CN116650633A

CN116650633A - coronavirus vaccine

Info

Publication number: CN116650633A
Application number: CN202211528863.7A
Authority: CN
Inventors: A·穆伊克; A·波兰; U·沙欣; K·A·斯旺森; Q·杨; H·蔡
Original assignee: Biotechnology Europe Inc
Current assignee: Biotechnology Europe Inc
Priority date: 2021-11-29
Filing date: 2022-11-29
Publication date: 2023-08-29

Abstract

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

Description

Coronavirus vaccine

The present disclosure relates to the field of RNA for preventing or treating coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccinating against coronavirus infection and inducing an effective coronavirus antigen-specific immune response such as an antibody and/or T cell response. These methods and agents are particularly useful for preventing or treating coronavirus infections. Administration of an RNA disclosed herein to a subject can protect the subject from coronavirus infection. In particular, in one embodiment, the present disclosure relates to a method comprising administering to a subject an 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 (particularly the S protein of SARS-CoV-2), i.e., a vaccine RNA encoding a vaccine antigen, in a subject. Administration of RNA encoding a vaccine antigen to a subject can provide (after expression of the RNA by appropriate target cells) a vaccine antigen for inducing an immune response against the vaccine antigen (and disease-associated antigen) in the subject.

Coronaviruses are viruses enveloped by a plus-sense single-stranded RNA ((+) ssRNA) that encode a total of four structural proteins, namely spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N). Spike proteins (S proteins) are responsible for receptor recognition, attachment to cells, infection via the endosomal pathway, and genome release driven by fusion of the virus and endosomal membrane. Although the sequences differ between the different family members, the presence of conserved regions and motifs within the S protein makes it possible to divide the S protein into two subdomains: s1 and S2. S2 together with its transmembrane domain is responsible for membrane fusion, whereas the S1 domain recognizes virus-specific receptors and binds to the target host cell. The Receptor Binding Domain (RBD) was identified within several coronavirus isolates and the general structure of the S protein was defined (fig. 1).

The gene sequence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (MN 908947.3) is available to the WHO and the public and is classified into the Beta coronavirus subfamily. Through sequence analysis, the evolutionary tree showed a closer relationship with Severe Acute Respiratory Syndrome (SARS) virus isolates than with another coronavirus, the Middle East Respiratory Syndrome (MERS) virus, that infects humans.

SARS-CoV-2 infection and the resulting disease COVID-19 have been spread worldwide, with more and more countries being affected. On day 3 and 11 of 2020, the WHO identified a covd-19 outbreak as pandemic. By 12 months and 1 day in 2020, globally diagnosed cases of covd-19 are over 6300 tens of thousands, and deaths are over 140 tens of thousands, affecting 191 countries/regions. Ongoing pandemics remain a significant challenge to public health and economic stability worldwide.

Since there is no pre-existing immunity to SARS-CoV-2, every individual is at risk of being infected. After infection, some but not all individuals develop protective immunity in terms of neutralizing antibody responses and cell-mediated immunity. However, it is currently not known to what extent and for how long this protection persists. According to WHO, 80% of infected individuals recover without hospital care, while 15% develop more severe disease and 5% require intensive care. Age and underlying medical conditions are considered risk factors for developing severe disease.

The manifestation of covd-19 is often accompanied by coughing and fever, and chest radiographs show a frosted glass-like or patch-like shadow. However, many patients exhibit no fever or imaging changes, and infections may be asymptomatic, which is relevant to control of transmission. For symptomatic subjects, progression of the disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multiple organ failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgia, nausea/vomiting or diarrhea, headache, weakness and rhinorrhea. In about 3% of individuals with covd-19, loss of smell (loss of sense of smell) or loss of taste (loss of sense of taste) may be the only symptoms represented.

The disease can occur at all ages, but the mortality rate (CFR) is significantly increased in people over 60 years of age. Complications have also been associated with elevated CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. The proportion of medical personnel in the patient with covd-19 is too high due to occupational exposure to the infected patient.

In most cases, molecular testing is used to detect SARS-CoV-2 and confirm infection. 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. The sample to be tested is collected from the nose and/or throat using a swab.

Furthermore, the present disclosure provides a profound understanding of immune responses elicited by exposure (e.g., by vaccination and/or infection) to different SARS-CoV-2 variants or immunogenic polypeptides (e.g., S protein) or immunogenic fragments thereof. For example, in some embodiments, administration of RNA encoding the S protein of ba.2 and/or ba.4/5 Omicron SARS-CoV-2 variants, or immunogenic fragments thereof, can result in an improvement in immune response, including, for example, improved neutralization of Omicron ba.4 and/or Omicron ba.5 SARS-CoV-2 variants and/or more extensive cross-neutralization of Omicron variants of interest (e.g., higher neutralization titers for a greater number of Omicron variants of interest). In some embodiments, the disclosure provides insight that a bivalent novel coronavirus vaccine (e.g., a bivalent ba.4/5 vaccine comprising a first RNA encoding the SARS-CoV-2MN S protein of the MN908947 strain or immunogenic fragment thereof and a second RNA encoding a mutated SARS-CoV-2S protein comprising one or more mutations characteristic of the ba.4/5 omacron variant or immunogenic fragment thereof) can provide a broader cross-neutralization of the SARS-CoV-2MN908947 strain and certain variants thereof (e.g., in some embodiments, variants that are prevalent and/or rapidly spread in the relevant jurisdiction, such as certain omacron variants) in certain subjects as compared to a monovalent coronavirus vaccine (e.g., a vaccine comprising RNA encoding the coronavirus strain or variant SARS-CoV-2S protein thereof). . In some embodiments, this broader cross-neutralization may be observed in non-vaccinated subjects. In some embodiments, this broader cross-neutralization can be observed in subjects not infected with coronavirus (e.g., SARS-CoV-2 infection). In some embodiments, this broader cross-neutralization can be observed in subjects who have been previously vaccinated with a SARS-CoV-2 vaccine (e.g., in some embodiments, an RNA vaccine encoding the SARS-CoV-2S protein, such as in some embodiments, an RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 strain). In some embodiments, this broader cross-neutralization can be observed 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 insight that exposure to at least two of certain SARS-CoV-2 variants or immunogenic polypeptides (e.g., S protein) or immunogenic fragments thereof can result in a synergistic improvement in immune response (e.g., higher neutralization titers, wider cross-neutralization, and/or immune response less susceptible to immune escape) than exposure to other combinations of one strain of SARS-CoV-2 and/or SARS-CoV-2 variants. In some embodiments, the present disclosure provides insight that exposure to SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain (e.g., by vaccination and/or infection) and exposure to SARS-CoV-2S protein or immunogenic fragment thereof from the Omicron ba.1 variant (e.g., by vaccination and/or infection) can result in a synergistic improvement in immune response (e.g., higher neutralization titers, wider cross-neutralization, and/or immune response less susceptible to immune escape) than exposure to other combinations of one strain of SARS-CoV-2 and/or SARS-CoV-2 variants. In some embodiments, the disclosure provides insight that exposure to SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain (e.g., by vaccination and/or infection) and exposure to SARS-CoV-2S protein or immunogenic fragment thereof of Omicron ba.4 or ba.5 variant (e.g., by vaccination and/or infection) can result in a synergistic improvement in immune response (e.g., higher neutralization titer, wider crossover and/or immune response less susceptible to immune escape) than exposure to other combinations of a SARS-CoV-2 strain and/or SARS-CoV-2 variant. In some embodiments, the present disclosure provides insight that (i) exposure to SARS-CoV-2S protein or immunogenic fragment thereof from a strain/variant selected from the group consisting of MN908947 strain, alpha variant, beta variant, delta variant, omicron ba.1 and a subline derived from any of the foregoing strains/variants (e.g., by vaccination and/or infection) in combination with (ii) exposure to SARS-CoV-2S protein or immunogenic fragment thereof from a strain/variant selected from Omicron ba.2, omicron ba.4, omicron ba.5 and a subline derived from any of the foregoing strains/variants (e.g., by vaccination and/or infection) can result in a synergistic improvement in immune response (e.g., higher neutralization titer, wider cross neutralization and/or immune response less susceptible to immune escape) as compared to exposure to other combinations of a SARS-CoV-2 strain and/or SARS-CoV-2 variant.

The present disclosure also provides important insight as to how to generate an immune response in a subject following exposure (e.g., vaccination and/or infection) to a plurality of different SARS-CoV-2 strains. Furthermore, it is disclosed herein that different combinations of SARS-CoV-2 variants are found to elicit different immune responses. In particular, the present disclosure provides insight that exposure to certain combinations of SARS-CoV-2 variants can elicit an improvement in immune response (e.g., higher neutralization titers, wider cross-neutralization, and/or immune response less susceptible to immune escape). In some embodiments, an improved immune response may result when a subject is 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 may result when a subject is delivered a combination of SARS-CoV-2S proteins (e.g., as polypeptides or RNAs encoding such polypeptides) that share no more than 50% (e.g., no more than 40%, no more than 30%, no more than 20%, or more) epitopes (including, e.g., amino acid mutations) that can be bound by neutralizing antibodies. In some embodiments, an improved immune response may be generated by delivering as polypeptides or RNAs encoding such polypeptides (a) the SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain, the Alpha variant, the Beta variant, or the Delta variant of SARS-CoV-2 omacron, and (b) the S protein or immunogenic fragment thereof from the SARS-CoV-2 omacron variant. In some embodiments, an improved immune response may be generated by delivering as polypeptides or RNAs encoding such polypeptides (a) the SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain, the Alpha variant, the Beta variant, or the Delta variant and (b) the S protein or immunogenic fragment thereof of the SARS-CoV-2 Omicron variant that is not a ba.1 Omicron variant. In some embodiments, an improved immune response may be generated by delivering as polypeptides or RNAs encoding such polypeptides (a) an S protein or immunogenic fragment thereof from a MN908947 strain, alpha variant, beta variant, delta SARS-CoV-2 variant or ba.1 Omicron variant and (b) an S protein or immunogenic fragment thereof that is a SARS-CoV-2 Omicron variant that is not a ba.1 Omicron variant. In some embodiments, an improved immune response may be generated by delivering as polypeptides or RNAs encoding such polypeptides (a) the SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain, alpha variant, beta variant, or Delta variant and (b) the S protein or immunogenic fragment thereof of the ba.2 or ba.4 or ba.5SARS-CoV-2 Omicron variant.

In some embodiments, the disclosure also provides 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) can provide certain beneficial effects on the affinity of the antibody to one or more SARS-CoV-2 strains, or variants thereof. In some embodiments, for antibodies directed against certain omacron variants, the beneficial effect of affinity for the antibody is observed. By way of example only, in some embodiments, the beneficial effects of antibody affinity may be observed for antibodies directed against a particular omacron variant (e.g., sharing at least one or more common epitopes with the MN908947 strain).

Also disclosed herein are compositions that can produce improved immune responses (e.g., immune responses that have broader cross-neutralizing activity, stronger neutralization, and/or are less susceptible to immune escape). In some embodiments, the compositions described herein comprise two or more antigens or nucleic acids (e.g., RNAs) encoding such antigens with few shared epitopes. In some embodiments, the compositions described herein deliver as a polypeptide or nucleic acid encoding such polypeptide a combination of SARS-CoV-2S protein or immunogenic fragment thereof that shares no more than 50% (e.g., no more than 40%, no more than 30%, no more than 20% or more) epitopes (including, e.g., amino acid mutations) that can be bound by neutralizing antibodies. In some embodiments, the compositions described herein comprise (a) RNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the MN908947 strain, alpha variant, beta variant, or Delta variant and (b) RNA encoding S protein from an Omicron variant of SARS-CoV-2 (e.g., in some embodiments, S protein from a ba.1, ba.2, or ba.4/5 Omicron variant) or an immunogenic fragment thereof. In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof from a MN908947 strain, alpha variant, beta variant, or Delta variant and (b) an RNA encoding an S protein or immunogenic fragment thereof that is not an Omicron variant of SARS-CoV-2 of a ba.1 Omicron variant. In some embodiments, the compositions described herein comprise (a) an RNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof that is a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 Omicron variant and (b) an RNA encoding an S protein or immunogenic fragment thereof that is not an Omicron variant of ba.1 Omicron variant. In some embodiments, the compositions described herein comprise (a) an RNA encoding the SARS-CoV-2S protein of the MN908947 strain, alpha variant, beta variant or Delta variant of SARS-CoV-2 and (b) an RNA encoding the S protein or immunogenic fragment thereof from the BA.2 or BA.4 or BA.5 Omicron variant of SARS-CoV-2. In some embodiments, the compositions described herein comprise RNA encoding the S protein from the ba.2 omacron variant of SARS-CoV-2 or an immunogenic fragment thereof. In some embodiments, the composition comprises RNA encoding the S protein from the 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 these spike proteins is a surface protein that binds to angiotensin converting enzyme 2 (ACE-2) present on host cells. Thus, spike proteins are considered to be relevant antigens for vaccine development.

BNT162b2 (SEQ ID NO: 20) is an mRNA vaccine for the prevention of COVID-19 and shows an efficacy of 95% or more in the prevention of COVID-19. The vaccine is made from 5' capped mRNA encoding full length SARS-CoV-2 spike glycoprotein (S) encapsulated in Lipid Nanoparticles (LNP). The finished product is in the form of a concentrate of the dispersion for injection, containing BNT162b2 as active substance. The other components are as follows: ALC-0315 (4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), ALC-0159 (2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide), 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 instead of PBS. In some embodiments, the buffer is formulated as a Tris-buffer 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-bitetradecylacetamide), DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine), cholesterol, sucrose, tromethamine (Tris), tromethamine hydrochloride, and water.

In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.1mg/ml. In some embodiments, about 30ug of RNA is administered by administering about 200uL of the RNA formulation. In some embodiments, the RNA in the pharmaceutical RNA formulation is diluted prior to administration (e.g., to a concentration of about 0.05 mg/ml). In some embodiments, the administration volume is between about 200 μl and about 300 μl. In some embodiments, the RNA in the pharmaceutical RNA formulation is formulated in about 10mM Tris buffer and about 10% sucrose.

In some embodiments, the RNA concentration in the pharmaceutical RNA formulation is about 0.1mg/ml, formulated in about 10mM Tris buffer, about 10% sucrose, and at a dose of about 10 μg, or the RNA is administered by diluting the pharmaceutical RNA formulation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA formulation. In some embodiments, the concentration of RNA in the pharmaceutical RNA formulation is about 0.1mg/ml, and the RNA in the pharmaceutical RNA formulation is formulated in about 10mM Tris buffer, about 10% sucrose, and a dose of about 10 μg of RNA is administered by diluting the pharmaceutical RNA formulation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA formulation.

The amino acid sequence of the S protein encoded by BNT162b2 was selected based on the sequence of "SARS-CoV-2 isolate Wuhan-Hu-1": genbank: MN908947.3 (complete genome) and Genbank: QHD43416.1 (spike surface glycoprotein). BNT162b2 active substance consists of single-stranded 5' -capped codon optimized mRNA translated into spike antigen of SARS-CoV-2. BNT162b2 encodes a spike antigen protein sequence containing two proline mutations which stabilizes improved pre-fusion validation (P2S) on the antigen. BNT162b2 does not contain any uridine; instead of uridine, modified N1-methyl pseudouridine was used in RNA synthesis. BNT162b2 mRNA was translated into SARS-CoV-2S protein in the host cell. The S protein is then expressed on the cell surface where it induces an adaptive immune response. The S protein encoded by BNT162b2 was identified as a target for neutralizing antibodies against viruses and was considered a relevant vaccine component. For adult unvaccinated subjects (i.e., subjects 16 years old and older and not previously administered a SARS-CoV-2 vaccine), the FDA approved dosing regimen for BNT162b2 includes two 30 μg doses of diluted vaccine solution administered Intramuscularly (IM) approximately 21 days apart.

The recent advent of novel cyclic variants of SARS-CoV-2 has raised significant concerns over the geographic and temporal efficacy of vaccine intervention. One of the earliest and rapidly leading variants worldwide is D614G.

Alpha variants (also known as B.1.1.7, VOC202012/01, 501Y.V1 or GRY) were initially detected. Alpha variants have a number of mutations, including several mutations in the S gene. It has been shown to be inherently more transmissible, with growth rates estimated 40-70% higher than other SARS-CoV-2 strains in various 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).

First the Beta variant (also called B.1.351 or GH/501Y.V2) was detected. Beta variants carry several mutations in the S gene. Three of these mutations are located at sites in RBD that are associated with immune evasion: N501Y (common to Alpha) and E484K and K417N.

Gamma variants (also known as P.1 or GR/501Y.V3) were first detected. Gamma variants carry several mutations affecting spike protein, including two common to Beta (N501Y and E484K) and a different mutation at position 417 (K417T).

The Delta variants (also known as B.1.617.2 or G/478K.V1) were first recorded. Delta variants have several point mutations affecting spike proteins, including P681R (a mutation site common to Alpha and adjacent to the furin cleavage site) and L452R, the latter in RBD and associated 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 become popular worldwide and become a major variant in the geographic area in which they were first identified.

On month 11 and 24 of 2021, the omacron (b.1.1.529) variant was first reported to WHO by south africa. Since its initial appearance at month 11 of 2021, SARS-CoV-2 Omicron and its sublines have had a significant impact on the epidemiological pattern of the COVID-19 pandemic (WHO Technical Advisory Group on SARS-CoV-2 Virus evaluation (TAG-VE): classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concem (2021); WHO Headquaters (HQ), WHO Health Emergencies Programme, enhancing Response to Omicron SARS-CoV-2 variant:Technical brief and priority actions for Member States (2022)). The significant changes in the spike (S) glycoprotein of the first Omacron variant BA.1 resulted in the loss of many neutralizing antibody epitopes (M.Hoffmann et al, "The Omicron variant is highly resistant against antibody mediated neutralization: implications for control of the COVID-19 pandemic", cell 185, 447-456.e11 (2022)), which enabled BA.1 to partially evade previously established immunity based on the SARS-CoV-2 wild-type strain (Wuhan-Hu-1) (V.Servelia et al, "Neutralizing 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", namre 602, 657-663 (2022)). Thus, breakthrough infections of individuals vaccinated with omacron are more common than previous variants of interest (VOCs). Although omacron ba.1 is replaced by ba.2 variants in many countries around the world, other variants such as ba.1.1 and ba.3 have gained temporary and/or localized momentum of development but have 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.greull et al," SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape pattems,Cell Host Microbe 7, 241 (2022)). Omicron BA.2.12.1 then replaced BA.2, which is predominant in the United states, whereas BA.4 and BA.5 replaced BA.2 in Europe, african parts and sub/Tai (H.Gruell et al, "SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape pattems," Cell Host Microbe, 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, "Next: real-time tracking of pathogen evolution," Bioinformation 34, 4121-4123 (2018)). Currently, omicron BA.5 is globally dominant, including in the United states (Centers for Disease Control and priority. COVID Data Tracker.Atlanta, GA: US Department of Health and Human Services, CDC;2022, 8, 12 th day https:// covid. CDC. Gov/covddata-tracker (2022)).

Omicron has obtained a number of changes (amino acid exchanges, insertions or deletions) in S glycoproteins, some of which are common among all Omicron VOCs, while others are unique to one or more Omicron sublines. Antigenically, BA.2.12.1 shows a high similarity to BA.2 but not to BA.1, whereas BA.4 and BA.5 differ considerably from their prototype BA.2 and even more from B A.1, which is consistent with their germ line (A.Z.Mykyn 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 in BA.1 from the remaining Omicron VOCs include DELTA143-145, L212I or ins214EPE in the N-terminal domain of the S glycoprotein and G446S or G496S in the Receptor Binding Domain (RBD). The amino acid changes T376A, D405N and R408S in RBD are, in turn, common to ba.2 and its progeny but not found in ba.1. In addition, some changes are unique to individual ba.2 progeny VOCs, including L4520 for ba.2.12.1 or L452R and F486V for ba.4 and ba.5 (ba.4 and ba.5 encode the same S sequence). Most of these common and VOC-specific alterations are shown to play an important role in immune escape against monoclonal antibodies and polyclonal serum raised against wild-type S glycoprotein. In particular, the specific changes in BA.4/BA.5 are closely related to immune escape of these VOCs (P.Wang et al, "Antibody resistance of SARS-CoV-2 variants B.1.35 1 and B.1.1.7.Namre 593.130A 135 (2021); Q.Wang et al," Antibody evasion by SARS-CoV-2 Omicron subvariants BA.2.12.1,BA.4, & BA.5.Nature 608, 603-608 (2022)).

Disclosure of Invention

The present disclosure generally relates to immunotherapy of a subject, comprising administering RNA encoding an amino acid sequence, e.g., a vaccine RNA, e.g., a vaccine antigen, i.e., an antigenic peptide or protein, comprising SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein, or an immunogenic variant thereof. Thus, the vaccine antigen comprises an epitope of the SARS-CoV-2S protein for inducing an immune response against the coronavirus S protein, in particular the SARS-CoV-2S protein, in a subject. RNA encoding the vaccine antigen is administered to provide (after expression of the polynucleotide by the appropriate target cell) an antigen for inducing (i.e., stimulating, eliciting and/or amplifying) an immune response, such as an antibody and/or immune effector cell targeting the target antigen (coronavirus S protein, particularly SARS-CoV-2S protein) or a processed product thereof. In one embodiment, the immune response 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 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.

The vaccines described herein comprise as active ingredient single stranded RNA which can be translated into the corresponding protein upon entry into the recipient's cells. In addition to the wild-type or codon-optimized sequence encoding the antigen sequence, the RNA may contain one or more structural elements (e.g., 5' cap, 5' utr, 3' utr, poly (a) tail, and combinations thereof) that are optimized for maximum efficacy of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA contains all of these elements. In one embodiment, cap1 structures may be used as specific capping structures at the 5' -end of the RNA drug substance. In one embodiment, beta-S-ARCA (D1) (m ₂ ^7,2’-O GppSpG) or m ₂ ^7,3'-O Gppp(m ₁ ^2'-O ) ApG can be used as a specific capping structure at the 5' -end of an RNA drug substance. As 5'-UTR sequences, the 5' -UTR sequences of human Alpha-globulin mRNA may be used, optionally with an optimized 'Kozak sequence' to increase translation efficiency (e.g., SEQ ID NO: 12). As 3' -UTR sequences, a combination of two sequence elements (FI elements) derived from a "split amino terminal enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I) (e.g., SEQ ID NO: 13) can be used, which is placed between the coding sequence and the poly (a) tail to ensure a higher maximum protein level and prolonged persistence of the mRNA. These features are confirmed by ex vivo selection methods of sequences that confer RNA stability and increase total protein expression (see WO 2017/060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeated 3' -UTRs of human Beta-globin mRNA. Additionally or alternatively, in some embodiments, the poly (a) -tail can comprise a length of at least 100 adenosine residues (including, for example, at least 110 adenosine residues, at least 120 adenosine residues, 130 adenosine residues or longer). In some embodiments, the poly (a) -tail may comprise a length of about 100 to about 150 adenosine residues. In some embodiments, the poly (a) -tail may comprise an interrupted poly (a) -tail. For example, in some such embodiments, a poly (A) tail of 110 nucleotides in length can be used that consists of a fragment of 30 adenosine residues followed by a 10 nucleotide linker sequence (of random nucleotides) and an additional 70 adenosine residues (e.g., SEQ ID NO: 14). This poly (A) tail sequence was designed to enhance RNA stability and translation efficiency.

Furthermore, in some embodiments, the nucleotide sequence encoding the secretion signal peptide (sec) may be fused to the antigen encoding region of the RNA, preferably in some embodiments in such a way that sec is translated into an N-terminal tag. In one embodiment, sec corresponds to the secretion signal peptide of the SARS-CoV-2S protein (e.g., of the MN908947 strain). In some embodiments, sequences encoding short-chain peptides consisting essentially of the amino acids glycine (G) and serine (S) commonly used in fusion proteins can be used as GS/linkers between sec and antigen.

The vaccine RNAs described herein may be complexed with proteins and/or lipids, preferably lipids, to produce RNA particles for administration. If a combination of different RNAs is used, the RNAs may be complexed together or separately with proteins and/or lipids to produce RNA particles for administration.

In one aspect, the disclosure relates to compositions or pharmaceutical preparations comprising RNA encoding an amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein or an immunogenic variant thereof.

In one embodiment, the immunogenic fragment of SARS-CoV-2S protein is the Receptor Binding Domain (RBD) of the S1 subunit of SARS-CoV-2S protein or the S1 subunit of SARS-CoV-2S protein.

In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof is capable of forming a multimeric complex, in particular a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof may comprise a domain that allows the formation of a multimeric complex, in particular a trimeric complex, comprising the amino acid sequence of the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof. In one embodiment, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain as described herein, e.g., a SARS-CoV-2S protein trimerization domain. In one embodiment, trimerization is achieved by adding a trimerization domain, such as a T4-fibrin (fibritin) derived "foldon" trimerization domain (e.g., SEQ ID NO: 10), particularly if the amino acid sequence of the immunogenic fragment constituting the SARS-CoV-2S protein, immunogenic variant thereof or SARS-CoV-2S protein or immunogenic variant thereof corresponds to the portion of the SARS-CoV-2S protein that does not comprise the SARS-CoV-2S protein trimerization domain.

In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein the codon optimization and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence. One skilled in the art will recognize that codon optimization involves selecting between or among alternative codons encoding the same amino acid residue. Codon optimization typically involves consideration of codons preferred by the particular host in which the sequence is to be expressed. In accordance with the present disclosure, in many embodiments, the preferred host is a human. In some embodiments, the preferred host may be a domestic animal. Alternatively or additionally, in some embodiments, the selection between or among the possible codons encoding the same amino acid may take into account one or more other features, such as total G/C content (as noted above) and/or similarity to a particular reference. For example, in some embodiments of the present disclosure, provided coding sequences encoding SARS-CoV-2S proteins or immunogenic variants thereof differ in amino acid sequence from the amino acid sequence encoded by the BNT162b2 construct described herein, utilizing codons at least one position of such a difference that retain greater similarity to the BNT162b2 construct sequence relative to at least one replacement codon encoding the same amino acid at such a position of difference.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9, nucleotides 979 to 1584, and 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 the nucleotide sequence of nucleotides 979 to 1584, or a nucleotide sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 979 to 1584 or a nucleotide sequence identical to SEQ ID NO: 2. 8 or 9, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1, amino acid sequence of amino acids 327 to 528, which corresponds to SEQ ID NO:1, 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 327 to 528 of SEQ ID NO:1 or amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, and an immunogenic fragment of 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.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:30, and nucleotide sequences of nucleotides 11 to 986, which correspond to SEQ ID NO:30, or 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, or SEQ ID N0:30 or a nucleotide sequence identical to nucleotides 111 to 986 of SEQ ID NO:30, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or amino acid sequence corresponding to amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of a chloro acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2055, and 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 the nucleotide sequence of nucleotides 49 to 2055, or a nucleotide sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2055 or a nucleotide sequence identical to SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 2055; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1, amino acid sequence of amino acids 17 to 685, which corresponds to SEQ ID NO:1, 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 685, or SEQ ID NO:1 or amino acid sequence corresponding to amino acids 17 to 685 of SEQ ID NO:1, and an immunogenic fragment of 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.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819, and 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 the nucleotide sequence of nucleotides 49 to 3819, or SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819 or from SEQ ID N0: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 3819; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1 or 7, amino acid 1 7 to 1273, and 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 the amino acid sequence of amino acids 1 7 to 1273 of SEQ ID NO:1 or 7 or amino acid sequence of amino acids 1 7 to 1273 or amino acid sequence identical to SEQ ID NO:1 or 7, and the amino acid sequence of amino acids 1 7 to 1273 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises a secretion signal peptide.

In one embodiment, the secretion signal peptide is fused to the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof, preferably an N-terminal fusion.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the secretion signal peptide comprises SEQ ID NO: 2. 8 or 9, and the nucleotide sequence of nucleotides 1 to 48 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 the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2. 8 or 9 or a nucleotide sequence identical to nucleotides 1 to 48 of SEQ ID NO: 2. 8 or 9, the nucleotide sequence of nucleotides 1 to 48 having a fragment of the nucleotide sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or

(ii) The secretion signal peptide comprises SEQ ID NO:1, and amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 16 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:1 or amino acid sequence corresponding to amino acids 1 to 16 of SEQ ID NO:1, the amino acid sequence of amino acids 1 to 16 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:6, and SEQ ID NO:6, 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:6 or a nucleotide sequence identical to SEQ ID NO:6, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:5, and 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 or an amino acid sequence identical to SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:30, and nucleotide sequence of nucleotides 54 to 986, which hybridizes with SEQ ID NO:30, or 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, or SEQ ID NO:30 or a nucleotide sequence that hybridizes to nucleotides 54 to 986 of SEQ ID NO:30, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 1 to 311, which corresponds to 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 amino acids 1 to 311 of SEQ ID NO:29 or amino acid sequence corresponding to amino acids 1 to 311 of SEQ ID NO:29, amino acid 1 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment, the RNA is a modified RNA, in particular a stable mRNA. In one embodiment, the RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, the RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U).

In one embodiment, the RNA comprises a modified nucleoside instead of uridine.

In one embodiment, the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

In one embodiment, the RNA comprises a 5' cap.

In one embodiment, the RNA encoding the amino acid sequence that comprises the immunogenic fragment of the SARS-CoV-2S protein, immunogenic variant thereof or SARS-CoV-2S protein or immunogenic variant thereof comprises: comprising SEQ ID NO:12 or a nucleotide sequence identical to SEQ ID NO:12, a 5' utr of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity.

In one embodiment, the RNA encoding the amino acid sequence that comprises the immunogenic fragment of the SARS-CoV-2S protein, immunogenic variant thereof or SARS-CoV-2S protein or immunogenic variant thereof comprises: comprising SEQ ID NO:13 or a nucleotide sequence identical to SEQ ID NO:13, has a 3' utr of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment, the RNA encoding the amino acid sequence that constitutes the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises a poly A sequence.

In one embodiment, the poly a sequence comprises at least 100 nucleotides.

In one embodiment, the poly a sequence comprises SEQ ID NO:14 or a nucleotide sequence consisting of SEQ ID NO: 14.

In one embodiment, the RNA is formulated or to be formulated as a liquid, a solid, or a combination thereof.

In one embodiment, the RNA is formulated or to be formulated for injection.

In one embodiment, the RNA is formulated or to be formulated for intramuscular administration.

In one embodiment, the RNA is formulated or to be formulated as particles.

In one embodiment, the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (1 ipoplex, lpx) particle.

In one embodiment, the LNP comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 2- [ (polyethylene glycol) -2000] -N, N-bistetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

In one embodiment, the RNA cationic lipid complex can be obtained by mixing RNA with a liposome. In one embodiment, the RNA cationic lipid complex particles can be obtained by mixing RNA with a lipid.

In one embodiment, the RNA is formulated or to be formulated as a colloid. In one embodiment, the RNA is formulated or to be formulated as particles forming a colloidal dispersed phase. In one embodiment, 50% or more, 75% or more, or 85% or more of the RNA is present in the dispersed phase. In one embodiment, the RNA is formulated or to be formulated as particles comprising RNA and lipid. In one embodiment, the particles are formed by contacting RNA dissolved in the aqueous phase with lipids dissolved in the organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, the particles are formed by contacting RNA dissolved in an aqueous phase with lipids dispersed in the aqueous phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.

In one embodiment, the RNA is mRNA or saRNA.

In one embodiment, the composition or pharmaceutical formulation is a pharmaceutical composition.

In one embodiment, the composition or pharmaceutical formulation is a vaccine.

In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

In one embodiment, the composition or pharmaceutical formulation is a kit.

In one embodiment, the RNA and optional particle-forming components are in separate vials.

In one embodiment, the kit further comprises instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in the subject.

In one aspect, the present disclosure relates to a composition or pharmaceutical formulation for pharmaceutical use as described herein.

In one embodiment, the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

In one embodiment, the pharmaceutical use includes therapeutic or prophylactic treatment of coronavirus infection.

In one embodiment, the composition or pharmaceutical formulation described herein is for administration to a human.

In one embodiment, the coronavirus is a Beta coronavirus.

In one embodiment, the coronavirus is sand Bei Bingdu (sarbecovirus).

In one embodiment, the coronavirus is SARS-CoV-2.

In one aspect, the disclosure relates to a method of inducing an immune response against a coronavirus in a subject, comprising administering to the subject a composition comprising RNA encoding an amino acid sequence that constitutes an immunogenic fragment of a SARS-CoV-2S protein, an immunogenic variant thereof, or a SARS-CoV-2S protein or an immunogenic variant thereof.

In one embodiment, the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of SARS-CoV-2S protein.

In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof is capable of forming a multimeric complex, in particular a trimeric complex. To this end, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof may comprise a domain that allows the formation of a multimeric complex, in particular a trimeric complex of amino acid sequences comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof. In one embodiment, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain as described herein.

In one embodiment, the amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein the codon optimization and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

In one embodiment of the present invention, in one embodiment,

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1, and 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 the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, and an immunogenic fragment of 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.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:30, and nucleotide sequence of nucleotides 111 to 986, which corresponds to SEQ ID NO:30, or 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, or the nucleotide sequence of SEQ ID NO:30 or a nucleotide sequence identical to nucleotides 111 to 986 of SEQ ID NO:30, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or amino acid sequence corresponding to amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment of the present invention, in one embodiment,

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819, and 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 the nucleotide sequence of nucleotides 49 to 3819, or SEQ ID NO: 2. 8 or 9 or a nucleotide sequence that is identical to nucleotide 49 to 3819 of SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 3819; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1 or 7, amino acid sequence of amino acids 17 to 1273, which corresponds to 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 the amino acid sequence of amino acids 17 to 1273, or SEQ ID NO:1 or 7 or amino acid sequence of amino acids 17 to 1273 or amino acid sequence identical to SEQ ID NO:1 or 7, and an immunogenic fragment of 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.

In one embodiment of the present invention, in one embodiment,

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:5, and SEQ ID NO:5, or a chloro 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 amino acid sequence identical to SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence.

In one embodiment of the present invention, in one embodiment,

In one embodiment, the RNA is a modified RNA, particularly a stable mRNA. In one embodiment, the RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, the RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U).

In one embodiment, the RNA comprises a modified nucleoside instead of uridine.

In one embodiment, the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U).

In one embodiment, the RNA comprises a cap.

In one embodiment, the RNA encoding the amino acid sequence that comprises the immunogenic fragment of the SARS-CoV-2S protein, immunogenic variant thereof or SARS-CoV-2S protein or immunogenic variant thereof comprises: comprising SEQ ID NO:13 or a nucleotide sequence which hybridizes with SEQ ID NO:13, has a 3' utr of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In one embodiment, the poly a sequence comprises at least 100 nucleotides.

In one embodiment, the RNA is formulated as a liquid, a solid, or a combination thereof.

In one embodiment, the RNA is administered by injection.

In one embodiment, the RNA is administered by intramuscular administration.

In one embodiment, the RNA is formulated as particles.

In one embodiment, the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

In one embodiment, the RNA cationic lipid complex particles can be obtained by mixing RNA with liposomes. In one embodiment, the RNA cationic lipid complex particles can be obtained by mixing RNA with a lipid.

In one embodiment, the RNA is formulated as a colloid. In one embodiment, the RNA is formulated as particles forming a colloidal dispersed phase. In one embodiment, 50% or more, 75% or more, or 85% or more of the RNA is present in the dispersed phase. In one embodiment, the RNA is formulated as particles comprising RNA and lipid. In one embodiment, the particles are formed by contacting RNA dissolved in the aqueous phase with lipids dissolved in the organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, the particles are formed by contacting RNA dissolved in an aqueous phase with lipids dispersed in the aqueous phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.

In one embodiment, the RNA is mRNA or saRNA.

In one embodiment, the methods disclosed herein are methods of vaccinating against coronaviruses.

In one embodiment, the methods disclosed herein are methods for the therapeutic or prophylactic treatment of coronavirus infections.

In one embodiment, the subject is a human.

In one embodiment, the coronavirus is a Beta coronavirus.

In one embodiment, the coronavirus is sand Bei Bingdu.

In one embodiment, the coronavirus is SARS-CoV-2.

In one embodiment of the methods described herein, the compositions described herein are administered to a subject.

In one aspect, the present disclosure relates to a composition or pharmaceutical formulation described herein for use in a method described herein.

Furthermore, the present disclosure teaches compositions comprising lipid nanoparticle encapsulated RNAs encoding at least a portion (e.g., being or comprising an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein) that can reach an antibody titer detectable against the epitope in serum within 7 days after administration to a population of adult subjects according to a regimen comprising administration of at least one dose of the composition. Furthermore, the present disclosure teaches the persistence of such antibody titers. In some embodiments, when modified mRNA is used, the antibody titer is increased compared to the titer achieved with the corresponding unmodified mRNA.

In some embodiments, the provided regimens include at least one dose. In some embodiments, the provided regimen comprises 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 than the at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, the provided regimen comprises two doses. In some embodiments, the provided regimen consists of two doses.

In certain embodiments, the immunogenic composition is formulated as a single dose in a container (e.g., vial). In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation comprises at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose volume in the vial is equal. In some embodiments, the first dose is different from the subsequent dose volumes.

A "stable" multi-dose formulation does not exhibit unacceptable levels of microbial growth and has substantially no or no decomposition or degradation of the active biomolecule components. As used herein, a "stable" immunogenic composition includes a formulation that is still capable of eliciting a desired immunological response when administered to a subject.

In some embodiments, the multi-dose formulation remains stable for a specified period of time with multiple or repeated inoculations/insertions into the multi-dose container. For example, in some embodiments, a multi-dose formulation may be stable for at least three days with up to ten uses when contained within a multi-dose container. In some embodiments, the multi-dose formulation remains stable with 2-20 inoculations/insertions.

In some embodiments, for example, according to the protocols described herein, administration of a composition comprising lipid nanoparticle encapsulated RNA (e.g., in some embodiments mRNA) encoding at least a portion (e.g., being or comprising an epitope) of a polypeptide encoded by SARS-CoV-2 (e.g., S protein encoded by SARS-CoV-2) can result in lymphopenia in some subjects (e.g., in most subjects, in about 50% or less, in about 40% or less, in about 25% or less, in about 20% or less, in about 15% or less, in about 10% or less, in about 5% or less, etc.). Furthermore, the present disclosure teaches that such lymphopenia may resolve over time. For example, in some embodiments, lymphopenia resolves within about 14 days, about 1O days, about 9 days, about 8 days, about 7 days, or less. In some embodiments, the lymphopenia is grade 3, grade 2, or lower.

Thus, in addition, the present disclosure provides compositions comprising lipid nanoparticle encapsulated RNAs (e.g., mrnas in some embodiments) that encode at least a portion (e.g., are or comprise epitopes) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein), the compositions being characterized as exhibiting certain characteristics (e.g., achieving certain effects) as described herein when administered to an associated adult population. In some embodiments, the provided compositions can be prepared, stored, transported, characterized, and/or used under conditions where the temperature does not exceed a particular threshold. Alternatively or additionally, in some embodiments, the provided compositions may be protected from light (e.g., certain wavelengths) during some or all of their preparation, storage, transportation, characterization, and/or use. In some embodiments, one or more characteristics of the provided compositions (e.g., RNA stability, as may be assessed, for example, by one or more of size, presence or modification of a particular moiety, or the like; lipid nanoparticle stability or aggregation, pH, or the like) may or may have been assessed at one or more points during preparation, storage, transport, and/or use prior to administration.

In addition, the present disclosure describes certain provided compositions in which nucleotides within the RNA (e.g., mRNA in some embodiments) are not modified (e.g., are naturally occurring A, U, C, G), and/or provided methods relating to such compositions, characterized (e.g., when administered to a related population, which may be or include an adult population in some embodiments) by an inherent adjuvant effect. In some embodiments, such compositions and/or methods can induce an antibody and/or T cell response. In some embodiments, such compositions and/or methods can induce a higher T cell response than conventional vaccines (e.g., non-RNA vaccines, such as protein vaccines).

Alternatively or additionally, the present disclosure describes compositions provided (e.g., compositions comprising lipid nanoparticle encapsulated RNAs encoding at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein)) wherein nucleotides within the RNAs are modified and/or methods related to such compositions, characterized (e.g., when administered to a related population, the related population may be or comprise an adult population in the absence of an inherent adjuvant effect or in the reduction of an inherent adjuvant effect as compared to an otherwise comparable composition (or method) whose results are unchanged). Alternatively or additionally, in some embodiments, such compositions (or methods) are characterized in that they (e.g., when administered to a related population, which may be or include an adult population in some embodiments) induce an antibody response and/or a cd4+ T cell response. Still further alternatively or additionally, in some embodiments, such compositions (or methods) are characterized in that they (e.g., when administered to a related population, which in some embodiments may be or include an adult population) induce a higher cd4+ T cell response than that observed with alternative vaccine forms (e.g., peptide vaccines). In some embodiments involving modified nucleotides, such modified nucleotides may be present in, for example, a 3'utr sequence, an antigen encoding sequence, and/or a 5' utr sequence. In some embodiments, the modified nucleotide is or includes one or more modified uracil residues and/or one or more modified cytosine residues.

Furthermore, the present disclosure describes compositions (e.g., compositions comprising lipid nanoparticle encapsulated RNAs encoding at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein)) and/or methods that are characterized by (e.g., when administered to a relevant population, the relevant population may be or include in some embodiments a sustained expression of a polypeptide encoded by the relevant population (e.g., SARS-CoV-2 encoded protein [ e.g., S protein ] or a portion thereof, which may be or comprise in some embodiments an epitope thereof)) and/or that include a lipid nanoparticle encapsulated RNA. For example, in some embodiments, such compositions and/or methods are characterized in that they achieve detectable polypeptide expression in a biological sample (e.g., serum) from a human when administered to such human, and in some embodiments, such expression is for a period of at least 36 hours or more, including, for example, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or more.

Those skilled in the art having read the present disclosure will recognize that various RNA constructs (e.g., mRNA constructs in some embodiments) encoding at least a portion (e.g., being or comprising an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein) are described. One of ordinary skill in the art will particularly recognize upon reading this disclosure that it describes various RNA (e.g., mRNA constructs in some embodiments) constructs encoding at least a portion of the SARS-CoV-2S protein (e.g., at least the RBD portion of the SARS-CoV-2S protein). Still further, one of ordinary skill in the art will recognize upon reading this disclosure that it describes certain features and/or advantages of an RNA construct (e.g., an mRNA construct in some embodiments) that encodes at least a portion (e.g., is or comprises an epitope) of a SARS-CoV-2 encoded polypeptide (e.g., SARS-CoV-2 encoded S protein). In some embodiments, the RNA construct (e.g., in some embodiments, the mRNA construct) can encode at least one domain of a polypeptide encoded by SARS-CoV-2 (e.g., one or more domains of a polypeptide encoded by SARS-CoV-2 as described in WO 2021/159740, including, for example, the N-terminal domain (NTD) of a SARS-CoV-2 spike protein, the Receptor Binding Domain (RBD) of a SARS-CoV-2 spike protein, the heptad repeat 1 (HR 1) of a SARS-CoV-2 spike protein, the heptad repeat 2 (HR 1) of a SARS-CoV-2 spike protein, and/or combinations thereof). Furthermore, the present disclosure particularly describes the unexpected and useful features and/or advantages of certain RNA constructs (e.g., mRNA constructs in some embodiments) that encode a SARS-CoV-2 RBD moiety and in some embodiments do not encode a full length SARS-CoV-2S protein. Without wishing to be bound by any particular theory, the present disclosure suggests that the provided RNA constructs encoding less than full length SARS-CoV-2S protein (e.g., mRNA constructs in some embodiments) and particularly RNA constructs encoding at least the RBD portion of such SARS-CoV-2S protein (e.g., mRNA constructs in some embodiments) may be particularly useful and/or effective for use as or in an immunogenic composition (e.g., vaccine) and/or for achieving an immunological effect as described herein (e.g., production of SARS-CoV-2 neutralizing antibodies and/or T cell responses (e.g., cd4+ and/or cd8+ T cell responses)).

In some embodiments, the disclosure provides RNAs (e.g., mrnas) comprising an open reading frame encoding a polypeptide comprising a receptor binding portion of a SARS-CoV-2S protein, the RNAs being suitable for intracellular expression of the polypeptide. In some embodiments, such encoded polypeptide does not comprise an intact S protein. In some embodiments, the encoded polypeptide comprises, for example, a polypeptide as set forth in SEQ ID NO:5, and a Receptor Binding Domain (RBD) shown in seq id no. In some embodiments, the encoded polypeptide comprises a polypeptide according to SEQ ID NO:29 or 31. In some embodiments, such RNA (e.g., mRNA) can be complexed with a cationic polymer(s), protein(s), or peptide(s). In some embodiments, such RNAs may be formulated in lipid nanoparticles (e.g., those described herein). In some embodiments, such RNAs (e.g., mrnas) may be particularly useful and/or effective for use as or in immunogenic compositions (e.g., vaccines) and/or for achieving an immunological effect as described herein (e.g., production of SARS-CoV-2 neutralizing antibodies and/or T cell responses (e.g., cd4+ and/or cd8+ T cell responses)). In some embodiments, such RNA (e.g., mRNA) can be used for vaccination of humans (including, for example, humans known to have been exposed to and/or infected with SARS-CoV-2, and/or humans unknown to have been exposed to SARS-CoV-2).

Those of skill in the art having read the present disclosure will further recognize that various mRNA constructs comprising nucleic acid sequences encoding full-length SARS-CoV-2 spike proteins are described (e.g., including embodiments wherein such encoded SARS-CoV-2 spike proteins may comprise at least one or more amino acid substitutions, e.g., proline substitutions as described herein, and/or embodiments wherein the mRNA sequences are codon optimized, e.g., for a mammal, e.g., a human subject). In some embodiments, such full-length SARS-CoV-2 spike protein can have the amino acid sequence of SEQ ID NO:7 or an amino acid sequence comprising SEQ ID NO: 7. Still further, one of ordinary skill in the art will recognize upon reading this disclosure that, in addition, certain features and/or advantages of certain mRNA constructs comprising a nucleic acid sequence encoding a full-length SARS-CoV-2 spike protein are described. Without wishing to be bound by any particular theory, the present disclosure suggests that the provided mRNA constructs encoding full-length SARS-CoV-2S protein may be particularly useful and/or effective for use as or in immunogenic compositions (e.g., vaccines) in a particular subject population (e.g., a particular age population). For example, in some embodiments, such mRNA compositions may be particularly useful in young (e.g., less than 25 years old, 20 years old, 1 8 years old, 15 years old, 10 years old, or younger) subjects; alternatively or additionally, in some embodiments, such mRNA compositions may be particularly useful in elderly subjects (e.g., over 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, or older). In certain embodiments, an immunogenic composition comprising such an mRNA construct provided herein exhibits a minimal to moderate increase (e.g., no more than a 30% increase, no more than a 20% increase, or no more than a 10% increase, or less) in dose level and/or dose-dependent systemic reactogenicity (e.g., fever, fatigue, headache, cold, diarrhea, muscle pain, and/or joint pain, etc.) and/or local tolerance (e.g., pain, redness, and/or swelling, etc.) at least in some subjects (e.g., in some subject age groups); in some embodiments, such reactivities and/or local tolerability are observed particularly in young groups (e.g., less than 25 years old, 20 years old, 1 8 years old, or younger) of subjects and/or in older (e.g., elderly) age groups (e.g., 65-85 years old). In some embodiments, the provided mRNA constructs encoding full-length SARS-CoV-2S protein may be particularly useful and/or effective for use as or in an immunogenic composition (e.g., a vaccine) that induces SARS-CoV-2 neutralizing antibody response levels in a population of subjects at high risk for serious diseases associated with SARS-CoV-2 infection (e.g., an aged population, e.g., 65-85 year old). In some embodiments, one of ordinary skill in the art having read the present disclosure will recognize that the mRNA constructs encoding full-length SARS-CoV-2S proteins provided herein, in addition, exhibit advantageous reactogenic characteristics in young and elderly populations (e.g., as described herein), which may be particularly useful and/or effective for use as or in an immunogenic composition (e.g., vaccine) that achieves an immunological effect as described herein (e.g., production of SARS-CoV-2 neutralizing antibodies and/or T cell responses (e.g., cd4+ and/or cd8+ T cell responses)). In some embodiments, the present disclosure also demonstrates that the provided mRNA constructs encoding full-length SARS-CoV-2S proteins can be particularly effective in protecting against SARS-CoV-2 infection, as characterized by early clearance of SARS-CoV-2 viral RNA in a non-human mammalian subject (e.g., rhesus monkey) immunized with an immunogenic composition comprising such mRNA constructs and subsequently challenged with a SARS-CoV-2 strain. In some embodiments, such early clearance of SARS-CoV-2 virus mRNA can be observed in the nose of a non-human mammalian subject (e.g., rhesus monkey) immunized with an immunogenic composition comprising such an MRNA construct and subsequently challenged with a SARS-CoV-2 strain.

In some embodiments, the disclosure provides RNAs (e.g., mrnas) comprising an open reading frame encoding a full-length SARS-CoV-2S protein (e.g., a full-length SARS-CoV-2S protein having one or more amino acid substitutions) that are suitable for intracellular expression of a polypeptide. In some embodiments, the encoded polypeptide comprises SEQ ID NO: 7. In some embodiments, such RNA (e.g., mRNA) can be complexed by a cationic polymer(s), protein(s), or peptide(s). In some embodiments, such RNAs may be formulated in lipid nanoparticles (e.g., those described herein).

In some embodiments, an immunogenic composition provided herein can comprise a plurality (e.g., at least two or more, including, for example, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, etc.) immunoreactive epitopes of a SARS-CoV-2 polypeptide or variant thereof. In some such embodiments, such multiple immunoreactive epitopes may be encoded by multiple RNAs (e.g., mrnas). In some such embodiments, such multiple immunoreactive epitopes may be encoded by a single RNA (e.g., mRNA). In some embodiments, nucleic acid sequences encoding multiple immunoreactive epitopes may be separated from each other in a single RNA (e.g., mRNA) by a linker (e.g., in some embodiments, a peptide linker). Without wishing to be bound by any particular theory, in some embodiments, when considering the genetic diversity of SARS-CoV-2 variants, the provided multi-epitope (polyepitope) immunogenic compositions (including, for example, those encoding full-length SARS-CoV-2 spike proteins) may be particularly useful in providing protection against numerous viral variants, and/or may provide better opportunities to develop diversity and/or otherwise robust (e.g., persistence) Long, e.g., detectable about 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days or more after administration of one or more doses), neutralizing antibodies and/or T cell responses, particularly robust T _H Type 1T cells (e.g., cd4+ and/or cd8+ T cells) react.

In some embodiments, the disclosure features provided compositions and/or methods that are characterized (e.g., when administered to a related population, which may be or include an adult population in some embodiments), that, in the case of a single administration, they achieve one or more specific therapeutic results (e.g., effective immune response and/or detectable expression of the encoded SARS-CoV-2S protein or immunogenic fragment thereof as described herein); in some such embodiments, the results can be evaluated, for example, compared to that observed in the absence of an RNA vaccine (e.g., an mRNA vaccine) as described herein. In some embodiments, specific results may be achieved at dosages lower than those required by one or more alternative strategies.

In some embodiments, the disclosure provides an immunogenic composition comprising an isolated messenger ribonucleic acid (mRNA) polynucleotide, wherein the isolated mRNA polynucleotide comprises an open reading frame encoding a polypeptide comprising a receptor binding portion of an SARs-CoV-2S protein, and wherein the isolated mRNA polynucleotide is formulated in at least one lipid nanoparticle. For example, in some embodiments, the molar ratio of such lipid nanoparticles can be 20-60% ionizable cationic lipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol or steroid, and O.5-15% polymer conjugated lipid (e.g., PEG modified lipid). In some embodiments, the sterol or steroid included in the lipid nanoparticle may be or comprise cholesterol. In some embodiments, the neutral lipid may be or comprise 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC). In some embodiments, the polymer conjugated lipid may be or comprise PEG2000 DMG. In some embodiments, such immunogenic compositions may comprise a total lipid content of about 1mg to 10mg or 3mg to 8mg or 4mg to 6 mg. In some embodiments, such immunogenic compositions can comprise a total lipid content of about 5mg/mL-15mg/mL or 7.5mg/mL-12.5mg/mL or 9-11 mg/mL. In some embodiments, such isolated mRNA polynucleotides are provided in an amount effective to induce an immune response in a subject administered at least one dose of the immunogenic composition. In some embodiments, the polypeptide encoded by the provided isolated mRNA polynucleotide does not comprise an intact S protein. In some embodiments, such isolated mRNA polynucleotides provided in the immunogenic composition are not self-replicating RNAs.

In some embodiments, the immune response may include generating a binding antibody titer against a SARS-CoV-2 protein (including, for example, in some embodiments, a stable pre-fusion spike trimer) or fragment thereof).

In some embodiments, the immune response can include generating neutralizing antibody titers against SARS-CoV-2 protein (including, for example, in some embodiments, stable pre-fusion spike trimers) or fragments thereof. In some embodiments, the immune response may include generating a neutralizing antibody titer against the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein. In some embodiments, it has been determined that the provided immunogenic compositions achieve neutralizing antibody titers in an appropriate system (e.g., in a human and/or population thereof infected with SARS-CoV-2 and/or in a model system therefor). For example, in some embodiments, such neutralizing antibody titers may have been demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus monkey), and/or a mouse model.

In some embodiments, the neutralizing antibody titer is a titer (e.g., has been determined to be) sufficient to reduce viral infection of B cells relative to that observed for an appropriate control (e.g., a control subject that is not vaccinated, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In some such embodiments, the reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In some embodiments, the neutralizing antibody titer is a titer (e.g., has been determined to be) sufficient to reduce the rate of asymptomatic viral infection relative to that observed for an appropriate control (e.g., a control subject that is not vaccinated, or a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine, or a combination thereof). In some such embodiments, the decrease is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, this decrease can be characterized by serological assessment of SARS-CoV-2N protein. Observations in real life also confirm significant protection against asymptomatic infections (see also Dagan N. Et al, N Engl J Med.2021, doi:10.1056/NEJMOA2101765.Epub head of print. PMID: 33626250)

In some embodiments, the neutralizing antibody titer is a titer (e.g., has been determined to be) sufficient to reduce or block fusion of virus to epithelial cells and/or B cells of a vaccinated subject relative to that observed for an appropriate control (e.g., a non-vaccinated control subject, or a subject vaccinated with a live attenuated virus vaccine, an inactivated virus vaccine, or a protein subunit virus vaccine, or a combination thereof). In some such embodiments, the reduction is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In some embodiments, induction of neutralizing antibody titers can be characterized by an increased number of B cells, which in some embodiments can include plasma cells, class-switched IgG1 and IgG2 positive B cells, and/or germinal center B cells. In some embodiments, it has been determined that the provided immunogenic compositions achieve such an increase in B cell numbers in a suitable system (e.g., in a human and/or population thereof infected with SARS-CoV-2 and/or in a model system therefor). For example, in some embodiments, such an increase in B cell number may have been demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus monkey), and/or a mouse model. In some embodiments, such an increase in B cell number may have been demonstrated in the draining lymph nodes and/or spleen of a mouse model following immunization of such a mouse model with the provided immunogenic composition (e.g., at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days later).

In some embodiments, induction of neutralizing antibody titers can be characterized by a reduction in the number of circulating B cells in the blood. In some embodiments, it has been determined that the provided immunogenic compositions achieve such a reduction in circulating B cell numbers in the blood of an appropriate system (e.g., in a human and/or population thereof infected with SARS-CoV-2 and/or in a model system therefor). For example, in some embodiments, such a reduction in the number of circulating B cells in the blood may have been demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus monkey), and/or a mouse model. In some embodiments, such a reduction in the number of circulating B cells in the blood may have been demonstrated in a mouse model following immunization of such a mouse model with the provided immunogenic composition (e.g., after at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days). Without wishing to be bound by theory, the reduction of circulating B cells in the blood may be due to homing of B cells to lymphoid compartments.

In some embodiments, by the provided exemption The immune response induced by the epidemic composition may include an increase in the number of T cells. In some embodiments, such an increase in T cell number may include T follicular assist (T _FH ) An increase in cell number, which in some embodiments may comprise one or more subsets of ICOS upregulation. Those skilled in the art will appreciate that T in a germinal center _FH Is necessary for the generation of an adaptive B-cell response and in humans, T occurs in the circulation after vaccination _FH Usually associated with a high frequency of antigen-specific antibodies. In some embodiments, it has been determined that the provided immunogenic composition achieves T-cell (e.g., T-cell in an appropriate system (e.g., in a human and/or population thereof infected with SARS-CoV-2 and/or in a model system therefor) _FH Cells) are used. For example, in some embodiments, T cells (e.g., T _FH Cells) may have been demonstrated in one or more of a human population, a non-human primate model (e.g., rhesus monkey), and/or a mouse model. In some embodiments, T cells (e.g., T _FH Cells) may have been demonstrated in the draining lymph nodes, spleen and/or blood of the mouse model after immunization of such mouse model with the provided immunogenic composition (e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days).

In some embodiments, the protective response against SARS-CoV-2 induced by the provided immunogenic composition has been established in an appropriate model system for SARS-CoV-2. For example, in some embodiments, such protective responses may have been demonstrated in animal models, such as non-human primate models (e.g., rhesus monkeys) and/or mouse models. In some embodiments, a non-human primate (e.g., rhesus monkey) or population thereof that has received at least one immunization with the provided immunogenic composition is challenged with SARS-CoV-2, e.g., by intranasal and/or intratracheal routes. In some embodiments, such challenge may be performed several weeks (e.g., 5-10 weeks) after at least one immunization (including, e.g., at least two immunizations) with the provided immunogenic compositions. In some embodiments, this challenge can be performed when a detectable level of neutralization titer (e.g., an antibody response to SARS-CoV-2 spike protein and/or fragments thereof, including, for example, but not limited to, a stable pre-fusion spike trimer, S-2P, and/or an antibody response to a receptor binding portion of SARS-CoV-2) has been achieved in a non-human primate (e.g., rhesus) that has been subjected to at least one immunization (including, for example, at least two immunizations) with the provided immunogenic composition. In some embodiments, the protective response is characterized by the absence or reduction of detectable viral RNA in bronchoalveolar lavage (BAL) and/or nasal swabs of an challenged non-human primate (e.g., rhesus). In some embodiments, the immunogenic compositions described herein can be characterized by a greater percentage of challenged animals (e.g., non-human primates) in a population (e.g., rhesus) that has been received at least one immunization (including, e.g., at least two immunizations) with the provided immunogenic composition (e.g., rhesus) showing no detectable RNA in their BAL and/or nasal swabs compared to a non-immunized animal population such as a non-human primate (e.g., rhesus). In some embodiments, an immunogenic composition described herein can be characterized in that an challenged animal, e.g., a non-human (e.g., rhesus monkey) in a population, that has been received at least one immunization (including, e.g., at least two immunizations) with the provided immunogenic composition, e.g., a non-human in the population (e.g., rhesus monkey), can exhibit clearance of viral RNA in a nasal swab no later than 10 days, including, e.g., no later than 8 days, no later than 6 days, no later than 4 days, etc.

In some embodiments, the immunogenic compositions described herein do not substantially increase the risk of vaccine-related enhanced respiratory disease when administered to a subject in need thereof. In some embodiments, such vaccine-associated enhanced respiratory disease may be associated with enhanced antibody-dependent replication and/or vaccine antigens that induce antibodies with poor neutralizing activity and Th 2-biased responses. In some embodiments, the immunogenic compositions described herein do not substantially increase the risk of antibody-dependent replication enhancement when administered to a subject in need thereof.

In some embodiments, a single dose of an RNA composition (e.g., mRNA formulated in a lipid nanoparticle) can induce a therapeutic antibody response in less than 10 days of vaccination. In some embodiments, such therapeutic antibody response may be characterized in that such RNA vaccines can induce the production of about 10-100ug/mL IgG when measured 10 days after vaccination at doses of 0.1 to 10ug or 0.2-5ug in animal models. In some embodiments, such therapeutic antibody response may be characterized by the induction of about 100-1000ug/mL IgG by such RNA vaccine measured at 20 days of vaccination at doses of 0.1 to 10ug or 0.2-5ug in animal models. In some embodiments, a single dose may induce a pseudovirus neutralization titer of 10-200pVN titer 15 days after vaccination, as measured in an animal model. In some embodiments, a single dose may induce a pseudovirus neutralization titer of 50-500pVN50 titers 15 days after vaccination, as measured in an animal model.

In some embodiments, a single dose of an RNA composition (e.g., an mRNA composition) can amplify an antigen-specific CD8 and/or CD 4T cell response by at least 50% or more (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) compared to that observed in the absence of such an RNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., a spike protein and/or receptor binding domain). In some embodiments, a single dose of the RNA composition can amplify an antigen-specific CD8 and/or CD 4T cell response by at least 1.5-fold or more (including, e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or more) compared to that observed in the absence of such RNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and/or receptor binding domain).

In some embodiments, the regimen (e.g., a single dose of an mRNA composition) can expand T cells that exhibit a Th1 phenotype (e.g., characterized by expression of IFN-Gamma, IL-2, IL-4, and/or IL-5) by at least 50% or more (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., a spike protein and/or receptor binding domain). In some embodiments, the regimen (e.g., a single dose of an mRNA composition) can expand T cells that exhibit a Th1 phenotype (e.g., characterized by expression of IFN-Gamma, IL-2, IL-4, and/or IL-5) by at least, e.g., at least 1.5-fold or more (including, e.g., at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more) compared to that observed in the absence of such an mRNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and/or receptor binding domain). In some embodiments, the T cell phenotype may be or comprise a Th 1-dominant cytokine profile (e.g., characterized by INF-Gamma positivity and/or IL-2 positivity), and/or no biologically insignificant secretion of IL-4.

In some embodiments, the protocols as described herein (e.g., one or more doses of an mRNA composition) induce and/or achieve the production of RBD-specific cd4+ T cells. Furthermore, the present disclosure describes that mRNA compositions encoding RBD-containing portions of SARS-CoV-2 spike protein (e.g., and not encoding full-length SARS-CoV-2 spike protein) may be particularly useful and/or effective in such induction and/or production of RBD-specific cd4+ T cells. In some embodiments, RBD-specific cd4+ T cells induced by the mRNA compositions described herein (e.g., mRNA compositions encoding SARS-CoV-2 spike protein and in some embodiments not encoding full-length SARS-CoV-2 spike protein) exhibit a Th 1-dominant cytokine profile (e.g., characterized by INF-Gamma positivity and/or IL-2 positivity), and/or by the absence of biologically insignificant IL-4 secretion.

In some embodiments, cd4+ and/or cd8+ T cell responses (e.g., as described herein) in a subject receiving an RNA composition (e.g., as described herein) can be characterized using PBMCs collected from the subject using an ex vivo assay.

In some embodiments, the immunogenicity of an RNA (e.g., mRNA) composition described herein can be assessed by one or more of the following serological immunogenicity assays: detecting the presence of IgG, igM and/or IgA directed against SARS-CoV-2S protein in a blood sample of a subject receiving the provided RNA composition and/or using a neutralization assay of SARS-CoV-2 pseudovirus and/or wild-type SARS-CoV-2 virus.

In some embodiments, the RNA composition (e.g., as described herein) provides relatively low side effects (e.g., grade 1-2 pain, redness, and/or swelling) within 7 days after vaccination at a dose of 10ug-100ug or 1ug-50 ug. In some embodiments, the RNA composition (e.g., as described herein) provides observations of relatively low systemic events (e.g., grade 1-2 fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, medication, and combinations thereof) within 7 days after vaccination at a dose of 10ug-100 ug.

In some embodiments, the RNA (e.g., mRNA) composition is characterized in that IgG against the SARS-CoV2 immunogenic protein or fragment thereof (e.g., spike protein and/or receptor binding domain) can be produced at a level of 100-100,000u/mL or 500-50,000u/mL 21 days after vaccination when administered to a subject at a dose of 10-100ug or 1ug-50 ug.

In some embodiments, the RNA (e.g., mRNA) encodes a naturally folded trimeric receptor binding protein of SARS-CoV-2. In some embodiments, the RNA (e.g., mRNA) encodes a variant of such a receptor binding protein such that the encoded variant binds ACE2 with a Kd of 10pM or less, including, for example, with a Kd of 9pM, 8pM, 7pM, 6pM, 5pM, 4pM or less. In some embodiments, the RNA (e.g., mRNA) encodes a variant of such a receptor binding protein such that the encoded variant binds ACE2 with a Kd of 5 pM. In some embodiments, the RNA (e.g., mRNA) encodes a trimeric receptor binding portion of SARS-CoV-2 that comprises an ACE2 receptor binding site. In some embodiments, the RNA (e.g., mRNA) comprises a coding sequence for a receptor binding portion of SARS-CoV-2 and a trimerization domain (e.g., the native trimerization domain (folder) of T4 fibrin) such that the coding sequence directs the expression of a trimeric protein that has an ACE2 receptor binding site and binds ACE 2. In some embodiments, the RNA (e.g., mRNA) encodes a trimeric receptor binding portion of SARS-CoV-2 or a variant thereof such that its Kd is less than the Kd of the monomeric Receptor Binding Domain (RBD) of SARS-CoV-2. For example, in some embodiments, the RNA (e.g., mRNA) encodes a trimeric receptor binding portion of SARS-CoV-2 or a variant thereof such that its Kd is at least 10-fold (including, e.g., at least 50-fold, at least 100-fold, at least 500-fold, at least 1-000-fold, etc.) less than the Kd of the RBD of SARS-CoV-2.

In some embodiments, when the trimeric receptor binding portion of SARS-CoV-2 encoded by RNA (e.g., as described herein) binds ACE2 and B in a closed conformation ⁰ AT1 neutral amino acid transporter complexing, its size can be determined to be about 3-4 angstroms, as characterized by electron cryomicroscopy (cryem). In some embodiments, the geometric mean SARS-CoV-2 neutralization titers characterized and/or achieved by the RNA compositions or methods as described herein can reach at least 1.5-fold, including at least 2-fold, at least 2.5-fold, at least 3-fold, or more, of a group of covd-19 convalescence personnel (e.g., a group of sera from covd-19 convalescence personnel obtained 20-40 days after onset of symptoms and at least 14 days after onset of an asymptomatic convalescence).

In some embodiments, RNA compositions as provided herein may be characterized in that a subject that has been treated with such compositions (e.g., with at least one dose, at least two doses, etc.) may exhibit reduced and/or shorter presence of viral RNA at a relevant site (e.g., the nose and/or lung, etc., and/or any other tissue susceptible to infection) compared to an appropriate control (e.g., a determined expected level of a comparable subject or population that has not been subjected to such treatment and is exposed to the virus under reasonably comparable exposure conditions).

In some embodiments, RBD antigens expressed by mRNA constructs (e.g., as described herein) can be modified by the addition of T4-fibrin-derived "foldback" trimerization domains, e.g., to increase their immunogenicity.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by certain local reactions (e.g., pain, redness and/or swelling, etc.) and/or systemic events (e.g., fever, fatigue, headache, etc.) that may occur and/or peak on day 2 after vaccination. In some embodiments, the RNA (e.g., mRNA) compositions described herein are characterized by certain local reactions (e.g., pain, redness and/or swelling, etc.) and/or systemic events (e.g., heat, fatigue, headache, etc.) that may subside by day 7 after vaccination.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that no grade 1 or greater change in conventional clinical laboratory values or laboratory abnormalities are observed in a subject receiving the RNA composition (e.g., as described herein). Examples of such clinical laboratory assays may include lymphocyte counts, hematology changes, and the like.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that the Geometric Mean Concentration (GMC) of IgG for the SARS-CoV-2S polypeptide or immunogenic fragment thereof (e.g., RBD) can reach 200-3000 units/mL or 500-2000 units/mL by 21 days after the first administration (e.g., 10-100ug (inclusive) or 1ug-50ug (inclusive)), as compared to 602 units/mL for the covd-19 convalescence human serum group. In some embodiments, the RNA (e.g., mRNA) compositions described herein are characterized in that the Geometric Mean Concentration (GMC) of IgG directed against SARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g., RBD) can be increased by at least 8-fold or greater, including, for example, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold or greater, 7 days after the second administration (e.g., 10-30ug (inclusive), or 1ug-50ug (inclusive)). In some embodiments, the RNA (e.g., mRNA) compositions described herein are characterized in that the Geometric Mean Concentration (GMC) of IgG for the SARS-CoV-2S polypeptide or immunogenic fragment thereof (e.g., RBD) can be increased to 1500 units/mL to 40,000 units/mL or 4000 units/mL to 40,000 units/mL by 7 days after the second administration (e.g., 10-30ug (inclusive), or 1ug-50ug (inclusive). In some embodiments, the concentration of antibodies described herein can last at least 20 days or more after a first administration, including, for example, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, or at least 10 days or more after a second administration, including, for example, at least 15 days, at least 20 days, at least 25 days or more. In some embodiments, the antibody concentration may last up to 35 days after the first administration, or at least 14 days after the second administration.

In some embodiments, the RNA (e.g., mRNA) compositions described herein are characterized in that the GMC of IgG directed against the SARS-CoV-2S polypeptide or immunogenic fragment thereof (e.g., RBD) is at least 30% higher (including, e.g., at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 95% higher) than the concentration of antibodies observed in the covd-19 convalescence human serogroup when measured 7 days after the second administration (e.g., 1-50ug (inclusive)). In many embodiments, the Geometric Mean Concentration (GMC) of IgG described herein is the GMC of RBD-bound IgG.

In some embodiments, the RNA (e.g., mRNA) compositions described herein are characterized in that the GMC of IgG directed against the SARS-CoV-2S polypeptide or immunogenic fragment thereof (e.g., RBD) is at least 1.1-fold higher (including, e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher) than the concentration of antibody observed in a covd-19 convalescence human serogroup when measured 7 days after the second administration (e.g., 1-50ug (inclusive)). In many embodiments, the Geometric Mean Concentration (GMC) of IgG described herein is the GMC of RBD-bound IgG.

In some embodiments, an RNA (e.g., mRNA) composition described herein is characterized in that the GMC of IgG directed against a SARS-CoV-2S polypeptide or immunogenic fragment thereof (e.g., RBD) is at least 5-fold higher (including, e.g., at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher) than the concentration of antibodies observed in a covd-19 convalescence human serogroup when measured 21 days after the second administration. In many embodiments, the Geometric Mean Concentration (GMC) of IgG described herein is the GMC of RBD-bound IgG.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that an increase (e.g., at least 30%, at least 40%, at least 50% or more) in the neutralization Geometric Mean Titer (GMT) of SARS-CoV-2 is observed 21 days after the first administration. In some embodiments, an RNA (e.g., mRNA) composition described herein is characterized in that substantially greater serum neutralization GMT is obtained 7 days after the subject receives a second dose (e.g., 10 μg to 30 μg (inclusive)), reaching 150-300 compared to 94 for the covd-19 recovery serogroup.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by a protective efficacy of at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95% 7 days after administration of the second dose. In one embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by a protective efficacy of at least 70% 7 days after administration of the second dose. In one embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by a protective efficacy of at least 80% 7 days after administration of the second dose. In one embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by a protective efficacy of at least 90% 7 days after administration of the second dose. In one embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized by a protective efficacy of at least 95% 7 days after administration of the second dose.

In some embodiments, the RNA compositions provided herein are characterized in that they induce an immune response against SARS-CoV-2 at least 7 days after administration (e.g., after a second administration). In some embodiments, the RNA compositions provided herein are characterized in that they induce an immune response against SARS-CoV-2 in less than 14 days after administration (e.g., after a second administration). In some embodiments, the RNA compositions provided herein are characterized in that they induce an immune response against SARS-CoV-2 at least 7 days after a vaccination regimen. In some embodiments, the vaccination regimen comprises a first dose and a second dose. In some embodiments, the first dose and the second dose are administered at least 21 days apart. In some such embodiments, an immune response is induced against SARS-CoV-2 at least 28 days after the first administration.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that the Geometric Mean Concentration (GMC) of antibodies against the SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) is significantly higher than that of the convalescence serogroup (e.g., as described herein) as measured in serum from a subject receiving the RNA (e.g., mRNA) compositions of the present disclosure (e.g., at a dose of 10-30 (inclusive)). In some embodiments in which the subject may receive a second dose (e.g., 21 days after the 1 st administration), the Geometric Mean Concentration (GMC) of antibodies to the SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) may be 8.0-50-fold higher than the convalescence serogroup GMC as measured in serum from the subject. In some embodiments in which the subject can receive a second dose (e.g., 21 days after the 1 st administration), the Geometric Mean Concentration (GMC) of antibodies to the SARS-CoV-2 spike polypeptide or immunogenic fragment thereof (e.g., RBD) can be at least 8.0-fold or higher, including, for example, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold or higher, as compared to the convalescence serogroup GMC, as measured in serum from the subject.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that the SARS-CoV-2 neutralization geometric mean titer can be at least 1.5-fold or higher (including, for example, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold or higher) as measured at 28 days after the first administration or 7 days after the second administration as compared to the neutralizing GMT of the convalescence serogroup.

In some embodiments, the regimen administered to the subject may be or comprise a single dose. In some embodiments, the regimen administered to the 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 administered at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, or longer. In some embodiments, such doses may be separated by 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. In some embodiments, the doses may be administered a few 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 days or more apart. In some embodiments, the 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, the dosages may be separated by a period of about 7 to about 60 days, such as about 14 to about 48 days, etc. In some embodiments, the 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 days or more. In some embodiments, the 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 days or less. In some embodiments, the doses may be about 21 to about 28 days apart. In some embodiments, the doses may be about 19 to about 42 days apart. In some embodiments, the doses may be about 7 to about 28 days apart. In some embodiments, the dosage may be about 14 to about 24 days. In some embodiments, the dosage may be about 21 to about 42 days.

In some embodiments, particularly for compositions established to achieve elevated antibody and/or T cell titers over a period of time longer than about 3 weeks, e.g., in some embodiments, provided compositions are established to achieve elevated antibody and/or T cell titers (e.g., specific for a relevant portion of SARS-CoV-2 spike protein) over a period of time longer than about 3 weeks; in some such embodiments, the dosing regimen may involve only a single dose, or may involve two or more doses, which in some embodiments may be separated from each other by a period of time longer than about 21 days or three weeks. For example, in some such embodiments, such a period of time may be about 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or longer, or about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer, or in some embodiments about one year or longer.

In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first dose and the second dose may be administered in deltoid muscle. In some embodiments, the first dose and the second 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., 0.3mL each) separated by 21 days. In some embodiments, each dose is about 30ug. In some embodiments, each dose may be greater than 30ug, for example, about 40ug, about 50ug, about 60ug. In some embodiments, each dose may be less than 30ug, e.g., about 20ug, about 10ug, about 5ug, etc. In some embodiments, each dose is about 3ug or less, e.g., about 1ug. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 16 years old or older (including, e.g., 16-85 years old). In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 18-55 years old. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject aged 56-85 years. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered as a single dose (e.g., by intramuscular injection).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that RBD-specific IgG (e.g., polyclonal reaction) induced by such RNA compositions and/or methods exhibit higher binding affinity to RBD than a reference human monoclonal antibody having SARS-CoV-2 RBD binding affinity (e.g., CR3022 as described in j.ter Meulen et al, PLOS med.3, e237 (2006)).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity in a set (e.g., at least 10, at least 15, or more) of SARs-CoV-2 thorn mutants (e.g., in a set of variants described herein). In some embodiments, such SARs-CoV-2 spike mutants include RBD mutations (e.g., but not limited to, Q321L, V341I, A348T, N354D, S359 compared to SEQ ID NO:1, 408N, V367F, K378R, R9869I, Q409E, A435S, N439K, K458R, 1472V, G476S, S477N, V483A, Y508H, H519P, etc.) and/or spike protein mutations (e.g., but not limited to, D614G, etc. compared to SEQ ID NO: 1). Those skilled in the art are aware of the various spike mutants and/or the resources that document them (e.g., a table of mutation sites in spikes found by the covd-19 virus genome analysis pipeline and found at https:// cov.lanl.gov/components/sequence/COV/int_sites_tbls.comp) (last visit is day 8, 24 of 2020), and will understand from reading this specification that the RNA compositions and/or methods described herein can be characterized as their ability to induce serum in vaccinated subjects that exhibits neutralizing activity to any or all such variants and/or combinations thereof.

In particular embodiments, the RNA composition encoding the RBD of SARS-CoV-2 spike protein is characterized in that serum of a vaccinated subject exhibits neutralizing activity in a set (e.g., at least 10, at least 15, or more) of SARS-CoV-2 spike mutants including RBD variants (e.g., but not limited to, Q321L, V341I, A348T, N354 (as compared to SEQ ID NO: 1), 359N, V F, K378R, R408I, Q409E, A S, N4397 (458R), 1472V, G476S, S477N, V483 508H, H (508H, H5P, etc.) and spike protein variants (e.g., but not limited to, as compared to SEQ ID NO: 1).

In particular embodiments, an RNA (e.g., mRNA) composition encoding a SARS-CoV-2 spike protein variant comprising two consecutive proline substitutions at amino acid positions 986 and 987 on top of the central helix of the S2 subunit is characterized in that the serum of the vaccinated subject exhibits neutralizing activity in a set (e.g., at least 10, at least 15, or more) of SARS-CoV-2 spike mutants comprising RBD variants (e.g., but not limited to, as compared to SEQ ID NO:1, Q321L, V341I, A348T, N354 823 354 359 8235 359 4637N, V367F, K R, R378 54408 6278 4638 4639 439 4398R, 1472V, G476S, S477N, V483A, Y508H, H P, etc.) and spike protein variants (e.g., but not limited to, as compared to SEQ ID NO:1, D614G). For example, in some embodiments, an RNA (e.g., mRNA) composition encoding SEQ ID NO 7 (S P2) elicits an immune response against any of the SARs-CoV-2 thorn mutants, including RBD variants (e.g., without limitation, Q321L, V341I, A348T, N354D, S359 6767367F, K378R, R378I, Q E, A435S, N439K, K458R, 1472V, G476 3834 477N, V483A, Y508H, H P, etc.) and spike protein variants (e.g., without limitation, D614G, as compared to SEQ ID NO 1).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a mutation at position 501 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising an N501Y mutation in the spike protein.

The one or more comprises a sequence that is identical to SEQ ID NO:1 or the one or more SARs-CoV-2 spike mutant comprising a mutation at position 501 compared to spike protein or a mutation at position 1 comprising a nucleotide sequence corresponding to SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant that is mutated as compared to N501Y in spike protein as set forth in SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, a570D, D614G, P681H, T716I, S A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L/a 243/L244 deletion, etc., as compared to SEQ ID No. 1).

In a particular embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of vaccinated subjects exhibits neutralizing activity against SARs-CoV-2 spike mutant "variant of interest 202012/01" (VOC-202012/O1; also referred to as lineage B.1.1.7). This variant was previously named by the public health department of the united kingdom (Public Health England) in month 12 as the first variant under investigation (Variant Under Investigation) (VUI one 202012/01), but was reclassified as the variant of interest (VOC-202012/01). VOC-202012/01 is a variant of SARS-CoV-2, and was first detected in the sample taken from the previous month during the broad epidemic of COVID-19 in the United kingdom at month 10 in 2020 and started spreading rapidly in the middle of 12 months. It is associated with a significant increase in the infection rate of british covd-19; this increase is thought to be due, at least in part, to the change in N501Y within the spike glycoprotein receptor binding domain, which is required for ACE2 binding in human cells. The VOC-202012/01 variant is defined by 23 mutations: 13 nonsensical mutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17 mutations that alter the protein and 6 mutations that do not alter the protein). Spike protein changes in VOC 202012/01 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H. One of the most important changes in VOC-202012/O1 appears to be N50lY, i.e., the change from asparagine (N) to tyrosine (Y) at amino acid position 501. Such mutations, alone or in combination with a deletion at position 69/70 in the N-terminal domain (NTD), may enhance viral transmissibility.

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO:1, deletions 69-70, deletions 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.

In particular embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against the SARs-CoV-2 spike mutant "501.v2". This variant was observed in a sample of the first 10 months of 2020, after which in south africa more than 300 cases of the 501.v2 variant have been confirmed by Whole Genome Sequencing (WGS), which is the major form of the virus in 12 months 2020. Preliminary results indicate that the variant may have increased transmissibility. The v2 variant is defined by a plurality of spike protein changes, including: d80A, D215G, E484K, N501Y and a701V, and there are other changes in recently collected viruses: L18F, R246I, K417N and deletions 242-244.

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y and a701V compared to 1, and optionally: and SEQ ID N0: L18F, R246I, K417N and deletions 242-244 of the 1-phase comparison. The SARs-CoV-2 mutant may further comprise a sequence that hybridizes to SE0 ID NO: 1D 614G mutation compared to 1.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a H69/V70 deletion in the spike protein.

In some embodiments, one or more of the amino acid sequences that are identical to SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant comprising a H69/V70 deletion in the spike protein as compared to SEQ ID NO:1 (e.g., but not limited to, a deletion of Y144, a deletion of N501Y, A570D, D G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L3518F, R I, K417N, L/a 243/L244, a deletion of Y453F, 1692V, S1147L, M1229I, etc. compared to SEQ ID NO: 1).

In a particular embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of vaccinated subjects exhibits neutralizing activity against SARs-CoV-2 spike mutant "variant of interest 202012/01" (VOC-202012/01; also referred to as lineage B.1.1.7).

In a particular embodiment, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that the serum of the vaccinated subject exhibits neutralizing activity against the SARs-CoV-2 spike mutant "Cluster 5", which is also referred to as Δfvi spike by the national serum institute of denmark (Danish State Serum Institute, SSI). It was found in North Jutland, denmark, and is believed to be transmitted from mink to humans through mink farms. In cluster 5, several different mutations in the spike protein of the virus have been demonstrated. Specific mutations include 69-70 Δhv (deletion of histidine and valine residues at positions 69 and 70 of the protein), Y453F (tyrosine to phenylalanine at position 453), I692V (isoleucine to valine at position 692), M1229I (methionine to isoleucine at position 1229) and optionally S1147L (serine to leucine at position 1147).

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO:1, deletion 69-70, Y453F, I692V, M1229I and optionally S1147L.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a mutation at the spike protein 614 position. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising the D614G mutation in the spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more SARs-CoV-2 spike mutant comprising a mutation at position 614 compared to spike protein or a mutation at position 614 compared to spike protein comprising a nucleotide sequence corresponding to SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant that is mutated as compared to D614G in spike protein as set forth in SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, N501Y, A570D, P681H, T716I, S982A, D11 18H, D80A, D215G, E484K, A701V, L F, R246I, K N, L/a 243/L244 deletion, Y453F, 1692V, S1147L, M1229I, etc., compared to SEQ ID No. 1).

