

<213> Artificial Sequence

<220>

<223> BNT162b3c

<400> 30 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug 60 uguuucuugu gcugcugccu cuugugucuu cucagugugu gaauuugaca gugagauuuc 120 caaauauuac aaaucugugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug 180 uguaugcaug gaauagaaaa agaauuucua auuguguggc ugauuauucu gugcuguaua 240 auagugcuuc uuuuuccaca uuuaaauguu auggaguguc uccaacaaaa uuaaaugauu 300 uauguuuuac aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga 360 uugcccccgg acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua 420 caggaugugu gauugcuugg aauucuaaua auuuagauuc uaaaguggga ggaaauuaca 480 auuaucugua cagacuguuu agaaaaucaa aucugaaacc uuuugaaaga gauauuucaa 540 cagaaauuua ucaggcugga ucaacaccuu guaauggagu ggaaggauuu aauuguuauu 600 uuccauuaca gagcuaugga uuucagccaa ccaauggugu gggauaucag ccauauagag 660 ugguggugcu gucuuuugaa cugcugcaug caccugcaac agugugugga ccuaaaggcu 720 cccccggcuc cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu 780 acguucguaa agauggcgaa uggguauuac uuucuaccuu uuuaggaagc ggcagcggau 840 cugaacagua cauuaaaugg ccuugguaca uuuggcuugg auuuauugca ggauuaauug 900 caauugugau ggugacaauu auguuauguu guaugacauc auguuguucu uguuuaaaag 960 gauguuguuc uuguggaagc uguuguugau gacucgagcu gguacugcau gcacgcaaug 1020 cuagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua 1080 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca 1140 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau 1200 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu 1260 ucgugccagc cacacccugg agcuagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca 1320 uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaa 1397

<210> 31

<211> 314

<212> PRT

<213> Artificial Sequence

<220>

<223> BNT162b3d

<400> 31

Met Asp Trp lie Trp Arg lie Leu Phe Leu Vai Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser Gin Met Gin Vai Arg Phe Pro Asn lie Thr Asn Leu Cys

20 25 30 Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

35 40 45

Trp Asn Arg Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

50 55 60

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro 65 70 75 80

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe

85 90 95

Vai lie Arg Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly

100 105 110

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

115 120 125

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn

130 135 140

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

145 150 155 160

Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys

165 170 175

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

180 185 190

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

195 200 205

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys

210 215 220 Gly Ser Pro Gly Ser Gly Ser Gly Ser Gly Tyr lie Pro Glu Ala Pro

225 230 235 240

Arg Asp Gly Gin Ala Tyr Vai Arg Lys Asp Gly Glu Trp Vai Leu Leu

245 250 255

Ser Thr Phe Leu Gly Ser Gly Ser Gly Ser Glu Gin Tyr lie Lys Trp

260 265 270

Pro Trp Tyr lie Trp Leu Gly Phe lie Ala Gly Leu lie Ala lie Vai

275 280 285

Met Vai Thr lie Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu

290 295 300

Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys

305 310

<210> 32

<211> 1406

<212> RNA

<213> Artificial Sequence

<220>

<223> BNT162b3d

<400> 32 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauggauu 60 ggauuuggag aauccuguuc cucgugggag ccgcuacagg agcccacucc cagaugcagg 120 ugagauuucc aaauauuaca aaucuguguc cauuuggaga aguguuuaau gcaacaagau 180 uugcaucugu guaugcaugg aauagaaaaa gaauuucuaa uuguguggcu gauuauucug 240 ugcuguauaa uagugcuucu uuuuccacau uuaaauguua uggagugucu ccaacaaaau 300 uaaaugauuu auguuuuaca aauguguaug cugauucuuu ugugaucaga ggugaugaag 360 ugagacagau ugcccccgga cagacaggaa aaauugcuga uuacaauuac aaacugccug 420 augauuuuac aggaugugug auugcuugga auucuaauaa uuuagauucu aaagugggag 480 gaaauuacaa uuaucuguac agacuguuua gaaaaucaaa ucugaaaccu uuugaaagag 540 auauuucaac agaaauuuau caggcuggau caacaccuug uaauggagug gaaggauuua 600 auuguuauuu uccauuacag agcuauggau uucagccaac caauggugug ggauaucagc 660 cauauagagu gguggugcug ucuuuugaac ugcugcaugc accugcaaca guguguggac 720 cuaaaggcuc ccccggcucc ggcuccggau cugguuauau uccugaagcu ccaagagaug 780 ggcaagcuua cguucguaaa gauggcgaau ggguauuacu uucuaccuuu uuaggaagcg 840 gcagcggauc ugaacaguac auuaaauggc cuugguacau uuggcuugga uuuauugcag 900 gauuaauugc aauugugaug gugacaauua uguuauguug uaugacauca uguuguucuu 960 guuuaaaagg auguuguucu uguggaagcu guuguugaug acucgagcug guacugcaug 1020 cacgcaaugc uagcugcccc uuucccgucc uggguacccc gagucucccc cgaccucggg 1080 ucccagguau gcucccaccu ccaccugccc cacucaccac cucugcuagu uccagacacc 1140 ucccaagcac gcagcaaugc agcucaaaac gcuuagccua gccacacccc cacgggaaac 1200 agcagugauu aaccuuuagc aauaaacgaa aguuuaacua agcuauacua accccagggu 1260 uggucaauuu cgugccagcc acacccugga gcuagcaaaa aaaaaaaaaa aaaaaaaaaa 1320 aaaaaagcau augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1380 aaaaaaaaaa aaaaaaaaaa aaaaaa 1406