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y, A V and D614G compared to 1, and optionally: and SEQ ID NO: L18F, R246I, K417N and deletions 242-244 of the 1-phase comparison.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising mutations at positions 501 and 614 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising an N501Y mutation and a D614G mutation in the spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more SARS-CoV-2 spike mutant comprising mutations compared to spike proteins 501 and 614 or the sequence comprising a sequence identical to SEQ ID NO:1 and the SARs-CoV-2 spike mutant of the N501Y mutation and the D614G mutation in the spike protein may comprise a sequence that is identical to SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, a570D, P681H, T716I, S A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, L242/a243/L244 deletion, Y453F, I692V, S1147L, M1229I, etc. as compared to SEQ ID No. 1).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a mutation at position 484 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising an E484K mutation in a spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more SARs-CoV-2 spike mutant comprising a mutation at position 484 compared to spike protein or the one or more SARs-CoV-2 spike mutant comprising a sequence as set forth in SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant that is mutated as compared to E484K in spike protein as set forth in SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80 56215V, L18F, R I, K N, L/a 243/L244 deletion, Y453F, I692V, S1147L, M1229I, T N, P26S, D138Y, R79190S, K T, H655Y, T1027I, V1176F, etc. as compared to SEQ ID No. 1).

In particular embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against the SARs-CoV-2 spike mutant "501.v2".

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y and a701V compared to 1, and optionally: and SEQ ID NO: l18F, R246I, K7N and deletions 242-244 of the 1-phase comparison. The SARs-CoV-2 mutant may further comprise a sequence identical to SEQ ID NO: 1D 614G mutation compared to 1.

Lineage b.1.1.248, known as the brazil variant, is a variant of SARS-CoV-2, designated as the p.1 lineage, and has 17 unique amino acid changes, 10 of which are in its spike protein, including N501Y and E484K. B.1.1.248 is derived from b.1.1.28. E484K is present in B.1.1.28 and B.1.1.248. B.1.1.248 has multiple S protein polymorphisms [ L18F, T N, P S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I, V1176F ], and is similar to variants described in south africa at some key RBD positions (K417, E484, N501).

In particular embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against the SARs-CoV-2 spike mutant "b.1.1.28".

In particular embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against the SARs-CoV-2 spike mutant "b.1.1.248".

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO:1, L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I and V1176F.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising mutations at positions 501 and 484 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising an N501Y mutation and an E484K mutation in the spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more SARs-CoV-2 spike mutant comprising mutations at positions 501 and 484 compared to spike protein or the one or more SARs-CoV-2 spike mutant comprising a sequence corresponding to SEQ ID NO:1 and the SARs-CoV-2 spike mutant of the N501Y mutation and the E484K mutation in the spike protein may comprise a sequence that is identical to SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, a570D, D614G, P681H, T716I, S A, D1118H, D80A, D215G, A V, L18F, R246I, K417N, L/a 243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, K417T, H655Y, T1027I, V1176F, etc. as compared to SEQ ID No. 1).

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y and a701V compared to 1, and optionally: and SEQ ID NO: L18F, R246I, K417N and deletions 242-244 of the 1-phase comparison. The SARs-CoV-2 mutant may further comprise a sequence identical to SEQ ID NO: 1D 614G mutation compared to 1.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising mutations at positions 501, 484 and 614 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, including the N501Y mutation, the E484K mutation, and the D614G mutation in the spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more comprise a SARs-CoV-2 spike mutant mutated at positions 501, 484 and 614 compared to spike protein or SEQ ID NO:1, the SARs-CoV-2 spike mutant compared to the N501Y mutation, the E484K mutation, and the D614G mutation in the spike protein may comprise a sequence that is identical to SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, a570D, P681H, T716I, S982A, D1118H, D80A, D215G, A701V, L18F, R246 48417N, L/a 243/L244 deletion, Y453F, I692V, S1147 569I, T20N, P26 79138 3558S, K417T, H655Y, T1027I, V1176F, etc. as compared to SEQ ID No. 1).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a deletion of L242/A243/L244 in the spike protein.

In some embodiments, one or more of the amino acid sequences that are identical to SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant comprising a deletion of L242/A243/L244 in the spike protein as compared to SEQ ID NO:1 (e.g., without limitation, a deletion of H69/V70, a deletion of Y144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A701V, L18F, R246I, K417N, Y453F, I692 1147 797 1229 7920N, P S, D Y, R S, K417S, K655Y, T1027I, V1176F, etc. as compared to SEQ ID NO: 1).

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y, A V and deletions 242-244 compared to 1, optionally: and SEQ ID NO: L18F, R246I and K417N compared to 1. The SARs-CoV-2 mutant may further comprise a sequence identical to SEQ ID NO: 1D 614G mutation compared to 1.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a mutation at position 417 of the spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a K417N or K417T mutation in a spike protein.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the one or more SARs-CoV-2 spike mutant comprising a mutation at position 417 compared to spike protein or spike mutant comprising a sequence as compared to SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant that is mutated compared to either K417N or K417T in spike protein as set forth in SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, E484K, A V, L18F, R246I, L242/a243/L244 deletion, Y453F, I692V, S1147L, M1229I, T20N, P26S, D138Y, R S, H655Y, T1027I, V1176F, etc. as compared to SEQ ID No. 1).

In certain embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: D80A, D215G, E484K, N501Y, A V and K417N compared to 1, optionally: and SEQ ID NO: L18F, R246I and deletions 242-244 for phase 1. The SARs-CoV-2 mutant may further comprise a sequence identical to SEQ ID NO: 1D 614G mutation compared to 1.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising mutations at positions 417 and 484 and/or 501 of spike protein. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against one or more SARs-CoV-2 spike mutations that are identical to SEQ ID NO:1, comprising a K417N or K417T mutation in the spike protein, an E484K and/or N501Y mutation.

In some embodiments, one or more comprises a nucleotide sequence that hybridizes to SEQ ID NO:1 or the SARs-CoV-2 spike mutant mutated at positions 417 and 484 and/or 501 compared to spike protein or the one or more comprises a sequence as set forth in SEQ ID NO:1 may comprise a SARs-CoV-2 spike mutant that is compared to the K417N or K417T mutation in the spike protein and the E484K and/or N501Y mutation as compared to SEQ ID NO:1 (e.g., without limitation, a H69/V70 deletion, a Y144 deletion, a570D, D614G, P681H, T716I, S982 38324H, D80A, D215G, A701V, L18F, R246/a 243/L244 deletion, Y453F, 1692V, S1147L, M1229I, T20N, P26S, D138Y, R190S, H655Y, T1027I, V1176F, etc. as compared to SEQ ID No. 1).

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 thorn mutant omacron (b.1.1.529) variants. The Omicron (b.1.1.529) variant is a SARS-CoV-2 variant detected in south africa. Various omacron variants or sublines have emerged, including, for example, the ba.1, ba.2, ba.2.12.1, ba.3, ba.4, ba.5, and ba.2.75 sublines. As used herein, unless otherwise indicated, "Omicron variant" refers to the first disclosed Omicron variant (ba.1) or any variant thereof that appears thereafter (e.g., omicron variants described herein). In some embodiments of the present invention, in some embodiments, spike protein changes in the Omicron (b.1.1.529) ba.1 variant include a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE (EPE inserted after amino acid 214), G339D, S L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493 496S, Q498R, N501Y, Y H, T547K, D G, H655 to be 5 769K, P681 764H, N796H, N954H, N969K and L981F. In some embodiments, spike protein changes in omacron (b.1.1.529) variants include a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE (EPE inserted after amino acid 214), G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q4963 496S, Q498R, N501Y, Y H, T547K, D614G, H655 38395 679K, P681 764K, D796 856K, Q954H, N969K, and L981F. In some embodiments of the present invention, in some embodiments, the spinodal mutations in the Omicron BA.2 variant include T19I, delta24-26 a27S, G142D, V213G, G339D, S371 373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N Y, Y505H, D G, H655Y, N679K, P681K, P764K, P796 5294 954K, P969K. In some embodiments, ba.4 and ba.5 have the same spike protein amino acid sequence, in which case "ba.4/5" is used for either omacron variant. In some embodiments, the thorn mutation in omacron ba.4/5 comprises: T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405 79408S, K417N, N K, L452R, S477N, T478K, E484A, F486V, Q R, N Y, Y505H, D614G, H655Y, N679 35681K, P681H, N764K, D796Y, Q954H and N969K. In some embodiments of the present invention, in some embodiments, the spinodal mutations in OmicronBA.2.75 include T19I, Δ24-26 a27S, G142D, K147E, W R, F52373R, F417R, F440R, F446R, F460R, F477R, F478R, F484R, F498R, F505R, F614R, F655R, F679R, F681R, F764R, F796 5297 954H and N969K.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations 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: and SEQ ID NO:1, T547K, H655Y, D614G, N679K, P681K, P969 373K, P371K, P52440K, P339K, P446K, P856K, P764K, P417K, P796K, P954K, P5295K, P67K, P981K, P477K, P496K, P498K, P493K, P484K, P375K, P505K, P143 del, H69del, V70del, N211del, L212K, P EPE, G142K, P del, Y145del, L141del, Y144K, P145K, P del.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations 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: and SEQ ID NO:1, T547K, H655Y, D614G, N679K, P681P, S K, S373P, S371L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954 5295I, A67V, L983 6767N, G496S, T478K, Q498R, Q493R, E484A. The SARs-CoV-2 mutant may include at least 1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations: and SEQ ID NO:1, N501Y, S375F, Y505H, V143del, H69del, V70del, 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: and SEQ ID NO:1, N211del, L212I, ins EPE, G142D, Y144del, Y145del. In some embodiments, the SARs-CoV-2 mutant can include at least 1, at least 2, at least 3, or all of the following mutations: and SEQ ID NO:1, L141del, Y144F, Y145D, G142del.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 stab mutations 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 of the following mutations: and SEQ ID NO:1, a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N K, G446S, S477N, T478K, E484 63493R, G496S, Q498R, N501Y, Y H, T547K, D614G, H655 679K, P681H, N764H, N796H, N856H, N954 5297 969K and L981F.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARS-CoV-2 spike mutations 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, or at least 31 of the following mutations: and SEQ ID NO:1, T19I, Δ24-26, a27S, G142 213G, G339D, S371 373 3575 371 373F, T37376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D G, H655Y, N679K, P681 764K, P796K, P954K, P969K.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARS-CoV-2 spike mutations 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, or at least 34 of the following mutations: and SEQ ID NO:1, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S F, T376A, D405 79408S, K417N, N K, L452R, S477N, T478K, E484A, F486V, Q498R, N Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796 5297 954H and N969K.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO:1, a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N K, G446S, S477N, T478K, E484 63493R, G496S, Q498R, N501Y, Y H, T547K, D614G, H655 679K, P681H, N764H, N796H, N856H, N954 5297 969K and L981F.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARS-CoV-2 spike mutations comprising: T19I, Δ24-26, a27S, G142 213G, G339D, S371 37373P, S375F, T A, D405N, R408 417N, N440K, S477N, T478K, E484 79484A, Q493R, Q498R, N501Y, Y505H, D G, H655Y, N679K, P681 764K, P764 796K, P954K, P969K. In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARS-CoV2 spike mutations comprising: and SEQ ID NO:1, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S F, T376A, D405 79408S, K417N, N K, L452R, S477N, T478K, E484A, F486V, Q498R, N Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796 5297 954H and N969K.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO:1, a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q4989 496S, Q498R, N501Y, Y505H, T547K, D614G, H655 3835 679K, P681 764K, D796Y, N856 9564H, N969K and L981F.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein are characterized in that serum of a vaccinated subject exhibits neutralizing activity against SARs-CoV-2 spike mutations comprising: and SEQ ID NO: in contrast to the 1-degree of freedom, T19I, delta24-26, A27S, G142D, K147E, W152R, F L1210V, V213G, G257S, G339S, G371S, G373S, G376S, G52405S, G52408 417S, G446S, G460S, G477S, G478S, G484S, G498S, G501S, G505S, G614 655S, G679S, G681S, G764S, G796S, G954H and N969K.

And SEQ ID NO:1, the SARs-CoV-2 spike protein encoded by an RNA (e.g., mRNA) described herein may or may not include the D614G mutation.

In some embodiments, the SARS-CoV-2 spike protein encoded by the RNA (e.g., mRNA) described herein comprises a mutation in the furin cleavage site (e.g., residues 682-685 of SEQ ID NO:1 in some embodiments). In some embodiments, the SARS-CoV-2 spike protein encoded by an RNA (e.g., mRNA) described herein comprises a mutation in a furin cleavage site that prevents cleavage by furin (e.g., human furin). In some embodiments, the SARS-CoV-2 protein described herein comprises a furin mutation (e.g., a GSAS mutation) as disclosed in WO2021163365 or WO2021243122, the contents of both of which are incorporated herein by reference in their entirety.

In some embodiments, the RNA (e.g., mRNA) compositions and/or methods described herein can provide protection against SARS-CoV-2 and/or reduce the severity of SARS-CoV-2 infection in at least 50% of subjects receiving such RNA (e.g., mRNA) compositions and/or methods.

In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject 18-55 years old. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject aged 56-85 years. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein includes elderly subjects (e.g., subjects over 60, 65, 70, 75, 80, 85 years old, etc., such as subjects aged 65-85 years old). In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject 18-85 years old. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject 18 years old or younger. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject 12 years old or younger. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein comprises a subject aged 10 years or younger. In some embodiments, the population to be treated with the RNA (e.g., mRNA) compositions described herein can include a population of adolescents (e.g., individuals from about 12 years to about 17 years old). In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein can include a population of children (e.g., as described herein). In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein includes infants (e.g., less than 1 year old). In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein excludes infants (e.g., less than 1 year old) whose mothers have received such an RNA (e.g., mRNA) composition described herein during pregnancy. Without wishing to be bound by any particular theory, a rat study suggests that SARS-CoV-2 neutralizing antibody responses induced in female rats administered such RNA (e.g., mRNA) compositions during pregnancy can be transferred to the fetus. In some embodiments, the population to be treated with an RNA (e.g., mRNA) composition described herein includes infants (e.g., less than 1 year old) whose mothers did not receive such an RNA (e.g., mRNA) composition described herein during pregnancy. In some embodiments, a population to be treated with an RNA (e.g., mRNA) composition described herein can include a pregnant woman; in some embodiments, an infant whose mother is vaccinated during gestation (e.g., receives at least one dose, or alternatively only two doses) is not vaccinated for the first few weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or more, or 1, 2, 3, 4, 5 years, or more) after birth. Alternatively or additionally, in some embodiments, an infant whose mother is vaccinated during gestation (e.g., receives at least one dose, or alternatively only two doses) receives a reduced vaccination (e.g., a lower dose and/or fewer administrations, e.g., a booster, and/or a lower total exposure in a given time period) after birth, e.g., for the first few weeks, months, or even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks, or longer, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or longer, or 1, 2, 3, 4, 5 years, or longer), or may require a reduced vaccination (e.g., a lower dose and/or fewer administrations, e.g., booster, in a given time period). In some embodiments, the compositions provided herein are administered to a population that does not include a pregnant woman.

In some particular embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen comprising administering a first dose after about 24 weeks of gestation (e.g., after about 22, 23, 24, 25, 26, 27, 28 or more weeks of gestation); in some embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen comprising administering a first dose prior to about 34 weeks of gestation (e.g., prior to about 30, 31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In some embodiments, the compositions provided herein are administered to a pregnant woman according to a regimen comprising administering a first dose after about 24 weeks of gestation (e.g., after about 27 weeks of gestation, e.g., between about 24 and 34 weeks of gestation, or between about 27 and 34 weeks of gestation) and a second dose after about 21 days; in some embodiments, both doses are administered prior to labor. Without wishing to be bound by any particular theory, it is proposed that such a regimen (e.g., comprising administering the first dose after about 24 weeks or 27 weeks of gestation and optionally before about 34 weeks of gestation and optionally administering the second dose within about 21 days of gestation (desirably before delivery)) has certain advantages in terms of safety (e.g., reducing the risk of premature birth or fetal morbidity or mortality) and/or efficacy (e.g., administering residual vaccination to infants) over alternative dosing regimens (e.g., dosing at any time during gestation, avoiding dosing during gestation, and/or dosing at e.g., late gestation such that only one dose is administered during gestation). In some embodiments, infants born to a mother vaccinated during gestation, e.g., according to the particular protocols described herein, may not need further vaccination for a period of time after birth (e.g., as described herein), or may need reduced vaccination (e.g., lower doses and/or fewer administration times, e.g., booster, and/or lower total exposure over a given period of time).

In some embodiments, the compositions provided herein are administered to a population in which it is recommended that females not be pregnant for a period of time following vaccination (e.g., after receiving the first dose of vaccine, after receiving the last dose of vaccine, etc.); in some such embodiments, the period of time may be 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, or longer, or 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, or longer.

In some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include one or more populations having one or more particularly high risk conditions or medical history, e.g., as described herein. For example, in some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include subjects whose occupational and/or environmental exposure can significantly increase the risk of infection with SARS-CoV-2 (including, for example, but not limited to, public transportation personnel, prisoners, grocery store workers, residents in long-term care facilities, butchers or other meat processing workers, medical personnel, and/or first responders, e.g., emergency responders). In certain embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include a healthcare worker and/or a first responder, such as an emergency responder. In some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include a population having a history of smoking or electronic smoking (e.g., over 6 months, 12 months, or more, including a history of chronic smoking or electronic smoking). In some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include certain populations that have been determined to be more susceptible to SARS-CoV-2 infection.

In some embodiments, populations treated with an RNA (e.g., mRNA) composition described herein can include certain populations having blood groups that may have been determined to be more susceptible to SARS-CoV-2 infection. In some embodiments, populations treated with RNA (e.g., mRNA) compositions described herein can include subjects with reduced immune function (e.g., subjects with HIV/AIDS, cancer patients (e.g., receiving anti-tumor therapy), patients taking certain immunosuppressive drugs (e.g., transplant patients, cancer patients, etc.), autoimmune diseases or other physiological conditions that are expected to require immunosuppressive therapy (e.g., within 3 months, within 6 months, or longer), and patients with genetic diseases affecting the immune system (e.g., congenital agaropectinemia, congenital IgA deficiency). In some embodiments, the population treated with an RNA (e.g., mRNA) composition described herein can include a population having an infectious disease. For example, in some embodiments, populations treated with RNA (e.g., mRNA) compositions described herein can include those infected with Human Immunodeficiency Virus (HIV) and/or hepatitis virus (e.g., HBV, HCV). In some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include a population having a fundamental medical condition. Examples of such basic medical conditions may include, but are not limited to, hypertension, cardiovascular disease, diabetes, chronic respiratory disease, such as chronic lung disease, asthma, etc., cancer, and other chronic diseases, such as lupus, rheumatoid arthritis, Chronic liver disease, chronic kidney disease (e.g., stage 3 or worse, such as in some embodiments with Glomerular Filtration Rate (GFR) of less than 60mL/min/1.73m ² Is characterized by the following steps). In some embodiments, a population treated with an RNA (e.g., mRNA) composition described herein can include overweight or obese subjects, for example, specifically including Body Mass Index (BMI) above about 30kg/m ² Is a subject of (a). In some embodiments, the population treated with an RNA (e.g., mRNA) composition described herein can include a subject with a prior diagnosis of covd-19, or with evidence of current or prior infection with SARS-CoV-2 (e.g., according to serology or nasal swabs). In some embodiments, the population treated comprises white and/or non-spanish/non-latin.

In some embodiments, certain RNA (e.g., mRNA) compositions described herein may be selected for administration to asian populations, or in particular embodiments, older asian populations (e.g., 60 years or older, e.g., 60-85 years or 65-85 years).

In some embodiments, RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been determined not to show past infection and/or evidence of current infection prior to administration, and/or are evaluated in such subjects; in some embodiments, the evidence of past infection and/or current infection may be or include evidence of the presence of intact virus or any viral nucleic acid, protein, lipid, etc., in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and/or tissue), and/or evidence of an immune response by the subject thereto. In some embodiments, RNA (e.g., mRNA) compositions provided herein are administered to subjects who have been determined to show past infection and/or evidence of current infection prior to administration, and/or are evaluated in such subjects; in some embodiments, the evidence of past infection and/or current infection may be or include evidence of the presence of intact virus or any viral nucleic acid, protein, lipid, etc., in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and/or tissue), and/or evidence of an immune response by the subject thereto. In some embodiments, the subject is considered to have a past infection based on having a positive N-binding antibody detection result or a positive nucleic acid amplification detection (NAAT) result on the day of dose 1.

In some embodiments, the RNA (e.g., mRNA) compositions provided herein are administered to a subject who has been informed of the risk of side effects, which may include, for example, one or more of the following: shivering, fever, headache, pain at the injection site, muscle pain, fatigue; in some embodiments, an RNA (e.g., mRNA) composition is administered to a subject, if one or more such side effects occur, either as exceeding mild or moderate, for more than a day or several days, or if any serious or unexpected event occurs that the subject has reasonably justified regarding receiving the composition, the subject is required to inform the healthcare provider. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject who has been required to inform a healthcare provider of a particular medical condition, which may include, for example, one or more of allergy, hemorrhagic disease, or administration of blood thinner drugs, breast feeding, fever, immunocompromised status, or administration of drugs affecting the immune system, pregnancy, or planned pregnancy, etc. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject who has been required to inform a healthcare provider that another covd-19 vaccine has been received. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject that does not have one of the following medical conditions: fever, treatment with immunosuppressants, treatment with anticoagulants, suffering from hemorrhagic diseases (e.g. diseases where intramuscular injection is contraindicated), or pregnancy and/or lactation/lactation. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject that does not receive another covd-19 vaccine. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject that is not allergic to any component of the RNA (e.g., mRNA) composition. Examples of such allergic reactions include, but are not limited to, dyspnea, swelling of the respiratory tract and/or throat, increased heartbeat, rash, dizziness, and/or weakness. In some embodiments, an RNA (e.g., mRNA) composition provided herein is administered to a subject that received a first dose and is free of an allergic reaction to the first dose (e.g., as described herein). In some embodiments in which an allergic reaction occurs after a subject receives a dose of an RNA (e.g., mRNA) composition provided herein, the subject may be subjected to one or more interventions, such as treatments, to control and/or alleviate symptoms of such allergic reaction, e.g., using antipyretics and/or anti-inflammatory agents.

In some embodiments, subjects who have received at least one dose of an RNA (e.g., mRNA) composition provided herein are notified to avoid exposure to coronavirus (e.g., SARS-CoV-2) unless and until a number of days have passed since the second dose was administered (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.). For example, a subject who has received at least one dose of an RNA (e.g., mRNA) composition provided herein is notified that measures to prevent SARS-CoV-2 infection (e.g., maintaining social distance, wearing masks, frequent hand washing, etc.) are taken unless and until days have passed since the second dose was administered (e.g., at least 7 days, at least 8 days, 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, etc.). Thus, in some embodiments, a method of administering an RNA (e.g., mRNA) composition provided herein comprises administering a second dose of such RNA (e.g., mRNA) composition provided herein to a subject that received the first dose and takes precautions to avoid exposure to coronavirus (e.g., SARS-CoV-2).

In some embodiments, the RNA compositions described herein can be delivered to a draining lymph node of a subject in need thereof, e.g., for vaccine priming. In some embodiments, such delivery may be by intramuscular administration of the provided RNA composition.

In some embodiments, different specific RNA compositions can be administered to different subject populations; alternatively or additionally, in some embodiments, different dosing regimens may be administered to different subject populations. For example, in some embodiments, R administered to a particular subject populationNA compositions may be characterized by one or more specific effects (e.g., incidence and/or degree of influence) in these subject populations. In some embodiments, such effects may be or include, for example, neutralizing antibodies and/or T cells (e.g., T _H T cells of type 1, e.g. CD4 ⁺ And/or CD8 ⁺ T cells), protection against challenge (e.g., by injection and/or nasal exposure, etc.), incidence, severity, and/or persistence of side effects (e.g., reactogenicity), etc.

In some embodiments, one or more RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to reduce incidence of covd-19 per 1000 person-years, e.g., based on laboratory tests such as nucleic acid amplification assays (NAAT). In some embodiments, one or more RNA (e.g., mRNA) compositions described herein can be administered according to established protocols established based on laboratory tests (e.g., nucleic acid amplification assays (NAATs)) in subjects receiving at least one dose of provided RNA (e.g., mRNA) composition and no serological or virologic evidence of past SARS-CoV-2 infection (e.g., up to 7 days after receiving the last dose) to reduce incidence of covd-19 per 1000 people. In some embodiments, one or more of the RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to reduce the incidence of severe covd-19 diagnosis every 1000 person-years. In some embodiments, one or more of the RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to reduce the incidence of severe covd-19 per 1000 person-years in subjects receiving at least one dose of the provided RNA (e.g., mRNA) composition without serological or virological evidence of past SARS-CoV-2 infection.

In some embodiments, one or more RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to produce neutralizing antibodies against SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD) over a period of time, as measured in serum of a subject that meets or exceeds a reference level (e.g., a reference level determined based on human SARS-CoV-2 infection/covd-19 convalescence serum), and/or to induce a cell-mediated immune response (e.g., T cell response against SARS-CoV-2) over a period of time, including, for example, in some embodiments, inducing T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD). In some such embodiments, the period of time may be at least 2 months, 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, one or more epitopes recognized by vaccine-induced T cells (e.g., cd8+ T cells) can be present on MHC class I alleles present in at least 50% of the subjects in the population, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more; in some such embodiments, MHC class I alleles may be HLA-B0702, HLA-a 2402, HLA-B3501, HLA-B4401, or HLA-a 0201. In some embodiments, the epitope may comprise HLA-A x 0201 YLQPRTFLL; HLA-A x 0201 RLQSLQTYV; HLA-A 2402 qyikwpi; HLA-A 2402 NYNYLYRLF; HLA-A 2402 kw pwyilgf; HLA-B3501 QPTE SIVRF; HLA-B3501 IPFAMQMAY; or HLA-B3501 LPFNDGVYF.

In some embodiments, efficacy is assessed as incidence of covd-19 per 1000 persons in individuals without serological or virological evidence of prior SARS-CoV-2 infection prior to and during the vaccination regimen; alternatively or additionally, in some embodiments, efficacy is assessed as incidence of covd-19 per 1000 person-year in subjects with and without evidence of prior SARS-CoV-2 infection prior to and during vaccination regimens. In some such embodiments, such incidence is the incidence of a diagnosis of covd-19 over a specified period of time after the last dose of vaccine (e.g., first dose in a single dose regimen; second dose in a two dose regimen, etc.); in some embodiments, such a period of time may be within a particular number of days (e.g., 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 days or more) (i.e., up to and including 7 days). In some embodiments, such a period of time may be within 7 days or within 14 days or within 21 days or within 28 days. In some embodiments, such a period of time may be within 7 days. In some embodiments, such a period of time may be within 14 days.

In some embodiments (e.g., in some embodiments of assessing efficacy), a subject is determined to be suffering from a covd-19 infection if one or more of the following holds: SARS-CoV-2 nucleic acid is detected in a sample from a subject, antibodies that specifically recognize SARS-CoV-2 (e.g., SARS-Co-V-2 spike protein), one or more symptoms of a COVID-19 infection, and combinations thereof are detected. In some such embodiments, detection of SARS-CoV-2 nucleic acid can include, for example, NAAT testing of a medium turbinate swab sample. In some such embodiments, detection of the relevant antibodies may include serological testing of the blood sample or portion thereof. In some such embodiments, the symptoms of a covd-19 infection may be or include: fever, a new or aggravated cough, a new or aggravated shortness of breath, chills, a new or aggravated muscle pain, a new gustation or olfaction loss, sore throat, diarrhea, vomiting, and combinations thereof. In some such embodiments, the symptoms of a covd-19 infection may be or include: fever, a new or aggravated cough, a new or aggravated shortness of breath, chills, a new or aggravated muscle pain, a new gustation or olfaction loss, sore throat, diarrhea, vomiting, fatigue, headache, nasal obstruction or running nose, nausea, and combinations thereof. In some such embodiments, if the subject develops one such symptom and either the SARS-CoV-2 nucleic acid detection is positive or the antibody detection is positive, or both, the subject is determined to develop a COVID-19 infection. In some such embodiments, if the subject develops one such symptom and the SARS-CoV-2 nucleic acid detection is positive, the subject is determined to have developed a COVID-19 infection. In some such embodiments, if the subject develops one such symptom and the SARS-CoV-2 antibody detects positive, then the subject is determined to have developed a COVID-19 infection.

In some embodiments (e.gIn some embodiments that evaluate efficacy), a subject is determined to have a severe covd-19 infection if the subject has one or more of the following: clinical signs at rest indicate severe systemic disease (e.g., one or more of respiratory rate greater than or equal to 30 beats per minute, heart rate 125 beats per minute or more, spO under sea level indoor air conditions) ₂ Less than or equal to 93%, or PaO ₂ /FiO ₂ Below 300 mmhg), respiratory failure (e.g., requiring one or more of high flow oxygen inhalation, noninvasive ventilation, mechanical ventilation, ECMO), signs of shock (systolic below 90 mmhg, diastolic below 60 mmhg, need booster medication), significant acute renal, hepatic or neurological dysfunction, entry into the intensive care unit, death, and combinations thereof.

In some embodiments, one or more RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to reduce the percentage of subjects reporting at least one of the following: (i) One or more local reactions up to 7 days after each administration (e.g., as described herein); (ii) One or more systemic events up to 7 days after each administration; (iii) Adverse events (e.g., as described herein) from the first dose to 1 month after the last dose; and/or (iv) a serious adverse event from the first dose to 6 months after the last dose (e.g., as described herein).

In some embodiments, one or more subjects (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, including, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more, including, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or more, including, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) that have received an RNA (e.g., mRNA) composition described herein can be monitored to assess, e.g., for the presence of an immune response to a component of the administered composition, evidence of exposure to SARS-CoV-2 or another coronavirus and/or an immune response thereto, evidence of any adverse event, and the like. In some embodiments, monitoring may be performed by remote access. Alternatively or additionally, in some embodiments, the monitoring may be performed in the presence.

In some embodiments, the therapeutic effect conferred by one or more of the RNA (e.g., mRNA) compositions described herein is characterized by: (i) SARS-CoV-2 anti-S1 binding antibody levels above a predetermined threshold; (ii) SARS-CoV-2 anti-RBD binding antibody levels above a predetermined threshold; and/or (iii) SARS-CoV-2 serum neutralization titers are above threshold levels, e.g., at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and/or 24 months after vaccination is complete. In some embodiments, the anti-S1 binding antibody and/or anti-RBD binding antibody levels and/or serum neutralization titers can be characterized by a Geometric Mean Concentration (GMC), geometric Mean Titer (GMT), or geometric mean fold increase (GMFR).

In some embodiments, the therapeutic effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized by a percentage of treated subjects exhibiting a SARS-CoV-2 serum neutralization titer that is above a predetermined threshold (e.g., 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and/or 24 months after vaccination completion) that is higher than a percentage of untreated subjects exhibiting a SARS-CoV-2 serum neutralization titer that is above such a predetermined threshold (e.g., as described herein). In some embodiments, serum neutralization titers can be characterized by Geometric Mean Concentration (GMC), geometric Mean Titer (GMT), or geometric mean fold increase (GMFR).

In some embodiments, the therapeutic effect conferred by one or more of the RNA (e.g., mRNA) compositions described herein can be characterized by detection of SARS-CoV-2 NVA-specific binding antibodies.

In some embodiments, the therapeutic effect conferred by one or more of the RNA (e.g., mRNA) compositions described herein can be characterized by SARS-CoV-2 detection by nucleic acid amplification detection.

In some embodiments, the therapeutic effect conferred by one or more of the RNA (e.g., mRNA) compositions described herein can be characterized by: induction of a cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, for example, in some embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within a SARS-CoV-2 spike polypeptide and/or immunogenic fragment thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., cd8+ T cells) can be present on MHC class I alleles present in at least 50% of the subjects in a population, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more; in some such embodiments, MHC class I alleles may be HLA-B0702, HLA-a 2402, HLA-B3501, HLA-B4401, or HLA-a 0201. In some embodiments, the epitope may comprise HLA-A 0201 YLQPRTFLL; HLA-A x 0201 RLQSLQTYV; HLA-A 2402 qyikwpi; HLA-A 2402 NYNYLYRLF; HLA-A 2402 kw pwyilgf; HLA-B3501 QPTE SIVRF; HLA-B3501 IPFAMQMAY; or HLA-B3501 LPFNDGVYF.

In some embodiments, when there is sufficient evidence (posterior probability) that primary VE1 or both primary VE1 and primary VE2 > 30% or more (including, for example, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or more), primary Vaccine Efficacy (VE) of one or more RNA (e.g., mRNA) compositions described herein can be established, wherein primary VE is defined as primary VE = 100× (1-IRR); whereas IRR is calculated as the ratio of the prevalence of covd-19 in the vaccine group to the corresponding prevalence in the placebo group. Primary VE1 represents VE of a prophylactic RNA (e.g., mRNA) composition described herein against diagnosed covd-19 in participants without evidence of infection prior to vaccination, and primary VE2 represents VE of a prophylactic RNA (e.g., mRNA) composition described herein against diagnosed covd-19 in all participants after vaccination. In some embodiments, primary VE1 and VE2 may be evaluated sequentially to control 2.5% of total type I error (hierarchical detection). In some embodiments where one or more of the RNA (e.g., mRNA) compositions described herein are demonstrated to achieve a primary VE endpoint as described above, secondary VE endpoints (e.g., severe covd-19 diagnosed in participants without evidence of infection prior to vaccination and severe covd-19 diagnosed in all participants) may be sequentially assessed, e.g., by the same methods as described above for primary VE endpoint assessment (fractionation detection). In some embodiments, the assessment of primary and/or secondary VE endpoints may be based on at least 20,000 or more subjects (e.g., at least 25,000 or more subjects) randomly allocated in a 1:1 ratio to vaccine or placebo group, e.g., based on the following assumptions: (i) A prevalence of 1.0% per year in the placebo group, and (ii) no assessment or serological evidence of past infection with SARS-CoV-2 by 20% of the participants, which could potentially make them immune to further infection.

In some embodiments, one or more RNA (e.g., mRNA) compositions described herein can be administered according to established protocols to achieve maintenance and/or continued boost of an immune response. For example, in some embodiments, the administration regimen may include a first dose, optionally followed by one or more subsequent doses; in some embodiments, the need, time, and/or amount of any such subsequent agent may be selected to maintain, enhance, and/or improve one or more immune responses or characteristics thereof. In some embodiments, the number, time, and/or amount of doses is determined to be effective when administered to the relevant population. In some embodiments, the number, time, and/or amount of agent runs may be adjusted for an individual subject; for example, in some embodiments, one or more characteristics of an immune response in an individual subject may be assessed at least once (and optionally more than once, e.g., multiple times, typically spaced apart, typically at preselected intervals) after receiving the first dose. For example, antibodies, B cells, and/or T cells (e.g., CD4 ⁺ And/or CD8 ⁺ T cells) and/or the presence of cytokines secreted thereby, and/or the extent of recognition of and/or response to a particular antigen and/or epitope. In some embodiments, the need, time, and/or amount of a subsequent dose may be determined from such an evaluation.

As described above, in some embodiments, one or more subjects who have received an RNA (e.g., mRNA) composition described herein can be monitored (e.g., maintained) from the time of receiving any particular doseFor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or longer, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or longer, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or longer, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or longer), to assess, for example, the presence of an immune response to a component of the administered composition, evidence of exposure to SARS-CoV-2 or another coronavirus and/or immune response thereto, evidence of any adverse event, and the like, including assessing one or more of the following: antibodies, B cells and/or T cells (e.g., CD4 ⁺ And/or CD8 ⁺ T cells), and/or the presence of cytokines secreted thereby, and/or the extent of recognition of and/or response to a particular antigen and/or epitope. The compositions described herein may be administered according to a regimen comprising one or more such monitoring steps.

For example, in some embodiments, the need, time, and/or amount of a second dose relative to a first dose (and/or a subsequent dose relative to a previous dose) is assessed, determined, and/or selected such that administration of such second (or subsequent) dose achieves an amplification or improvement in immune response observed after the first (or other previous) dose (e.g., as described herein). In some embodiments, such amplification of an immune response (e.g., an immune response described herein) may be 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 more compared to the level of immune response observed after the first dose. In some embodiments, such amplification of the immune response may be at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, or more compared to the immune response level observed after the first dose.

In some embodiments, the need, time, and/or amount of a second (or subsequent) dose relative to the first (or other previous) dose is assessed, determined, and/or selected such that administration of a later dose extends the duration of an immune response observed after the earlier dose (e.g., as described herein); in some such embodiments, the duration may be extended by 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, at least 6 months, at least 7 months, at least 8 months, at least 9 months, or more. In some embodiments, the immune response observed after the first dose may be characterized by: the production of neutralizing antibodies (measured in serum from a subject) against SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD), and/or induction of cell-mediated immune responses (e.g., T cell responses against SARS-CoV-2), including, for example, in some embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., cd8+ T cells) can be present on MHC class I alleles present in at least 50% of the subjects in a population, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more; in some such embodiments, MHC class I alleles may be HLA-B0702, HLA-a 2402, HLA-B3501, HLA-B4401, or HLA-a 0201. In some embodiments, the epitope may comprise HLA-A 0201 YLQPRTFLL; HLA-A x 0201 RLQSLQTYV; HLA-A 2402 qyikwpi; HLA-A 2402 NYNYLYRLF; HLA-A 2402 kw pwyilgf; HLA-B3501 QPTE SIVRF; HLA-B3501 IPFAMQMAY; or HLA-B3501 LPFNDGVYF.

In some embodiments, the need, time, and/or amount of a second dose relative to the first dose (or other subsequent doses relative to the previous dose) is assessed, determined, and/or selected such that administration of the second (or subsequent) dose maintains or exceeds a reference level of immune response; in some such embodiments, the reference level is determined based on human SARS-CoV-2 infection/convalescence-19 serum and/or PBMC samples taken from the subject (e.g., at least a period of time, e.g., at least 14 days or more, including, e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days or more, when the subject is asymptomatic) following a PCR-confirmed diagnosis. In some embodiments, the immune response may be characterized by the following list: the production of neutralizing antibodies (measured in serum from a subject) against SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD), and/or the production of cell-mediated immune responses (e.g., T cell responses against SARS-CoV-2), including, for example, in some embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within the SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., cd8+ T cells) can be present on MHC class I alleles present in at least 50% of the subjects in a population, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more; in some such embodiments, MHC class I alleles may be HLA-B0702, HLA-a 2402, HLA-B3501, HLA-B4401, or HLA-a 0201. In some embodiments, the epitope may comprise HLA-A 0201 YLQPRTFLL; HLA-A x 0201 RLQSLQTYV; HLA-A 2402 qyikwpi; HLA-A 2402 NYNYLYRLF; HLA-A 2402 kw pwyilgf; HLA-B3501 QPTE SIVRF; HLA-B3501 IPFAMQMAY; or HLA-B3501 LPFNDGVYF.

In some embodiments, determining the need, time, and/or amount for a second (or subsequent) dose may include one or more steps of evaluating after a first (or other previous) dose (e.g., after 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days, or more) of: the presence and/or expression level (measured in serum from a subject) of neutralizing antibodies against SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD), and/or induction of cell-mediated immune responses (e.g., T cell responses against SARS-CoV-2), including, for example, in some embodiments, induction of T cells that recognize at least one or more MHC-restricted (e.g., MHC class I-restricted) epitopes within SARS-CoV-2 spike polypeptide and/or immunogenic fragments thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., cd8+ T cells) can be present on MHC class I alleles present in at least 50% of the subjects in a population, including, for example, at least 60%, at least 70%, at least 80%, at least 90% or more; in some such embodiments, MHC class I alleles may be HLA-B0702, HLA-a 2402, HLA-B3501, HLA-B4401, or HLA-a 0201. In some embodiments, the epitope may comprise HLA-A 0201 YLQPRTFLL; HLA-A x 0201 RLQSLQTYV; HLA-A 2402 qyikwpi; HLA-A 2402 NYNYLYRLF; HLA-A 2402 kw pwyilgf; HLA-B3501 QPTE SIVRF; HLA-B3501 IPFAMQMAY; or HLA-B3501 LPFNDGVYF.

In some embodiments, the kits provided herein can include a real-time monitoring logging device, for example, in some embodiments, the device is capable of providing a delivery temperature, a delivery time, and/or a location.

In some embodiments, the RNA (e.g., mRNA) compositions described herein can be delivered, stored, and/or used in a container (e.g., vial or syringe), such as a glass container (e.g., glass vial or syringe), which in some embodiments can be a single dose container or a multi-dose container (e.g., can be arranged and configured to hold, and/or in some embodiments can hold, a single dose or multiple doses of a product for administration). In some embodiments, a multi-dose container (e.g., a multi-dose vial or syringe) may be arranged and configured to hold, and/or may hold, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses; in some particular embodiments, it may be designed to hold and/or may hold 5 doses. In some embodiments, single or multi-dose containers (e.g., single or multi-dose vials or syringes) may be arranged and configured to hold and/or may hold volumes or amounts greater than the indicated number of doses, e.g., so as to allow for some loss in transfer and/or administration. In some embodiments, the RNA (e.g., mRNA) compositions described herein can be transported, stored, and/or used in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single or multiple dose preservative-free glass vial or syringe). In some embodiments, the RNA (e.g., mRNA) compositions described herein can be transported, stored, and/or used in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single or multiple dose preservative-free glass vial or syringe) containing a frozen liquid, e.g., in some embodiments 0.45ml of frozen liquid (e.g., including 5 doses). In some embodiments, the RNA (e.g., mRNA) compositions and/or containers (e.g., vials or syringes) in which they are placed described herein can be transported, stored, and/or used at temperatures below room temperature, 4 ℃ or less, 0 ℃ or less, -20 ℃ or less, -60 ℃ or less, -70 ℃ or less, -80 ℃ or less, -90 ℃ or less, etc. In some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed may be kept at a temperature between-80 ℃ and-60 ℃ for shipping, storage, and/or use, and in some embodiments protected from light. In some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed can be kept for shipping, storage, and/or use at a temperature below about 25 ℃, and in some embodiments protected from light. In some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed can be transported, stored, and/or used at a temperature of less than about 5 ℃ (e.g., less than about 4 ℃) and, in some embodiments, protected from light. In some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed can be kept at a temperature below about-20 ℃ for shipping, storage, and/or use, and in some embodiments protected from light. In some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed can be kept in transit, stored, and/or used at a temperature above about-60 ℃ (e.g., at about-20 ℃ or above about-20 ℃ in some embodiments, at about 4-5 ℃ or above about 4-5 ℃ in some embodiments, optionally below about 25 ℃ in both cases), and protected from light in some embodiments, or without active measures (e.g., cooling measures) to substantially lower the storage temperature below about-20 ℃.

In some embodiments, the RNA (e.g., mRNA) compositions and/or containers (e.g., vials or syringes) in which they are placed described herein are transported, stored, and/or used with and/or in the context of a thermal protection material or container and/or a temperature regulating material. For example, in some embodiments, the RNA (e.g., mRNA) compositions described herein and/or containers (e.g., vials or syringes) in which they are placed are transported, stored, and/or used with ice and/or dry ice and/or with insulation materials. In some particular embodiments, a container (e.g., a vial or syringe) containing an RNA (e.g., mRNA) composition is placed in a tray or other holding device and further contacted with (or in the presence of) a temperature regulating (e.g., ice and/or dry ice) material and/or an insulating material. In some embodiments, multiple containers (e.g., multiple vials or syringes, such as single-use or multi-use vials or syringes described herein) with the provided RNA (e.g., mRNA) composition are placed together (e.g., in a common tray, rack, box, etc.) and packaged (or in the presence of) a temperature regulating (e.g., ice and/or dry ice) material and/or an insulating material. To name just one example, in some embodiments, multiple containers (e.g., multiple vials or syringes, such as single-use or multi-use vials or syringes described herein) with RNA (e.g., mRNA) compositions are placed in a common tray or rack, and multiple such trays or racks are stacked in a carton surrounded by a temperature regulating material (e.g., dry ice) in a hot (e.g., insulated) conveyor. In some embodiments, the temperature regulating material is periodically replenished (e.g., within 24 hours of arrival at the location, and/or every 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc.). Preferably, the heat carrier should be replaced infrequently, and ideally should not be more than twice a day. In some embodiments, the heat conveyor is turned back off within 5, 4, 3, 2, or 1 minutes or less after being turned on. In some embodiments, the provided RNA (e.g., mRNA) compositions are still useful for storage for a period of time (optionally within a specific temperature range) within a heat transporter. For example, in some embodiments, an RNA (e.g., mRNA) composition can be used for up to 10 days if a heat carrier containing the provided RNA (e.g., mRNA) composition as described herein is or has been maintained (e.g., stored) at a temperature in the range of about 15 ℃ to about 25 ℃; that is, in some embodiments, the provided RNA (e.g., mRNA) composition that has been maintained for no more than 10 days in a heat transporter (the heat transporter being at a temperature in the range of about 15 ℃ to about 25 ℃) is administered to a subject. Alternatively or additionally, in some embodiments, the provided RNA (e.g., mRNA) composition can be used for up to 10 days if it is or has been held (e.g., stored) within a heat carrier (the heat carrier has been held (e.g., stored) within a temperature range of about 15 ℃ to about 25 ℃); that is, in some embodiments, the provided RNA (e.g., mRNA) composition maintained in a heat transporter (the heat transporter having been maintained at a temperature in the range of about 15 ℃ to about 25 ℃ for no more than 10 days) is administered to a subject.

In some embodiments, provided RNA (e.g., mRNA) compositions are delivered and/or stored in a frozen state. In some embodiments, the provided RNA (e.g., mRNA composition) is delivered and/or stored in the form of a frozen suspension, which in some embodiments is preservative-free. In some embodiments, the frozen RNA (e.g., mRNA) composition is thawed. In some embodiments, the thawed RNA (e.g., mRNA) composition (e.g., suspension) can comprise white to off-white opaque amorphous particles. In some embodiments, if the thawed RNA (e.g., mRNA) composition is maintained (e.g., stored) at room temperature or below (e.g., below about 30 ℃, 25 ℃, 20 ℃, 15 ℃, 10 ℃, 8 ℃, 4 ℃, etc.), the composition can be used up to a short number of days (e.g., 1, 2, 3, 4, 5, or 6 days) after thawing. In some embodiments, the thawed RNA (e.g., mRNA) composition can be used after storage (e.g., for such a short period of time) at a temperature of about 2 ℃ to about 8 ℃; alternatively or additionally, the thawed RNA (e.g., mRNA) composition can be used within a short number of hours (e.g., 1, 2, 3, 4, 5, 6 hours) after thawing at room temperature. Thus, in some embodiments, the provided RNA (e.g., mRNA) composition that has been thawed and maintained at room temperature or a temperature below room temperature, in some embodiments between about 2 ℃ and about 8 ℃, for no more than 6, 5, 4, 3, 2, or 1 day is administered to the subject. Alternatively or additionally, in some embodiments, the provided RNA (e.g., mRNA) composition that has been thawed and maintained at room temperature for no more than 6, 5, 4, 3, 2, or 1 hour is administered to the subject. In some embodiments, the provided RNA (e.g., mRNA) composition is transported and/or stored in a concentrated state. In some embodiments, such concentrated compositions are diluted prior to administration. In some embodiments, the diluted composition is administered within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour after dilution; in some embodiments, such administration is within 6 hours after dilution. Thus, in some embodiments, a diluted formulation of a provided RNA (e.g., mRNA) composition is administered to a subject within 6 hours after dilution (e.g., after having been maintained at an appropriate temperature, e.g., at a temperature below room temperature, at 4 ℃ or less, at 0 ℃ or less, at-20 ℃ or less, at-60 ℃ or less, at-70 ℃ or less, at-80 ℃ or less, etc., as described herein, and typically at about 2 ℃ or more, e.g., between about 2 ℃ and about 8 ℃ or between about 2 ℃ and about 25 ℃). In some embodiments, unused composition is discarded within hours (e.g., about 10, about 9, about 8, about 7, about 6, about 5 hours, or less) after dilution; in some embodiments, unused composition is discarded within 6 hours of dilution.

In some embodiments, an RNA (e.g., mRNA) composition (e.g., frozen composition, liquid concentrate composition, diluted liquid composition, etc.) stored, shipped, or used may have been maintained at a temperature substantially above-60 ℃ for at least 1, 2, 3, 4, 5, 6, 7 days, or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or more, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more; in some such embodiments, such compositions may have been maintained at a temperature of about-20 ℃ or above for such a period of time, and/or at a temperature of up to or about 4-5 ℃, and/or may be maintained at a temperature of greater than about 4-5 ℃, and optionally at a temperature of about 25 ℃, for a period of time less than two (2) months, and/or optionally up to about one (1) month. In some embodiments, such compositions may not be stored, transported, or otherwise used (or otherwise exposed) at temperatures substantially above about 4-5 ℃, particularly at temperatures of about 25 ℃ or near about 25 ℃ for a period of up to about 2 weeks, or in some embodiments for a period of 1 week. In some embodiments, such compositions may not be stored, transported, or otherwise used (or otherwise exposed) at temperatures substantially above about-20 ℃, particularly at temperatures of about 4-5 ℃ or near about 4-5 ℃ for a period of time of about 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, or in some embodiments, for a period of time of up to about 8 weeks or 6 weeks or substantially more than about 2 months or in some embodiments 3 months or in some embodiments 4 months.

In some embodiments, the RNA (e.g., mRNA) composition (e.g., frozen composition, liquid concentrate composition, diluted liquid composition, etc.) stored, transported, or used can be protected from light. In some embodiments, one or more steps may be taken to reduce or minimize light exposure of such compositions (e.g., the compositions may be placed into a container such as a vial or syringe). In some embodiments, exposure to direct sunlight and/or ultraviolet light is avoided. In some embodiments, the diluted solution may be treated and/or used under normal indoor light conditions (e.g., no special steps are taken to minimize or reduce indoor light exposure). It will be appreciated that in the handling (e.g., dilution and/or administration) of RNA (e.g., mRNA) compositions described herein, strict adherence to aseptic techniques is required. In some embodiments, the RNA (e.g., mRNA) compositions described herein are not administered intravenously (e.g., not by intravenous injection). In some embodiments, the RNA (e.g., mRNA) compositions described herein are not administered intradermally (e.g., not injected intradermally). In some embodiments, the RNA (e.g., mRNA) compositions described herein are not administered subcutaneously (e.g., not subcutaneously). In some embodiments, the RNA (e.g., mRNA) compositions described herein are not administered in any manner intravenously, intradermally, or subcutaneously (e.g., are not injected in any manner intravenously, intradermally, or subcutaneously). In some embodiments, an RNA (e.g., mRNA) composition described herein is not administered to a subject having a known hypersensitivity reaction to any of its components. In some embodiments, a subject administered an RNA (e.g., mRNA) composition is monitored for the appearance of one or more signs of allergic reaction. In some embodiments, the subject administered the RNA (e.g., mRNA) composition has previously received at least one dose of a different SARS-CoV-2 vaccine; in some embodiments, the subject administered the RNA (e.g., mRNA) composition has not previously received a different SARS-CoV-2 vaccine. In some embodiments, the body temperature of the subject is measured rapidly prior to administration of the RNA (e.g., mRNA) composition (e.g., prior to thawing, diluting, and/or administering such composition or shortly thereafter); in some embodiments, if the subject is determined to be febrile, the administration is delayed or cancelled. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered to a subject that is undergoing anticoagulant therapy or is suffering from or susceptible to an bleeding disorder or condition that would be contraindicated for intramuscular injection. In some embodiments, the RNA (e.g., mRNA) compositions described herein are used by a medical professional who has informed about information related to side effects and risks to the subject receiving the composition. In some embodiments, the RNA (e.g., mRNA) compositions described herein are used by a medical professional who has agreed to submit an adverse event report for any serious adverse event, which may include one or more of the following: death, the occurrence of disability or congenital abnormalities/birth defects (e.g., in a subject's child), hospitalization (including prolonged existing hospitalization), life threatening events, drugs or surgical interventions to prevent death, sustained or significant or substantial disruption of the ability to perform normal living functions; or another important medical event that may jeopardize the individual and may require pharmaceutical or surgical intervention (treatment) to prevent one of the other outcomes.

In some embodiments, the provided RNA composition is administered to a population of individuals under 18 years old, or under 17 years old, or under 16 years old, or under 15 years old, or under 14 years old, or under 13 years old, e.g., according to an established regimen having an incidence of one or more of the local response events described below that does not exceed the incidence described below:

injection site pain (75% after the first dose and/or second dose, and/or lower incidence after the second dose, e.g., 65% after the second dose);

redness of the injection site (less than 5% after the first and/or second dose); and/or

Swelling at the injection site (less than 5% after the first dose and/or the second dose).

In some embodiments, the provided RNA composition is administered to a population of individuals under 18 years old, or under 17 years old, or under 16 years old, or under 15 years old, or under 14 years old, or under 13 years old, e.g., according to an established regimen having an occurrence rate for one or more systemic reaction events described below that does not exceed the occurrence rate described below:

fatigue (55% after the first dose and/or the second dose);

headache (50% after the first dose and/or the second dose);

muscle pain (40% after the first dose and/or the second dose);

Shivering (40% after the first dose and/or the second dose);

joint pain (20% after the first dose and/or the second dose);

fever (25% after the first dose and/or the second dose);

emesis (10% after the first dose and/or the second dose); and/or

Diarrhea (10% after the first dose and/or the second dose).

In some embodiments, a medicament that alleviates one or more symptoms of one or more local and/or systemic response events (e.g., as described herein) is administered to an individual under 18 years old, or under 17 years old, or under 16 years old, or under 15 years old, or under 14 years old, or under 13 years old, who has been administered the provided RNA composition and has developed (e.g., as described herein) one or more of the local and/or systemic response events. In some embodiments, an antipyretic and/or analgesic may be administered to the individuals.

Drawings

FIG. 1 is a schematic overview of the S protein organization of SARS-CoV-2S protein.

The sequence within the S1 subunit consists of a Signal Sequence (SS) and a Receptor Binding Domain (RBD), a key subunit within the S protein associated with binding to the human cell receptor ACE 2. The S2 subunit contains an S2 protease cleavage site (S2') followed by a Fusion Peptide (FP) for membrane fusion, heptad repeats (HR 1 and HR 2) with a Central Helical (CH) domain, a transmembrane domain (TM) and a Cytoplasmic Tail (CT).

FIG. 2. Exemplary SARS-CoV-2 vaccine constructs.

Based on the intact and wild-type S proteins, we have designed different constructs that encode (1) intact proteins with mutations in the first heptapeptide repeat (HRP 1) in close proximity, including stable mutations that retain the neutralizing sensitive site, (2) the S1 domain or (3) the RB domain only (RBD). In addition, to stabilize the protein fragment, a minor fibrin domain (F) was fused to the C-terminus. All constructs start with a Signal Peptide (SP) to ensure transport of the golgi to the cell membrane.

FIG. 3. General structure of RNA.

Schematic representation of the general structure of RNA vaccines with 5' -caps, 5' -and 3' -untranslated regions, coding sequences with intrinsic secretion signal peptide and GS-linker, and poly (a) -tails. Note that the individual elements are not drawn to scale entirely, as compared to their respective sequence lengths.

UTR = untranslated region; sec = secretion signal peptide; RBD = receptor binding domain; gs=glycine-serine linker.

FIG. 4. General structure of RNA.

Schematic representation of the general structure of RNA drug substances with 5' -cap, 5' -and 3' -untranslated regions, coding sequences with intrinsic secretion signal peptide and GS-linker, and poly (a) -tail. Note that the individual elements are not drawn to scale entirely, as compared to their respective sequence lengths.

Gs=glycine-serine linker; UTR = untranslated region; sec = secretion signal peptide; RBD = receptor binding domain.

FIG. 5. General structure of RNA.

Schematic representation of the general structure of Venezuelan Equine Encephalitis Virus (VEEV) RNA-dependent RNA polymerase replicase with 5' -cap, 5' -and 3' -untranslated regions, with intrinsic secretion signal peptide and GS-linker and the coding sequences for SARS-CoV-2 antigen, as well as RNA vaccines of poly (a) -tail. Note that the individual elements are not drawn to scale entirely, as compared to their respective sequence lengths.

FIG. 6 is a schematic overview of the S protein organization of SARS-CoV-2S protein and the constructs used to develop the SARS-CoV-2 vaccine.

Based on the wild-type S protein, we designed two different transmembrane anchored RBD-based vaccine constructs encoding RBD fragments fused to the T4 secondary fibrin trimerization domain (F) and the native (autochromus) transmembrane domain (TM). Construct (1) starts with SARS-CoV-2-S signal peptide (SP; AA1-19 of S protein) and construct (2) starts with human Ig heavy chain signal peptide (huSec) to ensure Golgi transport to cell membrane.

FIG. 7 anti-S protein IgG responses 6, 14 and 21 days after immunization with modRNA formulated with LNP-C12 encoding a transmembrane anchored RBD-based vaccine construct.

BALB/C mice were IM immunized once with 4 μg of LNP-C12 formulated transmembrane anchored RBD-based vaccine construct (instead of BNT162b3C/BNT162b3 d). On days 6, 14 and 21 after immunization, animals were bled and serum samples were analyzed for total amounts of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions were included for day 6 (1:50), day 14 (1:300) and day 21 (1:900). One dot in the graph represents one mouse, and each mouse sample was measured in duplicate (group size n=8; each group included mean+sem).

FIG. 8 neutralization of SARS-CoV-2 pseudovirus 6, 14 and 21 days after immunization with modRNA formulated with LNP-C12 encoding a transmembrane anchored RBD-based vaccine construct.

BALB/C mice were IM immunized once with 4 μg of LNP-C12 formulated transmembrane anchored RBD-based vaccine construct (instead of BNT162b3C/BNT162b3 d). Animals were bled and serum tested for neutralization of the SARSCOV-2 pseudovirus on days 6, 14 and 21 after immunization. The graph shows pVN serum dilutions (50% reduction in infection events compared to positive control without serum). One dot in the figure represents one mouse. Each mouse sample was measured in duplicate. Group size n=8. Mean + SEM are shown for each group with horizontal bars with whiskers. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

FIG. 9 50% pseudovirus neutralization titers (pVNT 50) of serum collected 21 days after the second dose of BNT162b2 and 1 month after the third dose of BNT162b2 against VSV-SARS-CoV-2-S pseudovirus harboring the MN908947 Hu-1 reference or Omicron lineage spike protein. N=19-20 serum from immunized subjects collected 21 days after the second BNT162b2 dose or 1 month after the third BNT162b2 dose was tested. LOD/2 values are plotted for values below the detection limit (LOD; 10). Group GMT (higher than bar value) with 95% confidence interval is shown.

Figure 10 cd8+ T cell epitopes in bnt162b2 vaccine were largely unaffected by Omicron ba.1 variant mutation. The number of previously identified MHC-I epitopes affected by various variants of interest (VOCs) is shown. Approximately 80% of the previously identified cd8+ epitopes were not affected by the ba.1 omacron variant mutation, suggesting that two doses of BNT162b2 may still induce protection against severe disease.

FIG. 11 neutralization of Omacron BA.1 after two doses of BNT162b2 and variant-specific enhancer. Neutralization of Omicron ba.1 variants from serum of patients administered with: two doses of BNT162b2 and (i) a third booster dose of BNT162b2, or (ii) a third booster dose of RNA encoding a spike protein having an alpha or delta variant mutation, or a third booster dose of spike protein comprising an alpha mutation and spike protein comprising a delta mutation. These values come from the separately neutralized GMT of the pseudovirus test. A schematic drawing depicting the process of developing a new SARS-CoV-2 variant specific vaccine is also shown.

FIG. 12A longitudinal analysis of the neutralizing antibody response of VSV-SARS-CoV-2-S pseudovirus with MN908947 or OmacronBA.1 variant spike protein in a subset of study participants. The sera of n=9 participants extracted 21 days after dose 2, before dose 3, 1 month after dose 3, and 3 months after dose 3 were tested. Each serum was tested in duplicate and a single geometric mean 50% pseudovirus neutralization titer (GMT) was calculated. LOD/2 values are assigned for values below the detection Limit (LOD). The group GMT (values in the table) and 95% confidence interval for each time point are shown.

FIG. 13 analysis of HLA class I T cell epitope conservation between MN908947 and Omacron BA.1 variants. HLA class I restricted spike protein epitopes are plotted according to their position along the spike protein (bottom row) (top row) with T cell reactivity identified based on their identity by cd8+ T cells and reported as IEDB (n=244). Epitope indication is defined by the amino acid position of the epitope center; the conserved epitopes in both variants are marked in light grey (n=208); whereas epitopes spanning the Omicron ba.1 mutation site are marked dark grey (n=36). NTD = N-terminal domain; RBD = receptor binding domain; fcs=furin cleavage site. The S1 and S2 regions of spike protein are indicated.

Fig. 14 is a schematic diagram of an exemplary vaccination regimen.

FIG. 15. The queuing, sampling and experimental apparatus for characterization of immune responses in Omicron breakthrough cases. Blood samples were drawn from the following four queues: individuals vaccinated with double or triple BNT162b2 without Omicron infection, and individuals vaccinated with double or triple BNT162b2 with subsequent Omicron BA.1 breakthrough infection. PBMCs and serum were isolated in the non-Omicron-infected cohort at the time points indicated after the last vaccination; for the convalescence cohort, the time from their last vaccination to infection with omacron ba.1 and to infection with PBMC and serum isolation (all values are designated as median range). Serum neutralization ability was assessed using pseudovirus and live virus neutralization tests; assessment of SARS-CoV-2 spike-specific B by flow cytometry-based B cell phenotyping using a plurality of PBMC _MEM And (3) cells. N/A, inapplicable.

FIG. 16 Omicron breakthrough infection induced extensive neutralization of Omicron BA.1, BA.2 and other VOCs in individuals vaccinated with BNT162b2 double and triple.

Serum was drawn from double vaccinated individuals 22 days after the second dose (BNT 162b 2) ² ) (open circles), triple vaccinated individuals 28 days after the third dose (BNT 162b 2) ³ ) (filled circles), double vaccinated individuals with omacron breakthrough infection at 46 days post-infection (BNT 162b 2) ² + Omi) (open triangles), and triple vaccinated individuals with omacron breakthrough infection at 44 days post-infection (BNT 162b 2) ³ + Omi) (closed triangle). Serum testing was performed in duplicate; (A) Shows 50% pseudovirus neutralization (pVN) ₅₀ ) Geometric Mean Titre (GMT), (B) shows 50% virus neutralization (VN ₅₀ ) GMT, and (C) shows SARS-CoV-2 variant of interest (VOC) versus MN908947VN ₅₀ Geometric mean ratio of GMT. LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of MN908947 neutralization group GMT with respect to designated variants and SARS-CoV-1. The multiple adjusted p-values are shown. (A) For MN908947, VOC and SApVN of RS-CoV-1 pseudovirus ₅₀ GMT. (B) VN against living SARS-CoV-2 MN908947, beta, delta and Omicron BA.1 ₅₀ GMT. (C) Group geometric mean ratio and 95% confidence interval for all queues shown in (B).

FIG. 17B of individuals vaccinated with BNT162B2 in duplicate and in triplicate _MEM Cells widely recognize VOCs and receive further boost due to Omicron breakthrough infection. Analysis of SARS-CoV-2 specific B by B-cell decoy staining via flow cytometry from PBMC samples _MEM Cells (B) _MEM -CD3-CD19+CD20+IgD-CD38 ^int/low ) Frequency: double vaccinated individuals (BNT 162b 2) 22 days after the second dose (open squares) and 5 months after the second dose (open circles) ² ) Triple vaccinated individuals 84 days after the third dose (BNT 162b 2) ³ ) (filled circles), double vaccinated individuals with omacron breakthrough infection at 46 days post-infection (BNT 162b 2) ² + Omi) (open triangles), and triple vaccinated individuals with omacron breakthrough infection at 44 days post-infection (BNT 162b 2) ³ + Omi) (closed triangle). (A) SARS-CoV-2S protein tetramer decoy pair B using fluorescent dye labeling that allows discrimination of variant recognition _MEM Schematic of one-dimensional staining of cells. Four groups of individuals were analyzed for MN908947 or VOC full-length S protein specificity B _MEM Cell (B) and RBD specific B _MEM Frequency of cells (C). Specific variant B _MEM Cell frequency was normalized to the frequency of S protein binding (D) and RBD binding (E) of MN 908947. (F) Depicts RBD protein-specific B _MEM Cell and full-length S protein specificity B _MEM Frequency ratio of cells.

FIG. 18 Omicron breakthrough infections of BNT162B2 double and triple vaccinated individuals were mainly enhanced with B _MEM Resistance to conserved epitopes widely shared between proteins of MN 908947S and other VOCs, rather than strict OmicronS-specific epitopes. Analysis of SARS-CoV-2 specific memory B cells (B) from the following PBMC samples by B cell decoy staining via flow cytometry _MEM -CD3-CD19+CD20+IgD-CD38 ^int/low ) Frequency ((schematic in a)): at the second stageDouble vaccinated individuals (BNT 162b 2) 22 days after dose (open square) and 5 months after second dose (open circle) ² ) Triple vaccinated individuals 84 days after the third dose (BNT 162b 2) ³ ) (filled circles), double vaccinated individuals with omacron breakthrough infection at 46 days post-infection (BNT 162b 2) ² + Omi) (open triangles), and triple vaccinated individuals with omacron breakthrough infection at 44 days post-infection (BNT 162b 2) ³ + Omi) (closed triangle). (B) Representative flowsheets of omacron and MN 908947S protein binding and RBD binding for each of the four groups of individuals investigated are shown. For vaccinators subjected to double and triple BNT162B2 vaccination with omacron as well as uninfected omacron, B _MEM The frequency of binding Omicron, MN908947, or both (shared) is shown in (C) for the full-length S protein and (D) for RBD. (E) Venn diagram visualization combination (Boolean) gating strategy to identify cross-reactions B that simultaneously recognize all four variants (all 4+ve) _MEM And B recognizing only Omicron (only Omi) or only MN908947 (only MN 908947) S protein _MEM . In four different groups of individuals investigated, these three B's were compared _MEM Frequency of full-length S protein (F) and RBD (G) of the subgroup. B (B) _MEM The RBD variant recognition pattern of (c) was evaluated by Boolean (boost) flow cytometry gating strategy, and the frequency of recognition of MN908947 and/or variant RBD combinations is shown in (H) for all omacron convalescence subjects (pooled double and triple vaccinators, n=13). (I) Specific B by RBD after Omicron breakthrough infection _MEM Conserved sites within the identified RBD domains. The average is indicated in C, D, F and G. n = number of individuals per group.

FIG. 19. Omicron breakthrough infection of individuals vaccinated with other approved covd-19 vaccines or cocktail protocols resulted in immune sera that extensively neutralized Omicron BA.1, BA.2 and other VOCs as well as SARS-CoV-1. Serum was drawn from 10 individuals vaccinated with other approved covd-19 vaccines or mixed regimens at a median day 43 post infection (diamonds). Serum was tested in duplicate; variants of BA.1 and BA.2 were plotted against SARS-CoV-2 MN908947, alpha, beta, delta and Omicron And single 50% pseudovirus neutralization of SARS-CoV-1 (pVN) ₅₀ ) Geometric Mean Titer (GMT). LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of MN908947 neutralization group GMT with respect to designated variants and SARS-CoV-1. The multiple adjusted p-values are shown. Approved vaccines include AZD1222, BNT162b2 (in some embodiments as part of a 4 dose series), ad26.cov2.s, mRNA-1273 (administered as a two or three dose series), and combinations thereof.

FIG. 20 50% neutralization titers of serum collected 1 month after the fourth dose of BNT162b2 or Omicron BA.1 specific enhancer. Subjects who had previously received two doses of BNT162b2 and a third (booster) dose of BNT162b2 (30 ug) received one dose (30 ug) (i) of RNA encoding SARS-CoV-2S protein from the omacron ba.1 variant (e.g., as described herein (referred to herein as an "omacron ba.1-specific RNA vaccine"), or (ii) BNT162b2 as a fourth (booster) dose, serum from subjects was collected 1 month after administration of the 4 th (booster) dose, group GMT (higher than the bar value) showing 95% confidence interval, "b2" refers to serum from subjects administered MN 908947-specific RNA vaccine as the 4 th (booster) dose BNT162b2, "OMI" refers to serum from subjects administered with omacron ba.1-specific 4 (booster) dose, also showing a change in titer (pre-vaccination/post-dose) from the 4 th dose administration to the 4 th dose, and an average ratio of geometric fold increase in the geometric fold ratio of the 2 nd to the 2 nd (gmb) of the carrier as compared to the geometric fold observed for the subjects as the ratio of the geometric fold increase in the 2-specific RNA vaccine to the 4 th dose to the subjects administered to the 2 nd (gmb). "FFRNT" refers to a fluorescence focus reduction neutralization test. Neutralization data were obtained using FFRNT assay with viral particles containing SARS-CoV-2S protein from the variants shown in the figures. (A) The neutralizing antibody titers against SARS-CoV-2-S pseudovirus comprising a SARS-CoV-2S protein having a mutation that is characteristic of an Omacron BA.1 variant were compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (B) The neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising a SARS-CoV-2S protein having a mutation that is characteristic of an omacron ba.1 variant in serum from a population including subjects previously or presently infected with SARS-CoV-2 (as determined by an antigen assay or PCR assay, respectively) were compared. (C) Neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising SARS-CoV-2S protein from the MN908947 strain were compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (D) The titers of neutralizing antibodies against SARS-CoV-2 pseudoviruses comprising SARS-CoV-2S protein from the MN908947 strain in serum from a population including individuals previously or presently infected with SARS-CoV-2 (as determined by an antigen assay or PCR assay, respectively) were compared. (E) Neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising a mutant SARS-CoV-2S protein that is characteristic of delta variants were compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (F) The neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising the SARS-CoV-2S protein having a mutation that is characteristic of a delta variant in serum from a population including subjects previously or presently infected with SARS-CoV-2 (as determined by an antigen assay or PCR assay, respectively) are compared.

FIG. 21 neutralization of SARS-CoV-2 pseudovirus with modRNA encoding variant specific S protein 7 days after immunization. Mice were immunized twice with an LNP formulated vaccine comprising: (i) BNT162B2 (encoding SARS-CoV-2S protein from MN908947 strain), (ii) RNA encoding SARS-CoV-2S protein having a mutation that is characteristic of Omicron ba.1 variant (Omi), (iii) RNA encoding mutant S protein having a mutation that is characteristic of Delta variant, (iv) combination of BNT162B2 with RNA encoding a protein having a mutation that is characteristic of Omicron ba.1 variant (b2+ Omi), or (v) RNA encoding SARS-CoV-2S protein having a mutation that is characteristic of Delta variant with RNA encoding SARS-CoV-2S protein having a mutation that is characteristic of Omicron ba.1 variant (delta+ Omi). At 7 days post second immunization, animals were bled and tested for neutralization of SARS-CoV-2-S pseudovirus in serum, which pseudovirus contained the SARS-CoV-2S protein from the MN908947 strain, or had the BA.1 variant as beta, delta or OmicronMutated SARS-CoV-2S protein characteristic of the body. Figure pVN ₅₀ Serum dilution (50% reduction in infection events compared to positive control without serum). One dot in the figure represents one mouse. Each mouse sample was measured in duplicate. Mean + SEM are shown for each group with horizontal bars with whiskers. LLOD, lower limit of detection. ULOD, upper limit of detection.

FIG. 22. RNA encoding SARS-CoV-2S protein with mutations characteristic of Beta variants increases neutralizing antibody titres against SARS-CoV-2S when administered to a patient previously administered two doses of vaccine encoding SARS-CoV-2 protein of MN908947 strain. The subjects previously administered two doses of the RNA vaccine encoding SARS-CoV-2S protein of MN908947 strain were administered a third dose and a fourth dose of the RNA vaccine encoding SARS-CoV-2S protein having a mutation that is characteristic of the Beta variant. Neutralizing antibody titers measured at the following times: one month (M1 PD 2) after administration of the second dose of RNA vaccine encoding SARS-CoV-2S protein of Wuhan strain, 1 month after administration of the third dose of RNA vaccine encoding SARS-CoV-2S protein having a mutation that is characteristic of SARS-CoV-2 Beta variant, and one month after administration of the fourth dose of RNA vaccine encoding SARS-CoV-2S protein having a mutation that is characteristic of SARS-CoV-2 Beta variant, prior to administration of the RNA vaccine encoding SARS-CoV-2S protein of Wuhan strain (prior to D1-vaccination). The third and fourth doses were administered 1 month apart. GMFR refers to the geometric mean fold increase, which is a measure of the increase in neutralizing antibody titer since the previous vaccine dose (e.g., GMFR after dose 2 (PD 2) is a measure of the increase in neutralizing antibody titer relative to before any vaccine administration (before vaccination). (A) Neutralizing antibody titers measured in virus neutralization assays using virus particles comprising the SARS-CoV-2S protein of the MN908947 strain. (B) Neutralizing antibody titers measured in virus neutralization assays using viral particles comprising SARS-CoV-2S protein with mutations that characterize Beta variants.

FIG. 23 50% neutralization titers of serum collected 7 days after the fourth dose of BNT162b2, omacron BA.1-specific enhancer, or bivalent vaccine. Subjects previously administered two doses of BNT162b2 (30 ug) and a third (booster) BNT162b2 (30 ug) received: (i) a 30ug dose of BNT162b2 (encoding SARS-CoV-2S protein from the MN908947 strain), (ii) a 60ug dose of BNT162b2, (iii) a 30ug dose of RNA encoding SARS-CoV-2S protein having a mutation that is characteristic of an Omicron ba.1 variant (e.g., as described herein (referred to as an "Omicron ba.1 specific RNA vaccine")), (iii) a 60ug dose of RNA encoding SARS-CoV-2S protein having a mutation that is characteristic of an Omicron ba.1 variant, (iv) a 30ug dose of bivalent vaccine (comprising 15ug BNT162b2 and 15ug encoding RNA comprising a mutation that is characteristic of an Omicron ba.1 variant), or (v) a 60ug dose of bivalent vaccine (comprising 30ug BNT162b2 and 30ug encoding RNA comprising a mutation that is characteristic of an Omicron ba.1 variant. Geometric Mean Ratio (GMR) of titers in serum from subjects was collected 7 days after dose 4 administration. "b2" refers to serum from a subject administered MN908947 specific RNA vaccine as dose 4 BNT162b 2. "OMI" refers to serum from subjects administered omacron BA.1 specific dose 4. "bivalent" refers to serum from a subject administered a composition comprising BNT162b2 and RNA encoding SARS-CoV-2S protein comprising a mutation that is characteristic of the Omicron BA.1 variant as dose 4. Fold increases in titres from before dose 4 to 7 days after dose 4 are also shown (fold increases). "FFRNT" refers to a fluorescence focus reduction neutralization test. Neutralization data were obtained using FFRNT assay with viral particles containing SARS-CoV-2S protein with mutations that are characteristic of the variants shown in the figures. LLOQ is a specified lower limit and ULOQ is a specified upper limit. (A) The neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising a SARS-CoV-2S protein having a mutation that is characteristic of an Omacron BA.1 variant were compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (B) The neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising a mutant SARS-CoV-2S protein having a characteristic as an omacron ba.1 variant in serum from a population comprising subjects previously or presently infected with SARS-CoV-2 (e.g., as determined by an antigen test or PCR assay, respectively) were compared. (C) The neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising the SARS-CoV-2S protein of the MN908947 strain were compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (D) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising the SARS-CoV-2S protein of the MN908947 strain in serum from a population comprising individuals previously or presently infected with SARS-CoV-2. (E) Neutralizing antibody titers against SARS-CoV-2 pseudoviruses comprising a SARS-CoV-2S protein having a mutation that is characteristic of the Delta variant are compared. Serum from subjects previously or currently infected with SARS-CoV-2 was excluded. (F) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudovirus comprising a SARS-CoV-2S protein having a mutation that is characteristic of a Delta variant in serum from a population comprising subjects previously or presently infected with SARS-CoV-2. (G) The geometric mean increase in neutralizing antibodies observed in subjects administered with the following was compared to the geometric mean increase observed in subjects administered with 30ug bnt162b2 as dose 4: 60ug BNT162b2, 30ug encoding RNA of SARS-CoV-2S protein having a mutation that is characteristic of an Omicron BA.1 variant (OMI 30 ug), 60ug encoding RNA of SARS-CoV-2S protein having a mutation that is characteristic of an Omicron BA.1 variant (OMI 60 ug), 30ug comprising 15ug BNT162b2 and 15ug encoding a bivalent vaccine of RNA of SARS-CoV-2S protein having a mutation that is characteristic of an Omicron BA.1 variant (bivalent 30 ug), or 60ug comprising 30ug BNT162b2 and 30ug encoding RNA bivalent vaccine of SARS-CoV-2S protein having a mutation that is characteristic of an RNA Omicron BA.1 variant (bivalent 60 ug). Results are shown for the population pool excluding subjects previously or currently infected with SARS-CoV-2 and the population pool including those subjects.

Figure 24 reactogenicity of certain exemplary RNAs (formulated in LNP) at given doses: subjects administered a 60ug dose of RNA encoding SARS-CoV-2S protein are more likely to exhibit higher injection site pain and exhibit a systemic response similar to those administered a 30ug dose of RNA. The subjects were administered 30ug or 60ug of RNA encoding SARS-CoV-2S protein from MN908947 strain (BNT 162b2, corresponding to G1 and G2 groups, respectively), 30ug or 60ug of RNA encoding SARS-CoV-2 protein with mutations characteristic of Omicron ba.1 variant (BNT 162b2OMI, corresponding to G3 and G4 groups, respectively), 30ug of RNA encoding SARS-CoV-2S protein from MN908947 strain and 15ug of bivalent vaccine encoding SARS-CoV-2S protein with mutations characteristic of Omicron ba.1 variant (BNT 162b2 (15 ug) +bnt162b2OMI (15 ug), corresponding to G5 group), or 60ug of RNA encoding SARS-CoV-2 protein from MN908947 strain and 30ug of RNA encoding SARS-CoV-2 protein with mutations characteristic of Omicron ba.1 (BNT 2) (b 2 of BNT162b2, corresponding to G5 group), 30ug of bivalent vaccine (162 ug). (A) Local reactions observed within 7 days after injection, including redness, swelling and pain at the injection site. Pain at the injection site was found to be increased in subjects injected with 60ug of RNA or bivalent vaccine encoding SARS-CoV-2S protein comprising a mutation that is characteristic of the omacron ba.1 variant, compared to the other test doses. (B) Systemic reactions observed within 7 days after injection, including fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain and medication. Systemic reactions were observed to be approximately similar between the different groups within 7 days. The fatigue tendency was found to be higher after the administration of the 60ug dose compared to the 30ug dose.

FIG. 25 50% neutralization titers of serum collected 1 month after the fourth dose of BNT162b2, omacron BA.1 specific enhancer, or bivalent vaccine against the Omacron BA.1 variant. Subjects previously administered two doses of BNT162b2 (30 ug) and a third (booster) BNT162b2 (30 ug) were administered: (i) a 30ug dose of BNT162b2 (encoding SARS-CoV-2S protein from MN908947 strain), (ii) a 60ug dose of BNT162b2, (iii) a 30ug dose of RNA encoding SARS-CoV-2S protein with mutations characteristic of Omicron ba.1 variant ("BNT 162b2 OMI"), (iii) a 60ug dose of BNT162b2OMI, (iv) a 30ug dose of bivalent vaccine ("bivalent") comprising 15ug BNT162b2 and 15ug BNT162b2OMI, or (v) a 60ug dose of bivalent vaccine comprising 30ug t162b2 and 30ug BNT162b2 OMI. The Geometric Mean Titer (GMT) of neutralizing antibodies was measured in serum from subjects collected 1 month after dose 4 administration (GMT indicated above each column). The geometric fold increase (GMFR) of titers from pre-dose 4 (pre-vaccination) to 1 month post-dose 4 (1 MPD) is shown below the x-axis for each patient group. Neutralization data were obtained using a neutralization assay using viral particles containing the SARS-CoV-2S protein having a mutation that is characteristic of the OmicronBA.1 variant.

Figure 26. Omicron ba.1 breakthrough infection in BNT162b2 double and triple vaccinated individuals induced extensive neutralization of Omicron ba.1, ba.2 and other VOCs, but less neutralization of ba.4 and ba.5. The figure is an extension of figure 16, including data neutralization activity against Omicron ba.4 and ba.5. Serum was tested in duplicate as described in fig. 16; 50% pseudovirus neutralization was plotted (pVN) ₅₀ ) Geometric Mean Titre (GMT) (in a and B), and for MN908947 pVN ₅₀ GMT normalized SARS-CoV-2 variant of interest (VOC) and SARS-CoV-1pVN ₅₀ Geometric mean ratio of GMT (in C). LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of MN908947 neutralization group GMT with respect to designated variants and SARS-CoV-1. The multiple adjusted p-values are shown. (A) pVN against MN908947, VOC and SARS-CoV-1 pseudovirus in patients receiving two or three doses of BNT162b2 ₅₀ GMT. (B) pVN against MN908947, VOC and SARS-CoV-1 pseudovirus in patients receiving two or three doses of BNT162b2 and having been previously infected with the Omicron BA.1 variant of SARS-CoV-2 ₅₀ GMT. (C) Group geometric mean ratio and 95% confidence interval for all the queues shown in (a) and (B).

FIG. 27 Omicron BA.1 breakthrough infection of individuals vaccinated with other approved covd-19 vaccines or mixed regimens resulted in extensive neutralization of Omicron BA.1, BA.2 and other VOCs, but less immune serum neutralization of BA. The figure is an extension of figure 19, including data neutralization activity against Omicron ba.4 and ba.5. Serum was tested in duplicate as depicted in fig. 19; single 50% pseudovirus neutralization for SARS-CoV-2MN908947, alpha, beta, delta and Omicron BA.1, BA.2 and BA.4/5 variants are plotted (pVN) ₅₀ ) Geometric Mean Titer (GMT). LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. Non-parameter with Dunn multiple comparison correctionThe friedman test was used to compare titers of MN908947 neutralization group GMT with respect to the specified variants and SARS-CoV-1. The multiple adjusted p-values are shown.

FIG. 28 sequence of RBD of SARS-COV-2MN908947 strain and variants thereof. Variant specific amino acid changes are indicated in bold red font and original MN908947 amino acids are highlighted in bold blue font.

Fig. 29. Queue and sampling of the study described in example 14. Schematic diagrams for testing the immune response of triple vaccinated patients who were (i) not contacted with Omicron, (ii) infected with Omicron ba.1 variant, or (iii) infected with Omicron ba.2 variant are shown. Blood samples were drawn from the following three queues: non-Omicron infected individuals triple vaccinated with BNT162b2 (BNT 162b 2) ³ Green), and individuals vaccinated with homologous or heterologous three dose regimens that subsequently developed omacron breakthrough infections when ba.1 was dominant (11 months 2021 to 1 month 2022; all vaccine + ba.1, purple) or individuals who developed Omicron breakthrough infection when germany ba.2 was dominant (2022, 3 months to 5 months; all vaccine + ba.2, blue). Serum (droplets) was isolated in the non-Omicron infected cohort at the time point indicated after the last vaccination; for the convalescence cohort, the time from their last vaccination to infection by omacron and from infection to serum isolation is indicated. All values are designated as median ranges. Serum neutralization capacity was assessed using a pseudovirus neutralization test.