<210> 33

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 33

Gly Ser Pro Gly Ser Gly Ser Gly Ser

1 5

<210> 34

<211> 6

<212> PRT

<213> Artificial Sequence <220>

<223> Linker

<400> 34

Gly Ser Gly Ser Gly Ser

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 35

Tyr Leu Gin Pro Arg Thr Phe Leu Leu

1 5

<210> 36

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 36

Arg Leu Gin Ser Leu Gin Thr Tyr Vai

1 5

<210> 37

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 37

Gin Tyr lie Lys Trp Pro Trp Tyr lie

1 5 <210> 38

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 38

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe

1 5

<210> 39

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 39

Lys Trp Pro Trp Tyr lie Trp Leu Gly Phe

1 5 10

<210> 40

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 40

Gin Pro Thr Glu Ser lie Vai Arg Phe

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial Sequence

<220> <223> Epitope

<400> 41 lie Pro Phe Ala Met Gin Met Ala Tyr

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 42

Leu Pro Phe Asn Asp Gly Vai Tyr Phe

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 43

Gly Vai Tyr Phe Ala Ser Thr Glu Lys

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400 > 44

Cys Vai Ala Asp Tyr Ser Vai Leu Tyr

1 5 <210> 45

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 45

Lys Cys Tyr Gly Vai Ser Pro Thr Lys

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 46

Phe Gin Pro Thr Asn Gly Vai Gly Tyr

1 5

<210> 47

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope

<400> 47

Gly Thr His Trp Phe Vai Thr Gin Arg

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Epitope <400> 48

Vai Tyr Asp Pro Leu Gin Pro Glu Leu 1 5

<210> 49

<211> 1270

<212> PRT

<213> Artificial Sequence

<220>

<223> Vaccine sequence

<400> 49

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu

35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Vai lie Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro 65 70 75 80

Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser lie Glu Lys Ser

85 90 95

Asn lie lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser Lys Thr

100 105 110

Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie Lys Vai

115 120 125

Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn

130 135 140 Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr Ser Ser Ala Asn Asn 145 150 155 160

Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu Met Asp Leu Glu Gly

165 170 175

Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe Vai Phe Lys Asn lie

180 185 190

Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr Pro lie lie Vai Arg

195 200 205

Glu Pro Glu Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu Pro Leu Vai

210 215 220

Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr Leu Leu Ala

225 230 235 240

Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr

245 250 255

Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro Arg Thr Phe

260 265 270

Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala Vai Asp Cys

275 280 285

Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr

290 295 300

Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai Gin Pro Thr 305 310 315 320

Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Asp

325 330 335 Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg

340 345 350

Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Leu

355 360 365

Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu

370 375 380

Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg 385 390 395 400

Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Asn lie Ala

405 410 415

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala

420 425 430

Trp Asn Ser Asn Lys Leu Asp Ser Lys Vai Ser Gly Asn Tyr Asn Tyr

435 440 445

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

450 455 460 lie Ser Thr Glu lie Tyr Gin Ala Gly Asn Lys Pro Cys Asn Gly Vai

465 470 475 480

Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro

485 490 495

Thr Tyr Gly Vai Gly His Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe

500 505 510

Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys Lys Ser Thr

515 520 525

Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn Gly Leu Lys

530 535 540 Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gin

545 550 555 560

Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai Arg Asp Pro

565 570 575

Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe Gly Gly Vai

580 585 590

Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai Ala Vai Leu

595 600 605

Tyr Gin Gly Vai Asn Cys Thr Glu Vai Pro Vai Ala lie His Ala Asp

610 615 620

Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser Asn Vai Phe

625 630 635 640

Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu Tyr Vai Asn Asn Ser

645 650 655

Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala Ser Tyr Gin

660 665 670

Thr Gin Thr Lys Ser His Arg Arg Ala Arg Ser Vai Ala Ser Gin Ser

675 680 685 lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Vai Ala Tyr

690 695 700

Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie Ser Vai Thr

705 710 715 720

Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai Asp Cys Thr

725 730 735 Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gin

740 745 750

Tyr Gly Ser Phe Cys Thr Gin Leu Lys Arg Ala Leu Thr Gly lie Ala

755 760 765

Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin Vai Lys Gin

770 775 780 lie Tyr Lys Thr Pro Pro lie Lys Tyr Phe Gly Gly Phe Asn Phe Ser

785 790 795 800

Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe lie Glu

805 810 815

Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly Phe lie Lys

820 825 830

Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp Leu lie Cys

835 840 845

Ala Gin Lys Phe Lys Gly Leu Thr Vai Leu Pro Pro Leu Leu Thr Asp

850 855 860

Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly Thr lie Thr

865 870 875 880

Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie Pro Phe Ala

885 890 895

Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr Gin Asn Vai

900 905 910

Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn Ser Ala lie

915 920 925

Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys

930 935 940 Leu Gin Asp Vai Vai Asn His Asn Ala Gin Ala Leu Asn Thr Leu Vai 945 950 955 960

Lys Gin Leu Ser Ser Lys Phe Gly Ala lie Ser Ser Vai Leu Asn Asp

965 970 975 lie Phe Ser Arg Leu Asp Pro Pro Glu Ala Glu Vai Gin lie Asp Arg

980 985 990

Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai Thr Gin Gin

995 1000 1005

Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn Leu Ala Ala

1010 1015 1020

Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys Arg Vai Asp 1025 1030 1035

Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gin Ser Ala

1040 1045 1050

Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai Pro Ala Gin 1055 1060 1065

Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His Asp Gly Lys 1070 1075 1080

Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn Gly Thr His 1085 1090 1095

Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin lie lie Thr

1100 1105 1110

Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai Vai lie Gly

1115 1120 1125 lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro Glu Leu Asp 1130 1135 1140

Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser

1145 1150 1155

Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn Ala Ser Vai

1160 1165 1170

Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu Vai Ala Lys

1175 1180 1185

Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu Gly Lys Tyr

1190 1195 1200

Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu Gly Phe lie

1205 1210 1215

Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met Leu Cys Cys 1220 1225 1230

Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly 1235 1240 1245

Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Vai Leu Lys 1250 1255 1260

Gly Vai Lys Leu His Tyr Thr

1265 1270

<210> 50

<211> 3816

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding sequence

<400> 50 auguucgugu uccuggugcu gcugccucug guguccagcc agugugugaa ccugaccacc 60 agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgac 120 aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180 aacgugaccu gguuccacgu gaucuccggc accaauggca ccaagagauu cgacaacccc 240 gugcugcccu ucaacgacgg gguguacuuu gccagcaucg agaaguccaa caucaucaga 300 ggcuggaucu ucggcaccac acuggacagc aagacccaga gccugcugau cgugaacaac 360 gccaccaacg uggucaucaa agugugcgag uuccaguucu gcaacgaccc cuuccuggac 420 cacaagaaca acaagagcug gauggaaagc gaguuccggg uguacagcag cgccaacaac 480 ugcaccuucg aguacguguc ccagccuuuc cugauggacc uggaaggcaa gcagggcaac 540 uucaagaacc ugcgcgaguu cguguuuaag aacaucgacg gcuacuucaa gaucuacagc 600 aagcacaccc cuaucaucgu gagagagccc gaggaucugc cucagggcuu cucugcucug 660 gaaccccugg uggaucugcc caucggcauc aacaucaccc gguuucagac acugcuggcc 720 cugcacagaa gcuaccugac accuggcgau agcagcagcg gauggacagc uggugccgcc 780 gcuuacuaug ugggcuaccu gcagccuaga accuuccugc ugaaguacaa cgagaacggc 840 accaucaccg acgccgugga uugugcucug gauccucuga gcgagacaaa gugcacccug 900 aaguccuuca ccguggaaaa gggcaucuac cagaccagca acuuccgggu gcagcccacc 960 gaauccaucg ugcgguuccc caauaucacc aaucugugcc ccuucgacga gguguucaau 1020 gccaccagau ucgccucugu guacgccugg aaccggaagc ggaucagcaa uugcguggcc 1080 gacuacuccg ugcuguacaa ccuggccccc uucuucaccu ucaagugcua cggcgugucc 1140 ccuaccaagc ugaacgaccu gugcuucaca aacguguacg ccgacagcuu cgugauccgg 1200 ggagaugaag ugcggcagau ugccccugga cagacaggca acaucgccga cuacaacuac 1260 aagcugcccg acgacuucac cggcugugug auugccugga acagcaacaa gcuggacucc 1320 aaagucagcg gcaacuacaa uuaccuguac cggcuguucc ggaaguccaa ucugaagccc 1380 uucgagcggg acaucuccac cgagaucuau caggccggca acaagccuug uaacggcgug 1440 gccggcuuca acugcuacuu cccacugcgg uccuacagcu uuaggcccac auacggcgug 1500 ggccaccagc ccuacagagu gguggugcug agcuucgaac ugcugcaugc cccugccaca 1560 gugugcggcc cuaagaaaag caccaaucuc gugaagaaca aaugcgugaa cuucaacuuc 1620 aacggccuga agggcaccgg cgugcugaca gagagcaaca agaaguuccu gccauuccag 1680 caguuuggcc gggauaucgc cgauaccaca gacgccguua gagaucccca gacacuggaa 1740 auccuggaca ucaccccuug cagcuucggc ggagugucug ugaucacccc uggcaccaac 1800 accagcaauc agguggcagu gcuguaccag ggcgugaacu guaccgaagu gcccguggcc 1860 auucacgccg aucagcugac accuacaugg cggguguacu ccaccggcag caauguguuu 1920 cagaccagag ccggcugucu gaucggagcc gaguacguga acaauagcua cgagugcgac 1980 auccccaucg gcgcuggaau cugcgccagc uaccagacac agacaaagag ccaccggaga 2040 gccagaagcg uggccagcca gagcaucauu gccuacacaa ugucucuggg cgccgagaac 2100 agcguggccu acuccaacaa cucuaucgcu auccccacca acuucaccau cagcgugacc 2160 acagagaucc ugccuguguc caugaccaag accagcgugg acugcaccau guacaucugc 2220 ggcgauucca ccgagugcuc caaccugcug cugcaguacg gcagcuucug cacccagcug 2280 aaaagagccc ugacagggau cgccguggaa caggacaaga acacccaaga gguguucgcc 2340 caagugaagc agaucuacaa gaccccuccu aucaaguacu ucggcggcuu caauuucagc 2400 cagauucugc ccgauccuag caagcccagc aagcggagcu ucaucgagga ccugcuguuc 2460 aacaaaguga cacuggccga cgccggcuuc aucaagcagu auggcgauug ucugggcgac 2520 auugccgcca gggaucugau uugcgcccag aaguuuaagg gacugacagu gcugccuccu 2580 cugcugaccg augagaugau cgcccaguac acaucugccc ugcuggccgg cacaaucaca 2640 agcggcugga cauuuggagc aggcgccgcu cugcagaucc ccuuugcuau gcagauggcc 2700 uaccgguuca acggcaucgg agugacccag aaugugcugu acgagaacca gaagcugauc 2760 gccaaccagu ucaacagcgc caucggcaag auccaggaca gccugagcag cacagcaagc 2820 gcccugggaa agcugcagga cguggucaac cacaaugccc aggcacugaa cacccugguc 2880 aagcagcugu ccuccaaguu cggcgccauc agcucugugc ugaacgauau cuucagcaga 2940 cuggacccuc cugaggccga ggugcagauc gacagacuga ucacaggcag acugcagagc 3000 cuccagacau acgugaccca gcagcugauc agagccgccg agauuagagc cucugccaau 3060 cuggccgcca ccaagauguc ugagugugug cugggccaga gcaagagagu ggacuuuugc 3120 ggcaagggcu accaccugau gagcuucccu cagucugccc cucacggcgu gguguuucug 3180 cacgugacau augugcccgc ucaagagaag aauuucacca ccgcuccagc caucugccac 3240 gacggcaaag cccacuuucc uagagaaggc guguucgugu ccaacggcac ccauugguuc 3300 gugacacagc ggaacuucua cgagccccag aucaucacca ccgacaacac cuucgugucu 3360 ggcaacugcg acgucgugau cggcauugug aacaauaccg uguacgaccc ucugcagccc 3420 gagcuggaca gcuucaaaga ggaacuggac aaguacuuua agaaccacac aagccccgac 3480 guggaccugg gcgauaucag cggaaucaau gccagcgucg ugaacaucca gaaagagauc 3540 gaccggcuga acgagguggc caagaaucug aacgagagcc ugaucgaccu gcaagaacug 3600 gggaaguacg agcaguacau caaguggccc ugguacaucu ggcugggcuu uaucgccgga 3660 cugauugcca ucgugauggu cacaaucaug cuguguugca ugaccagcug cuguagcugc 3720 cugaagggcu guuguagcug uggcagcugc ugcaaguucg acgaggacga uucugagccc 3780 gugcugaagg gcgugaaacu gcacuacaca ugauga 3816