FIG. 30 from BNT162b2 ³ And 50% pseudovirus neutralization of the total vaccine+omiba.1 breakthrough infection cohort (pVN) ₅₀ ) Geometric Mean Titer (GMT). Serum was drawn from a three-vaccinated individuals with BNT162b2 (BNT 162b 2) that were not infected with Omacron 28 days after the third dose ³ Circles), and vaccinated individuals who subsequently developed omacron ba.1 breakthrough infection at a median day after infection (total vaccine+omiba.1, triangles). 50% pseudovirus neutralization of individuals not infected with Omicron (pVN ₅₀ ) Geometric Mean Titers (GMT) are plotted in (a), while GMT of individuals presenting ba.1 breakthrough infections are plotted in (B). This data was previously published in Quandt et al (Omi)cron ba.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved peptides, "Science immunology, eabq2427 (2022), doi: 10.1126/sciimmunol.abq2427), except for ba.2.12.1 neutralization data. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of MN908947 neutralization group GMT with respect to designated variants and SARS-CoV-1. The multiple adjusted p-values are shown.

FIG. 31. Omicron BA.2 breakthrough infection of previously immunized individuals refocused neutralization against Omicron BA.2 and BA.2 derived subvariants BA.2.12.1 and BA.4/BA.5. Serum was drawn from individuals vaccinated with BNT162b2 triple vaccination who subsequently developed an Omacron BA.1 breakthrough infection at a median day 44 post-infection (BNT 162b2 ³ +omiba.1, triangle), and individuals who subsequently developed Omicron ba.2 breakthrough infections 38 days post infection (BNT 162b 2) with triple BNT162b2 vaccination ³ + Omi ba.2, square). 50% pseudovirus neutralization was plotted (pVN) ₅₀ ) Geometric Mean Titre (GMT) (in A, B), and variants of SARS-CoV-2 interest (VOC) and SARS-CoV-1pVN normalized to MN908947 pVN GMT ₅₀ Geometric mean ratio of GMT (in C). non-Omicron infected individuals triple vaccinated with BNT162b2 (BNT 162b2 ³ Circle) and pVN of individuals vaccinated with BNT162b2 triple vaccine who developed Omicon BA.1 breakthrough infection ₅₀ GMT and geometric mean ratio data were previously published in Quandt et al (Omicron BA.1 breakthrough infection drives cross-variant neutralization and memory B cell formation against conserved epops. "Science immunology, eabq2427 (2022), doi: 10.1126/sciimmunol.abq2427), with the exception of BA.2.12.1 neutralization data. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). The values above the violin graph represent the group GMT. A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of MN908947 neutralization group GMT with respect to designated variants and SARS-CoV-1. Showing the followingMultiple adjusted p-values. (A, B) pVN GMT against MN908947, VOC and SARS-CoV-1 pseudovirus. (C) group geometric mean ratio with 95% confidence interval.

FIG. 32 characterization of SARS-CoV-2S glycoprotein used in neutralization assays based on VSV-SARS-CoV-2 pseudovirus. The sequence of the SARS-CoV-2S glycoprotein of the MN908947-Hu-1 isolate (GenBank: QHD 43416.1) was used as a reference. Amino acid positions, amino acid descriptions (one letter code) and mutation types (substitutions, deletions, insertions) are indicated. NTD, N-terminal domain; RBD, receptor binding domain, delta, deletion; ins, inserting; * A cytoplasmic domain truncated at the C-terminal 19 amino acids.

FIG. 33 changes in the amino acid sequence of the spike glycoprotein of the SARS-CoV-2 Omacron subline. Amino acid positions, amino acid descriptions (one letter code) and mutation types (substitutions, deletions, insertions) are indicated. The white letters in the boxes indicate amino acid substitutions for each subline; delta, absence; ins, inserting; NTD, N-terminal domain; RBD, receptor binding domain.

Fig. 34 immunization protocol for study with VOC boosters. According to the indicated schedule, BALB/c mice were immunized with two doses (1 ug per dose) of the original BNT162b2 vaccine, and then at least one dose (1 ug total) of the following monovalent, divalent, or trivalent booster: (a) raw BNT162b2 ("BNT 162b 2"); (b) bnt162b2omiba.1 ("omiba.1"); (c) BNT162b2OMIBA.4/5 ("OMIBA.4/5"); or a combination thereof.

Fig. 35, baseline grouping and GMT. The geometric mean titers of neutralizing antibodies against various strains were assessed for sera drawn from mice immunized as shown in figure 34 (day 104, pre-boost). The data is grouped by queue.

Fig. 36, baseline staggering neutralizes GMT. The geometric mean titers of neutralizing antibodies against various strains were assessed for sera drawn from mice immunized as shown in figure 34 (day 104, pre-boost). The data is presented in an interleaved format (i.e., per strain evaluated for neutralization).

Fig. 37, baseline cross-neutralization. Geometric mean titers of neutralizing antibodies against various strains were assessed for sera drawn from mice immunized as shown in fig. 34 (day 104, prior to boosting). The cross-neutralization results are presented as calculated variant/MN 908947 reference GMT ratio.

Figure 38 geometric mean fold increase after boosting of gmt. Geometric mean fold increases in GMT for neutralizing antibodies to various strains were assessed for sera drawn from mice immunized as shown in figure 34 (day 111, day 7 post boost).

Fig. 39. Neutralizing GMT of the post-emphasis packet. Geometric mean fold increases in GMT for neutralizing antibodies to various strains were assessed for sera drawn from mice immunized as shown in figure 34 (day 111, day 7 post boost). The data is grouped by queue.

Fig. 40 cross-neutralization after reinforcement. Geometric mean fold increases in GMT for neutralizing antibodies to various strains were assessed for sera drawn from mice immunized as shown in figure 34 (day 111, day 7 post boost). The cross-neutralization results are presented as calculated variant/MN 908947 reference GMT ratio.

Fig. 41, queues and sampling. Serum samples (droplets) were drawn from the following three queues: triple vaccinated individuals with BNT162b2 (BNT 162b 2) not infected with SARS-CoV-2 at the time of sampling ³ ) And subsequent Omacron breakthrough infection (month 11 of 2021 to month 1 of 2022) at ba.1 predominance with three doses of mRNA COVID-19 vaccine (BNT 1162b2/mRNA-1273 homologous or heterologous regimen); mRNA vaccine ³ +ba.1) or when ba.2 is dominant (3 months to 5 months of 2022; mRNA vaccine ³ +ba.2) individuals who developed Omicron breakthrough infection. For the convalescence cohort, relevant intervals between critical events (such as recent vaccination, SARS-CoV-2 infection and serum separation) are indicated. All values are designated as median ranges. N/A, inapplicable.

FIG. 42. Omicron BA.2 breakthrough infection of triple mRNA vaccinated individuals induced extensive neutralization of SARS-CoV-2 variant pseudoviruses including Omicron BA.4/5. The queues and serum samples are as described in figure 41. (A) 50% pseudovirus neutralization for indicated SARS-CoV-2 variants of interest (VOCs) or SARS-CoV-1 pseudoviruses (pVN) ₅₀ ) Geometric Mean Titer (GMT). Small handleThe values above the organ plot represent group Geometric Mean Titer (GMT). BNT162b2 ³ Represents a triple vaccinated individual not infected with SARS-CoV-2: mRNA vaccine ³ +ba.1 represents a triple vaccinated individual who subsequently developed a breakthrough infection with the omicronba.1 variant; and mRNA-vaccine ³ +ba.2 represents a triple vaccinated individual who subsequently developed a breakthrough infection with the Omicron ba.2 variant. (B) Against wild-type strain pVN ₅₀ GMT normalized SARS-CoV-2 variant of interest (VOC) pVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratios with 95% confidence intervals are shown in the figure and listed in the table. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of wild-type strain neutralization group GMT with respect to indicated variants and SARS-CoV-1. The multiple adjusted p-values are shown.

FIG. 43. Omicron BA.2 breakthrough infection of previously vaccinated individuals induced extensive neutralization of authentic live SARS-CoV-2 variants, including Omicron BA.4/5. The queues and serum samples are as described in figure 41. (A) 50% Virus Neutralization (VN) for indicated SARS-CoV-2 variants of interest (VOCs) ₅₀ ) Geometric Mean Titer (GMT). The values listed above the violin diagram represent the group GMT. (B) For wild strain VN ₅₀ GMT normalized SARS-CoV-2 VOC VN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratios with 95% confidence intervals are shown in the figure and listed in the table. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of wild-type strain neutralization group GMT with respect to indicated variants and SARS-CoV-1. The multiple adjusted p-values are shown.

FIG. 44 neutralization of Omacron BA.2 and BA.4/5 by serum of individuals in the recovery phase of BA.2 vaccinated with triple mRNA was mediated to a large extent by NTD targeting antibodies. The queues and serum samples are as described in figure 41. (A) Serum samples (n=6 per cohort) were depleted of RBD binding antibodies or NTD binding antibodies. RBD and NTD depleted serum was calculated (unconsumedpVN of the control serum of Dragon's blood ₅₀ Titres were set to 100%) relative neutralization activity against wild-type strains ba.1, ba.2 and ba.4/5 and group geometric mean with 95% confidence interval was shown. (B) 50% pseudovirus neutralization against Omicron BA.4/5 and Omicron BA.1-BA.4/5 hybrid pseudoviruses (pVN) ₅₀ ) Geometric Mean Titer (GMT). The numbers above the figures represent the fold change in group Geometric Mean Titer (GMT) and GMT between ba.4/5 and the hybrid pseudovirus. LOD/2 values are plotted for titer values below the limit of detection (LOD).

FIG. 45 Omacron BA.2 breakthrough infection of individuals triple vaccinated with BNT162b2 induced extensive neutralization of VOCs including Omacron BA.4/BA.5. The queues and serum samples are as described in figure 41. (A) - (B) 50% pseudovirus neutralization (pVN) against indicated SARS-CoV-2 variants of interest (VOCs) or SARS-CoV-1 pseudoviruses ₅₀ ) Geometric Mean Titer (GMT). The values listed above the violin diagram represent the group GMT. (C) Against wild-type strain pVN ₅₀ GMT normalized SARS-CoV-2VOC pVN ₅₀ GMT ratio. The ratio of the geometric mean of the cohorts for occurrence of Omicron ba.2 breakthrough infections to BNT162b2 ³ And BNT162b2 ³ +BA.1 was compared. Group geometric mean ratio with 95% confidence interval is shown. (D) - (E) BNT162b2 ³ +BA.2 and BNT162b2 ³ 50% virus neutralization of +BA.1 (VN ₅₀ ) GMT. The values listed above the violin diagram represent the group GMT. (F) For wild strain VN ₅₀ GMT is normalized to the SARS-CoV-2 VOCGMT ratio. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). A nonparametric friedemann test with Dunn multiple comparison correction was used to compare group GMT with wild type strain and group GMT with indicated variants and SARS-CoV-1. The multiple adjusted p-values are shown.

Figure 46.50% pseudovirus neutralization (pVN ₅₀ ) With 50% active SARS-CoV-2 neutralization (VN ₅₀ ) Titer data was related. VSV-SARS-CoV-2 pVN of n=45 serum samples taken from the following individuals ₅₀ And living SARS-CoV 2 VN ₅₀ Non-parametric Spearman correlation of titers: BNT16 of uninfected SARS-CoV-2 after the third dose2b2 triple vaccinated individuals (BNT 162b2 ³ The method comprises the steps of carrying out a first treatment on the surface of the n=18), individuals vaccinated with mRNA triple and subsequently developed omacron ba.1 breakthrough infection after infection (mRNA-vaccine ³ +ba.1; n=14), and individuals vaccinated with triple mRNA and subsequently developed omacron ba.2 breakthrough infections after infection (mRNA-vaccine ³ +ba.2; n=13). Correlations were plotted for each SARS-CoV-2 variant. The correlation coefficient r, the two-tailed P value and the linear equation are given.

Fig. 47 rbd-binding antibodies and NTD-binding antibodies can be depleted in human serum. Serum was drawn from BNT162b2 triple vaccinated individuals not infected with SARS-CoV-2 (BNT 162b 2) ³ The method comprises the steps of carrying out a first treatment on the surface of the n=6), and Omicron ba.1 infection (mRNA-vaccine) occurred ³ +ba.1; n=6) or Omicron ba.2 infected triple RNA vaccinated individuals (mRNA-vaccine) ³ +ba.2; n=6). The magnetic bead technique is used to deplete RBD binding antibodies or NTD binding antibodies in serum, or to mimic depletion. Schematic of the depletion of (a) antibodies from serum. (B) The relative concentrations of RBD-binding antibodies and NTD-binding antibodies are determined by multiplex electrochemiluminescence immunoassay. A relative decrease in antibody concentration in the depleted serum compared to the mock-depleted serum is shown. The numbers above the graph show the geometric mean reduction in the group.

FIG. 48 characterization of SARS-CoV-2S glycoprotein used in a live-based assay for authentic SARS-CoV-2. The sequence of the SARS-CoV-2S glycoprotein of the MN908947-Hu-1 isolate (GenBank: QHD 43416.1) was used as a reference. Amino acid positions, amino acid descriptions (one letter code) and types of changes (substitutions, deletions, insertions) are indicated. NTD, N-terminal domain; RBD, receptor binding domain, delta, deletion; ins, inserting; * A cytoplasmic domain truncated at the C-terminal 19 amino acids.

FIG. 49 BA.4/5-breakthrough infection and BA.4/5-booster study design. (a) The effect of Omicron ba.4/ba.5 breakthrough infection on serum neutralization activity was evaluated in individuals vaccinated with three doses of mRNA covd-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) and subsequently subjected to Omicron ba.4 or ba.5 infection. The intervals between vaccination, breakthrough infection and sampling are expressed as median/range. (b) The effect of omacron ba.4/ba.5 adaptive boost on serum neutralization activity was investigated in mice vaccinated twice with BNT162b2 (21 days between vaccine) followed by a booster dose of ba.4/ba.5 adaptive vaccine after 3.5 months. Neutralization activity was assessed 7, 21 and 35 days (D7D 3, D21D3, D35D3, respectively) before (before D3) and after (after) the boost. (c) The effect of omacron ba.4/ba.5 adaptive vaccine on serum neutralization activity was investigated in unvaccinated mice vaccinated with two ba.4/ba.5 adaptive vaccines (21 days between vaccine). Neutralization activity was assessed 14 days after the second dose administration (D14D 2).

FIG. 50. Omicron BA.4/BA.5 breakthrough infection of individuals vaccinated with triple mRNA mediated broad Omicron neutralization. The queues and serum samples are as described in figure 53. (a) mRNA vaccine ³ 50% pseudovirus neutralization in +BA.4/BA.5 serum against indicated SARS-CoV-2 variants of interest (VOCs) or SARS-CoV-1 pseudoviruses (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. (b) mRNA vaccine ³ +BA.4/BA.5 against wild type strain pVN ₅₀ GMT normalized SARS-CoV-2VOC pVN ₅₀ GMT (ratio of VOC to wild type) and reference queues are summarized in fig. 53. Group geometric mean ratio with 95% confidence interval is shown. (c) mRNA vaccine ³ 50% Virus Neutralization (VN) in +BA.4/BA.5 serum against indicated SARS-CoV-2VOC ₅₀ ) GMT. (d) For wild strain VN ₅₀ GMT normalized SARS-CoV-2VOC VN ₅₀ GMT (VOC to wild type ratio). Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). A nonparametric friedemann test with Dunn multiple comparison correction was used to compare titers of wild-type strain neutralization group GMT with respect to indicated variants and SARS-CoV-1. The multiple adjusted p-values are shown.

FIG. 51. Booster immunization with Omicron BA.4/BA.5S glycoprotein-adapted RNA vaccine mediated pan Omicron neutralization in double vaccinated mice. BALB/c mice (n=8) were given two doses of 1 μg BNT162b2 intramuscularly at 21 days intervals, and the mice were given a third dose of BNT162b2 (1 μg) or indicated monovalent intramuscularly 104 days after the first vaccination (1. Mu.g) or bivalent (0.5. Mu.g each) Omacron BA.1 or BA.4/5-adapted vaccine. (a) 50% pseudovirus neutralization of indicated SARS-CoV-2 variants of interest (VOCs) in serum collected 21 days after third vaccination (D21D 3) (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. (b) pVN at D21D3 ₅₀ Geometric mean fold increase in titer (GMFI) relative to baseline titer before third vaccination. The value above the bar represents the group GMFI. (c) Against wild-type strain pVN ₅₀ GMT normalized SARS-CoV-2VOCpVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. (d) pVN against BA.1 and BA.4/5 over time relative to baseline titers prior to boost with BNT162b2/BA.1 or BNT162b2/BA.4/5 bivalent vaccine ₅₀ Titer GMFI. LOD/2 values are plotted for titer values below the limit of detection (LOD). Error bars represent 95% confidence intervals. (e) 50% Virus Neutralization (VN) of indicated SARS-CoV-2VOC at D21D3 ₅₀ ) GMT. The value above the bar represents the group GMT. Error bars represent 95% confidence intervals. (f) For wild strain VN ₅₀ GMT normalized SARS-CoV-2VOC VN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). Error bars represent 95% confidence intervals.

FIG. 52 immunization with BNT162b2RNA vaccine supplemented with Omicron BA.4/BA.5S glycoprotein driven pan-Omicron neutralization in previously unvaccinated mice. Two doses of BNT162b2 (1 μg) or indicated monovalent (1 μg) or bivalent (0.5 μg each) Omicron ba.1 or ba.4/5 adaptive vaccine were injected intramuscularly to unvaccinated BALB/c mice (n=5) 21 days apart. (a) 50% pseudovirus neutralization of indicated SARS-CoV-2 variants of interest (VOCs) in serum collected 14 days after the second vaccination (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. Error bars represent 95% confidence intervals. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). "wild-type" refers to SARS-CoV-2S comprising the original MN908947 variantNeutralization titer of pseudoviruses of the protein.

Fig. 53 queues and samples. Serum samples (droplets) were from the following four queues: individuals vaccinated with three doses of mRNA COVID-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) and subsequently developed an Omacron BA.4/BA.5 breakthrough infection (mRNA-vaccine) ³ +BA.4/BA.5). Breakthrough infection occurred when ba.4/ba.5 was dominant and/or was confirmed as a variation by genomic sequencing. The study included three cohorts as references: triple mRNA vaccination with predominance of BA.2 (3 months 2022 to 5 months 2022; mRNA vaccine) ³ +ba.2) or ba.1 is dominant (month 11 of 2021 to month 1 of 2022; mRNA vaccine ³ +BA.1) individuals who underwent breakthrough infection, or individuals who were triple vaccinated with BNT162b2 (BNT 162b 2) without SARS-CoV-2 infection at the time of sampling ³ ). For the convalescence cohort, relevant intervals between critical events (such as recent vaccination, SARS-CoV-2 infection and serum separation) are indicated. All values are designated as median ranges. N/A, inapplicable.

FIG. 54 design of Omicon adaptive vaccine. Characterization of SARS-CoV-2S glycoprotein encoded by variant specific RNA vaccines (e.g., mRNA vaccines in some embodiments). The sequence of the SARS-CoV-2S glycoprotein of the MN908947-Hu-1 isolate (GenBank: QHD 43416.1) was used as a reference. Amino acid positions, amino acid descriptions (one letter code) and types of changes (substitutions, deletions, insertions) are indicated. NTD, N-terminal domain; RBD, receptor binding domain, delta, deletion; ins, insert. In some embodiments, the variant specific vaccine further comprises a mutation that stabilizes the pre-fusion conformation (e.g., a proline mutation at positions corresponding to residues 986 and 987 of SEQ ID NO: 1) and/or does not comprise a C-terminal truncation.

Figure 55.Omicron specific vaccine showed comparable RNA purity and integrity, as well as in vitro expression of antigen. (a) liquid capillary electrophoresis of in vitro transcribed samples. (b) Surface expression of BNT162b2 and omacron-adapted vaccine in HEK293T cells was measured using mFc-labeled human ACE-2 as detection reagent, which was then analyzed in flow cytometry. HEK293T cells were transfected with: BNT162b2 or Omicron-adapted vaccine formulated as lipid nanoparticles or vaccine RNA mixed with commercial transfection reagents, or no vaccine/RNA (untransfected). Bar height represents the mean of the technical replicates.

FIG. 56 the breadth and intensity of neutralization activity against SARS-CoV-2 variant in mice vaccinated with BNT162b2 prior to booster vaccination was comparable. BALB/c mice were injected intramuscularly with two doses of 1 μg BNT162b2 at 21 days intervals. Mice were assigned to each group (n=8) prior to administration of booster-time indicator vaccine. On day 104 after the first vaccination, serum was collected from mice prior to the boost vaccine injection. (a) 50% pseudovirus neutralization for indicated SARS-CoV-2 variants of interest (VOCs) (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. Error bars represent 95% confidence intervals. (b) S against wild strain VN ₅₀ GMT normalized SARS-CoV-2VOC pVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD).

FIG. 57. Booster immunization with Omicron BA.4/BA.5S glycoprotein-adapted mRNA vaccine mediated pan Omicron neutralization in mice 7 days after administration of the booster. BALB/c mice (n=8) were given two doses of 1 μg BNT162b2 (21 days interval between doses) and the mice were given a third dose of BNT162b2 (1 μg) or indicated monovalent (1 μg) or bivalent (0.5 μg each) Omicron ba.1 or ba.4/5 compliant vaccine (104 days after the first vaccination). (a) 50% pseudovirus neutralization of indicated SARS-CoV-2 variants of interest (VOCs) in serum collected 7 days after third vaccination (D7D 3) (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. (b) pVN at D7D3 ₅₀ Geometric mean fold increase in titer (GMFI) relative to baseline titer before third vaccination. The value above the bar represents the group GMFI. (c) Against wild-type strain pVN ₅₀ GMT normalized SARS-CoV-2VOCpVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). Error bar Indicating a 95% confidence interval.

FIG. 58. Booster immunization with Omicron BA.4/BA.5S glycoprotein adaptive vaccine mediated pan Omicron neutralization in mice 35 days after the booster. Two doses of 1 μg BNT162b2 (21 days between doses) were injected intramuscularly into BALB/c mice (n=8), and the mice were injected intramuscularly with a third dose of BNT162b2 (1 μg) or indicated monovalent (1 μg) or bivalent (0.5 μg each) Omicron ba.1 or ba.4/5 compliant vaccine 104 days after the first vaccination. (a) 50% pseudovirus neutralization of indicated SARS-CoV-2 variants of interest (VOCs) in serum collected 35 days after third vaccination (D35D 3) (pVN) ₅₀ ) Geometric Mean Titer (GMT). The value above the bar represents the group GMT. (b) pVN at D35D3 ₅₀ Geometric mean fold increase in titer (GMFI) relative to baseline titer before third vaccination. The value above the bar represents the group GMFI. (c) Against wild-type strain pVN ₅₀ GMT normalized SARS-CoV-2VOCpVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. (d) 50% Virus Neutralization (VN) at D35D3 against indicated SARS-CoV-2VOC ₅₀ ) GMT. The value above the bar represents the group GMT. Error bars represent 95% confidence intervals. (e) For wild strain VN ₅₀ GMT normalized SARS-CoV-2 VOC VN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio is shown. Serum was tested in duplicate. LOD/2 values are plotted for titer values below the limit of detection (LOD). Error bars represent 95% confidence intervals.

FIG. 59. Omicron BA.4/BA.5 breakthrough infection of triple mRNA vaccinated individuals induced cross-neutralization of Omicron BA.4.6 and BA.2.75. The queues and serum samples are as described in figure 61. (a) 50% pseudovirus neutralization against indicated SARS-CoV-2 wild-type strain or Omicron variant of interest (VOC) (pVN ₅₀ ) Geometric Mean Titer (GMT). The values above the bar graph represent the group GMT. LOD/2 values are plotted for titer values below the limit of detection (LOD). The nonparametric Friedman test with Dunn multiple comparison correction was used to compare the neutralization titers against Omacron BA.4/BA.5 pseudoviruses (which represent the currently dominant BA.5) to titers against other pseudovirusesCompared with the prior art. The multiple adjusted p-values are shown. (b) S against wild strain VN ₅₀ GMT normalized SARS-CoV-2 VOC pVN ₅₀ GMT (VOC to wild type ratio). Group geometric mean ratio with 95% confidence interval is shown. A nonparametric Kruskal-Wallis test with Dunn multiple comparison correction was used to compare the VOC GMT ratio between the queues. * P < 0.0001; * P < 0.01; * P is less than 0.05. Serum was tested in duplicate.

FIG. 60 changes in the amino acid sequence of the spike glycoprotein of the SARS-CoV-2 Omacron subline. The white letters in the boxes indicate amino acid substitutions for each subline; delta, absence; ins, inserting; NTD, N-terminal domain; RBD, receptor binding domain.

Fig. 61, queues and sampling. Serum samples were drawn from the following five queues: triple vaccination with BNT162b2 (BNT 162b 2) ³ ) Or individuals with non-infected SARS-CoV-2 that were quadrupled with BNT162b2 (BNT 162b 24), and vaccinated with three doses of mRNA COVID-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) and subsequently developed Omicron BA.1 breakthrough infection (mRNA-vaccine ³ +BA.1), the occurrence of BA.2 breakthrough infection (mRNA-vaccine ³ +BA.2) or the occurrence of a BA.4/BA.5 breakthrough infection (mRNA-vaccine) ³ +BA.4/5). Breakthrough infections occur when the corresponding VOCs predominate (BA.1:2021, 11 months to 2021, 1 month, BA.2:2022, 3 months to 2022, 5 months, BA.4/5:2022, 6 middle ten to 7 middle ten) and/or are confirmed as variants by genomic sequencing. For the convalescence cohort, relevant intervals between critical events (such as recent vaccination, SARS-CoV-2 infection and serum separation) are indicated. All values are designated as median ranges. N/A, inapplicable; ^§ Serum withdrawal was performed between 28 and 35 days post vaccination according to the protocol.

FIG. 62 characterization of SARS-CoV-2S glycoprotein used in a VSV-SARS-CoV-2 variant pseudovirus neutralization assay. Mutation positions are shown relative to the reference MN908947-Hu-1 isolate SARS-CoV-2S glycoprotein (GenBank: QHD 43416.1). Amino acid positions, amino acid descriptions (one letter code) and types of changes (substitutions, deletions, insertions) are indicated. NTD, N-terminal domain; RBD, receptor binding domain, delta, deletion; ins, inserting; * A cytoplasmic domain truncated at the C-terminal 19 amino acids.

FIG. 63 Omacron BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1 and XBB.1 neutralization reactions in the case of bivalent (BA.4/5-adapted RNA+BNT162b 2) or BNT16b2 monovalent enhancers. The bar height and the number directly above the bar represent the geometric mean of the neutralization titer (GMT). 95% CI is indicated. The bar labeled "bivalent" represents the neutralization titer of subjects administered as dose 4 bivalent vaccine (RNA comprising SARS-CoV-2S protein encoding the MN908947 strain and RNA comprising SARS-CoV-2S protein comprising one or more mutations that characterize the ba.4/5 omacron variant); the bar labeled "monovalent" represents the neutralization titer of subjects administered a monovalent vaccine (comprising RNA encoding the SARS-CoV-2S protein of the MN908947 strain) as dose 4. FFRNT50 for USA-WA1/2020 spike, BA.4/5 spike, BA.4.6 spike, BA.2.75.2 spike, BQ.1.1 spike, XBB.1 spike for a divalent or monovalent enhancer is shown. The "Pre" samples correspond to serum samples collected on the day of booster administration; the "1MPD4" sample corresponds to a serum sample collected one month after dose 4 (i.e., one month after booster administration). GMFR corresponds to a GMT increase factor and is calculated as a ratio of 1mpd4 GMT to the previous GMT (Pre GMT). The number above GMFR represents the ratio between divalent GMFR and monovalent GMFR. (A) FFRNT50 of all subjects, regardless of the infection status. P-value of GMFR for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike for divalent or monovalent booster (two-tailed, wilcoxon matched pair symbol rank test): all < 0.0001. P-value of GMFR ratio of divalent enhancer to monovalent enhancer for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, BQ 1.1-spike, xbb.1-spike (dual tail, mann-Whitney test): 0.0061, < 0.0001. (B) FFRNT50 of all subjects with no evidence of prior infection with SARS-CoV-2. P-value of GMFR for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike for bivalent enhancer (two-tailed, wilcoxon matched pair symbol rank test): all < 0.0001. P-value of GMFR for the monovalent booster for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike (two-tailed, wilcoxon matched pair symbol rank test): 0.013, 0.0006, < 0.0001, 0.013, 0.016. P-value of GMFR ratio of divalent enhancer to monovalent enhancer for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike (dual tail, mann-Whitney test): 0.035, < 0.0001. (C) FFRNT50 of all subjects with evidence of previous infection with SARS-CoV-2. P-value of GMFR for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike for bivalent enhancer (two-tailed, wilcoxon matched pair symbol rank test): all < 0.0001. P-value of GMFR for the monovalent booster for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike (two-tailed, wilcoxon matched pair symbol rank test): 0.0016, 0.0003, 0.0026, 0.0011, < 0.0001. Divalent enhancer p-value of GMFR ratio for USA-WA1/2020, ba.4/5-spike, ba.4.6-spike, ba.2.75.2-spike, bq.1.1-spike, xbb.1-spike compared to monovalent enhancer (dual tail, mann-Whitney test): 0.065, 0.025, 0.0032, 0.0086, 0.0064, 0.0006.

FIG. 64 shows that vaccine-induced and convalescent immune sera had significant cross-neutralization on the Omacron sub-line. A) 50% pseudovirus neutralization of designated SARS-CoV-2 wild-type strain or Omicron target variant strain (VOC) in the following individuals (pVN) ₅₀ ) Geometric mean titer (gmt): at the time of sampling, she was not infected with SARS-CoV-2 (i) triple vaccinated with BNT162b2 or (ii) quadrupled vaccinated with BNT162b2, and from the following individuals: 3 doses of mRNACOVID-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) were vaccinated with (iii) the predominance of BA.1, (iv) the predominance of BA.2 or (v) the predominance of BA.4/5, followed by an Omicron breakthrough infection. The values above the histogram represent the group gmt. LOD/2 values are plotted for titer values below the limit of detection (LOD). Comparison against Omicron BA.4/BA.5 pseudovirus (representing the currently prevailing BA) using the nonparametric Friedman test with Dunn multiple comparison correctionNeutralization titers of 5) and titers against other pseudoviruses. The p-value for the multiple correction is given. (B) SARS-CoV-2 VOC pVN ₅₀ GMT relative to wild strain pVN ₅₀ GMT normalization (ratio of VOC to wild type). The group geometric mean ratio and its 95% confidence interval are shown. Comparison of the inter-queue VOC GMT ratios was corrected using the nonparametric Kruskal-Wallis test and Dunn multiple comparisons. * P < 0.0001; * P < 0.001; * P < 0.01; * P is less than 0.05. Serum assays were performed in duplicate.

FIG. 65. Changes in amino acid sequence of SARS-CoV-2 Omacron subline spike glycoprotein. Amino acid positions, amino acid descriptions (one letter code) and mutation types (substitutions, deletions, insertions) are indicated. The white letters in the boxes indicate amino acid substitutions for each sub-lineage; delta, absence; ins, inserting; NTD, N-terminal domain; RBD, receptor binding domain.

FIG. 66T cell epitope and B cell epitope retention in the S protein of certain SARS-CoV-2 variants of interest. All potential T cell epitopes were retrieved from the immune epitope database (Immune Epitope Database, IEDB) at 11/2022 and the percentage of unchanged linear T cell epitopes of S protein (compared to the epitopes present in BNT162b 2) in each variant strain was calculated. The percentage of unchanged B cell neutralizing epitopes (NTD and RBD) in each variant strain compared to BNT162B2 was estimated using an automated pre-warning system (described in Beguir, karim, et al, "Early computational detection of potential high risk SARS-CoV-2 derivatives," bioRxiv (2021)). And (A) radar and (B) line pattern analysis of conservation of T cell and neutralizing B cell epitopes in different SARS-CoV-2 target variant S proteins. The percentage of unchanged T cell epitopes and the percentage of unchanged B cell neutralizing epitopes are shown. In (B), the top number corresponding to each variant represents the number of mutations of the spike protein compared to the MN908947 strain. The variants were arranged in ascending order of mutation number.

Detailed Description

Although the present disclosure is described in detail below, it is to be understood that the present disclosure is not limited to the particular methods, 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 meaning as commonly understood by one of ordinary skill in the art.

Preferably, terms such as "A multilingual glossary of biotechnologicalterms" are used herein: (IUPAC Recommendations) ", H.G.W.Leuenberger, B.Nagel and H.K6lbl, helvetica Chimica Acta, CH-4010Basel, 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 (see, e.g., molecular Cloning: ALa boratory Manual, 2 nd edition, J. Sambrook et al, eds., cold Spring HarborLaboratory Press, cold Spring Harbor 1989).

Elements of the present disclosure will be described below. 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 examples and embodiments described previously should not be construed as limiting the disclosure to only the explicitly described embodiments. The specification should be understood to disclose and cover embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Moreover, any arrangement and combination of all described elements should be considered as disclosed by the specification unless the context indicates otherwise.

Several documents are cited throughout this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's patent specifications, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior invention. .

Definition of the definition

Hereinafter, definitions applicable to all aspects of the present disclosure will be provided. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its accepted meaning.

The term "about" means approximately or near, and in one embodiment in the context of the values or ranges shown herein, means +20%, ±10%, ±5% or ±3% of the recited or claimed value or range.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following 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 are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were 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 illuminate 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 as essential to the practice of the disclosure.

The term "comprising" in the context of this document is used to mean that in addition to the list members introduced by "comprising" further members may optionally be present, unless explicitly stated otherwise. However, as a specific embodiment of the present disclosure, it is contemplated that the term "comprising" encompasses the possibility that no further members are present, i.e., for the purposes of this embodiment "comprising" may be understood to have the meaning of "consisting of.

As used herein, terms such as "reduce", "inhibit" or "damage" relate to the ability to reduce or cause a total reduction, preferably by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or even more in level. These terms include complete or substantially complete inhibition, i.e., a decrease to 0 or substantially to 0.

Terms such as "increasing", "enhancing" or "exceeding" preferably relate to increasing or enhancing 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 present disclosure, the term "peptide" encompasses oligopeptides and polypeptides, and refers to substances comprising about 2 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, which are linked to each other by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 150 amino acids, but the terms "peptide", "protein" and "polypeptide" are generally used synonymously herein.

When provided to a subject in a therapeutically effective amount, a "therapeutic protein" has a positive or beneficial effect on the subject's condition or disease state. In one embodiment, the therapeutic protein has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Therapeutic proteins may have prophylactic properties and may be used to delay the onset of a disease or to reduce the severity of such disease or pathological conditions. The term "therapeutic protein" includes intact proteins or peptides, and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of the protein. Examples of therapeutically active proteins include, but are not limited to, antigens and immunostimulants such as cytokines for vaccination.

With respect to amino acid sequences (peptides or proteins), "fragments" relate to a portion of an amino acid sequence, i.e. a sequence representing an amino acid sequence shortened at the N-terminal and/or C-terminal end. The shortened fragment at the C-terminus (N-terminal fragment) is obtainable, for example, by translation of a truncated open reading frame lacking the 3' -end of the open reading frame. The shortened fragment at the N-terminus (C-terminal fragment) is obtainable, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, provided that the truncated open reading frame comprises the initiation codon for initiation of translation. Fragments of an amino acid sequence comprise, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. Fragments of an amino acid sequence preferably comprise 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 the amino acid sequence.

"variant" as used herein refers to an amino acid sequence that differs from a parent amino acid sequence by 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 form of a wild-type amino acid. 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 compared to the parent, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications.

"wild-type" or "WT" or "natural" as used herein refers to amino acid sequences found in nature, including allelic variations. A wild-type amino acid sequence, peptide or protein has a fragment of an amino acid sequence that has not been deliberately modified.

In some embodiments, the disclosure relates to SARS-CoV-2 variants that are prevalent and/or rapidly spread in a relevant jurisdiction. In some embodiments, such variants may be based on publicly available data (e.g., at GISAID Initiative database:https：//www.gisaid.organd/or WHO (e.g., as provided inhttps：//www.who.int/activities/tracking-SARS-CoV-2-variantsData at) to identify. In some embodiments, such variants refer to the variants disclosed herein.

For the purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) comprises an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant, and/or an amino acid substitution variant. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants and species homologs, particularly those that occur naturally. In particular, the term "variant" includes fragments of an amino acid sequence.

Amino acid insertion variants include insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of variants with an inserted amino acid sequence, one or more amino acid residues are inserted into a specific site in the amino acid sequence, however random insertion of the resulting product is also possible, suitably screened. Amino acid addition variants comprise amino-and/or carboxy-terminal fusions of one or more amino acids (e.g., 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 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions may be in any position of the protein. Amino acid deletion variants comprising deletions at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue at its position. Modifications in positions in the amino acid sequence that are not conserved between homologous proteins or peptides are preferred and/or amino acids are replaced with other amino acids having similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitution of one of the related families of amino acids in the side chain. Naturally occurring amino acids are generally divided into 4 families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (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 collectively classified as aromatic amino acids. In one embodiment, the conservative amino acid substitutions include substitutions within the following group: glycine, alanine;

Valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine; and

phenylalanine, tyrosine.

Preferably, the degree of similarity between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence, preferably the degree of identity 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 preferably given for an amino acid region that comprises 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, it is preferred that the degree of similarity or identity is given 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, and in some embodiments for consecutive amino acids. In some embodiments, the degree of similarity or identity is given with respect to the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity, may be accomplished using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings (preferably EMBOSS:: needle, matrix: blosum62, gapOpen 10.0, gap Extend0.5).

"sequence similarity" means the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences. "sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

The terms "% identity", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in the optimal alignment between the sequences to be compared. The percentages are purely statistical and the differences between the two sequences may, but need not, be randomly distributed over the length of the sequences to be compared. The comparison of two sequences is typically performed by comparing the sequences after optimal alignment of the segments or "comparison windows" in order to identify local regions of the respective sequences. The optimal alignment for comparison can be performed manually or by means of a local homology algorithm of Smith and Waterman,1981,Ads App Math.2, 482, by means of Neddleman and Wunsch,1970, a local homology algorithm of j.mol.biol.48, 443, by means of a similarity search algorithm of Pearson and Lipman,1988,Proc.Natl Acad.Sci.USA 88, 2444, or by means of a computer program (Wisconsin Genetics Software Package, geneticsComputer Group,575Science Drive,Madison,Wis. GAP, BESTFIT, FASTA, BLAST P, BLAST N and tfast a) using said algorithms. In some embodiments, the percent identity of the two sequences is determined using a BLASTN or BLASTP algorithm as available at the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.lm.nih.gov/blast.cgiepage_type=blastsearch & blast_spec=blast 2seq & LIN k_loc=align 2 seq). In some embodiments, the algorithm parameters for the BLASTN algorithm on the NCBI website include: (i) the desired threshold is set to 10; (ii) word length is set to 28; (iii) the maximum match in the query range is set to 0; (iv) match/mismatch score is set to 1, -2; (v) notch cost is set to be linear; and (vi) filters for low complexity regions are used. In some embodiments, the algorithm parameters for the BLASTP algorithm on the NCBI website include: (i) the desired threshold is set to 10; (ii) the word length is set to 3; (iii) the maximum match in the query range is set to 0; (iv) the matrix is set to BLOSUM62; (v) notch cost is set to exist: 11, extension: 1, a step of; and (vi) conditional component scoring matrix adjustment.

The percent identity is obtained by determining the number of identical positions to which the sequences to be compared correspond, dividing this number by the number of positions being 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 that 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 for consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

According to the present disclosure, the homologous amino acid sequence exhibits 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 skilled artisan can readily prepare amino acid sequence variants described herein, for example, by recombinant DNA procedures. For example, sambrook et al (1989) describe in detail the manipulation of DNA sequences for the preparation of peptides or proteins with substitutions, additions, insertions or deletions. Furthermore, the peptides and amino acid variants described herein can be readily prepared by known peptide synthesis techniques (such as, for example, by solid phase synthesis and similar methods).

In one embodiment, the 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 refers to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which the fragment or variant is derived, i.e., it is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is the one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant" as used herein refers in particular to a variant molecule or sequence comprising an amino acid sequence that has been altered in one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of performing one or more of the functions of the parent molecule or sequence (e.g., inducing an immune response). In one embodiment, 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 various embodiments, the function of the functional fragment or functional variant may be reduced but still be significantly present, e.g., the 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, the 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) that "derives from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the particular amino acid sequence or fragment thereof. The amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or fragment thereof. For example, one of ordinary skill in the art will appreciate that an antigen suitable for use herein may be altered such that the antigen differs in sequence from the naturally occurring sequence or native sequence from which the antigen originates, while retaining the desired activity of the native sequence.

As used herein, "instructional material" or "instructions" includes publications, records, diagrams, or any other presentation medium that can be used to convey the availability of the compositions and methods of the present disclosure. The instructional material of the kits of the present disclosure can, for example, be adhered to or transported with a container containing the compositions of the present disclosure. Alternatively, the instructional material may be shipped separately from the container for the purpose of allowing the recipient to cooperatively use the instructional material and the composition.

"isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from its naturally occurring coexisting materials, is "isolated. The isolated nucleic acid or protein may be present in a substantially pure form, or may be present in a non-natural environment, such as, for example, a host cell.

The term "recombinant" in the context of the present disclosure means produced by "genetic engineering". Preferably, a "recombinant body" such as a recombinant nucleic acid is not naturally occurring in the context of the present disclosure.

The term "naturally occurring" as used herein refers to the fact that an object may be found in nature. For example, peptides or nucleic acids that are present in organisms (including viruses) and that can be isolated from natural sources and that have not been intentionally modified by man in the laboratory are naturally occurring.

As used herein, "physiological pH" refers to a pH of about 7.5.

The term "genetic modification" or simply "modification" includes transfection of a cell with a nucleic acid. The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into cells. For the purposes of this disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, cells for transfection of nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and/or organism of a patient. Transfection may be transient or stable in accordance with the present disclosure. 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 the protein it encodes. Because nucleic acids introduced during transfection are not normally integrated into the nuclear genome, foreign nucleic acids may be diluted or degraded by mitosis. Cells that allow free amplification of nucleic acids greatly reduce the dilution rate. If the nucleic acid desired to be transfected is actually retained in the genome of the cell and its daughter cells, stable transfection must be performed. Such stable transfection may be achieved by transfection using a viral-based system or a transposon-based system. Typically, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

The term "seroconversion" includes an increase of ≡4 fold from before vaccination to 1 month after dose 2.

Coronavirus

Coronaviruses are enveloped, sense, single stranded RNA ((+) ssRNA) viruses. They have the largest genome (26-32 kb) among known RNA viruses and are phylogenetically divided into 4 genera (Alpha, beta, gamma and 6), while Beta coronaviruses are further subdivided into 4 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 disease, although the severity may be higher in infants, the elderly, and immunocompromised persons. Middle east respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) belonging to Beta coronavirus lineages C and B, respectively, are highly pathogenic. Both viruses entered the human population from animal hosts over the last 15 years and resulted in outbreaks of high mortality. Since the middle 12 th 2019, the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes atypical pneumonia (2019 coronavirus disease; COVID-19), persists in China and has evolved into an emergent public health event of international concern. SARS-CoV-2 (MN 908947.3) belongs to the Beta coronavirus lineage B. It has at least 70% sequence similarity with SARS-CoV.

In general, coronaviruses have 4 structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N) and spike (S). The E and M proteins have important functions in viral assembly, while the N protein is essential for viral RNA synthesis. The key 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 in the producer cell by furin-like host protease into two non-covalently associated subunits S1 and S2. The S1 subunit contains a Receptor Binding Domain (RBD) that recognizes host cell receptors. The S2 subunit contains a 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 large pre-fusion spikes.

The S precursor protein of SARS-CoV-2 can be proteolytically cleaved into the S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit comprises a Receptor Binding Domain (RBD) that mediates viral entry into sensitive cells via the host angiotensin converting enzyme 2 (ACE 2) receptor.

Antigens

The present disclosure includes the use of RNAs encoding amino acid sequences comprising SARS-CoV-2S protein, immunogenic variants thereof, or immunogenic fragments of the SARS-CoV-2S protein or immunogenic variants thereof. Thus, the RNA encodes a peptide or protein comprising at least the epitope SARS-CoV-2S protein or an immunogenic variant thereof for use in inducing an immune response against coronavirus S protein, in particular SARS-CoV-2S protein, in a subject. The amino acid sequence comprising the SARS-CoV-2S protein, immunogenic variant thereof, or an immunogenic fragment (i.e., an antigenic peptide or protein) of said SARS-CoV-2S protein or immunogenic variant thereof is also designated herein as a "vaccine antigen", "peptide and protein antigen", "antigenic molecule" or simply as an "antigen". The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of said SARS-CoV-2S protein or immunogenic variant thereof is also designated herein as an "antigenic peptide or protein" or "antigenic sequence".

The full length spike (S) protein of SARS-CoV-2 coronavirus from the first detected strain of SARS-CoV-2 (referred to herein as the MN908947 strain) consists of 1273 amino acids and has a sequence according to SEQ ID NO:1, amino acid sequence:

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for the purposes of this disclosure, the above sequences are considered wild-type or MN908947 SARS-CoV-2S protein amino acid sequences. Unless otherwise indicated, the position numbers in the SARS-CoV-2S proteins given herein correspond to the positions according to SEQ ID NO:1 and related amino acid sequences. Those of skill in the art will appreciate upon reading this disclosure that variants of SARS-CoV-2S protein are based on the amino acid sequence relative to SEQ ID NO:1, and the corresponding position of the position number of the amino acid sequence of 1.

In particular embodiments, the full length spike (S) protein encoded by the RNAs described herein may be modified in a manner that stabilizes the pre-fusion conformation of the prototype. Certain mutations that stabilize the presusion conformation are known in the art, for example, 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 of which are incorporated herein by reference in their entirety. In some embodiments, the SARS-CoV-2S protein can be stabilized by introducing one or more proline mutations. In some embodiments, the SARS-CoV-2S protein corresponds to SEQ ID NO: residues 986 and/or 987 of 1 comprise a proline substitution. In some embodiments, the SARS-CoV-2S protein corresponds to SEQ ID NO: residues 817, 892, 899 and 942 of 1 comprise a proline substitution at one or more positions. In some embodiments, the SARS-CoV-2S protein corresponds to SEQ ID NO: residues 817, 892, 899 and 942 of 1 each comprise a proline substitution at a position of each. In some embodiments, the SARS-CoV-2S protein corresponds to SEQ ID NO: residues 817, 892, 899, 942, 986 and 987 of 1 comprise a proline substitution at the position of each.

In some embodiments, stabilization of the pre-fusion conformation may be achieved by introducing two consecutive proline substitutions at residues 986 and 987 in the full length spike protein. Specifically, spike (S) protein stabilized protein variants are obtained in such a way that the amino acid residue at position 986 is exchanged for proline and the amino acid residue at position 987 is also exchanged for proline. In one embodiment, the variant SARS-CoV-2S protein in which the pre-prototypic conformation is stabilized comprises the amino acid sequence of SEQ ID NO: 7:

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those skilled in the art are aware of the various spike mutants and/or record their resources. For example, strains whose SARS-CoV-2S protein amino acid sequence, particularly their modifications compared to the wild-type SARS-CoV-2S protein amino acid sequence (e.g., compared to SEQ ID NO: 1), are useful herein.

B.1.1.7 ("202012/01 of interest" (variant of VOC-202012/01)

B.1.1.7 ("Alpha variant") is a variant of SARS-CoV-2 that was first detected in the sample taken during the UK COVID-19 pandemic at 10 months 2020 and rapidly started to spread in the 12 th month. This is associated with a significant increase in the infection rate of british covd-19; this increase is thought to be due, at least in part, to the N501Y change in the spike glycoprotein receptor binding domain necessary for ACE2 binding in human cells. B.1.1.7 variants were defined by the following 23 mutations: 13 nonsensical mutations, 4 deletions, and 6 synonymous mutations (i.e., 17 mutations change protein, 6 mutations do not change protein). The spike protein changes in b.1.1.7 included deletions 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.

B.1.351(501.V2)

B.1.351 lineage ("Beta variant"), commonly known as south Africa COVID-19 variant, is a variant of SARS-CoV-2. Preliminary results indicate that the variant may have increased transmission capacity. B.1.351 variants are defined by a plurality of spike protein changes including: L18F, D80A, D G, deletions 242-244, R246I, K417N, E484K, N501Y, D G and A701V. B.1.351 there are three mutations of particular interest in the spike region of the genome: K417N, E484K, N501Y.

B.1.1.298(Cluster 5)

B.1.1.298 was found in the peninsula of solar-ridland, denmark, and is believed to be transmitted from mink to humans through mink farms. Several different mutations of the spike protein of the virus have been demonstrated. Specific mutations include deletions 69-70, Y453F, D614G, I692V, M1229I and optionally S1147L.

P.1(B.1.1.248)

Lineage b.1.1.248 ("Gamma variant"), known as brazil variant, is one of the variants of SARS-CoV-2, which has been designated the p.1 lineage. P.1 there are many modifications of the S protein [ L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N Y, D614G, H655Y, T1027I, V1176F ], and at some key RBD positions (K417, E484, N501) are similar to variant B.1.351 from south Africa.

B.1.427/B.1.429(CAL.20C)

Lineage b.1.427/b.1.429 ("epsilon variant"), also known as cal.20c, is defined by the following modifications in the S protein: S13I, W152C, L452R and D614G, wherein the L452R modification is of particular interest. CDC has listed b.1.427/b.1.429 as a "variant of interest".

B.1.525

B.1.525 The ("eta variants") carry the same E484K modification as found in the p.1 and b.1.351 variants and also carry the same Δh69/Δv70 deletion as found in b.1.1.7 and b.1.1.298. It also carries modifications D614G, Q677H and F888L.

B.1.526

B.1.526 ("iota variants") were detected as new emerging viral isolate lineages in New York region sharing mutations with previously reported variants. The most common spike sets in this lineage are L5F, T95I, D253G, E484K, D614G and a701V.

The following table shows an overview of the circulating SARS-CoV-2 strain as VOI/VOC.

B.1.1.529

B.1.529 ("omacron variant") was first discovered in south africa at month 11 in 2021. Omicron propagates approximately 70-fold faster than the Delta variant and rapidly becomes the major strain of SARS-CoV-2 worldwide. Since the initial detection, many omacron sublines have emerged. The omacron variants of current interest are listed below, along with certain characteristic mutations associated with the S protein of each variant. The S proteins of ba.4 and ba.5 have the same set of characteristic mutations, which is why there is only one row for "ba.4 or ba.5" in the following table, and why the disclosure mentions "ba.4/5"S protein in some embodiments. Similarly, the S proteins of the ba.4.6 and bf.7omicron variants have the same set of characteristic mutations, which is why the following table has only one row for "ba.4.6 or bf.7".

Table 2: omicron variants and characteristic mutations of interest

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In some embodiments, the RNAs described herein comprise a nucleotide sequence encoding a SARS-CoV-2S protein that comprises one or more mutations (including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations) that are characteristic of an Omicron variant (e.g., one or more mutations of an Omicron variant listed in table 2). In some embodiments, such RNAs further comprise one or more mutations that stabilize the S protein in the pre-fusion conformation (e.g., in some embodiments, such RNAs further comprise proline residues at positions corresponding to residues 986 and 987 of SEQ ID No. 1). In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations listed in table 2 (including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more mutations). In some such embodiments, the one or more mutations may be from two or more variants listed in table 2. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising each of the mutations identified in table 2 as characteristic of a particular omacron variant (e.g., in some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising each of the mutations listed in table 2 as characteristic of omacron ba.1, ba.2, ba.2.12.1, ba.4/5, ba.2.75, ba.2.75.1, ba.4.6, bq.1.1, XBB, xbb.1, xbb.2, or xbb.1.3 variants).

In some embodiments, the RNAs disclosed herein comprise a nucleotide sequence that encodes an immunogenic fragment (e.g., RBD) of a SARS-CoV-2S protein and the immunogenic fragment comprises one or more mutations that characterize a SARS-CoV-2 variant (e.g., an Omicron variant as described herein). For example, in some embodiments, the RNA comprises a nucleotide sequence that encodes the RBD of the S protein of a SARS-CoV-2 variant (e.g., a region of the S protein that corresponds to amino acids 327 through 528 of SEQ ID NO:1 and that comprises one or more mutations that characterize a variant of interest within that region of the S protein).

In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises a subset of the mutations listed in Table 2. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises the most prevalent mutation in a particular variant listed in table 2 (e.g., a mutation that has 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 the sequences that have been collected so far for a given variant that has been sequenced). Mutation prevalence may be determined, for example, based on published sequences (e.g., sequences collected by GISAID and provided to the public).

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize a ba.4/5 variant. In some embodiments, the one or more mutations that characterize the ba.4/5 variant include T19I, Δ24-26, a27S, Δ69/70, G142D, V213 42339 339D, S371F, S373P, S375F, T376A, D405 56417N, N452R, S477N, T478K, E484A, F486V, Q498 501Y, Y614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that are characteristic of ba.4/5 variants and do not include R408S. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of ba.4/5 variants and do not include R408S.

In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of a ba.2.75 variant. In some embodiments, the one or more mutations that are characteristic of the ba.2.75 variant include T19I, Δ24-26, a27S, G142D, K147E, W152R, F157L, I210V, V213G, G339H, S371F, S373F, S375F, S376F, S52405F, S408 417F, S440 446F, S460F, S477 478 484F, S498F, S501F, S614F, S655 679F, S681F, S764F, S954H, and N969K. In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises 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 mutations) that are characteristic of the ba.2.75 variant and that do not include N354D. In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises 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 mutations) that are characteristic of the ba.2.75 variant and that do not include D796Y. In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises 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 mutations) that are characteristic of the ba.2.75 variant and that do not include D796Y and N354D.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize the ba.2.75.2 variant. In some embodiments, the one or more mutations that characterize the ba.2.75.2 variant include T19I, Δ24-26, a27 142 147 152 157 210, 257 339 371 373 375 376 405 408 417 440 446 477 478 484 486 498 501 505 614 655 679 681 764 679 6816. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of the ba.2.75.2 variant and do not include R346T.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein comprising one or more mutations that characterize ba.4.6 or bf.7 variants. In some embodiments, the one or more mutations that are characteristic of the ba.4.6 or bf.7 variant include T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, R346T, S371 56373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T K, E484 486 6783 4983 498R, N501Y, Y614G, H655 35655Y, N679K, P681H, N764K, D796Y, Q H and N969K. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of ba.4.6 or bf.7 variants and do not include R408S. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of ba.4.6 or bf.7 variants and do not include N658S. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of ba.4.6 or bf.7 variants and do not include N658S and R408S.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize omacron XBB variants. In some embodiments of the present invention, in some embodiments, the one or more mutations that characterize the Omicron XBB variant include T19I, Δ24-26, a27S, V83S, V142D, Δ144H 146S, V183S, V339S, V52368S, V373S, V405S, V417S, V445 446S, V460) S, V477S, V478S, V484S, V486S, V490S, V498S, V501S, V505S, V655S, V679S, V681 764S, V796S, V954H and N969K.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize omacron xbb.1 variants. In some embodiments, the one or more mutations that characterize the omacron xbb.1 variant comprise G252V. In some embodiments, the one or more mutations that characterize the omacron xbb.1 variant include T19I, Δ24-26, a27 83 142D, Δ144, H146 213 339 368 373 375 376 405 408 417 440 445 446 460 477 478 484 486 498 505 614 67 655 679 681 764 796 954H and N969K. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of omacron xbb.1 variants and do not include Q493R. In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises 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 mutations) that are characteristic of Omicron XBB variants and that do not include Q493R and G252V.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize omacron xbb.2 variants. In some embodiments, the one or more mutations that characterize the omacron xbb.2 variant comprise D253G. In some embodiments, the one or more mutations that characterize the omacron xbb.2 variant include T19I, Δ24-26, a27 83 142D, Δ144, H146 213 253 339 368 373 375 376 405 408 417 440 445 446 477 478 484 486 490 493 498 501 505 614 655 679 681 764 796 954H and N969K.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize an omacron xbb.1.3 variant. In some embodiments, the one or more mutations that characterize the omacron xbb.1.3 variant include G252V and a484T. In some embodiments, the one or more mutations that characterize the omacron xbb.1.3 variant include T19I, Δ24-26, a27 83 142D, Δ144, H146 213 252 339 346 368 371 373 376 405 408 417 440 446 460 477 478 486 490 493 498 501 505 614 655 679 681 764 796 954H, and N969K.

In some embodiments, the RNAs described herein encode SARS-CoV-2S protein that comprises one or more mutations that characterize the bq.1.1 variant. In some embodiments of the present invention, in some embodiments, the one or more mutations that characterize the BQ.1.1 variant include T19I, Δ24-26, A27S, Δ69/70G 142D, V213G, G339D, S F, S373P, S375F, T376A, D N, R408S, K417N, N440K, K444 452R, N463K, S477N, T478K, E486V, Q498R, N and R, N614 and 655R, N and R, N679R, N and 681R, N764 and R, N796 and R, N954H and N969K. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins that comprise 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 mutations) that are characteristic of a bq.1.1 variant.

In one embodiment, the vaccine antigens described herein comprise, consist essentially of, or consist of the spike protein (S) of SARS-CoV-2, variants thereof, or immunogenic fragments thereof [ e.g., without limitation, RBD ].

In one embodiment, the vaccine antigen comprises SEQ ID NO:1 or 7, amino acid sequence of amino acids 17 to 1273, which corresponds to 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 the amino acid sequence of amino acids 17 to 1273, or SEQ ID NO:1 or 7 or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 or a sequence identical to SEQ ID NO:1 or 7, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 or 7 from amino acid 17 to 1273.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819, and 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 the nucleotide sequence of nucleotides 49 to 3819, or SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819 or a fragment of the nucleotide sequence of SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 3819; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 or 7, amino acid sequence of amino acids 17 to 1273, which corresponds to 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 the amino acid sequence of amino acids 17 to 1273, or SEQ ID NO:1 or 7 or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 or a sequence identical to SEQ ID NO:1 or 7, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to nucleotide 3819; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 or 7 from amino acid 17 to 1273.

In one embodiment, the vaccine antigen comprises a SARS-CoV-2 spike S1 fragment (S1) (S1 subunit of the spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof, consisting essentially of a SARS-CoV-2 spike S1 fragment (S1), a variant thereof, or a fragment thereof, or a SARS-CoV-2 spike S1 fragment (S1), a variant thereof, or a fragment thereof.

In one embodiment, the vaccine antigen comprises SEQ ID NO:1, amino acid sequence of amino acids 17 to 683, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 17 to 683 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or SEQ ID NO:1 or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 or a sequence identical to SEQ ID NO:1, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 from amino acid 17 to 683.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2049, and 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 the nucleotide sequence of nucleotide 49 to 2049 of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2049 or a fragment of the nucleotide sequence of SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 2049; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, amino acid sequence of amino acids 17 to 683, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 17 to 683 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or SEQ ID NO:1 or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 or a sequence identical to SEQ ID NO:1, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to nucleotide 2049; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 from amino acid 17 to 683.

In one embodiment, the vaccine antigen comprises SEQ ID NO:1, amino acid sequence of amino acids 17 to 685, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 17 to 685 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 from amino acids 17 to 685.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2055, and 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 the nucleotide sequence of nucleotides 49 to 2055, or a nucleotide sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 2055 or a fragment of the nucleotide sequence of SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 2055; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, amino acid sequence of amino acids 17 to 685, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 17 to 685 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 49 to nucleotide 2055; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 from amino acids 17 to 685.

In one embodiment, the vaccine antigen comprises, consists essentially of, or consists of the Receptor Binding Domain (RBD) of the S1 subunit of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof. SEQ ID NO:1, or a variant thereof, or a fragment thereof, is also referred to herein as an "RBD" or "RBD domain".

In one embodiment, the vaccine antigen comprises SEQ ID NO:1, amino acid sequence of amino acids 327 to 528, which corresponds to SEQ ID NO:1, or 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, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 to 528.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9, nucleotides 979 to 1584, and SEQ ID NO: 2. 8 or 9, nucleotide 979 to 1584, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO: 2. 8 or 9 from nucleotide 979 to 1584 or a fragment of the nucleotide sequence of SEQ ID NO: 2. 8 or 9, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, amino acid sequence of amino acids 327 to 528, which corresponds to SEQ ID NO:1, or 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, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 979 to 1584; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 to 528.

According to certain embodiments, the signal peptide is fused to the SARS-CoV-2S protein, variant thereof, or fragment thereof, i.e., antigenic peptide or protein, either directly or through a linker. Thus, in one embodiment, the signal peptide is fused to the above-described amino acid sequence contained in the above-described vaccine antigen derived from SARS-CoV-2S protein or an immunogenic fragment thereof (antigenic peptide or protein).

Such signal peptides are sequences that generally exhibit a length of about 15-30 amino acids and are preferably located at the N-terminus of an antigenic peptide or protein, but are not limited thereto. The signal peptide as defined herein preferably allows for the transport of an antigenic peptide or protein as encoded by said RNA to a defined cell compartment, preferably a cell surface, endoplasmic Reticulum (ER) or endosomal-lysosomal compartment. In one embodiment, the signal peptide sequence as defined herein includes, but is not limited to, a signal peptide sequence of SARS-CoV-2S protein, in particular a signal peptide sequence comprising SEQ ID NO:1 or the amino acid sequence of amino acids 1 to 16 or 1 to 19 or a functional variant thereof.

In one embodiment, the signal sequence comprises SEQ ID NO:1, and amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 16 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, the amino acid sequence of amino acids 1 to 16 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the signal sequence comprises SEQ ID NO:1 to 16.

In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO: 2. 8 or 9, and the nucleotide sequence of nucleotides 1 to 48 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 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 or a nucleotide sequence identical to SEQ ID NO: 2. 8 or 9, the nucleotide sequence of nucleotides 1 to 48 having a fragment of the nucleotide sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, and amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 16 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, the amino acid sequence of amino acids 1 to 16 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO: 2. 8 or 9 from nucleotide 1 to 48; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 to 16.

In one embodiment, the signal sequence comprises SEQ ID NO:1, and amino acid sequence of amino acids 1 to 19, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 19 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, the amino acid sequence of amino acids 1 to 19 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the signal sequence comprises SEQ ID NO:1 to 19.

In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO: 2. 8 or 9, and nucleotide sequences of nucleotides 1 to 57 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 the nucleotide sequence of nucleotides 1 to 57, or SEQ ID NO: 2. 8 or 9 or a fragment of the nucleotide sequence of nucleotides 1 to 57 or a fragment of the sequence corresponding to SEQ ID NO: 2. 8 or 9, the nucleotide sequence of nucleotides 1 to 57 having a fragment of the nucleotide sequence at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, and amino acid sequence of amino acids 1 to 19, which corresponds to SEQ id no:1, or the amino acid sequence of amino acids 1 to 19 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, the amino acid sequence of amino acids 1 to 19 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO: 2. 8 or 9 from nucleotide 1 to 57; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 to 19.

In some embodiments, the signal peptide as defined herein includes, but is not limited to, a signal peptide sequence of an immunoglobulin, such as a signal peptide sequence of an immunoglobulin heavy chain variable region, wherein the immunoglobulin may be a human immunoglobulin. In particular, in some embodiments, the signal peptide sequence as defined herein includes a sequence comprising SEQ ID NO:31 or a functional variant thereof.

In some embodiments, the signal peptide sequence is functional in mammalian cells.

In some embodiments, the signal sequence used is "native" in that it is essentially associated with (e.g., linked to) the encoded polypeptide.

In some embodiments, the signal sequence utilized is heterologous to the encoded polypeptide, e.g., the signal sequence utilized is not a natural component of the polypeptide (e.g., protein) whose sequence is contained in the encoded polypeptide.

In some embodiments, the signal peptide is a sequence generally characterized by a length of about 15 to 30 amino acids.

In many embodiments, the signal peptide is located at the N-terminus of the encoded polypeptide as described herein, but is not limited thereto. In some embodiments, the signal peptide preferably allows for the transport of polypeptides encoded by RNAs of the present disclosure associated therewith to a particular cell compartment (preferably a cell surface, endoplasmic Reticulum (ER) or endosomal-lysosomal compartment).

In some embodiments, the signal sequence is selected from the group consisting of S1S2 signal peptide (aa 1-19), immunoglobulin secretion signal peptide (aa 1-22), HSV-1gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), HSV-2gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA); human SPARC signal peptide, human insulin subtype 1 signal peptide, human albumin signal peptide, and the like. Those skilled in the art will be aware of other secretion signal peptides, for example, as disclosed in WO2017/081082 (e.g., SEQ ID NOs: 1-1115 and 1728, or fragment variants thereof) and as disclosed in WO 2019008001.

In some embodiments, the RNA sequence encodes an epitope that may comprise or otherwise be linked to a signal sequence (e.g., a secretion sequence) (e.g., those listed in table a) or a sequence having at least 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 up to 5 amino acid differences relative thereto is used. In some embodiments, sequences such as MFVFLVLLPLVSSQCVNLT or sequences having 1, 2, 3, 4, or up to 5 amino acid differences relative thereto are used.

In some embodiments, the signal sequences are selected from those included in table a below and/or those encoded by the sequences in table B below:

table a: exemplary Signal sequence

Table B: exemplary nucleotide sequences encoding Signal sequences

In some embodiments, the RNA used as described herein encodes a multimerization element (e.g., a heteromultimerization element). In some embodiments, the heteromultimerization element comprises a dimerisation, trimerisation or tetramerisation element.

In some embodiments, the multimerization element is the element described in WO2017/081082 (e.g., SEQ ID NOS: 1116-1167, or fragments or variants thereof).

Exemplary trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibrin folding subdomain from enterophage T4, GCN4pll, GCN4-pll, and p53.

In some embodiments, the provided encoded polypeptides are capable of forming a trimeric complex. For example, the encoded polypeptides utilized may comprise domains that allow for the formation of multimeric complexes, such as particular trimeric complexes comprising the amino acid sequences of the encoded polypeptides as described herein. In some embodiments, the domain that allows for the formation of a multimeric complex comprises a trimerization domain, e.g., a trimerization domain as described herein.

In some embodiments, the encoded polypeptide may be modified, e.g., to increase its immunogenicity, by the addition of a T4-fibrin-derived "folder" trimer domain.

In some embodiments, the RNAs described herein encode a membrane-binding element (e.g., a heterologous membrane-binding element), such as a transmembrane domain.

The transmembrane domain may be at the N-terminus, C-2 terminus or within the encoded polypeptide. The coding sequence of the transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or within the coding sequence of the sequence to which it is linked (e.g., the sequence encoding the polypeptide).

In some embodiments, the transmembrane domain comprises or is the Hemagglutinin (HA) of influenza virus, env of HIV-1, equine Infectious Anemia Virus (EIAV), murine Leukemia Virus (MLV), murine mammary tumor virus, G protein of Vesicular Stomatitis Virus (VSV), the transmembrane domain of rabies virus, or the receptor of seven transmembrane domains.

In one embodiment, the signal sequence comprises SEQ ID NO:31, and amino acid sequence of amino acids 1 to 22, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 22 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or a functional fragment of the amino acid sequence of amino acids 1 to 22 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 22 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the signal sequence comprises SEQ ID NO:31 from amino acid 1 to 22.

In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO:32, and nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO:32, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to nucleotide 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, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, and amino acid sequence of amino acids 1 to 22, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 22 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or a functional fragment of the amino acid sequence of amino acids 1 to 22 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 22 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a signal sequence comprises SEQ ID NO:32 from nucleotide 54 to 119; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 1 to 22.

Such signal peptides are preferably used to facilitate secretion of the encoded antigenic peptide or protein. More preferably, the signal peptide as defined herein is fused to a coded antigenic peptide or protein as defined herein.

Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, preferably fused to the antigenic peptide or protein, more preferably fused to the N-terminus of the antigenic peptide or protein as described herein.

In one embodiment, the vaccine antigen comprises SEQ ID NO:1 or 7, and 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 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, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 or 7.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9, and 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 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, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 or 7, and 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 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, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 or 7.

In one embodiment, the vaccine antigen comprises SEQ ID NO:7, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:7, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO: 7.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 15. 16, 19, 20, 24 or 25, and SEQ ID NO: 15. 16, 19, 20, 24 or 25, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to 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, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:7, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:7, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 15. 16, 19, 20, 24 or 25; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 7.

In one embodiment, the vaccine antigen comprises SEQ ID NO:1, amino acid sequence of amino acids 1 to 683, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 683 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 to 683.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9, and nucleotide 1 to 2049, and 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 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 or a fragment of the sequence corresponding to 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 nucleotide 1 to 2049; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, amino acid sequence of amino acids 1 to 683, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 683 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 1 to nucleotide 2049; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 to 683.

In one embodiment, the vaccine antigen comprises SEQ ID NO:1, and amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 685 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%90%, 85% or 80% identity, or SEQ ID NO:1 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:1 to 685.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9, and nucleotide 1 to 2055, and 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 the nucleotide sequence of nucleotides 1 to 2055, or a nucleotide sequence of SEQ ID NO: 2. 8 or 9 or a fragment of the nucleotide sequence of nucleotides 1 to 2055 or a nucleotide sequence identical to SEQ ID NO: 2. 8 or 9, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, and amino acid sequence of amino acids 1 to 685 of SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 685 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 2. 8 or 9 from nucleotide 1 to 2055; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 to 685.

In one embodiment, the vaccine antigen comprises SEQ ID NO:3, and SEQ ID NO:3, 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:3 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:3, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:3, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:4, and SEQ ID NO:4, 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:4 or a fragment of the nucleotide sequence of SEQ ID NO:4, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:3, and SEQ ID NO:3, 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:3 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:3, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:4, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:3, and a sequence of amino acids.

In one embodiment, the vaccine antigen comprises SEQ ID NO:29, amino acid 1 to 221, and SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 221 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 or a sequence identical to SEQ ID NO:29, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:29 from amino acid 1 to 221.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO:30, or a nucleotide sequence of nucleotides 54 to 716 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:30 or a fragment of the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO:30, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, amino acid 1 to 221, and SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 221 has an amino acid sequence of at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 or a sequence identical to SEQ ID NO:29, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30 from nucleotide 54 to 716; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29 from amino acid 1 to 221.

In one embodiment, the vaccine antigen comprises SEQ ID NO:31, amino acid sequence of amino acids 1 to 224, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 224 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 224 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:31 from amino acid 1 to 224.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO:32, or a nucleotide sequence of nucleotide 54 to 725 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 nucleotides 54 to 725 or a nucleotide sequence identical to SEQ ID NO:32, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, amino acid sequence of amino acids 1 to 224, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 224 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 224 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32 from nucleotide 54 to 725; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 1 to 224.

According to certain embodiments, the trimerization domain is fused to the SARS-CoV-2S protein, variant thereof, or fragment thereof, i.e., an antigenic peptide or protein, either directly or through a linker (e.g., glycine/serine linker). Thus, in one embodiment, the trimerization domain is fused to the above-described amino acid sequence (which may optionally be fused to a signal peptide as described above) comprised by the above-described vaccine antigen, which is derived from the SARS-CoV-2S protein or an immunogenic fragment thereof (antigenic peptide or protein).

Such trimerization domains are preferably located at the C-terminus of the antigenic peptide or protein, but are not limited thereto. The trimerization domain as defined herein preferably allows trimerization of an antigenic peptide or protein as encoded by said RNA. Examples of trimerization domains as defined herein include, but are not limited to, the native trimerization domain of the foldon (T4 fibrin). The C-terminal domain of T4 fibrin (folder) is essential for the formation of fibrin trimer structures and can be used as an artificial trimerization domain. In one embodiment, the trimerization domain as defined herein comprises, but is not limited to, a sequence comprising SEQ ID NO:10 or a functional variant thereof. In one embodiment, the trimerization domain as defined herein comprises, but is not limited to, a sequence comprising SEQ ID NO:10 or a functional variant thereof.

In one embodiment, the trimerization domain comprises SEQ ID NO:10, amino acid sequence of amino acids 3 to 29, which corresponds to SEQ ID NO:10, or the amino acid sequence of amino acids 3 to 29 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:10 or a functional fragment of the amino acid sequence of amino acids 3 to 29 or a sequence identical to SEQ ID NO:10, the amino acid sequence of amino acids 3 to 29 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the trimerization domain comprises SEQ ID NO:10 from amino acids 3 to 29.

In one embodiment, RNA (i) encoding the trimerization domain comprises SEQ ID NO:11, and nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO:11, or a nucleotide sequence of nucleotide 7 to 87 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:11 or a fragment of the nucleotide sequence of nucleotides 7 to 87 or a nucleotide sequence identical to SEQ ID NO:11, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:10, amino acid sequence of amino acids 3 to 29, which corresponds to SEQ ID NO:10, or the amino acid sequence of amino acids 3 to 29 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:10 or a functional fragment of the amino acid sequence of amino acids 3 to 29 or a sequence identical to SEQ ID NO:10, the amino acid sequence of amino acids 3 to 29 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding the trimerization domain comprises SEQ ID NO:11 from nucleotide 7 to 87; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:10 from amino acids 3 to 29.

In one embodiment, the trimerization domain comprises SEQ ID NO:10, and SEQ ID NO:10, 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:10 or a functional fragment of the amino acid sequence of SEQ ID NO:10, a functional fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the trimerization domain comprises SEQ ID NO: 10.

In one embodiment, RNA (i) encoding the trimerization domain comprises SEQ ID NO:11, and SEQ ID NO:11, 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:11 or a fragment of the nucleotide sequence of SEQ ID NO:11, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:10, and SEQ ID NO:10, 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:10 or a functional fragment of the amino acid sequence of SEQ ID NO:10, a functional fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding the trimerization domain comprises SEQ ID NO:11, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 10.

Such trimerization domains are preferably used to promote trimerization of the encoded antigenic peptide or protein. More preferably, the trimerization domain as defined herein is fused to an antigenic peptide or protein as defined herein.

Thus, in a particularly preferred embodiment, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerization domain as defined herein, preferably fused to the antigenic peptide or protein, more preferably to the C-terminus of the antigenic peptide or protein.

In one embodiment, the vaccine antigen comprises SEQ ID NO:5, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO: 5.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:6, and SEQ ID NO:6, 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:6 or a fragment of the nucleotide sequence of SEQ ID NO:6, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:5, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:6, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 5.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 17. 21 or 26, and SEQ ID NO: 17. 21 or 26, 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: 17. 21 or 26 or a fragment of the nucleotide sequence of SEQ ID NO: 17. 21 or 26, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:5, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 17. 21 or 26; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 5.

In one embodiment, the vaccine antigen comprises SEQ ID NO:18, and SEQ ID NO:18, 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:18 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:18, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:18, and a sequence of amino acids.

In one embodiment, the vaccine antigen comprises SEQ ID NO:29, amino acid sequence of amino acids 1 to 257, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 257 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 or a sequence identical to SEQ ID NO:29, and an immunogenic fragment of 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. In one embodiment, the vaccine antigen comprises SEQ ID NO:29 from amino acids 1 to 257.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO:30, or a nucleotide sequence of nucleotide 54 to 824 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:30 or a fragment of the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO:30, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, amino acid sequence of amino acids 1 to 257, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 257 has an amino acid sequence that is at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 or a sequence identical to SEQ ID NO:29, and an immunogenic fragment of 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. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30 from nucleotide 54 to 824; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29 from amino acids 1 to 257.

In one embodiment, the vaccine antigen comprises SEQ ID NO:31, amino acid sequence of amino acids 1 to 260, which corresponds to SEQ ID NO:31, 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 1 to 260, or SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 260 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:31 from amino acid 1 to 260.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and nucleotide sequence of nucleotides 54 to 833, which corresponds to SEQ ID NO:32, or a nucleotide sequence of nucleotide 54 to 833 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:32 or a fragment of the nucleotide sequence of nucleotides 54 to 833 or a nucleotide sequence identical to SEQ ID NO:32, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, amino acid sequence of amino acids 1 to 260, which corresponds to SEQ ID NO:31, 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 1 to 260, or SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 260 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32 from nucleotide 54 to 833; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 1 to 260.

In one embodiment, the vaccine antigen comprises SEQ ID NO:29, amino acids 20 to 257, and SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 257 has an amino acid sequence that is at least 99%, 98%, 97%, 96%95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 or a sequence identical to SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 257 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:29 from amino acids 20 to 257.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO:30, or a nucleotide sequence of nucleotides 111 to 824 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:30 or a fragment of the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO:30, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, amino acids 20 to 257, and SEQ id no:29, or the amino acid sequence of amino acids 20 to 257 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 or a sequence identical to SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 257 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30 from nucleotide 111 to 824; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29 from amino acids 20 to 257.

In one embodiment, the vaccine antigen comprises SEQ ID NO:31, amino acid sequence of amino acids 23 to 260, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 23 to 260 has an amino acid sequence that is at least 99%, 98%, 97%, 96%95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 23 to 260 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:31 from amino acid 23 to 260.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and nucleotide sequence of nucleotides 120 to 833, which corresponds to SEQ ID NO:32, or a nucleotide sequence of nucleotide 120 to 833 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:32 or a fragment of the nucleotide sequence of nucleotides 120 to 833 or a nucleotide sequence identical to SEQ ID NO:32, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, amino acid sequence of amino acids 23 to 260, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 23 to 260 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 23 to 260 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32 from nucleotide 120 to 833; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 23 to 260.

According to certain embodiments, the transmembrane domain is fused to the SARS-CoV-2S protein, variant thereof, or fragment thereof, i.e., an antigenic peptide or protein, either directly or through a linker (e.g., glycine/serine linker). Thus, in one embodiment, the transmembrane domain is fused to the above-described amino acid sequence (which may optionally be fused to a signal peptide and/or trimerization domain as described above) comprised by the above-described vaccine antigen, derived from the SARS-CoV-2S protein or an immunogenic fragment thereof (antigenic peptide or protein).

Such a transmembrane domain is preferably located at the C-terminus of the antigenic peptide or protein, but is not limited thereto. Preferably, such a transmembrane domain is located at the C-terminus of the trimerization domain (if present), but is not limited thereto. In one embodiment, the trimerization domain is present between the SARS-CoV-2S protein, variant thereof, or fragment thereof (i.e., antigenic peptide or protein) and the transmembrane domain.

The transmembrane domain as defined herein preferably allows the anchoring of an antigenic peptide or protein as encoded by said RNA into the cell membrane.

In one embodiment, the transmembrane domain sequence as defined herein includes, but is not limited to, a transmembrane domain sequence of SARS-CoV-2S protein, in particular comprising the amino acid sequence of SEQ ID NO:1 or a functional variant thereof.

In one embodiment, the transmembrane domain sequence comprises SEQ ID NO:1, amino acid 1207 to 1254, and SEQ ID NO:1, or the amino acid sequence of amino acids 1207 to 1254 has an amino acid sequence of at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:1 or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 or a sequence identical to SEQ ID NO:1, and the amino acid sequence of amino acids 1207 to 1254 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity. In one embodiment, the transmembrane domain sequence comprises SEQ ID NO:1 from amino acids 1207 to 1254.

In one embodiment, RNA (i) encoding a transmembrane domain sequence comprises SEQ ID NO: 2. 8 or 9 from nucleotide 3619 to 3762, and SEQ ID NO: 2. 8 or 9 from nucleotide 3619 to 3762, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ id no: 2. 8 or 9 from nucleotide 3619 to 3762 or a fragment of the nucleotide sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 3619 to 3762, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1, amino acid 1207 to 1254, and SEQ ID NO:1, or the amino acid sequence of amino acids 1207 to 1254 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:1 or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 or a sequence identical to SEQ ID NO:1, and the amino acid sequence of amino acids 1207 to 1254 has at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identity. In one embodiment, RNA (i) encoding a transmembrane domain sequence comprises SEQ ID NO: 2. 8 or 9 from nucleotide 3619 to nucleotide 3762; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:1 from amino acids 1207 to 1254.

In one embodiment, the vaccine antigen comprises SEQ ID NO:29, amino acid sequence of amino acids 1 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 311 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, amino acid 1 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:29 from amino acid 1 to 311.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and nucleotide sequence of nucleotides 54 to 986, which hybridizes with SEQ ID NO:30, or a nucleotide sequence of nucleotide 54 to 986 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:30 or a fragment of the nucleotide sequence of nucleotides 54 to 986 or a nucleotide sequence identical to SEQ ID NO:30, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, amino acid sequence of amino acids 1 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 311 has an amino acid sequence of at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO:29, amino acid 1 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30 from nucleotide 54 to 986; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29 from amino acid 1 to 311.

In one embodiment, the vaccine antigen comprises SEQ ID NO:31, amino acid sequence of amino acids 1 to 314, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 314 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 314 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:31 from amino acid 1 to 314.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and nucleotide sequences of nucleotides 54 to 995, which correspond to SEQ ID NO:32, or a nucleotide sequence of nucleotide 54 to 995 having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:32 or a fragment of the nucleotide sequence of nucleotides 54 to 995 or a nucleotide sequence identical to SEQ ID NO:32, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, amino acid sequence of amino acids 1 to 314, which corresponds to SEQ ID NO:31, or the amino acid sequence of amino acids 1 to 314 has an amino acid sequence that is at least 99%, 98%97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 or a sequence identical to SEQ ID NO:31, the amino acid sequence of amino acids 1 to 314 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32 from nucleotide 54 to 995; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 1 to 314.

In one embodiment, the vaccine antigen comprises SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence of at least 99%, 98%, 97%, 96%95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:29 from amino acids 20 to 311.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and nucleotide sequence of nucleotides 111 to 986, which corresponds to SEQ ID NO:30, or 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, or the nucleotide sequence of SEQ ID NO:30 or a fragment of the nucleotide sequence of nucleotides 111 to 986 or a nucleotide sequence identical to SEQ ID NO:30, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ id no:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity, or SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30 from nucleotide 111 to 986; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29 from amino acids 20 to 311.

In one embodiment, the vaccine antigen comprises SEQ ID NO:31, amino acid sequence of amino acids 23 to 314, which corresponds to SEQ ID NO:31 from amino acids 23 to 314, or an amino acid sequence having at least 99%, 98%, 97%, 96%95%, 90%, 85% or 80% identity to SEQ ID NO:31 or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314 of SEQ ID NO:31 from amino acids 23 to 314 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the vaccine antigen comprises SEQ ID NO:31 from amino acid 23 to 314.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and nucleotide sequences of nucleotides 120 to 995, which correspond to SEQ ID NO:32, or 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, or a nucleotide sequence of SEQ ID NO:32 or a fragment of the nucleotide sequence of nucleotides 120 to 995 or a nucleotide sequence identical to SEQ ID NO:32, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, amino acid sequence of amino acids 23 to 314, which corresponds to SEQ id no:31, or the amino acid sequence of amino acids 23 to 314 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence 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 from amino acids 23 to 314 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32 from nucleotide 120 to 995; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31 from amino acid 23 to 314.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, and SEQ ID NO:30, 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:30 or a fragment of the nucleotide sequence of SEQ ID NO:30, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, and SEQ id no:29, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:29 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:29, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:30, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 29.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, and SEQ ID NO:32, 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:32 or a fragment of the nucleotide sequence of SEQ ID NO:32, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:31, and SEQ ID NO:31, 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:31 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:31, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:32, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 31.

In one embodiment, the vaccine antigen comprises SEQ ID NO:28, and SEQ ID NO:28, 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:28 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:28, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:28, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:27, and SEQ ID NO:27, 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:27 or a fragment of the nucleotide sequence of SEQ ID NO:27, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:28, and SEQ id no:28, 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:28 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:28, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:27, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:28, and a sequence of amino acids.