<210> 51

<211> 4274

<212> RNA

<213> Artificial Sequence

<220>

<223> Vaccine RNA

<400> 51 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguucg 60 uguuccuggu gcugcugccu cuggugucca gccagugugu gaaccugacc accagaacac 120 agcugccucc agccuacacc aacagcuuua ccagaggcgu guacuacccc gacaaggugu 180 ucagauccag cgugcugcac ucuacccagg accuguuccu gccuuucuuc agcaacguga 240 ccugguucca cgugaucucc ggcaccaaug gcaccaagag auucgacaac cccgugcugc 300 ccuucaacga cgggguguac uuugccagca ucgagaaguc caacaucauc agaggcugga 360 ucuucggcac cacacuggac agcaagaccc agagccugcu gaucgugaac aacgccacca 420 acguggucau caaagugugc gaguuccagu ucugcaacga ccccuuccug gaccacaaga 480 acaacaagag cuggauggaa agcgaguucc ggguguacag cagcgccaac aacugcaccu 540 ucgaguacgu gucccagccu uuccugaugg accuggaagg caagcagggc aacuucaaga 600 accugcgcga guucguguuu aagaacaucg acggcuacuu caagaucuac agcaagcaca 660 ccccuaucau cgugagagag cccgaggauc ugccucaggg cuucucugcu cuggaacccc 720 ugguggaucu gcccaucggc aucaacauca cccgguuuca gacacugcug gcccugcaca 780 gaagcuaccu gacaccuggc gauagcagca gcggauggac agcuggugcc gccgcuuacu 840 augugggcua ccugcagccu agaaccuucc ugcugaagua caacgagaac ggcaccauca 900 ccgacgccgu ggauugugcu cuggauccuc ugagcgagac aaagugcacc cugaaguccu 960 ucaccgugga aaagggcauc uaccagacca gcaacuuccg ggugcagccc accgaaucca 1020 ucgugcgguu ccccaauauc accaaucugu gccccuucga cgagguguuc aaugccacca 1080 gauucgccuc uguguacgcc uggaaccgga agcggaucag caauugcgug gccgacuacu 1140 ccgugcugua caaccuggcc cccuucuuca ccuucaagug cuacggcgug uccccuacca 1200 agcugaacga ccugugcuuc acaaacgugu acgccgacag cuucgugauc cggggagaug 1260 aagugcggca gauugccccu ggacagacag gcaacaucgc cgacuacaac uacaagcugc 1320 ccgacgacuu caccggcugu gugauugccu ggaacagcaa caagcuggac uccaaaguca 1380 gcggcaacua caauuaccug uaccggcugu uccggaaguc caaucugaag cccuucgagc 1440 gggacaucuc caccgagauc uaucaggccg gcaacaagcc uuguaacggc guggccggcu 1500 ucaacugcua cuucccacug cgguccuaca gcuuuaggcc cacauacggc gugggccacc 1560 agcccuacag agugguggug cugagcuucg aacugcugca ugccccugcc acagugugcg 1620 gcccuaagaa aagcaccaau cucgugaaga acaaaugcgu gaacuucaac uucaacggcc 1680 ugaagggcac cggcgugcug acagagagca acaagaaguu ccugccauuc cagcaguuug 1740 gccgggauau cgccgauacc acagacgccg uuagagaucc ccagacacug gaaauccugg 1800 acaucacccc uugcagcuuc ggcggagugu cugugaucac cccuggcacc aacaccagca 1860 aucagguggc agugcuguac cagggcguga acuguaccga agugcccgug gccauucacg 1920 ccgaucagcu gacaccuaca uggcgggugu acuccaccgg cagcaaugug uuucagacca 1980 gagccggcug ucugaucgga gccgaguacg ugaacaauag cuacgagugc gacaucccca 2040 ucggcgcugg aaucugcgcc agcuaccaga cacagacaaa gagccaccgg agagccagaa 2100 gcguggccag ccagagcauc auugccuaca caaugucucu gggcgccgag aacagcgugg 2160 ccuacuccaa caacucuauc gcuaucccca ccaacuucac caucagcgug accacagaga 2220 uccugccugu guccaugacc aagaccagcg uggacugcac cauguacauc ugcggcgauu 2280 ccaccgagug cuccaaccug cugcugcagu acggcagcuu cugcacccag cugaaaagag 2340 cccugacagg gaucgccgug gaacaggaca agaacaccca agagguguuc gcccaaguga 2400 agcagaucua caagaccccu ccuaucaagu acuucggcgg cuucaauuuc agccagauuc 2460 ugcccgaucc uagcaagccc agcaagcgga gcuucaucga ggaccugcug uucaacaaag 2520 ugacacuggc cgacgccggc uucaucaagc aguauggcga uugucugggc gacauugccg 2580 ccagggaucu gauuugcgcc cagaaguuua agggacugac agugcugccu ccucugcuga 2640 ccgaugagau gaucgcccag uacacaucug cccugcuggc cggcacaauc acaagcggcu 2700 ggacauuugg agcaggcgcc gcucugcaga uccccuuugc uaugcagaug gccuaccggu 2760 ucaacggcau cggagugacc cagaaugugc uguacgagaa ccagaagcug aucgccaacc 2820 aguucaacag cgccaucggc aagauccagg acagccugag cagcacagca agcgcccugg 2880 gaaagcugca ggacgugguc aaccacaaug cccaggcacu gaacacccug gucaagcagc 2940 uguccuccaa guucggcgcc aucagcucug ugcugaacga uaucuucagc agacuggacc 3000 cuccugaggc cgaggugcag aucgacagac ugaucacagg cagacugcag agccuccaga 3060 cauacgugac ccagcagcug aucagagccg ccgagauuag agccucugcc aaucuggccg 3120 ccaccaagau gucugagugu gugcugggcc agagcaagag aguggacuuu ugcggcaagg 3180 gcuaccaccu gaugagcuuc ccucagucug ccccucacgg cgugguguuu cugcacguga 3240 cauaugugcc cgcucaagag aagaauuuca ccaccgcucc agccaucugc cacgacggca 3300 aagcccacuu uccuagagaa ggcguguucg uguccaacgg cacccauugg uucgugacac 3360 agcggaacuu cuacgagccc cagaucauca ccaccgacaa caccuucgug ucuggcaacu 3420 gcgacgucgu gaucggcauu gugaacaaua ccguguacga cccucugcag cccgagcugg 3480 acagcuucaa agaggaacug gacaaguacu uuaagaacca cacaagcccc gacguggacc 3540 ugggcgauau cagcggaauc aaugccagcg ucgugaacau ccagaaagag aucgaccggc 3600 ugaacgaggu ggccaagaau cugaacgaga gccugaucga ccugcaagaa cuggggaagu 3660 acgagcagua caucaagugg cccugguaca ucuggcuggg cuuuaucgcc ggacugauug 3720 ccaucgugau ggucacaauc augcuguguu gcaugaccag cugcuguagc ugccugaagg 3780 gcuguuguag cuguggcagc ugcugcaagu ucgacgagga cgauucugag cccgugcuga 3840 agggcgugaa acugcacuac acaugaugac ucgagcuggu acugcaugca cgcaaugcua 3900 gcugccccuu ucccguccug gguaccccga gucucccccg accucggguc ccagguaugc 3960 ucccaccucc accugcccca cucaccaccu cugcuaguuc cagacaccuc ccaagcacgc 4020 agcaaugcag cucaaaacgc uuagccuagc cacaccccca cgggaaacag cagugauuaa 4080 ccuuuagcaa uaaacgaaag uuuaacuaag cuauacuaac cccaggguug gucaauuucg 4140 ugccagccac acccuggagc uagcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagcauau 4200 gacuaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaa 4274