In one embodiment, the vaccine antigen comprises SEQ ID NO:49, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:49, and a sequence of amino acids. The SEQ ID NO:49 corresponds to the amino acid sequence of the full-length S protein from omacron ba.1, which amino acid sequence comprises an amino acid sequence corresponding to SEQ ID NO:1 (residues 983 and 984 of SEQ ID NO: 49).

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:50, and SEQ ID NO:50, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:50, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and a sequence of amino acids. The SEQ ID NO:50 is a nucleotide sequence designed to encode an amino acid sequence of a full-length S protein from Omicron ba.1 which corresponds to SEQ ID NO:1 (residues 983 and 984 of SEQ ID NO: 49) has a proline residue at the position of residues 986 and 987.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:51, and SEQ ID NO:51, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:49, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:51, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and a sequence of amino acids. The SEQ ID NO:51 corresponds to an RNA construct (e.g., comprising a 5'utr, an S protein coding sequence, a 3' utr, and a poly a tail) that encodes an amino acid sequence of a full-length S protein from omacron ba.1 that corresponds to SEQ ID NO:1 (residues 983 and 984 corresponding to SEQ ID NO: 49) has a proline residue at the position of residues 986 and 987.

In one embodiment, the vaccine antigen comprises SEQ ID NO:55, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO: 55.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:56, and SEQ ID NO:56, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:55, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:56, a nucleotide sequence of 56; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 55.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:57, and SEQ ID NO:57, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:55, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:55, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:57, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 55.

In one embodiment, the vaccine antigen comprises SEQ ID NO:58, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:58, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:59, and SEQ ID NO:59, or 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 having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO: 59; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:60, and SEQ ID NO:60, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:58, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:60, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, and a sequence of amino acids.

In one embodiment, the vaccine antigen comprises SEQ ID NO:61, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:61, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:62a, and SEQ ID NO:62a, or 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:62a or a fragment of the nucleotide sequence of SEQ ID NO:62a, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the nucleotide sequence; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:62 a; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, and a sequence of amino acids.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:63a, and SEQ ID NO:63a, or 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:63a or a fragment of the nucleotide sequence of SEQ ID NO:63a, a fragment of 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; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:61, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:63 a; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, and a sequence of amino acids.

In one embodiment, the vaccine antigen comprises SEQ ID NO:52, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, the vaccine antigen comprises SEQ ID NO:52, and an amino acid sequence of seq id no.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:53, and SEQ ID NO:53, or 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 having a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:53, a nucleotide sequence of 53; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and an amino acid sequence of seq id no.

In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:54, and SEQ ID NO:54, or 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, a fragment of a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and 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 or an immunogenic fragment of the amino acid sequence of SEQ ID NO:52, an immunogenic fragment of an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity to the amino acid sequence. In one embodiment, RNA (i) encoding a vaccine antigen comprises SEQ ID NO:54, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and an amino acid sequence of seq id no.

In one embodiment, the vaccine antigen comprises a contiguous sequence of SARS-CoV-2 coronavirus spike (S) protein consisting of or consisting essentially of the above-described amino acid sequence derived from SARS-CoV-2S protein or an immunogenic fragment thereof (antigenic peptide or protein) comprised by the above-described vaccine antigen. In one embodiment, the vaccine antigen comprises 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, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), described herein as BNT162b1 (RBP 020.3), BNT162b2 (RBP 020.1 or RBP 020.2), or BNT162b3 (e.g., BNT162b3 c). In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as RBP 020.2. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), described herein as BNT162b3 (e.g., BNT162b3 c).

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:21, and SEQ ID NO:21, and/or (ii) encodes a nucleotide sequence comprising 95%, 90%, 85% or 80% identity to the nucleotide sequence of seq id no: SEQ ID NO:5, or with SE0 ID NO:5 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:21, and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 5.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:19 or 20, and SEQ ID NO:19 or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:7, or an amino acid sequence that hybridizes to SEQ ID NO:7 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:19 or 20; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 7.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:20, and SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:7, or an amino acid sequence that hybridizes to SEQ ID NO:7 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:20, and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 7.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:30, and SEQ ID NO:30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:29, or an amino acid sequence that hybridizes to SEQ ID NO:29, has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:30, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 29.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:50, and SEQ ID NO:50, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, or an amino acid sequence that hybridizes to SEQ ID NO:49 has an amino acid sequence that is at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:50, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and a sequence of amino acids.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:51, and SEQ ID NO:51 has a nucleotide sequence having at least 99.5%,99%,98.5%,98%,98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, or an amino acid sequence that hybridizes to SEQ ID NO:49 has an amino acid sequence that is at least 99.5%,99%,98.5%,98%,98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:51, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:49, and a sequence of amino acids.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:57, and SEQ ID NO:57, a nucleotide sequence having at least 99.5%,99%,98.5%,98%,98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:55, or an amino acid sequence that hybridizes to SEQ ID NO:55 has an amino acid sequence that is at least 99.5%,99%,98.5%, 98.98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:57, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO: 55.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:60, and SEQ ID NO:60, a nucleotide sequence having at least 99.5%,99%,98.5%,98%,98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, or an amino acid sequence that hybridizes to SEQ ID NO:58 has an amino acid sequence that is at least 99.5%,99%,98.5%, 98.98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:60, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:58, and a sequence of amino acids.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:63a, and SEQ ID NO:63a, a nucleotide sequence having at least 99.5%,99%,98.5%,98%,98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, or an amino acid sequence that hybridizes to SEQ ID NO:61 has an amino acid sequence that is at least 99.5%,99%,98.5%, 98.98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:63 a; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:61, and a sequence of amino acids.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:53, and SEQ ID NO:53 has a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, or an amino acid sequence that hybridizes to SEQ ID NO:52 has an amino acid sequence that is at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:53, a nucleotide sequence of 53; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and an amino acid sequence of seq id no.

In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:54, and SEQ ID NO:54, a nucleotide sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identity; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, or an amino acid sequence that hybridizes to SEQ ID NO:52 has an amino acid sequence that is at least 99.5%, 99%, 98.5%, 98%, 98.5% or 97% identical. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:54, a nucleotide sequence of seq id no; and/or (ii) encodes an amino acid sequence comprising: SEQ ID NO:52, and an amino acid sequence of seq id no.

As used herein, the term "vaccine" refers to a composition that induces an immune response upon vaccination into a subject. In some embodiments, the induced immune response provides protective immunity.

In one embodiment, the RNA encoding the antigenic molecule is expressed in cells of the subject to provide the antigenic molecule. In one embodiment, the expression of the antigenic 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 antigenic molecule is transiently expressed in the cells of the subject. In one embodiment, expression of the RNA encoding the antigen molecule occurs in the muscle after administration of the RNA encoding the antigen molecule, in particular after intramuscular administration of the RNA encoding the antigen molecule. In one embodiment, expression of the RNA encoding the antigen molecule occurs in the spleen after administration of the RNA encoding the antigen molecule. In one embodiment, expression of the RNA encoding the antigen molecule occurs in antigen presenting cells (preferably professional antigen presenting cells) after administration of the RNA encoding the antigen molecule. 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 antigenic molecule, no or substantially no expression of the RNA encoding the antigenic molecule occurs in the lung and/or liver. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in the spleen is at least 5 times greater than the amount expressed in the lung.

In some embodiments, the methods and agents described herein (e.g., RNA compositions) result in delivery of RNA encoding a vaccine antigen to lymph nodes and/or spleen following administration to a subject, particularly following intramuscular administration. In some embodiments, the RNA encoding the vaccine antigen is detectable in the lymph nodes and/or spleen 6 hours or later, and preferably up to 6 days or more after administration.

In some embodiments, the methods and agents described herein (e.g., mRNA compositions) result in delivery of RNA encoding a vaccine antigen to B cell follicles, subintimal lymphatics, and/or T cell regions, particularly to B cell follicles of lymph nodes and/or subintimal lymphatics of lymph nodes, following administration to a subject, particularly following intramuscular administration.

In some embodiments, following administration to a subject, particularly following intramuscular administration, the methods and agents described herein (e.g., mRNA compositions) result in delivery of RNA encoding a vaccine antigen to T cell regions of lymph nodes and B cells in the middle sinus (cd19+), subintimal sinus macrophages (cd169+) and/or dendritic cells (cd11c+), particularly B cells of lymph nodes (cd19+) and/or subintimal sinus macrophages (cd169+).

In some embodiments, the methods and agents described herein (e.g., mRNA compositions) result in delivery of RNA encoding a vaccine antigen to the white marrow of the spleen after administration to a subject, particularly after intramuscular administration.

In some embodiments, the methods and agents described herein (e.g., mRNA compositions) result in delivery of RNA encoding a vaccine antigen to B cells, DCs (cd11c+), particularly those surrounding B cells, and/or macrophages of the spleen, particularly B cells and/or DCs (cd11c+), following administration to a subject, particularly following intramuscular administration.

In one embodiment, the vaccine antigen is expressed in the lymph nodes and/or spleen, in particular in the cells of the above lymph nodes and/or spleen.

Peptide and protein antigens suitable for use in accordance with the present disclosure generally include peptides or proteins comprising an epitope of the SARS-CoV-2S protein or a functional fragment thereof for inducing an immune response. The peptide or protein or epitope may be derived from a target antigen, i.e. an antigen against which an immune response is elicited. For example, a peptide or protein antigen or an epitope contained within a 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 a SARS-CoV-2S protein.

An antigen molecule or processed product thereof, e.g., a fragment thereof, may bind to an antigen receptor carried by an immune effector cell, such as BCR or TCR, or to an antibody.

The peptide or protein antigen provided to a subject by administration of RNA encoding the peptide and protein antigen according to the present disclosure, i.e., a vaccine antigen, preferably results in induction of an immune response, e.g., a humoral and/or cellular immune response, in the subject provided with the peptide or protein antigen. The immune response is preferably directed against a target antigen, in particular the coronavirus S protein, in particular the SARS-CoV-2S protein. Thus, a vaccine antigen may comprise a target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragments or variants are immunologically equivalent to the target antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" refers to an agent that results in the induction of an immune response that targets the antigen, i.e., the target antigen. Thus, a vaccine antigen may correspond to or may comprise a target antigen, may correspond to or may comprise a fragment of a target antigen, or may correspond to or may comprise an antigen homologous to a target antigen or fragment thereof. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence homologous to an immunogenic fragment of a target antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to an antigen fragment capable of inducing an immune response against a 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 substantially the same immunological properties and/or exerts the same or substantially the same immunological effect (e.g., with respect to the type of immunological effect). In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or property of an antigen or antigen variant for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if the amino acid sequence induces an immune response having specificity for reacting with the reference amino acid sequence upon exposure to the immune system of a subject.

As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector function. The term "activated immune effector cells" refers in particular to immune effector cells that undergo cell division.

The term "priming" refers to a process in which immune effector cells, such as T cells, are first contacted with their specific antigen and caused to differentiate into effector cells, such as effector T cells.

The term "clonal amplification" or "amplification" refers to a process in which a specific entity is increased. 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 expand with specific immune effector cells recognizing the antigen. Preferably, clonal expansion results in differentiation of immune effector cells.

The term "antigen" relates to an agent comprising an epitope against which an immune response can be generated. In particular, the term "antigen" includes proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system (such as antigen presenting cells, e.g., dendritic cells or macrophages). In one embodiment, the antigen or a processed product thereof, such as a T-cell epitope, binds to a T-or B-cell receptor, or to an immunoglobulin molecule, such as an antibody. Thus, the antigen or processed product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, the antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS-CoV-2S protein, and the epitope is derived from such an antigen.

The term "viral antigen" refers to any viral component having antigenic properties, i.e. capable of eliciting an immune response in an individual. The viral antigen may be a coronavirus S protein, for example, SARS-CoV-2S protein.

The term "expressed on the surface of a cell" or "associated with the surface of a cell" means that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, wherein at least a portion of the molecule faces the extracellular space of the cell and is accessible from the outside of the cell, e.g., by antibodies located outside the cell. In this context, a fraction 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 through one or more transmembrane domains, one or more lipid anchors, or through interactions with any other protein, lipid, sugar, or other structure that may be found on the outer leaflet of the cytoplasmic membrane. For example, the molecule associated with the cell surface may be a transmembrane protein having an extracellular portion, or may be a protein associated with the cell surface by interacting with another protein that is a transmembrane protein.

"cell surface" or "surface of a cell" is used in its normal sense in the art and thus includes the exterior of a cell that is accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if the antigen is located on the surface and can be accessed for binding by, for example, an antigen-specific antibody added to the cell.

The term "extracellular portion" or "extracellular domain" in the context of the present disclosure refers to a portion of a molecule, such as a protein, which faces the extracellular space of a cell and is preferably accessible from outside said cell, e.g. by a binding molecule (such as an antibody) located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, an epitope may be recognized by a T cell, B cell, or antibody. Epitopes of an antigen may include contiguous or non-contiguous portions of the antigen and may be from about 5 to about 100, such as from about 5 to about 50, more preferably from about 8 to about 30, and most preferably from about 8 to about 25 amino acids in length, for example, epitopes may preferably be 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, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in an immune response, where they bind peptide epitopes and present them for T cell receptor recognition on T cells. MHC-encoded proteins are expressed on the cell surface and display self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC/peptide complexes, the binding peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptide is typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids, while longer or shorter peptides may be effective.

Peptide and protein antigens may be 2-100 amino acids in length, 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 some embodiments, the peptide may be greater than 50 amino acids in length. In some embodiments, the peptide may be greater than 100 amino acids in length.

The peptide or protein antigen may be any peptide or protein that can induce or increase the ability of the immune system to produce antibodies and T cell responses to the peptide or protein.

In one embodiment, the vaccine antigen is recognized by immune effector cells. Preferably, if the vaccine antigen is recognized by immune effector cells, it is capable of inducing stimulation, priming and/or expansion of immune effector cells carrying antigen receptors recognizing the vaccine antigen in the presence of an appropriate co-stimulatory signal. In the context of 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, the antigen is presented by a diseased cell (e.g., a virus-infected cell). In one embodiment, the antigen receptor is a TCR that binds to an epitope presented in the context of MHC. In one embodiment, the binding of a TCR to an antigen presented by a cell (e.g., an antigen presenting cell) results in stimulation, priming, and/or expansion of the T cell when expressed by and/or present on the T cell. In one embodiment, binding of the TCR to an antigen presented on a diseased cell results in cytolysis and/or apoptosis of the diseased cell when expressed by and/or present on the T cell, wherein the T cell preferably releases cytotoxic factors such as perforin and granzyme.

In one embodiment, the antigen receptor is an antibody or B cell receptor that binds to an epitope in an antigen. In one embodiment, the antibody or B cell receptor binds to a native epitope of the antigen.

Nucleic acid

As used herein, the term "polynucleotide" or "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The 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, the polynucleotides are preferably isolated.

The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to the skilled artisan, including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as retroviral vectors, adenoviral vectors, or baculovirus vectors), or artificial chromosome vectors (such as Bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC), or P1 Artificial Chromosome (PAC)). The vector comprises an expression vector and a cloning vector. Expression vectors include plasmids as well as viral vectors and typically comprise the desired coding sequences and appropriate DNA sequences necessary for expression of the operably linked coding sequences in a particular host organism (e.g., bacteria, yeast, plant, insect or mammalian) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify certain desired DNA fragments and may lack the functional sequences required for expression of the desired DNA fragments.

In one embodiment of all aspects of the disclosure, the RNA encoding the vaccine antigen is expressed in cells, such as antigen presenting cells of a subject receiving treatment 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 comprising ribonucleotide residues. In preferred embodiments, the RNA comprises all or most ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide that has a hydroxy group at the 2' -position of the β -D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may be directed to internal RNA nucleotides or the addition of non-nucleotide species at one or more ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNAs.

In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As determined in the art, mRNA will typically comprise 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, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides.

In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of a suitable DNA template. The promoter controlling transcription may be any promoter of any RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In certain embodiments of the present disclosure, the RNA is a "replicon RNA" or simply "replicon", particularly a "self-replicating RNA" or a "self-amplifying RNA". In a 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 alphavirus. Alphaviruses are typically representative of positive strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for reviews of the life cycle of alphaviruses, see Jos e et al, future microbiol.,2009, volume 4, pages 837-856). The total genomic length of many alphaviruses is typically between 11,000 and 12,000 nucleotides, and genomic RNAs typically have a 5 '-cap and a 3' -poly (a) tail. The genome of alphaviruses encodes nonstructural proteins (involved in transcription, modification and replication of viral RNA, and protein modification) and structural proteins (forming viral particles). There are typically two Open Reading Frames (ORFs) in the genome. Four nonstructural proteins (nsP 1-nsP 4) are usually encoded together by a first ORF starting near the 5 'end of the genome, while the alphavirus structural proteins are encoded together by a second ORF that is present downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is greater than the second ORF in a ratio of about 2:1. In cells infected with an alphavirus, only the nucleic acid sequence encoding the nonstructural protein is translated from genomic RNA, while the genetic information encoding the structural protein is translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; gould et al, 2010,Antiviral Res, vol. 87, pages 111-124). After infection, i.e., early in the viral life cycle, (+) strand genomic RNA is used directly like messenger RNA to translate the open reading frame encoding the nonstructural polyprotein (nsP 1234). Alphavirus-derived vectors have been proposed for delivering exogenous genetic information into target cells or organisms. In a simple method, the open reading frame encoding the alphavirus structural protein is replaced by the open reading frame encoding the 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 trans-replicated by the replicase (hence the name trans-replication system). Trans-replication requires the simultaneous presence of both nucleic acid molecules in a given host cell. Nucleic acid molecules capable of being trans-replicated by replicases must contain certain alphavirus sequence elements to allow recognition by and synthesis of RNA by the alphavirus replicases.

In one embodiment, the RNAs described herein can have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside that replaces at least one (e.g., each) uridine.

As used herein, the term "uracil" describes one of the nucleobases that may be present in an RNA nucleic acid. The uracil has the structure:

as used herein, the term "uridine" describes one of the nucleosides that can be present 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 an example of a modified nucleoside that is an isomer of uridine, in which uracil is attached to the pentose ring through a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m 1 ψ), which has the following structure:

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

another exemplary modified nucleoside is 5-methyluridine (m 5U), which has the following structure:

in some embodiments, one or more uridine in the RNAs described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

In some embodiments, the RNA comprises a modified nucleoside that replaces at least one uridine. In some embodiments, the RNA comprises a modified nucleoside that replaces each uridine.

In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m 5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides include pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides include pseudouridine (ψ) and 5-methyluridine (m 5U). In some embodiments, the modified nucleosides include N1-methyl-pseudouridine (m 1 ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

In some embodiments, the modified nucleoside that replaces one or more (e.g., all) uridine in the RNA can be one or more of the following modified nucleosides: 3-methyl-uridine (m) ³ U), 5-methoxy-uridine (mo) ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s) ² U), 4-thio-uridine(s) ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho) ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo) ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo) ⁵ U), 5-carboxymethyl-uridine (cm) ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm) ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm) ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) ⁵ s ² U), 5-aminomethyl-2-thiouridine (nm) ⁵ s ² U), 5-methylaminomethyl-uridine (mn) ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mn) ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mn) ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm) ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm) ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm) ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm) ⁵ U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m) ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m) ¹ s ⁴ Psi), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m) ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydro-uridine (D), dihydro-pseudouridine (m 5S2S 7), 5, 6-dihydro-uridine, 5-methyl-dihydro-uridine (m) ⁵ D) 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methylOxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp) ³ U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp) ³ Psi), 5- (isopentenyl aminomethyl) uridine (mm) ⁵ U), 5- (isopentenyl aminomethyl) -2-thio-uridine (inm) ⁵ s ² U), alpha-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m) ⁵ Um), 2' -O-methyl-pseudouridine _ψ m), 2-thio-2' -O-methyl-uridine(s) ² Um), 5-methoxycarbonylmethyl-2' -O-methyl-uridine (mcm) ⁵ Um), 5-carbamoylmethyl-2' -O-methyl-uridine (ncm) ⁵ Um), 5-carboxymethylaminomethyl-2' -O-methyl-uridine (cmnm) ⁵ Um), 3,2' -O-dimethyl-uridine (m) ³ Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm) ⁵ Um), 1-thio-uridine, deoxythymidine, 2' -F-ara-uridine, 2' -F-uridine, 2' -OH-ara-uridine, 5- (2-carbomethoxyvinyl) uridine, 5- [3- (1-E-propenyl amino) uridine, or any other modified uridine known in the art.

In one embodiment, the RNA comprises other modified nucleosides or comprises further modified nucleosides, such as modified cytidine. For example, in one embodiment, 5-methylcytidine is partially or fully substituted with cytidine, preferably fully substituted with cytidine, in RNA. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In one embodiment, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

In some embodiments, an 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 can be modified with a 5' -cap analog. The term "5 '-cap" refers to a structure present on the 5' -end of an mRNA molecule, typically consisting of guanosine nucleotides attached to the mRNA by 5 '-to 5' -triphosphate linkages. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' -cap or 5' -cap analogue may be accomplished by in vitro transcription, wherein the 5' -cap is co-transcribed into the RNA strand, or may be post-transcriptionally linked to the RNA using a capping enzyme.

In some embodiments, the RNA (e.g., mRNA) comprises cap0, cap1, or cap2, preferably comprises cap1 or cap2, more preferably comprises cap1. According to the present disclosure, the term "cap0" includes the structure "m ⁷ Gppppn ", where N is any nucleoside bearing an OH moiety in the 2' position. According to the present disclosure, the term "cap1" includes the structure "m ⁷ GpppNm ", wherein Nm is OCH-carrying at the 2' -position ₃ Part of any nucleoside. According to the present disclosure, the term "cap2" includes the structure "m ⁷ GpppNmNM ", wherein each Nm independently is OCH-carrying at the 2' -position ₃ Part of any nucleoside.

In some embodiments, the building block cap of the RNA is m ₂ ^7，3’-O Gppp(m ₁ ^2’-O ) ApG (sometimes also referred to as m) ₂ ^7，3’O G(5’)ppp(5’)m ^2’-O ApG) having the following structure:

the following is an exemplary Cap1 RNA comprising RNA and m ₂ ^7，3’O G(5’)ppp(5’)m ^2’-O ApG：

The following is another exemplary Cap1 RNA (Cap-less analogue):

in some embodiments, a "Cap0" structure is utilized (in one embodimentIn the case, a cap analogue anti-reverse cap ARCA cap (m) ₂ ^7，3’O G (5 ') ppp (5') G)) modified RNA:

the following is a RNA and m ₂ ^7，3’O G(5’)ppp(5’)G：

Is described below for an exemplary Cap0 RNA.

In some embodiments, a cap analog Beta-S-ARCA (m ₂ ^7，2’O G (5 ') ppSp (5') G) results in a "Cap0" structure:

the following is a composition comprising Beta-S-ARCA (m ₂ ^7，2’O Exemplary Cap0 RNA of G (5 ') ppSp (5') G) and RNA:

the "D1" diastereomer of Beta-S-ARCA or "Beta-S-ARCA (D1)" is the diastereomer of Beta-S-ARCA, which elutes first on the HPLC column as compared to the D2 diastereomer of Beta-S-ARCA (D2), and thus exhibits a shorter retention time (see WO 2011/015347, incorporated herein by reference).

Particularly preferred caps are Beta-S-ARCA (D1) (m ₂ ^7，2′-O GppSpG) or m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG。

In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and/or a 3' -UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) may be present 5 '(upstream) of the open reading frame (5' -UTR) and/or 3 '(downstream) of the open reading frame (3' -UTR). The 5'-UTR (if present) is located at the 5' end upstream of the start codon of the protein coding region. The 5' -UTR is located downstream (if present) of the 5' -cap, for example immediately adjacent to the 5' -cap. The 3' -UTR, if present, is located at the 3' end of the protein coding region downstream of the stop codon, but the term "3' -UTR" preferably excludes the poly (A) sequence. Thus, the 3' -UTR is located upstream of the poly (a) sequence (if present), e.g. immediately adjacent to the poly (a) sequence.

In some embodiments, the RNA comprises a 5' -UTR comprising the sequence of SEQ ID NO:12, or a nucleotide sequence that hybridizes to SEQ ID NO:12, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

In some embodiments, the RNA comprises a 3' -UTR comprising the sequence of SEQ ID NO:13, or a nucleotide sequence that hybridizes to SEQ ID NO:13, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

Particularly preferred 5' -UTRs comprise the nucleotide sequence of 12. Particularly preferred 3' -UTRs comprise the nucleotide sequence of 13.

In some embodiments, an 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 intermittent sequence of adenylate residues that is typically located at the 3' end of an RNA molecule. The poly (A) sequence is known to those skilled in the art and may follow the 3' -UTR in the RNAs described herein. The uninterrupted poly (A) sequence is characterized by consecutive adenylate residues. In nature, uninterrupted poly (A) sequences are typical. The RNAs disclosed herein may have a poly (a) sequence that is linked 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.

A poly (a) sequence of about 120 nucleotides has been shown to have beneficial effects on RNA levels in transfected eukaryotic cells and levels of protein translated from the open reading frame present upstream (5') of the poly (a) sequence (Holtkamp et al, 2006, blood, volume 108, pages 4009-4017).

The poly (A) sequence may have any length. In some embodiments, the poly (a) sequence comprises, or consists essentially 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, in particular about 120 a nucleotides. Herein, "consisting essentially of" means that most of the 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 the nucleotides in the poly (a) sequence are a nucleotides, but the remaining nucleotides are allowed to be nucleotides other than a nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid) or C nucleotides (cytidylic acid). In the present specification, "consisting of" means that all nucleotides in the poly (a) sequence, i.e. 100% by number of the nucleotides in the poly (a) sequence are a nucleotides. The term "a nucleotide" or "a" refers to an adenylate.

In some embodiments, the poly (a) sequence is ligated during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylates) in the strand complementary to the coding strand. The DNA sequence encoding the poly (A) sequence (coding strand) is referred to as the poly (A) cassette.

In some embodiments, the poly (a) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cartridge is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any of the poly (A) cassettes disclosed in WO 2016/005324 A1 may be used in the present disclosure. Included are poly (a) cassettes consisting essentially of dA nucleotides, but interrupted by random sequences having an equivalent distribution of four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides, showing constant amplification of plasmid DNA in e.coli at the DNA level and still being related to the beneficial properties at the RNA level related to supporting RNA stability and translation efficiency. Thus, in some embodiments, the poly (a) sequence contained in the RNA molecules described herein consists essentially of a nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, no nucleotide is flanking the 3 'end of the poly (a) sequence other than the a nucleotide, 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 at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly (a) sequence may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100 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 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, the poly (a) sequence included in the RNAs described herein is an interrupted poly (a) sequence, e.g., as described in WO2016/005324, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the poly (a) sequence comprises a stretch of at least 20 adenosine residues (including, for example, at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues) followed by a linker sequence (including, for example, non-a nucleotides in some embodiments) and another stretch of at least 20 adenosine residues (including, for example, at least 30, at least 40, at least 50, at least 60, at least 70, or more adenosine residues). In some embodiments, such linker sequences may be 3-50 nucleotides in length, or 5-25 nucleotides in length, or 10-15 nucleotides in length. In some embodiments, the poly (a) sequence comprises a stretch of about 30 adenosine residues followed by a linker sequence of about 5-15 nucleotides in length (e.g., including non-a nucleotides in some embodiments) and another stretch of about 70 adenosine residues.

In some embodiments, the RNA comprises a poly (a) sequence comprising SEQ ID NO:14, or a nucleotide sequence that hybridizes to SEQ ID NO:14, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

Particularly preferred poly (a) sequences comprise SEQ ID NO: 14.

According to the present disclosure, vaccine antigens are preferably administered in the form of single-stranded, 5' -capped RNA (e.g., mRNA) that is translated into the corresponding protein upon entry into cells of a subject to whom the RNA is administered. Preferably, the RNA comprises structural elements (5 ' -cap, 5' -UTR, 3' -UTR, poly (a) sequences) optimized for maximum efficacy of the RNA in terms of stability and translation efficiency.

In one embodiment, beta-S-ARCA (D1) is used as a specific capping structure at the 5' end of the RNA. In one embodiment, m ₂ ^7，3’-O Gppp(m ₁ ^2’-O ) ApG is used as a specific capping structure at the 5' -end of the RNA. In one embodiment, the 5' -UTR sequence is derived from human α -globin mRNA and optionally has an optimized ' Kozak sequence ' to increase translation efficiency. In one embodiment, a combination of two sequence elements (FI elements) derived from a "split amino terminal enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I) is placed between the coding sequence and the poly (a) sequence to ensure higher maximum protein levels and prolonged persistence of the RNA (e.g., mRNA). In one implementation In the scheme, two repeated 3' -UTRs derived from human β -globin mRNA are placed between the coding sequence and the poly (a) sequence to ensure higher maximum protein levels and prolonged persistence of RNA (e.g., mRNA). In one embodiment, a 110 nucleotide long poly (A) sequence is used, consisting of a stretch of 30 adenosine residues, followed by a linker sequence (e.g., a 10 nucleotide linker sequence), and another 70 adenosine residues. The poly (A) sequences are designed to enhance RNA stability and translation efficiency.

In one embodiment of all aspects of the disclosure, the RNA encoding the vaccine antigen is expressed in cells of the subject receiving the treatment to provide the vaccine antigen. In one embodiment of all aspects of the disclosure, the RNA is transiently expressed in the cells of the subject. In one embodiment of all aspects of the disclosure, the RNA is in vitro transcribed RNA. In one embodiment of all aspects of the disclosure, the vaccine antigen is expressed on the cell surface. In one embodiment of all aspects of the disclosure, the vaccine antigen is expressed and presented in the context of MHC. In one embodiment of all aspects of the disclosure, the expression of the vaccine antigen is in the extracellular space, i.e. the vaccine antigen is secreted.

In the context of the present disclosure, the term "transcription" relates to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into peptides or proteins.

According to the present disclosure, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" relates to a process of synthesizing RNA, in particular mRNA, in a cell-free system, preferably in vitro using a suitable cell extract. Preferably, cloning vectors are used to produce transcripts. These cloning vectors are often referred to as transcription vectors, and are encompassed by the term "vector" in accordance with the present disclosure. In accordance with the present disclosure, the RNA used in the present disclosure is preferably in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of a suitable DNA template. The promoter controlling transcription may be any promoter of any RNA polymerase. Specific examples of RNA polymerase are T7, T3 and SP6 RNA polymerase. Preferably, in vitro transcription according to the present disclosure is controlled by the T7 or SP6 promoter. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In the case of RNA, the term "expression" or "translation" refers to a process in the ribosomes of cells by which a strand of mRNA directs the assembly of amino acid sequences to produce a peptide or protein.

In one embodiment, after administration of the RNAs described herein (e.g., formulated as RNA lipid particles), at least a portion of the RNAs are delivered to the target cells. In one embodiment, at least a portion of the RNA is delivered into 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 described herein, such as RNA lipid particles, can be used to deliver RNA to such target cells. Thus, the present disclosure also relates to methods for delivering RNA to a target cell in a subject, the methods comprising administering to the subject an RNA particle described herein. In one embodiment, the RNA is delivered into the cytosol of the target cell. In one embodiment, the RNA is translated by a target cell to produce a peptide or protein encoded by the RNA.

"coding" refers to the inherent nature of a particular nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand whose nucleotide sequence is identical to the mRNA sequence and which is generally provided in the sequence listing, and the non-coding strand which serves as a transcription template for a gene or cDNA, may both be referred to as a protein or other product encoding the gene or cDNA.

In one embodiment, the RNA encoding the vaccine antigen administered according to the present disclosure is non-immunogenic. RNA encoding an immunostimulant may be administered according to the present disclosure to provide an adjuvant effect. The RNA encoding the immunostimulant may be standard RNA or non-immunogenic RNA.

The term "non-immunogenic RNA" as used herein refers to an RNA that does not induce a response by the immune system when administered to, for example, a mammal, or that differs therefrom only in that it has not undergone the same RNA-induced response that renders the non-immunogenic RNA non-immunogenic and treated, i.e., is weaker than that induced by standard RNA (stdna). In a preferred embodiment, the non-immunogenic RNA, which is also referred to herein as modified RNA (modRNA), is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and removing double stranded RNA (dsRNA).

In order to render a non-immunogenic RNA non-immunogenic by incorporating a modified nucleoside, any modified nucleoside may be used as long as it reduces or inhibits the immunogenicity of the RNA. Particularly preferred are modified nucleosides that inhibit RNA-mediated activation of an innate immune receptor. In one embodiment, the modified nucleoside comprises replacing one or more uridine with a nucleoside comprising a modified nucleobase. In one embodiment, the modified nucleobase is a modified uracil. In one embodiment, the nucleoside comprising the modified nucleobase is selected from the group consisting of: 3-methyl-uridine (m) ³ U), 5-methoxy-uridine (mo) ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s) ² U), 4-thio-uridine(s) ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho) ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo) ⁵ U), uridine 5-oxyacetic acid methyl ester (mcmo) ⁵ U), 5-carboxymethyl-uridine (cm) ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (ch)m ⁵ U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm) ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm) ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm) ⁵ s ² U), 5-aminomethyl-2-thiouridine (nm) ⁵ s ² U), 5-methylaminomethyl-uridine (mn) ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mn) ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mn) ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm) ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm) ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm) ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm) ⁵ U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m) ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m) ¹ s ⁴ Psi), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m) ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydro-uridine (D), dihydro-pseudouridine (m 5S2S 7), 5, 6-dihydro-uridine, 5-methyl-dihydro-uridine (m) ⁵ D) 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 (acp) ³ U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp) ³ Psi), 5- (isopentenyl aminomethyl) uridine (mm) ⁵ U), 5- (isopentenyl aminomethyl) -2-thio-uridine (inm) ⁵ s ² U), alpha-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m) ⁵ Um), 2 '-O-methyl-pseudouridine (ψm), 2-thio-2' -O-methyl-uridine(s) ² Um), 5-methoxycarbonylmethyl-2' -O-methyl-uridine (mcm) ⁵ Um), 5-carbamoylmethyl-2' -O-methyl-uridine (ncm) ⁵ Um), 5-carboxymethylaminomethyl-2' -O-methyl-uridine (cmnm) ⁵ Um)、3，2' -O-dimethyl-uridine (m) ³ Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm) ⁵ Um), 1-thio-uridine, deoxythymidine, 2' -F-ara-uridine, 2' -F-uridine, 2' -OH-ara-uridine, 5- (2-carbomethoxyvinyl) uridine and 5- [3- (1-E-propenyl amino) uridine. In a particularly preferred embodiment, the nucleoside comprising the modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ) or 5-methyl-uridine (m 5U), in particular N1-methyl-pseudouridine.

In one embodiment, replacing one or more uridine with a nucleoside comprising a modified nucleobase comprises replacing 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 uridine.

During the synthesis of RNA (e.g., mRNA) by In Vitro Transcription (IVT) using T7 RNA polymerase, a number of abnormal products, including double-stranded RNA (dsRNA), are produced due to the unusual activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes, resulting in inhibition of protein synthesis. dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reverse phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using E.coli (E.Coli) RNase III that specifically hydrolyzes dsRNA but not ssRNA can be used to eliminate dsRNA contaminants from IVT RNA formulations. Furthermore, dsRNA can be isolated from ssRNA by using cellulosic material. In one embodiment, the RNA formulation is contacted with the cellulosic material under conditions that allow binding of dsRNA to the cellulosic material but not ssRNA to the cellulosic material, and the ssRNA is isolated from the cellulosic material.

As used herein, the term "removing" or "removal" refers to the feature of the contiguous separation of a population of a first substance, such as non-immunogenic RNA, from a population of a second substance, such as dsRNA, wherein the population of the first substance is not necessarily devoid of the second substance and the population of the second substance is not necessarily devoid of the first substance. However, the population of removed first material characterized by a population of second material has a measureable lower content of second material than the non-separated mixture of first material and second material.

In one embodiment, removing dsRNA from a non-immunogenic RNA comprises removing 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 substantially 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 translation of the non-immunogenic RNA in the cell is more efficient than a standard RNA having the same sequence. In one embodiment, translation is enhanced by a factor of 2 relative to its unmodified counterpart. In one embodiment, translation is enhanced by a factor of 3. In one embodiment, translation is enhanced by a factor of 4. In one embodiment, translation is enhanced by a factor of 5. In one embodiment, translation is enhanced by a factor of 6. In one embodiment, translation is enhanced by a factor of 7. In one embodiment, translation is enhanced by a factor of 8. In one embodiment, translation is enhanced by a factor of 9. In one embodiment, translation is enhanced by a factor of 10. In one embodiment, translation is enhanced by a factor of 15. In one embodiment, translation is enhanced by a factor of 20. In one embodiment, translation is enhanced by a factor of 50. In one embodiment, translation is enhanced by a factor of 100. In one embodiment, translation is enhanced by a factor of 200. In one embodiment, translation is enhanced by a factor of 500. In one embodiment, translation is enhanced by a factor of 1000. In one embodiment, translation is enhanced by a factor of 2000. In one embodiment, the factor is 10-1000 times. In one embodiment, the factor is 10-100 times. In one embodiment, the factor is 10-200 times. In one embodiment, the factor is 10-300 times. In one embodiment, the factor is 10-500 times. In one embodiment, the factor is 20-1000 times. In one embodiment, the factor is 30-1000 fold. In one embodiment, the factor is 50-1000 times. In one embodiment, the factor is 100-1000 times. In one embodiment, the factor is 200-1000 times. In one embodiment, translation enhances any other significant amount or range of amounts.

In one embodiment, the non-immunogenic RNA exhibits significantly lower innate immunogenicity as compared to a standard RNA having the same sequence. In one embodiment, the non-immunogenic RNA exhibits a 2-fold lower innate immune response than its unmodified counterpart. In one embodiment, the innate immunogenicity is reduced by a factor of 3. In one embodiment, the innate immunogenicity is reduced by a factor of 4. In one embodiment, the innate immunogenicity is reduced by a factor of 5. In one embodiment, the innate immunogenicity is reduced by a factor of 6. In one embodiment, the innate immunogenicity is reduced by a factor of 7. In one embodiment, the innate immunogenicity is reduced by a factor of 8. In one embodiment, the innate immunogenicity is reduced by a factor of 9. In one embodiment, the innate immunogenicity is reduced by a factor of 10. In one embodiment, the innate immunogenicity is reduced by a factor of 15. In one embodiment, the innate immunogenicity is reduced by a factor of 20. In one embodiment, the innate immunogenicity is reduced by a factor of 50. In one embodiment, the innate immunogenicity is reduced by a factor of 100. In one embodiment, the innate immunogenicity is reduced by a factor of 200. In one embodiment, the innate immunogenicity is reduced by a factor of 500. In one embodiment, the innate immunogenicity is reduced by a factor of 1000. In one embodiment, the innate immunogenicity is reduced by a factor of 2000.

The term "exhibiting 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 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 the production of proteins encoded by the non-immunogenic RNA. In one embodiment, the reduction allows for repeated administration of the non-immunogenic RNA without eliciting an innate immune response sufficient to eliminate detectable production of proteins encoded by the non-immunogenic RNA.

"immunogenicity" is the ability of a foreign substance (such as RNA) to elicit an immune response in a human or other animal. The innate immune system is a relatively nonspecific and direct component of the immune system. Which is one of the two major components of the vertebrate immune system and the adaptive immune system.

As used herein, "endogenous" refers to any substance from or produced within an organism, cell, tissue, or system.

As used herein, the term "exogenous" refers to any substance introduced or produced from 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 "fused" are used interchangeably. These terms refer to two or more elements or components or domains that are joined together.

Codon optimization/increase in G/C content

In some embodiments, the amino acid sequences described herein comprising SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein or an immunogenic variant thereof are encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon optimized and/or have an increased G/C content compared to the corresponding sequence region of the wild-type coding sequence. In one embodiment, codon optimisation and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

The term "codon optimized" refers to a change in codons reflecting the typical codon usage (codon usage) of a host organism, and not preferably a change in codons of the coding region of a nucleic acid molecule that alters the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, the coding region is preferably codon optimized for optimal expression in a subject treated with an RNA molecule described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA appearance in the cell. Thus, the RNA sequence can be modified such that codons are inserted that can give rise to frequently occurring trnas instead of "rare codons".

In some embodiments of the disclosure, the G/C content of the coding region of the RNAs described herein is increased as compared to the guanosine/cytosine (G/C) content of the corresponding coding sequence of the wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably unmodified as 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 the mRNA. Sequences with increased G (guanosine)/C (cytosine) content are more stable than sequences with increased A (adenosine)/U (uracil) content. With respect to the fact that several codons encode the same amino acid (so-called degeneracy of the genetic code), the most advantageous codons for stability (so-called selective codon usage) can be determined. Depending on the amino acids encoded by the RNA, there are a number of possibilities for modification of the RNA sequence compared to the wild-type sequence of the RNA sequence. In particular, codons containing a and/or U nucleotides can be modified by replacing these codons with other codons encoding the same amino acid but not containing a and/or U or containing a lower content of a and/or U nucleotides.

In various embodiments, the G/C content of the coding region of an 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, as compared to the G/C content of the coding region of a wild-type RNA. In some embodiments, the G/C content of the coding region is increased by about 10% to about 60% (e.g., about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, or about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, or about 60%) as compared to the G/C content of the coding region of the wild-type RNA.

In some embodiments, the RNAs disclosed herein comprise a sequence disclosed herein (e.g., SEQ ID NO: 9) that has been modified to encode one or more mutational features of a SARS-CoV-2 variant (e.g., variants described herein, including but not limited to ba.2 or ba.4/5 omacron variants). In some embodiments, the RNA can be modified to encode one or more mutant features of the SARS-CoV-2 variant by making as few nucleotide changes as possible. In some embodiments, the RNA may be modified to encode one or more mutations that are characteristic of the SARS-CoV-2 variant by introducing a mutation that results in a Gao Mima sub-optimization and/or increased G/C content.

In some embodiments, one or more of the mutational characteristics of the SARS-CoV-2 variant is introduced into a full-length S protein (e.g., an S protein comprising SEQ ID NO: 1). In some embodiments, one or more mutational features of the SARS-CoV-variant are introduced onto the full-length S protein with one or more proline mutations that enhance the stability of the pre-fusion confirmation. For example, in some embodiments, in a sequence that hybridizes to SEQ ID NO: proline substitutions were made at positions corresponding to positions 986 and 987 of 1. In some embodiments, at least 4 proline substitutions are made. In some embodiments, at least 4 such proline mutations are included in a sequence corresponding to SEQ ID NO:1, for example as described in WO 2021243122 A2, the entire contents of which are incorporated herein by reference. In some embodiments, such SARS-CoV-2S protein is comprised in a polypeptide corresponding to SEQ ID NO:1, which may further comprise a proline substitution at positions corresponding to residues 817, 892, 899 and 942 of SEQ ID NO: proline substitutions at positions 986 and 987 of residue 1. In some embodiments, one or more of the mutational characteristics of the SARS-CoV-2 variant are introduced into an immunogenic fragment of the S protein (e.g., the RBD of SEQ ID NO: 1.

Embodiments of administered RNA

In some embodiments, the disclosure provides an RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide comprising at least a portion of a SARS-CoV-2S protein. RNA is suitable for intracellular expression of the polypeptide. In some embodiments, such encoded polypeptide comprises a sequence corresponding to an intact S protein. In some embodiments, such encoded polypeptide does not comprise a sequence corresponding to the complete S protein. In some embodiments, the encoded polypeptide comprises a sequence corresponding to a Receptor Binding Domain (RBD). In some embodiments, the encoded polypeptide comprises a sequence corresponding to RBD, and further comprises a trimerization domain (e.g., a trimerization domain as disclosed herein, such as a fibrin domain). In some embodiments, the RBD includes a signaling domain (e.g., a signaling domain disclosed herein). In some embodiments, the RBD comprises a transmembrane domain (e.g., a transmembrane domain disclosed herein). In some embodiments, the RBD comprises a signaling domain and a trimerization domain. In some embodiments, the RBD comprises a signaling domain, a trimerization domain, and a transmembrane domain.

In some embodiments, the encoded polypeptide comprises sequences corresponding to two receptor binding domains. In some embodiments, the encoded polypeptide comprises a sequence corresponding to two receptor binding domains in tandem in an amino acid chain, e.g., as described 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 herein by reference in their entirety).

In some embodiments, the SARS-CoV-2S protein, or an immunogenic fragment thereof, comprises one or more mutations to alter or remove the glycosylation site, e.g., as described in WO2022221835A2, US20220323574A1 or WO2022195351A 1.

In some embodiments, the compositions or pharmaceutical formulations described herein comprise RNA encoding an amino acid sequence comprising SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein or an immunogenic variant thereof. Also, the methods described herein include administering such RNA.

The active platform used herein is based on an antigen-encoding RNA vaccine that induces robust neutralizing antibodies and concomitant T cell responses, thereby achieving protective immunity 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 a particularly preferred embodiment, the RNA is in vitro transcribed RNA. In some embodiments, the uRNA is mRNA. In some embodiments, the modRNA is mRNA.

Hereinafter, embodiments of these three different RNA platforms are described, wherein certain terms used when describing elements thereof have the following meanings:

S1S2 protein/S1S 2 RBD: sequences encoding the corresponding antigens of SARS-CoV-2.

nsP1, nsP2, nsP3, and nsP4: wild-type sequences and subgenomic promoters encoding Venezuelan Equine Encephalitis Virus (VEEV) RNA-dependent RNA polymerase replicase plus conserved sequence elements supporting replication and translation.

virUTR: a viral untranslated region encoding a portion of a subgenomic promoter, and replication and translation supporting sequence elements.

hAg-Kozak: 5' -UTR sequences of human alpha-globin mRNA with optimized ' Kozak sequences ' to increase translation efficiency.

Sec: sec corresponds to a secretion signal peptide (Sec) that directs the migration of nascent polypeptide chains into the endoplasmic reticulum. In some embodiments, such secretion signal peptides include an intrinsic S1S2 secretion signal peptide. In some embodiments, such secretion signal peptide is a secretion signal peptide from a non-S1S 2 protein. For example, an immunoglobulin secretion signal peptide (aa 1-22), an HSV-1gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY), an HSV-2gD 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): short linker encoding amino acids glycine (G) and serine (S)PeptidesIs generally used for fusion proteins。

Fibrin: partial sequence of T4 fibrin (folder) used as artificial trimerization domain.

TM: the TM sequence corresponds to the transmembrane portion of the protein. The transmembrane domain may be at the N-terminus, C-terminus or within the encoded polypeptide. The coding sequence of a transmembrane element is typically located in-frame (i.e., in the same reading frame), 5', 3', or internal to the coding sequence of the sequence to which it is linked (e.g., the sequence encoding a polypeptide). In some embodiments, the transmembrane domain comprises or is the transmembrane domain of Hemagglutinin (HA) of influenza virus, env of HIV-1, equine Infectious Anemia Virus (EIAV), murine Leukemia Virus (MLV), murine mammary tumor virus, G protein of Vesicular Stomatitis Virus (VSV), rabies virus, or seven transmembrane domain receptor. In some embodiments, the transmembrane portion of the protein is from an S1S2 protein.

FI element: the 3' -UTR is a combination of two sequence elements derived from the "split amino terminal enhancer" (AES) mRNA (referred to as F) and the mitochondrially encoded 12S ribosomal RNA (referred to as I). These elements are identified by an ex vivo selection process of sequences that confer RNA stability and increase total protein expression.

a30L70: a poly (a) tail of 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 designed to enhance the stability and translation efficiency of RNA in dendritic cells.

In some embodiments, the vaccine RNAs described herein can comprise one of the following structures from 5 'to 3':

cap-5 '-UTR-vaccine antigen coding sequence-3' -UTR-poly (A)

Or (b)

Cap-hAg-Kozak-vaccine antigen coding sequence-FI-A30L 70.

In some embodiments, the vaccine antigens described herein can comprise a full-length S protein or an immunogenic fragment thereof (e.g., RBD). In some embodiments in which the vaccine antigen comprises a full-length S protein, its secretion signal peptide and/or transmembrane domain may be replaced with a heterologous secretion signal peptide (e.g., as described herein) and/or a heterologous transmembrane domain (e.g., as described herein).

In some embodiments, the vaccine antigens described herein may comprise one of the following structures from N-terminus to C-terminus:

signal sequence-RBD-trimerization domain

Or (b)

Signal sequence-RBD-trimerization domain-transmembrane domain.

The RBD and trimerisation domains can be separated by a linker, in particular a GS linker (such as a linker having the amino acid sequence GSPGSGSGS). The trimerisation domain and the transmembrane domain may be separated by a linker, in particular a GS linker (such as a linker having the amino acid sequence gsgsgsgs).

The signal sequence may be a signal sequence as described herein. As described herein, an RBD may be an RBD domain. The trimerization domain may be a trimerization domain as described herein. The transmembrane domain may be a transmembrane domain as described herein.

In one embodiment of the present invention, in one embodiment,

the signal sequence comprises SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO:31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence,

the RBD comprises SEQ ID NO:1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence,

the trimerization domain comprises SEQ ID NO:10 or amino acid sequence of amino acids 3 to 29 of SEQ ID NO:10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence; and

the transmembrane domain comprises SEQ ID NO:1, 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 1207 to 1254 of the amino acid sequence

In one embodiment, the signal sequence comprises SEQ ID NO:1, or the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO:31 from amino acid 1 to 22,

the RBD comprises SEQ ID NO:1 from amino acids 327 to 528,

the trimerization domain comprises SEQ ID NO:10 or amino acid sequence of amino acids 3 to 29 of SEQ ID NO:10, an amino acid sequence of seq id no; and

the transmembrane domain comprises SEQ ID NO:1 from amino acids 1207 to 1254.

In some embodiments, an RNA polynucleotide comprising an open reading frame (such as the nucleotide sequence of SEQ ID NO:50 or the nucleotide sequence of SEQ ID NO:53, variants or fragments thereof) encoding a vaccine antigen (e.g., a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2S protein or an immunogenic variant thereof), or comprising a sequence encoding a vaccine antigen (e.g., a 3'utr sequence comprising the nucleotide sequence of SEQ ID NO: 13), a 3' utr sequence (e.g., a poly a sequence comprising the nucleotide sequence of SEQ ID NO: 14), and a poly a sequence (e.g., a sequence comprising the nucleotide sequence of SEQ ID NO: 14) further comprises a 5 'Cap (e.g., a 5' Cap comprising Cap1 structure). In some embodiments, the RNA is formulated in a composition comprising ((4-hydroxybutyl) azadiyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), cholesterol, distearoyl phosphatidylcholine, and (2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide).

The RNA described herein or encoding the vaccine antigens described herein may be unmodified uridine-containing RNA (uRNA), nucleoside-modified RNA (modRNA), or self-amplifying RNA (saRNA). In some embodiments, the uRNA is mRNA. In some embodiments, the modRNA is mRNA. In one embodiment, the RNA described herein or encoding the vaccine antigens described herein is nucleoside modified RNA (modRNA).

Variant specific vaccine

In some embodiments, the RNAs disclosed herein encode an S protein comprising one or more mutations that are characteristic of SARS-CoV-2 variants. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of Alpha variants. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of the Beta variant. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of a Delta variant. In some embodiments, the RNA encodes a SARS-CoV-2S 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.4/5Omicron variant). In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of the BA.1 Omacron variant. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of the BA.2 Omacron variant. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of the BA.2.12.1Omicron variant. In some embodiments, the RNA encodes a SARS-CoV-2S protein that comprises one or more mutations characteristic of the BA.3 Omacron variant. In some embodiments, the RNA encodes a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.4 or ba.5omicron variants.

Unmodified uridine RNAs (uRNAs)

In some embodiments, the non-modifiedUridine RNAs are messenger RNAs. In some embodiments, the unmodified message RNA is caused to beThe active ingredient of the drug substance is single-chain mRNA which is translated after entering cells. In addition to the sequences encoding coronavirus vaccine antigens (i.e. open reading frames),each uRNA preferably comprises a generic structural element that is optimized for the maximum efficacy of the RNA in terms of stability and translation efficiency (including, for example, 5' -cap, 5' -UTR, 3' -UTR, poly (a) -tail, as described herein). A preferred 5' cap structure is Beta-SS-ARCA (D1) (m ₂ ^7，2′ - ^O GppSpG). Preferred 5 '-UTRs and 3' -UTRs comprise SEQ ID NOs: 12 and SEQ ID NO:13. preferred poly (a) tails comprise SEQ ID NO: 14.

Different embodiments of the 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

Viral spike protein (S1S 2 protein) of the encoded antigen SARS-CoV-2 (S1S 2 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

BNT162a1；RBL063.3(SEQ ID NO：17；SEQ ID NO：5)

Structure Beta-S-ARCA (D1) -hAg-Kozak-RBD-GS-Fibritin-FI-A30L70

Viral spike protein (S protein) of the encoded antigen SARS-CoV-2 (partial sequence, receptor Binding Domain (RBD) of S1S2 protein)

FIG. 3 graphically shows the general structure of RNA encoding an antigen.

Nucleoside modified RNA (modRNA)

In some embodiments, the nucleoside modified RNA is mRNA. In some embodiments, the active ingredient of the nucleoside modified RNA (modRNA) drug substance is a single stranded RNA (e.g., mRNA) that can be translated upon entry into a cell. In addition to the sequences encoding coronavirus vaccine antigens (i.e., open reading frames), each modRNA also contains universal structural elements (5 ' -cap, 5' -UTR, 3' -UTR, poly (A) optimized for maximum efficacy of RNA as uRNATail). In contrast to uRNA, the modRNA comprises at least one nucleotide modification (e.g., as described herein). In some embodiments, the modRNA comprises 1-methyl-pseudouridine instead of uridine. Preferred 5' cap structures are m ₂ ^7，3’-O Gppp(m ₁ ^2’-O ) ApG. Preferred 5 '-UTRs and 3' -UTRs comprise SEQ ID NOs: 12 and SEQ ID NO: 13. Preferred poly (a) -tails comprise SEQ ID NO: 14. An additional purification step is performed on the modRNA to reduce dsRNA contaminants generated during the in vitro transcription reaction.

Different embodiments of the platform are as follows:

BNT162b2；RBP020.1(SEQ ID NO：19；SEQ ID NO：7)

structure m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

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

Structure m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG)-hAg-Kozak-S 1S2-PP-FI-A30L70

BNT162b1；RBP020.3(SEQ ID NO：21；SEQ ID NO：5)

Structure m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG)-hAg-Kozak-RBD-GS-Fibritin-FI-A3OL70

Viral spike protein (S1S 2 protein) of the encoded antigen SARS-CoV-2 (partial sequence, receptor Binding Domain (RBD) of the S1S2 protein fused to fibrin)

FIG. 4 graphically shows the general structure of RNA encoding an antigen.

BNT162b3c(SEQ ID NO：29；SEQ ID NO：30)

Structure m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70

The viral spike protein (S1S 2 protein) of the encoded antigen SARS-CoV-2 (partial sequence, receptor Binding Domain (RBD) of S1S2 protein fused to fibrin fused to transmembrane domain (TM) of S1S2 protein; intrinsic S1S2 protein secretion signal peptide (aa 1-19) at N-terminus of antigen sequence

BNT162b3d(SEQ ID NO：31；SEQ ID NO：32)

The virus spike protein (S1S 2 protein) of the encoded antigen SARS-CoV-2 (partial sequence, receptor Binding Domain (RBD) of S1S2 protein fused to fibrin fused to transmembrane domain (TM) of S1S2 protein; immunoglobulin secretion signal peptide (aa 1-22) at N-terminal end of antigen sequence.

BNT162b2-Beta variant; RBP020.11 (SEQ ID NO:57; SEQ ID NO: 55)

A viral spike protein (S1S 2 protein) (S1S 2 full-length protein, sequence variant) encoding an antigen SARS-CoV-2 comprising the mutational characteristics of the Beta variant of SARS-CoV-2

BNT162b2-Alpha variant; RBP020.14 (SEQ ID NO:60; SEQ ID NO: 58)

A viral spike protein (S1S 2 protein) (S1S 2 full-length protein, sequence variant) encoding an antigen SARS-CoV-2 comprising the mutational characteristics of the Alpha variant of SARS-CoV-2

BNT162b2-Delta variant; RBP020.16 (SEQ ID NO:63a; SEQ ID NO: 61)

A viral spike protein (S1S 2 protein) (S1S 2 full-length protein, sequence variant) encoding an antigen SARS-CoV-2 comprising the mutant characteristics of the Delta variant of SARS-CoV-2

Nucleotide sequence of RBP020.11 (Beta-specific vaccine)

The nucleotide sequence is shown with individual sequence elements indicated by bold letters. In addition, the sequence of the translated protein is shown in italics under the coding nucleotide sequence (=stop codon). The red text indicates point mutations in the nucleotide and amino acid sequences.

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The sequence of RBP020.11 is also shown in table 3.

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Nucleotide sequence of RBP020.14 (Alpha-specific vaccine)

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The sequence of BP020.14 is also shown in table 4.

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Nucleotide sequence of RBP020.16 (Delta specific vaccine)

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The sequence of BP020.14 is also shown in table 5.

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Tables 3-16 show the amino acid sequences of SARS-CoV-2S proteins encoded by RNAs from different variants described herein, which correspond to SEQ ID NOs: 1 and V987P have 2 proline substitutions at positions. In some embodiments, the RNAs described herein encode SARS-CoV-2S proteins described herein (e.g., in some embodiments, as described in tables 3-16) that are encoded in a sequence corresponding to SEQ ID NO:1 and V987P do not have 2 proline substitutions at positions. In some embodiments, such RNAs described herein encode SARS-CoV-2S proteins that comprise one or more variant-characteristic mutations described herein, and further comprise at least four (including, for example, at least five, at least six, or more) proline mutations. In some embodiments, at least four such proline mutations include a sequence corresponding to SEQ ID NO:1, for example, as described in WO 2021243122A2 (the entire contents of which are incorporated herein by reference) at positions 817, 892, 899 and 942. In some embodiments, this corresponds to SEQ ID NO: residues 817, 892, 899 and 942 of 1, the entire contents of which contain a proline substitution, are incorporated herein by reference in their entirety, can also be found in a polypeptide corresponding to SEQ ID NO: residues 986 and 987 of 1 comprise a proline substitution.

Self-amplified RNA (saRNA)

The active ingredient of self-amplifying RNA (saRNA) drug substance is single-stranded RNA, which self-amplifies upon entry into cells, and then coronavirus vaccine antigen is translated. In contrast to uRNA and modRNA, which preferably encode a single protein, the coding region of saRNA comprises two Open Reading Frames (ORFs). The 5' -ORF encodes an 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 an antigen. In addition, the saRNA UTR comprises 5 'and 3' Conserved Sequence Elements (CSEs) required for self-amplification. saRNA comprises general structural elements (including, e.g., 5' -cap, 5' -UTR, 3' UTR, poly (a) tail) optimized for maximum efficacy of RNA as uRNA. In some embodiments, the saRNA preferably contains uridine. In some embodiments, the saRNA comprises one or more nucleoside modifications as described herein. A preferred 5' cap structure is Beta-S-ARCA (D1) (m ₂ ^7，2-O Gpp SpG)。

Cytoplasmic transmission of saRNA initiates an alphavirus-like life cycle. However, saRNA does not encode the structural proteins of alphaviruses required for genome packaging or cell entry, and thus the production of replication-competent viral particles is highly unlikely or impossible. Replication does not involve any intermediate steps to generate DNA. Thus, the use/uptake of saRNA does not pose the risk of genomic integration or other permanent genetic modification within the target cell. In addition, saRNA itself effectively activates the innate immune response by recognizing dsRNA intermediates, thereby preventing its continued replication.

Different embodiments of the platform are as follows:

RBS004.1(SEQ ID NO：24；SEQ ID NO：7)

structure Beta-S-ARCA (D1) -replicase-S1S 2-PP-FI-A30L70

Viral spike protein (S protein) of the encoded antigen SARS-CoV-2 (S1S 2 full-length protein, sequence variant)

RBS004.2(SEQ ID NO：25；SEQ ID NO：7)

Structure Beta-S-ARCA (D1) -replicase-S1S 2-PP-FI-A30L70

BNT162c1；RBS004.3(SEQ ID NO：26；SEQ ID NO：5)

Structure Beta-S-ARCA (D1) -replicase-RBD-GS-fibrin-FI-A30L 70

RBS004.4(SEQ ID NO：27；SEQ ID NO：28)

Structure Beta-S-ARCA (D1) -replicase-RBD-GS-fibrin-TM-FI-A30L 70

FIG. 5 graphically shows the general structure of RNA encoding an antigen.

In some embodiments, the vaccine RNAs described herein comprise nucleotides selected from the group consisting of: SEQ ID NO: 15. 16, 17, 19, 20, 21, 24, 25, 26, 27, 30 and 32. Particularly preferred vaccine RNAs described herein comprise a nucleotide sequence selected from the group consisting of: SEQ ID NO: 15. 17, 19, 21, 25, 26, 30 and 32, such as a nucleotide sequence selected from the group consisting of: SEQ ID NO: 17. 19, 21, 26, 30 and 32.

In some embodiments, the RNA described herein is formulated in a lipid nanoparticle, a lipid complex (lipoplex), a multimeric complex (PLX), a lipidated multimeric complex (LPLX), a liposome, or a polysaccharide nanoparticle. In some embodiments, the RNAs described herein are preferably formulated in Lipid Nanoparticles (LNPs). In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and 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 intramuscular administration in an aqueous cryoprotectant buffer. The drug is preferably a preservative-free RNA sterile dispersion for intramuscular administration formulated in Lipid Nanoparticles (LNP) in an aqueous cryoprotectant buffer.

In various embodiments, the pharmaceutical product preferably comprises the components shown below in the proportions or concentrations shown below:

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

[2] ALC-0159=2- [ (polyethylene glycol) -2000] -N, N-ditetradecylacetamide/2- [2- (ω -methoxy (polyvinyl alcohol 2000) ethoxy ] -N, N-ditetradecylacetamide

[3] Dspc=1, 2-distearoyl-sn-glycero-3-phosphatidylcholine

Right amount = sufficient amount (as satisfied as possible)

ALC-0315：

ALC-0159：

DSPC：

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Cholesterol:

in some embodiments, the particles disclosed herein are formulated in a solution comprising 10mm tris and 10% sucrose and optionally having a pH of about 7.4. In some embodiments, the particles disclosed herein are formulated in a solution comprising about 103mg/ml sucrose, about 0.20mg/ml tromethamine (Tris base), and about 1.32mg/ml Tris.

In some embodiments, the composition comprises:

(a) About 0.1mg/mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof,

(b) About 1.43mg/ml ALC-0315,

(c) About 0.18mg/ml ALC-0159,

(d) About 0.31mg/ml of DSPC,

(e) About 0.62mg/ml of cholesterol,

(f) About 103mg/ml of sucrose was used,

(g) About 0.20mg/ml tromethamine (Tris base),

(h) About 1.32mg/ml Tris (hydroxymethyl) aminomethane hydrochloride (Tris HCl), and

(i) And a proper amount of water.

In one embodiment, the ratio of RNA (e.g., mRNA) to total lipid (N/P) is between 6.0 and 6.5, e.g., about 6.0 or about 6.3.

Nucleic acid-containing particles

Nucleic acids described herein, such as RNA encoding vaccine antigens, can be formulated into particles for administration.

In the present disclosure, the term "particle" relates to a structured entity formed by a molecule or a molecular complex. In one embodiment, the term "particle" relates to a micro-or nano-sized structure, such as a micro-or nano-sized dense structure dispersed in a medium. In one embodiment, the particles are nucleic acid-containing particles, such as particles comprising DNA, RNA, or mixtures thereof.

The electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids participate in the formation of particles. This results in complex formation and spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

As used in this disclosure, "nanoparticle" refers to particles having an average diameter suitable for parenteral administration.

"nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., a cell, tissue, organ, etc.). The nucleic acid particles may be formed from at least one cationic lipid or cationic-ionizable lipid-like substance, at least one cationic polymer such as protamine or mixtures thereof, and a nucleic acid. In some embodiments, exemplary nanoparticles include lipid nanoparticles, multimeric complexes (PLX), lipidated multimeric complexes (LPLX), liposomes, or polysaccharide nanoparticles.

In some embodiments, RNA encoding an amino acid sequence comprising a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of a SARS-CoV-2S protein or an immunogenic variant thereof is formulated as LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids; one or more neutral lipids (e.g., sterols, such as cholesterol, and/or phospholipids in some embodiments), 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-phosphorylcholine), cholesterol, sucrose, tromethamine (Tris), tromethamine hydrochloride, and water.

RNA particles as described herein include nanoparticles. In some embodiments, exemplary nanoparticles include lipid nanoparticles, lipid complexes, multimeric complexes (PLX), lipidated multimeric complexes (LPLX), liposomes, or polysaccharide nanoparticles.

Polymeric complexes (PLX), polysaccharide nanoparticles and liposomes are all delivery techniques well known to those skilled in the art. See, for example, respectively Ulrich and Ernst wagner. "Nucleic acid therapeutics using polyplexes: a journ ey of 50years (and beyond) "Chemical reviews 115.19 (2015): 11043-11078; plucinski, alexander, san Lyu and Bernhard VKJ Schmidt, "Polysaccharide nanoparticles: from fabrication to applications, "Journal of MaterialsChemistry B (2021); and Tenchov, rumia et al, "Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement," ACS nano 15.11 (2021): 16982-17015, each of which is incorporated herein by reference in its entirety.

In some embodiments, the concentration of RNA in the RNA pharmaceutical formulation is about 0.1mg/ml. In some embodiments, the concentration of RNA in the RNA pharmaceutical formulation is about 30 μg/ml to about 100 μg/ml. In some embodiments, the concentration of RNA in the RNA pharmaceutical formulation is about 50 μg/ml to about 100 μg/ml.

Without wishing to be bound by any theory, it is believed that the cationic lipid or cationically ionizable lipid or lipid-like substance and/or cationic polymer combines with the nucleic acid to form an aggregate, and the aggregate produces colloidally stable particles.

In one embodiment, the particles described herein further comprise at least one lipid or lipid-like substance other than a cationic lipid or a lipid-like substance that ionizes a cation, at least one polymer other than a cationic polymer, or a mixture thereof.

In some embodiments, the nucleic acid particles comprise more than one type of nucleic acid molecule, wherein the molecular parameters of the nucleic acid molecules (e.g., in terms of molar mass or basic structural elements (such as molecular structure), capping, coding regions, or other characteristics) may be similar to or different from each other.

In one embodiment, the nucleic acid particles described herein can have an average diameter of about 30nm to about 1000nm, about 50nm to about 800nm, about 70nm to about 600nm, about 90nm to about 400nm, or about 100nm to about 300 nm.

The nucleic acid particles described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. For example, the nucleic acid particles can exhibit a polydispersity index in the 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 number of nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is related to the charge ratio because nitrogen atoms (depending on the pH) are usually positively charged, while phosphate groups are negatively charged. The N/P ratio at charge balance depends on the pH. Lipid formulations are typically formed at N/P ratios greater than 4 to 12, as positively charged nanoparticles are believed to facilitate transfection. In this case, the RNA is considered to be fully bound to the nanoparticle.

The nucleic acid particles described herein can be prepared using a variety of methods, which can include obtaining a colloid from at least one cationic lipid or cationic-ionizable lipid-or lipid-like substance and/or at least one cationic polymer, and mixing the colloid with a nucleic acid to obtain a nucleic acid particle.

The term "colloid" as used herein relates to a type of homogeneous mixture in which the dispersed particles do not precipitate out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 nm and 1000 nm. The mixture may be referred to as a colloid or colloid suspension. Sometimes the term "colloid" refers only to the particles in the mixture, not the entire suspension.

For the preparation of colloids comprising at least one cationic lipid or cationically ionizable lipid-like substance and/or at least one cationic polymer, methods conventionally used for the preparation of liposome vesicles and suitably adapted are suitable herein. The most common methods for preparing liposome vesicles share the following basic stages: (i) dissolution of lipids in organic solvents, (ii) drying of the resulting solution, and (iii) hydration of anhydrous lipids (using various aqueous media).

In the membrane hydration process, the lipids are first dissolved in a suitable organic solvent and dried to produce a membrane at the bottom of the flask. The obtained lipid membrane is hydrated using a suitable aqueous medium to produce a liposome dispersion. In addition, an additional downsizing step may be included.

Reverse phase evaporation is an alternative method for the preparation of thin film hydration of liposome vesicles, which involves the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Short sonications of the mixture are required to homogenize the system. The organic phase was removed under reduced pressure to give a milky gel which subsequently became a liposome suspension.

The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. In general, the RNA lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. In one embodiment, such colloidal liposome dispersions are formed using ethanol injection techniques as follows: an ethanol solution comprising lipids (such as cationic lipids and additional lipids) is injected into the aqueous solution with stirring. In one embodiment, the RNA lipid complex particles described herein are obtained without an extrusion step.

The term "extrusion" or "extrusion" refers to the production of particles having a fixed cross-sectional profile. In particular, it refers to a reduction in the size of the particles, thereby forcing the particles through a filter having defined pores.

Other methods having the characteristic of being free of organic solvents may also be used to prepare colloids in accordance with the present disclosure.

LNP typically comprises four components: ionizable cationic lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer conjugated lipids such as polyethylene glycol (PEG) -lipids. Each component is responsible for the protection of the payload and enables efficient intracellular delivery. LNP can be prepared by rapid mixing of lipids dissolved in ethanol with nucleic acids in an aqueous buffer.

The term "average diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS), wherein data analysis is performed using a so-called cumulant algorithm, which results in a so-called Z with a length dimension _{Average of} And a dimensionless Polydispersity Index (PI) (Koppel, d., j.chem. Phys.57, 1972, pages 4814-4810, ISO 13321). Here, the "average diameter", "diameter" or "size" of the particles is equal to the Z _{Average of} Are used synonymously.

The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis mentioned in the definition of "average diameter". Under certain preconditions, it may serve as a measure of the size distribution of the assembly of nanoparticles.

Different types of nucleic acid-containing particles have been previously described as being suitable for delivering nucleic acids in particulate form (e.g., kaczmark, j.c. et al, 2017,Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acids from degradation and, depending on the particular chemistry, can aid in cellular uptake and endosomal escape.

The present disclosure describes particles comprising nucleic acid, at least one cationic lipid or cationically ionizable lipid or lipid-like substance, and/or at least one cationic polymer, which particles are associated with nucleic acid to form nucleic acid particles and compositions comprising such particles. The nucleic acid particles may comprise nucleic acids complexed in different forms by non-covalent interactions with the particles. The particles described herein are not viral particles, in particular infectious viral particles, i.e. they are not capable of virally infecting cells.

Suitable cationic lipids or cationically ionizable lipids or lipid-like substances and cationic polymers are those that form nucleic acid particles and are included in the term "particle-forming component" or "particle former". The term "particle-forming component" or "particle former" refers to any component that associates with a nucleic acid to form a nucleic acid particle. Such components include any component that may be part of a nucleic acid particle.

In some embodiments, the nucleic acid-containing particles (e.g., lipid Nanoparticles (LNPs)) comprise two or more RNA molecules, each comprising a different nucleic acid sequence. In some embodiments, the nucleic acid-containing particles comprise two or more RNA molecules, each encoding a different immunogenic polypeptide or immunogenic fragment thereof. In some embodiments, the two or more RNA molecules present in the nucleic acid-containing particle comprise: a first RNA molecule encoding an immunogenic polypeptide from a coronavirus or an immunogenic fragment thereof, and a second RNA molecule encoding an immunogenic polypeptide from an infectious disease pathogen (e.g., virus, bacteria, parasite, etc.) or an immunogenic fragment thereof. For example, in some embodiments, two or more RNA molecules present in the 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-2MN908947 strain or variant thereof, e.g., SARS-CoV-2 having one or more mutations characteristic of omacron variants), and a second RNA molecule encoding an immunogenic polypeptide or immunogenic fragment thereof from an influenza virus. In some embodiments, the two or more RNA molecules present in the nucleic acid-containing particle comprise: 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, the first coronavirus is different from the second coronavirus. In some embodiments, the first and/or second coronaviruses are independently from a SARS-CoV-2MN908947 strain or variant thereof, e.g., SARS-CoV-2 having one or more mutations characteristic of an omacron variant.

In some embodiments, two or more RNA molecules present in the nucleic acid-containing particle each encode an immunogenic polypeptide or immunogenic fragment thereof from the same and/or different strains and/or variants of coronavirus (e.g., SARS-CoV-2 strain or variant in some embodiments). For example, in some embodiments, two or more RNA molecules present in the 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 nonstructural protein, and/or a coronavirus helper protein. In some embodiments, such immunogenic polypeptides, or immunogenic fragments thereof, may be from the same or different coronaviruses (e.g., SARS-CoV-2MN908947 strain or variants thereof in some embodiments, e.g., variants having mutations characteristic of one or more prevalent variants (such as Omicron variants) in some embodiments). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein or immunogenic fragment thereof from a first strain or variant, and a second RNA molecule encoding a SARS-CoV-2S protein or 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, the nucleic acid-containing particle (e.g., in some embodiments, LNP as described herein) comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain and a second RNA molecule encoding a mutated SARS-CoV-2S protein comprising one or more features that are omacron variants (e.g., ba.10 microvariant, ba.2 omacron variant, ba.3 omacron variant, ba.4 omacron variant, or ba.50micron variant).

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of an omacron ba.1 variant. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.1 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.1 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.1 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of an omacron ba.2 variant. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.2 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.2 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the mutated SARS-CoV-2S protein comprising one or more features that are omacron ba.2 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of an omacron ba.3 variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of omacron ba.4 or ba.5 variants. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations that are characteristic of omacron ba.4 or ba.5 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations that are characteristic of omacron ba.4 or ba.5 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein from the MN908947 strain to the second RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations that are characteristic of omacron ba.4 or ba.5 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a first omacron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a second omacron variant.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.1omicron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of the ba.2omicron variant. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations characteristic of the ba.1Omicron variant strain to the second RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron ba.2 variant is 1:1. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.2 variant is 1:2. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.2 variant is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.1omicron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.3omicron variant. In some embodiments, the ratio of the first RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations characteristic of the ba.1Omicron variant strain to the second RNA molecule encoding the SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron ba.3 variant is 1:1. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.3 variant is 1:2. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.3 variant is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.1 omacron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4 or ba.5 omacron variant. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.4 or BA.5 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.4 or BA.5 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the BA.1Omicron variant strain to the second RNA molecule encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the Omicron BA.4 or BA.5 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.2omicron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.3omicron variant. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutant comprising one or more features that are Omicron ba.3 variant is 1:1. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.3 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.3 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.2 omacron variant and a second-RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4 or ba.5 omacron variant. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.2omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:3.

In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from a ba.3omicron variant and a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4 or ba.5omicron variant. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.3Omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:1. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.3Omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:2. In some embodiments, the ratio of the first RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations specific for the ba.3Omicron variant strain to the second RNA molecule encoding a mutation that is characteristic of one or more Omicron ba.4 or ba.5 variants is 1:3.

In some embodiments, the nucleic acid-containing particle (e.g., in some embodiments, an LNP as described herein) comprises three or more RNA molecules, each RNA molecule encoding a mutated SARS-CoV-2 protein comprising a different SARS-CoV-2 variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain, a second RNA molecule encoding a mutant SARS-CoV-2S protein comprising one or more features that are features of an omacron variant (e.g., ba.1 omacron variant, ba.2 omacron variant, ba.3 omacron variant, ba.4 omacron variant, or ba.5 omacron variant), and a third RNA molecule encoding a mutant SARS-CoV-2S protein comprising one or more features that are features of an omacron variant (e.g., ba.1 omacron variant, ba.2 omacron variant, ba.3 omacron variant, ba.4 omacron variant, or ba.5 omacron variant), wherein the second RNA molecule and the third RNA molecule encode a SARS-2S comprising one or more different Omicron sub-variants (subvariants). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain, a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.1omicron variant, and a third RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.2omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain, a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.1omicron variant, and a third RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.2omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain, a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.1Omicron variant, and a third RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4/5Omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain, a second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.2Omicron variant, and a third RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4/5Omicron variant. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule encoding a mutated SARS-CoV-2S protein that comprises a feature of a ba.1Omicron variant, a second RNA molecule encoding a mutated SARS-CoV-2S protein that comprises one or more features of a ba.2Omicron variant, and a third RNA molecule encoding a mutated SARS-CoV-2S protein that comprises one or more features of a ba.4/5Omicron variant.

In some embodiments, the nucleic acid-containing particles comprising two or more RNA molecules (e.g., in some embodiments, LNPs as described herein) comprise each RNA molecule in the same amount (i.e., in a 1:1 ratio).

In some embodiments, a nucleic acid-containing particle comprising two or more RNA molecules (e.g., in some embodiments, an LNP as described herein) comprises different amounts of each RNA molecule. For example, in some embodiments, the nucleic acid-containing particles comprise a first RNA molecule and a second RNA molecule, wherein the first RNA molecule is present in an amount of 0.01 to 100 fold that of the second RNA molecule (e.g., wherein the amount of the first RNA molecule is 0.01 to 50 fold, 0.01 to 4 fold, 0.01 to 30 fold, 0.01 to 25 fold, 0.01 to 20 fold, 0.01 to 15 fold, 0.01 to 10 fold, 0.01 to 9 fold, 0.01 to 8 fold, 0.01 to 7 fold, 0.01 to 6 fold, 0.01 to 5 fold, 0.01 to 4 fold, 0.01 to 3 fold, 0.01 to 2 fold, 0.01 to 1.5 fold, 1 to 50 fold, 1 to 4 fold, 1 to 30 fold, 1 to 25 fold, 1 to 20 fold, 1 to 15 fold, 1 to 10 fold, 1 to 9 fold, 1 to 7 fold, 1 to 1, 1 to 2 fold, 1 to 1, 1 to 5 fold, 1 to 2 fold). In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 10 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 5 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 1 to 3 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 2 times the concentration of the second RNA molecule. In some embodiments, the nucleic acid-containing particle comprises a first RNA molecule and a second RNA molecule, wherein the concentration of the first RNA molecule is 3 times the concentration of the second RNA molecule.

In some embodiments, the nucleic acid-containing particles comprising three RNA molecules (e.g., in some embodiments, LNPs as described herein) comprise each RNA molecule in the same amount (i.e., in a 1:1 ratio).

In some embodiments, the nucleic acid-containing particles comprising three RNA molecules (e.g., in some embodiments, LNPs as described herein) comprise different amounts of each RNA molecule. For example, in some embodiments, the first RNA molecule: a second RNA molecule: the ratio of the third RNA molecules is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-9;1:0.01-9:0.01-9;1:0.01-8:0.01-8;1:0.01-7:0.01-7;1:0.01-6:0.01-6;1:0.01-5:0.01-5;1:0.01-4:0.01-4;1:0.01-3; 1:0.01-2:0.01-2, or 1.01-5:0.01). In some embodiments, the ratio of the first RNA molecule to the second RNA molecule to the third RNA molecule is 1:1:3. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule to the third RNA molecule is 1:3:3.

In some embodiments, the first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain comprises a nucleic acid sequence encoding SEQ ID NO:7, and a nucleotide sequence of the amino acid sequence of seq id no. In some embodiments, the first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain comprises a sequence identical to SEQ ID NO:9 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain comprises a sequence identical to SEQ ID NO:20 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the first RNA molecule encoding a SARS-CoV-2S protein from the MN908947 strain comprises a sequence identical to SEQ ID NO:7 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a mutated SARS-CoV-2S protein having one or more features that are omacron variants comprises a sequence encoding SEQ ID NO:49, and a nucleotide sequence of an amino acid sequence of seq id no. In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:50 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:51 has a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:49 (e.g., at least 85%, at least 90%, at least 1%, 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).

In some embodiments, the second RNA molecule encoding a mutated SARS-CoV-2S protein having one or more features that are omacron variants comprises a sequence encoding SEQ ID NO:64, and a nucleotide sequence of the amino acid sequence of 64. In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:65 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:67 has a nucleotide sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence encoding a sequence that hybridizes to SEQ ID NO:64 (e.g., at least 85%, at least 90%, at least 1%, 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).

In some embodiments, the second RNA molecule encoding a mutated SARS-CoV-2S protein having one or more features that are omacron variants comprises a sequence encoding SEQ ID NO: 69. In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:70 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:72 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence encoding a sequence that hybridizes to SEQ ID NO:69 (e.g., at least 85%, at least 90%, at least 1%, 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).

In some embodiments, the second RNA molecule encoding a mutated SARS-CoV-2S protein having one or more features that are omacron variants comprises a sequence encoding SEQ ID NO:74, and a nucleotide sequence of the amino acid sequence of 74. In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:75 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence that hybridizes to SEQ ID NO:77 (e.g., at least 85%, at least 90%, at least 1%, 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). In some embodiments, the second RNA molecule encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an omacron variant comprises a sequence encoding a sequence that hybridizes to SEQ ID NO:74 (e.g., at least 85%, at least 90%, at least 1%, 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).