<210> 52

<211> 1270

<212> PRT

<213> Artificial Sequence

<220>

<223> Vaccine sequence

<400> 52

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30 Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu

35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Vai lie Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro 65 70 75 80

Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser lie Glu Lys Ser

85 90 95

Asn lie lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser Lys Thr

100 105 110

Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie Lys Vai

115 120 125

Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn

130 135 140

Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr Ser Ser Ala Asn Asn

145 150 155 160

Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu Met Asp Leu Glu Gly

165 170 175

Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe Vai Phe Lys Asn lie

180 185 190

Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr Pro lie lie Vai Arg

195 200 205

Glu Pro Glu Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu Pro Leu Vai

210 215 220

Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr Leu Leu Ala

225 230 235 240 Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr

245 250 255

Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro Arg Thr Phe

260 265 270

Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala Vai Asp Cys

275 280 285

Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr

290 295 300

Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai Gin Pro Thr 305 310 315 320

Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys Pro Phe Asp

325 330 335

Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg

340 345 350

Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Leu

355 360 365

Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu

370 375 380

Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg 385 390 395 400

Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly Lys lie Ala

405 410 415

Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Vai lie Ala

420 425 430 Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr

435 440 445

Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp

450 455 460 lie Ser Thr Glu lie Tyr Gin Ala Gly Asn Lys Pro Cys Asn Gly Vai

465 470 475 480

Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro

485 490 495

Thr Tyr Gly Vai Gly His Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe

500 505 510

Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys Lys Ser Thr

515 520 525

Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn Gly Leu Lys

530 535 540

Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gin 545 550 555 560

Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai Arg Asp Pro

565 570 575

Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe Gly Gly Vai

580 585 590

Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai Ala Vai Leu

595 600 605

Tyr Gin Gly Vai Asn Cys Thr Glu Vai Pro Vai Ala lie His Ala Asp

610 615 620

Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser Asn Vai Phe

625 630 635 640 Gin Thr Arg Ala Gly cys Leu lie Gly Ala Glu Tyr Vai Asn Asn Ser

645 650 655

Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala Ser Tyr Gin

660 665 670

Thr Gin Thr Lys Ser His Arg Arg Ala Arg Ser Vai Ala Ser Gin Ser

675 680 685 lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Vai Ala Tyr

690 695 700

Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie Ser Vai Thr

705 710 715 720

Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai Asp Cys Thr

725 730 735

Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gin

740 745 750

Tyr Gly Ser Phe Cys Thr Gin Leu Lys Arg Ala Leu Thr Gly lie Ala

755 760 765

Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin Vai Lys Gin

770 775 780 lie Tyr Lys Thr Pro Pro lie Lys Tyr Phe Gly Gly Phe Asn Phe Ser

785 790 795 800

Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe lie Glu

805 810 815

Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly Phe lie Lys

820 825 830 Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp Leu lie Cys

835 840 845

Ala Gin Lys Phe Lys Gly Leu Thr Vai Leu Pro Pro Leu Leu Thr Asp

850 855 860

Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly Thr lie Thr

865 870 875 880

Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie Pro Phe Ala

885 890 895

Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr Gin Asn Vai

900 905 910

Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn Ser Ala lie

915 920 925

Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys

930 935 940

Leu Gin Asp Vai Vai Asn His Asn Ala Gin Ala Leu Asn Thr Leu Vai 945 950 955 960

Lys Gin Leu Ser Ser Lys Phe Gly Ala lie Ser Ser Vai Leu Asn Asp

965 970 975 lie Phe Ser Arg Leu Asp Pro Pro Glu Ala Glu Vai Gin lie Asp Arg

980 985 990

Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai Thr Gin Gin

995 1000 1005

Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn Leu Ala Ala

1010 1015 1020

Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys Arg Vai Asp 1025 1030 1035 Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gin Ser Ala

1040 1045 1050

Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai Pro Ala Gin 1055 1060 1065

Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His Asp Gly Lys 1070 1075 1080

Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn Gly Thr His 1085 1090 1095

Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin lie lie Thr

1100 1105 1110

Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai Vai lie Gly

1115 1120 1125 lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro Glu Leu Asp

1130 1135 1140

Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser

1145 1150 1155

Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn Ala Ser Vai

1160 1165 1170

Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu Vai Ala Lys

1175 1180 1185

Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu Gly Lys Tyr

1190 1195 1200

Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu Gly Phe lie

1205 1210 1215 Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met Leu Cys Cys

1220 1225 1230

Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly

1235 1240 1245

Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Vai Leu Lys

1250 1255 1260

Gly Vai Lys Leu His Tyr Thr

1265 1270

<210> 53

<211> 3816

<212> RNA

<213> Artificial Sequence

<220>

<223> Coding sequence

<400> 53 auguucgugu uccuggugcu gcugccucug guguccagcc agugugugaa ccugaccacc 60 agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgac 120 aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180 aacgugaccu gguuccacgu gaucuccggc accaauggca ccaagagauu cgacaacccc 240 gugcugcccu ucaacgacgg gguguacuuu gccagcaucg agaaguccaa caucaucaga 300 ggcuggaucu ucggcaccac acuggacagc aagacccaga gccugcugau cgugaacaac 360 gccaccaacg uggucaucaa agugugcgag uuccaguucu gcaacgaccc cuuccuggac 420 cacaagaaca acaagagcug gauggaaagc gaguuccggg uguacagcag cgccaacaac 480 ugcaccuucg aguacguguc ccagccuuuc cugauggacc uggaaggcaa gcagggcaac 540 uucaagaacc ugcgcgaguu cguguuuaag aacaucgacg gcuacuucaa gaucuacagc 600 aagcacaccc cuaucaucgu gagagagccc gaggaucugc cucagggcuu cucugcucug 660 gaaccccugg uggaucugcc caucggcauc aacaucaccc gguuucagac acugcuggcc 720 cugcacagaa gcuaccugac accuggcgau agcagcagcg gauggacagc uggugccgcc 780 gcuuacuaug ugggcuaccu gcagccuaga accuuccugc ugaaguacaa cgagaacggc 840 accaucaccg acgccgugga uugugcucug gauccucuga gcgagacaaa gugcacccug 900 aaguccuuca ccguggaaaa gggcaucuac cagaccagca acuuccgggu gcagcccacc 960 gaauccaucg ugcgguuccc caauaucacc aaucugugcc ccuucgacga gguguucaau 1020 gccaccagau ucgccucugu guacgccugg aaccggaagc ggaucagcaa uugcguggcc 1080 gacuacuccg ugcuguacaa ccuggccccc uucuucaccu ucaagugcua cggcgugucc 1140 ccuaccaagc ugaacgaccu gugcuucaca aacguguacg ccgacagcuu cgugauccgg 1200 ggagaugaag ugcggcagau ugccccugga cagacaggca agaucgccga cuacaacuac 1260 aagcugcccg acgacuucac cggcugugug auugccugga acagcaacaa ccuggacucc 1320 aaagucggcg gcaacuacaa uuaccuguac cggcuguucc ggaaguccaa ucugaagccc 1380 uucgagcggg acaucuccac cgagaucuau caggccggca acaagccuug uaacggcgug 1440 gccggcuuca acugcuacuu cccacugcgg uccuacagcu uuaggcccac auacggcgug 1500 ggccaccagc ccuacagagu gguggugcug agcuucgaac ugcugcaugc cccugccaca 1560 gugugcggcc cuaagaaaag caccaaucuc gugaagaaca aaugcgugaa cuucaacuuc 1620 aacggccuga agggcaccgg cgugcugaca gagagcaaca agaaguuccu gccauuccag 1680 caguuuggcc gggauaucgc cgauaccaca gacgccguua gagaucccca gacacuggaa 1740 auccuggaca ucaccccuug cagcuucggc ggagugucug ugaucacccc uggcaccaac 1800 accagcaauc agguggcagu gcuguaccag ggcgugaacu guaccgaagu gcccguggcc 1860 auucacgccg aucagcugac accuacaugg cggguguacu ccaccggcag caauguguuu 1920 cagaccagag ccggcugucu gaucggagcc gaguacguga acaauagcua cgagugcgac 1980 auccccaucg gcgcuggaau cugcgccagc uaccagacac agacaaagag ccaccggaga 2040 gccagaagcg uggccagcca gagcaucauu gccuacacaa ugucucuggg cgccgagaac 2100 agcguggccu acuccaacaa cucuaucgcu auccccacca acuucaccau cagcgugacc 2160 acagagaucc ugccuguguc caugaccaag accagcgugg acugcaccau guacaucugc 2220 ggcgauucca ccgagugcuc caaccugcug cugcaguacg gcagcuucug cacccagcug 2280 aaaagagccc ugacagggau cgccguggaa caggacaaga acacccaaga gguguucgcc 2340 caagugaagc agaucuacaa gaccccuccu aucaaguacu ucggcggcuu caauuucagc 2400 cagauucugc ccgauccuag caagcccagc aagcggagcu ucaucgagga ccugcuguuc 2460 aacaaaguga cacuggccga cgccggcuuc aucaagcagu auggcgauug ucugggcgac 2520 auugccgcca gggaucugau uugcgcccag aaguuuaagg gacugacagu gcugccuccu 2580 cugcugaccg augagaugau cgcccaguac acaucugccc ugcuggccgg cacaaucaca 2640 agcggcugga cauuuggagc aggcgccgcu cugcagaucc ccuuugcuau gcagauggcc 2700 uaccgguuca acggcaucgg agugacccag aaugugcugu acgagaacca gaagcugauc 2760 gccaaccagu ucaacagcgc caucggcaag auccaggaca gccugagcag cacagcaagc 2820 gcccugggaa agcugcagga cguggucaac cacaaugccc aggcacugaa cacccugguc 2880 aagcagcugu ccuccaaguu cggcgccauc agcucugugc ugaacgauau cuucagcaga 2940 cuggacccuc cugaggccga ggugcagauc gacagacuga ucacaggcag acugcagagc 3000 cuccagacau acgugaccca gcagcugauc agagccgccg agauuagagc cucugccaau 3060 cuggccgcca ccaagauguc ugagugugug cugggccaga gcaagagagu ggacuuuugc 3120 ggcaagggcu accaccugau gagcuucccu cagucugccc cucacggcgu gguguuucug 3180 cacgugacau augugcccgc ucaagagaag aauuucacca ccgcuccagc caucugccac 3240 gacggcaaag cccacuuucc uagagaaggc guguucgugu ccaacggcac ccauugguuc 3300 gugacacagc ggaacuucua cgagccccag aucaucacca ccgacaacac cuucgugucu 3360 ggcaacugcg acgucgugau cggcauugug aacaauaccg uguacgaccc ucugcagccc 3420 gagcuggaca gcuucaaaga ggaacuggac aaguacuuua agaaccacac aagccccgac 3480 guggaccugg gcgauaucag cggaaucaau gccagcgucg ugaacaucca gaaagagauc 3540 gaccggcuga acgagguggc caagaaucug aacgagagcc ugaucgaccu gcaagaacug 3600 gggaaguacg agcaguacau caaguggccc ugguacaucu ggcugggcuu uaucgccgga 3660 cugauugcca ucgugauggu cacaaucaug cuguguugca ugaccagcug cuguagcugc 3720 cugaagggcu guuguagcug uggcagcugc ugcaaguucg acgaggacga uucugagccc 3780 gugcugaagg gcgugaaacu gcacuacaca ugauga 3816