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 (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% or more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 (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% or more) identity; and a second RNA molecule comprising SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 has 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%, or at least 99% or more) identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:5Q has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20 (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% or more) identical; a second RNA molecule comprising SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:51 has 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%, or at least 99% or more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:51 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:51 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:51 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:64 or an amino acid sequence identical to SEQ ID NO:64 has 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%, or at least 99% or more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, said first RNA molecule encodes SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:64 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:64 or an amino acid sequence identical to SEQ ID NO:64 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:64 or an amino acid sequence identical to SEQ ID NO:64 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 (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% or more) identity; and a second RNA molecule comprising SEQ ID NO:65 or a nucleotide sequence identical to SEQ ID NO:65 has 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%, or at least 99% or more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:65 or a nucleotide sequence identical to SEQ ID NO:65 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:65 or a nucleotide sequence identical to SEQ ID NO:65, in some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:65 or a nucleotide sequence identical to SEQ ID NO:65 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:67 has 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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:67 or a nucleotide sequence identical to SEQ ID NO:67 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:67 or a nucleotide sequence identical to SEQ ID NO:67 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:67 or a nucleotide sequence identical to SEQ ID NO:67 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:69 or amino acid sequence identical to SEQ ID NO:69 has 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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:69 or amino acid sequence identical to SEQ ID NO:69 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:69 or amino acid sequence identical to SEQ ID NO:69 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:69 or amino acid sequence identical to SEQ ID NO:69 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 (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% or more) identity; and a second RNA molecule comprising SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70 has 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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70, and a sequence having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70, and a sequence having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:70, and a sequence having at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:72 or a nucleotide sequence that hybridizes to SEQ ID NO:72 has 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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:72 or a nucleotide sequence that hybridizes to SEQ ID NO:72 having a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:72 or a nucleotide sequence that hybridizes to SEQ ID NO:72 having a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:72 or a nucleotide sequence that hybridizes to SEQ ID NO:72 having a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:74 or amino acid sequence corresponding to SEQ ID NO:74 (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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:74 or amino acid sequence corresponding to SEQ ID NO:74 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:74 or amino acid sequence corresponding to SEQ ID NO:74 has a sequence of at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7, said second RNA molecule encodes a sequence of at least 80% identity to SEQ ID NO:74 or amino acid sequence corresponding to SEQ ID NO:74 has a sequence of at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 (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% or more) identity; and a second RNA molecule comprising SEQ ID NO:75 or a nucleotide sequence that hybridizes to SEQ ID NO:75 has 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 more) identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:75 or a nucleotide sequence that hybridizes to SEQ ID NO:75, and a sequence having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:75 or a nucleotide sequence that hybridizes to SEQ ID NO:75, and a sequence having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:75 or a nucleotide sequence that hybridizes to SEQ ID NO:75, and a sequence having at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SE0 ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:77 or a nucleotide sequence identical to SEQ ID NO:77 has 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 more) identical. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:1, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:77 or a nucleotide sequence identical to SEQ ID NO:77 having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:2, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:77 or a nucleotide sequence identical to SEQ ID NO:77 having at least 80% identity. In some embodiments, the ratio of the first RNA molecule to the second RNA molecule is 1:3, the first RNA molecule comprising the amino acid sequence of SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20, said second RNA molecule comprises a sequence of at least 80% identity to SEQ ID NO:77 or a nucleotide sequence identical to SEQ ID NO:77 having at least 80% identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:7 or an amino acid sequence identical to SEQ ID NO:7 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO: 55. 58 or 61 or an amino acid sequence identical to SEQ ID NO: 55. 58 or 61 has 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 more) identity

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 (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 more) identical; and a second RNA molecule comprising SEQ ID NO: 56. 59 or 62a or a nucleotide sequence identical to SEQ ID NO: 56. 59 or 62a has 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 more) identical

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:20 or a nucleotide sequence identical to SEQ ID NO:20 (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 more) identical; and a second RNA molecule comprising SEQ ID NO: 57. 60 or 63a or a nucleotide sequence identical to SEQ ID NO: 57. 60 or 63a has 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 more) identical

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:58 or amino acid sequence identical to SEQ ID NO:58 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO: 49. 55 or 61 or an amino acid sequence identical to SEQ ID NO: 49. 55 or 61 has 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 more) identity

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:59 or a nucleotide sequence identical to SEQ ID NO:59 (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 more) identical; and a second RNA molecule comprising SEQ ID NO: 50. 56 or 62a or a nucleotide sequence identical to SEQ ID NO: 50. 56 or 62a has 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 more) identical.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:60 or a nucleotide sequence identical to SEQ ID NO:60 (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 more) identical; and a second RNA molecule comprising SEQ ID NO: 51. 57 or 63a or a nucleotide sequence identical to SEQ ID NO: 51. 57 or 63a has 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 more) identical.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:55 or 61 or an amino acid sequence identical to SEQ ID NO:55 or 61 has 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 more) identity

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:56 or 62a or a nucleotide sequence identical to SEQ ID NO:56 or 62a has 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 more) identical

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:51 or a nucleotide sequence identical to SEQ ID NO:51 has 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 more) identical; and a second RNA molecule comprising SEQ ID NO:57 or 63a or a nucleotide sequence identical to SEQ ID NO:57 or 63a has 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 more) identical

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising a sequence encoding SEQ ID NO:55 or amino acid sequence identical to SEQ ID NO:55 (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 more) identical. And a second RNA molecule comprising a sequence encoding SEQ ID NO:61 or amino acid sequence identical to SEQ ID NO:61 has an amino acid sequence having 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 more) identity.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:56 or a nucleotide sequence identical to SEQ ID NO:56 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:62a or a nucleotide sequence identical to SEQ ID NO:62a has 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 more) identical.

In some embodiments, the nucleic acid-containing particles (e.g., in some embodiments, LNPs as described herein) comprise: a first RNA molecule comprising SEQ ID NO:57 or a nucleotide sequence identical to SEQ ID NO:57 (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 more) identical; and a second RNA molecule comprising SEQ ID NO:63a or a nucleotide sequence identical to SEQ ID NO:63a has 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 more) identical.

In some embodiments, particles (e.g., LNPs in some embodiments) comprising nucleic acids (e.g., RNAs) encoding different polypeptides can be formed by mixing a plurality (e.g., at least two, at least three, or more) RNA molecules with a particle-forming component (e.g., a lipid). In some embodiments, nucleic acids (e.g., RNAs) encoding different polypeptides may be mixed (e.g., in substantially equal proportions in some embodiments, e.g., in a 1:1 ratio when two RNA molecules are present) prior to mixing with the particle-forming component (e.g., lipid).

In some embodiments, two or more RNA molecules, each encoding a different polypeptide (e.g., as described herein), may be mixed with a particle former to form a nucleic acid-containing particle as described above. In alternative embodiments, two or more RNA molecules, each encoding a different polypeptide (e.g., as described herein), may be formulated into separate particle compositions, which are then mixed together. For example, in some embodiments, individual populations comprising nucleic acid particles (each population comprising RNA molecules encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., as described herein)) can be formed separately and then mixed together (e.g., by a health care professional of administration), e.g., prior to filling into vials during manufacture, or at the point of administration. Thus, in some embodiments, described herein are compositions comprising two or more populations of particles (e.g., lipid nanoparticles in some embodiments), each population comprising at least one RNA molecule encoding a different immunogenic polypeptide or immunogenic fragment thereof (e.g., SARS-CoV-2S protein or fragment thereof from a different variant). In some embodiments, each population may be provided in the composition in a desired ratio (e.g., in some embodiments, each population may be provided in the composition in an amount that provides the same amount of RNA molecules).

Cationic polymers

In view of its high degree of chemical flexibility, polymers are common materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically agglomerate negatively charged nucleic acids into nanoparticles. These positively charged groups typically consist of amines that change their protonation state in the pH range of 5.5 to 7.5, which is believed to cause ionic imbalance, leading to endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethylenimine, as well as naturally occurring polymers such as chitosan, have all been applied for nucleic acid delivery and are suitable as cationic polymers herein. In addition, some researchers have synthesized polymers that are specific for nucleic acid delivery. In particular poly (β -amino esters), have found wide application in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

As used herein, "polymer" is given its ordinary meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeat units may all be the same or, in some cases, more than one type of repeat unit may be present in the polymer. In some cases, the polymer is of biological origin, i.e., a biopolymer, such as a protein. In some cases, additional moieties may also be present in the polymer, for example targeting moieties (such as those described herein).

A polymer is said to be a "copolymer" if more than one type of repeating unit is present in the polymer. It should be understood that the polymer used herein may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, the repeating units may be arranged in random order, alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeating unit (e.g., a first block), and one or more regions each comprising a second repeating unit (e.g., a second block), and so forth. The block copolymer may have two (diblock copolymer), three (triblock copolymer) or a greater number of different blocks.

In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that do not generally lead to significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is capable of chemical and/or biological degradation in a physiological environment (such as in vivo).

In certain embodiments, the polymer may be a protamine or a polyalkyleneimine, particularly protamine.

The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and that are found to specifically associate with DNA in place of the somatic histones in sperm cells of various animals (e.g., fish). In particular, the term "protamine" refers to a protein found in fish sperm that has a strong alkalinity, is soluble in water, is not thermally coagulated, and when hydrolyzed primarily produces arginine. They are used in purified form for long acting insulin preparations and to neutralize the anticoagulant effect of heparin.

According to the present disclosure, the term "protamine" as used herein is meant to include any protamine amino acid sequence obtained from or derived from a natural or biological source, including fragments thereof and multimeric forms of said amino acid sequence or fragments thereof, as well as (synthetic) polypeptides, which are artificial and specifically designed for a specific purpose and which cannot be isolated from a natural or biological source.

In a real worldIn embodiments, the polyalkyleneimine comprises a polyethyleneimine and/or a polypropyleneimine, preferably a polyethyleneimine. The preferred polyalkyleneimine is Polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75.10 ² To 10 ⁷ Da, preferably from 1000 to 10 ⁵ Da, more preferably 10000 to 40000Da, more preferably 15000 to 30000Da, even more preferably 20000 to 25000Da.

Preferred in accordance with the present disclosure are linear polyalkyleneimines, such as linear Polyethyleneimine (PEI).

Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer capable of electrostatically binding nucleic acids. In one embodiment, it is contemplated that the cationic polymer used herein includes any cationic polymer with which a nucleic acid can associate (e.g., by forming a complex with a nucleic acid or forming a vesicle in which a nucleic acid is blocked or encapsulated).

The particles described herein may also comprise polymers other than cationic polymers, i.e. non-cationic polymers and/or anionic polymers. The anionic polymer and the neutral polymer are collectively referred to as non-cationic polymers.

Lipid and lipid-like substance

The terms "lipid" and "lipid-like substance" are defined herein broadly as molecules comprising one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic portion and a hydrophilic portion are also often referred to as amphiphiles. Lipids are generally poorly soluble in water. In an aqueous environment, amphiphilic properties allow molecules to self-assemble into organized structures and distinct phases. One of these phases consists of lipid bilayers because they are present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment. Hydrophobicity may be imparted by the inclusion of non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups may include polar and/or charged groups and include carbohydrates, phosphates, carboxyl groups, sulfate groups, amino groups, mercapto groups, nitro groups, hydroxyl groups, and other similar groups.

As used herein, the term "amphiphilic" refers to a molecule having a polar portion and a non-polar portion. Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water and the non-polar moiety is insoluble in water. In addition, the polar moiety may bear a formal positive or formal negative charge. Alternatively, the polar moiety may have a formal positive charge and a formal negative charge and be a zwitterionic or internal salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or more natural or unnatural lipids and lipid-like compounds.

The term "lipid-like substance", "lipid-like compound" or "lipid-like molecule" relates to substances that are structurally and/or functionally related to lipids but are not considered to be lipids in a strict sense. For example, the term includes compounds capable of forming amphiphilic layers, as they are present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment, including surfactants, or synthetic compounds having hydrophilic and hydrophobic portions. In general, the term refers to molecules comprising a hydrophilic portion and a hydrophobic portion having different structural organization, which may or may not be similar to the structural organization of lipids. As used herein, the term "lipid" will be interpreted to include lipids and lipid-like substances unless the context clearly contradicts or otherwise indicates.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized as insoluble in water, but soluble in many organic solvents. In general, lipids can be divided into eight classes: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and isoprenoids (derived from condensation of isoprenoid subunits). While the term "lipid" is sometimes used as a synonym for fat, fat is a subset of lipids, called triglycerides. Lipids also include molecules such as fatty acids and derivatives thereof (including triglycerides, diglycerides, 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 from hydrocarbon chains ending in carboxylic acid groups; this arrangement imparts a polar hydrophilic end and a non-polar hydrophobic end that is insoluble in water to the molecule. The carbon chain, which is typically between 4 and 24 carbons in length, may be saturated or unsaturated and may be attached to functional groups containing oxygen, halogen, nitrogen and sulfur. If the fatty acid contains a double bond, there is a possibility of cis or trans geometric isomerism, which can significantly affect the configuration of the molecule. Cis double bonds cause bending of the fatty acid chains, an effect that complexes with more double bonds in the chain. Other major lipid classes in the fatty acid class are fatty esters and fatty amides.

Glycerides consist of mono-, di-and tri-substituted glycerols, most notably fatty acid triesters of glycerol, known as triglycerides. The term "triacylglycerols" is sometimes synonymous with "triglycerides". In these compounds, the three hydroxyl groups of glycerol are typically each esterified with a different fatty acid. Other subclasses of glycerides are represented by glycosylglycerols, characterized by the presence of one or more sugar residues linked to the glycerol by glycosidic linkages.

Glycerophospholipids are amphiphilic molecules (comprising a hydrophobic region and a hydrophilic region) comprising a glycerol core linked by an ester linkage to two fatty acid-derived "tails" and linked by a phosphate linkage to one "head" group. Glycerophospholipids, commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids), examples of which 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, the sphingosine base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subset of sphingoid base derivatives with amide linked fatty acids. Fatty acids are generally saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The main sphingomyelins (phosphinoglycolins) of mammals are sphingomyelins (ceramide phosphorylcholine (ceramide phosphocholines)), whereas insects mainly contain ceramide phosphoethanolamine (ceramide phosphoethanolamines), and fungi have autonomic ceramide phosphoinositides (phytoceramide phosphoinositols) and mannose-containing headgroups. Glycosphingolipids (glycophilippides) are a diverse family of molecules consisting of one or more sugar residues linked to a sphingosine base via glycosidic linkages. Examples of such substances are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, along with glycerophospholipids and sphingomyelins are important components of membrane lipids.

Glycolipids describe compounds in which fatty acids are directly attached to the sugar backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone in glycerolipids and glycerophospholipids. The most common glycolipids are acylated glucosamine precursors of the lipid a component of lipopolysaccharides in gram-negative bacteria. A typical lipid a molecule is a disaccharide of glucosamine, which is derivatized with up to 7 fatty acyl chains. The smallest lipopolysaccharide required for growth in E.coli (E.coli) is Kdo 2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octanoonic acid (Kdo) residues.

Polyketides are synthesized by classical enzymes and repetitive and multi-modular enzymes with common mechanical characteristics with fatty acid synthases, polymerizing acetyl and propionyl subunits. They contain a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources and have a great structural diversity. Many polyketides are cyclic molecules whose backbone is often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

According to the present disclosure, lipids and lipid-like substances may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in the form of uncharged or neutral zwitterionic at a selected pH.

Cationic or ionizable cationic lipids or lipid-like substances

The nucleic acid particles described herein may comprise at least one cationic or ionizable cationic lipid or lipid-like material as a particle former. Cationic or ionizable cationic lipid or lipid-like materials contemplated for use herein include any cationic or ionizable cationic lipid or lipid-like material capable of electrostatically binding nucleic acids. In one embodiment, the cationic or ionizable cationic lipid or lipid-like material contemplated for use herein may be associated with a nucleic acid, for example, by forming a complex with the nucleic acid or forming vesicles in which the nucleic acid is encapsulated or encapsulated.

As used herein, "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 acids through electrostatic interactions. Typically, cationic lipids have 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 a positive charge.

In certain embodiments, the cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, and preferably has no net positive charge, preferably is uncharged, i.e. neutral, at a different, preferably higher pH such as physiological pH. This ionisable behaviour is believed to enhance efficacy by helping endosomes escape and reducing toxicity compared to particles that remain cationic at physiological pH.

For the purposes of this disclosure, the term "cationic lipid or lipid-like material" includes such "ionizable cationic" lipids or lipid-like materials unless contradicted by context.

In one embodiment, the cationic or ionizable cationic lipid or lipid-like material comprises a head group comprising at least one positively charged or capable of being protonated nitrogen atom (N).

Examples of cationic lipids include, but are not limited to, 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP); n, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA), 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), dimethyl Dioctadecyl Ammonium (DDAB); 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP); 1, 2-diacyloxy-3-dimethylammonium propane; 1, 2-dialkoxy-3-dimethylammonium propane; dioctadecyl dimethyl ammonium chloride (DODAC), 1, 2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 2, 3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethyl azonia (DMRIE), 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine (DMEPC), 1, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) and 2, 3-dioleoyloxy-N- [2 (spermidine) ethyl ] -N, N-dimethyl-1-trifluoroacetate propylamine (DOSPA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA), dioctadecyl amidoglycinamide (DOGS), 3-dimethylamino-2- (cholest-5-en-3-beta-oxybutynin-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), 2- [5'- (cholest-5-en-3-beta-oxy) -3' -oxapentoxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienoxy) propane (CpLinDMA), N-dimethyl-3, 4-Dioleoyloxybenzylamine (DMOBA), 1,2-N, N '-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2, 3-dioleoyloxy-N, N-dimethylpropylamine (DLinDAP), 1,2-N, N' -diiodocarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1, 2-diiodocarbamoyl-3-dimethylaminopropane (DLinCDAP), 2-diiodo-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2-diiodo-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-K-XTC 2-DMA), 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), thirty-seven carbons-6, 9, 28, 31-tetralin-19-yl-4- (dimethylamino) butanoate (DLin-MC 3-DMA), N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanammonium bromide (dmriie), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (cis-9-tetradecyloxy) -1-propanammonium bromide (GAP-DMORIE), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (dodecyloxy) -1-propanammonium bromide (GAP-DLRIE), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanammonium bromide (GAP-dmriie), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanammonium bromide (. Beta. -AE-dmriie), N- (4-carboxybenzyl) -N, N-dimethyl-2, 3-bis (dodecoyl) propanammonium bromide (GAP-DLRIE), (±) -N- (3-aminopropyl) -2, 3-bis (tetradecyloxy) -1-propanammonium bromide (βae-dmriie), N- (2-aminoethyl) -N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanamide (βae-dmriie) 2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadec-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA), 1, 2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1, 2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-amino-propyl) amino ] butylcarboxamido) ethyl ] -3, 4-bis [ oleyloxy ] -benzamide (MVL 5), 1, 2-dioleoyl-sn-glycero-3-ethyl phosphorylcholine (DOEPC), 2, 3-bis (dodecyloxy) -N- (2-hydroxyethyl) -N, N-dimethylpropan-1-ammonium bromide (DLP), N- (2-aminoethyl) -N, N-dimethyl-2, 3-propanediol-1-amine (RIE), RIE (RIE), 8' - ((((2 (dimethylamino) ethyl) thio) carbonyl) azanedioctanoic acid di ((Z) -non-2-en-1-yl) ester (ATX), N-dimethyl-2, 3-bis (dodecyloxy) propan-1-amine (DLDMA), N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-amine (DMDMA), di ((Z) -non-2-en-1-yl) -9- ((4- (dimethylamino butyryl) oxy) heptadecanedioate (L319), N-dodecyl-3- ((2-dodecylcarbamoyl-ethyl) - {2- [ (2-dodecylcarbamoyl-ethyl) -2- { (2-dodecylcarbamoyl-ethyl) - [2- (2-dodecylcarbamoyl-ethylamino) -ethyl ] -amino } -ethylamino) propanamide (lipid 98N 12-5), 1- [2- [ bis (2-hydroxydodecyl) amino ] ethyl- [2- [4- [2- [ bis (2-hydroxydodecyl) amino ] ethyl ] piperazin-1-yl ] ethyl ] amino ] dodeca-2-ol (lipid C12-200).

In some embodiments, the cationic lipid may comprise from about 10mol% to about 100mol%, from about 20mol% to about 100mol%, from about 30mol% to about 100mol%, from about 40mol% to about 100mol%, or from about 50mol% to about 100mol% of the total lipid present in the particle.

Additional lipid or lipid-like material

The particles described herein may also comprise lipids or lipid-like materials other than cationic or ionizable cationic lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-ionizable cationic lipids or lipid-like materials). Anionic and neutral lipids or lipid-like materials are collectively referred to herein as non-cationic lipids or lipid-like materials. In addition to ionizable/cationic lipids or lipid-like materials, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties such as cholesterol and lipids can enhance particle stability and efficacy of nucleic acid delivery.

Additional lipids or lipid-like materials may be incorporated that 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, for example, one or more anionic lipids and/or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In a preferred embodiment, the additional lipid comprises one of the following neutral lipid components: (1) phospholipids, (2) cholesterol or derivatives thereof; or (3) a mixture of phospholipids and cholesterol or derivatives thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, cholestanol, cholestyl-2 '-hydroxyethyl ether, cholestyl-4' -hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

Specific phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids include inter alia diacyl phosphatidyl choline, such as distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dimyristoyl phosphatidyl choline (DMPC), eicosapentaerytyl phosphatidyl choline, dilauroyl phosphatidyl choline, dipalmitoyl phosphatidyl choline (DPPC), didecyl phosphatidyl choline (DAPC), dibbehenyl phosphatidyl choline (DBPC), ditrianoyl phosphatidyl choline (DTPC), ditetradecyl phosphatidyl choline (DLPC), palmitoyl oleoyl-phosphatidyl choline (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphoryl choline (18:0 diether PC), 1-oleoyl-2-cholesteryl hemi-succinyl-sn-3-phosphoryl choline (ochepc), 1-hexadecyl-sn-glycero-3-phosphoryl choline (C16 lysopc), and phosphatidyl ethanolamine, in particular diacyl phosphatidyl ethanolamine, such as dioleoyl phosphatidyl ethanolamine (DOPC), stearoyl phosphatidyl ethanolamine (DPPE), and phosphatidyl ethanolamine (DPPE) with the other phosphatidyl choline having the same hydrophobicity.

In certain preferred embodiments, the additional lipids are DSPC or DSPC and cholesterol.

In certain embodiments, the nucleic acid particles comprise a cationic lipid and an additional lipid.

In one embodiment, the particles described herein comprise a polymer conjugated lipid, such as a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of at least one cationic lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and biological activity of the nucleic acid, as compared to the amount of at least one additional lipid. Thus, 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, from about 4:1 to about 1:2, or from about 3:1 to about 1:1.

In some embodiments, the non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and/or cholesterol), may comprise from about 0mol% to about 90mol%, from about 0mol% to about 80mol%, from about 0mol% to about 70mol%, from about 0mol% to about 60mol%, or from about 0mol% to about 50mol% of the total lipids present in the particles.

Lipid complex particles

In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipid complex particles.

In the context of the present disclosure, the term "RNA lipid complex particles" relates to particles containing lipids, in particular cationic lipids and RNA. The electrostatic interaction between positively charged liposomes and negatively charged RNAs results in the complexation and spontaneous formation of RNA lipid complex particles. Positively charged liposomes can generally be synthesized using cationic lipids (such as DOTMA) and additional lipids (such as DOPE). In one embodiment, the RNA lipid complex particles are nanoparticles.

In certain embodiments, the RNA lipid complex particles comprise a cationic lipid and an additional lipid. In one 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, from about 4:1 to about 1:2, or from about 3:1 to about 1:1. In particular 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.

The average diameter of the RNA lipid complex particles described herein is in one embodiment in the range of about 200nm to about 1000nm, about 200nm to about 800nm, about 250 to about 700nm, about 400nm to about 600nm, about 300nm to about 500nm, or about 350nm to about 400nm. In specific embodiments, the RNA lipid complex particles have an average diameter of about 200nm, about 225nm, about 250nm, about 275nm, about 300nm, about 325nm, about 350nm, about 375nm, about 400nm, about 425nm, about 450nm, about 475nm, about 500nm, about 525nm, about 550nm, about 575nm, about 600nm, about 625nm, about 650nm, about 700nm, about 725nm, about 750nm, about 775nm, about 800nm, about 825nm, about 850nm, about 875nm, about 900nm, about 925nm, about 950nm, about 975nm, or about 1000nm. In one embodiment, the average diameter of the RNA lipid complex particles is in the range of about 250nm to about 700 nm. In another embodiment, the average diameter of the RNA lipid complex particles is in the range of about 300nm to about 500 nm. In an exemplary embodiment, the average diameter of the RNA lipid complex particles is about 400nm.

The RNA lipid complex particles and compositions comprising the RNA lipid complex particles described herein can be used to deliver RNA to a target tissue following parenteral administration, particularly following intravenous administration. RNA lipid complex particles may be prepared using liposomes which are obtainable by injection of a solution of the lipid 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, for example, in an amount of about 5 mM. Liposomes can be used to prepare RNA lipid complex particles by mixing the liposomes with RNA. In one embodiment, the liposome and RNA lipid complex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), and/or 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposome and RNA lipid complex particles comprise 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE).

Spleen-targeted RNA lipid complex particles are described in WO 2013/143683, which is incorporated herein by reference. RNA lipid complex particles with a net negative charge have been found to be useful for preferentially targeting spleen tissue or spleen cells such as antigen presenting cells, particularly dendritic cells. Thus, RNA accumulation and/or RNA expression occurs in the spleen following administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver following administration of the RNA lipid complex particles. In one embodiment, RNA accumulation and/or RNA expression occurs in professional antigen presenting cells in antigen presenting cells such as the spleen after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

Lipid Nanoparticles (LNP)

In one embodiment, a nucleic acid, such as RNA, described herein is administered in the form of a Lipid Nanoparticle (LNP). 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 RNA encapsulated within or associated with the lipid nanoparticle.

In one embodiment, the LNP comprises 40 to 55mol%, 40 to 50mol%, 41 to 49mol%, 41 to 48mol%, 42 to 48mol%, 43 to 48mol%, 44 to 48mol%, 45 to 48mol%, 46 to 48mol%, 47 to 48mol%, or 47.2 to 47.8mol% 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 48.0mol% cationic lipid.

In one embodiment, the neutral lipid is present at a concentration in the range of 5 to 15mol%, 7 to 13mol%, or 9 to 11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10, or 10.5 mol%.

In one embodiment, the steroid is present at a concentration in the range of 30 to 50 mole%, 35 to 45 mole%, or 38 to 43 mole%. In one embodiment, the steroid is present at a concentration of about 40, 41, 42, 43, 44, 45 or 46 mole%.

In one embodiment, the LNP comprises 1 to 10mol%, 1 to 5mol%, or 1 to 2.5mol% polymer conjugated lipid.

In one embodiment, the LNP comprises 40 to 50mol% cationic lipid; 5 to 15mol% of neutral lipids; 35 to 45 mole% of a steroid; 1 to 10 mole% of a polymer conjugated lipid; and RNA encapsulated within or associated with the lipid nanoparticle.

In one embodiment, the mole percent is determined based on the total moles 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:

R ¹² And R is ¹³ Each independently is a linear 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 linkages; and w has a value in the range of 30 to 60Average value. In one embodiment, R ¹² And R is ¹³ Each independently is a straight saturated alkyl chain containing from 12 to 16 carbon atoms. In one embodiment, w has an average value in the range of 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R ¹² And R is ¹³ Each independently is a straight saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

In one embodiment, the pegylated lipid is DMG-PEG 2000, e.g., having the structure:

in some embodiments, the cationic lipid component of the LNP has the structure of formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L ¹ or L ² One of them is-O (c=o) -, - (c=o) O-, -C (=o) -, -O-, -S (O) _x -、-S-S-、-C(＝O)S-、SC(＝O)-、-NR ^a C(＝O)-、-C(＝O)NR ^a -、NR ^a C(＝O)NR ^a 、-OC(＝O)NR ^a -or-NR ^a C (=o) O-, and L ¹ Or L ² The other of them is-O (c=o) -, - (c= =o) O-, -C (=o) -, -O-, -S (O) _x -、-S-S-、-C(＝O)S-、SC(＝O)-、-NR ^a C(＝O)-、-C(＝O)NR ^a -、NR ^a C(＝O)NR ^a -、-OC(＝O)NR ^a -or-NR ^a C (=o) O-or a direct bond;

G ¹ And G ² Each independently is unsubstituted C ₁ -C ₁₂ Alkylene or C ₁ -C ₁₂ Alkenylene;

G ³ is C ₁ -C ₂₄ Alkylene, C ₁ -C ₂₄ Alkenylene, C ₃ -C ₈ Cycloalkylene, C ₃ -C ₈ A cycloalkenyl group;

R ^a is H or C ₁ -C ₁₂ An alkyl group;

R ¹ and R is ² Each independently is C ₆ -C ₂₄ Alkyl or C ₆ -C ₂₄ Alkenyl groups;

R ³ h, OR of a shape of H, OR ⁵ 、CN、-C(＝O)OR ⁴ 、-OC(＝O)R ⁴ or-NR ⁵ C(＝O)R ⁴ ；

R ⁴ Is C ₁ -C ₁₂ An alkyl group;

R ⁵ is H or C ₁ -C ₆ An alkyl group; and is also provided with

x is 0, 1 or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

wherein:

a is a 3 to 8 membered cycloalkyl or cycloalkylene ring;

R ⁶ at each occurrence independently H, OH or C ₁ -C ₂₄ An alkyl group;

n is an integer in the range of 1 to 15.

In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

wherein y and z are each independently integers in the range of 1 to 12.

In any of the foregoing embodiments of formula (III),L ¹ or L ² One of them is-O (c=o) -. For example, in some embodiments, L ¹ And L ² is-O (c=o) -. In some of the various embodiments of any of the foregoing, L ¹ And L ² Each independently is- (c=o) O-or-O (c=o) -. For example, in some embodiments, L ¹ And L ² Is- (c=o) O-.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

in some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

in some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, e.g., 2 to 8 or 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 in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

In some of the foregoing embodiments of formula (III), R ⁶ H. In other preceding embodiments, R ⁶ Is C ₁ -C ₂₄ An alkyl group. In other embodiments, R ⁶ Is OH.

In some embodiments of formula (III), G ³ Is unsubstituted. In other embodiments, G3 is substituted. In various embodiments, G ³ Is straight-chain C ₁ -C ₂₄ Alkylene or straight-chain C ₁ -C ₂₄ Alkenylene radicals.

In some other of the foregoing embodiments of formula (III), R ¹ Or R is ² Or both are C ₆ -C ₂₄ Alkenyl groups. For example, in some embodiments, R ¹ And R is ² Each independently has the following structure:

wherein:

R ^7a and R is ^7b At each occurrence independently H or C ₁ -C ₁₂ An alkyl group; and is also provided with

a is an integer of 2 to 12,

wherein R is ^7a 、R ^7b And a are each selected such that R ¹ And R is ² Each independently comprising 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

In some of the foregoing embodiments of formula (III), R is present at least once ^7a H. For example, in some embodiments, R ^7a H at each occurrence. In various other embodiments of the foregoing, R is present at least once ^7b Is C ₁ -C ₈ An alkyl group. For example, in some embodiments, C ₁ -C ₈ Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.

In various embodiments of formula (III), R ¹ Or R is ² Or both have one of the following structures:

in some of the foregoing embodiments of formula (III), R ³ Is OH, CN, -C (=O) OR ⁴ 、-OC(＝O)R ⁴ or-NHC (=o) R ⁴ . In some embodiments, R ⁴ Is methyl or ethyl.

In various embodiments, the cationic lipid of formula (III) has one of the structures listed in the following table.

Table 17: representative compounds of formula (III).

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In some embodiments, the LNP comprises a lipid of formula (III), RNA, neutral lipids, steroids, and pegylated lipids. 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 of about 40 mole% to about 50 mole%. In one embodiment, the neutral lipid is present in the LNP in an amount of about 5 mole% to about 15 mole%. In one embodiment, the steroid is present in the LNP in an amount of about 35 mole% to about 45 mole%. In one embodiment, the pegylated lipid is present in the LNP in an amount of about 1 mole% to about 10 mole%.

In some embodiments, the LNP comprises compound III-3 in an amount of about 40 to about 50 mole%, DSPC in an amount of about 5 to about 15 mole%, cholesterol in an amount of about 35 to about 45 mole%, and ALC-0159 in an amount of about 1 to about 10 mole%.

In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mole%, DSPC in an amount of about 10 mole%, cholesterol in an amount of about 40.7 mole%, and ALC-0159 in an amount of about 1.8 mole%.

In various embodiments, the cationic lipid has one of the structures listed in the following table.

Table 18: representative cationic lipids.

In some embodiments, the LNP comprises a cationic lipid (e.g., a cationic lipid of formula (B) or formula (D), particularly a cationic lipid of formula (D)), RNA, a neutral lipid, a steroid, and a pegylated lipid as shown in the above table. 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 (lipid). 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 is in the range of 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.

The LNPs described herein may have an average diameter in one embodiment in the range of about 30nm to about 200nm, or about 60nm to about 120 nm.

RNA targeting

Some aspects of the disclosure relate to targeted delivery of RNAs disclosed herein (e.g., RNAs encoding vaccine antigens and/or immunostimulants).

In one embodiment, the disclosure relates to targeting the lung. If the RNA administered is RNA encoding a vaccine antigen, it is particularly preferred to target the lung. The RNA can be delivered to the lung, for example, by inhalation administration of RNA that can be formulated into particles (e.g., lipid particles) as described herein.

In one embodiment, the present disclosure relates to targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. If the RNA administered is RNA encoding a vaccine antigen, it is particularly preferred to target the lymphatic system, in particular the secondary lymphoid organs, more particularly the spleen.

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 a part of the circulatory system and is also an important component of the immune system, including the lymphatic network that carries the lymph. The lymphatic system consists of lymphoid organs, lymphatic vessel conduction networks and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. Thymus and bone marrow constitute the primary lymphoid organ. Secondary or peripheral lymphoid organs, including lymph nodes and spleen, maintain mature naive lymphocytes and elicit an adaptive immune response.

RNA can be delivered to the spleen by a so-called liposome complex formulation, wherein the RNA is bound to liposomes comprising cationic lipids and optionally additional or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injection of a solution of the lipid in ethanol into water or a suitable aqueous phase. RNA lipid complex particles can be prepared by mixing liposomes with RNA. Spleen-targeted RNA lipid complex particles are described in WO 2013/143683, which is incorporated herein by reference. RNA lipid complex particles with a net negative charge have been found to be useful for preferentially targeting spleen tissue or spleen cells such as antigen presenting cells, particularly dendritic cells. Thus, RNA accumulation and/or RNA expression occurs in the spleen following administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver following administration of the RNA lipid complex particles. In one embodiment, RNA accumulation and/or RNA expression occurs in professional antigen presenting cells in antigen presenting cells such as the spleen after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

The charge of the RNA lipid complex particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in RNA. The charge ratio is the ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: charge ratio = [ (cationic lipid concentration (mol)) (total number of positive charges in cationic lipid) ]/[ (RNA concentration (mol)) (total number of negative charges in RNA) ].

The spleen-targeted RNA lipid complex particles described herein preferably have a net negative charge at physiological pH, such as a charge ratio of positive to negative charges of about 1.9:2 to about 1:2, or about 1.6:2 to about 1.1:2. In particular embodiments, the charge ratio of positive to negative charges in the RNA lipid complex 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.

The immunostimulant may be provided to a subject by administering to the subject an RNA encoding the immunostimulant in a formulation for preferential delivery of the RNA to the liver or liver tissue. The delivery of RNA to such target organs or tissues is particularly preferred if it is desired to express a high amount of immunostimulant and/or if it is desired or required that the immunostimulant is present systemically, in particular in significant amounts.

RNA delivery systems have an inherent preference for the liver. This relates 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 liver vasculature or 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 to the liver by preventing degradation of the RNA. For example, a multimeric complex nanomicelle consisting of a poly (ethylene glycol) (PEG) coated surface and a core containing RNA (e.g., mRNA) is a useful system because nanomicelle provides excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the multimeric complex nanomicelle surface consisting of a dense PEG palisade effectively evade host immune defenses.

Examples of suitable immunostimulants for targeting the 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, the RNA encoding the immunostimulant may be administered in a formulation to preferentially deliver the RNA to the lymphatic system, particularly the secondary lymphoid organs, more particularly the spleen. If an immunostimulant is desired to be present in the organ or tissue (e.g. for inducing an immune response, in particular in case an immunostimulant such as a cytokine is required during T cell priming or for activating resident immune cells), whereas an immunostimulant is not desired to be present systemically, in particular in significant amounts (e.g. because the immunostimulant is systemically toxic), it is particularly preferred to deliver the immunostimulant to such target tissue.

Examples of suitable immunostimulants are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN- α or IFN- β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

Immunostimulant

In one embodiment, the RNA encoding the vaccine antigen may be non-immunogenic. In this and other embodiments, the RNA encoding the 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 (hereinafter referred to as an "extended Pharmacokinetic (PK)" immunostimulant. The methods and agents described herein are particularly effective if the immunostimulant is administered in the form of RNA encoding the immunostimulant. In one embodiment, the RNA targets the liver for systemic use. Hepatocytes can be transfected efficiently and are capable of producing large amounts of proteins.

An "immunostimulant" is any substance that stimulates the immune system by inducing the activation or increasing the activity of any component of the immune system, in particular immune effector cells. The immunostimulant may be pro-inflammatory.

According to one aspect, the immunostimulant is a cytokine or variant thereof. Examples of cytokines include interferons such as interferon-alpha (IFN-alpha) or interferon-gamma (IFN-gamma); interleukins, such as IL2, IL7, IL12, IL15 and IL23; colony stimulating factors such as M-CSF and GM-CSF; tumor necrosis factor. According to another aspect, the immunostimulant comprises an adjuvant-type immunostimulant, such as an APC Toll-like receptor agonist or a co-stimulatory/cell adhesive membrane protein. Examples of Toll-like receptor agonists include co-stimulatory/adhesion proteins such as CD80, CD86 and ICAM-1.

Cytokines are a class of small proteins (about 5-20 kDa) important in cell signaling. Their release has an effect on the behaviour of the cells surrounding them. Cytokines are involved as immunomodulators in autocrine signaling, paracrine signaling and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in terms). Cytokines are produced by a wide range of cells including immune cells such as 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 particularly important in the immune system; cytokines regulate the balance between humoral and cell-based immune responses, and they regulate maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the effects of other cytokines in a complex manner.

According to the present disclosure, the cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, in particular any mammal. One particularly preferred cytokine is interferon- α.

Interferon

Interferon (IFN) is a group of signal proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites and tumor cells. In a typical case, virus-infected cells will release interferon, causing nearby cells to enhance their antiviral defenses.

Interferons are generally classified into three classes based on the type of receptor through which they signal: type I interferon, type II interferon, and type III interferon.

All type I interferons bind to a specific cell surface receptor complex called the IFN- α/β receptor (IFNAR) consisting of IFNAR1 and IFNAR2 chains.

Type I interferons present in humans are IFN alpha, IFN beta, IFN epsilon, IFN kappa and IFN alpha omega. Typically, type I interferon is produced when the body recognizes a virus that has invaded the body. They are produced by fibroblasts and monocytes. Once released, the type I interferon binds to specific receptors on the target cells, which results in the 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 (pDC). They are mainly involved in innate immunity against viral infections. Genes responsible for their synthesis have 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are present together in clusters on chromosome 9.

IFN beta protein by fibroblasts mass production. They have antiviral activity mainly involved in innate immune responses. Two types of IFN beta, IFN beta 1 and IFN beta 3 have been described. The natural and recombinant forms of IFN beta 1 have antiviral, antibacterial and anticancer properties.

Type II interferons (ifnγ in humans) are also known as immunointerferons and are activated by IL 12. In addition, type II interferons are released by cytotoxic T cells and T helper cells.

Type III interferons signal through a receptor complex consisting of IL10R2 (also known as CRF 2-4) and IFNLR1 (also known as CRF 2-12). Although more recently discovered than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of viral or fungal infections.

In general, type I and type II interferons are responsible for modulating and activating immune responses.

According to the present disclosure, the type I interferon is preferably IFN alpha or IFN beta, more preferably IFN alpha.

According to the present disclosure, the interferon may be a naturally occurring interferon or a functional fragment or variant thereof. The interferon may be human interferon and may be derived from any vertebrate, particularly any mammal.

Interleukin

Interleukins (IL) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups according to 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 present disclosure, the interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be human interleukin and may be derived from any vertebrate, in particular any mammal.

Extended PK groups

The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulant may be fused to an extended PK group, which increases the circulatory half-life. Non-limiting examples of extended PK groups are described below. It is understood that other PK groups that increase the circulation half-life of an immunostimulant such as a cytokine or variant thereof are also suitable for use in 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 the properties of a compound, including, for 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 circulatory half-life of a bioactive molecule when fused or administered together with the bioactive molecule. Examples of extended PK groups 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 us publication 2005/0287153 and 2007/0003549). Other exemplary extended PK groups are disclosed in Kontermann, expert Opin Biol Ther, month 7 of 2016; 16 (7): 903-15, which are incorporated herein by reference in their entirety. As used herein, an "extended PK" immunostimulant refers to an immunostimulant moiety combined with an extended PK group. In one embodiment, the extended PK immunostimulant is a fusion protein in which the immunostimulant moiety is linked or fused to an extended PK group.

In certain embodiments, the serum half-life of the extended PK immunostimulatory agent is increased relative to the immunostimulatory agent alone (i.e., an immunostimulatory agent 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 it takes for the serum or plasma concentration of a compound, such as a peptide or protein, to decrease by 50% in vivo, for example, due to degradation and/or clearance or sequestration by natural mechanisms. Extended PK immunostimulants suitable for use herein are stable in vivo and have increased half-lives by, for example, fusion with anti-degradation and/or clearance or chelating serum albumin (e.g. HSA or MSA). Half-life may be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally involve the following steps: appropriately administering to the subject an appropriate dose of the amino acid sequence or compound; collecting a blood sample or other sample from the subject at regular intervals; determining the level or concentration of an amino acid sequence or compound in the blood sample; and calculating from the data thus obtained the time after administration until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level. Further details are provided, for example, in standard handbooks such as Kenneth, a. Et al, chemical Stability of Pharmaceuticals: a Handbook for Pharmacists and Peters et al Pharmacokinetic Analysis: a Practical Approach (1996). Reference is also made to Gibaldi, M.et al, pharmacokinetics, revision 2, marcel Dekker (1982).

In certain embodiments, the extended PK groups include serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in us publication 20070048282.

As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one albumin molecule (or fragment or variant thereof) with at least one protein molecule, such as a therapeutic protein, in particular an immunostimulant. Albumin fusion proteins may be produced by translation of a nucleic acid wherein a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding albumin. Therapeutic proteins and albumin, once part of an albumin fusion protein, may each be referred to as a "portion," "region," or "portion" of an albumin fusion protein (e.g., a "therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, the albumin fusion protein comprises at least one therapeutic protein molecule (including but not limited to a mature form of a therapeutic protein) and at least one albumin molecule (including but not limited to a mature form of albumin). In one embodiment, the albumin fusion protein is processed by a host cell, such as a cell of a target organ for administration of RNA, e.g., a hepatocyte, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathway of a host cell for expressing RNA can include, but is not limited to, signal peptide cleavage; formation of disulfide bonds; correctly folding; carbohydrate addition and processing (e.g., N-and O-linked glycosylation); specific proteolytic cleavage; and/or assembled into multimeric proteins. Albumin fusion proteins are preferably encoded by RNA in a non-processed form, in particular having a signal peptide at their N-terminus, and are preferably present in a processed form after secretion by a cell, in which in particular the signal peptide has been excised. In the most preferred embodiment, the "processed form of an albumin fusion protein" refers to an albumin fusion protein product that has undergone cleavage by the N-terminal signal peptide, also referred to herein as a "mature albumin fusion protein".

In a preferred embodiment, the albumin fusion protein comprising the therapeutic protein has a higher plasma stability than the plasma stability of the same therapeutic protein not fused to albumin. Plasma stability generally refers to the period of time between when a therapeutic protein is administered in vivo and carried into the blood stream and when the therapeutic protein is degraded and cleared from the blood stream to the organ, such as the kidney or liver, which ultimately clears the therapeutic protein from the body. Plasma stability is calculated from the half-life of the therapeutic protein in the blood stream. The half-life of a therapeutic protein in the blood stream can be readily determined by routine assays known in the art.

As used herein, "albumin" is collectively referred to as an albumin protein or amino acid sequence, or fragment or variant of albumin having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, especially a mature form of human albumin, or albumin from other vertebrates or a fragment thereof, or a variant of these molecules. Albumin may be derived from any vertebrate, in particular any mammal, for example human, bovine, ovine or porcine. Non-mammalian albumin includes, but is 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 a fragment or variant 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, an albumin fragment sufficient to extend the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment that is sufficient in length or structure to stabilize or extend the therapeutic activity or plasma stability of the protein such that the plasma stability of the therapeutic protein portion of the albumin fusion protein is extended or extended as compared to the plasma stability in the non-fused state.

The albumin portion of the albumin fusion protein may comprise the full length of the albumin sequence, or may comprise one or more fragments thereof capable of stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise about 15, 20, 25, 30, 50 or more contiguous amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is a mature form of HSA.

In general, fragments or variants of albumin will be at least 100 amino acids long, preferably at least 150 amino acids long.

According to the present disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. The albumin may be human albumin and may be derived from any vertebrate, in particular any mammal.

Preferably, the albumin fusion protein comprises albumin as the N-terminal part and a therapeutic protein as the C-terminal part. Alternatively, albumin fusion proteins 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-terminus and the C-terminus are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are different therapeutic proteins. In one embodiment, the different therapeutic proteins are cytokines.

In one embodiment, the therapeutic protein is conjugated to albumin through a peptide linker. The linker peptide between the fusion moieties may provide greater physical separation between the moieties and thus maximize accessibility of the therapeutic protein moiety, e.g., for binding to its cognate receptor. The linker peptide may be composed of amino acids such that it is flexible or more rigid. The linker sequence may be cleaved by protease or chemical means.

As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed from the corresponding Fc domains (or Fc portions) 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, the Fc domain begins at the hinge region just upstream of the papain cleavage site and terminates at the C-terminus of the antibody. Thus, the complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain comprises at least one of the following: a hinge (e.g., upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or variants, portions or fragments thereof. In certain embodiments, the Fc domain comprises an intact Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, the Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, the Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, the Fc domain lacks at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2 and/or CH3 domains, as well as fragments of such peptides comprising only, for example, the hinge, CH2 and CH3 domains. The Fc domain may be derived from immunoglobulins of any species and/or subtype, including but not limited to human IgG1, igG2, igG3, igG4, igD, igA, igE, or IgM antibodies. Fc domains include native Fc and Fc variant molecules. As described herein, one of ordinary skill in the art will appreciate that any Fc domain may be modified such that it differs in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., fcγr binding).

The Fc domains of the polypeptides described herein may be derived from different immunoglobulin molecules. For example, the Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

In certain embodiments, the extended PK group comprises an Fc domain or fragment thereof, or a variant of an Fc domain or fragment thereof (all of which are included in the term "Fc domain" for purposes of this disclosure). The Fc domain does not contain a variable region that binds to an antigen. Fc domains suitable for use in the present disclosure may be obtained from a number of different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgG1 constant region. However, it is understood that the Fc domain may be derived from an immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, cynomolgus monkey).

Furthermore, the Fc domain (or fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, igG, igD, igA and IgE, as well as any immunoglobulin isotype, including IgG1, igG2, igG3, and IgG4.

Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in publicly available deposit forms. Constant region domains comprising Fc domain sequences that lack specific effector functions and/or have specific modifications to reduce immunogenicity may be selected. Numerous sequences of antibodies and antibody-encoding genes have been disclosed, and suitable Fc domain sequences (e.g., hinge, CH2 and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art-recognized techniques.

In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422, US 2010/013339, WO2009/083804, and WO2009/133208, which are incorporated herein 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 incorporated herein 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 incorporated herein by reference in their entirety. In certain embodiments, the extended PK group is a fibronectin (Fn) -based scaffold domain protein that binds serum albumin, such as those disclosed in US2012/0094909, which is incorporated herein by reference in its entirety. Methods of making fibronectin based scaffold domain proteins are also disclosed in US 2012/0094909. A non-limiting example of an extended PK group based on Fn3 is Fn3 (HSA), an Fn3 protein that binds human serum albumin.

In certain aspects, extended PK immunostimulants suitable for use according to the present disclosure may employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that links two or more domains (e.g., an extended PK moiety and an immunostimulatory moiety) in the linear amino acid sequence of a polypeptide chain. For example, peptide linkers can be used to link the immunostimulatory moiety to the HSA domain.

Linkers suitable for fusing extended PK groups to, for example, immunostimulants 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 consisting of glycine and serine residues.

In addition to or in lieu of the heterologous polypeptides described above, the immunostimulatory polypeptides described herein may contain sequences encoding "markers" or "reporter molecules". Examples of markers or reporter genes include beta-lactamase, chloramphenicol Acetyl Transferase (CAT), adenosine Deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine Kinase (TK), beta-galactosidase, and xanthine guanine phosphoribosyl transferase (XGPRT).

Pharmaceutical composition

The agents described herein may be administered in the form of a pharmaceutical composition or medicament, 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 disclosure, components described herein, such as RNA encoding a vaccine antigen, may be administered in a pharmaceutical composition that may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers, and the like. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatment, e.g., for treatment or prevention of 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. The pharmaceutical composition may be used to treat, prevent, or reduce the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

The pharmaceutical compositions of the present disclosure may comprise or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g. Freund's adjuvant), mineral compounds (such as alum), bacterial products (such as pertussis toxin) or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, cpG oligodeoxynucleotides, growth factors and cytokines, such as monokines, lymphokines, interleukins, chemokines. The cytokine may be IL1, IL2, IL3, IL4, IL5. IL6, IL7, IL8, IL9, IL10, IL12, IFN alpha, IFN gamma, GM-CSF, LT-alpha. Further known adjuvants are aluminium hydroxide, freund's adjuvant or oils such asISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

Pharmaceutical compositions according to the present disclosure are generally administered in "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations".

The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the action of the active components of the pharmaceutical composition.

The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that alone or in combination with other dosages achieves the desired response or desired effect. In the case of treating a particular disease, the desired response preferably involves inhibiting the progression of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be to delay the onset of the disease or the condition or to prevent the onset of the disease or the condition. The effective amount of the compositions described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, body type and weight), the duration of the treatment, the type of concomitant therapy (if present), the particular route of administration, and the like. Thus, the dosage of the compositions described herein to be administered may depend on a variety of such parameters. In cases where the response in the patient is insufficient at the initial dose, a higher dose (or effectively a higher dose 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, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents or coloring agents.

The term "diluent" refers to diluents and/or diluents. Furthermore, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and/or a mixing medium. Examples of suitable diluents include ethanol, glycerol and water.

The term "carrier" refers to a component that may be natural, synthetic, organic, inorganic, wherein the active components are combined so as 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 suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, ringer's solution of lactic acid, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, mack Publishing co. (A.R Gennaro editions 1985).

The pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical administration or systemic administration. Systemic administration may include enteral administration involving absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than by 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-administration" as used herein refers to the process of administering different compounds or compositions (e.g., an antigen-encoding RNA and an immunostimulant-encoding RNA) to the same patient. The different compounds or compositions may be administered simultaneously, substantially simultaneously or sequentially.

The pharmaceutical compositions and products described herein may be provided as a frozen concentrate of a solution for injection, for example, at a concentration of 0.50 mg/mL. In one embodiment, to prepare a solution for injection, the drug product is thawed and diluted with an isotonic sodium chloride solution (e.g., 0.9% nacl, saline), for example, by a one-step dilution method. In some embodiments, a bacteriostatic sodium chloride solution (e.g., 0.9% nacl, saline) cannot be used as a diluent. In some embodiments, the diluted pharmaceutical product is an off-white suspension. The concentration of the final solution for injection varies according to the respective dosage level to be administered.

In one embodiment, due to the risk of microbial contamination and considering the multi-dose method of the preparation process, the administration is performed within 6 hours after the start of the preparation. In one embodiment, during this period of 6 hours, two conditions are allowed: room temperature preparation, handling and transfer, and storage at 2 ℃ to 8 ℃.

The compositions described herein may be transported and/or stored under temperature controlled conditions, such as about 4-5 ℃ or less, about-20 ℃ or less, -70 ℃ ± 10 ℃ (e.g., 80 ℃ to 60 ℃), for example, using a cooling system (e.g., which may be or include dry ice) to maintain a desired temperature. In one embodiment, the compositions described herein are transported in a temperature controlled hot-box. Such shipping boxes may contain GPS-enabled thermal sensors to track the location and temperature of each piece of cargo. The composition may be stored by refilling with, for example, dry ice.

Treatment of

The present disclosure provides methods and agents for inducing an adaptive immune response against coronaviruses in a subject comprising administering an effective amount of a composition comprising RNA encoding a coronavirus vaccine antigen as described herein.

In one embodiment, the methods and agents described herein provide immunity to coronaviruses, coronavirus infections, or to diseases or disorders associated with coronaviruses in a subject. Accordingly, the present disclosure provides methods and agents for treating or preventing infections, diseases or disorders associated with coronaviruses.

In one embodiment, the methods and agents described herein are administered to a subject suffering from an infection, disease or disorder associated with coronavirus. In one embodiment, the methods and agents described herein are administered to a subject at risk of developing an infection, disease or disorder associated with coronavirus. For example, the methods and agents described herein can be administered to a subject at risk of contact with coronavirus. In one embodiment, the methods and agents described herein are administered to a subject living in, traveling to, or expected to travel to, a geographical area where coronaviruses are prevalent. In one embodiment, the methods and agents described herein are administered to a subject in contact with or expected to be in contact with another person living in, traveling to, or expected to travel to a geographical area where coronaviruses are prevalent. In one embodiment, the methods and agents described herein are administered to a subject who is intentionally exposed to coronavirus by occupational or other contact thereof. In one embodiment, the coronavirus is SARS-CoV-2. In some embodiments, the methods and agents described herein are administered to subjects with evidence of prior exposure to or cross-reaction with SARS-CoV-2 and/or an antigen or epitope thereof. For example, in some embodiments, the methods and agents described herein are administered to a subject in which antibodies, B cells, and/or T cells reactive with one or more epitopes of SARS-CoV-2 spike protein can be detected and/or have been detected.

For use as a vaccine, the composition must induce an immune response against the coronavirus antigen in a cell, tissue, or subject (e.g., human). In some embodiments, the composition induces an immune response against a coronavirus antigen in a cell, tissue, or subject (e.g., human). In some cases, the vaccine induces a protective immune response in the mammal. The therapeutic compounds or compositions of the present disclosure can be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to a subject suffering from, or at risk of developing, a disease or disorder. Such subjects can be identified using standard clinical methods. In the context of the present disclosure, prophylactic administration occurs before the manifestation of the obvious clinical symptoms of the disease, such that the disease or disorder is prevented or alternatively delayed in its progression. In the context of the pharmaceutical field, the term "prevention" encompasses any activity that reduces the burden of mortality or morbidity of the disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of disease, secondary and tertiary prevention levels encompass activities aimed at preventing the progression of disease and the appearance of symptoms, as well as reducing the negative impact of established disease, by restoring function and reducing disease-related complications.

The term "dose" as used herein generally refers to a "dose amount" which relates to the amount of RNA administered per administration, i.e. per administration.

In some embodiments, administration of the immunogenic compositions or vaccines of the present disclosure can be enhanced by a single administration or by multiple administrations.

In some embodiments, the regimens described herein comprise at least one dose. In some embodiments, the regimen comprises a first dose and at least one subsequent dose. In some embodiments, the first dose is the same amount as the 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 than the at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, the regimen comprises two doses. In some embodiments, the provided regimen consists of two doses. In some embodiments, the regimen comprises three doses.

In one embodiment, the present disclosure contemplates administration of a single dose. In one embodiment, the present disclosure contemplates administration of a priming dose followed by administration of one or more boosting doses. The booster dose or first booster dose may be administered 7 to 28 days or 14 to 24 days after administration of the priming dose. In some embodiments, the 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) after administration of the priming dose. In some embodiments, the subsequent booster dose may be administered at least 1 week or more after the previous booster dose, including, for example, 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 more. 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 boost dose (e.g., after the first boost dose) may be administered at least 3 months or more after the previous dose, including, for example, 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 more.

In some embodiments, the subsequent dose administered to the individual (e.g., as part of a primary regimen or a boost regimen) may have the same amount of RNA as previously administered to the individual. In some embodiments, the amount of RNA administered to the subject at a subsequent dose (e.g., as part of a primary regimen or a boost regimen) may be different compared to the amount previously administered to the subject. For example, in some embodiments, the subsequent dose may be higher or lower than the preceding dose, e.g., based on various factors including, for example, immunogenicity and/or reactogenicity induced by the preceding dose, prevalence of the disease, etc. In some embodiments, the subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more higher than the prior dose. In some embodiments, the subsequent dose may be at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, or more higher than the prior dose. In some embodiments, the subsequent dose may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more higher than the prior dose. In some embodiments, the subsequent dose may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or less lower than the prior dose. In some embodiments, an amount of RNA described herein of 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 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 can be administered per dose (e.g., in a given dose).

In some embodiments, an amount of RNA described herein of 60 μg or less, 55 μg or less, 50 μg or less, 45 μg or less, 40 μg or less, 35 μg or less, 30 μg or less, 25 μg or less, 20 μg or less, 15 μg or less, 10 μg or less, 5 μg or less, 3 μg or less, 2.5 μg or less, or 1 μg or less can be administered per dose (e.g., in a given dose).

In some embodiments, an amount of 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 can be administered per dose (e.g., in a given dose). In some embodiments, an amount of at least 3ug of an RNA described herein may be administered in at least one given dose. In some embodiments, an amount of at least 10ug of an RNA described herein can be administered in at least one given dose. In some embodiments, an amount of at least 15ug of an RNA described herein may be administered in at least one given dose. In some embodiments, an amount of at least 20ug of an RNA described herein can be administered in at least one given dose. In some embodiments, an amount of at least 25ug of an RNA described herein can be administered in at least one given dose. In some embodiments, an amount of at least 30ug of an RNA described herein can be administered in at least one given dose. In some embodiments, an amount of at least 50ug of an RNA described herein may be administered in at least one given dose. In some embodiments, an amount of at least 60ug of an RNA described herein can be administered in at least one given dose. In some embodiments, a combination of the above amounts may be administered in a regimen comprising two or more doses (e.g., a preceding dose and a subsequent dose may have different amounts as described herein). In some embodiments, a combination of the above amounts can be administered in a primary regimen and a boost regimen (e.g., different dosages can be administered in the primary regimen and the boost regimen).

In some embodiments, the RNA described herein may be administered in an amount 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 per dose. In some embodiments, the RNA described herein in an amount of 3 μg to 30 μg can be administered in at least one given dose. In some embodiments, the RNA described herein in an amount of 3 μg to 20 μg can be administered in at least one given dose. In some embodiments, the RNA described herein in an amount of 3 μg to 15 μg can be administered in at least one given dose. In some embodiments, the RNA described herein in an amount of 3 μg to 10 μg can be administered in at least one given dose. In some embodiments, the RNA described herein in an amount of 10 μg to 30 μg can be administered in at least one given dose.

In some embodiments, the regimen administered to the subject can include multiple doses (e.g., at least two doses, at least three doses, or more). In some embodiments, the regimen administered to the subject may comprise a first dose and a second dose administered at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, or more apart. In some embodiments, such doses may be spaced 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. In some embodiments, the dose may be administered on several days, such as on 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. In some embodiments, the doses may be administered about 1 week to about 3 weeks apart, or about 1 week to about 4 weeks apart, or about 1 week to about 5 weeks apart, or about 1 week to about 6 weeks apart, or about 1 week to more than 6 weeks apart. In some embodiments, the doses may be spaced apart for a period of about 7 days to about 60 days, such as about 14 days to about 48 days, and the like. In some embodiments, the 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, the 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 days or less. In some embodiments, the doses may be spaced about 21 days to about 28 days apart. In some embodiments, the doses may be spaced about 19 days to about 42 days apart. In some embodiments, the doses may be spaced about 7 days to about 28 days apart. In some embodiments, the doses may be spaced about 14 to about 24 days apart. In some embodiments, the doses may be spaced about 21 to about 42 days apart.

In some embodiments, the vaccination regimen comprises a first dose and a second dose. In some embodiments, the first dose and the second dose are administered at least 21 days apart. In some embodiments, the first dose and the second dose are administered at least 28 days apart.

In some embodiments, the 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, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is different from the amount of RNA administered in the second dose.

In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is less than the amount of RNA 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, the first dose comprises less than about 30ug of RNA and the second dose comprises at least about 30ug of RNA. In some embodiments, the first dose comprises about 1 to less than about 30ug RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or less than about 30ug RNA), and the second dose comprises about 30 to about 100ug RNA (e.g., about 30, about 40, about 50, or about 60ug RNA). In some embodiments, the first dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA, and the second dose comprises about 30 to about 60ug of RNA.

In some embodiments, the first dose comprises about 1 to about 10ug RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10ug RNA) and the second dose comprises about 30 to about 60ug RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60ug RNA).

In some embodiments, the first dose comprises about 1ug of RNA and the second dose comprises about 30ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 30ug of RNA. In some embodiments, the first dose comprises about 5ug of RNA and the second dose comprises about 30ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 30ug of RNA. In some embodiments, the first dose comprises about 15ug of RNA and the second dose comprises about 30ug of RNA.

In some embodiments, the first dose comprises about 1ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 5ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 6ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 15ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 20ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 25ug of RNA and the second dose comprises about 60ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 60ug of RNA.

In some embodiments, the first dose comprises less than about 10ug of RNA and the second dose comprises at least about 10ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 10ug 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 10ug RNA), and the second dose comprises about 10 to about 30ug RNA (e.g., about 10, about 15, about 20, about 25, or about 30ug RNA). In some embodiments, the first dose comprises about 0.1 to about 10ug of RNA, about 1 to about 5ug of RNA, or about 0.1 to about 3ug of RNA, and the second dose comprises about 10 to about 30ug of RNA.

In some embodiments, the first dose comprises about 0.1 to about 5ug RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, about 5ug RNA), and the second dose comprises about 10 to about 20ug RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20ug RNA).

In some embodiments, the first dose comprises about 0.1ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 0.3ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 1ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 10ug of RNA.

In some embodiments, the first dose comprises less than about 3ug of RNA and the second dose comprises at least about 3ug of RNA. In some embodiments, the first dose comprises about 0.1 to less than about 3ug 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 2.5ug RNA), and the second dose comprises about 3 to about 10ug RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug RNA). In some embodiments, the first dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA, and the second dose comprises about 3 to about 10ug of RNA.

In some embodiments, the first dose comprises about 0.1 to about 1.0ug 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 1.0ug RNA), and the second dose comprises about 1 to about 3ug RNA (e.g., about 1.0, about 1.5, about 2.0, about 2.5, or about 3.0ug RNA).

In some embodiments, the first dose comprises about 0.1ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 0.3ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 0.5ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 1ug of RNA and the second dose comprises about 3ug of RNA.

In some embodiments, the vaccination regimen comprises a first dose and a second dose, wherein the amount of RNA administered in the first dose is greater than the amount of RNA 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, the first dose comprises at least about 30ug of RNA and the second dose comprises less than about 30ug of RNA. In some embodiments, the first dose comprises about 30 to about 100ug RNA (e.g., about 30, about 40, about 50, or about 60ug RNA) and the second dose comprises about 1 to about 30ug RNA (e.g., about 0.1, about 1, about 3, about 5, about 10, about 15, about 20, about 25, or about 30ug RNA). In some embodiments, the second dose comprises about 1 to about 20ug RNA, about 1 to about 10ug RNA, or about 1 to 5ug RNA. In some embodiments, the first dose comprises about 30 to about 60ug of RNA and the second dose comprises about 1 to about 20ug of RNA, about 1 to about 10ug of RNA, or about 0.1 to about 3ug of RNA.

In some embodiments, the first dose comprises about 30 to about 60ug RNA (e.g., about 30, about 35, about 40, about 45, about 50, about 55, or about 60ug RNA), and the second dose comprises about 1 to about 10ug RNA (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10ug RNA).

In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 30ug of RNA and the second dose comprises about 15ug of RNA.

In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 3ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 5ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 6ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 10ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 15ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 20ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 25ug of RNA. In some embodiments, the first dose comprises about 60ug of RNA and the second dose comprises about 30ug of RNA.

In some embodiments, the first dose comprises at least about 10ug of RNA and the second dose comprises less than about 10ug of RNA. In some embodiments, the first dose comprises about 10 to about 30ug RNA (e.g., about 10, about 15, about 20, about 25, or about 30ug RNA) and the second dose comprises about 0.1 to less than about 10ug 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 10ug RNA). In some embodiments, the first dose comprises about 10 to about 30ug of RNA, or about 0.1 to about 3ug of RNA, and the second dose comprises about 1 to about 10ug of RNA, or about 1 to about 5ug of RNA.

In some embodiments, the first dose comprises about 10 to about 20ug RNA (e.g., about 10, about 12, about 14, about 16, about 18, about 20ug RNA), and the second dose comprises about 0.1 to about 5ug RNA (e.g., about 0.1, about 0.5, about 1, about 2, about 3, about 4, or about 5ug RNA).

In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 0.1ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 0.3ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 1ug of RNA. In some embodiments, the first dose comprises about 10ug of RNA and the second dose comprises about 3ug of RNA.

In some embodiments, the first dose comprises at least about 3ug of RNA and the second dose comprises less than about 3ug of RNA. In some embodiments, the first dose comprises about 3 to about 10ug RNA (e.g., about 3, about 4, about 5, about 6, or about 7, about 8, about 9, or about 10ug RNA), and the second dose comprises 0.1 to less than about 3ug 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 2.5ug RNA). In some embodiments, the first dose comprises about 3 to about 10ug of RNA and the second dose comprises about 0.1 to about 3ug of RNA, about 0.1 to about 1ug of RNA, or about 0.1 to about 0.5ug of RNA.

In some embodiments, the first dose comprises about 1 to about 3ug RNA (e.g., about 1, about 1.5, about 2.0, about 2.5, or about 3.0ug RNA), and the second dose comprises about 0.1 to 0.3ug 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 1.0ug RNA).

In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.1ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.3ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 0.6ug of RNA. In some embodiments, the first dose comprises about 3ug of RNA and the second dose comprises about 1ug of RNA.

In some embodiments, the vaccination regimen comprises at least two doses, including, for example, at least three doses, at least four doses, or more. In some embodiments, the vaccination regimen comprises three doses. In some embodiments, the time interval between the first dose and the second dose may 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 may be longer than the time interval between the second dose and the third dose, e.g., 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 may be shorter than the time interval between the second dose and the third dose, e.g., 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 may be shorter than the time interval between the second dose and the third dose by, for example, at least 1 month (including, for example, 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, the last dose of the primary regimen and the first dose of the boosting regimen are administered 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 apart. In some embodiments, the primary regimen may include two doses. In some embodiments, the primary regimen may include three doses.

In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered by intramuscular injection. In some embodiments, the first dose and the second dose (and/or other subsequent doses) may be administered in deltoid muscles. In some embodiments, the first dose and the second dose (and/or other subsequent doses) 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) in a series of two doses (e.g., 0.3mL each) at 21 day intervals. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) in a series of two doses (e.g., 0.2mL each) at 21 day intervals. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) in a series of three doses (e.g., 0.3mL or less, including, e.g., 0.2 mL), wherein the doses are administered at least 3 weeks apart. In some embodiments, the first dose and the second dose may be administered 3 weeks apart, while the second dose and the third dose may be administered longer than the time interval between the first dose and the second dose, 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 apart or longer). In some embodiments, each dose is about 60ug. In some embodiments, each dose is about 50ug. In some embodiments, each dose is about 30ug. In some embodiments, each dose is about 25ug. In some embodiments, each dose is about 20ug. In some embodiments, each dose is about 15ug. In some embodiments, each dose is about 10ug. In some embodiments, each dose is about 3ug.

In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 60ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 50ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 30ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 25ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 20ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 15ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 10ug. In some embodiments, at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3ug.

In one embodiment, the RNA described herein is administered in an amount of about 60 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 50 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 30 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 25 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 20 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 15 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 10 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 5 μg per dose. In one embodiment, the RNA described herein is administered in an amount of about 3 μg per dose. In one embodiment, at least two such doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, the efficacy of an RNA vaccine described herein (e.g., administered in two doses, wherein the second dose can be administered about 21 days after administration of the first dose, and for example, administered in an amount of about 30 μg/dose) begins 7 days after administration of the second dose (e.g., 28 days after administration of the first dose if the second dose is administered 21 days after administration of the first dose) by at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, such efficacy is observed in a population at least 50, at least 55, at least 60, at least 65, at least 70 years old or older. In some embodiments, the efficacy of an RNA vaccine described herein (e.g., administered in two doses, wherein the second dose can be administered about 21 days after administration of the first dose, and for example, administered in an amount of about 30 μg/dose) begins 7 days after administration of the second dose (e.g., if the second dose is administered 21 days after administration of the first dose, then begins 28 days after administration of the first dose) in a population at least 65 years old, such as 65 to 80 years old, 65 to 75 years old, or 65 to 70 years old, 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 a period of up to 1 month, 2 months, 3 months, 6 months or even longer.

In one embodiment, vaccine efficacy is defined as the percentage of decrease in the number of subjects with signs of infection (vaccinated versus unvaccinated subjects).

In one embodiment, efficacy is assessed by monitoring potential cases of covd-19. If at any time the patient develops an acute respiratory illness, for purposes herein, the patient may be considered potentially suffering from a covd-19 illness. The evaluation may include a nasal (middle turbinate) 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 care tests may be evaluated.

In some embodiments, efficacy assessment can utilize the definition of SARS-CoV-2 related cases, wherein:

certified covd-19: during the symptomatic phase or 4 days before and after the symptomatic phase, there are at least 1 of the following symptoms and positive for SARS-CoV-2 NAAT (nucleic acid amplification based test): heating; new or added cough; new or increased shortness of breath; shivering; new or increased muscle pain; loss of new taste or smell; sore throat; diarrhea; vomiting.

Alternatively or additionally, in some embodiments, efficacy assessment may utilize the definition of SARS-CoV-2 related cases, where one or more of the following additional symptoms defined by CDC may be considered: fatigue; headache; nasal congestion or runny nose; nausea.

In some embodiments, efficacy assessment can utilize the definition of severe cases related to SARS-CoV-2

Serious covd-19 confirmed: the confirmed covd-19 and at least 1 of the following are present: resting clinical signs indicating severe systemic disease (e.g., RR.gtoreq.30 breaths/min, HR.gtoreq.125 beats/min, at sea level to obtain SpO in indoor air) ₂ Less than or equal to 93 percent, or PaO ₂ /FiO ₂ < 300mm Hg); respiratory failure (which may be defined as requiring high flow of oxygen, non-invasive ventilation, mechanical ventilation, or ECMO); evidence of shock (e.g., SBP < 90mm Hg, DBP < 60mm Hg, or vasopressors are required); severe acute kidney, liver or nerve dysfunction; check in ICU; death.

Alternatively or additionally, in some embodiments, the serological definition may be for patients that do not clinically present covd-19: for example, conversion of serum to SARS-CoV-2 was confirmed without confirmed COVID-19: for example, positive N-binding antibodies result in patients having previously had negative N-binding antibody results.

In some embodiments, any or all of the following assays may be performed on the serum sample: SARS-CoV-2 neutralization assay; s1-binding IgG level assay; RBD-binding IgG level assay; n-binding antibody assay.

In one embodiment, the methods and agents described herein are administered to a pediatric population. In various embodiments, the pediatric population comprises or consists of subjects under 18 years old, e.g., 5 to less than 18 years old, 12 to less than 18 years old, 16 to less than 18 years old, 12 to less than 16 years old, 5 to less than 12 years old, or 6 months to less than 12 years old. In various embodiments, the pediatric population comprises or consists of subjects under 5 years of age, 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 a subject less than 2 years old, e.g., 6 months to less than 2 years old. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject for less than 6 months, e.g., 1 month to less than 4 months. In some embodiments, the dosing regimen (e.g., dose and/or dosing schedule) for the pediatric population may vary with different age groups. For example, in some embodiments, a subject from 6 months to 4 years old may be administered according to a primary regimen comprising at least three doses, wherein the first two doses are administered at least 3 weeks apart (including, for example, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer), followed by a third dose administered at least 8 weeks after the second dose (including, for example, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, or longer). In some such embodiments, the at least one dose administered is 3ug RNA as described herein. In some embodiments, subjects aged 5 and older may be administered according to a primary regimen comprising at least two doses, wherein the two doses are administered at least 3 weeks apart (including, for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer). In some such embodiments, at least one dose administered is 10ug RNA as described herein. In some embodiments, immunocompromised subjects aged 5 years and older (e.g., in some embodiments, subjects who have undergone solid organ transplantation or are diagnosed with a condition believed to have comparable immunocompromised) may be administered according to a primary regimen comprising at least three doses, wherein the first two doses are administered at least 3 weeks apart (including, e.g., at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, or longer), followed by a third dose administered at least 4 weeks after the second dose (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).

In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and each dose is about 30ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older (including, e.g., 18 years old or older), and each dose is greater than 30ug, including, e.g., 35ug, 40ug, 45ug, 50ug, 55ug, 60ug, 65ug, 70ug, or higher. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and each dose is about 60ug. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and each dose is about 50ug. In one embodiment, the pediatric population comprises or consists of subjects from 12 to less than 18 years old, including subjects from 16 to less than 18 years old and/or subjects from 12 to less than 16 years old. In this embodiment, the treatment 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., patients aged 12 years or older) compared to younger children or infants (e.g., 2 to less than 5 years, 6 months to less than 2 years, or less than 6 months). In some embodiments, a higher dose is administered to children from 2 to less than 5 years old as compared to infants and/or infants (e.g., infants and/or infants from 6 months to less than 2 years old, or less than 6 months old).

In one embodiment, the pediatric population comprises or consists of subjects from 5 to less than 18 years old, including subjects from 12 to less than 18 years old and/or subjects from 5 to less than 12 years old. In this embodiment, the treatment 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 of RNA per dose, for example by intramuscular administration. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject aged 5 to 11 years, and each dose is about 10ug

In some embodiments, each dose comprises about 5ug of RNA encoding the SARS-CoV-2S protein of the first variant and about 5ug of RNA encoding the SARS-CoV-2S protein of the second variant. In some embodiments, each dose comprises about 5ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain and about 5ug of RNA encoding SARS-CoV-2S protein of the Omicron variant. In some embodiments, each dose comprises about 5ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain (e.g., comprising RNA of SEQ ID NO: 20) and about 5ug of RNA encoding SARS-CoV-2S protein of the BA.1 Omicron variant (e.g., comprising RNA of SEQ ID NO: 51). In some embodiments, each dose comprises about 5ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain (e.g., comprising RNA of SEQ ID NO: 20) and about 5ug of RNA encoding SARS-CoV-2S protein of the BA.4/5 Omacron variant (e.g., comprising RNA of SEQ ID NO: 72).

In one embodiment, the pediatric population comprises or consists of subjects less than 5 years old, including subjects from 2 to less than 5 years old, subjects from 12 to less than 24 months old, subjects from 7 to less than 12 months old, subjects from 6 to less than 12 months old, and/or subjects less than 6 months old. In this embodiment, the treatment may comprise 2 vaccinations, e.g., 21 to 42 days apart, e.g., 21 days apart, wherein in various embodiments the vaccine is administered in an amount of 3 μg, 10 μg, 20 μg, or 30 μg of RNA per dose, e.g., by intramuscular administration. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from 2 to less than 5 years old, and each dose is about 3ug. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from about 6 months to less than about 5 years old, and each dose is about 3ug.

In some embodiments, each dose comprises about 1.5ug of RNA encoding the SARS-CoV-2S protein of the first variant and about 1.5ug of RNA encoding the SARS-CoV-2S protein of the second variant. In some embodiments, each dose comprises about 1.5ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain and about 1.5ug of RNA encoding SARS-CoV-2S protein of the Omacron variant. In some embodiments, each dose comprises about 1.5ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain (e.g., comprising RNA of SEQ ID NO: 20) and about 1.5ug of RNA encoding SARS-CoV-2S protein of the BA.1 Omicron variant (e.g., comprising RNA of SEQ ID NO: 51). In some embodiments, each dose comprises about 1.5ug of RNA encoding SARS-CoV-2S protein of MN908947 strain (e.g., comprising RNA of SEQ ID NO: 20) and about 1.5ug of RNA encoding SARS-CoV-2S protein of BA.4/5 Omicron variant (e.g., comprising RNA of SEQ ID NO: 72).

In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 60ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 30ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject 12 years old or older, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 15ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from 5 years to less than 12 years old, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 10ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from 2 years to less than 5 years old, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3ug. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to a subject from 6 months to less than 2 years old, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3ug or less, including, for example, 2ug, 1ug, or less. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered to an infant less than 6 months, and at least one dose administered in an vaccination regimen (e.g., a primary vaccination regimen and/or a booster vaccination regimen) is about 3ug or less, including, for example, 2ug, 1ug, 0.5ug or less.

In some embodiments, an RNA (e.g., mRNA) composition described herein is administered as a single dose (e.g., by intramuscular injection). In some embodiments, a single dose comprises a single RNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof (e.g., RBD domain). In some embodiments, a single dose comprises at least two RNAs described herein, e.g., each RNA encodes SARS-CoV-2S protein or an immunogenic fragment thereof (e.g., RBD domain) from a different strain. 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 (e.g., mRNA) compositions described herein can be mixed prior to injection to produce a single mixture. In some embodiments, such at least two RNAs described herein can be administered as two separate compositions, which can be administered, for example, at different injection sites (e.g., on different arms, or at different sites on the same arm).

In some embodiments, the dose administered to a subject in need thereof may comprise administering a single RNA (e.g., mRNA) composition described herein.

In some embodiments, the dose administered to a subject in need thereof may comprise administration of at least two or more (including, for example, at least three or more) different pharmaceutical products/formulations. For example, in some embodiments, at least two or more different pharmaceutical products/formulations can 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, the RNA (e.g., mRNA) compositions disclosed herein can be administered in combination with a vaccine targeting a different infectious agent. In some embodiments, the different infectious agent is an infectious agent that increases the likelihood of a subject experiencing a detrimental symptom when co-infected with SARS-CoV-2 and the infectious agent. In some embodiments, the infectious agent is an infectious agent that increases the infectivity of SARS-CoV-2 when the subject is co-infected with SARS-CoV-2 and the infectious agent. In some embodiments, at least one RNA (e.g., mRNA) composition described herein can be administered in combination with a vaccine that targets influenza. In some embodiments, at least two or more different pharmaceutical products/formulations can comprise at least one RNA (e.g., mRNA) composition described herein and a vaccine (e.g., influenza vaccine) that targets different infectious agents. In some embodiments, the different pharmaceutical products/formulations are administered separately. In some embodiments, such different pharmaceutical products/formulations are administered separately at the same time (e.g., during the same vaccination period) at different sites of the subject (e.g., at different arms of the subject).

In one embodiment, at least two doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, at least one single dose is administered. In some embodiments, such a single dose is administered to a subject, e.g., who may have previously received one or more doses or complete regimens of SARS-CoV-2 vaccine (e.g., BNT162b2 vaccine [ including, e.g., as described herein ], mRNA-1273 vaccine, A26. CoV2.S vaccine, chAdxOx1 vaccine, NVX-CoV2373 vaccine, cvnCoV vaccine, GAM-COVID0Vac vaccine, coronaVac vaccine, BBIBP-CorV vaccine, ad5-nCoV vaccine, zf2001 vaccine, SCB-2019 vaccine, or other approved RNA (e.g., mRNA) 2019 vaccine, or the like).

In some embodiments, wherein at least one single dose is administered to a subject who has 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 vaccine (e.g., BNT162b 2) having the same activity; in some such embodiments, such subjects do not receive a complete regimen of such prior vaccines and/or have undergone one or more undesired responses or effects to one or more received doses of such prior vaccines. In some embodiments, such prior vaccines are or comprise a higher dose of the same active substance (e.g., BNT162b 2). Alternatively or additionally, in some such embodiments, such subjects are exposed to and/or infected with SARS-CoV-2 prior to completion of the complete regimen of such prior vaccine (but in some embodiments, after initiation).

In one embodiment, at least three doses are administered. In some embodiments, such a 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 dose and the second dose. For example, in some embodiments, the third dose may be administered about 21 days after the second dose is administered. In some embodiments, the third dose is administered relative to the second dose after a longer period of time than the second dose relative to the first dose. In some embodiments, the 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 immunosuppressive therapy (e.g., an organ transplant patient). In some embodiments, the length of time between the second dose and the third dose (e.g., the second dose and the third dose administered to an immunocompromised patient) is at least about 21 days (e.g., at least about 28 days).

In some embodiments, the vaccination regimen comprises administering the same amount of RNA at different doses (e.g., at a first dose and/or a second dose and/or a third dose and/or a subsequent dose). In some embodiments, the vaccination regimen comprises administering different amounts of RNA at different doses. In some embodiments, one or more later doses are greater than one or more earlier doses (e.g., where vaccine efficacy is observed to be attenuated from one or more earlier doses and/or where epidemic and/or rapidly spreading variants (e.g., one described herein) are observed to escape from immunity in the relevant jurisdiction upon administration). In some embodiments, one or more later doses may be 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 more greater than one or more earlier doses, provided that the safety and/or tolerability of such doses is clinically acceptable. In some embodiments, one or more later doses may be at least 1.1-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more greater than one or more earlier doses, provided that the safety and/or tolerability of such doses is clinically acceptable. In some embodiments, the one or more later doses are less than the one or more earlier doses (e.g., undergo a negative response after the one or more earlier doses and/or if exposed to and/or infected with SARS-CoV-2 between the earlier and later doses). In some embodiments, one or more later doses may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or more smaller than one or more earlier doses. In some embodiments, when different dosages are used, they are related to each other by being the same and/or diluted as the common stock described herein.

In some embodiments, when at least two or more doses are administered (e.g., at least two doses in a primary regimen, at least two doses in a boosting regimen, or at least one dose in a primary regimen and at least one dose in a boosting regimen), the same RNA composition described herein can be administered in such doses, and each of such doses can be the same or different (as described herein). In some embodiments, when at least two or more doses (e.g., at least two doses in a primary regimen, at least two doses in a boost regimen, or at least one dose in a primary regimen and at least one dose in a boost regimen) are administered, different RNA compositions described herein (e.g., different encoded viral polypeptides, e.g., from different coronavirus clades, or different strains from the same coronavirus clade, different construct elements, such as 5' caps, 3' utrs, 5' utrs, etc., different formulations, e.g., different excipients and/or buffers (e.g., PBS versus Tris), different LNP compositions, or combinations thereof) can be administered at such doses, and each of such doses can be the same or different (e.g., as described herein).

In some embodiments, two or more RNAs are administered to the subject (e.g., as part of a primary regimen or a boost regimen), wherein the two or more RNAs are administered on the same day or at the same visit. In some embodiments, two or more RNAs are administered in separate compositions, e.g., by administering each RNA to a separate portion of the subject (e.g., by intramuscular administration to a different arm of the subject or to a different site of the same arm of the subject). In some embodiments, the two or more RNAs are mixed prior to administration (e.g., immediately prior to administration, e.g., by an administration practitioner). In some embodiments, 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, such that each LNP comprises two or more RNAs). In some embodiments, the two or more RNAs comprise RNAs encoding coronavirus S proteins or immunogenic fragments thereof (e.g., RBDs or other related domains) from one strain (e.g., MN908947 strain) and variants (e.g., variants described herein) that are prevalent and/or rapidly spread in the relevant jurisdiction upon administration. In some embodiments, such variants are omacron variants (e.g., ba.1, ba.2, or ba.3 variants). 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 a subject. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from the MN908947 strain and an RNA encoding a SARS-CoV-2S protein from the ba.1 omacron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from the MN908947 strain and an RNA encoding a SARS-CoV-2S protein from the ba.2 omacron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from the MN908947 strain and an RNA encoding a SARS-CoV-2S protein from a ba.4 or ba.5 omacron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from a ba.1 Omicron variant and an RNA encoding a SARS-CoV-2S protein from a ba.2 Omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from a ba.1 Omicron variant and an RNA encoding a SARS-CoV-2S 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-2S protein from a ba.2 Omicron variant and an RNA encoding a SARS-CoV-2S protein from a ba.4 or 5 Omicron variant. In some embodiments, the two or more RNAs comprise an RNA encoding a SARS-CoV-2S protein from the MN908947 strain, an alpha variant, a beta variant, or a delta variant, or a subline derived therefrom; and RNA encoding SARS-CoV-2S protein from BA.2, BA.4 or 5 Omacron variants or sublines derived therefrom.

In some embodiments, any of combinations 1 through 66 listed in the following table may be administered to a subject. In some embodiments, such combinations can be administered using LNP formulations, wherein 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 administration to a subject at separate sites).

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¹ The listed RNAs encode SARS-CoV-2S protein having the mutational characteristics of the indicated SARS-CoV-2 variant.

In some embodiments, the first RNA and the second RNA are administered to the subject in the same amount (i.e., 1:1 ratio) each.

In some embodiments, the first RNA and the second RNA are administered to the subject in different amounts each. For example, in some embodiments, the first RNA is administered to the subject in an amount that is 0.01 to 100 times the amount of the second RNA (e.g., wherein the amount of the first RNA is 0.01 to 50, 0.01 to 4, 0.01 to 30, 0.01 to 25, 0.01 to 20, 0.01 to 15, 0.01 to 10, 0.01 to 9, 0.01 to 8, 0.01 to 7, 0.01 to 6, 0.01 to 5, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.01 to 1.5, 1 to 50, 1 to 4, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 to 1.5 times the amount of the second RNA). In some embodiments, the first RNA and the second RNA are administered to the subject, wherein the concentration of the first RNA is 1 to 10 times that of the second RNA. In some embodiments, the first RNA and the second RNA are administered to the subject, wherein the amount of the first RNA is 1 to 5 times the amount of the second RNA. In some embodiments, the first RNA and the second RNA are administered to the subject, wherein the concentration of the first RNA is 1 to 3 times that of the second RNA. In some embodiments, the first RNA and the second RNA are administered to the subject, wherein the amount of the first RNA is 2 times the amount of the second RNA. In some embodiments, the first RNA and the second RNA are administered to the subject, wherein the concentration of the first RNA is 3-fold that of the second RNA.

In some embodiments, three RNAs are administered to a subject, each encoding a SARS-CoV-2S protein comprising one or more mutational features of a different SARS-CoV-2 variant and each in the same amount (i.e., 1:1:1 ratio).

In some embodiments, three RNAs are administered to the subject, each encoding a SARS-CoV-2S protein comprising one or more mutational features of a different SARS-CoV-2 variant, wherein the amount of each RNA is different (e.g., one RNA is present in a different amount than the other two RNAs, or all three RNAs are present in different amounts). For example, in some embodiments, the ratio of the first RNA to the second RNA to the third RNA is 1:0.01-100:0.01-100 (e.g., 1:0.01-50:0.01-50; 1:0.01-40:0.01-40; 1:0.01-30:0.01-25; 1:0.01-25:0.01-25; 1:0.01-20:0.01-20; 1:0.01-15:0.01-15; 1:0.01-10:0.01-9;1:0.01-9:0.01-9;1:0.01-8:0.01-8;1:0.01-7:0.01-7;1:0.01-6:0.01-6;1:0.01-5:0.01-5;1:0.01-4:0.01-4;1:0.01-3:0.01-3; 1:0.01-2.01-2:1.01-2.01). In some embodiments, three RNAs are administered to a subject in a ratio of 1:1:3. In some embodiments, three RNAs are administered to a subject in a ratio of 1:3:3.

In some embodiments, the vaccination regimen comprises a first vaccination regimen (e.g., a primary regimen) comprising at least two doses of an RNA composition as described herein, e.g., wherein the second dose may be administered about 21 days after administration of the first dose, and a second vaccination regimen (e.g., a booster regimen) comprising a single dose or multiple doses, e.g., two doses, of an RNA composition as described herein. In some embodiments, the dose of the boosting regimen is related to the dose of the primary regimen by being the same as or diluted from the co-parent as described herein. In various embodiments, a boosting regimen is administered (e.g., started) 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 the primary regimen comprising at least two doses is administered, e.g., after completion of the primary regimen. In various embodiments, a boosting regimen is administered (e.g., initiated) after administration of the primary regimen, e.g., 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 completion of the primary regimen comprising at least two doses. In various embodiments, the boosting regimen is administered (e.g., started) after administration of the primary regimen, e.g., 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 completion of the two-dose primary regimen. In some embodiments, each dose of the primary regimen is about 60 μg/dose. In some embodiments, each dose of the primary regimen is about 50 μg/dose. In some embodiments, each dose of the primary regimen is about 30 μg/dose. In some embodiments, each dose of the primary regimen is about 25 μg/dose. In some embodiments, each dose of the primary regimen is about 20 μg/dose. In some embodiments, each dose of the primary regimen is about 15 μg/dose. In some embodiments, each dose of the primary regimen is about 10 μg/dose. In some embodiments, each dose of the primary regimen is about 3 μg/dose. In some embodiments, each dose of the boosting regimen is the same as the dose of the primary regimen. In some embodiments, each dose of the boosting regimen comprises the same amount of RNA as the dose administered in the primary regimen. In some embodiments, at least one dose of the boosting regimen is the same as the dose of the primary regimen. In some embodiments, at least one dose of the boosting regimen comprises the same amount of RNA as at least one dose of the primary regimen. In some embodiments, at least one dose of the boosting regimen is lower than the dose of the primary regimen. In some embodiments, at least one dose of the boosting regimen comprises a lower amount of RNA than the primary regimen. In some embodiments, at least one dose of the boosting regimen is higher than the dose of the primary regimen. In some embodiments, at least one dose of the boosting regimen comprises a higher amount of RNA than the primary regimen.

In some embodiments, a boosting regimen (e.g., as described herein) is administered to a pediatric patient (e.g., a patient 2 to 5 years old, a patient 5 to 11 years old, or a patient 12 to 15 years old). In some embodiments, a boost regimen is administered to pediatric patients ranging from 6 months to less than 2 years old. In some embodiments, a boost regimen is administered to pediatric patients for less than 6 months. In some embodiments, a boost regimen is administered to pediatric patients ranging from 6 months to less than 5 years old. In some embodiments, the boost regimen is administered to pediatric patients between 2 years old and less than 5 years old. In some embodiments, the boost regimen is administered to pediatric patients between 5 years old and less than 12 years old. In some embodiments, the boost regimen is administered to pediatric patients between 12 years old and less than 16 years old. In some embodiments, each dose of the pediatric boosting regimen comprises about 3 μg of RNA. In some embodiments, each dose of the pediatric boosting regimen comprises about 10 μg of RNA. In some embodiments, each dose of the pediatric boosting regimen comprises about 15 μg of RNA. In some embodiments, each dose of the pediatric boosting regimen comprises about 20 μg RNA. In some embodiments, each dose of the pediatric boosting regimen comprises about 25 μg RNA. In some embodiments, each dose of the pediatric boosting regimen comprises about 30 μg of RNA. In some embodiments, the boost regimen is administered to a non-pediatric patient (e.g., a patient aged 16 years or older, a patient aged 18 to 64 years, and/or a patient aged 65 years or older). In some embodiments, each dose of the non-pediatric boosting regimen comprises about 3ug of RNA, about 10ug of RNA, about 25ug of RNA, about 30ug of RNA, about 40ug of RNA, about 45ug of RNA, about 50ug of RNA, about 55ug of RNA, or about 60ug of RNA. In some embodiments, the same boost regimen may be administered to both pediatric and non-pediatric patients (e.g., to patients 12 years old or older). In some embodiments, the boosting regimen administered to a non-pediatric patient is administered in a formulation and dosage that is the same as or related to the formulation and dosage of the primary regimen previously received by the patient, as described herein, by dilution. In some embodiments, a non-pediatric patient receiving a lower dose than the boosting regimen of the primary regimen may have experienced adverse effects on one or more doses of such primary regimen, and/or may have been exposed to and/or infected with SARS-CoV-2 between such primary regimen and such boosting regimen, or between doses of such primary regimen and/or such boosting regimen. In some embodiments, pediatric and non-pediatric patients may receive higher dose boost regimens than the primary regimen when reduced vaccine efficacy at lower doses is observed, and/or when immune evasion of variants that are prevalent and/or rapidly spread in the relevant jurisdiction at the time of administration is observed.

In some embodiments, the one or more doses of the boosting regimen are different from the one or more doses of the primary regimen. For example, in some embodiments, the administered dose may correspond to the age of the subject, and the patient may be aged beyond one treatment age group and into the next treatment age group. Alternatively or additionally, in some embodiments, the administered dose may correspond to a condition (e.g., an immunocompromised state) of the patient, and a different dose than the primary regimen may be selected for one or more doses of the boost regimen (e.g., due to intervening cancer treatment, HIV infection, receiving immunosuppressive therapy, e.g., associated with organ transplantation). In some embodiments, at least one dose of the boosting regimen may comprise a higher amount of RNA than at least one dose administered in the primary regimen (e.g., where vaccine efficacy is observed to be attenuated from one or more earlier doses and/or where epidemic and/or rapidly spreading variants (e.g., one described herein) are observed in the relevant jurisdiction at the time of administration).

In some embodiments, the primary regimen may involve one or more 3ug doses, and the boosting regimen may involve one or more 10ug doses, and/or one or more 20ug doses, or one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 3ug doses and the boosting regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve two or more 3ug doses (e.g., at least two doses, each comprising 3ug RNA, and administered about 21 days after each other), and the boosting regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses, and the boosting regimen may involve one or more 20ug doses, and/or one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses, and the boosting regimen may involve one or more 10ug doses. In some embodiments, the primary regimen may involve two or more 10ug doses (e.g., two doses, each containing 10ug RNA, administered about 21 days apart), and the boosting regimen may involve one or more 10ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses and the boosting regimen may involve one or more 30ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses and the boosting regimen may involve one or more 20ug doses. In some embodiments, the primary regimen may involve one or more 30ug doses, and the boosting regimen may also involve one or more 30ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses, each containing 30ug RNA, administered about 21 days apart), and the boosting regimen may also involve one or more 30ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses, each containing 30ug RNA, administered about 21 days apart), and the boosting regimen may involve one or more 50ug doses. In some embodiments, the primary regimen may involve two or more 30ug doses (e.g., two doses, each containing 30ug RNA, administered about 21 days apart), and the boosting regimen may involve one or more 60ug doses.

In some embodiments, a boosting regimen comprising at least one 30ug dose of RNA is administered to a subject. In some embodiments, a boosting regimen is administered to a subject that includes at least one 30ug dose of RNA encoding SARS-CoV-2S protein from a strain of SARS-CoV-2MN908947 (e.g., BNT162b 2). In some embodiments, a boosting regimen is administered to a subject that includes at least one dose of 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, a boosting regimen is administered to a subject that includes at least one dose of 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an omacron variant (e.g., ba.1, ba.2, ba.3, or ba.4, or ba.5 omacron variant). In some embodiments, a boosting regimen comprising at least one dose of 30ug of RNA is administered to the subject, wherein the 30ug of RNA comprises RNA encoding SARS-CoV-2S protein from the MN908947 strain and RNA encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the SARS-CoV-2 variant (e.g., in some embodiments, the boosting regimen comprises at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the omacron variant). In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 20ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.1 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 20ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.2Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 20ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants.

In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the ba.1omicron variant and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the ba.1 omacron variant and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2 omacron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the ba.1 omacron variant and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 omacron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the ba.1 omacron variant and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 omacron variant.

In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from a ba.2Omicron variant and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from a ba.2Omicron variant and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the ba.2 omacron variant and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.3 Omicron variant and 15ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 10ug of RNA encoding SARS-CoV-2S protein from a ba.3 Omicron variant and 20ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 7.5ug of RNA encoding SARS-CoV-2S protein from the ba.3 omacron variant and 22.5ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 omacron variant.

In some embodiments, a boosting regimen is administered to a subject that includes two or more doses of 30ug RNA administered at least two months apart from each other. For example, in some embodiments, a boosting regimen is administered to a subject that includes two doses of 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an omacron variant (e.g., ba.1, ba.2, or ba.4 or ba.5 omacron variant).

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a SARS-CoV-2 omicn variant (e.g., ba.1, ba.2, ba.3, ba.4, or ba.5 omicn variant), wherein the boosting 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, the subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having omicton variant characteristics.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.1 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.2 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug doses of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of SARS-CoV-2ba.4 or ba.5 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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, the subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the ba.4 or ba.5 omacron variant characteristics.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 15ug of RNA encoding 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.2 Omicron variant and 15ug of RNA encoding 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, the subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising at least one 30ug dose of RNA encoding SARS-CoV-2S protein from a non-ba.1 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising at least two 30ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins characteristic of Omicron variants, wherein the boosting regimen is administered at least two months (including, for example, 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 boosting doses are administered at least two months apart.

In some embodiments, a boosting regimen comprising at least one 50ug dose of RNA is administered to a subject. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose of 50ug of RNA encoding SARS-CoV-2S protein from a MN908947 strain (e.g., BNT162b 2). In some embodiments, a boosting regimen is administered to a subject that includes at least one dose of 50ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, a boosting regimen is administered to a subject that includes at least one dose of 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins with the characteristics of an omacron variant. In some embodiments, a boosting regimen comprising at least one 50ug dose of RNA is administered to the subject, wherein the 50ug of RNA comprises RNA encoding SARS-CoV-2S protein from the MN908947 strain and RNA encoding SARS-CoV-2S protein comprising one or more mutations characteristic of the SARS-CoV-2 variant (e.g., in some embodiments, the boosting regimen is administered to the subject comprising a 50ug dose of RNA comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the omacron variant).

In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants.

In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins that have the characteristics of the ba.2 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.1omicron variant and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant.

In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from a ba.2 Omicron variant and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 25ug of RNA encoding SARS-CoV-2S protein from a ba.3Omicron variant and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered about 21 days apart, and (ii) a boosting regimen comprising at least one 50ug dose of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a SARS-CoV-2 Omicron variant (e.g., ba.1, ba.2, ba.4, or ba.5Omicron variant), wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered about 21 days apart, and (ii) a boosting regimen comprising at least one 50ug dose of RNA, wherein the 50ug RNA comprises 25ug of RNA encoding SARS-CoV-2S protein of the MN908947 strain and 25ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of an omacron variant (e.g., ba.1, ba.2, ba.4, or ba.5 variant), wherein the boosting regimen is administered at least two months (including, for example, at least three months, at least four months, at least five months, at least six months, or more) after completion of the first boosting regimen.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 50ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.1 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 50ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.2 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 50ug doses of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of SARS-CoV-2ba.4 or ba.5 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the ba.4 or ba.5 omacron variant characteristics.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 25ug of RNA encoding 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 25ug of RNA encoding 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 25ug of RNA encoding SARS-CoV-2S protein from the ba.2 Omicron variant and 25ug of RNA encoding 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, a boosting regimen comprising at least one 60ug dose of RNA is administered to a subject. In some embodiments, a boosting regimen is administered to a subject that includes 60ug of RNA encoding SARS-CoV-2S protein from a variant of MN 908947. In some embodiments, 60ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a SARS-CoV-2 variant (e.g., a variant described herein) is administered to a subject. In some embodiments, a boosting regimen is administered to a subject that includes 60ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an omacron variant (e.g., ba.1, ba.2, ba.4, or ba.5 omacron variant). In some embodiments, a boosting regimen comprising 60ug of RNA is administered to a subject, wherein the RNA comprises a first RNA encoding a SARS-CoV-2S protein from the MN908947 strain and at least one additional RNA encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of a SARS-CoV-2 variant (e.g., in some embodiments, a boosting regimen comprising 30ug of RNA encoding a SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding a SARS-CoV-2S protein having one or more mutations characteristic of an Omicron variant (e.g., ba.1, ba.2, ba.4, or ba.5 variant) is administered to the subject.

In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 40ug of RNA encoding one or more mutated SARS-CoV-2S proteins that have the characteristics of a ba.1 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 45ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 40ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 45ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 40ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 45ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.3Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of ba.4 or ba.5 Omicron variants.

In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the ba.1omicron variant and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.2Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.3 Omicron variant. In some embodiments, a boosting regimen is administered to the subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.1Omicron variant and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5 Omicron variant.

In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from a ba.2Omicron variant and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from a ba.2Omicron variant and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from a ba.2Omicron variant and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 30ug of RNA encoding SARS-CoV-2S protein from a ba.3 Omicron variant and 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 20ug of RNA encoding SARS-CoV-2S protein from a ba.3 Omicron variant and 40ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a ba.4 or ba.5Omicron variant. In some embodiments, a boosting regimen is administered to a subject that includes at least one dose comprising 15ug of RNA encoding SARS-CoV-2S protein from the ba.3 Omicron variant and 45ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of the ba.4 or ba.5Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising two 30ug doses of RNA encoding SARS-CoV-2S protein from SARS-CoV-2MN908947 strain, wherein the two doses are administered about 21 days apart, and (ii) a boosting regimen comprising at least one 60ug dose of RNA encoding SARS-CoV-2S protein from SARS-CoV-2MN908947 strain, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered about 21 days apart, and (ii) a boosting regimen comprising at least one 60ug dose of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a SARS-CoV-2 Omicron variant (e.g., ba.1, ba.2, ba.4, or ba.5Omicron variant), wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of a SARS-CoV-2 Omicron variant and 30ug of RNA encoding at least one 60ug dose of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 60ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.1 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.1 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 60ug doses of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of SARS-CoV-2ba.2 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of a ba.2 omacron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 60ug doses of RNA encoding SARS-CoV-2S protein having one or more mutations characteristic of SARS-CoV-2ba.4 or ba.5 Omicron variant, wherein the boosting regimen is administered at least two months (including, for example, 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 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the ba.4/5 omacron variant characteristics.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 30ug of RNA encoding 60ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.2 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.1 Omicron variant and 30ug of RNA encoding 60ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, a subject is administered (i) a primary regimen comprising at least two 30ug doses of RNA encoding SARS-CoV-2S protein from the MN908947 strain, wherein the two doses are administered at least about 21 days apart, and (ii) a boosting regimen comprising 30ug of RNA encoding SARS-CoV-2S protein from the ba.2 Omicron variant and 30ug of RNA encoding 60ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of the ba.4 or ba.5 Omicron variant.

In some embodiments, the patient is administered a primary regimen comprising two 30ug doses administered about 21 days apart and a boosting regimen comprising at least one 60ug dose of RNA (e.g., 60ug of RNA encoding SARS-CoV-2S protein from MN908947 strain, 60ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant (e.g., ba.1, ba.2, ba.4, or ba.5 Omicron variant), or 30ug of RNA encoding SARS-CoV-2S protein from MN908947 strain and 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant). In some embodiments, the patient is administered a primary regimen comprising two 30ug doses administered about 21 days apart and a boosting regimen comprising at least one 50ug dose of RNA (e.g., 50ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain, 50ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant, or 25ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 25ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant). In some embodiments, the patient is administered a primary regimen comprising two 30ug doses administered about 21 days apart and a boosting regimen comprising at least one 30ug dose of RNA (e.g., 30ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain, 30ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant, or 15ug of RNA encoding SARS-CoV-2S protein from the MN908947 strain and 15ug of RNA encoding one or more mutated SARS-CoV-2S proteins having the characteristics of an Omicron variant).

In some embodiments, the primary regimen may involve one or more 30ug doses, and the boosting regimen may involve one or more 20ug doses, one or more 10ug doses, and/or one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 20ug doses, and the boosting regimen may involve one or more 10ug doses, and/or one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 10ug doses and the boosting regimen may involve one or more 3ug doses. In some embodiments, the primary regimen may involve one or more 3ug doses, and the boosting regimen may also involve one or more 3ug doses.

In some embodiments, the boosting regimen includes a single dose, e.g., for a patient experiencing an adverse reaction upon receiving the primary regimen.

In some embodiments, the same RNA as used in the primary protocol is used in the boosting protocol. In some embodiments, the RNA used in the primary and boosting regimen is BNT162b2.

In some embodiments, different RNAs are used in the boosting regimen relative to the RNAs used in the primary regimen administered to the same subject. In some embodiments, BNT162b2 is used in a primary regimen but not in a boosting regimen. In some embodiments, BNT162b2 is used in a boosting regimen but not in a prime regimen. In some embodiments, similar BNT162b2 constructs can be used in both the primary and boosting protocols, except that the RNA constructs used in the primary and boosting protocols encode SARS-CoV-2S proteins (or immunogenic portions thereof) of different SARS-CoV-2 strains (e.g., as described herein).

In some embodiments, when BNT162b2 is used in the primary or boost regimen, but not both, and in another regimen a different RNA is used, such different RNA can be an RNA encoding the same SARS-CoV-2S protein but with a different codon optimization or other different RNA sequence. In some embodiments, such different RNAs can encode different SARS-CoV-2 strains, such as SARS-CoV-2S protein (or immunogenic portions thereof) of the variant strains discussed herein. In some such embodiments, such variant strains are prevalent or rapidly spread in the relevant jurisdiction. In some embodiments, such different RNAs may be RNAs encoding SARS-CoV-2S protein or variants thereof (or immunogenic portions of either) comprising one or more mutations of an S protein variant described herein, such as a SARS-CoV-2S protein variant, particularly a naturally occurring S protein variant; in some such embodiments, the SARS-CoV-2 variant can be selected from the group consisting of VOC-202012/01, 501.V2, cluster 5 and B.1.1.248. In some embodiments, the SARS-CoV-2 variant can be selected from the group consisting of VOC-202012/01, 501.V2, cluster 5, and B.1.1.248, B.1.1.7, B.1.617.2, and B.1.1.529. In some embodiments, the boosting regimen comprises at least one dose of RNA encoding a SARS-CoV-2S protein (or immunogenic fragment thereof) that is a variant that rapidly spreads in the relevant jurisdiction upon administration. In some such embodiments, the variant encoded by RNA administered in the boosting regimen may be different from the variant encoded by RNA administered in the primary regimen.

In some embodiments, the boosting regimen comprises administering (i) a dose of RNA encoding the same SARS-CoV-2S protein (or immunogenic fragment thereof) as the RNA administered in the primary regimen (e.g., RNA encoding the SARS-CoV-2S protein (or immunogenic fragment thereof) from the SARS-CoV-2MN908947 strain), and (ii) a dose of RNA encoding the SARS-CoV-2S protein (or immunogenic fragment thereof) that is a variant that rapidly propagates in the relevant jurisdiction upon administration (e.g., the SARS-CoV-2S protein (or immunogenic fragment thereof) from one of the SARS-CoV-2 variants discussed herein).

In some embodiments, the boosting regimen includes multiple doses (e.g., at least two doses, at least three doses, or more). For example, in some embodiments, a first dose of the boosting regimen may comprise RNA encoding the same SARS-CoV-2S protein (or immunogenic fragment thereof) administered in the primary regimen, and a second dose of the boosting regimen may comprise RNA encoding a SARS-CoV-2S protein that rapidly propagates variants in the relevant jurisdiction upon administration. In some embodiments, a first dose of the boosting regimen may comprise RNA encoding a SARS-CoV-2S protein (or immunogenic fragment thereof) that is a variant that rapidly propagates in the relevant jurisdiction upon administration, and a second dose of the boosting regimen may comprise RNA encoding the same SARS-CoV-2S protein (or immunogenic fragment thereof) administered in the primary regimen. In some embodiments, the boosting regimen includes multiple doses, and RNA encoding the S protein of the variant that rapidly propagates in the relevant jurisdiction is administered in a first dose, and RNA encoding the S protein administered in the primary regimen is administered in a second dose.

In some embodiments, the doses in the boosting regimen (e.g., the first dose and the second dose or any two consecutive doses) are administered at least 2 weeks apart, including, for example, 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, at least 9 weeks apart, at least 10 weeks apart, at least 11 weeks apart, at least 12 weeks apart, at least 13 weeks apart, at least 14 weeks apart, at least 15 weeks apart, at least 16 weeks apart, or longer. In some embodiments, the doses in the boost regimen (e.g., the first dose and the second dose or any two consecutive doses) are administered about 2 to 168 weeks apart. In some embodiments, the doses in the boost regimen (e.g., the first dose and the second dose or any two consecutive doses) are administered about 3 to 12 weeks apart. In some embodiments, the doses in the boost regimen (e.g., the first dose and the second dose or any two consecutive doses) are administered about 4 to 10 weeks apart. In some embodiments, the doses in the boost regimen (e.g., the first dose and the second dose or any two consecutive doses) are administered about 6 to 8 weeks apart. (e.g., about 21 days apart, or about 6 to 8 weeks apart). In some embodiments, the first dose and the second dose are administered on the same day (e.g., by intramuscular injection at different sites in the subject).

In such embodiments, the boosting regimen may optionally further comprise a third dose and a fourth dose administered about 2 to 8 weeks after the first dose and the second dose (e.g., about 21 days after the first dose and the second dose, or about 6 weeks to about 8 weeks after the first dose and the second dose), wherein the third dose and the fourth dose are also administered on the same day (e.g., by intramuscular injection at different sites in the subject), and comprise the same RNA administered in the first dose and the second dose of the boosting regimen.

In some embodiments, multiple boosting regimens may be applied. In some embodiments, the boosting regimen is administered to a patient who has previously administered the boosting regimen.

In some embodiments, the second boosting regimen is administered to a patient who has previously received the first boosting regimen, and the amount of RNA administered in the at least one dose of the second boosting regimen is greater than the amount of RNA administered in the at least one dose of the first boosting regimen.

In some embodiments, the second boosting regimen comprises administering at least one dose of 3ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 5ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 10ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 15ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 20ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 25ug RNA. In some embodiments, the second boosting regimen includes administering at least one dose of 30ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 50ug RNA. In some embodiments, the second boosting regimen comprises administering at least one dose of 60ug RNA.

In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart and a boosting regimen comprising at least one dose of about 30ug of RNA are administered to the subject. In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart and a boosting regimen comprising at least one dose of about 50ug of RNA are administered to the subject. In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart and a boosting regimen comprising at least one dose of about 60ug of RNA are administered to the subject.

In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart, a first boosting regimen comprising at least one dose of about 30ug of RNA, and a second boosting regimen comprising at least one dose of about 30ug of RNA is administered to the subject. In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart, a first boosting regimen comprising at least one dose of about 30ug of RNA, and a second boosting regimen comprising at least one dose of about 50ug of RNA are administered to the subject. In some embodiments, a primary regimen comprising two doses of 30ug of RNA administered about 21 days apart, a first boosting regimen comprising at least one dose of about 30ug of RNA, and a second boosting regimen comprising at least one dose of about 60ug of RNA are administered to the subject. In some embodiments, the first boosting regimen comprises two doses of RNA, wherein each dose comprises RNA encoding spike protein from a different SARS-CoV-2 variant. In some embodiments, the first boosting regimen comprises two doses of RNA, wherein each dose comprises RNA encoding 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 RNA encoding a spike protein from a first SARS-CoV-2 variant with a second composition comprising RNA encoding a spike protein from a second SARS-CoV-2 variant).

In some embodiments, a boosting regimen is administered to a subject, the boosting regimen comprising a first dose comprising RNA encoding a spike protein from a strain of SARS-CoV-2MN908947 and a second dose comprising RNA encoding a spike protein comprising mutations that are prevalent and/or rapidly spread variants in the relevant jurisdiction upon administration of the boosting regimen, wherein the first dose and the second dose of RNA can be administered on the same day. In some embodiments, a boosting regimen is administered to a subject, the boosting regimen comprising a first dose comprising RNA encoding a spike protein from a strain of SARS-CoV-2MN908947 and a second dose comprising RNA encoding a spike protein comprising a mutation from a variant of SARS-CoV-2 a, wherein the first dose and the second dose can be administered on the same day. In some embodiments, a boosting regimen is administered to a subject, the boosting regimen comprising a first dose comprising RNA encoding a spike protein from a strain of SARS-CoV-2MN908947 and a second dose comprising RNA encoding a spike protein comprising a mutation from a variant of SARS-CoV-2 β, wherein the first dose and the second dose can be administered on the same day. In some embodiments, a boosting regimen is administered to a subject, the boosting regimen comprising a first dose comprising RNA encoding a spike protein from a strain of SARS-CoV-2MN908947 and a second dose comprising RNA encoding a spike protein comprising a mutation from a variant of SARS-CoV-2 delta, wherein the first dose and the second dose can be administered on the same day. In some embodiments, a boosting regimen is administered to a subject, the boosting regimen comprising a first dose comprising RNA encoding a spike protein from a strain of SARS-CoV-2MN908947 and a second dose comprising RNA encoding a spike protein comprising a mutation from a variant of SARS-CoV-2 Omicron, wherein the first dose and the second dose can be administered on the same day. Such booster dosing regimens may be administered, for example, 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 first boosting regimen comprising a first dose of 15ug of RNA encoding spike protein from a MN908947 variant and a second dose of 15ug of RNA encoding spike protein from a SARS-CoV-2 omacron variant is administered to a subject, wherein the first dose and the second dose are administered on the same day (e.g., wherein a composition comprising RNA is mixed prior to administration and then the mixture is administered to a patient). In some embodiments, a first boosting regimen comprising a first dose of 25ug of RNA encoding spike protein from a MN908947 variant and a second dose of 25ug of RNA encoding spike protein from a SARS-CoV-2 Omicron variant is administered to the subject. In some embodiments, the first dose and the second dose are optionally administered on the same day. In some embodiments, a first boosting regimen comprising a first dose of 25ug of RNA encoding spike protein from a MN908947 variant and a second dose of 25ug of RNA encoding spike protein from a SARS-CoV-2 Omicron variant is administered to the subject. In some embodiments, the first dose and the second dose are administered on the same day. In some embodiments, a first boosting regimen comprising a first dose of 30ug of RNA encoding spike protein from a MN908947 variant and a second dose of 30ug of RNA encoding spike protein from a SARS-CoV-2 omacron variant is administered to the subject, wherein the first dose and the second dose are optionally administered on the same day (e.g., administered separately or as a multivalent vaccine). In some embodiments, such a first boosting regimen is administered to a subject who has previously administered a primary regimen comprising two doses of 30ug RNA administered about 21 days apart, wherein the first boosting regimen is administered at least 3 months (e.g., at least 4, at least 5, or at least 6 months) after administration of the primary regimen.

In some embodiments, a second boost regimen comprising a first dose of 15ug of RNA encoding spike protein from the MN908947 variant and a second dose of 15ug of RNA encoding spike protein from the SARS-CoV-2Omicron variant is administered to the subject, wherein the first dose and the second dose are administered on the same day (e.g., wherein the RNA-containing composition is mixed to form a multivalent vaccine prior to administration, and then the mixture is administered to the patient). In some embodiments, a second boosting regimen comprising a first dose of 25ug RNA encoding spike protein from a MN908947 variant and a second dose of 25ug RNA encoding spike protein from a SARS-CoV-2 omacron variant is administered to the subject, wherein the first dose and the second dose are optionally administered on the same day (e.g., by administration of a multivalent vaccine or by administration of separate compositions). In some embodiments, the subject is administered a second boosting regimen comprising a first dose of 25ug of RNA encoding spike protein from a variant of MN908947 and a second dose of 25ug of RNA encoding spike protein from a variant of SARS-CoV-2 Omicron. In some embodiments, a second boosting regimen comprising a first dose of 30ug RNA encoding spike protein from the MN908947 variant and a second dose of 30ug RNA encoding spike protein from the SARS-CoV-2 omacron variant is administered to the subject, wherein the first dose and the second dose are optionally administered on the same day (e.g., by administration of a multivalent vaccine or by administration of separate compositions). In some embodiments, such a second boosting regimen is administered to a subject who has previously administered an initial regimen comprising two doses of 30ug RNA administered about 21 days apart. In some embodiments, such a second boosting regimen is administered to a subject who has previously administered a primary regimen comprising two doses of 30ug RNA administered about 21 days apart and a first boosting regimen comprising 30ug RNA, wherein the second boosting regimen is administered at least 3 months (e.g., at least 4, at least 5, or at least 6 months) after administration of the first boosting regimen.

In some embodiments, for example, after administration of one or more doses, one or more specific conditions of a patient receiving a dose of an RNA composition as described herein are monitored. In some embodiments, such conditions may be or include allergic reactions (particularly in subjects with associated allergies or history of allergic reactions), myocarditis (myocardial inflammation, particularly where the subject is a young man and/or may have undergone such inflammation), pericarditis (epicardial inflammation, particularly where the subject is a young man and/or may have undergone such inflammation), fever, bleeding (particularly where the subject is known to have a hemorrhagic disorder or is receiving treatment with a blood diluent). Alternatively or additionally, a patient that may be more closely monitored may be or include a patient who is immunocompromised or is receiving treatment with a drug that affects the immune system, is pregnant or is scheduled to become pregnant, is breast fed, has received another covd-19 vaccine, and/or has had syncope associated with injection. In some embodiments, the patient is monitored for myocarditis after administration of one of the compositions disclosed herein. In some embodiments, the patient is monitored for pericarditis after administration of one of the compositions disclosed herein. Current standard of care may be used to monitor and/or treat a patient's condition.

In some embodiments, the efficacy of an RNA (e.g., mRNA) composition described in the pediatric population (e.g., as described herein) can be assessed by various metrics described herein (including, for example, but not limited to, the incidence of COVID-19 per 1000 person-year in subjects without serological or virological evidence of past SARS-CoV-2 infection; geometric mean ratio of SARS CoV-2 neutralization titers (GMR) measured, for example, 7 days after the second dose; etc.)

In some embodiments, after administration of an RNA composition (e.g., mRNA) described herein, the pediatric population (e.g., 12 years to less than 16 years old) described herein may be monitored for the occurrence of multiple systemic inflammatory syndromes (MIS) (e.g., inflammation in different body parts such as, for example, heart, lung, kidney, brain, skin, eye, and/or gastrointestinal organs). Exemplary symptoms of child MIS may include, but are not limited to, fever, abdominal pain, vomiting, diarrhea, cervicodynia, rash, conjunctival congestion, oversensitive fatigue, and combinations thereof.

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP 020.3), BNT162b2 (RBP 020.1 or RBP 020.2), or BNT162b3 (e.g., BNT162b3 c). In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA) described herein as RBP 020.2. In one embodiment, the RNA encoding the vaccine antigen is a nucleoside modified messenger RNA (modRNA) described herein as BNT162b3 (e.g., BNT162b3 c).

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:21, a nucleotide sequence identical to SEQ ID NO:21, and/or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:5, or an amino acid sequence identical to SEQ ID NO:5, having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:21, a nucleotide sequence of seq id no; and/or (ii) encodes a polypeptide comprising SEQ ID NO:5, and a polypeptide sequence of the amino acid sequence of 5.

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:19 or 20, and SEQ ID NO:19 or 20, and/or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence comprising SEQ ID NO:7, or an amino acid sequence identical to SEQ ID NO:7, an amino acid sequence having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:19 or 20; and/or (ii) encodes a polypeptide comprising SEQ ID NO:7, and a sequence of amino acids of the amino acid sequence of 7.

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:20, and SEQ ID NO:20, and/or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO:7, or an amino acid sequence identical to SEQ ID NO:7, an amino acid sequence having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:20, a nucleotide sequence of 20; and/or (ii) encodes a polypeptide comprising SEQ ID NO:7, and a sequence of amino acids of the amino acid sequence of 7.

In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:30, a nucleotide sequence identical to SEQ ID NO:30, and/or (ii) a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence comprising SEQ ID NO:29, or an amino acid sequence that hybridizes to SEQ ID NO:29, has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical. In one embodiment, the RNA administered as described above is a nucleoside modified messenger RNA (modRNA), and (i) comprises the sequence of SEQ ID NO:30, a nucleotide sequence of seq id no; and/or (ii) encodes a polypeptide comprising SEQ ID NO:29, and a polypeptide comprising the amino acid sequence of 29.

In one embodiment, the RNA administered is a nucleoside modified messenger RNA (modRNA) that (i) comprises the amino acid sequence of SEQ ID NO:20, a nucleotide sequence of 20; and/or (ii) encodes a polypeptide comprising SEQ ID NO:7 and is administered in an amount of about 30 μg per dose. In one embodiment, at least two such doses are administered. For example, the second dose may be administered about 21 days after administration of the first dose.

In some embodiments, the population treated with RNA described herein comprises, consists essentially of, or consists of a subject having an age of at least 50, at least 55, at least 60, or at least 65. In some embodiments, the population treated with RNA described herein comprises, consists essentially of, or consists of subjects between 55 and 90, 60 and 85, or 65 and 85 in age.

In some embodiments, the period of time between doses administered is at least 7 days, at least 14 days, or at least 21 days. In some embodiments, the period of time between 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 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, and/or number of administrations) such that Adverse Events (AEs), i.e., any undesirable medical event in the patient, such as any adverse and unexpected signs, symptoms, or diseases associated with the use of a medical product, whether or not associated with a medical product, are mild or moderate in intensity. In some embodiments, the methods and agents described herein are administered such that Adverse Events (AEs) can be managed with interventions such as treatment with acetaminophen or other drugs (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin, ibuprofen, and naproxen) that provide analgesic, antipyretic (reduced fever), and/or anti-inflammatory effects. Acetaminophen or "acetaminophen," which is not classified as an NSAID, exerts a weak anti-inflammatory effect and may be administered as an analgesic of the present disclosure.

In some embodiments, the methods and agents described herein provide neutralization in a subject suffering from a coronavirus, a coronavirus infection, or a disease or disorder associated with a coronavirus.

In some embodiments, the methods and agents described herein induce an immune response that blocks or neutralizes coronavirus in a subject after administration to the subject. In some embodiments, the methods and agents described herein induce the production of antibodies, such as IgG antibodies that block or neutralize coronaviruses in a subject, upon administration to the subject. In some embodiments, the methods and agents described herein induce an immune response that blocks or neutralizes the coronavirus S protein that binds ACE2 in a subject after administration to the subject. In some embodiments, the methods and agents described herein induce the production of antibodies that block or neutralize ACE 2-binding coronavirus S protein in a subject after administration to the subject.

In some embodiments, the methods and agents described herein induce a Geometric Mean Concentration (GMC) of RBD domain-binding antibodies, such as IgG antibodies, of at least 500U/ml, 1000U/ml, 2000U/ml, 3000U/ml, 4000U/ml, 5000U/ml, 10000U/ml, 15000U/ml, 20000U/ml, 25000U/ml, 30000U/ml, or even higher after administration to a subject. In some embodiments, the elevated GMC of the RBD domain binding antibody lasts 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 induce a Geometric Mean Titer (GMT) of neutralizing antibodies, such as IgG antibodies, of at least 100U/ml, 200U/ml, 300U/ml, 400U/ml, 500U/ml, 1000U/ml, 1500U/ml, or even higher after administration to a subject. In some embodiments, the elevated GMT of the neutralizing antibody lasts 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 a binding agent, such as an antibody, binds to a biologically active site of a virus, such as a receptor binding protein, thereby inhibiting viral infection of a cell. As used herein, the term "neutralization" with respect to coronaviruses, particularly coronavirus S proteins, refers to an event in which a binding agent, such as an antibody, binds to the RBD domain of the S protein, thereby inhibiting viral infection of a cell. In particular, the term "neutralization" refers to an event in which the binding agent eliminates or significantly reduces the virulence (e.g., the ability to infect cells) of the virus of interest.

The type of immune response generated in response to antigen challenge can generally be distinguished by the subpopulations of helper T (Th) cells that are involved in the response. Immune responses can be broadly divided into two types: th1 and Th2.Th1 immune activation is optimal for intracellular infections such as viruses, while Th2 immune response is optimal for humoral (antibody) response. Th1 cells produce interleukin 2 (IL-2), tumor necrosis factor (TNF. Alpha.) and interferon gamma (IFN. Gamma.). Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13.Th1 immune activation is most desirable in many clinical situations. Vaccine compositions that specifically elicit Th2 or humoral immune responses are generally ineffective against most viral diseases.

In some embodiments, the methods and agents described herein induce or promote a Th 1-mediated immune response in a subject after administration to the subject. In some embodiments, the methods and agents described herein induce or promote a cytokine profile typical of a Th 1-mediated immune response in a subject after administration to the subject. In some embodiments, the methods and agents described herein induce or promote the production of interleukin 2 (IL-2), tumor necrosis factor (tnfα), and/or interferon gamma (ifnγ) in a subject following administration to the subject. In some embodiments, the methods and agents described herein induce or promote the production of interleukin 2 (IL-2) and interferon gamma (IFNgamma) in a subject following administration to the subject. In some embodiments, the methods and agents described herein do not induce or promote a Th 2-mediated immune response in a subject after administration to a subject, or induce or promote a Th 2-mediated immune response to a significantly lower extent in a subject than a Th 1-mediated immune response. In some embodiments, the methods and agents described herein do not induce or promote a cytokine profile typical of a Th 2-mediated immune response in a subject after administration to a subject, or induce or promote a cytokine profile typical of a Th 2-mediated immune response in a subject to a significantly lower extent than a cytokine profile typical of a Th 1-mediated immune response. In some embodiments, the methods and agents described herein do not induce or promote IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13 production in a subject after administration to a subject, or induce or promote IL-4, IL-5, IL-6, IL-9, IL-10, and/or IL-13 production in a subject to a significantly lower extent than induction or promotion of interleukin 2 (IL-2), tumor necrosis factor (TNF. Alpha.) and/or interferon gamma (IFN. Gamma.) in a subject. In some embodiments, the methods and agents described herein do not induce or promote IL-4 production after administration to a subject, or induce or promote IL-4 production to a significantly lesser extent in a subject than does interleukin 2 (IL-2) and interferon gamma (IFNgamma) in a subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets a diverse set of S protein variants such as SARS-CoV-2S protein variants, particularly naturally occurring S protein variants. In some embodiments, the set 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 include variants having amino acid modifications in the RBD domain and/or variants having amino acid modifications outside of the RBD domain. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof of S. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 321 (q) is L. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof of amino acid position 341 (V) in 1. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 348 (a) is a naturally occurring variant thereof of T. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 354 (N) is D. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 359 (S) is a naturally occurring variant thereof of N. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 367 (V) is F. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof of S. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 378 (K) is R. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 408 (R) is a naturally occurring variant thereof of I. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 409 (Q) is a naturally occurring variant thereof of E. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 435 (a) is a naturally occurring variant thereof of S. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 439 (N) is K. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 458 (K) is R. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 472 (I) is V. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof of S. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 477 (S) is N. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 483 (V) is a. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 508 (Y) is H. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1 is a naturally occurring variant thereof in which the amino acid at position 519 (H) is P. In one embodiment, such S protein variants comprise SARS-CoV-2S protein or a variant thereof corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is a naturally occurring variant thereof of G.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, a naturally occurring S protein variant comprising a mutation at position 501 (N). In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y.

Said sequence corresponding to SEQ ID NO: the S protein variant comprising a mutation at position 501 (N) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted.

In some embodiments, the methods and agents described herein induce a VOC-202012/01-targeting antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: deletions 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets 501.v2 after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: d80A, D215G, E484K, N501Y and a701V, and optionally: L18F, R246I, K417N and deletions 242-244. The S protein variant may also be found in a polypeptide corresponding to SEQ ID NO:1 comprises a D- > G mutation at position 614.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1 comprising a deleted naturally occurring S protein variant at positions 69 (H) and 70 (V).

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variant comprising a deletion at positions 69 (H) and 70 (V) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets "Cluster 5" after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: deletions 69-70, Y453F, 1692V, M1229I and optionally S1 147L.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, comprising a mutant naturally occurring S protein variant at position 614 (D). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variant comprising a mutation at position 614 (D) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: d80A, D215G, E484K, N501Y, D614G and a701V, and optionally: L18F, R246I, K417N and deletions 242-244.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1 (D) and 614 (D) comprises a mutated naturally occurring S protein variant at position 501 (N) and 614 (D). In one embodiment, the sequence corresponding to SEQ ID NO:1 is Y and corresponds to the amino acid at position 501 (N) in SEQ ID NO:1, the amino acid at position 614 (D) is G.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variants comprising mutations at positions 501 (N) and 614 (D) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ id no:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, a naturally occurring S protein variant comprising a mutation at position 484 (E). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variant comprising a mutation at position 484 (E) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is T. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets "b.1.1.28" after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets "b.1.1.248" after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: l18F, T20N, P S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T1027I and V1176F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, and positions 501 (N) and 484 (E) in 1, comprises a mutated naturally occurring S protein variant. In one embodiment, the sequence corresponding to SEQ ID NO:1 is Y and corresponds to the amino acid at position 501 (N) in SEQ ID NO:1, the amino acid at position 484 (E) is K.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variants comprising mutations at positions 501 (N) and 484 (E) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is T. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1 (a), 484 (E), and 614 (D) at positions 501 (N), 484 (E), and 614 (D) in 1. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 501 (N) is Y, corresponding to SEQ ID NO:1 is K and corresponds to amino acid 484 (E) in SEQ ID NO:1, the amino acid at position 614 (D) is G.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variants comprising mutations at positions 501 (N), 484 (E) and 614 (D) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is T. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: d80A, D215G, E484K, N501Y, A V and D614G, and optionally: L18F, R246I, K417N and deletions 242-244.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, positions 242 (L), 243 (a) and 244 (L) comprise deleted naturally occurring S protein variants.

In some embodiments, at a position corresponding to SEQ ID NO: the S protein variants comprising deletions at positions 242 (L), 243 (a) and 244 (L) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is T. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: D80A, D215G, E484K, N501Y, A V and deletions 242-244, and optionally L18F, R246I and K417N. The S protein variant may also be found in a polypeptide corresponding to SEQ ID NO:1 comprises a D- > G mutation at position 614.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1, comprising a mutant naturally occurring S protein variant at position 417 (K). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 417 (K) is T.

In some embodiments, at a position corresponding to SEQ ID NO:1 (K), the S protein variant comprising a mutation at position 417 (K) in 1 may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 484 (E) is K. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant that is in a position corresponding to SEQ ID NO:1 comprises the following mutations at the positions: d80A, D215G, E484K, N501Y, A V and K417N, and optionally: L18F, R246I and deletions 242-244. The S protein variant may also be found in a polypeptide corresponding to SEQ ID NO:1 comprises a D- > G mutation at position 614.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject after administration to the subject that targets an S protein variant such as a SARS-CoV-2S protein variant, particularly at a position corresponding to SEQ ID NO:1 and 484 (E) and/or 501 (N) comprises mutated naturally occurring S protein variants at positions 417 (K) and 484 (E). In one embodiment, the sequence corresponding to SEQ ID NO:1 is N and corresponds to the amino acid at position 417 (K) of SEQ ID NO:1 is K and/or corresponds to the amino acid at position 484 (E) of SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1 is T and corresponds to amino acid 417 (K) in SEQ ID NO:1 is K and/or corresponds to the amino acid at position 484 (E) of SEQ ID NO: the amino acid at position 501 (N) in 1 is Y.

In some embodiments, at a position corresponding to SEQ ID NO:1 and 484 (E) and/or 501 (N), and/or the S protein variant comprising a mutation at position (S) 417 (K) and 484 (E) may comprise one or more additional mutations. Such one or more additional mutations may be selected from the group consisting of those corresponding to SEQ ID NOs: 1, a mutation at the position of: 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 sequence corresponding to SEQ ID NO:1, the amino acid at position 69 (H) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 70 (V) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 144 (Y) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 570 (a) is D. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 614 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 681 (P) is H. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 716 (T) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 982 (S) is a. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1118 (D) in 1 is H. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 80 (D) is A. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 215 (D) is G. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 701 (a) is V. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 18 (L) is F. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 246 (R) is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, amino acid at position 242 (L). In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 243 (a) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 244 (L) is deleted. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 453 (Y) in 1 is F. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 692 (I) in 1 is V. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1147 (S) in 1 is L. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid position 1229 (M) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 20 (T) is N. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 26 (P) is S. In one embodiment, the sequence corresponding to SEQ ID NO:1 and the amino acid at position 138 (D) is Y. In one embodiment, the sequence corresponding to SEQ ID NO:1, the amino acid at position 190 (R) is S. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 655 (H) in 1 is Y. In one embodiment, the sequence corresponding to SEQ ID NO: amino acid at position 1027 (T) in 1 is I. In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 1176 (V) in 1 is F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets omacron (b.1.1.529) variants after administration to the subject.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681K, P969 373K, P339 440K, P339K, P446K, P856K, P764K, P417K, P796 5297 954 5297K, P5297 981K, P477 496K, P478K, P498 493K, P484K, P375K, P505K, P143 del H69del, V70del, N211del, L212K, P214 EPE, G142K, P del, Y145del, L141del, Y144K, P145K, P142 del.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L F, S477N, G496S, T478K, Q498R, Q493R, E484A. The S protein variant may include a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: N501Y, S375F, Y505H, V143del, H69del, V70del, and/or may include the amino acid sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: n211del, L212I, ins2 14EPE, G142D, Y144del, Y145del. In some embodiments, the S protein variant may include a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, or all of the following mutations compared to 1: l141del, Y144F, Y145D, G142del.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1:

a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N K, G446S, S477N, T478K, E484A, Q493 496S, Q498R, N501Y, Y H, T547K, D614G, H655 to 679K, P681 764H, N796H, N856 954 5297 969K and L981F.

In some embodiments, the methods and agents described herein induce an antibody response, particularly a neutralizing antibody response, in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 1:

a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371 373P, S375F, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T7958 655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F.

In some embodiments, the methods and agents described herein induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in a subject that targets Omicron (b.1.1.529) variants after administration to the subject.

In some embodiments, the methods and agents described herein induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681K, P969 373K, P339 440K, P339K, P446K, P856K, P764K, P417K, P796 5297 954 5297K, P5297 981K, P477 496K, P478K, P498 493K, P484K, P375K, P505K, P143 del H69del, V70del, N211del, L212K, P214 EPE, G142K, P del, Y145del, L141del, Y144K, P145K, P142 del.

In some embodiments, the methods and agents described herein induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L F, S477N, G496S, T478K, Q498R, Q493R, E484A. The S protein variant may include a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: N501Y, S375F, Y505H, V143del, H69del, V70del, and/or may include the amino acid sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: n211del, L212I, ins214EPE, G142D, Y144del, Y145del. In some embodiments, the S protein variant may include a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, or all of the following mutations compared to 1: l141del, Y144F, Y145D, G142del.

In some embodiments, the methods and agents described herein induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1:

In some embodiments, the methods and agents described herein induce an immune response (cellular and/or antibody response, particularly a neutralizing antibody response) in a subject that targets an S protein variant comprising an amino acid sequence as set forth in SEQ ID NO: 1:

As used herein, the term "amino acid corresponding to position..is intended to mean an amino acid position number corresponding to an amino acid position number in a SARS-CoV-2S protein, in particular the amino acid sequence of SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1. The phrase "as with SEQ ID NO:1 is "equivalent to" a sequence corresponding to SEQ ID NO:1 is at the position below in 1. By binding to the SARS-CoV-2S protein, in particular SEQ ID NO:1, and the corresponding amino acid positions in other coronavirus S protein variants such as SARS-CoV-2S protein variants may be found. It is considered well known in the art how to align sequences or fragments thereof and thereby determine the position in the sequence corresponding to the amino acid position according to the present disclosure. Standard sequence alignment programs, such as ALIGN, clustalW or similar programs, may be used, typically by default.

In some embodiments, the set of different S protein variants targeted by the antibody response 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, N D, S359N, V367F, K378S, R, I, Q E, A435S, K458R, I, V, G476S, V483A, Y508H, H519P and D614G variants described above. In some embodiments, the set of different S protein variants targeted by the antibody response is selected from all S protein variants of the set consisting of the q321S, V341I, A348T, N354 359N, V367F, K378S, R408I, Q409E, A435S, K458 472V, G476S, V483A, Y508H, H519P and D614G variants described above.

In some embodiments, the set of different S protein variants targeted by the antibody response 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, N359D, S359N, V367F, K378R, R I, Q3749 439K, K458R, I472V, G476S, S474 3933A, Y508H, H519P and D614G variants described above. In some embodiments, the set of different S protein variants targeted by the antibody response is selected from all S protein variants of the set consisting of the q321L, V341I, A348T, N D, S359 described above, 367F, K378R, R408I, Q409E, A435S, N439K, K458R, I V, G476S, S477N, V483A, Y508H, H519P and D614G variants.

In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by the RNAs described herein, comprises one or more of the mutations described herein for an S protein variant, such as a SARS-CoV-2S protein variant, particularly a naturally occurring S protein variant. In one embodiment, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof as encoded by the RNAs described herein is encoded by a polypeptide corresponding to the sequence of SEQ ID NO:1, comprising a mutation at position 501 (N). In one embodiment, the sequence corresponding to SEQ ID NO: the amino acid at position 501 (N) in 1 is Y. In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof as encoded by the RNAs described herein comprises one or more mutations, such as all mutations, of the SARS-CoV-2S protein selected from the group consisting of the SARS-CoV-2 variant of VOC-202012/01, 501.V2, cluster 5 and B.1.1.248. In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by the RNAs described herein, is comprised in SEQ ID NO:1, an amino acid sequence having an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242 to 244, and a proline substitution at positions 986 and 987.

In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by an RNA described herein comprises an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681K, P969 373K, P339 440K, P339K, P446K, P856K, P764K, P417K, P796 5297 954 5297K, P5297 981K, P477 496K, P478K, P498 493K, P484K, P375K, P505K, P143 del H69del, V70del, N211del, L212K, P214 EPE, G142K, P del, Y145del, L141del, Y144K, P145K, P142 del. In some embodiments, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprising the mutation as encoded by the RNAs described herein comprises the amino acid sequence as set forth in SEQ ID NO:1, K986P and V987P.

In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by an RNA described herein comprises an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1: T547K, H655Y, D614G, N679K, P681H, N969K, S373P, S L, N440K, G339D, G446S, N856K, N764K, K417N, D796Y, Q954H, T95I, A67V, L F, S477N, G496S, T478K, Q498R, Q493R, E484A. The SARs-CoV-2S protein, variant or fragment may comprise a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: N501Y, S375F, Y505H, V143del, H69del, V70del, and/or may include the amino acid sequence as set forth in SEQ ID NO:1, at least 2, at least 3, at least 4, at least 5, or all of the following mutations compared to 1: n211del, L212I, ins214EPE, G142D, Y144del, Y145del. In some embodiments, the SARs-CoV-2S protein, variant or fragment can comprise a sequence as set forth in SEQ ID NO:1, at least 2, at least 3, or all of the following mutations compared to 1: l141del, Y144F, Y145D, G142del. In some embodiments, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprising the mutation as encoded by the RNAs described herein comprises the amino acid sequence as set forth in SEQ ID NO:1, K986P and V987P.

In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by an RNA described herein comprises an amino acid sequence as set forth in SEQ ID NO: 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 compared to 1:

a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N K, G446S, S477N, T478K, E484A, Q493 496S, Q498R, N501Y, Y H, T547K, D614G, H655 to 679K, P681 764H, N796H, N856 954 5297 969K and L981F. In some embodiments, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprising the mutation as encoded by the RNAs described herein comprises the amino acid sequence as set forth in SEQ ID NO:1, K986P and V987P.

In some embodiments, for example, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof, as encoded by an RNA described herein comprises an amino acid sequence as set forth in SEQ ID NO: 1:

In some embodiments, the SARS-CoV-2S protein, immunogenic variant thereof, or immunogenic fragment of the SARS-CoV-2S protein or immunogenic variant thereof comprising the mutation as encoded by the RNAs described herein comprises the amino acid sequence as set forth in SEQ ID NO:1, K986P and V987P.

a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N, 440K, G446S, S477N, T478K, E484 353R, G496S, Q498R, N501Y, Y547Y, Y655 and 655 679Y, Y681 764Y, Y796Y, Y856Y, Y954 5297 981Y, Y986P and V987P.

a67V, Δ69-70, T95 4815D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484 35A, Q493R, G496S, Q498R, N501Y, Y505H, T547 5483 614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981 5297 986P and V987P.

In some embodiments, the thorn mutation in the omacron ba.2 variant comprises a mutation as set forth in SEQ ID NO: T19I, Δ24-26, a27S, G142D, V, G, G, 339D, S, 371, F, S373, P, S, 375, F, T, A, D, 405, 408, S, K, 417, N, N, 440, 477, N, T, 478, K, E, 484, 493, 4982, R, N, 501, Y, Y, H, D, 679, H, D, 681, 764, H, D, 796, 5297, 969, H, D, 986P, and V987P compared to 1.

T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614H, D655 679H, D681H, D764H, D796 954 5297 969 52986P and V987P. In some embodiments, administration of a variant specific vaccine (e.g., a variant specific vaccine disclosed herein) can produce an improved immune response in a patient as compared to administration of a vaccine encoding or comprising SARS-CoV-2S protein, or an immunogenic fragment thereof, from the MN908947 strain. In some embodiments, administration of the variant-specific vaccine can result in inducing a broader immune response in the subject (e.g., inducing a stronger neutralization response against a greater number of SARS-CoV-2 variants and/or recognizing an epitope in a greater number of SARS-CoV-2 variants) than a patient administered a vaccine comprising or encoding the SARS-CoV-2S protein (or immunogenic fragment thereof) from the MN908947 strain. In particular embodiments, a broader immune response can be induced when the variant-specific vaccine is administered in combination with a vaccine comprising or encoding a SARS-CoV-2S protein from a different variant or from a strain of MN908947 (e.g., in some embodiments, a broader immune response can be induced when the variant-specific vaccine is administered in combination with a vaccine comprising or encoding a SARS-CoV-2S protein from a strain of MN908947 or a vaccine comprising or encoding a SARS-CoV-2S protein comprising different mutant characteristics of the SARS-CoV-2 variant). For example, when an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain is administered in combination with an RNA vaccine encoding SARS-CoV-2S protein having the characteristics of an Omacron variant mutation, a broader immune response can be induced. In another embodiment, a broader immune response can be induced when an RNA vaccine encoding a SARS-CoV-2S protein comprising one or more delta variant mutation characteristics is administered in combination with an RNA vaccine encoding a SARS-CoV-2S protein comprising one or more omacron variant mutation characteristics. In such embodiments, a "broader" immune response can be defined relative to a patient administered a vaccine comprising or encoding a SARS-CoV-2S protein from a single variant (e.g., an RNA vaccine encoding a SARS-CoV-2S protein from a MN908947 strain). Vaccines comprising or encoding the S protein or immunogenic fragments thereof from different SARS-CoV-2 variants may be administered in combination by administration at different time points (e.g., a vaccine encoding the SARS-CoV-2S protein from the MN908947 strain and a vaccine encoding the SARS-CoV-2S protein having one or more variant strain mutation characteristics, e.g., both administered as part of a primary regimen or as part of a boosting regimen, or one administered as part of a primary regimen and the other administered as part of a boosting regimen). In some embodiments, vaccines comprising or encoding the S protein or immunogenic fragment thereof from different SARS-CoV-2 variants can be administered in combination by administering a multivalent vaccine (e.g., a composition comprising RNA encoding the SARS-CoV-2S protein from the MN908947 strain and RNA encoding the SARS-CoV-2S protein having the mutant characteristics of an omacron variant). In some embodiments, variant-specific vaccines can induce an excellent immune response against the variant to which the vaccine is specifically designed to immunize (e.g., induce a higher concentration of neutralizing antibodies), as well as an immune response against one or more other variants. In some such embodiments, the immune response against other variants may be comparable to or higher than that observed with vaccines encoding or comprising SARS-CoV-2S protein from the MN908947 strain.

In some embodiments, the Geometric Mean Ratio (GMR) or geometric mean fold increase (GMFR) of the variant-specific vaccine-induced neutralizing antibodies is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, or 2.0-fold (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) higher than the GMR or GMFR of the non-variant-specific vaccine-induced neutralizing antibodies (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, an immunogenic fragment of a SARS-CoV-2S protein, immunogenic variant thereof, or SARS-CoV-2S protein or immunogenic variant thereof, comprising the mutation, e.g., as encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:49, amino acid sequence identical to SEQ ID NO:49, or an amino acid sequence having at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85% or 80% identity to SEQ ID NO:49, or an amino acid sequence that hybridizes to SEQ ID NO:49, an immunogenic fragment of 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. In some embodiments, an immunogenic fragment of a SARS-CoV-2S protein, immunogenic variant thereof, or SARS-CoV-2S protein or immunogenic variant thereof, comprising the mutation, e.g., as encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:49, and a sequence of amino acids.

In some embodiments, an immunogenic fragment of a SARS-CoV-2S protein, immunogenic variant thereof, or SARS-CoV-2S protein or immunogenic variant thereof, comprising the mutation, e.g., as encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:52, amino acid sequence identical to SEQ ID NO:52 has an amino acid sequence that is at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an amino acid sequence of SEQ ID NO:52, or an amino acid sequence that hybridizes to SEQ ID NO:52 has an amino acid sequence that is at least 99.5%, 99%, 98.5%, 98%, 98.5%, 97%, 96%, 95%, 90%, 85% or 80% identical. In some embodiments, an immunogenic fragment of a SARS-CoV-2S protein, immunogenic variant thereof, or SARS-CoV-2S protein or immunogenic variant thereof, comprising the mutation, e.g., as encoded by an RNA described herein, comprises the amino acid sequence of SEQ ID NO:52, and an amino acid sequence of seq id no.

In some embodiments, the methods and reagents described herein (e.g., RNA (e.g., mRNA) compositions) induce a cell-mediated immune response (e.g., cd4+ and/or cd8+ T cell response) upon administration to a subject. In some embodiments, T cells are induced that recognize one or more epitopes (e.g., MHC class I restricted epitopes) selected from the group consisting of: LPFNDGVYF, GVYFASTEK, YLQPRTFLL, QPTESIVRF, CVADYSVLY, KCYGVSPTK, NYNYLYRLF, FQPTNGVGY, IPFAMQMAY, RLQSLQTYV, GTHWFVTQR, VYDPLQPEL, QYIKWPWYI and KWPWYIWLGF. In one embodiment, T cells recognizing epitope YLQPRTFLL are induced. In one embodiment, T cells recognizing epitope NYNYLYRLF are induced. In one embodiment, T cells recognizing epitope QYIKWPWYI are induced. In one embodiment, T cells recognizing epitope KCYGVSPTK are induced. In one embodiment, T cells recognizing epitope RLQSLQTYV are induced. In some embodiments, the methods and reagents described herein (e.g., RNA (e.g., mRNA) compositions) are administered according to a regimen that achieves induction of such T cells.

In some embodiments, the methods and agents described herein (e.g., RNA (e.g., mRNA) compositions) induce a cell-mediated immune response (e.g., cd4+ and/or cd8+ T cell response) after administration to a subject 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 RNA described herein (wherein the second dose can be administered about 21 days after administration of the first dose). In some embodiments, the methods and reagents described herein (e.g., RNA (e.g., mRNA) compositions) are administered according to a regimen that achieves induction of such cell-mediated immune responses.

In one embodiment, vaccination against coronaviruses described herein, e.g., using RNAs described herein, the RNAs may be administered in amounts and regimens described herein, e.g., at two doses of 30 μg/dose, e.g., 21 days apart, may be repeated after a period of time using the same or different vaccine as the first vaccination, e.g., once a protective attenuation against coronavirus infection is observed. Such a 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 with a lower frequency of administration. For example, a first vaccination may comprise a vaccination with a dose of about 30 μg/dose, wherein in one embodiment at least two such doses are administered (e.g., a second dose may be administered about 21 days after administration of the first dose) and a second or further vaccination may comprise a vaccination with a dose of less than about 30 μg/dose, wherein in one embodiment only one such dose is administered. In one embodiment, RNA that is different from that 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 with at least two doses of an RNA described herein, e.g., two doses of an RNA described herein (wherein a second dose may be administered about 21 days after administration of the first dose), and a second vaccination with a single dose or multiple doses, e.g., two doses, of an 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 the administration of the first vaccination, e.g., after the initial double dose regimen. The amount of RNA used in each dose of the second vaccination may be the same as or different from 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/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 the second vaccination is BNT162b2.

In some embodiments, when the RNA used for the first vaccination and the second vaccination is BNT162b2, the purpose is to induce an immune response targeting SARS-CoV-2 variants, including but not limited to Omicron (b.1.1.529) variants. Thus, in some embodiments, when the RNA used for the first vaccination and the second vaccination is BNT162b2, the purpose is to protect the subject from infection with SARS-CoV-2 variants, including but not limited to Omicron (b.1.1.529) variants.

In one embodiment, RNA that is different from that 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 an RNA encoding SARS-CoV-2S protein of a variant strain of SARS-CoV-2 (e.g., a strain as discussed herein). In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is an RNA encoding SARS-CoV-2S protein of a SARS-CoV-2 variant strain that is prevalent or rapidly transmitted during 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 SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein or an immunogenic variant thereof comprising one or more of the mutations described herein for an S protein variant (such as a SARS-CoV-2S protein variant, in particular a naturally occurring S protein variant). In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is an RNA encoding SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of SARS-CoV-2S protein or an immunogenic variant thereof, said immunogenic variant comprising one or more mutations, such as all mutations of SARS-CoV-2S protein of a SARS-CoV-2 variant selected from the group consisting of VOC-202012/01, 501.V2, cluster 5, B.1.1.248 and Omicron (B.1.1.529).

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and the amino acid sequence substituted with a proline residue at positions 986 and 987 of SEQ ID NO: an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242 to 244, and a proline substitution at positions 986 and 987.

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 and the amino acid sequence substituted with a proline residue at positions 986 and 987 of SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

And SEQ ID NO:1, a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L2121, ins214EPE, G339D, S371L, S373P, S375F, K417N, N K, G446S, S4783 478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614 3563 655 679Y, N681Y, N764Y, N796Y, N856Y, N954 5297 969Y, N981 5297P and V987P.

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, the following mutations in 1: in SEQ ID NO:1 and the RNA used for the second vaccination is an RNA encoding a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

and SEQ ID NO: in contrast to the 1-degree of freedom, T19I, delta24-26 a27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D655H, D679 681H, D764H, D954H, D969H, D986P and V987P.

And SEQ ID NO:1, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498 82 501Y, Y505H, D614H, D655H, D679H, D681 764H, D796 5297 954 5297 986P.

In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

the RNA used for the second vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

And SEQ ID NO:1, T19I, Δ24-26, a27S, G142D, V, G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440, 477N, T478K, E484A, Q493R, Q R, N501Y, Y505H, D655 679H, D681H, D764H, D796 5297 954 5297 969 52986P and V987P, the RNA used for the second vaccination encodes a polypeptide comprising a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 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 a dna encoding a dna comprising SEQ ID NO:49, and a polypeptide having the amino acid sequence of seq id no.

In one embodiment, the RNA used for the first vaccination comprises SEQ ID NO:20, and the RNA used for the second vaccination is a dna comprising the nucleotide sequence of SEQ ID NO:51, and a nucleotide sequence of a nucleic acid sequence of seq id no.

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 a dna encoding a dna comprising SEQ ID NO: 55. 58 or 61, and a polypeptide of amino acid sequence of 58 or 61.

In one embodiment, the RNA used for the first vaccination comprises SEQ ID NO:20, and the RNA used for the second vaccination is a dna comprising the nucleotide sequence of SEQ ID NO: 57. 60 or 63 a.

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 a dna encoding a dna comprising SEQ ID NO: 49. 55 or 61, and a polypeptide having the amino acid sequence of 55 or 61.

In one embodiment, the RNA used for the first vaccination comprises SEQ ID NO:60, and the RNA used for the second vaccination is a dna comprising the nucleotide sequence of SEQ ID NO: 51. 57 or 63 a.

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 a dna encoding a dna comprising SEQ ID NO:55 or 61, and a polypeptide having the amino acid sequence of 55 or 61.

In one embodiment, the RNA used for the first vaccination comprises SEQ ID NO:51, and the RNA used for the second vaccination is a dna comprising SEQ ID NO:57 or 63 a.

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 a dna encoding a dna comprising SEQ ID NO:61, and a polypeptide of amino acid sequence.

In one embodiment, the RNA used for the first vaccination comprises SEQ ID NO:57, and the RNA used for the second vaccination is a dna comprising the nucleotide sequence of SEQ ID NO:63 a.

and SEQ ID NO:1, a67V, Δ69-70, T95 48135D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, S4775 478K, E484A, Q493 496S, Q498R, N501Y, Y H, T547K, D614G, H655Y, N679K, P79681H, N764K, D796Y, N856 35K, Q954H, N969K, L981 5297 986P and V987P. In some embodiments, the RNA encoding polypeptide used for the second vaccination is further comprised in a polypeptide corresponding to SEQ ID NO: proline residue substitutions at positions 986 and 987 of 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 a dna encoding a dna comprising SEQ ID NO:52, and a polypeptide having the amino acid sequence of seq id no.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one dose of 50 μg of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one dose of 60 μg of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:49, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen comprising at least two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least two 30 μg doses 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 boosting 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 as a booster dose a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one dose of 50 μg of RNA comprising a nucleotide sequence encoding SEQ ID NO:49, wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, said boosting regimen comprising at least one dose of 60 μg of RNA comprising a nucleotide sequence encoding SEQ ID NO:49, wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In some embodiments, a primary regimen is administered to a subject, the primary regimen comprising two doses of 30 μg of RNA (e.g., administered about 21 days after each other), wherein each 30 μg dose of RNA comprises 15 μg of RNA comprising a sequence encoding SEQ ID NO:7 and 15 μg of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, and a nucleotide sequence of the amino acid sequence of seq id no. In some embodiments, such a primary regimen is administered to a non-vaccinated subject.

In some embodiments, a primary regimen is administered to a subject, the primary regimen comprising two doses of 30 μg of RNA (e.g., administered about 21 days after each other), wherein each 30 μg dose of RNA comprises a sequence encoding SEQ ID NO:49, and a nucleotide sequence of an amino acid sequence of seq id no. In some embodiments, such a primary regimen is administered to a non-vaccinated subject.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting regimen comprises at least one 30 μg dose of RNA, wherein the 30 μg of RNA comprises 15 μg of RNA comprising a nucleotide sequence encoding SEQ ID NO:7 and 15 μg of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, wherein both RNAs are optionally administered in the same composition, and wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting regimen comprises at least one dose of 50 μg of RNA, wherein the 50 μg of RNA comprises 25 μg of RNA comprising a nucleotide sequence encoding SEQ ID NO:7 and 25 μg of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, wherein both RNAs are optionally administered in the same composition (e.g., a formulation comprising both RNAs), and wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, a primary regimen and a boosting regimen are administered to a subject, the primary regimen comprising two 30 μg doses of RNA comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:7, wherein the boosting regimen comprises at least one dose of 60 μg of RNA, wherein the 60 μg of RNA comprises 30 μg of RNA comprising a nucleotide sequence encoding SEQ ID NO:7 and 30 μg of RNA comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:49, wherein both RNAs are optionally administered in the same composition, and wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg 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 SEQ ID NO:20, and the RNA used for the second vaccination is a dna comprising the nucleotide sequence of SEQ ID NO:54, and a nucleotide sequence of 54.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA comprising the nucleotide sequence of SEQ ID NO:20, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one dose of 50 μg of RNA comprising the nucleotide sequence of SEQ ID NO:20, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one dose of 60 μg of RNA comprising the nucleotide sequence of SEQ ID NO:20, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA comprising the nucleotide sequence of SEQ ID NO:51, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA, wherein said 30 μg of RNA comprises 15 μg of a nucleotide sequence comprising SEQ ID NO:20 and 15 μg of RNA comprising the nucleotide sequence of SEQ ID NO:51, wherein the two RNAs are optionally administered in the same composition, and wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one RNA comprising a dose of 50 μg, wherein said 50 μg RNA comprises 25 μg of a nucleic acid comprising SEQ ID NO:20 and 25 μg of RNA comprising the nucleotide sequence of SEQ ID NO:51, wherein the two RNAs are optionally administered in the same composition, and wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one RNA comprising a dose of 60 μg, wherein said 60 μg RNA comprises 30 μg of a nucleic acid comprising SEQ ID NO:20 and 30 μg of an RNA comprising the nucleotide sequence of SEQ ID NO:51, wherein the two RNAs are optionally administered in the same composition, and wherein the boosting 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 initial regimen, and wherein the subject optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA comprising the nucleotide sequence of SEQ ID NO:57, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA comprising the nucleotide sequence of SEQ ID NO:60, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one 30 μg dose of RNA comprising the nucleotide sequence of SEQ ID NO:63a, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:20, said boosting regimen comprising at least one dose of 50 μg of RNA comprising the nucleotide sequence of SEQ ID NO:63a, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid sequence of SEQ ID NO: 20.

In one embodiment, a primary regimen comprising two 30 μg doses of RNA comprising the amino acid sequence of SEQ ID NO:63a, said boosting regimen comprising at least one dose of 60 μg of RNA comprising the nucleotide sequence of SEQ ID NO:57, wherein the boosting 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 optionally has previously administered a first boosting regimen comprising a 30 μg dose of RNA comprising the amino acid 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 the amino acid sequence set forth in SEQ ID NO:1 and at positions 986 and 987, said two doses being administered about 21 days apart, said second vaccination using a single dose or multiple doses of RNA encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and at positions 986 and 987, said single dose or multiple doses being administered after administration of the first vaccination, i.e. about 4 to 12 months, 5 to 12 months or 6 to 12 months after the initial two dose regimen. In one embodiment, each RNA dose comprises 30 μg of RNA. In this embodiment, the aim of one embodiment is to induce an immune response targeting SARS-CoV-2 variants, including but not limited to omacron (b.1.1.529) variants. Thus, in this embodiment, the object of one embodiment is to protect a subject from infection by a variant of SARS-CoV-2, including but not limited to the Omacron (B.1.1.529) variant.

In one embodiment, the vaccination regimen comprises a first vaccination using two doses of RNA encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and at positions 986 and 987, said two doses being administered about 21 days apart, said second vaccination using a single dose or multiple doses of RNA encoding a polypeptide comprising a polypeptide having a sequence set forth in SEQ ID NO: an alanine substitution at position 80, a glycine substitution at position 215, a lysine substitution at position 484, a tyrosine substitution at position 501, a valine substitution at position 701, a phenylalanine substitution at position 18, an isoleucine substitution at position 246, an asparagine substitution at position 417, a glycine substitution at position 614, a deletion at positions 242 to 244, and a proline substitution at positions 986 and 987 of 1, said single dose or multiple doses being administered after administration of the first vaccination, i.e. about 6 to 12 months after the first two doses regimen. In one embodiment, each RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination using two doses of RNA encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and at positions 986 and 987, said two doses being administered about 21 days apart, said second vaccination using a single dose or multiple doses of RNA encoding a polypeptide comprising a polypeptide having a sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

and SEQ ID NO:1, a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y547Y, Y614Y, Y655 679Y, Y681 764Y, Y796Y, Y856Y, Y9537 5297 969Y, Y981Y, Y986P and V987P, for example, after administration of the first vaccination, i.e. about 6 to 12 months after the first two dose regimen. In one embodiment, each RNA dose comprises 30 μg of RNA.

And SEQ ID NO:1, a67V, Δ69-70, T95 48135D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, S477N, T478K, E484A, Q493 496S, Q498R, N Y, Y H, T547K, D614G, H655Y, N679K, P79681H, N764K, D796Y, N856 95182H, N969K, L981F, K986P and V987P, for example, after administration of the first vaccination, i.e. about 6 to 12 months after the first two dose regimen. In one embodiment, each RNA dose comprises 30 μg of RNA. In some embodiments, the encoded polypeptide is further comprised in a polypeptide corresponding to SEQ ID NO: proline residue substitutions at positions 986 and 987 of 1.

In one embodiment, the vaccination regimen comprises a first vaccination involving at least two doses of RNA encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and at positions 986 and 987, said two doses being administered about 21 days apart, said second vaccination involving a single dose or multiple doses of RNA encoding a polypeptide comprising a polypeptide having a sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

And SEQ ID NO:1, T19I, Δ24-26, a27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440 477N, T478K, E484A, Q493 4982R, N501Y, Y505H, D655 679H, D681 764H, D796 954 5297 969 5297 986P and V987P, for example, after administration of the first vaccination, i.e. about 6 to 12 months after the first two dose regimen. In one embodiment, each or at least one RNA dose comprises 30 μg of RNA.

and SEQ ID NO:1, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614H, D655 679H, D681 764H, D796 5297 954H, D969 5298P and V987P, for example, after administration of the first vaccination, i.e. about 6 to 12 months after the first two dose regimen. In one embodiment, each or at least one RNA dose comprises 30 μg of RNA.

In one embodiment, the vaccination regimen comprises a first vaccination involving at least two doses of RNA encoding a polypeptide comprising a polypeptide having a sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

a67V, Δ69-70, T95 37142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y547Y, Y614Y, Y655 679Y, Y681 764Y, Y796Y, Y856 954 969Y, Y981 986P 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 a polypeptide having a sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

and SEQ ID NO:1, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614H, D655 679H, D681 764H, D796 5297 954H, D969 5298P and V987P, for example, after administration of the first vaccination, i.e. about 6 to 12 months after the first two dose regimen. In one embodiment, each or at least one RNA dose comprises 30 μg RNA.

and SEQ ID NO:1, T19I, delta24-26, A27S, G142D, V, G, G, D, S, 371, F, S, 373, P, S, 375, F, T, A, D, N, R, 408, 417, N, N, 440, 477, N, T, 478, K, E, 484, 493, 4982, R, N, 501, Y, Y, 505, H, D, 655, 679, H, D, 681, H, D, 764, H, D, 796, 954, 5297, 969, 5297, 986P 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 a polypeptide having a sequence set forth in SEQ ID NO:1, the amino acid sequence of the following mutation in 1:

In one embodiment, a vaccination regimen comprises (i) a first vaccination comprising at least three doses of RNA described herein (e.g., wherein each dose comprises about 30 μg of RNA comprising the nucleotide sequence of SEQ ID NO: 20), wherein a second dose may be administered about 21 days after administration of the first dose, and a third dose may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second dose; and (ii) a second vaccination comprising at least one dose of RNA described herein (e.g., wherein each dose comprises about 30 μg of RNA per dose). In some embodiments, the second vaccination comprises at least one dose of a bivalent vaccine described herein, e.g., about 30 μg total of the bivalent vaccine, e.g., a bivalent vaccine (b2+ Omi) comprising about 15 μg of RNA encoding SARS-CoV-2S protein from Wuhan strain and about 15 μg of RNA encoding SARS-CoV-2S protein comprising a mutation characteristic of an Omicron variant. In some embodiments, the bivalent vaccine comprises about 15 μg of RNA encoding SARS-CoV-2S protein from the Wuhan strain and about 15 μg of RNA encoding SARS-CoV-2S protein comprising a mutation characteristic of the ba.1omicron variant (e.g., 15 μg of RNA comprising the sequence of SEQ ID NO:20 and 15 μg of RNA comprising the sequence of SEQ ID NO: 51). In some embodiments, the bivalent vaccine comprises about 15 μg of RNA encoding SARS-CoV-2S protein from the Wuhan strain and about 15 μg of RNA encoding SARS-CoV-2S protein comprising a mutation characteristic of the BA.4/5Omicron variant (e.g., 15 μg of RNA comprising the sequence of SEQ ID NO:20 and 15 μg of RNA comprising the sequence of SEQ ID NO: 72). In some embodiments, the vaccination regimen is administered to a subject at least about 12 years old. In some embodiments, the vaccination regimen is administered to a subject at least about 6 months old to less than about 12 years old.

In one embodiment, the second vaccination results in a boost of the immune response.

In one embodiment, the RNAs described herein are 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 epitope. In some embodiments, the RNA described herein is co-administered with one or more T cell epitopes or RNA encoding such epitopes, M protein derived from SARS-CoV-2, N protein, and/or ORF1ab protein, e.g., the compositions disclosed in WO2021188969, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the RNA described herein (e.g., RNA encoding SARS-CoV-2S protein comprising a mutation characteristic of the BA.1, BA.2 or BA.4/5 omacron variant, optionally administered with RNA encoding SARS-CoV-2S protein of the Wuhan variant) is co-administered with a T-string construct described in WO2021188969 (e.g., RNA encoding SEQ ID NO: RS C7p2full of WO 2021/188969). In some embodiments, the RNA described herein and the T-string construct described in WO2021188969 are administered in a combination of up to about 100 μg of RNA in total. In some embodiments, at least 2 doses of an RNA described herein (e.g., in some embodiments, 30 μg per dose) are administered to a subject in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO 2021/188969), e.g., an RNA described herein and an RNA encoding SEQ ID NO: the combination of RNA of RS C7p2full total up to about 100 μg of RNA, wherein the two doses are administered, for example, at least 4 weeks or longer (including, for example, 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 each other. In some embodiments, at least 3 doses of an RNA described herein (e.g., in some embodiments, 30 μg per dose) are administered to a subject in combination with a T-string construct (e.g., an RNA encoding SEQ ID NO: RS C7p2full of WO 2021/188969), e.g., an RNA described herein and an RNA encoding SEQ ID NO: the combination of RNA of the RS C7p2full total up to about 100 μg of RNA, wherein the first and second doses and the second and third doses are each independently administered at least 4 weeks or more (including, for example, 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 more) from each other. In some embodiments, the RNA and T-string constructs described herein can be co-administered as separate formulations (e.g., formulations administered to different injection sites on the same day). In some embodiments, the RNA and T-string constructs described herein can be co-administered as a co-formulation (e.g., a formulation comprising the RNA and T-string constructs described herein as a separate LNP formulation or as an LNP formulation comprising both the T-string construct and the RNA described herein).

In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against non-SARS-CoV-2 disease. In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against non-SARS-COV-2 viral diseases. In some embodiments, the RNA compositions described herein are co-administered with one or more vaccines against non-SARS-CoV-2 respiratory disease. In some embodiments, the non-SARS-CoV-2 respiratory disease is a non-SARS-CoV-2 coronavirus, influenza virus, pneumoviridae (Pneumoviridae) virus, or Paramyxoviridae (Paramyxoviridae) virus. In some embodiments, the pneumoviridae virus is respiratory syncytial virus or metapneumovirus. In some embodiments, the metapneumovirus is human metapneumovirus (hMPV). In some embodiments, the paramyxoviridae virus is a parainfluenza virus or henipav virus. In some embodiments, the parainfluenza virus is PIV3. In some embodiments, the non-SAR-CoV-2 coronavirus is a type B coronavirus (e.g., SARS-CoV-1). In some embodiments, the non-SARS-CoV-2 coronavirus is a Marbaceae virus (Merbecovims) (e.g., MERS-CoV virus).

In some embodiments, the RNA compositions described herein are co-administered with an RSV vaccine (e.g., an RSV a or RSV B vaccine). In some embodiments, the RSV vaccine comprises an RSV fusion protein (F), an RSV attachment protein (G), an RSV minihydrophobic protein (SH), an RSV matrix protein (M), an RSV nucleoprotein (N), an RSV M2-1 protein, an RSV macropolymerase (L), and/or an RSV phosphoprotein (P), or immunogenic fragments of their immunogenic variants, or a nucleic acid (e.g., RNA) encoding any of them.

In some embodiments, the RNA compositions described herein are co-administered with an influenza vaccine. In some embodiments, the influenza vaccine is an influenza a virus, an influenza b virus, an influenza y virus, or an influenza delta 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 the group consisting of 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 thereof, or a nucleic acid (e.g., RNA) encoding any of them. 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 thereof, or a nucleic acid (e.g., RNA) encoding any of them. In some embodiments, the influenza vaccine comprises at least one influenza virus Hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS 1), nonstructural protein 2 (NS 2), nuclear Export Protein (NEP), polymerase acidic Protein (PA), polymerase basic proteins PB1, PB1-F2, and/or polymerase basic protein 2 (PB 2), or immunogenic fragments or variants thereof, or a nucleic acid (e.g., RNA) encoding any of them.

In some embodiments, the RNA compositions provided herein and the other injectable vaccine or vaccines are administered at different times. In some embodiments, the RNA compositions provided herein are administered concurrently with other one or more injectable vaccines. In some such embodiments, the RNA composition provided herein and the at least one additional injectable vaccine are administered at different injection sites. In some embodiments, the RNA compositions provided herein are not mixed with any other vaccine in the same syringe. In some embodiments, the RNA compositions provided herein are not combined with other coronavirus vaccines as part of vaccination against coronaviruses (e.g., SARS-CoV-2).

The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is generally interpreted as a medical condition associated with a particular symptom and sign. The disease may be caused by exogenous factors, such as infectious diseases, or by internal dysfunction, such as autoimmune diseases. In humans, "disease" is generally used more broadly to direct the death of a painful, dysfunctional, painful, social or affected individual, or any condition that is similar to a problem for a person in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical variations in structure and function, while in other cases and for other purposes these may be considered distinguishable categories. Diseases often affect individuals not only physically, but also emotionally, because infection and suffering from many diseases can alter a person's opinion of life and a person's personality.