<210> 54

<211> 4274

<212> RNA

<213> Artificial Sequence

<220>

<223> Vaccine RNA

<400> 54 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguucg 60 uguuccuggu gcugcugccu cuggugucca gccagugugu gaaccugacc accagaacac 120 agcugccucc agccuacacc aacagcuuua ccagaggcgu guacuacccc gacaaggugu 180 ucagauccag cgugcugcac ucuacccagg accuguuccu gccuuucuuc agcaacguga 240 ccugguucca cgugaucucc ggcaccaaug gcaccaagag auucgacaac cccgugcugc 300 ccuucaacga cgggguguac uuugccagca ucgagaaguc caacaucauc agaggcugga 360 ucuucggcac cacacuggac agcaagaccc agagccugcu gaucgugaac aacgccacca 420 acguggucau caaagugugc gaguuccagu ucugcaacga ccccuuccug gaccacaaga 480 acaacaagag cuggauggaa agcgaguucc ggguguacag cagcgccaac aacugcaccu 540 ucgaguacgu gucccagccu uuccugaugg accuggaagg caagcagggc aacuucaaga 600 accugcgcga guucguguuu aagaacaucg acggcuacuu caagaucuac agcaagcaca 660 ccccuaucau cgugagagag cccgaggauc ugccucaggg cuucucugcu cuggaacccc 720 ugguggaucu gcccaucggc aucaacauca cccgguuuca gacacugcug gcccugcaca 780 gaagcuaccu gacaccuggc gauagcagca gcggauggac agcuggugcc gccgcuuacu 840 augugggcua ccugcagccu agaaccuucc ugcugaagua caacgagaac ggcaccauca 900 ccgacgccgu ggauugugcu cuggauccuc ugagcgagac aaagugcacc cugaaguccu 960 ucaccgugga aaagggcauc uaccagacca gcaacuuccg ggugcagccc accgaaucca 1020 ucgugcgguu ccccaauauc accaaucugu gccccuucga cgagguguuc aaugccacca 1080 gauucgccuc uguguacgcc uggaaccgga agcggaucag caauugcgug gccgacuacu 1140 ccgugcugua caaccuggcc cccuucuuca ccuucaagug cuacggcgug uccccuacca 1200 agcugaacga ccugugcuuc acaaacgugu acgccgacag cuucgugauc cggggagaug 1260 aagugcggca gauugccccu ggacagacag gcaagaucgc cgacuacaac uacaagcugc 1320 ccgacgacuu caccggcugu gugauugccu ggaacagcaa caaccuggac uccaaagucg 1380 gcggcaacua caauuaccug uaccggcugu uccggaaguc caaucugaag cccuucgagc 1440 gggacaucuc caccgagauc uaucaggccg gcaacaagcc uuguaacggc guggccggcu 1500 ucaacugcua cuucccacug cgguccuaca gcuuuaggcc cacauacggc gugggccacc 1560 agcccuacag agugguggug cugagcuucg aacugcugca ugccccugcc acagugugcg 1620 gcccuaagaa aagcaccaau cucgugaaga acaaaugcgu gaacuucaac uucaacggcc 1680 ugaagggcac cggcgugcug acagagagca acaagaaguu ccugccauuc cagcaguuug 1740 gccgggauau cgccgauacc acagacgccg uuagagaucc ccagacacug gaaauccugg 1800 acaucacccc uugcagcuuc ggcggagugu cugugaucac cccuggcacc aacaccagca 1860 aucagguggc agugcuguac cagggcguga acuguaccga agugcccgug gccauucacg 1920 ccgaucagcu gacaccuaca uggcgggugu acuccaccgg cagcaaugug uuucagacca 1980 gagccggcug ucugaucgga gccgaguacg ugaacaauag cuacgagugc gacaucccca 2040 ucggcgcugg aaucugcgcc agcuaccaga cacagacaaa gagccaccgg agagccagaa 2100 gcguggccag ccagagcauc auugccuaca caaugucucu gggcgccgag aacagcgugg 2160 ccuacuccaa caacucuauc gcuaucccca ccaacuucac caucagcgug accacagaga 2220 uccugccugu guccaugacc aagaccagcg uggacugcac cauguacauc ugcggcgauu 2280 ccaccgagug cuccaaccug cugcugcagu acggcagcuu cugcacccag cugaaaagag 2340 cccugacagg gaucgccgug gaacaggaca agaacaccca agagguguuc gcccaaguga 2400 agcagaucua caagaccccu ccuaucaagu acuucggcgg cuucaauuuc agccagauuc 2460 ugcccgaucc uagcaagccc agcaagcgga gcuucaucga ggaccugcug uucaacaaag 2520 ugacacuggc cgacgccggc uucaucaagc aguauggcga uugucugggc gacauugccg 2580 ccagggaucu gauuugcgcc cagaaguuua agggacugac agugcugccu ccucugcuga 2640 ccgaugagau gaucgcccag uacacaucug cccugcuggc cggcacaauc acaagcggcu 2700 ggacauuugg agcaggcgcc gcucugcaga uccccuuugc uaugcagaug gccuaccggu 2760 ucaacggcau cggagugacc cagaaugugc uguacgagaa ccagaagcug aucgccaacc 2820 aguucaacag cgccaucggc aagauccagg acagccugag cagcacagca agcgcccugg 2880 gaaagcugca ggacgugguc aaccacaaug cccaggcacu gaacacccug gucaagcagc 2940 uguccuccaa guucggcgcc aucagcucug ugcugaacga uaucuucagc agacuggacc 3000 cuccugaggc cgaggugcag aucgacagac ugaucacagg cagacugcag agccuccaga 3060 cauacgugac ccagcagcug aucagagccg ccgagauuag agccucugcc aaucuggccg 3120 ccaccaagau gucugagugu gugcugggcc agagcaagag aguggacuuu ugcggcaagg 3180 gcuaccaccu gaugagcuuc ccucagucug ccccucacgg cgugguguuu cugcacguga 3240 cauaugugcc cgcucaagag aagaauuuca ccaccgcucc agccaucugc cacgacggca 3300 aagcccacuu uccuagagaa ggcguguucg uguccaacgg cacccauugg uucgugacac 3360 agcggaacuu cuacgagccc cagaucauca ccaccgacaa caccuucgug ucuggcaacu 3420 gcgacgucgu gaucggcauu gugaacaaua ccguguacga cccucugcag cccgagcugg 3480 acagcuucaa agaggaacug gacaaguacu uuaagaacca cacaagcccc gacguggacc 3540 ugggcgauau cagcggaauc aaugccagcg ucgugaacau ccagaaagag aucgaccggc 3600 ugaacgaggu ggccaagaau cugaacgaga gccugaucga ccugcaagaa cuggggaagu 3660 acgagcagua caucaagugg cccugguaca ucuggcuggg cuuuaucgcc ggacugauug 3720 ccaucgugau ggucacaauc augcuguguu gcaugaccag cugcuguagc ugccugaagg 3780 gcuguuguag cuguggcagc ugcugcaagu ucgacgagga cgauucugag cccgugcuga 3840 agggcgugaa acugcacuac acaugaugac ucgagcuggu acugcaugca cgcaaugcua 3900 gcugccccuu ucccguccug gguaccccga gucucccccg accucggguc ccagguaugc 3960 ucccaccucc accugcccca cucaccaccu cugcuaguuc cagacaccuc ccaagcacgc 4020 agcaaugcag cucaaaacgc uuagccuagc cacaccccca cgggaaacag cagugauuaa 4080 ccuuuagcaa uaaacgaaag uuuaacuaag cuauacuaac cccaggguug gucaauuucg 4140 ugccagccac acccuggagc uagcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagcauau 4200 gacuaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4260 aaaaaaaaaa aaaa 4274

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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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 cationica I ly 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;

(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:

(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 SE 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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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:

(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 SE ID NO:69; and

(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;

(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

(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:

(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:

(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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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

(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;

(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:

(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 NQ:70; and

(1) D982P, V984P, F814P, A889P, A896P, and A939P;

(2) K983P, V984P, F814P, A889P, A896P, and A939P;

(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:

(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 NQ:104 or SEQ ID NQ:105; and

(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;

(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:

(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;

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 NQ: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:

(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

(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

(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

(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

(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

(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

(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:

(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:

(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 NQ:70, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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:

(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:

(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:

(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:

(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:

(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

(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:

(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 NQ: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:

(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 NQ:104 or 105, and comprises one of the following sets of amino substitutions relative to SEQ ID NQ: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:

(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 NQ: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:

(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:

(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:

(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:

(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:

(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

(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

(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

(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

(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

(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:

(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:

(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:

(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:

(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:

(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 NQ:70, and comprises the following substitutions relative to SEQ ID NQ:70: D982P, V984P, F814P, A889P, A896P, and A939P; and

(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

(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

(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 NQ:70, and comprises the following substitutions relative to SEQ ID NQ: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 NQ: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

(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 m₂ ⁷'^3'-OGppp(m₁ ^2'-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 cationica I ly 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 NQ:70, or does not comprise a D980P substitution relative to SEQ ID NQ:104 or SEQ ID NQ: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 NQ:70, or does not comprise a D980P substitution relative to SEQ ID NQ:104 or SEQ ID NQ: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.