In the context of this document, the term "treatment (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 a full spectrum of treatments for a given condition to which a subject is exposed, such as administration of a therapeutically effective compound to alleviate symptoms or complications, delay of progression of the disease, disorder or condition, alleviate or relieve symptoms and complications, and/or cure or eliminate the disease, disorder or condition, as well as prevention of the condition, wherein prevention is understood to be the management and care of an individual for the purpose of combating the disease, disorder or condition, and includes administration of an active compound to prevent the onset of symptoms or complications.

The term "therapeutic treatment" relates to any treatment that improves the health condition and/or prolongs (increases) the life of an individual. The treatment may eliminate a disease in an individual, prevent or slow the progression of a disease in an individual, inhibit or slow the progression of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and/or reduce relapse in an individual who is currently suffering from or has previously suffered from a disease.

The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

The terms "individual" and "subject" are used interchangeably herein. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may or may not have a disease or disorder. In various embodiments, the subject is a human. Unless otherwise indicated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderly people, children, and newborns. In some embodiments, the term "subject" includes a human having an age of at least 50 years, at least 55 years, at least 60 years, at least 65 years, at least 70 years, or older. In some embodiments, the term "subject" includes a human having an age of at least 65 years, such as 65 to 80 years, 65 to 75 years, or 65 to 70 years. In embodiments of the present disclosure, an "individual" or "subject" is a "patient.

The term "patient" means an individual or subject for treatment, particularly an individual or subject suffering from a disease.

In one embodiment of the present disclosure, it is an object to provide an immune response against coronaviruses, and to prevent or treat coronavirus infections.

A pharmaceutical composition comprising RNA encoding a peptide or protein comprising an epitope may be administered to a subject to elicit an immune response in the subject against an antigen comprising the epitope, which immune response may be therapeutic or partially or fully protective. One skilled in the art will appreciate that one of the principles of immunotherapy and vaccination is based on the fact that: that is, an immunoprotection response against a disease may be generated by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease to be treated. Thus, the pharmaceutical compositions described herein are useful for inducing or enhancing an immune response. The pharmaceutical compositions described herein are therefore useful for the prophylactic and/or therapeutic treatment of diseases involving antigens or epitopes.

As used herein, "immune response" refers to a general 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 the more primitive innate immune system and the vertebrate acquired or adaptive immune system, each of which contains both humoral and cellular components.

"cell-mediated immunity", "cellular immune response" or similar terms are intended to include a cellular response to a cell characterized by expression of an antigen, in particular by presentation of an antigen with class I or class II MHC. Cellular responses involve immune effector cells, particularly cells known as T cells or T lymphocytes, which act as "helper" or "woundguard". Helper T cells (also known as CD4 ⁺ T cells) play a central role by modulating immune responses, killer cells (also known as cytotoxic T cells, cytolytic T cells, CD 8) ⁺ T cells or CTLs) kill diseased cells, such as virus-infected cells, preventing the production of more diseased cells.

Immune effector cells include any cell that is responsive to a vaccine antigen. Such reactivity 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 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 destruction of the target cell. For example, cytotoxic T cells trigger the destruction of target cells by either or both of the following. First, upon activation, T cells release cytotoxins such as perforins, granzymes, and granulysins. Perforin and granulysin create pores in the target cells, and granzyme enters the cells and triggers a caspase cascade in the cytoplasm, thereby inducing apoptosis (programmed cell death). Second, apoptosis can be induced by Fas-Fas ligand interaction between T cells and target cells.

In the context of the present disclosure, the term "effector function" includes any function mediated by components of the immune system that results in, for example, neutralization of pathogens (such as viruses) and/or killing of diseased cells (such as virus-infected cells). In one embodiment, in the context of the present disclosure, the effector function is a T cell mediated effector function. These functions include, for example, the expression of helper T cells (CD 4 ⁺ T cells), cytokine release and/or CD8 ⁺ Activation of lymphocytes (CTLs) and/or B cells, and, in the case of CTLs, elimination of cells, i.e., cells characterized by expression of an antigen, e.g., by apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN- γ and TNF- α, and specific cell lysis killing of target cells expressing the antigen.

In the context of the present disclosure, the term "immune effector cell" or "immune response cell" relates to a cell that performs an effector function during an immune response. In one embodiment, an "immune effector cell" is capable of binding an antigen, such as an antigen presented in the context of MHC on a cell or an antigen expressed on the surface of a cell and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in In the context of the present disclosure, an "immune effector cell" is a T cell, preferably CD4 ⁺ And/or CD8 ⁺ T cells, most preferably CD8 ⁺ T cells. According to the present disclosure, the term "immune effector cell" also includes cells that can mature into immune cells (such as T cells, in particular T helper cells, or cytolytic T cells) under appropriate stimulation. Immune effector cells include CD34 ⁺ Hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. Differentiation of T cell precursors to cytolytic T cells upon exposure to antigen is analogous to clonal selection of the immune system.

A "lymphoid cell" is a cell capable of generating an immune response, such as a cellular immune response, or a precursor cell of such a cell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells may be immune effector cells as described herein. Preferred lymphoid cells are T cells.

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), including cytolytic T cells. The term "antigen-specific T cell" or similar terms relate to T cells that recognize an antigen to which the T cell is directed and preferably exert an effector function of the T cell.

T cells belong to a group of leukocytes called lymphocytes and play a central role in cell-mediated immunity. They differ from other lymphocyte types, such as B cells and natural killer cells, in the presence of specific receptors on their cell surface, known as T Cell Receptors (TCRs). Thymus is the major organ responsible for T cell maturation. Several different T cell subsets have been found, each with unique functions.

T helper cells assist in other leukocytes during the immune process, including B cell maturation into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also called cd4+ T cells because they express CD4 glycoproteins on the surface. Helper T cells are activated when they present peptide antigens via MHC class II molecules that are expressed on the surface of Antigen Presenting Cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in the active immune response.

Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. These cells are also called cd8+ T cells because they express CD8 glycoproteins on the surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of almost every cell of the body.

Most T cells have T Cell Receptors (TCRs) in the form of complexes of several proteins. TCRs of T cells are capable of interacting with immunogenic peptides (epitopes) that bind to Major Histocompatibility Complex (MHC) molecules and are presented on the surface of target cells. Specific binding of TCRs triggers a signaling cascade within T cells that results in proliferation and differentiation into mature effector T cells. The actual T cell receptor consists of two separate peptide chains that are produced from separate T cell receptor alpha and beta (TCR alpha and TCR beta) genes, known as the alpha-TCR chain and the beta-TCR chain. γδ T cells (γδ T cells) represent a small subset of T cells, which possess unique T Cell Receptors (TCRs) on their surface. However, in γδ T cells, the TCR is composed of one γ chain and one δ chain. This group of T cells is much fewer than αβ T cells (2% of total T cells).

"humoral immunity" or "humoral immune response" is an immune aspect mediated by macromolecules present in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It is in contrast to cell-mediated immunity. Aspects of which reference to antibodies are commonly referred to as antibody-mediated immunity.

Humoral immunity refers to antibody production and its accompanying accessory processes, including: th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell production. It also refers to effector functions of antibodies, including pathogen neutralization, classical complement activation, and the promotion of phagocytosis and pathogen elimination by the regulatory element.

In humoral immune response, B cells first mature in bone marrow and acquire a large number of B Cell Receptors (BCR) that are displayed on the cell surface. These membrane-bound protein complexes have antigen detection specific antibodies. Each B cell has a unique antibody that binds to the antigen. Mature B cells migrate from the bone marrow to lymph nodes or other lymphoid organs where they begin to encounter pathogens. When a B cell encounters an antigen, the antigen binds to the receptor and enters the B cell by endocytosis. Antigens are processed again by MHC-II proteins and presented on the surface of B cells. B cells wait for helper T cells (TH) to bind to the complex. This binding activates TH cells which then release cytokines that induce rapid B cell division, producing thousands of identical B cell clones. These daughter cells become plasma cells or memory cells. Where the memory B cells remain inactive; subsequently, when these memory B cells encounter the same antigen due to reinfection, they divide and form plasma cells. In another aspect, the plasma cells produce a plurality of antibodies that are free to be released into the circulatory system. These antibodies will encounter antigens and bind to them. This will interfere with the chemical interaction between the host and the foreign cell, or they may form a bridge between their antigenic sites, impeding their normal function, or their presence will attract macrophages or killer cells to attack and phagocytose them.

The term "antibody" includes immunoglobulins comprising at least two heavy (H) chains and two light (L) chains attached to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. VH and VL regions can be further subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), 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 carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Antibodies bind, preferably specifically bind, to an antigen.

Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptor. Five members included in this class of proteins are IgA, igG, igM, igD and IgE. IgA is the primary antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the primary immunoglobulin produced in the primary immune response of most subjects. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody reactions, and has an important role in protecting against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody functions, but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering mediator release from mast cells and basophils upon exposure to allergens.

As used herein, "antibody heavy chain" refers to the larger of two types of polypeptide chains that are present in a naturally occurring conformation in an antibody molecule.

As used herein, "antibody light chain" refers to the smaller of two types of polypeptide chains that are present in the naturally occurring conformation in an antibody molecule, and kappa and lambda light chains refer to the two major antibody light chain isoforms.

The present disclosure contemplates immune responses that may be protective, prophylactic, preventative, and/or therapeutic. As used herein, "inducing an immune response" may mean that there is no immune response to a particular antigen prior to induction, or it may mean that there is a basal level of immune response to a particular antigen prior to induction, which is enhanced after induction. Thus, "inducing an immune response" includes "enhancing an immune response".

The term "immunotherapy" relates to the treatment of a disease or disorder by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

The term "immunization" or "vaccination" describes the process of administering an antigen to an individual for the purpose of inducing an immune response, e.g., for therapeutic or prophylactic reasons.

The term "macrophage" refers to a subset of phagocytes produced by the differentiation of monocytes. Macrophages activated by inflammation, immune cytokines, or microbial products non-specifically phagocytose and kill foreign pathogens within the macrophages through hydrolytic and oxidative attack, resulting in degradation of the pathogens. Peptides from degraded proteins are displayed on the surface of macrophages where they can be recognized by T cells and where they can interact directly with antibodies on the surface of B cells, resulting in T and B cell activation and further stimulation of immune responses. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen presenting cells. In one embodiment, the dendritic cells are derived from hematopoietic myeloid progenitor cells. These progenitor cells are initially transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they come into contact with the presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small pieces, which are presented on the cell surface using MHC molecules at maturity. At the same time, they up-regulate 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 up-regulate CCR7, a chemotactic receptor that induces dendritic cells to enter the spleen through blood flow or into the lymph nodes through the lymphatic system. In this context, they act as antigen presenting cells and activate helper and killer T cells and B cells by presenting their antigen as well as non-antigen specific costimulatory signals. Thus, dendritic cells can actively induce T cell or B cell related immune responses. In one embodiment, the dendritic cell is a spleen dendritic cell.

The term "antigen presenting cell" (APC) is one of a variety of cells capable of displaying, retrieving and/or presenting at least one antigen or antigen fragment on (or at) the cell surface. Antigen presenting cells can be classified into professional antigen presenting cells and non-professional antigen presenting cells.

The term "professional antigen presenting cells" relates to antigen presenting cells that constitutively express the major histocompatibility complex class II (MHC class II) molecules required for interaction with the naive T cells. If the T cells interact with MHC class II molecule complexes on the antigen presenting cell membrane, the antigen presenting cells produce costimulatory molecules, thereby inducing activation of the T cells. Professional antigen presenting cells include dendritic cells and macrophages.

The term "non-professional antigen presenting cells" relates to antigen presenting cells that do not constitutively express MHC class II molecules, but constitutively express MHC class II molecules under 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 processing products (e.g., the degradation of proteins into peptides) and the association (e.g., by binding) of one or more of these fragments with MHC molecules to facilitate presentation of cells (such as antigen presenting cells) to specific T cells.

The term "antigen-related disease" refers to any disease in which an antigen is involved, e.g., a disease characterized by the presence of an antigen. The disease involving the antigen may be an infectious disease. As described above, the antigen may be a disease-associated antigen, such as a viral antigen. In one embodiment, the disease involving the antigen is a disease involving cells expressing the antigen (preferably on the cell surface).

The term "infectious disease" refers to any disease that can be transmitted between individuals or organisms and is caused by microbial agents (e.g., the common cold). Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases or parasitic diseases, which are caused by viruses, bacteria and parasites, respectively. In this regard, the infectious disease may be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV/acquired immunodeficiency syndrome (AIDS), diphtheria toxin, hepatitis b, hepatitis c, cholera, severe Acute Respiratory Syndrome (SARS), avian influenza, and influenza.

Exemplary dosing regimen

In some embodiments, the compositions and methods disclosed herein can be used according to an exemplary vaccination regimen as shown in fig. 14.

Primary dosing regimen

In some embodiments, the subject is administered a primary dosing regimen. The primary dosing regimen may include one or more doses. For example, in some embodiments, the primary dosing regimen comprises a single dose (PD ₁ ). In some embodiments, the primary dosing regimen comprises a first dose (PD ₁ ) And a second dose (PD ₂ ). In some embodiments, the primary dosing regimen includes a first dose, a second dose, and a third dose (PD ₃ ). In some embodiments, the primary dosing regimen comprises a first dose, a second dose, a third dose, and one or more additional doses (PD) of any of the pharmaceutical compositions described herein _n )。

In some embodiments, the PD ₁ Comprising administering 1 to 100ug of RNA. In some embodiments, the PD ₁ Comprising administering 1 to 60ug of RNA. In some embodiments, the PD ₁ Comprising administering 1 to 50ug of RNA. In some embodiments, the PD ₁ Comprising administering 1 to 30ug of RNA. In some embodiments, the PD ₁ Comprising administering about 3ug of RNA. In some embodiments, the PD ₁ Comprising administering about 5ug of RNA. In some embodiments，PD ₁ Comprising administering about 10ug of RNA. In some embodiments, the PD ₁ Comprising administering about 15ug of RNA. In some embodiments, the PD ₁ Comprising administering about 20ug of RNA. In some embodiments, the PD ₁ Comprising administering about 30ug of RNA. In some embodiments, the PD ₁ Comprising administering about 50ug of RNA. In some embodiments, the PD ₁ Comprising administering about 60ug of RNA.

In some embodiments, the PD ₂ Comprising administering 1 to 100ug of RNA. In some embodiments, the PD ₂ Comprising administering 1 to 60ug of RNA. In some embodiments, the PD ₂ Comprising administering 1 to 50ug of RNA. In some embodiments, the PD ₂ Comprising administering 1 to 30ug of RNA. In some embodiments, the PD ₂ Including administration of about 3ug. In some embodiments, the PD ₂ Comprising administering about 5ug of RNA. In some embodiments, the PD ₂ Comprising administering about 10ug of RNA. In some embodiments, the PD ₂ Comprising administering about 15ug of RNA. In some embodiments, the PD ₂ Comprising administering about 20ug of RNA. In some embodiments, the PD ₂ Comprising administering about 30ug of RNA. In some embodiments, the PD ₂ Comprising administering about 50ug of RNA. In some embodiments, the PD ₂ Comprising administering about 60ug of RNA.

In some embodiments, the PD ₃ Comprising administering 1 to 100ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 60ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 50ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 30ug of RNA. In some embodiments, the PD ₃ Comprising administering about 3ug of RNA. In some embodiments, the PD ₃ Comprising administering about 5ug of RNA. In some embodiments, the PD ₃ Comprising administering about 10ug of RNA. In some embodiments, the PD ₃ Comprising administering about 15ug of RNA. In some embodiments, the PD ₃ Comprising administering about 20ug of RNA. In some embodiments, the PD ₃ Comprising administering about 30ug of RNA. In some embodiments, the PD ₃ Comprising administering about 50ug of RNA.In some embodiments, the PD ₃ Comprising administering about 60ug of RNA.

In some embodiments, the PD _n Comprising administering 1 to 100ug of RNA. In some embodiments, the PD _n Comprising administering 1 to 60ug of RNA. In some embodiments, the PD _n Comprising administering 1 to 50ug of RNA. In some embodiments, the PD _n Comprising administering 1 to 30ug of RNA. In some embodiments, the PD _n Comprising administering about 3ug of RNA. In some embodiments, the PD _n Comprising administering about 5ug of RNA. In some embodiments, the PD _n Comprising administering about 10ug of RNA. In some embodiments, the PD _n Comprising administering about 15ug of RNA. In some embodiments, the PD _n Comprising administering about 20ug of RNA. In some embodiments, the PD _n Comprising administering about 30ug of RNA. In some embodiments, the PD _n Comprising administering about 50ug of RNA. In some embodiments, the PD _n Comprising administering about 60ug of RNA.

In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD ₁ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₁ Comprising encoding a strain from MN908947An RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD ₂ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₂ Comprising encoding SARS-CoV-2 spike protein or a variant thereof comprising one or more mutations from a Beta variantRNA of the immunogenic fragment. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD ₃ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₃ Comprising encoding SARS-CoV-2 spike comprising one or more mutations from Alpha variantsRNA of a synaptorin or an immunogenic fragment thereof. In some embodiments, the PD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD _n Comprising coding for SARS-CoV-2 spike from MN908947 strain RNA of a synaptorin or an immunogenic fragment thereof. In some embodiments, the PD _n Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD _n Comprising encoding SARS-CoV-2 spike protein from MN908947 strain or immunization thereofRNA of the immunogenic fragment, and RNA encoding SARS-CoV-2 spike protein or immunogenic fragment thereof comprising one or more mutations from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant).

In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And PD _n Each may independently include a plurality (e.g., at least two) of the mRNA compositions described herein. In some embodiments, the PD ₁ 、PD ₂ 、PD ₂ And PD _n Each may independently include a first mRNA composition and a second mRNA composition. In some embodiments, at least one of the plurality of mRNA compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof from a different SARS-CoV-2 variant. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the MN908947 strain SARS-CoV-2. In some embodiments, at least one of the plurality of mRNA compositions comprises RNA encoding SARS-CoV-2S protein or immunogenic fragment thereof that comprises one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from Alpha variants. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The various mRNA compositions given can each independently comprise at least two different mRNA constructs(e.g., differing at least in protein coding sequence). For example, in some embodiments, a PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, from the MN908947 strain SARS-CoV-2, and mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, derived from the MN908947SARS-CoV-2 strain, and mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some such embodiments, the variant may be an Alpha variant. In some such embodiments, the variant may be a Delta variant. In some such embodiments, the variant may be an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the plurality of mRNA compositions given can independently comprise at least two mrnas, each encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from different variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the various mRNA compositions presented can independently comprise mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the Alpha variant and mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the various mRNA compositions given can independently comprise a protein encoding SARS-CoV-2S from Alpha variants ormRNA for an immunogenic fragment thereof, and mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1Omicron variant). In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the various mRNA compositions given can independently comprise mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from a Delta variant, and mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1Omicron variant).

In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each comprising a plurality of mRNA compositions, wherein each mRNA composition is administered to a subject separately. For example, in some embodiments, each mRNA composition is administered via intramuscular injection at a different injection site. For example, in some embodiments, a PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The given first and second mRNA compositions were administered separately to different arms of the subject via intramuscular injection.

In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Comprising administering a plurality of RNA molecules, wherein each RNA molecule encodes a spike protein comprising a mutation from a different SARS-CoV-2 variant, and wherein the plurality of RNA molecules are administered to a subject in a single formulation. In some embodiments, the single formulation comprises RNA encoding a spike protein from the MN908947 strain or an immunogenic variant thereof, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the single formulation comprises RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, a single formulation comprises a nucleic acid encoding a strain from MN908947 An RNA of SARS-CoV-2 spike protein or an immunogenic fragment thereof, and an RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, a single formulation comprises RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₁ With PD ₂ Length of time between (PI) ₁ ) For at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, PI ₁ From about 1 week to about 12 weeks. In some embodiments, PI ₁ From about 1 week to about 10 weeks. In some embodiments, PI ₁ From about 2 weeks to about 10 weeks. In some embodiments, PI ₁ From about 2 weeks to about 8 weeks. In some embodiments, PI ₁ From about 3 weeks to about 8 weeks. In some embodiments, PI ₁ From about 4 weeks to about 8 weeks. In some embodiments, PI ₁ From about 6 weeks to about 8 weeks. In some embodiments, PI ₁ From about 3 weeks to about 4 weeks. In some embodiments, PI ₁ About 1 week. In some embodiments, PI ₁ About 2 weeks. In some embodiments, PI ₁ About 3 weeks. In some embodiments, PI ₁ About 4 weeks. In some embodiments, PI ₁ About 5 weeks. In some embodiments, PI ₁ About 6 weeks. In some embodiments, PI ₁ About 7 weeks. In some embodiments, PI ₁ About 8 weeks. In some embodiments, PI ₁ About 9 weeks. In some embodiments, PI ₁ About 10 weeks. In some embodiments, PI ₁ About 11 weeks. In some embodiments, PI ₁ About 12 weeks.

In some embodiments, the PD ₂ With PD ₃ Length of time between (PI) ₂ ) For at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodimentsIn the case of PI ₂ From about 1 week to about 12 weeks. In some embodiments, PI ₂ From about 1 week to about 10 weeks. In some embodiments, PI ₂ From about 2 weeks to about 10 weeks. In some embodiments, PI ₂ From about 2 weeks to about 8 weeks. In some embodiments, PI ₂ From about 3 weeks to about 8 weeks. In some embodiments, PI ₂ From about 4 weeks to about 8 weeks. In some embodiments, PI ₂ From about 6 weeks to about 8 weeks. In some embodiments, PI ₂ From about 3 weeks to about 4 weeks. In some embodiments, PI ₂ About 1 week. In some embodiments, PI ₂ About 2 weeks. In some embodiments, PI ₂ About 3 weeks. In some embodiments, PI ₂ About 4 weeks. In some embodiments, PI ₂ About 5 weeks. In some embodiments, PI ₂ About 6 weeks. In some embodiments, PI ₂ About 7 weeks. In some embodiments, PI ₂ About 8 weeks. In some embodiments, PI ₂ About 9 weeks. In some embodiments, PI ₂ About 10 weeks. In some embodiments, PI ₂ About 11 weeks. In some embodiments, PI ₂ About 12 weeks.

In some embodiments, the PD ₃ Between subsequent doses as part of the initial dosing regimen, or beyond PD ₃ The length of time between doses (PI) _n ) Each independently and independently selected from: about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, PI _n From about 1 week to about 12 weeks. In some embodiments, PI _n From about 1 week to about 10 weeks. In some embodiments, PI _n From about 2 weeks to about 10 weeks. In some embodiments, PI _n From about 2 weeks to about 8 weeks. In some embodiments, PI _n From about 3 weeks to about 8 weeks. In some embodiments, PI _n From about 4 weeks to about 8 weeks. In some embodiments, PI _n From about 6 weeks to about 8 weeks. In some embodiments, PI _n From about 3 weeks to about 4 weeks. In some embodiments, PI ₂ About 1 week. In some implementationsIn embodiments, PI _n About 2 weeks. In some embodiments, PI _n About 3 weeks. In some embodiments, PI _n About 4 weeks. In some embodiments, PI _n About 5 weeks. In some embodiments, PI _n About 6 weeks. In some embodiments, PI _n About 7 weeks. In some embodiments, PI _n About 8 weeks. In some embodiments, PI _n About 9 weeks. In some embodiments, PI _n About 10 weeks. In some embodiments, PI _n About 11 weeks. In some embodiments, PI _n About 12 weeks.

In some embodiments, with PD ₁ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with PD ₂ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with PD ₃ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with PD _n The composition or compositions administered are formulated in Tris buffer.

In some embodiments, the primary dosing regimen comprises administering two or more mRNA compositions described herein, as well as at least two of the mRNA compositions having different formulations. In some embodiments, the primary dosing regimen comprises PD ₁ And PD ₂ Wherein PD ₁ Comprising administering mRNA and PD formulated in Tris buffer ₂ Including administration of mRNA formulated in PBS buffer. In some embodiments, the primary dosing regimen comprises PD ₁ And PD ₂ Wherein PD ₁ Comprising administering mRNA and PD formulated in PBS buffer ₂ Comprising administering mRNA formulated in Tris buffer.

In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n One or more of the mRNA compositions given may be administered in combination with another vaccine. In some embodiments, another vaccine is for a disease other than COVID-19. In some embodiments, the disease is a disease that is caused by simultaneous infection in a subjectDiseases that increase the detrimental effects of SARS-CoV-2 when the disease is combined with SARS-CoV-2. In some embodiments, the disease is a disease that increases the transmissibility of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the other vaccine is a different commercially 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 in PD ₁ 、PD ₂ 、PD ₃ And/or PD _n One or more mRNA compositions given are administered separately at different injection sites, e.g., via intramuscular injection in some embodiments. For example, in some embodiments, influenza vaccines and PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Given one or more of the SARS-CoV-2 mRNA compositions described herein are administered separately to different arms of a subject via intramuscular injection.

Enhanced dosing regimen

In some embodiments, the vaccination methods disclosed herein include one or more booster dosing regimens. The booster dosing regimen disclosed herein includes one or more doses. In some embodiments, the booster dosing regimen is administered to a patient who has already been administered a primary dosing regimen (e.g., as described herein). In some embodiments, the booster dosing regimen is administered to a patient who has not yet received the pharmaceutical composition disclosed herein. In some embodiments, the booster dosing regimen is administered to a patient who has previously been vaccinated with a different covd-19 vaccine than the vaccine administered in the initial dosing regimen.

In some embodiments, the length of time between the primary and booster dosing regimens 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 initial and the booster dosing regimen is about 1 month. In some embodiments, the length of time between the initial and the booster dosing regimen is at least about 2 months. In some embodiments, the length of time between the initial and the booster dosing regimen is at least about 3 months. In some embodiments, the length of time between the initial and the booster dosing regimen is at least about 4 months. In some embodiments, the length of time between the initial and the booster dosing regimen is at least about 5 months. In some embodiments, the length of time between the initial and the booster dosing regimen is at least about 6 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 1 month to about 48 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 1 month to about 36 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 1 month to about 24 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 2 months to about 24 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 3 months to about 24 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 3 months to about 18 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 3 months to about 12 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 6 months to about 12 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 3 months to about 9 months. In some embodiments, the length of time between the initial and the booster dosing regimen is from about 5 months to about 7 months. In some embodiments, the length of time between the initial and the booster dosing regimen is about 6 months.

In some embodiments, the subject is administeredThe dosing regimen is enhanced. The booster dosing regimen may include one or more doses. For example, in some embodiments, the booster dosing regimen comprises a single dose (BD ₁ ). In some embodiments, the booster dosing regimen comprises a first dose (BD ₁ ) And a second dose (BD ₂ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, and a third dose (BD ₃ ). In some embodiments, the booster dosing regimen comprises a first dose, a second dose, a third dose, and one or more additional doses (BD) of any of the pharmaceutical compositions described herein _n )。

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

In some embodiments, BD ₂ Comprising administering 1 to 100ug of RNA. In some embodiments, BD ₂ Comprising administering 1 to 60ug of RNA. In some embodiments, BD ₂ Comprising administering 1 to 50ug of RNA. In some embodiments, BD ₂ Comprising administering 1 to 30ug of RNA. In some embodiments, BD ₂ Including administration of about 3ug. In some embodiments, BD ₂ Comprising administering about 5ug of RNA. In some embodiments, BD ₂ Comprising administering about 10ug of RNA. In some embodiments, BD ₂ Comprising administering about 15ug of RNA. In some embodimentsIn the case of BD ₂ Comprising administering about 20ug of RNA. In some embodiments, BD ₂ Comprising administering about 30ug of RNA. In some embodiments, BD ₂ Comprising administering about 50ug of RNA. In some embodiments, BD ₂ Comprising administering about 60ug of RNA.

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

In some embodiments, BD _n Comprising administering 1 to 100ug of RNA. In some embodiments, BD _n Comprising administering 1 to 60ug of RNA. In some embodiments, BD _n Comprising administering 1 to 50ug of RNA. In some embodiments, BD _n Comprising administering 1 to 30ug of RNA. In some embodiments, BD _n Comprising administering about 3ug of RNA. In some embodiments, BD _n Comprising administering about 5ug of RNA. In some embodiments, BD _n Comprising administering about 10ug of RNA. In some embodiments, BD _n Comprising administering about 15ug of RNA. In some embodiments, BD _n Comprising administering about 20ug of RNA. In some embodiments, BD _n Comprising administering about 30ug of RNA. In some embodiments, BD _n Comprising administering about 60ug of RNA. In some embodiments, BD _n Comprising administering about 50ug of RNA.

In some embodimentsIn BD (BD) ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD ₁ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some implementationsIn embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD ₂ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein from MN908947 strain or an immunogenic fragment thereof, and encoding a polypeptide comprising one or more variants from DeltaRNA of mutant SARS-CoV-2 spike protein or an immunogenic fragment thereof. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD ₃ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₃ Including encoding strains from MN908947And an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD _n Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BO.10 microvariant) or an immunogenic fragment thereof.

In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a plurality (e.g., at least two) of the mRNA compositions described herein. In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a first mRNA composition and a second mRNA composition. In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a plurality (e.g., at least two) of mRNA compositions, wherein at least one of the plurality of mRNA compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of mRNA compositions comprises a coding derived from a non-coding genemRNA of a SARS-CoV-2S protein or immunogenic fragment thereof that is a variant of SARS-CoV-2 (e.g., a variant that is prevalent or rapidly spreading in a relevant jurisdiction, such as the variants disclosed herein). In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the MN908947 strain SARS-CoV-2. In some embodiments, at least one of the plurality of mRNA compositions comprises RNA encoding SARS-CoV-2S protein or immunogenic fragment thereof that comprises one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from Alpha variants. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given can each independently comprise at least two different mRNA constructs (e.g., mRNA constructs having different protein coding sequences). For example, in some embodiments, as BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, from the MN908947 strain SARS-CoV-2, and mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n Each of the various mRNA compositions presented can independently comprise a polypeptide encoding SARS-CoV-2 from the MN908947SARS-CoV-2 strainAn mRNA of an S protein or immunogenic fragment thereof, and an mRNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from variants that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some such embodiments, the variant may be an Alpha variant. In some such embodiments, the variant may be a Delta variant. In some such embodiments, the variant may be an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise at least two mrnas, each encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from different variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein or immunogenic fragment thereof from the Alpha variant, and mRNA encoding the SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given can each independently comprise mRNA encoding a SARS-CoV-2S protein from an Alpha variant or an immunogenic fragment thereof, and mRNA encoding a SARS-CoV-2S protein from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant) or an immunogenic fragment thereof. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given can each independently comprise mRNA encoding a SARS-CoV-2S protein from a Delta variant or an immunogenic fragment thereof, and mRNA encoding a SARS-CoV-2S protein from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given are administered to the subject separately at different injection sites, e.g., via intramuscular injection in some embodiments. For example, in some embodiments, as BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The given first and second mRNA compositions were administered separately to different arms of the subject via intramuscular injection.

In some embodiments, BD ₁ And BD (BD) ₂ Length of time between (BI ₁ ) Is at least about ₁ A week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, BI ₁ From about 1 week to about 12 weeks. In some embodiments, BI ₁ From about 1 week to about 10 weeks. In some embodiments, BI ₁ From about 2 weeks to about 10 weeks. In some embodiments, BI ₁ From about 2 weeks to about 8 weeks. In some embodiments, BI ₁ From about 3 weeks to about 8 weeks. In some embodiments, BI ₁ From about 4 weeks to about 8 weeks. In some embodiments, BI ₁ From about 6 weeks to about 8 weeks. In some embodiments, BI ₁ From about 3 weeks to about 4 weeks. In some embodiments, BI ₁ About 1 week. In some embodiments, BI ₁ About 2 weeks. In some embodiments, BI ₁ About 3 weeks. In some embodiments, BI ₁ About 4 weeks. In some embodiments, BI ₁ About 5 weeks. In some embodiments, BI ₁ About 6 weeks. In some embodiments, BI ₁ About 7 weeks. In some embodiments, BI ₁ About 8 weeks. In some embodiments, BI ₁ About 9 weeks. In some embodiments, BI ₁ About 10 weeks.

In some embodiments, BD ₂ And BD (BD) ₃ Length of time between (BI ₂ ) For at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, BI ₂ From about 1 week to about 12 weeks. In some embodiments, BI ₂ From about 1 week to about 10 weeks. In some embodiments, BI ₂ About 2 weeks to about 1And 0 weeks. In some embodiments, BI ₂ From about 2 weeks to about 8 weeks. In some embodiments, BI ₂ From about 3 weeks to about 8 weeks. In some embodiments, BI ₂ From about 4 weeks to about 8 weeks. In some embodiments, BI ₂ From about 6 weeks to about 8 weeks. In some embodiments, BI ₂ From about 3 weeks to about 4 weeks. In some embodiments, BI ₂ About 1 week. In some embodiments, BI ₂ About 2 weeks. In some embodiments, BI ₂ About 3 weeks. In some embodiments, BI ₂ About 4 weeks. In some embodiments, BI ₂ About 5 weeks. In some embodiments, BI ₂ About 6 weeks. In some embodiments, BI ₂ About 7 weeks. In some embodiments, BI ₂ About 8 weeks. In some embodiments, BI ₂ About 9 weeks. In some embodiments, BI ₂ About 10 weeks.

In some embodiments, BD ₃ Between subsequent doses as part of a booster regimen, or beyond BD ₃ The length of time between doses (BI) _n ) Each independently and independently selected from: about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, BI _n From about 1 week to about 12 weeks. In some embodiments, BI _n From about 1 week to about 10 weeks. In some embodiments, BI _n From about 2 weeks to about 10 weeks. In some embodiments, BI _n From about 2 weeks to about 8 weeks. In some embodiments, BI _n From about 3 weeks to about 8 weeks. In some embodiments, BI _n From about 4 weeks to about 8 weeks. In some embodiments, BI _n From about 6 weeks to about 8 weeks. In some embodiments, BI _n From about 3 weeks to about 4 weeks. In some embodiments, BI _n About 1 week. In some embodiments, BI _n About 2 weeks. In some embodiments, BI _n About 3 weeks. In some embodiments, BI _n About 4 weeks. In some embodiments, BI _n About 5 weeks. In some embodiments, BI _n About 6 weeks. In some embodimentsIn BI (BI) _n About 7 weeks. In some embodiments, BI _n About 8 weeks. In some embodiments, BI _n About 9 weeks. In some embodiments, BI _n About 10 weeks.

In some embodiments, with BD ₁ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with BD ₂ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with BD ₃ The composition or compositions administered are formulated in Tris buffer. In some embodiments, with BD ₃ The composition or compositions administered are formulated in Tris buffer.

In some embodiments, the booster dosing regimen comprises administering two or more mRNA compositions described herein, as well as at least two of the mRNA compositions having different formulations. In some embodiments, the enhanced dosing regimen comprises BD ₁ And BD (BD) ₂ Wherein BD is ₁ Comprising administering mRNA and BD formulated in Tris buffer ₂ Including administration of mRNA formulated in PBS buffer. In some embodiments, the enhanced dosing regimen comprises BD ₁ And BD (BD) ₂ Wherein BD is ₁ Comprising administering mRNA and BD formulated in PBS buffer ₂ Comprising administering mRNA formulated in Tris buffer.

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n One or more of the mRNA compositions given may be administered in combination with another vaccine. In some embodiments, another vaccine is for a disease other than COVID-19. In some embodiments, the disease is a disease that increases the detrimental effects of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the disease is a disease that increases the transmissibility of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the other vaccine is a different commercially available vaccine. In some embodiments, the different commercially available vaccine is an RNA vaccine. In some embodiments, different vendorsThe purchased vaccine is a polypeptide-based vaccine. In some embodiments, another vaccine (e.g., as described herein) and as BD ₁ 、BD ₂ 、BD ₃ And/or BD _n One or more mRNA compositions given are administered separately at different injection sites, e.g., via intramuscular injection in some embodiments. For example, in some embodiments, influenza vaccines and BD ₁ 、BD ₂ 、BD ₃ And/or BD _n Given one or more of the SARS-CoV-2mRNA compositions described herein are administered separately to different arms of a subject via intramuscular injection.

Additional reinforcement scheme

In some embodiments, the vaccination methods disclosed herein comprise administering more than one booster dosing regimen. In some embodiments, it may be desirable to administer more than one booster dosing regimen to increase the neutralizing antibody response. In some embodiments, more than one booster dosing regimen may be required to combat the SARS-CoV-2 strain that has been shown to be highly likely to escape the immune response elicited by the previously received vaccine by the patient. In some embodiments, additional boost dosing regimens are administered to patients who have been determined to produce low concentrations of neutralizing antibodies. In some embodiments, an additional boost dosing regimen is administered to a patient (e.g., an immunocompromised patient, a cancer patient, and/or an organ transplant patient) who has been determined to be highly likely to be susceptible to SARS-CoV-2 infection despite prior vaccination.

The description provided above for the first booster dosing regimen also describes one or more additional booster dosing regimens. The time interval between the first boost regimen and the second boost regimen, or between subsequent boost regimens, may be any of the acceptable time intervals described above between the initial and first boost regimens.

In some embodiments, the dosing regimen includes a primary regimen and a boost regimen, wherein at least one dose given in the primary regimen and/or boost regimen includes a composition comprising RNA encoding an S protein or immunogenic fragment thereof from a variant (e.g., an Omicron variant as described herein) that is prevalent or rapidly spread in the relevant jurisdiction. For example, in some embodiments, the initial regimen comprises at least 2 doses of BNT162b2 (e.g., encoding the MN908947 strain), e.g., given at least 3 weeks apart, and the boosting regimen comprises at least 1 dose of a composition comprising RNA encoding an S protein or immunogenic fragment thereof from variants that are prevalent or rapidly spread in the relevant jurisdiction (e.g., omacron variants as described herein). In some such embodiments, this dose of boosting regimen may also include RNA encoding the S protein or immunogenic fragment thereof from the MN908947 strain, which RNA may be administered as a single mixture, or as two separate compositions (e.g., at a 1:1 weight ratio), along with RNA encoding the S protein or immunogenic fragment thereof from variants that are prevalent or rapidly spread in the relevant jurisdiction (e.g., omacron variants as described herein). In some embodiments, the boosting regimen may further comprise at least 1 dose of BNT162b2, which may be administered as a first boosting dose or a subsequent boosting dose.

In some embodiments, the RNA compositions described herein are given as a booster at a higher dose than the dose given during the primary regimen (primary dose) and/or the dose given for the first booster (if any). For example, in some embodiments, this dose may be 60ug; or in some embodiments, this dose may be greater than 30ug and less than 60ug (e.g., 55ug, 50ug, or less). In some embodiments, the RNA compositions described herein are given as a booster at least 3 to 12 months or 4 to 12 months, or 5 to 12 months, or 6 to 12 months after the last dose (e.g., the last dose of the initial regimen or the first dose of the boosting regimen). In some embodiments, the primary dose and/or the first booster dose (if any) may comprise BNT162b2, e.g., 30ug per dose.

In some embodiments, the RNA compositions described herein comprise a nucleic acid sequence encoding a nucleic acid sequence 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 identity to SEQ ID NO:49, 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 more). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 50). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 51).

In some embodiments, the RNA compositions described herein comprise a nucleic acid sequence encoding a nucleic acid sequence 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 identity to SEQ ID No. 55, 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 more). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 56). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 57).

In some embodiments, the RNA compositions described herein comprise a nucleic acid sequence encoding a nucleic acid sequence 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 identity to SEQ ID NO:58, 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 more). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 59). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID NO: 60).

In some embodiments, the RNA compositions described herein comprise a nucleic acid sequence encoding a nucleic acid sequence 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 identity to SEQ ID NO:61, 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 more). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID No. 62). In some embodiments, the RNA composition comprises a nucleic acid sequence comprising 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 more identity to SEQ ID No. 63).

In some embodiments, the formulations disclosed herein can be used to implement any of the dosing regimens described in (below) table C.

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In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given at the first dose of the initial regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given in a second dose of the initial regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, 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 the primary regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given at the first dose of the boosting regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given in a second dose of the boosting regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given in a first dose and a second dose of the boosting regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding a variant described herein) are given in a first dose and a second dose of the primary regimen and also in at least one dose of the boosting regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding the variants described herein) are given in at least one dose (including, e.g., at least two doses) of the boosting regimen, and BNT162b2 is given in the initial regimen. In some embodiments of certain exemplary dosing regimens as described above in table C, the RNA compositions described herein (e.g., comprising RNA encoding the variants described herein) are given in a second dose of the boosting regimen, and BNT162b2 is given in the first regimen and in the first dose of the boosting regimen. In some embodiments, an RNA composition described herein (e.g., comprising RNA encoding a variant described herein) comprises a nucleic acid sequence encoding a nucleic acid sequence 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 identity to SEQ ID NO:49, 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 more). In some embodiments, an RNA composition described herein (e.g., comprising RNA encoding a variant described herein) comprises an RNA comprising 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 more 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 comprising 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 more identity to SEQ ID NO: 51).

In some embodiments, such RNA compositions described herein (e.g., comprising RNA encoding variants described herein) can further comprise RNA encoding the S protein or immunogenic fragment thereof of a different strain (e.g., MN908947 strain). By way of example, in some embodiments, the second dose of the boosting regimen of regimens #9-11 as described above in table C may comprise an RNA composition described herein (e.g., comprising RNA encoding a variant such as Omicron described herein, e.g., in one embodiment, as described in this example) and a BNT162b2 construct, e.g., in a weight ratio of 1:1.

In some embodiments of regimen #6 as described above in table C, the first and second doses of the primary regimen and the first and second doses of the boosting regimen each comprise an RNA composition described herein (e.g., comprising RNA encoding a variant such as omacron described herein, e.g., in one embodiment, RNA as described in this example). In some such embodiments, a second dose of the boosting regimen may not be necessary.

In some embodiments of regimen #6 as described above in table C, the first dose and the second dose of the primary regimen each comprise BNT162b2 construct, and the first dose and the second dose of the boosting regimen each comprise an RNA composition described herein (e.g., comprising RNA encoding a variant such as Omicron described herein, e.g., in one embodiment, RNA as described in the present example). In some such embodiments, a second dose of the boosting regimen may not be necessary.

In some embodiments of regimen #6 as described above in table C, the first dose and the second dose of the primary regimen and the first dose of the boosting regimen each comprise BNT162b2 construct, and the second dose of the boosting regimen comprises an RNA composition described herein (e.g., comprising RNA encoding a variant such as omacron described herein, e.g., in one embodiment, RNA as described in the present example).

The following certain exemplary embodiments are also within the scope of the present disclosure:

1. a composition or pharmaceutical formulation comprising RNA that comprises a nucleotide sequence encoding a SARS-CoV-2S protein or immunogenic fragment thereof that comprises a mutation characteristic of one or more SARS-CoV-2 variants (e.g., in some embodiments, SARS-CoV-2 Omicron variants).

2. The composition or pharmaceutical formulation of embodiment 1, wherein the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

3. The composition or pharmaceutical formulation of embodiment 1 or 2, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations that are characteristic of ba.1, ba.2, ba.4/5, XBB, xbb.1 or bq.1.1 Omicron variants or sublines thereof.

4. The composition or pharmaceutical preparation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characterized by the BA.4/5 variant, wherein the one or more mutations are selected from T19I, DELTA-26, a27S, DELTA69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D N, R S, K35417N, N440K, L452R, S477N, T478 484N, T486 498N, T501N, T614N, T655N, T679N, T681N, T764N, T796N, T954H and N969K.

5. The composition or pharmaceutical formulation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characteristic of the ba.1 variant, wherein the one or more mutations are selected from the group consisting of a67V, DELTA69-70, T95I, G D, DELTA-145, DELTA211, L212I, ins EPE, G339D, S371L, S373P, S375 48417 417N, N440, 478N, N484N, N493 496N, N498 52505N, N547N, N614N, N679N, N764N, N796N, N856 9556 959 52937 52980F.

6. The composition or pharmaceutical formulation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characteristic of the ba.1 variant, wherein the one or more mutations are selected from the group consisting of T19I, DELTA24-26, a27S, G142D, V213G, G339D, S373P, S373 5483 376 63376A, D405 79408S, K417N, N K, S357N, T478K, E484K, E493R, Q498R, N501, 505R, N614 655R, N681R, N764R, N796R, N954H and N969K.

7. The composition or pharmaceutical preparation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations that are characteristic of the BA.2.75.1 variant, wherein the one or more mutations are selected from T19I, DELTA-26, a27S, G142D, V213G, G339D, S371P, S375F, T376A, D405N, R408S, K N, N440K, S477N, T478K, E484A, Q493R, Q498R, N614 655R, N679R, N681 764R, N796R, N954H and N969K.

8. The composition or pharmaceutical formulation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characteristic of the ba.4.6/bf.7 variant, wherein the one or more mutations are selected from the group consisting of T19I, DELTA-26, a27S, DELTA/70, G142D, V213G, G339D, R3535346T, S373P, S375F, T376F, T, F, T417F, T, 452F, T, F, T477, F, T484 486F, T498F, T501F, T, F, T614, F, T658 52679F, T681F, T764F, T796F, T H954 and N969K.

9. The composition or pharmaceutical formulation of embodiment 3, wherein the SARS-CoV-2 protein or immunogenic fragment thereof comprises one or more mutations characteristic of an XBB variant, wherein the one or more mutations are selected from the group consisting of T19 24-26, a27 83 142 144, H146 183 213 339 to 373 375 376 405 408 417 440 445 446 460 477 to 478 484 486 493 498 501 to 505 614 655 679 681 764 to 796 954H and N969K.

The composition or pharmaceutical formulation of embodiment 3, wherein the SARS-CoV-2 protein or immunogenic fragment thereof comprises one or more mutations characteristic of the xbb.1 variant, wherein the one or more mutations are selected from the group consisting of T19 24-26, a27 83 142 144, H146 183 213 252 339 368 373 375 405 408 417 440 445 446 460 477 478 486 490 493 498 501 505 614 655 679 681 764 796 954H and N969K.

11. The composition or pharmaceutical preparation of embodiment 3, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characterized by the BQ.1.1 variant, wherein the one or more mutations are selected from the group consisting of T19I, DELTA-26, A27S, DELTA/70G 142D, V52213D, V339D, V371D, V373D, V376D, V52408D, V52417D, V52440D, V444D, V52452D, V52460D, V477 52478D, V484D, V486D, V498D, V52501D, V52679D, V681 764D, V796D, V954D, V969K.

12. The composition or pharmaceutical formulation of any of embodiments 1-11, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations characteristic of an omacron variant is encoded by a sequence that is codon optimized (e.g., codon optimized for expression in human cells) and/or has an increased G/C content as compared to a wild-type encoding sequence.

13. The composition or pharmaceutical formulation of embodiment 3 or 4, wherein the SARS-CoV-2S protein comprises one or more mutations characterized by the omacron ba.4/5 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:69 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:70 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or with SEQ ID NO:71 has a nucleotide sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity.

14. The composition or pharmaceutical formulation of embodiment 3 or 5, wherein the SARS-CoV-2S protein comprises one or more mutations that are characteristic of the omacron ba.1 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:49 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:50 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity and/or NO:51 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

15. The composition or pharmaceutical formulation of embodiment 3 or 6, wherein the SARS-CoV-2S protein comprises one or more mutations characterized by the omacron ba.2 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:64 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:65 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity and/or NO:66 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

16. The composition or pharmaceutical formulation of embodiment 3 or 7, wherein the SARS-CoV-2S protein comprises one or more mutations characterized by the omacron ba.2.75 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:80 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:81 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity and/or NO:83 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

17. The composition or pharmaceutical formulation of embodiment 3 or 8, wherein the SARS-CoV-2S protein comprises one or more mutations characterized by the omacron ba.4.6 or bf.7 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:90 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:91 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity and/or NO:92 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

18. The composition or pharmaceutical formulation of embodiment 3 or 9, wherein the SARS-CoV-2S protein comprises one or more mutations that are characteristic of an omacron XBB variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:95 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or an immunogenic fragment thereof; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:96 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or with SEQ ID NO:98 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

19. The composition or pharmaceutical formulation of embodiment 3 or 10, wherein the SARS-CoV-2S protein comprises one or more mutations that are characteristic of the omacron bq.1.1 variant, and wherein:

a) The SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:100, or an immunogenic fragment thereof, having an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or

b) Said RNA encoding said SARS-CoV-2S protein comprises a sequence identical to SEQ ID NO:101 has at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity and/or NO:102 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

20. The composition or pharmaceutical formulation of any one of embodiments 1-19, wherein the SARS-CoV-2S protein comprises one or more mutations that improve expression, stability and/or immunogenicity.

21. The composition or pharmaceutical formulation of embodiment 20, wherein the SARS-CoV-2S protein comprises one or more mutations that stabilize the prefusion conformation.

22. The composition or pharmaceutical formulation of embodiment 20, wherein the SARS-CoV-2S protein is contained in a polypeptide corresponding to SEQ ID NO: proline mutations at positions 986 and 987 of residue 1.

23. The composition or pharmaceutical formulation of embodiment 21 or 22, wherein the SARS-CoV-2S protein is contained in a polypeptide corresponding to SEQ ID NO:1, 892, 899 and/or 942.

24. The composition or pharmaceutical formulation of any one of embodiments 20-23, wherein the SARS-CoV-2S protein comprises a mutation that prevents furin cleavage.

25. The composition or pharmaceutical formulation of embodiment 24, wherein the SARS-CoV-2S protein is contained in a polypeptide corresponding to SEQ ID NO:1 (e.g., GSAS mutations) at positions 682-685 of residues that prevent cleavage by furin.

26. The composition or pharmaceutical formulation of any of embodiments 20-25, wherein the SARS-CoV-2S protein comprises one or more mutations that reduce sloughing of the S protein (e.g., an aspartic acid mutation to glycine at a position corresponding to residue 614 of SEQ ID NO: 1).

27. The composition or pharmaceutical formulation of any one of embodiments 1-26, wherein the RNA comprises a modified nucleoside in place of uridine.

28. The composition or pharmaceutical formulation of embodiment 27, wherein the RNA comprises a modified nucleoside in place of each uridine.

29. The composition or pharmaceutical formulation of embodiment 27 or 28, wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

30. The composition or pharmaceutical formulation of embodiment 29, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

31. The composition or pharmaceutical formulation of any one of embodiments 1-30, wherein the RNA comprises a 5' cap.

32. The composition or pharmaceutical formulation of embodiment 31, wherein the 5' cap is or comprises a cap1 structure.

33. The composition or pharmaceutical formulation of embodiment 32, wherein the RNA comprises or comprises m ₂ ^7，3’ -OGppp(m ₁ ^2’-O ) ApG.

34. The composition or pharmaceutical formulation of any of embodiments 1-33, wherein the composition comprises a poly (a) sequence.

35. The composition or pharmaceutical formulation of embodiment 34, wherein the poly (a) sequence comprises at least 100 a nucleotides.

36. The composition or pharmaceutical formulation of embodiment 34 or 35, wherein the poly (a) sequence is an interrupt sequence for a nucleotide.

37. The composition or pharmaceutical formulation of embodiment 36, 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.

38. The composition or pharmaceutical formulation of embodiment 37, wherein the poly (a) sequence comprises SEQ ID NO:14 or a nucleotide sequence identical to SEQ ID NO:14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical thereto.

39. The composition or pharmaceutical formulation of any one of embodiments 1-38, wherein the RNA comprises a 5'-UTR that is or comprises a modified human Alpha-globulin 5' -UTR.

40. The composition or pharmaceutical formulation of embodiment 39, wherein the 5' utr comprises the amino acid sequence of SEQ ID NO:12 or a nucleotide sequence which is identical to SEQ ID NO:12, at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

41. The composition or pharmaceutical formulation of any one of embodiments 1-40, wherein the RNA comprises a 3' -UTR that is or comprises a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA.

42. The composition or pharmaceutical formulation of embodiment 41, wherein the RNA comprises: comprising SEQ ID NO:13 or a nucleotide sequence identical to SEQ ID NO:13, has a 3' utr of a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

43. The composition or pharmaceutical formulation of any one of embodiments 1-42, wherein the RNA is formulated or to be formulated as particles.

44. The composition or pharmaceutical formulation of embodiment 43, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

45. The composition or pharmaceutical formulation of embodiment 44, wherein the LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate.

46. The composition or pharmaceutical formulation of embodiment 45, wherein the neutral lipid is present at a concentration in the range of about 5 to about 15 mole% of the total lipid.

47. The composition or pharmaceutical formulation of embodiment 45 or 46, wherein the cationizable lipid is present at a concentration in the range of about 40 to about 50 mole% of the total lipid.

48. The composition or pharmaceutical formulation of any one of embodiments 45-47, wherein the sterol is present at a concentration in the range of about 30 to about 50 mole% of total lipids.

49. The composition or pharmaceutical formulation of any one of embodiments 45-48, wherein the polymer-lipid conjugate is present at a concentration in the range of about 1 to about 10 mole% of total lipid.

50. The composition or pharmaceutical formulation of any one of embodiments 45-49, wherein the lipid nanoparticle comprises about 40 to about 50 mole% of the cationically ionizable lipid; about 5 to about 15 mole% of the neutral lipid; about 35 to about 45 mole% of the sterol; and about 1 to about 10 mole% of the polymer conjugated lipid.

51. The composition or pharmaceutical formulation of embodiment 44, wherein said RNA cationic lipid complex particles are obtainable by mixing said RNA with a liposome.

52. A composition or pharmaceutical formulation comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of an Omicron ba.1 variant and comprising the amino acid sequence of SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 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 97%, at least 98%, or 99% or more) identical, and/or (b) comprises the amino acid sequence of SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

Wherein the LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate.

53. The composition or pharmaceutical formulation of embodiment 52, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

54. The composition or pharmaceutical formulation of embodiments 52 or 53, wherein the modified uridine is each N1-methyl-pseudouridine.

55. The composition or pharmaceutical formulation of any one of embodiments 52-54, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

56. The composition or pharmaceutical formulation of embodiment 55, 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.

57. The composition or pharmaceutical formulation of embodiment 56, wherein the poly a sequence comprises SEQ ID NO:14.

58. the composition or pharmaceutical formulation of any one of embodiments 52-57, wherein the RNA comprises the amino acid sequence of SEQ ID NO:51.

59. A composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a mutation comprising one or more Beta variants characteristic and comprising the amino acid sequence of SEQ ID NO:55 or amino acid sequence identical to SEQ ID NO:55 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 97%, at least 98%, or 99% or more) of the SARS-CoV-2S protein that is identical in amino acid sequence, and/or (b) comprises the amino acid sequence of SEQ ID NO:56 or a nucleotide sequence identical to SEQ ID NO:56 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

60. The composition or pharmaceutical formulation of embodiment 59, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

61. The composition or pharmaceutical formulation of embodiments 59 or 60, wherein each of the modified uridine is N1-methyl-pseudouridine.

62. The composition or pharmaceutical formulation of any one of embodiments 59 to 61, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

63. The composition or pharmaceutical formulation of embodiment 62, 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.

64. The composition or pharmaceutical formulation of embodiment 63, wherein the poly a sequence comprises SEQ ID NO:14.

65. the composition or pharmaceutical formulation of any one of embodiments 59-64, wherein the RNA comprises the amino acid sequence of SEQ ID NO:57.

66. a composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a mutation comprising one or more Alpha variants characteristic and comprising the amino acid sequence of SEQ ID NO:58 or a polypeptide that hybridizes to SEQ ID NO:58 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 97%, at least 98%, or 99% or more) SARS-CoV-2S protein that is identical in amino acid sequence, and/or (b) comprises the amino acid sequence of SEQ ID NO:59 or a nucleotide sequence identical to SEQ ID NO:59 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

67. The composition or pharmaceutical preparation of embodiment 66, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

68. The composition or pharmaceutical formulation of embodiment 66 or 67, wherein each of the modified uridine is N1-methyl-pseudouridine.

69. The composition or pharmaceutical formulation of any one of embodiments 66-68, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

70. The composition or pharmaceutical formulation of embodiment 69, 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.

71. The composition or pharmaceutical formulation of embodiment 70, wherein the poly a sequence comprises SEQ ID NO:14.

72. the composition or pharmaceutical formulation of any one of embodiments 66-71, wherein the RNA comprises the amino acid sequence of SEQ ID NO:60.

73. A composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence that (a) encodes a SARS-CoV-2S protein comprising one or more mutations characteristic of Delta variants and comprises the amino acid sequence of SEQ ID NO:61 or amino acid sequence identical to SEQ ID NO:61 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (b) comprising SEQ ID NO:62a or a nucleotide sequence identical to SEQ ID NO:62a 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

74. The composition or pharmaceutical preparation of embodiment 73, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

75. The composition or pharmaceutical formulation of embodiment 73 or 74, wherein the modified uridine is each N1-methyl-pseudouridine.

76. The composition or pharmaceutical formulation of any one of embodiments 73-75, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

77. The composition or pharmaceutical formulation of embodiment 76, 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.

78. The composition or pharmaceutical formulation of embodiment 77, wherein the poly a sequence comprises SEQ ID NO:14.

79. the composition or pharmaceutical formulation of any one of embodiments 73-78, wherein the RNA comprises the amino acid sequence of SEQ ID NO:63a.

80. A composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence that (a) encodes a SARS-CoV-2S protein comprising one or more mutations characteristic of a ba.2 omacron variant and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:64 or an amino acid sequence identical to SEQ ID NO:64 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (b) a polypeptide comprising the amino acid sequence of SEQ ID NO:65 or a nucleotide sequence identical to SEQ ID NO:65 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

81. The composition or pharmaceutical formulation of embodiment 80, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

82. The composition or pharmaceutical formulation of embodiment 80 or 81, wherein each of the modified uridine is N1-methyl-pseudouridine.

83. The composition or pharmaceutical formulation of any one of embodiments 80-82, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

84. The composition or pharmaceutical formulation of embodiment 83, 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.

85. The composition or pharmaceutical formulation of embodiment 84, wherein the poly a sequence comprises the amino acid sequence of SEQ ID NO:14.

86. the composition or pharmaceutical formulation of any one of embodiments 80-85, wherein the RNA comprises the amino acid sequence of SEQ ID NO:67.

87. A composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a polypeptide comprising one or more mutations characteristic of a ba.2.75 omacron variant and comprising the amino acid sequence of SEQ ID NO 80 or a sequence identical to SEQ ID NO:80 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 97%, at least 98%, or 99% or more) of a SARS-COV-2S protein that is identical in amino acid sequence, and/or (b) comprises the amino acid sequence of SEQ ID NO:81 or a nucleotide sequence identical to SEQ ID NO:81 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

88. The composition or pharmaceutical formulation of embodiment 87, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

89. The composition or pharmaceutical formulation of embodiments 86 or 88, wherein each of the modified uridine is N1-methyl-pseudouridine.

90. The composition or pharmaceutical formulation of any one of embodiments 87-89, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

91. The composition or pharmaceutical formulation of embodiment 90, 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.

92. The composition or pharmaceutical formulation of embodiment 91, wherein the poly a sequence comprises SEQ ID NO:14.

93. the composition or pharmaceutical formulation of any one of embodiments 90-92, wherein the RNA comprises the amino acid sequence of SEQ ID NO:83.

94. a composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence encoding a SARS-COV-2S protein comprising one or more mutations characteristic of a ba.2.75.2 omacron variant and comprising the amino acid sequence of SEQ ID NO 85 or an amino acid sequence identical to SEQ ID NO:85 (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 97%, at least 98%, or 99% or more) identical, and/or (b) comprises the amino acid sequence of SEQ ID NO:86 or a nucleotide sequence which hybridizes with SEQ ID NO:86 (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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

95. The composition or pharmaceutical preparation of embodiment 94, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

96. The composition or pharmaceutical formulation of embodiments 94 or 95, wherein each of the modified uridine is N1-methyl-pseudouridine.

97. The composition or pharmaceutical formulation of any one of embodiments 94-96, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

98. The composition or pharmaceutical formulation of embodiment 97, 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.

99. The composition or pharmaceutical formulation of embodiment 98, wherein the poly a sequence comprises SEQ ID NO:14.

100. the composition or pharmaceutical formulation of any one of embodiments 94-99, wherein the RNA comprises the amino acid sequence of SEQ ID NO:88.

101. A composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of omacron ba.4/5 variants and comprising the amino acid sequence of SEQ ID NO:69 or amino acid sequence identical to SEQ ID NO:69 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 97%, at least 98%, or 99% or more) identical, and/or (b) comprises the amino acid sequence of SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

102. The composition or pharmaceutical formulation of embodiment 101, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

103. The composition or pharmaceutical formulation of embodiment 101 or 102, wherein each of the modified uridine is N1-methyl-pseudouridine.

104. The composition or pharmaceutical formulation of any one of embodiments 101-103, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

105. The composition or pharmaceutical formulation of embodiment 104, 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.

106. The composition or pharmaceutical formulation of embodiment 105, wherein the poly a sequence comprises SEQ ID NO:14.

107. the composition or pharmaceutical formulation of any one of embodiments 101-106, wherein the RNA comprises the amino acid sequence of SEQ ID NO:72.

108. a composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a mutation comprising one or more omacron ba.4.6 or bf.7 variants and comprising the amino acid sequence of SEQ ID NO:90 or an amino acid sequence identical to SEQ ID NO:90 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 97%, at least 98%, or 99% or more) of a SARS-CoV-2S protein that is identical in amino acid sequence, and (b) comprises the amino acid sequence of SEQ ID NO:91 or a nucleotide sequence identical to SEQ ID NO:91 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

109. The composition or pharmaceutical preparation of embodiment 108, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

110. The composition or pharmaceutical formulation of embodiments 108 or 109, wherein each of the modified uridine is N1-methyl-pseudouridine.

111. The composition or pharmaceutical formulation of any one of embodiments 108-110, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

112. The composition or pharmaceutical formulation of embodiment 111, 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.

113. The composition or pharmaceutical formulation of embodiment 112, wherein the poly a sequence comprises the amino acid sequence of SEQ ID NO:14.

114. The composition or pharmaceutical formulation of any one of embodiments 107-112, wherein the RNA comprises the amino acid sequence of SEQ ID NO:93.

115. a composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence that (a) encodes a SARS-CoV-2S protein comprising one or more mutations characteristic of an XBB omacron variant and comprises the amino acid sequence of SEQ ID NO 85 or an amino acid sequence identical to SEQ ID NO:95 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (b) comprises the amino acid sequence of SEQ ID NO:96 or a nucleotide sequence identical to SEQ ID NO:96 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

116. The composition or pharmaceutical formulation of embodiment 115, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

117. The composition or pharmaceutical formulation of embodiment 115 or 116, wherein each of the modified uridine is N1-methyl-pseudouridine.

118. The composition or pharmaceutical formulation of any one of embodiments 115-117, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

119. The composition or pharmaceutical formulation of embodiment 118, 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.

120. The composition or pharmaceutical formulation of embodiment 119, wherein the poly a sequence comprises SEQ ID NO:14.

121. the composition or pharmaceutical formulation of any one of embodiments 114-120, wherein the RNA comprises the amino acid sequence of SEQ ID NO:98.

122. a composition or pharmaceutical formulation comprising a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA comprises a nucleotide sequence (a) encoding a polypeptide comprising one or more mutations characteristic of bq.1.1 omacron variants and comprising the amino acid sequence of SEQ ID NO 100 or a sequence identical to SEQ ID NO:100 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 97%, at least 98%, or 99% or more) of a SARS-CoV-2S protein that is identical in amino acid sequence, and/or (b) comprises the amino acid sequence of SEQ ID NO:101 or a nucleotide sequence identical to SEQ ID NO:101 (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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

123. The composition or pharmaceutical preparation of embodiment 122, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

124. The composition or pharmaceutical formulation of embodiment 122 or 123, wherein each of the modified uridine is N1-methyl-pseudouridine.

125. The composition or pharmaceutical formulation of any one of embodiments 122-124, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

126. The composition or pharmaceutical formulation of embodiment 125, 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.

127. The composition or pharmaceutical formulation of embodiment 126, wherein the poly a sequence comprises SEQ ID NO:14.

128. The composition or pharmaceutical formulation of any one of embodiments 121-127, wherein the RNA comprises the amino acid sequence of SEQ ID NO:103.

129. the composition or pharmaceutical formulation of any one of embodiments 1-128, wherein the RNA is present in the composition in an amount ranging from 1 μg to about 100 μg per dose.

130. The composition or pharmaceutical formulation of embodiment 129, wherein the RNA is present in the composition in an amount ranging from about 1 μg to about 60 μg per dose.

131. The composition or pharmaceutical formulation of embodiment 130, wherein the RNA is present in the composition in an amount of about 1.5 μg, about 2.5 μg, about 3.O μg, about 5.O μg, about 10 μg, about 15 μg, about 30 μg, or about 60 μg per dose.

132. The composition or pharmaceutical formulation of any one of embodiments 1-131, further comprising a second RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or immunogenic fragment thereof, wherein the SARS-CoV-2S protein comprises one or more mutations characteristic of a second SARS-CoV-2 variant.

133. A composition or pharmaceutical formulation comprising:

(a) A first RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or immunogenic fragment thereof of a first strain or variant; and

(b) A second RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or an immunogenic fragment thereof of the second variant,

wherein the first variant is different from the second variant, and optionally wherein the first and/or second variant is an Omicron variant.

134. The composition or pharmaceutical formulation of embodiment 133, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of omacron variants or an immunogenic fragment thereof.

135. The composition or pharmaceutical formulation of embodiment 133 or 134, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1.1 Omicron variants or sublines thereof.

136. The composition or pharmaceutical formulation of embodiment 133, wherein the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein, or an immunogenic fragment thereof, that is antigenically different from the S protein encoded by the first RNA.

137. A composition or pharmaceutical formulation comprising (a) a first RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or an immunogenic fragment thereof of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 Omicron variant, and (b) a second RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations that are not characteristic of an Omicron variant of a ba.1 Omicron variant.

138. The composition or pharmaceutical formulation of any one of embodiments 133-137, wherein said second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more ba.4/5 Omicron variants or variants evolved from ba.4/5 Omicron variants.

139. The composition or pharmaceutical formulation of any one of embodiments 133-135, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of a ba.1 Omicron variant or an immunogenic fragment thereof.

140. The composition or pharmaceutical formulation of any one of embodiments 133-138, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.2 Omicron variants or an immunogenic fragment thereof.

141. The composition or pharmaceutical formulation of any one of embodiments 133-138, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.4 or ba.5 Omicron variants or an immunogenic fragment thereof.

142. The composition or pharmaceutical formulation of any one of embodiments 133-138, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.2 Omicron variants or an immunogenic fragment thereof.

143. The composition or pharmaceutical formulation of any one of embodiments 133-138, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of BAA or ba.5 omacron variants or an immunogenic fragment thereof.

144. The composition or pharmaceutical formulation of any one of embodiments 133-143, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of XBB Omicron variants or an immunogenic fragment thereof.

145. The composition or pharmaceutical formulation of any one of embodiments 133-138, wherein the first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain or an immunogenic fragment thereof and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more bq.1.1 omacron variants or mutations characteristic of sublines thereof or an immunogenic fragment thereof.

146. The composition or pharmaceutical formulation of any one of embodiments 133-145, wherein the immunogenic fragment of the SARS-CoV-2S protein encoded by the first RNA and/or the second RNA comprises the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein or the S1 subunit of the SARS-CoV-2S protein.

147. The composition or pharmaceutical formulation of any one of embodiments 133-146, wherein each of the first RNA and the second RNA is codon optimized (e.g., codon optimized for expression in a human cell) and/or has an increased G/C content compared to a wild-type coding sequence.

148. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

a) The first RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of a MN908947 strain, wherein (i) the SARS-CoV-2S protein of the MN908947 strain comprises the nucleotide sequence of SEQ ID NO:7 or with SEQ ID NO:7 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the first RNA comprises the amino acid sequence of SEQ ID NO:9 or 20 or a nucleotide sequence identical to SEQ ID NO:9 or 20 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 97%, at least 98% or 99% or more) identical, and

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of ba.4/5 Omicron variants, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of ba.4/5 Omicron variants comprises the nucleotide sequence of SEQ ID NO:90 or an amino acid sequence identical to SEQ ID NO:90 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:91 or a nucleotide sequence identical to SEQ ID NO:91 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 97%, at least 98%, or 99% or more) identical.

149. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of ba.1 Omicron variants, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of ba.1 Omicron variants comprises the nucleotide sequence of SEQ ID NO:49 or amino acid sequence identical to SEQ ID NO:49 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70 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 97%, at least 98%, or 99% or more) identical.

150. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of ba.2 Omicron variants, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of ba.2 Omicron variants comprises the nucleotide sequence of SEQ ID NO:64 or an amino acid sequence identical to SEQ ID NO:69 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 97%, at least 98%, or 99% or more) identical, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:70 or a nucleotide sequence identical to SEQ ID NO:70 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 97%, at least 98%, or 99% or more) identical.

151. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of a ba.2.75 Omicron variant, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of a ba.2.75 Omicron variant comprises the nucleotide sequence of SEQ ID NO:80 or an amino acid sequence identical to SEQ ID NO:80 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises SEQ ID NO:81 or a nucleotide sequence identical to SEQ ID NO:81 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 97%, at least 98%, or 99% or more) identical.

152. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of the ba.2.75.2 Omicron variant, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of the ba.2.75.2 Omicron variant comprises the nucleotide sequence of SEQ ID NO:85 or an amino acid sequence identical to SEQ ID NO:85 (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 97%, at least 98%, or 99% or more) identical, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:86 or a nucleotide sequence which hybridizes with SEQ ID NO:86 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 97%, at least 98%, or 99% or more) identical.

153. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of a ba.4.6 or b.7 omicron variant, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of a ba.4.6 or b.7 omicron variant comprises the amino acid sequence of SEQ ID NO:90 or an amino acid sequence identical to SEQ ID NO:90 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:91 or a nucleotide sequence identical to SEQ ID NO:91 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 97%, at least 98%, or 99% or more) identical.

154. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of an XBB Omicron variant, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of an XBB Omicron variant comprises the nucleotide sequence of SEQ ID NO:95 or an amino acid sequence identical to SEQ ID NO:95 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:96 or a nucleotide sequence identical to SEQ ID NO:96 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 97%, at least 98%, or 99% or more) identical.

155. A composition or pharmaceutical formulation comprising a first RNA and a second RNA, wherein:

b) The second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 protein comprising one or more mutations characteristic of the bq.1.1 Omicron variant, wherein (i) the SARS-CoV-2S protein comprising one or more mutations characteristic of the bq.1.1 Omicron variant comprises the nucleotide sequence of SEQ ID NO:100 or an amino acid sequence identical to SEQ ID NO:100 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 97%, at least 98%, or 99% or more) identical amino acid sequence, and/or (ii) the second RNA comprises the amino acid sequence of SEQ ID NO:100 or a nucleotide sequence identical to SEQ ID NO:100 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 97%, at least 98%, or 99% or more) identical.

156. The composition or pharmaceutical preparation of any one of embodiments 133-155, wherein each of the first RNA and the second RNA encodes a SARS-CoV-2S protein comprising one or more mutations that improve expression, stability, and/or immunogenicity.

157. The composition or pharmaceutical formulation of embodiment 156, wherein each of the first RNA and the second RNA encodes a SARS-CoV-2S protein comprising one or more stable prefused conformations.

158. The composition or pharmaceutical formulation of embodiment 157, wherein each of the first RNA and the second RNA is encoded in a sequence corresponding to SEQ ID NO:1 and SARS-CoV-2S protein comprising a proline mutation at the positions of residues 986 and 987 of 1.

159. The composition or pharmaceutical formulation of embodiment 157 or 158, wherein each of the first RNA and the second RNA is encoded in a sequence corresponding to SEQ ID NO:1, SARS-CoV-2S protein comprising one or more proline mutations at positions 817, 892, 899 and/or 942.

160. The composition or pharmaceutical preparation of any one of embodiments 156-159, wherein each of the first RNA and the second RNA encodes a SARS-CoV-2S protein comprising a mutation that prevents furin cleavage.

161. The composition or pharmaceutical formulation of embodiment 160, wherein each of the first RNA and the second RNA is encoded in a sequence corresponding to SEQ ID NO:1 comprises a SARS-CoV-2S protein that comprises a mutation (e.g., a GSAS mutation) that prevents cleavage by furin.

162. The composition or pharmaceutical formulation of any one of embodiments 156-161, wherein each of the first RNA and the second RNA encodes a SARS-CoV-2S protein comprising one or more mutations that reduce S protein shedding (e.g., an aspartic acid mutation to glycine at a position corresponding to residue 614 of SEQ ID NO: 1).

163. The composition or pharmaceutical formulation of any one of embodiments 133-162, wherein each of the first RNA and the second RNA comprises a modified nucleoside in place of uridine.

164. The composition or pharmaceutical formulation of embodiment 163, wherein each of the first RNA and the second RNA comprises a modified nucleoside in place of each uridine.

165. The composition or pharmaceutical formulation of embodiment 163 or 164, wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

166. The composition or pharmaceutical formulation of embodiment 165, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

167. The composition or pharmaceutical formulation of any one of embodiments 133-166, wherein the first RNA and the second RNA each comprise a 5' cap.

168. The composition or pharmaceutical formulation of embodiment 167, wherein the 5' cap is or comprises a cap structure.

169The composition or pharmaceutical formulation of embodiment 168, wherein the 5' -cap is or comprises m ₂ ^7，3’ -OGppp(m ₁ ^2’-O )ApG。

170. The composition or pharmaceutical formulation of any one of embodiments 133-169, wherein the first RNA and the second RNA each comprise a poly (a) sequence.

171. The composition or pharmaceutical formulation of embodiment 170, wherein the poly (a) sequence comprises at least 100 a nucleotides.

172. The composition or pharmaceutical formulation of embodiment 170 or 171, wherein the poly (a) sequence is an interrupt sequence for an a nucleotide.

173. The composition or pharmaceutical formulation of embodiment 172, 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.

174. The composition or pharmaceutical formulation of any one of embodiments 170-173, wherein the poly (a) sequence comprises the amino acid sequence of SEQ ID NO:14 or a nucleotide sequence identical to SEQ ID NO:14, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical thereto.

175. The composition or pharmaceutical formulation of any one of embodiments 133-174, wherein the first RNA and the second RNA each comprise a 5'-UTR that is or comprises a modified human Alpha-globulin 5' -UTR.

176. The composition or pharmaceutical formulation of embodiment 175, wherein the 5' utr comprises SEQ ID NO:12 or a nucleotide sequence which is identical to SEQ ID NO:12, at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

178. The composition or pharmaceutical formulation of any one of embodiments 133-176, wherein each of the first RNA and the second RNA comprises a 3' -UTR that is or comprises a first sequence from a split amino-terminal enhancer (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA.

179. The composition or pharmaceutical formulation of embodiment 178, wherein the first RNA and the second RNA each comprise a 3' utr comprising the amino acid sequence of SEQ ID NO:13 or a nucleotide sequence identical to SEQ ID NO:13, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

180. The composition or pharmaceutical formulation of any one of embodiments 133-179, wherein each of the first RNA and the second RNA is formulated or to be formulated as particles.

181. The composition or pharmaceutical formulation of embodiment 180, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

182. The composition or pharmaceutical formulation of embodiment 181, wherein the LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate.

183. The composition or pharmaceutical formulation of embodiment 181 or 182, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

184. The composition or pharmaceutical formulation of embodiment 181 or 182, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

185. The composition or pharmaceutical formulation of any one of embodiments 182-184, wherein the neutral lipid is present at a concentration ranging from about 5 to about 15 mole percent of the total lipid.

186. The composition or pharmaceutical formulation of any one of embodiments 182-185, wherein the cationically ionizable lipid is present at a concentration in a range of about 40 to about 50 mole% of the total lipid.

187. The composition or pharmaceutical formulation of any one of embodiments 182-186, wherein the sterol is present at a concentration ranging from about 30 to about 50 mole% of the total lipids.

188. The composition or pharmaceutical formulation of any one of embodiments 182-187, wherein the polymer-lipid conjugate is present at a concentration in the range of about 1 to about 1O mole% of total lipid.

189. The composition or pharmaceutical formulation of any one of embodiments 182-188, wherein the lipid nanoparticle comprises about 40 to about 50 mole% of the cationically ionizable lipid; about 5 to about 15 mole% of the neutral lipid; about 35 to about 45 mole% of the sterol; and about 1 to about 10 mole% of the polymer conjugated lipid.

190. The composition or pharmaceutical formulation of embodiment 181, wherein the RNA cationic lipid complex particle is obtainable by mixing the first RNA and the second RNA with a liposome.

191. The composition or pharmaceutical formulation of any one of embodiments 133-190, wherein the first RNA and the second RNA are present in a combined amount ranging from about 1 μg to about 100 μg per dose.

192. The composition or pharmaceutical formulation of any one of embodiments 133-191, wherein the first RNA and the second RNA are present in a combined amount ranging from about 1 μg to about 60 μg per dose.

193. The composition or pharmaceutical formulation of any one of embodiments 133-192, wherein the first RNA and the second RNA are present in a combined amount of about 3.O μg, about 1O μg, about 30 μg, or about 60 μg per dose.

194. The composition or pharmaceutical formulation of any one of embodiments 133-193, wherein the ratio of the first RNA to the second RNA is about 1:10 to about 10:1.

195. The composition or pharmaceutical formulation of any one of embodiments 133-194, wherein the ratio of the first RNA to the second RNA is about 1:1.

196. The composition or pharmaceutical formulation of any one of embodiments 133-195, wherein:

a) The first RNA and the second RNA are each present in the composition in an amount of about 1.5 μg per dose;

b) The first RNA and the second RNA are each present in the composition in an amount of about 5 μg per dose;

c) The first RNA and the second RNA are each present in the composition in an amount of about 15 μg per dose; or (b)

d) The first RNA and the second RNA are each present in the composition in an amount of about 15 μg per dose.

197. The composition or pharmaceutical preparation of any of embodiments 124-169, further comprising an RNA encoding one or more T cell epitopes of SARS-CoV-2.

198. The composition or pharmaceutical preparation of embodiment 170, wherein the RNA encoding one or more T cell epitopes of SARS-CoV-2 comprises one or more epitopes of each of the ORF1ab, M, and N regions of the SARS-CoV-2 genome.

199. The composition or pharmaceutical formulation of any one of embodiments 1-198, formulated or to be formulated as a liquid, solid, or combination thereof.

200. The composition or pharmaceutical formulation of any one of embodiments 1-199, which is formulated or to be formulated for injection.

201. The composition or pharmaceutical formulation of any one of embodiments 1-200, wherein the composition or pharmaceutical formulation is formulated or to be formulated for intramuscular administration.

202. The composition or pharmaceutical formulation of any one of embodiments 1-201, which is a pharmaceutical composition.

203. The composition or pharmaceutical formulation of any one of embodiments 1-202, which is a vaccine.

204. The composition or pharmaceutical formulation of embodiment 202 or 203, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

205. The composition or pharmaceutical formulation of any one of embodiments 1-204, which is a kit.

206. The composition or pharmaceutical formulation of embodiment 205, further comprising instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in a subject.

207. The composition or pharmaceutical formulation of any one of embodiments 1-206, for pharmaceutical use.

208. The composition or pharmaceutical formulation of embodiment 207, wherein the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

209. The composition or pharmaceutical formulation of embodiment 207 or 208, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus infection.

210. The composition or pharmaceutical formulation of embodiment 208 or 209, wherein said coronavirus is sand Bei Bingdu.

211. The composition or pharmaceutical formulation of any one of embodiments 208-211, wherein the coronavirus is a Beta coronavirus.

212. The composition or pharmaceutical preparation of any of embodiments 208-212, wherein the coronavirus is SARS-CoV-2.

213. The composition or pharmaceutical formulation of any one of embodiments 1-212, for administration to a human.

214. A method of eliciting an immune response against SARS-CoV-2 in a subject, comprising administering the composition or pharmaceutical formulation of any one of embodiments 1-213.

215. The method of embodiment 214, wherein the omacron variant against SARS-CoV-2 elicits the immune response.

216. The method of embodiment 214 or 215, wherein the subject has been previously infected with SARS-CoV-2 or vaccinated against SARS-CoV-2.

217. The method of any one of embodiments 214-216, wherein an antigen of the MN908947 strain of SARS-CoV-2 (e.g., as a polypeptide or RNA encoding such polypeptide) has been previously delivered to the subject.

218. The method of any of embodiments 214-217, wherein the subject has previously been administered RNA encoding the SARS-CoV-2S protein of the MN908947 strain.

219. The method of any of embodiments 214-218, wherein the subject has previously been administered two or more doses of RNA encoding SARS-CoV-2S protein of the MN908947 strain.

220. The method of embodiment 219, wherein the RNA of any one of embodiments 1-206 is administered at least about 2 months after the two or more doses of RNA encoding SARS-CoV-2S protein of MN908947 strain.

221. The method of any one of embodiments 214-220, wherein the method comprises administering RNA encoding an antigen of a SARS-CoV-2 virus that is not a ba.1 omacron variant.

222. The method of any one of embodiments 214-221, wherein the method comprises administering an RNA comprising a nucleotide sequence encoding a SARS-Cov-2S protein, said SARS-Cov-2S protein comprising one or more mutations characteristic of an omacron variant, wherein the omacron variant is not a ba.1 omacron variant.

223. The method of any of embodiments 214-222, wherein the method comprises administering an RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein, said SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.2 omacron variant.

224. The method of any of embodiments 214-222, wherein the method comprises administering an RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein, said SARS-CoV-2S protein comprising one or more mutations that are characteristic of a ba.4 or ba.5 omacron variant.

225. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein of the MN908947 strain, or comprising one or more mutations that are characteristic of Alpha, beta, or Delta variants, and (b) a second RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations that are not characteristic of an Omicron variant of the ba.1 Omicron variant.

226. The method of embodiment 225, wherein the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein of a ba.2 omacron variant.

227. The method of embodiment 225, wherein the second RNA comprises a nucleotide sequence encoding an S protein comprising one or more mutations characteristic of a ba.4 or ba.5qmicron variant.

228. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein of an MN908947 strain, or comprising one or more mutations characteristic of Alpha variants, beta variants, delta variants, or ba.1 omacron variants, and (b) a second RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein that is antigenically different from the S protein encoded by the first RNA.

229. The method of embodiment 228, wherein the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations that are not characteristic of an Omicron variant of a ba.1 Omicron variant.

230. The method of embodiment 228, wherein the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of a ba.2 omacron variant.

231. The method of embodiment 228, wherein the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2S protein comprising one or more mutations characteristic of ba.4 or ba.5 omacron variants.

232. The method of any one of embodiments 214-231, wherein said first RNA and said second RNA are encapsulated in separate LNPs.

233. The method of any one of embodiments 214-231, wherein said first RNA and said second RNA are encapsulated in the same LNP.

234. The method of any one of embodiments 214-232, wherein said first RNA and said second RNA are administered separately, e.g., at different injection sites.

235. The method of any of embodiments 214-234, further comprising administering an RNA comprising a nucleotide sequence encoding one or more T cell epitopes of SARS-CoV-2.

236. The method of embodiment 235, wherein the nucleotide sequence encoding one or more T cell epitopes of SARS-CoV-2 encodes one or more epitopes of each of the ORF1 ab, M and N regions of the SARS-CoV-2 genome.

237. The method of embodiment 235 or 236, wherein said first RNA, said second RNA, and said RNA encoding one or more T cell epitopes of SARS-CoV-2 are each formulated in separate LNPs.

238. The method of embodiment 235 or 236, wherein the first RNA and the second RNA are co-formulated in the same LNP and the RNA encoding one or more T cell epitopes of SARS-CoV-2 are formulated in separate LNPs.

239. The method of embodiment 235 or 236, wherein each of said first RNA, said second RNA, and said RNA encoding one or more T cell epitopes of SARS-CoV-2 are co-formulated in the same LNP.

240. The method of any one of embodiments 235-239, wherein the first RNA, the second RNA, and the RNA encoding one or more T cell epitopes of SARS-CoV-2 are administered at a dose comprising:

(a) 30 μg of the first RNA and the second RNA in combination (e.g., 15 μg of the first RNA and 15 μg of the second RNA) and 5 μg of the RNA encoding one or more T cell epitopes of SARS-CoV-2;

(b) 30 μg of the first RNA and the second RNA in combination (e.g., 15 μg of the first RNA and 15 μg of the second RNA), 10 μg of the RNA encoding one or more T cell epitopes of SARS-CoV-2; or (b)

(c) 30pg of the first RNA and the second RNA in combination (e.g., 15pg of the first RNA and 15pg of the second RNA) and 15pg of the RNA encoding one or more T cell epitopes of SARS-CoV-2.

241. The method of any one of embodiments 235-240, wherein RNA encoding one or more T cell epitopes of SARS-CoV-2 encodes SEQ ID NO: polypeptide sequence of RS C7p2 full.

242. The method of any of embodiments 207-241, further comprising administering one or more vaccines against respiratory diseases.

243. The method of embodiment 242, wherein the respiratory disease is RSV or influenza.

244. The method of embodiment 242 or 243, comprising administering a vaccine against RSV and a vaccine against influenza.

245. The method of any of embodiments 242-244, wherein the one or more vaccines against respiratory diseases are RNA vaccines each comprising one or more RNAs encoding antigenic proteins.

246. The method of embodiment 245, comprising administering one or more RNAs encoding antigens from influenza virus and/or one or more RNAs encoding antigens from RSV.

247. The method of embodiment 246, wherein each RNA is formulated in a separate LNP.

248. The method of embodiment 246, wherein each RNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof is co-formulated in the same LNP, each RNA encoding an antigenic protein from influenza virus is co-formulated in the same LNP, and each RNA encoding an antigenic protein from RSV is co-formulated in the same LNP.

249. The method of embodiment 246, wherein each RNA encoding SARS-CoV-2S protein or immunogenic fragment thereof, each RNA encoding an antigenic protein from influenza virus, and each RNA encoding an antigenic protein from RSV are co-formulated together in the same LNP.

250. A composition or pharmaceutical formulation comprising RNA encoding an amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof.

251. The composition or pharmaceutical preparation of embodiment 250, wherein the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

252. The composition or pharmaceutical formulation of embodiment 250 or 251, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

253. The composition or pharmaceutical formulation of any one of embodiments 250 to 252, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9, nucleotides 979 to 1584, and 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 the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2. 8 or 9 from nucleotide 979 to 1584 or a nucleotide sequence identical to SEQ ID NO: 2. 8 or 9, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1, amino acid sequence of amino acids 327 to 528, which corresponds to SEQ ID NO:1, or the amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1 or amino acid sequence of amino acids 327 to 528 of SEQ ID NO:1, and an immunogenic fragment of 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.

254. The composition or pharmaceutical formulation of any one of embodiments 250-253, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:30, and nucleotide sequence of nucleotides 111 to 986, which corresponds to SEQ ID NO:30 or 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, or the nucleotide sequence of SEQ ID NO:30 or a nucleotide sequence identical to nucleotides 111 to 986 of SEQ ID NO:30, a fragment of 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; and/or

(ii)SARS-C _o The V-2S protein, immunogenic variant thereof, or immunogenic fragment of said SARS-CoV-2S protein or immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or amino acid sequence corresponding to amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

255. The composition or pharmaceutical formulation of any one of embodiments 250 to 254, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO: 2. 8 or 9 from nucleotide 49 to 3819, and 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 the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2. 8 or 9 or a nucleotide sequence that is identical to nucleotide 49 to 3819 of SEQ ID NO: 2. 8 or 9, and a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 3819; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:1 or 7, amino acid sequence of amino acids 17 to 1273, which corresponds to 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 the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO:1 or 7 or amino acid sequence of amino acids 17 to 1273 or amino acid sequence identical to SEQ ID NO:1 or 7, and an immunogenic fragment of 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.

256. The composition or pharmaceutical formulation of any one of embodiments 250 to 255, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a secretion signal peptide.

257. The composition or pharmaceutical formulation of embodiment 256, wherein the secretion signal peptide is fused to a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof, preferably an N-terminal fusion.

258. The composition or pharmaceutical formulation of embodiment 256 or 257, wherein

(i) The RNA encoding the secretion signal peptide comprises SEQ ID NO: 2. 8 or 9, and the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2. 8 or 9, or the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2. 8 or 9 or a nucleotide sequence identical to nucleotides 1 to 48 of SEQ ID NO: 2. 8 or 9, the nucleotide sequence of nucleotides 1 to 48 having a fragment of the nucleotide sequence of at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity; and/or

(ii) The secretion signal peptide comprises SEQ ID NO:1, and amino acid sequence of amino acids 1 to 16 of SEQ ID NO:1, or the amino acid sequence of SEQ ID NO:1 or amino acid sequence corresponding to amino acids 1 to 16 of SEQ ID NO:1, the amino acid sequence of amino acids 1 to 16 has a functional fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

259. The composition or pharmaceutical formulation of any one of embodiments 250 to 258, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:6, and SEQ ID NO:6, 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:6 or a nucleotide sequence identical to SEQ ID NO:6, a fragment of a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:5, and 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 or an amino acid sequence identical to SEQ ID NO:5, an immunogenic fragment of an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the amino acid sequence.

260. The composition or pharmaceutical formulation of any one of embodiments 250 to 259, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:30, and nucleotide sequence of nucleotides 54 to 986, which hybridizes with SEQ ID NO:30, or 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 nucleotide sequence that hybridizes to nucleotides 54 to 986 of SEQ ID NO:30, a fragment of 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; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 1 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 1 to 311 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or amino acid sequence corresponding to amino acids 1 to 311 of SEQ ID NO:29, amino acid 1 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

261. The composition or pharmaceutical formulation of any one of embodiments 250 to 258, wherein the RNA comprises a modified nucleoside in place of uridine, particularly wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U), particularly wherein the modified nucleoside is N1-methyl-pseudouridine (m 1 ψ).

262. The composition or pharmaceutical formulation of any one of embodiments 250 to 261, wherein the RNA comprises a 5' cap.

263. The composition or pharmaceutical formulation of any one of embodiments 250 to 262, wherein RNA encoding an amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a 5' utr comprising the amino acid sequence of SEQ ID NO:12 or a nucleotide sequence identical to SEQ ID NO:12, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

264. The composition or pharmaceutical formulation of any one of embodiments 250 to 263, wherein the RNA encoding the amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a 3' utr comprising the amino acid sequence of SEQ ID NO:13 or a nucleotide sequence identical to SEQ ID NO:13, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

265. The composition or pharmaceutical formulation of any one of embodiments 250 to 264, wherein the RNA encoding the amino acid sequence that comprises the SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises a poly a sequence.

266. The composition or pharmaceutical formulation of embodiment 265, wherein the poly a sequence comprises at least 100 nucleotides.

267. The composition or pharmaceutical formulation of embodiment 265 or 266, wherein the poly a sequence comprises the amino acid sequence of SEQ ID NO:14 or a nucleotide sequence consisting of SEQ ID NO: 14.

268. The composition or pharmaceutical formulation of any one of embodiments 250 to 267, wherein the RNA is formulated or to be formulated as a liquid, a solid, or a combination thereof.

269. The composition or pharmaceutical formulation of any one of embodiments 250 to 268, wherein the RNA is or is to be formulated for injection.

270. The composition or pharmaceutical formulation of any one of embodiments 250 to 269, wherein the RNA is formulated or to be formulated for intramuscular administration.

271. The composition or pharmaceutical formulation of any one of embodiments 250 to 270, wherein the RNA is formulated or to be formulated as particles.

272. The composition or pharmaceutical formulation of embodiment 271, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

273. The composition or pharmaceutical formulation of embodiment 272, wherein the LNP particles comprise ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), 2- [ (polyethylene glycol) -2000] -N, N-bis-tetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

274. The composition or pharmaceutical formulation of embodiment 272, wherein the RNA cationic lipid complex particles are obtainable by mixing the RNA with a liposome.

275. The composition or pharmaceutical formulation of any one of embodiments 250 to 274, wherein the RNA is mRNA or saRNA.

276. The composition or pharmaceutical formulation of any one of embodiments 250 to 275, which is a pharmaceutical composition.

277. The composition or pharmaceutical formulation of any one of embodiments 250 to 276, which is a vaccine.

278. The composition or pharmaceutical formulation of embodiment 276 or 277, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

279. The composition or pharmaceutical formulation of any one of embodiments 250 to 275, which is a kit.

280. The composition or pharmaceutical formulation of embodiment 279, wherein the RNA and optional particle-forming component are in separate vials.

281. The composition or pharmaceutical formulation of embodiment 279 or 280, further comprising instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in a subject.

282. The composition or pharmaceutical formulation of any one of embodiments 250 to 281 for pharmaceutical use.

283. The composition or pharmaceutical formulation of embodiment 282, wherein the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

284. The composition or pharmaceutical formulation of embodiment 282 or 283, wherein the pharmaceutical use comprises therapeutic or prophylactic treatment of a coronavirus infection.

285. The composition or pharmaceutical formulation of any one of embodiments 250 to 284, for administration to a human.

286. The composition or pharmaceutical formulation of any one of embodiments 282 to 285, wherein the coronavirus is a Beta coronavirus.

287. The composition or pharmaceutical formulation of any one of embodiments 282 to 286, wherein the coronavirus is sand Bei Bingdu.

288. The composition or pharmaceutical formulation of any one of embodiments 282 to 287, wherein the coronavirus is SARS-CoV-2.

289. A method of inducing an immune response against a coronavirus in a subject, comprising administering to the subject a composition comprising RNA encoding an amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof.

290. The method of embodiment 289, wherein the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

291. The method of embodiment 289 or 290, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content compared to a wild-type coding sequence, wherein said codon optimization and/or said increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

292. The method of any one of embodiments 289-291,

293. The method of any one of embodiments 289-292, wherein

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the amino acid sequence of SEQ ID NO:29, amino acid sequence of amino acids 20 to 311, which corresponds to SEQ ID NO:29, or the amino acid sequence of amino acids 20 to 311 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or the amino acid sequence of SEQ ID NO:29 or amino acid sequence corresponding to amino acids 20 to 311 of SEQ ID NO:29, and the amino acid sequence of amino acids 20 to 311 has an immunogenic fragment of an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

294. The method of any one of embodiments 289-293, wherein

295. The method of any one of embodiments 289-294, wherein an amino acid sequence comprising a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a secretion signal peptide.

296. The method of embodiment 295, wherein the secretion signal peptide is fused to a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof, preferably an N-terminal fusion.

297. The method of embodiment 295 or 297, wherein

298. The method of any one of embodiments 289-297, wherein

299. The method of any one of embodiments 289-298, wherein

300. The method of any one of embodiments 289-298, wherein said RNA comprises a modified nucleoside in place of uridine, particularly wherein said modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U), particularly wherein said modified nucleoside is N1-methyl-pseudouridine (m 1 ψ).

301. The method of any one of embodiments 289-300, wherein the RNA comprises a cap.

302. The method of any one of embodiments 289-301, wherein an RNA encoding an amino acid sequence constituting a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a 5' utr comprising the amino acid sequence of SEQ ID NO:12 or a nucleotide sequence identical to SEQ ID NO:12, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

303. The method of any one of embodiments 289-302, wherein an RNA encoding an amino acid sequence constituting a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a 3' utr comprising the amino acid sequence of SEQ ID NO:13 or a nucleotide sequence identical to SEQ ID NO:13, has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.

304. The method of any one of embodiments 289-303, wherein an RNA encoding an amino acid sequence constituting a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a poly a sequence.

305. The method of embodiment 304, wherein the poly-a sequence comprises at least 100 nucleotides.

306. The method of embodiment 304 or 305, wherein the poly a sequence comprises SEQ ID NO:14 or a nucleotide sequence consisting of SEQ ID NO: 14.

307. The method of any one of embodiments 289-306, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

308. The method of any one of embodiments 289-307, wherein the RNA is administered by injection.

309. The method of any one of embodiments 289-308, wherein the RNA is administered by intramuscular administration.

310. The method of any one of embodiments 289-309, wherein the RNA is formulated as particles.

311. The method of embodiment 310, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

312. The method of embodiment 311 wherein the LNP particles comprise ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

313. The method of any one of embodiment 311, wherein said RNA cationic lipid complex particle is obtainable by mixing said RNA with a liposome.

314. The composition or pharmaceutical formulation of any one of embodiments 289-313, wherein the RNA is mRNA or saRNA.

315. The method of any one of embodiments 289-314, which is a method of vaccinating against coronavirus.

316. The method of any one of embodiments 289-315, which is a method for therapeutic or prophylactic treatment of a coronavirus infection.

317. The method of any one of embodiments 289-316, wherein the subject is a human.

318. The method of any one of embodiments 289-317, wherein the coronavirus is a Beta coronavirus.

319. The method of any one of embodiments 289-318, wherein the coronavirus is sand Bei Bingdu.

320. The method of any one of embodiments 289-319, wherein the coronavirus is SARS-CoV-2.

321. The method of any one of embodiments 289-320, wherein the composition is the composition of any one of embodiments 1-39.

322. The composition or pharmaceutical formulation of any one of embodiments 250 to 288 for use in the method of any one of embodiments 289 to 320.

323. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising RNA, wherein said RNA encodes SEQ ID NO:49, and comprises the polypeptide of SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

Wherein the LNP comprises ((4-hydroxybutyl) azetidinediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

324. The immunogenic composition of embodiment 323, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

325. The immunogenic composition of embodiment 323 or 324, wherein each of the modified uridine is N1-methyl-pseudouridine.

326. The immunogenic composition of any one of embodiments 323-325, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

327. The immunogenic composition of embodiment 326, 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.

328. The immunogenic composition of embodiment 326, wherein the poly a sequence comprises SEQ ID NO:14.

329. the immunogenic composition of any one of embodiments 323-328, wherein the RNA comprises SEQ ID NO:51.

330. an immunogenic composition comprising a Lipid Nanoparticle (LNP) comprising RNA, wherein the RNA encodes SEQ ID NO:55, and comprises the polypeptide of SEQ ID NO:56 or a nucleotide sequence identical to SEQ ID NO:56 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

331. The immunogenic composition of embodiment 330, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

332. The immunogenic composition of embodiment 330 or 331, wherein the modified uridine is each N1-methyl-pseudouridine.

333. The immunogenic composition of any one of embodiments 330-332, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

334. The immunogenic composition of embodiment 333, 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.

335. The immunogenic composition of embodiment 334, wherein the poly a sequence comprises SEQ ID NO:14.

336. The immunogenic composition of any one of embodiments 330-335, wherein the RNA comprises the amino acid sequence of SEQ ID NO:57.

337. an immunogenic composition comprising a Lipid Nanoparticle (LNP) comprising RNA, wherein the RNA encodes SEQ ID NO:58, and comprises the polypeptide of SEQ ID NO:59 or a nucleotide sequence identical to SEQ ID NO:59 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

338. The immunogenic composition of embodiment 337, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

339. The immunogenic composition of embodiment 337 or 338, wherein the modified uridine is each N1-methyl-pseudouridine.

340. The immunogenic composition of any one of embodiments 337-339, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

341. The immunogenic composition of embodiment 340, 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.

342. The immunogenic composition of embodiment 341, wherein the poly a sequence comprises SEQ ID NO:14.

343. the immunogenic composition of any one of embodiments 337-342, wherein the RNA comprises SEQ ID NO:60.

344. an immunogenic composition comprising a Lipid Nanoparticle (LNP) comprising RNA, wherein the RNA encodes SEQ ID NO:61 and comprises the polypeptide of SEQ ID NO:62a or a nucleotide sequence identical to SEQ ID NO:62a 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 97%, at least 98%, or 99% or more) identical, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

345. The immunogenic composition of embodiment 344, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

346. The immunogenic composition of embodiment 344 or 345, wherein the modified uridine is each N1-methyl-pseudouridine.

347. The immunogenic composition of any one of embodiments 344-346, wherein the RNA further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

348. The immunogenic composition of embodiment 347, 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.

349. The immunogenic composition of embodiment 348, wherein the poly a sequence comprises SEQ ID NO:14.

350. The immunogenic composition of any one of embodiments 344-349, wherein the RNA comprises the amino acid sequence of SEQ ID NO:63a.

351. An immunogenic composition comprising a first RNA and a second RNA, wherein:

the first RNA encodes SEQ ID NO:7, and comprises the polypeptide of SEQ ID NO:9 or a nucleotide sequence identical to SEQ ID NO:9 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 97%, at least 98% or 99% or more) identical, and

the second RNA encodes SEQ ID NO:49, and comprises the polypeptide of SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 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 97%, at least 98% or 99% or more) of the same nucleotide sequence, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

wherein the first RNA and the second RNA are formulated in a Lipid Nanoparticle (LNP), wherein the LNP comprises ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylamide, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and cholesterol.

352. The immunogenic composition of embodiment 351, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

353. The immunogenic composition of embodiment 351, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

354. The immunogenic composition of any one of embodiments 351-353, wherein each of the first RNA and the second RNA comprises a modified uridine instead of all uridine.

355. The immunogenic composition of any one of embodiments 351-354, wherein the modified uridine is each N1-methyl-pseudouridine.

356. The immunogenic composition of any one of embodiments 351-355, wherein each of the first RNA and the second RNA independently comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

357. The immunogenic composition of embodiment 357, 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.

358. The immunogenic composition of embodiment 357, wherein the poly a sequence comprises SEQ ID NO:14.

359. the immunogenic composition of any one of embodiments 351-358, wherein the first RNA comprises SEQ ID NO:20, and the second RNA comprises SEQ ID NO:51.

360. an immunogenic composition comprising a first RNA and a second RNA, wherein:

the second RNA encodes SEQ ID NO: 55. 58 or 61, and comprises the polypeptide of SEQ ID NO: 56. 59 or 62a or a nucleotide sequence identical to SEQ ID NO: 56. 59 or 62a 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 97%, at least 98%, or 99% or more) identical, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

361. The immunogenic composition of embodiment 360, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

362. The immunogenic composition of embodiment 360, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

363. The immunogenic composition of any one of embodiments 360-362, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

364. The immunogenic composition of any one of embodiments 360-363, wherein the modified uridine is each N1-methyl-pseudouridine.

365. The immunogenic composition of any one of embodiments 360-364, wherein the first RNA and the second RNA each independently further comprise at least one, at least two, or all of the following features:

Comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

366. The immunogenic composition of any one of embodiments 360-365, 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.

367. The immunogenic composition of embodiment 366, wherein the poly a sequence comprises SEQ ID NO:14.

368. the immunogenic composition of any one of embodiments 360-367, wherein the first RNA comprises SEQ ID NO:9, and the second RNA comprises SEQ ID NO:56.

369. the immunogenic composition of any one of embodiments 360-368, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:9, and the second RNA comprises SEQ ID NO:59.

370. the immunogenic composition of any one of embodiments 360-368, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:9, and the second RNA comprises SEQ ID NO:62a.

371. The immunogenic composition of any one of embodiments 360-368, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:20, and the second RNA comprises SEQ ID NO:57.

372. The immunogenic composition of any one of embodiments 360-368, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:20, and the second RNA comprises SEQ ID NO:60.

373. the immunogenic composition of any one of embodiments 360-368, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:20, and the second RNA comprises SEQ ID NO:63a.

374. An immunogenic composition comprising a first RNA and a second RNA, wherein:

the first RNA encodes SEQ ID NO:58, and comprises the polypeptide of SEQ ID NO:59 or a nucleotide sequence identical to SEQ ID NO:59 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 97%, at least 98% or 99% or more) identical, and

the second RNA encodes SEQ ID NO: 49. 55 or 61, and comprises the polypeptide of SEQ ID NO: 50. 56 or 62a or a nucleotide sequence identical to SEQ ID NO: 50. 56 or 62a (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 97%, at least 98%, or 99% or more) identical, and

Wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

375. The immunogenic composition of embodiment 374, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

376. The immunogenic composition of embodiment 374, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

377. The immunogenic composition of any one of embodiments 374-376, wherein each of the first RNA and the second RNA comprises a modified uridine instead of all uridine.

378. The immunogenic composition of any one of embodiments 374-377, wherein the modified uridine are each N1-methyl-pseudouridine.

379. The immunogenic composition of any one of embodiments 374-378, wherein each of the first RNA and the second RNA, independently, further comprises at least one, at least two, or all of the following features:

Comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

380. The immunogenic composition of embodiment 379, 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.

381. The immunogenic composition of embodiment 379, wherein the poly a sequence comprises SEQ ID NO:14.

382. the immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:59, and the second RNA comprises SEQ ID NO:50.

383. the immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:59, and the second RNA comprises SEQ ID NO:56.

384. the immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:59, and the second RNA comprises SEQ ID NO:62a.

385. The immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:60, and the second RNA comprises SEQ ID NO:51.

386. The immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:60, and the second RNA comprises SEQ ID NO:57.

387. the immunogenic composition of any one of embodiments 374-381, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:60, and the second RNA comprises SEQ ID NO:63a.

69. An immunogenic composition comprising a first RNA and a second RNA, wherein:

the first RNA encodes SEQ ID NO:49, and comprises the polypeptide of SEQ ID NO:50 or a nucleotide sequence identical to SEQ ID NO:50 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 97%, at least 98% or 99% or more) of the same nucleotide sequence, and

the second RNA encodes SEQ ID NO:55 or 61, and comprises the polypeptide of SEQ ID NO:56 or 62a or a nucleotide sequence identical to SEQ ID NO:56 or 62a (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 97%, at least 98%, or 99% or more) identical, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

389. The immunogenic composition of embodiment 388, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

390. The immunogenic composition of embodiment 388, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

391. The immunogenic composition of any one of embodiments 388-390, wherein each of the first RNA and the second RNA comprises a modified uridine instead of all uridine.

392. The immunogenic composition of any one of embodiments 388-391, wherein the modified uridine is each N1-methyl-pseudouridine.

393. The immunogenic composition of any one of embodiments 388-392, 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:

Comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

394. The immunogenic composition of embodiment 393, 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.

395. The immunogenic composition of embodiment 393, wherein the poly a sequence comprises the amino acid sequence of SEQ ID NO:14.

396. the immunogenic composition of any one of embodiments 388-395, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:50, and the second RNA comprises SEQ ID NO:56.

397. the immunogenic composition of any one of embodiments 388-395, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:50, and the second RNA comprises SEQ ID NO:62a.

398. The immunogenic composition of any one of embodiments 388-395, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:51, and the second RNA comprises SEQ ID NO:57.

399. the immunogenic composition of any one of embodiments 388-395, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:51, and the second RNA comprises SEQ ID NO:63a.

400. An immunogenic composition comprising a first RNA and a second RNA, wherein:

the first RNA encodes SEQ ID NO:55, and comprises the polypeptide of SEQ ID NO:56 or a nucleotide sequence identical to SEQ ID NO:56 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 97%, at least 98% or 99% or more) identical, and

the second RNA encodes SEQ ID NO:61 and comprises the polypeptide of SEQ ID NO:62a or a nucleotide sequence identical to SEQ ID NO:62a 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 97%, at least 98% or 99% or more) identical, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

401. The immunogenic composition of embodiment 400, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

402. The immunogenic composition of embodiment 400, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

403. The immunogenic composition of any one of embodiments 400-402, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

404. The immunogenic composition of any one of embodiments 400-403, wherein the modified uridine is each N1-methyl-pseudouridine.

405. The immunogenic composition of any one of embodiments 400-404, wherein each of the first RNA and the second RNA, independently, further comprises at least one, at least two, or all of the following features:

comprising SEQ ID NO:12 (UTR);

comprising SEQ ID NO:13 (UTR); and

a poly a sequence of at least 100 a nucleotides.

406. The immunogenic composition of embodiment 405, 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.

407. The immunogenic composition of embodiment 405, wherein the poly a sequence comprises SEQ ID NO:14.

408. the immunogenic composition of any one of embodiments 400-407, wherein the first RNA comprises the amino acid sequence of SEQ ID NO:57, and the second RNA comprises SEQ ID NO:63a.

409. The immunogenic composition of any one of embodiments 323-408, wherein the 5' -cap is or comprises m ₂ ^7，3’-O Gppp(m ₁ ^2’-O )ApG。

410. The immunogenic composition of any one of embodiments 323-409 wherein the LNP comprises about 40 to about 50 mole% ((4-hydroxybutyl) azanediyl) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate), about 35 to about 45 mole% cholesterol, about 5 to about 15 mole% 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, and about 1 to about 10 mole% 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide.

411. The immunogenic composition of any one of embodiments 323-410, wherein the composition comprises a plurality of LNPs, wherein the plurality of LNPs has an average diameter of about 30nm to about 200nm or about 60nm to about 120nm (e.g., as determined by dynamic light scattering measurements).

412. A method of eliciting an immune response against SARS-CoV-2, comprising administering the immunogenic composition of any one of embodiments 74-162.

413. The method of embodiment 413, wherein the omacron variant against SARS-CoV-2 elicits the immune response.

414. The method of embodiment 412, wherein the Beta variant against SARS-CoV-2 elicits the immune response.

415. The method of embodiment 412, wherein the Alpha variant against SARS-CoV-2 elicits the immune response.

416. The method of embodiment 412, wherein the Delta variant against SARS-CoV-2 elicits the immune response.

417. The method of embodiment 412, wherein the MN908947 strain, omacron variant, beta variant, alpha variant and Delta variant directed against SARS-CoV-2 elicit the immune response.

418. A method of inducing an immune response in a subject, wherein the method comprises delivering (e.g., as a polypeptide or RNA encoding such polypeptide) an antigen of a SARS-CoV-2 virus that is not a ba.1 omacron variant of SARS-CoV-2.

419. The method of embodiment 418, wherein the subject has been previously infected with SARS-CoV-2 or vaccinated against SARS-CoV-2.

420. The method of embodiment 418 or 419, wherein the subject has previously been delivered (e.g., as a polypeptide or RNA encoding such polypeptide) an antigen of the MN908947 strain of SARS-CoV-2.

421. The method of any of embodiments 418-420, wherein the subject has previously been administered RNA encoding SARS-CoV-2S protein of the MN908947 strain.

422. The method of any of embodiments 418-421, wherein the subject has previously been administered two or more doses of RNA encoding SARS-CoV-2S protein of the MN908947 strain.

423. The method of any one of embodiments 418-422, wherein the method comprises administering RNA encoding an antigen of a SARS-CoV-2 virus that is not a ba.1 omacron variant.

424. The method of any of embodiments 418-423, wherein the method comprises administering RNA encoding SARS-CoV-2S protein from a SARS-CoV-2 variant that is a ba.1 omacron variant.

425. The method of any one of embodiments 418-424, wherein the method comprises administering RNA encoding the S protein of an Omicron variant of SARS-CoV-2, wherein the Omicron variant is not a ba.1 Omicron variant.

426. The method of any of embodiments 418-425, wherein the method comprises administering RNA encoding the S protein of the ba.2 omacron variant of SARS-CoV-2.

427. The method of any of embodiments 418-425, wherein the method comprises administering RNA encoding the S protein of ba.4 or ba.5 omacron variant of SARS-CoV-2.

428. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, or Delta variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is not an Omicron variant of a ba.1 Omicron variant.

429. The method of embodiment 428, wherein the second RNA encodes an S protein of a ba.2omicron variant.

430. The method of embodiment 428, wherein the second RNA encodes an S protein of ba.4 or ba.5 omacron variant.

431. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 omacron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is antigenically different from the S protein encoded by the first RNA.

432. The method of embodiment 431, wherein the second RNA encodes a SARS-CoV-2S protein that is not an omacron variant of the ba.1 omacron variant.

433. The method of embodiment 431, wherein the second RNA encodes a SARS-CoV-2S protein of the ba.2omicron variant.

434. The method of embodiment 431, wherein the second RNA encodes a SARS-CoV-2S protein of ba.4 or ba.5 omacron variant.

435. The method of any one of embodiments 418-434, wherein the first RNA and the second RNA are encapsulated in separate LNPs.

436. The method of any one of embodiments 418-435, wherein the first RNA and the second RNA are encapsulated in the same LNP.

437. The method of any one of embodiments 418-436, wherein said first RNA and said second RNA are administered separately, e.g., at different injection sites.

438. A composition comprising (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 omacron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is antigenically different from the S protein encoded by the first RNA.

439. A composition comprising (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 Omicron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is not an Omicron variant of a ba.1 Omicron variant.

440. The composition of embodiment 439, wherein said second RNA encodes SARS-CoV-2S protein of the ba.2 omacron variant.

441. The composition of embodiment 49, wherein said second RNA encodes SARS-CoV-2S protein of the BA.4 or BA.5 Omacron variant.

442. The composition of embodiment 439, wherein said first RNA encodes SARS-CoV-2S protein of the MN908947 strain and said second RNA encodes SARS-CoV-2S protein of the ba.2 omacron variant.

443. The composition of embodiment 439, wherein said first RNA encodes SARS-CoV-2S protein of the MN908947 strain and said second RNA encodes SARS-CoV-2S protein of a ba.4 or ba.5 Omicron variant.

444. The composition of embodiment 439, wherein said first RNA encodes a SARS-CoV-2S protein of a ba.1 Omicron variant and said second RNA encodes a SARS-CoV-2S protein of a ba.2Omicron variant.

445. The composition of embodiment 439, wherein said first RNA encodes a SARS-CoV-2S protein of a ba.1 Omicron variant and said second RNA encodes a SARS-CoV-2S protein of a ba.4 or ba.5 Omicron variant.

446. The composition of any one of embodiments 438-445, wherein the first RNA and the second RNA are administered in a form encapsulated in the same LNP.

447. The composition of any one of embodiments 438-446, wherein said first RNA and said second RNA are encapsulated in separate LNPs.

448. The method of any one of embodiments 214 to 249, 289 to 321, or 412 to 437, wherein the composition is the composition of any one of embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448.

448. The composition or pharmaceutical formulation of any one of embodiments 1-213, 250-288, 322-411, or 438-448 for use in the method of any one of embodiments 214-249, 289-321, or 412-437.

449. The composition or pharmaceutical formulation of any one of embodiments 1-213, 250-288, 322-411, or 438-448, which is a pharmaceutical composition.

450. The composition or pharmaceutical formulation of any one of embodiments 1-213, 250-288, 322-411, or 438-448, which is a vaccine.

451. The composition or pharmaceutical formulation of embodiment 449 or 450, wherein the pharmaceutical composition or vaccine further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

452. The composition or pharmaceutical formulation of any one of embodiments 1-213, 250-288, 322-411, or 438-448, which is a kit.

453. The composition or pharmaceutical formulation of embodiment 452, wherein the RNA and optionally the particle-forming component are in separate vials.

454. The composition or pharmaceutical formulation of embodiment 452 or 453, further comprising instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in a subject.

455. The composition or pharmaceutical formulation of any one of embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448 for pharmaceutical use.

456. The composition or pharmaceutical formulation of embodiment 455, wherein the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

457. The composition or pharmaceutical formulation of embodiments 455 or 456, wherein said pharmaceutical use comprises therapeutic or prophylactic treatment of a coronavirus infection.

458. A composition or pharmaceutical formulation as described in any one of embodiments 1 to 213, 250 to 288, 322 to 411 or 438 to 448 for use in the preparation of a medicament.

459. The composition or pharmaceutical formulation of embodiment 458, wherein the medicament is for inducing an immune response against a coronavirus in a subject.

460. The composition or pharmaceutical formulation of embodiment 458 or 459, wherein the medicament is for the therapeutic or prophylactic treatment of a coronavirus infection.

461. The composition or pharmaceutical formulation of any one of embodiments 1 to 213, 250 to 288, 322 to 411, or 438 to 448 for administration to a human.

462. The composition or pharmaceutical formulation of any one of embodiments 454, 456, 457, 459, or 460, wherein the coronavirus is a Beta coronavirus.

463. The composition or pharmaceutical formulation of embodiment 462, wherein the coronavirus is sand Bei Bingdu.

464. The composition or pharmaceutical preparation of embodiment 463, wherein the coronavirus is SARS-CoV-2.

Citation of documents and studies herein is not intended as an admission that any of the foregoing is prior art with respect to the present document. All statements as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the contents of these documents.

The following description is presented to enable one of ordinary skill in the art to make and use various embodiments. Descriptions of specific devices, techniques and applications are provided only as examples. Various modifications to the embodiments described herein will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and illustrated herein, but are to be accorded the scope consistent with the claims.

Examples

Example 1: immunogenicity studies of BNT162b3 variants BNT162b3c and BNT162b3d

To understand the potential efficacy of transmembrane anchored RBD-based vaccine antigens (schematic in FIG. 6; BNT162b3C (1) and BNT162b3d (2)), BALB/C mice were immunized intramuscularly with 4. Mu.g of LNP-C12 formulated mRNA or with buffer as control. Non-clinical LNP-C12 formulated mRNA was used as a surrogate for BNT162b3 variant BNT162b3C and BNT162b3 d. The immunogenicity of RNA vaccines is investigated by focusing on the antibody immune response.

ELISA data at days 6, 14 and 21 after the first immunization showed early dose-dependent immune activation against the S1 protein and receptor binding domain (FIG. 7). Serum obtained 6, 14 and 21 days after immunization showed high neutralization of SARS-CoV-2 pseudovirus, associated with an increase in IgG antibody titer (fig. 8).

Example 2: neutralization of SARS-CoV-2 BA.1Omicron lineage (also referred to as B.1.1.529) pseudoviruses by human serum elicited by BNT162b2 vaccine

Materials and methods:

according to the disclosed pseudotyping protocol, SARS-CoV-2 spike (S) was isolated with the Wuhan-Hu-1 isolate (GenBank: QHD 43416.1) and variant spikes with mutations (a 67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N505 527 547R, N614R, N655 856 679 856R, N764R, N796R, N954R, N9537 969 5297 969 981F) present in the S protein of the omacron (b.1.1.529) ba.1 lineage pseudotype recombinant replication defective VSV vectors encoding Green Fluorescent Protein (GFP) and luciferase (Luc) but not VSV-glycoprotein (VSV-G). Briefly, HEK293T/17 monolayers transfected to express the corresponding SARS-CoV-2S protein (SARS-CoV-2-S (CΔ19)) with C-terminal truncated cytoplasmic 19 amino acids were inoculated with a VSVΔG-GFP/Luc vector. After incubation for 2 hours at 37 ℃, the inoculum was removed, the cells were washed with PBS, and then medium supplemented with anti-VSV-G antibody (clone 8G5f11, kerafast) was added to neutralize the residual input virus. The medium containing VSV-SARS-CoV-2 pseudovirus was collected 20 hours after inoculation, filtered at 0.2 μm and stored at-80 ℃.

For the pseudovirus neutralization assay, 40,000 Vero 76 cells were seeded per 96 well. Serum was serially diluted 1:2 in culture medium starting from a 1:10 dilution (dilution range 1:10 to 1: 10,240). VSV-SARS-CoV-2-S pseudoparticles were diluted in medium for fluorescent focusing unit (ffu) counting in an assay of about 200 TU. Serum dilutions were mixed with pseudovirus 1:1 for 30 min at room temperature, then added to Vero 76 cell monolayers in 96-well plates, and incubated for 16-24 hours at 37 ℃. The supernatant was removed and the cells were lysed with luciferase reagent (Promega). Luminescence was recorded and neutralization titers were calculated as the reciprocal of the highest serum dilution that still resulted in 50% reduction in luminescence. Results are reported as duplicate GMTs. If no neutralization is observed, an arbitrary titer value of 5 (half of the limit of detection [ LOD ]) is reported.

Serum was collected from subjects 21 days after receiving the second 30 μg dose of BNT162b2 or one month after receiving the third 30 μg dose of BNT162b2 (n=19-20). Each serum was tested for neutralizing antibody titers against wild-type SARS-CoV-2Wuhan Hu-1 and the Omicron BA.1 lineage (B.1.1.529) spike protein pseudotyped VSV by a 50% neutralization assay (pVNT 50). Compared to the Wuhan reference, the omacron ba.1 strain spike protein used in the neutralization assay had the following amino acid changes: a67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371 37373P, S F, K417N, N, K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N Y, Y H, T547 48614G, H655G, H679G, H681 764G, H796G, H856 954 5297 969 5297 981F.

BNT162b2 immune serum generated 21 days after the second dose showed potent neutralization of the SARS-CoV-2Wuhan Hu-1 pseudotyped reference. However, a neutralization titer reduction of more than 25-fold was observed for the Omicron ba.1 variant (geometric mean titer GMT of 6 and 155) compared to Wuhan reference. Importantly, the third dose significantly increased the neutralizing antibody titer against omacron ba.1 strain pseudovirus by a factor of 25. Thus, the neutralization titers against omacron ba.1 variant pseudovirus after three doses of BNT162b2 were comparable to the neutralization titers against wild-type strains observed in the serum of individuals receiving two doses of BNT162b2 (GMT 154 and 155).

Example 3: other data on neutralization of SARS-CoV-2 Omicron BA.1 lineage (also referred to as B.1.1.529) pseudoviruses by human serum elicited by BNT162b2 vaccine

In addition to the study and data described in example 2, a longitudinal analysis of neutralization titers was performed in separate smaller subsets of subjects. Serum extracted 21 days after dose 2 exhibited 19.6-fold reduction in GMT against Omicron ba.1 variant compared to Wuhan reference pseudovirus (fig. 12; GMT 6 and 118). Serum obtained from study participants before receiving the third dose of BNT162b2 (median 251 days after dose 2) significantly reduced the neutralization titer against Wuhan pseudovirus (GMT 14), while Omicron ba.1-specific titers were less than the detection limit. The third dose of BNT162b2 significantly increased the neutralization titer against Wuhan pseudovirus (GMT 254) and increased the neutralization titer against Omicron ba.1 by > 26.6-fold 1 month after dose 3 compared to the titer 21 days after dose 2 (GMT 160 and 6). In all 9 subjects, a decrease but effective neutralization of Omicron ba.1 was observed up to 3 months after the third dose (3.2 fold decrease compared to 1 month after dose 3; GMT 50 and 160), while Wuhan-specific neutralization GMT remained stable.

In summary, the third dose of BNT162b2 enhanced Omicron ba.1 neutralization capacity to a level similar to that observed after two doses against Wuhan pseudovirus. Thus, the data indicate that providing a third dose of BNT162b2 can improve protection against omacron ba.1 variant infection.

Example 4: neutralization of human serum raised by BNT162b2 vaccine against pseudoviruses of other SARS-CoV-2 lineage

As described in example 2 and example 3, the reaction mixture was also assayed by 50% neutralization assay (pVNT ₅₀ ) To test the neutralizing antibody titres of each serum against Beta and Delta lineage spike protein pseudotyped VSV (data not shown).

Recombinant replication defective VSV vectors encoding Green Fluorescent Protein (GFP) and luciferase (Luc) but not VSV-glycoprotein (VSV-G) were pseudotyped with variant spikes present in the S proteins of the Wuhan-Hu-1 isolate SARS-CoV-2 spike (S) (GenBank: QHD 43416.1) according to the disclosed pseudotyping protocol with Beta lineages (mutation: L18F, D80A, D215G, R I, delta242-244, K417N, E484K, N501Y, D G, A V) and Delta lineages (mutation: T19R, G142D, delta157/158, K417N, L452R, T478K, D614G, P681R, D950N, K986P, V987P).

For sera collected 21 days after the second dose of BNT162b2, PVNT for Beta variant compared to Wuhan variant ₅₀ About 6.7-fold reduction (GMT of 24 and 155) and about 2.2-fold reduction (GMT of 73 and 155) for Delta variants, but significantly higher than the neutralization reaction for Delta variants. The third dose of BNT162b2 also increased the neutralizing activity against the Beta and Delta pseudoviruses, GMT 279 and 413 respectively.

Example 5: t cell epitope conservation in Omicron BA.1 spike mutant

In addition to humoral immunity, T cell mediated immunity is another layer of defense, particularly for the prevention of severe COVID-19. Previous observations indicate that efficacy against disease has been determined about 12 days after the first dose of BNT162b2, before the second dose is administered and before the onset of high neutralization titers, which further highlights the potential protective effect of T cell responses. Previous reports showed that CD 8T cell responses of individuals vaccinated with BNT162b2 are multi-epitope.

To assess the risk of immune escape of cd8+ T cell responses generated by omacron ba.1, a set of HLA class I restricted T cell epitopes from Wuhan spike protein sequences, reported as immunogenic in the immune epitope database (IEDB, n=244) (procedure for identifying these epitopes is described in the following paragraphs) was studied. Although there were a large number of mutations in Omicron ba.1 spike protein, 85.25% (n=208) of the epitope was not affected at the amino acid sequence level, suggesting that the target of the vast majority of T cell responses elicited by BNT162b2 may remain conserved in Omicron ba.1 variants (fig. 13). Early laboratory studies demonstrated that cd8+ T cell recognition of Omicron epitopes remained in the early pandemic exposed covd-19 recovered individuals, and omicronba.1 VOC had not evolved a broad range of T cell escape mutations at present.

To estimate the non-synonymous mutation rate of T cell epitopes in spike glycoprotein, the immune epitope database (https:// www.iedb.org /) was used to obtain epitopes that were confirmed to be T cell reactive in experimental assays. The database was screened using the following criteria: organisms: SARS-COV2; antigen: a spike glycoprotein; a forward assay; b cell free assay; MHC-free assay; MHC restriction types: class I; and (3) a host: homo sapiens (human). The results table was screened by removing epitopes "deduced from the reactive overlapping peptide pool" and epitopes longer than 14 amino acids, limiting the dataset to only the minimum identified epitopes. Of the 251 unique epitope sequences obtained in this way, 244 were found in the Wuhan strain spike glycoprotein. Of these, 36 epitopes (14.75%) contained the positions reported by the sequence analysis disclosed herein at which mutations occurred in omacron. The results are summarized in fig. 10. Also shown is the number of predicted MHC-I epitopes mutated in each of the Alpha, beta, gamma, delta SARS-CoV-2 variants. FIG. 13 depicts the position of T cell epitopes within the spike protein and indicates epitopes that are conserved or mutated in spike protein from the Omacron BA.1 variant.

Example 6: exemplary dosing regimen

Primary dosing regimen

In some embodiments, the PD ₁ Comprising administering 1 to 100ug of RNA. In some embodiments, the PD ₁ Comprising applying 1 to60ug of RNA. In some embodiments, the PD ₁ Comprising administering 1 to 50ug of RNA. In some embodiments, the PD ₁ Comprising administering 1 to 30ug of RNA. In some embodiments, the PD ₁ Comprising administering about 3ug of RNA. In some embodiments, the PD ₁ Comprising administering about 5ug of RNA. In some embodiments, the PD ₁ Comprising administering about 10ug of RNA. In some embodiments, the PD ₁ Comprising administering about 15ug of RNA. In some embodiments, the PD ₁ Comprising administering about 20ug of RNA. In some embodiments, the PD ₁ Comprising administering about 30ug of RNA. In some embodiments, the PD ₁ Comprising administering about 50ug of RNA. In some embodiments, the PD ₁ Comprising administering about 60ug of RNA.

In some embodiments, the PD ₃ Comprising administering 1 to 100ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 60ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 50ug of RNA. In some embodiments, the PD ₃ Comprising administering 1 to 30ug of RNA. In some embodiments, the PD ₃ Comprising administering about 3ug of RNA. In some embodiments, the PD ₃ Comprising administering about 5ug of RNA. In some embodiments, the PD ₃ Comprising administering about 10ug of RNA. In some embodiments, the PD ₃ Comprising administering about 15ug of RNA. In some embodiments, the PD ₃ Comprising administering about 20ug of RNA. In some embodiments, the PD ₃ Comprising administering about 30ug of RNA. In some embodiments, the PD ₃ Comprising administering about 50ug of RNA. In some embodiments, the PD ₃ Comprising administering about 60ug of RNA.

In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD ₁ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₁ Comprising encoding SARS-CoV-2 spike protein comprising one or more mutations from a Beta variant or its immunogenicity RNA of the fragment. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD ₂ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₂ Comprising encoding SARS-CoV-2 spike protein or comprising one or more mutations from Alpha variantsRNA of an immunogenic fragment thereof. In some embodiments, the PD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₃ Comprising coding for SARS-CoV-2 spike protein or from the MN908947 strainRNA of an immunogenic fragment thereof. In some embodiments, the PD ₃ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD ₃ Comprising coding for SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strainAnd an RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant).

In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, the PD _n Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more additional RNAs encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strains that are prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, the PD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And PD _n Each may independently include a plurality (e.g., at least two) of the mRNA compositions described herein. In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And PD _n Each may independently include a first mRNA composition and a second mRNA composition. In some embodiments, at least one of the plurality of mRNA compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof from a different SARS-CoV-2 variant. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the MN908947 strain SARS-CoV-2. In some embodiments, at least one of the plurality of mRNA compositions comprises RNA encoding SARS-CoV-2S protein or immunogenic fragment thereof that comprises one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from Alpha variants. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, at least one of the plurality of mRNA compositions comprises a nucleic acid encoding a polypeptide comprising a polypeptide from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or Bq.1omicron variant) or an immunogenic fragment thereof.

In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The various mRNA compositions given can each independently comprise at least two different mRNA constructs (e.g., differing at least in protein coding sequence). For example, in some embodiments, a PD ₁ 、PD ₂ 、PD ₃ And/or PD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, from the MN908947 strain SARS-CoV-2, and mRNA encoding the SARS-CoV-2S protein, or an immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or the various mRNA compositions given for PDn may each independently comprise mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, derived from the MN908947SARS-CoV-2 strain, and mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some such embodiments, the variant may be an Alpha variant. In some such embodiments, the variant may be a Delta variant. In some such embodiments, the variant may be an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the plurality of mRNA compositions given can independently comprise at least two mrnas, each encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from different variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the plurality of mRNA compositions provided can independently comprise mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the Alpha variant, and encodingmRNA comprising one or more mutations from the Delta variant SARS-CoV-2S protein or an immunogenic fragment thereof. In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the various mRNA compositions given can independently comprise mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the Alpha variant, and mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Omicron variant (e.g., BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1Omicron variant). In some embodiments, with PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Each of the various mRNA compositions given can independently comprise mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from a Delta variant, and mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1, or BQ.1Omicron variant).

In some embodiments, the PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Comprising administering a plurality of RNA molecules, wherein each RNA molecule encodes a spike protein comprising a mutation from a different SARS-CoV-2 variant, and wherein the plurality of RNA molecules are administered to a subject in a single formulation. In some embodiments, the single formulation comprises RNA encoding a spike protein from the MN908947 strain or an immunogenic variant thereof, and RNA encoding a SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some casesIn embodiments, a single formulation comprises RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Beta variant. In some embodiments, the single formulation comprises RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, a single formulation comprises RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, the PD ₁ With PD ₂ Length of time between (PI) ₁ ) For at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, PI ₁ From about 1 week to about 12 weeks. In some embodiments, PI ₁ From about 1 week to about 10 weeks. In some embodiments, PI ₁ From about 2 weeks to about 10 weeks. In some embodiments, PI ₁ From about 2 weeks to about 8 weeks. In some embodiments, PI ₁ From about 3 weeks to about 8 weeks. In some embodiments, PI ₁ From about 4 weeks to about 8 weeks. In some embodiments, PI ₁ From about 6 weeks to about 8 weeks. In some embodiments, PI ₁ From about 3 weeks to about 4 weeks. In some embodiments, PI ₁ About 1 week. In some embodiments, PI ₁ About 2 weeks. In some embodiments, PI ₁ About 3 weeks. In some embodiments, PI ₁ About 4 weeks. In some embodiments, PI ₁ About 5 weeks. In some embodiments, PI ₁ About 6 weeks. In some embodiments, PI ₁ About 7 weeks. In some embodiments, PI ₁ About 8 weeks. In some embodiments, PI ₁ About 9 weeks. In some embodiments, PI ₁ About 10 weeks.In some embodiments, PI ₁ About 11 weeks. In some embodiments, PI ₁ About 12 weeks.

In some embodiments, the PD ₂ With PD ₃ Length of time between (PI) ₂ ) For at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, PI ₂ From about 1 week to about 12 weeks. In some embodiments, PI ₂ From about 1 week to about 10 weeks. In some embodiments, PI ₂ From about 2 weeks to about 10 weeks. In some embodiments, PI ₂ From about 2 weeks to about 8 weeks. In some embodiments, PI ₂ From about 3 weeks to about 8 weeks. In some embodiments, PI ₂ From about 4 weeks to about 8 weeks. In some embodiments, PI ₂ From about 6 weeks to about 8 weeks. In some embodiments, PI ₂ From about 3 weeks to about 4 weeks. In some embodiments, PI ₂ About 1 week. In some embodiments, PI ₂ About 2 weeks. In some embodiments, PI ₂ About 3 weeks. In some embodiments, PI ₂ About 4 weeks. In some embodiments, PI ₂ About 5 weeks. In some embodiments, PI ₂ About 6 weeks. In some embodiments, PI ₂ About 7 weeks. In some embodiments, PI ₂ About 8 weeks. In some embodiments, PI ₂ About 9 weeks. In some embodiments, PI ₂ About 10 weeks. In some embodiments, PI ₂ About 11 weeks. In some embodiments, PI ₂ About 12 weeks.

In some embodiments, the PD ₃ Between subsequent doses as part of the initial dosing regimen, or beyond PD ₃ The length of time between doses (PI) _n ) Each independently and independently selected from: about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, PI _n From about 1 week to about 12 weeks. In some embodiments, PI _n From about 1 week to about 10 weeks. In some embodiments, PI _n From about 2 weeks to about 10 weeks. In some embodiments, PI _n From about 2 weeks to about 8 weeks. In some implementationsIn embodiments, PI _n From about 3 weeks to about 8 weeks. In some embodiments, PI _n From about 4 weeks to about 8 weeks. In some embodiments, PI _n From about 6 weeks to about 8 weeks. In some embodiments, PI _n From about 3 weeks to about 4 weeks. In some embodiments, PI ₂ About 1 week. In some embodiments, PI _n About 2 weeks. In some embodiments, PI _n About 3 weeks. In some embodiments, PI _n About 4 weeks. In some embodiments, PI _n About 5 weeks. In some embodiments, PI _n About 6 weeks. In some embodiments, PI _n About 7 weeks. In some embodiments, PI _n About 8 weeks. In some embodiments, PI _n About 9 weeks. In some embodiments, PI _n About 10 weeks. In some embodiments, PI _n About 11 weeks. In some embodiments, PI _n About 12 weeks.

In one placeIn some embodiments, PD ₁ 、PD ₂ 、PD ₃ And/or PD _n One or more of the mRNA compositions given may be administered in combination with another vaccine. In some embodiments, another vaccine is for a disease other than COVID-19. In some embodiments, the disease is a disease that increases the detrimental effects of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the disease is a disease that increases the transmissibility of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the other vaccine is a different commercially 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 in PD ₁ 、PD ₂ 、PD ₃ And/or PD _n One or more mRNA compositions given are administered separately at different injection sites, e.g., via intramuscular injection in some embodiments. For example, in some embodiments, influenza vaccines and PD ₁ 、PD ₂ 、PD ₃ And/or PD _n Given one or more of the SARS-CoV-2 mRNA compositions described herein are administered separately to different arms of a subject via intramuscular injection.

Enhanced dosing regimen

In some embodiments, a booster dosing regimen is administered to the subject. The booster dosing regimen may include one or more doses. For example, in some embodiments, the booster dosing regimen comprises a single dose (BD ₁ ). In some embodiments, the booster dosing regimen comprises a first dose (BD ₁ ) And a second dose (BD ₂ ). In some embodiments, the booster dosing regimen includes a first dose, a second dose, and a third dose (BD ₃ ). In some embodiments, the booster dosing regimen comprises a first dose, a second dose, a third dose, and one or more additional doses (BD) of any of the pharmaceutical compositions described herein _n )。

In some embodiments, BD ₂ Comprising administering 1 to 100ug of RNA. In some embodiments, BD ₂ Comprising administering 1 to 60ug of RNA. In some embodiments, BD ₂ Comprising administering 1 to 50ug of RNA. In some embodiments, BD ₂ Comprising the application of1 to 30ug of RNA was used. In some embodiments, BD ₂ Including administration of about 3ug. In some embodiments, BD ₂ Comprising administering about 5ug of RNA. In some embodiments, BD ₂ Comprising administering about 10ug of RNA. In some embodiments, BD ₂ Comprising administering about 15ug of RNA. In some embodiments, BD ₂ Comprising administering about 20ug of RNA. In some embodiments, BD ₂ Comprising administering about 30ug of RNA. In some embodiments, BD ₂ Comprising administering about 50ug of RNA. In some embodiments, BD ₂ Comprising administering about 60ug of RNA.

In some embodiments, BD _n Comprising administering 1 to 100ug of RNA. In some embodiments, BD _n Comprising administering 1 to 60ug of RNA. In some embodiments, BD _n Comprising administering 1 to 50ug of RNA. In some embodiments, BD _n Comprising administering 1 to 30ug of RNA. In some embodiments, BD _n Comprising administering about 3ug of RNA. In some embodiments, BD _n Comprising administering about 5ug of RNA. In some embodiments, BD _n Comprising administering about 10ug of RNA. In some embodiments, BD _n Comprising administering about 15ug of RNA. In some embodiments of the present invention, in some embodiments, BD _n Comprising administering about 20ug of RNA. In some embodiments, BD _n Comprising administering about 30ug of RNA. In some embodiments, BD _n Comprising administering about 60ug of RNA. In some embodiments, BD _n Comprising administering about 50ug of RNA.

In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD ₁ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD ₁ Comprising RNA encoding SARS-CoV-2 spike protein from MN908947 strain or an immunogenic fragment thereof, and encoding SARS-CoV comprising one or more mutations from a Delta variant-2 RNA of a spike protein or an immunogenic fragment thereof. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₁ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD ₂ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant). In some embodiments, BD2 comprises RNA encoding a SARS-CoV-2 spike protein, or an immunogenic fragment thereof, from a MN908947 strain, and one or more RNAs encoding spike proteins, or immunogenic fragments thereof, from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₂ Comprising RNA encoding SARS-CoV-2 spike protein from MN908947 strain or an immunogenic fragment thereof, andan 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 ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₂ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD ₃ Comprising coding for SARS-C from MN908947 strain _o RNA of V-2 spike protein or an immunogenic fragment thereof. In some embodiments, BD ₃ Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD ₃ Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain, and one or more RNAs encoding spike protein or an immunogenic fragment thereof from a SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD3 includes RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD ₃ Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof from the MN908947 strain. In some embodiments, BD _n Including RNA encoding spike proteins or immunogenic fragments thereof from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Alpha variant. In some embodiments, BD _n Comprising RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD _n Comprising encoding SARS-CoV-2 comprising one or more mutations from a Beta variantRNA of a spike protein or an immunogenic fragment thereof. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, the BDn comprises RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and one or more RNAs encoding spike protein from SARS-CoV-2 strain that is prevalent and/or rapidly transmitted in the relevant jurisdiction. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from the Alpha variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Delta variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein or an immunogenic fragment thereof comprising one or more mutations from a Beta variant. In some embodiments, BD _n Including RNA encoding SARS-CoV-2 spike protein from the MN908947 strain or an immunogenic fragment thereof, and RNA encoding SARS-CoV-2 spike protein comprising one or more mutations from an omacron variant (e.g., a BA.4/5, BA.1, BA.2, XBB, XBB.1 or BQ.1 omacron variant) or an immunogenic fragment thereof.

In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a plurality (e.g., at least two) of the mRNA compositions described herein. In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a first mRNA composition and a second mRNA composition. In some embodiments, BD ₁ 、BD ₂ 、BD ₃ And BD (BD) _n Each may independently include a plurality (e.g., at least two) of mRNA compositions, wherein at least one of the plurality of mRNA compositions comprises BNT162b2 (e.g., as described herein). In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof from a different SARS-CoV-2 variant (e.g., a variant that is prevalent or rapidly spreading in a relevant jurisdiction, such as the variants disclosed herein). In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof from the MN908947 strain SARS-CoV-2. In some embodiments, at least one of the plurality of mRNA compositions comprises RNA encoding SARS-CoV-2S protein or immunogenic fragment thereof that comprises one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from Alpha variants. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, at least one of the plurality of mRNA compositions comprises mRNA encoding SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given can each independently comprise at least two different mRNA constructs (e.g., mRNA constructs having different protein coding sequences). For example, in some embodiments, as BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions presented can each independently comprise mRNA encoding SARS-CoV-2S protein or immunogenic fragment thereof from the MN908947 strain SARS-CoV-2, and encoding a polypeptide comprising a variant that is prevalent and/or rapidly transmitted in the relevant jurisdictionmRNA of one or more mutated SARS-CoV-2S proteins or immunogenic fragments thereof in the body. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, derived from the MN908947SARS-CoV-2 strain, and mRNA encoding the SARS-CoV-2S protein, or immunogenic fragment thereof, comprising one or more mutations from variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some such embodiments, the variant may be an Alpha variant. In some such embodiments, the variant may be a Delta variant. In some such embodiments, the variant may be an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise at least two mrnas, each encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from different variants that are prevalent and/or rapidly spread in the relevant jurisdiction. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given may each independently comprise mRNA encoding the SARS-CoV-2S protein or immunogenic fragment thereof from the Alpha variant, and mRNA encoding the SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from the Delta variant. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n The various mRNA compositions given can each independently comprise mRNA encoding a SARS-CoV-2S protein from an Alpha variant or an immunogenic fragment thereof, and mRNA encoding a SARS-CoV-2S protein from an omacron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1 omacron variant) or an immunogenic fragment thereof. In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n Each of the various mRNA compositions presented can independently comprise a protein encoding SARS-CoV-2S from the Delta variant or a variant thereof An mRNA of an immunogenic fragment, and an mRNA encoding a SARS-CoV-2S protein or immunogenic fragment thereof comprising one or more mutations from an Omicron variant (e.g., ba.4/5, ba.1, ba.2, XBB, xbb.1, or bq.1Omicron variant).

In some embodiments, BD ₁ And BD (BD) ₂ Length of time between (BI ₁ ) For at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks. In some embodiments, BI ₁ From about 1 week to about 12 weeks. In some embodiments, BI ₁ From about 1 week to about 10 weeks. In some embodiments, BI ₁ From about 2 weeks to about 10 weeks. In some embodiments, B1 ₁ From about 2 weeks to about 8 weeks. In some embodiments, BI ₁ From about 3 weeks to about 8 weeks. In some embodiments, BI ₁ From about 4 weeks to about 8 weeks. In some embodiments, BI ₁ From about 6 weeks to about 8 weeks. In some embodiments, BI ₁ From about 3 weeks to about 4 weeks. In some embodiments, BI ₁ About 1 week. In some embodiments, B1 ₁ About 2 weeks. In some embodiments, BI ₁ About 3 weeks. In some embodiments, BI ₁ About 4 weeks. In some embodiments, BI ₁ About 5 weeks. In some embodiments, BI ₁ About 6 weeks. In some embodiments, BI ₁ About 7 weeks. In some embodiments, BI ₁ About 8 weeks. In some embodiments, BI ₁ About 9 weeks. In some embodiments, BI ₁ About 10 weeks.

In some embodiments, BD ₂ And BD (BD) ₃ Between which are locatedLength of time (BI) ₂ ) For at least about 1 week, at least about 2 weeks, or at least about 3 weeks. In some embodiments, BI ₂ From about 1 week to about 12 weeks. In some embodiments, BI ₂ From about 1 week to about 10 weeks. In some embodiments, BI ₂ From about 2 weeks to about 10 weeks. In some embodiments, BI ₂ From about 2 weeks to about 8 weeks. In some embodiments, BI ₂ From about 3 weeks to about 8 weeks. In some embodiments, BI ₂ From about 4 weeks to about 8 weeks. In some embodiments, BI ₂ From about 6 weeks to about 8 weeks. In some embodiments, BI ₂ From about 3 weeks to about 4 weeks. In some embodiments, BI ₂ About 1 week. In some embodiments, BI ₂ About 2 weeks. In some embodiments, BI ₂ About 3 weeks. In some embodiments, BI ₂ About 4 weeks. In some embodiments, BI ₂ About 5 weeks. In some embodiments, BI ₂ About 6 weeks. In some embodiments, BI ₂ About 7 weeks. In some embodiments, BI ₂ About 8 weeks. In some embodiments, BI ₂ About 9 weeks. In some embodiments, BI ₂ About 10 weeks.

In some embodiments, BD ₃ Between subsequent doses as part of a booster regimen, or beyond BD ₃ The length of time between doses (BI) _n ) Each independently and independently selected from: about 1 week or more, about 2 weeks or more, or about 3 weeks or more. In some embodiments, BI _n From about 1 week to about 12 weeks. In some embodiments, BI _n From about 1 week to about 10 weeks. In some embodiments, BI _n From about 2 weeks to about 10 weeks. In some embodiments, BI _n From about 2 weeks to about 8 weeks. In some embodiments, BI _n From about 3 weeks to about 8 weeks. In some embodiments, BI _n From about 4 weeks to about 8 weeks. In some embodiments, BI _n From about 6 weeks to about 8 weeks. In some embodiments, BI _n From about 3 weeks to about 4 weeks. In some embodiments, BI _n About 1 week. In some embodimentsIn BI (BI) _n About 2 weeks. In some embodiments, BI _n About 3 weeks. In some embodiments, BI _n About 4 weeks. In some embodiments, BI _n About 5 weeks. In some embodiments, BI _n About 6 weeks. In some embodiments, BI _n About 7 weeks. In some embodiments, BI _n About 8 weeks. In some embodiments, BI _n About 9 weeks. In some embodiments, BI _n About 10 weeks.

In some embodiments, with BD ₁ 、BD ₂ 、BD ₃ And/or BD _n One or more of the mRNA compositions given may be administered in combination with another vaccine. In some embodiments, another vaccine is for a disease other than COVID-19. In some embodiments, the disease is a disease that increases the detrimental effects of SARS-CoV-2 when a subject is infected with the disease and SARS-CoV-2 simultaneously. In some embodiments, the disease is in a subjectDiseases that increase the transmission rate of SARS-CoV-2 when a subject infects both the disease and SARS-CoV-2. In some embodiments, the other vaccine is a different commercially 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 as BD ₁ 、BD ₂ 、BD ₃ And/or BD _n One or more mRNA compositions given are administered separately at different injection sites, e.g., via intramuscular injection in some embodiments. For example, in some embodiments, influenza vaccines and BD ₁ 、BD ₂ 、BD ₃ And/or BD _n Given one or more of the SARS-CoV-2mRNA compositions described herein are administered separately to different arms of a subject via intramuscular injection.

Additional reinforcement scheme

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Example 7: omicron ba.1 breakthrough infection promotes cross variant neutralization and memory B cell formation

This example shows that Omicron ba.1 breakthrough infection of individuals vaccinated with BNT162B2 double and triple vaccination motivates cross variant neutralization and memory B cell formation, including production of neutralizing antibodies and B cell responses to Omicron ba.1 variants. Those of ordinary skill in the art reading this example will appreciate that such findings can be extended to the administration of an mRNA vaccine comprising RNA encoding a SARS-CoV-2S protein having a mutation unique to the Omicron ba.1 variant (e.g., a mutation as described herein) to a subject previously administered two or three doses of a SARS-CoV-2 vaccine (e.g., developed based on the S protein from the MN908947-Hu-1 strain in some embodiments).

Omicron is by far the most evolutionarily unique variant of SARS-CoV-2 interest (VOC). To clarify how an omacron breakthrough infection could potentially alter SARS-CoV-2 recognition in vaccinated individuals, serum neutralization and B in omacron ba.1 breakthrough infection in BNT162B2 double and triple vaccinated individuals was studied _{Male men} Influence of cellular antigen recognition. The Omicron ba.1 breakthrough infection induced extensive neutralization of VOCs including Omicron ba.1, which was significantly stronger than the duplex and triple vaccines that did not infect Omicron ba.1. Omicron ba.1 breakthrough infection enhanced B in individuals vaccinated with BNT162B2 double and triple vaccination _{Male men} cell-to-VOC broad recognition, where recognition is mainly directed to conserved epitopes widely shared between variants, rather than Omicron ba.1 specific epitopes. The data presented herein demonstrate that Omicron ba.1 breakthrough infection effectively expandsNeutralizing antibodies and/or B cell responses to various variants were shown, and it was shown that vaccines that were adapted to omacron ba.1S protein might be able to reconstitute the immune repertoire.

Introduction to the invention

Suppressing the current covd-19 pandemic requires the development of a durable and broad enough immunity that provides protection against both in-transit and future SARS-CoV-2 variants. The titer of neutralizing antibodies relative to SARS-CoV-2, and binding of antibodies to spike (S) glycoprotein and its Receptor Binding Domain (RBD) are considered factors relevant for protection against infection (D.S. Khory et al, "Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 interaction," Nature medium.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). The currently available vaccines are based on the prototype MN908947-Hu-1 strain and elicit antibodies (K) with a neutralizing capacity that exceeds the breadth elicited by infection with the MN908947 strain, or variants of interest (VOCs). 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 decrease 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/scitranslamed. Abn8057; S.Yamayoshi et al, "Antibody titers against SARS-CoV-2 decline,but do not disappear for several months," Eclinical Medicine,32, 100734 (2021), doi: 10.1016/j.eclin.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: 10.10)16/S2666-5247 (21) 00025-2; goldberg et al, "Waning Immunity after the BNT162b2 Vaccine in Israel," The New England joumal of media.385, e85 (2021), doi:10.1056/NEJMoa 2114228) and it is believed that conventional booster vaccination is required to trigger recall immunity and maintain efficacy against new VOCs (a.r. false et al, "SARS-CoV-2 Neutralization with BNT162b2 Vaccine Dose 3," The New England joumal ofmedia.385, 1627-1629 (2021), doi:10.1056/NEJMc2113468; choi et al, "Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccine boosters in healthy adults," Nature media.27, 2025-2031 (2021), doi:10.1038/s41591-021-01527-y; andrews et al, "Effectiveness of COVID-19 booster vaccines against Covid-19 related symptoms,hospitalisation and death in England," Nature media (2022), doi:10.1038/s41591-022-01699-1.

Longevity memory B (B) _{Male men} ) Cells are the basis for recall responses when antigens are again subjected to infection or booster vaccination. The cells play a central role in the maintenance and evolution of antiviral antibody responses against variants, due to the low affinity selection mechanism and B during germinal center reactions _{Male men} Sustained hypermutation of cells expands the breadth of virus 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 Joumal of experimental medicine,208, 2599-2606 (2011) doi: 10.1084/jem.201110140; and Y.Adachi et al, "Distinct germinal center selection at local sites shapes Memory B cell response to viral escape," The Journal of experimental media.212, 1709-1723 (2015), doi: 10.1084/jem.20142284).

How the protective immunity mediated by the vaccine will evolve over time, and how the protective immunity will change due to repeated exposure to the covd-19 vaccine and increasingly diverse viral variant infections is particularly relevant to the appearance of antigenically distinct VOCs. Omicron is the most evolutionarily distant recorded VOC, with an unprecedented number of amino acid changes in its S glycoprotein, including at least 15 amino acid changes in RBD and extensive changes in the N-terminal domain (NTD). These changes are expected to affect most neutralizing antibody epitopes. In addition, omicron is highly transmissible, and its sub-lines BA.1 and BA.2 have spread rapidly around the world, defeating Delta within weeks to become the primary spreading VOC (W.Dejniratisai et al, "SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses," cell.185, 467-484.e15 (2022), doi:10.1016/j.cell.2021.12.046; and M.Hoffmann et al, "The Omicron variant is highly resistant against antibody-mediated neutralization," cell.185, 447-456.e11 (2022), doi: 10.1016/j.cell.2021.12.032).

To date, more than 10 million people worldwide have been vaccinated with the mRNA-based covd-19 vaccine BNT162b2 and received a major 2 dose series or additional booster. The vaccine makes a significant contribution to the group immunity pattern in many regions, and the further immune editing and effects of the currently spreading variants will be based on the group immunity pattern.

To characterize the serum neutralization activity and B of omacron ba.1 breakthrough infection _{Male men} The effect of the magnitude and breadth of the cells, blood samples from individuals vaccinated with BNT162b2 double or triple vaccination were studied.

Since understanding of antigen-specific B memory cell pools is a critical determinant of an individual's ability to respond to emerging variants, this data can help guide vaccine development.

Results and discussion

Grouping and sampling

The blood samples were derived from a collection of biological samples from the BNT162b2 vaccine test, as well as from a pool of biological samples from prospective collections of vaccinated individuals with subsequent SARS-CoV-2 Omicron BA.1 breakthrough infections. Samples were selected to study biomarkers in four independent groups, i.e., at sample collection (BNT 162b2 ² 、BNT162b2 ³ ) Individuals who had not been previously or breakthrough vaccinated either (i) double vaccinated with BNT162b2 or (ii) triple vaccinated, and (iii) double vaccinated with BNT162b2 or (iv) triple vaccinated, and experienced SARS-CoV-2 Omicron BA.1 variant (BNT 162b2, respectively, after an average of about 5 months or 4 weeks ² +Omi，BNT162b2 ³ + Omi) (see materials and methods below). Immune serum was used to characterize the changes in magnitude and breadth of serum neutralization activity associated with Omicron ba.1 infection. PBMC are used for identifying peripheral B of corresponding full-length SARS-CoV-2S protein or RBD thereof _{Male men} The VOC specificity of the cells was characterized (fig. 15).

The Omicron breakthrough infection of individuals vaccinated with BNT162b2 double and triple vaccinations induced extensive neutralization of Omicron BA.1, BA.2 and other VOCs

To evaluate the neutralizing activity of immune serum, two orthogonal assay systems were used: a well-characterized pseudovirus neutralization assay (pnnt) for studying the breadth of inhibition of viral entry in a propagation-defective environment; and a live SARS-CoV-2 neutralization assay (VNT) designed to evaluate the neutralization of antibodies maintained throughout the entire assay period during multicycle replication of the authentic virus. For the former, the breadth was assessed using pseudoviruses carrying S protein containing mutations specific for the Omacron sub-line BA.1 or BA.2, other SARS-CoV-2 VOCs (MN 908947, alpha, beta, delta), while the potential panoxa Bei Bingdu (Pan-Sarbevirus et al, "Pan-Sarbecovirus Neutralizing Antibodies in BNT b2-Immunized SARS-CoV-1 survivinrs," The New England journal of medicine,385, 1401-1406 (2021), doi:10.1056/NEJMoa 2108453) was tested using pseudoviruses carrying S protein 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).

As previously reported (a.r. false 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; c. -W.tan et al, "Pan-Sarbecovirus Neutralizing Antibodies in BNT162b2-Immunized SARS-CoV-1 vitamins," The New England journal of media.385, 1401-1406 (2021), doi:10.1056/NEJMoa 2108453), 50% pseudovirus neutralization of Beta and Delta VOCs in duplicate vaccinated individuals not infected with omacron (pVN) ₅₀ ) Geometric Mean Titres (GMT) decreased and neutralization by two omacron sublines was virtually undetectable. pVN against all tested VOCs in the omacron-uninfected triple vaccinated individuals ₅₀ GMT is significantly higher, with strong neutralization of Alpha, beta and Delta variants. GMT for Omicron ba.1 was significantly lower than MN908947 (GMT 160 vs 398), while titres for Omicron ba.2 were also greatly reduced to 211. Thus, three doses of vaccination induced similar levels of neutralization for two Omicron sub-lines (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 BNT162b 2-immunozed SARS-CoV-1 survivinrs," The New England joumal of medicine,385, 1401-1406 (2021), doi:10.1056/NEJMoa2108453; J. Liu et al, "BNT162b2-Elicited neutralization of B.1.617and other SARS-CoV-2 variants," Nature,596, 273-275 (2021), doi:10.1038/s41586-021-03693-y; A. Muik et al, "Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-95," Muik et al, "N.support" support of the same principles as used in conjunction with the following claims, "N.support of the same principles as used in conjunction with the three doses (FIG. 16, A.)," Lei et al, "BNT162b2-Elicited neutralization of B.1.617and other than three doses of eggs, N.Y.)," Lei..

Omicron ba.1 breakthrough infection had a significant effect on both the magnitude and breadth of neutralizing antibody responses in both double-vaccinated and triple-vaccinated individuals, with slightly higher pVN observed in triple-vaccinated individuals ₅₀ GMT (fig. 16, a). pVN50 GMT of individuals vaccinated with double vaccinations against breakthrough infections of Omicron BA.1 and BA.2 was 100-fold and more than 35-fold higher than the GMT of individuals vaccinated with double vaccinations without Omicron BA.1. Immune sera from individuals vaccinated with both breakthrough infections had broad neutralization activity with pVN GMT against Beta and Delta higher than pVN GMT observed in individuals vaccinated with triple vaccine without omacron (GMT 740 vs 222 and 571 vs 370).

The effect of Omicron ba.1 breakthrough infection on neutralization of Omicron ba.1 and ba.2 pseudoviruses was less pronounced when triple vaccinated individuals were observed (about 7-fold and 4-fold increase in neutralization compared to non-Omicron infected triple vaccinated individuals). pVN for Omicron BA.1, BA.2 and Delta ₅₀ GMT was 1029, 836 and 1103 in the Omicron breakthrough individuals of triple vaccination, which was compared to 160, 211 and 370 in the non-Omicron infected triple vaccinated individuals. GMT for all SARS-CoV-2 VOCs including Beta and Omicron was close to titres for the MN908947 reference, but significantly reduced in triple vaccinated uninfected Omicron individuals.

Also, although serum from vaccinated uninfected omacron individuals had no detectable or only a weak pVN for distant SARS-CoV-1 ₅₀ Titer, but double vaccinations and even more significantly triple vaccinated convalescence sera of Omicron infected individuals were able to strongly neutralize SARS-CoV-1 pseudovirus (fig. 16, a and B). SARS-CoV-1 pVN50GMT in 9 of 18 breakthrough infected individuals (4 double vaccinated and 5 triple vaccinated) was comparable to or higher than pVN GMT (GMT. Gtoreq.120) against the MN908947 reference in duplicate vaccinated individuals not infected with Omacron.

A similar finding was also shown for the real live SARS-CoV-2 virus neutralization assay with MN908947, beta, delta and Omicron BA.1 pseudoviruses (FIG. 16, B). In individuals vaccinated with BNT162b2 double and triple vaccination, omicron BA.1 infection was greatly increased compared to that against Omicron BA.1Is associated with a neutralization activity of 50% of the virus neutralization (VN ₅₀ ) GMT is within the same range as GMT for MN908947 strain (fig. 16, b; GMT 493 vs 381 and GMT 538 vs 613). Similarly, individuals subjected to double vaccination and triple vaccination during omacron ba.1 recovery also showed comparable levels of neutralization for other variants (e.g., GMT 493 and 729 for Beta), indicating a broad breadth of neutralization activity.

Taken together, these data demonstrate that SARS-CoV-2 Omicron BA.1 breakthrough infection induces neutralization activity with a great breadth in individuals receiving the vaccine, which is further subject to VN against the MN908947 strain and SARS-CoV-2VOC ₅₀ The calculated ratio of GMT (fig. 16, c) supports discovery. Although uninfected Omicron individuals vaccinated with BNT162b2 and to a lesser extent also triple vaccinated exhibited significant differences in neutralizing capacity against VOCs, the neutralizing activity of Omicron ba.1 convalescence subjects reached levels of nearly the same high performance range for all variant strains tested.

Likewise, omacron BA.1 breakthrough infection had similarly broad neutralization enhancing effects in individuals vaccinated with other approved COVID-19 vaccines or heterologous regimens (FIG. 19; table 20).

TABLE 20 individuals were vaccinated with other approved COVID-19 vaccines or cocktail regimens following subsequent Omacron BA.1 breakthrough infections

n/a, inapplicable; N/A, inapplicable; AZ, astraZeneca AZD1222; BNT, biontech/Pfizer BNT162b2; j (J)&J，Johnson&Johnson Ad26.COV2.S；MOD，Modema mRNA-1273；BNT ⁴ BNT162b2 four dose series; MOD (Metal oxide semiconductor) ² Two dose series of mRNA-1273; MOD (Metal oxide semiconductor) ³ mRNA-1273 three dose series

B of individuals vaccinated with BNT162B2 double and triple vaccination _{Male men} Cells widely recognize VOCs and are further enhanced by omicon breakthrough infections

Subsequently, the phenotype and number of SARS-CoV-2S protein-specific B cells was studied. Flow cytometry-based B cell phenotypic assays are used for differential detection of variant specific S protein binding B cells in a large number of PBMCs. It was found that all S protein and RBD specific B cells in peripheral blood had B _{Male men} Phenotype (B) _{Male men} ；CD ^{20 height of} CD ^{38 integer/negative} ) As no antigen-specific plasmablasts or primary B cells were detected (data not shown). Thus, the assay allows for B of the intact S protein or RBD thereof at a hotspot identified as an amino acid change _{Male men} Each of the SARS-CoV-2 variants was distinguished between the cell and the variant-specific epitope (FIG. 17, A).

Antigen specificity B _{Male men} The total frequency of cells varies between groups. B in individuals vaccinated with two non-Omicron vaccines _{Male men} The frequency of cells was low at the initial time point after vaccination and increased over time: at 5 months, protein S specific B compared to 3 weeks after the second BNT162B2 dose _{Male men} Almost three times the cells, RBD specific B _{Male men} Cells were doubled over all VOCs, reaching a number similar to that observed in the non-omacron triple vaccinated individuals (fig. 17, b and C).

Individuals with BNT162B2 double or triple vaccinations with SARS-CoV-2 Omicron BA.1 breakthrough infection showed a greatly increased frequency of B _{Male men} Cells, which are higher than B of a triple vaccinated individual not infected with Omicron _{Male men} Cells (FIGS. 17, B and D).

B against Omicron BA.1S protein in all groups including uninfected Omicron and Omicron-infected individuals _{Male men} Detectable frequency of cells and B for MN908947 and other test VOCs _{Male men} Cell equivalents (FIGS. 17, B and D), B against Omicron BA.1 RBD _{Male men} The frequency of the cells was slightly lower than the other variants (fig. 17, c and E).

The ratio of RBD protein to S protein binding was then compared across the different groups and S protein recognition biased towards Omicron ba.1 VOC was found to be especially true in the uninfected Omicron group (fig. 17, f). In the group that underwent Omicron ba.1, this ratio was higher, indicating that Omicron ba.1 breakthrough infection increased Omicron ba.1 RBD recognition.

Omacron ba.1 breakthrough infection in BNT162B2 double and triple vaccinated individuals largely potentiated B against a conserved epitope widely shared between MN908947 and the S protein of other VOCs, rather than strictly against omacron S-specific epitopes _{Male men} And (3) cells.

These findings indicate that Omicron ba.1 infection in vaccinated individuals not only enhances neutralization activity and B against Omicron ba.1 _{Male men} Cells, and broadly enhance immunity against various VOCs. To study the specificity of the antibody response at a cellular level, B staining with fluorescently labeled variant-specific S or RBD proteins was performed _{Male men} Multiparameter analysis of cells.

Use of combinatorial gating strategies to discriminate B, which can only identify single variant specific epitopes of MN908947, alpha, delta or Omicron BA.1 _{Male men} Cell subpopulations, with B identifiable in any given combination thereof _{Male men} Cell subsets (FIG. 18, A).

In the first analysis, MN908947 and Omicron BA.1S and RBD protein B were evaluated _{Male men} Cell recognition (FIGS. 18, B, C and D). The SARS-CoV-2 Omicron BA.1 variant has 37 amino acid changes in the S protein compared to the MN908947 parent strain, 15 of which are in RBD, which are immunodominant targets for neutralizing antibodies induced by either the COVID-19 vaccine or SARS-CoV-2 infection.

Staining with full-length S protein showed a majority of B from individuals vaccinated twice with uninfected omacron, and even more predominantly from individuals vaccinated triple _{Male men} The cells were all directed against an epitope shared by the MN908947 and Omicron BA.1 SARS-CoV-2 variants. And (3) withThe observation that BNT162B2 vaccination could elicit an immune response against a wild-type epitope that does not recognize the corresponding altered epitope in the Omacron BA.1S protein (FIGS. 18, B and C) was consistent with the finding that in most individuals a smaller but clearly detectable proportion of B was found _{Male men} Cells recognize only MN908947S protein or RBD. Consistent with lack of exposure, no B specifically binding to Omicron BA.1S or RBD proteins was detected in these uninfected Omicron individuals _{Male men} And (3) cells.

B recognizing an S protein epitope shared between MN908947 and Omicron BA.1 in an Omicron BA.1 convalescent individual _{Male men} The frequency of cells was significantly higher than in individuals not infected with omacron (fig. 18, b and C). In a large part of these subjects, a small proportion of B was found that was entirely specific for the MN908947S protein _{Male men} Cells, and less frequently B with full Omicron BA.1 variant S protein specificity _{Male men} And (3) cells.

By staining B cells with labeled RBD protein, a similar but slightly different pattern was observed (fig. 18, B and D). Again, it was found that Omicron ba.1 breakthrough infections in double vaccinated/triple vaccinated individuals largely potentiated B _{Male men} Reactivity of cells with conserved epitopes. A modest increase in MN908947 specific reactivity was observed; however, only a small amount of Omicron-RBD specific B was detected in the test individuals _{Male men} Cells (FIG. 18, D).

Next, B binding to S protein or RBD was identified using a combinatorial gating approach _{Male men} Specifically binding to MN908947 or Omicron ba.1, or to a subset of common epitopes that are widely conserved across all four variants (MN 908947, alpha, delta and Omicron ba.1) in cells (fig. 18, e). In all four study groups, B was found to recognize the S protein epitope _{Male men} The frequency of the cells is conserved across all test variants, accounting for B binding to S protein _{Male men} The largest part of the cell pool (FIG. 18, F, all 4+ve). The S protein of the MN908947 strain does not have a specific amino acid change that distinguishes it from the spike protein of Alpha, delta or Omicron ba.1 VOC. Thus, at any point B, which specifically recognizes MN 908947S protein, is difficult to detect in individuals _{Male men} Cells (FIG. 18, F). In several individuals with Omicron BA.1 breakthrough infection, a small proportion of B was detected _{Male men} Cells specifically bound to Omicron ba.1S protein (fig. 18, f), whereas almost none of the individuals showed a response strictly specific for Omicron ba.1 RBD (fig. 18, g).

These findings indicate that SARS-CoV-2 Omicron BA.1 breakthrough infection in vaccinated individuals largely expands the broad range of target conserved S protein and RBD epitopes, rather than including a large number of Omicron-specific B _{Male men} B of cells _{Male men} Cell banks.

To further parse this reaction, B for RBD _{Male men} The subpopulations were characterized. Use of the combinatorial boolean gate approach to discriminate B with different binding patterns in the range of strictly variant specific epitopes and common epitopes shared by several variants _{Male men} And (3) cells. Multiple sequence alignment revealed that Omicron ba.1 RBD differs from the RBD sequence regions conserved in MN908947, alpha and Delta by 13 single amino acid changes. All Omicron BA.1 convalescent individuals were found to have a B that identified the stable frequency of MN908947, alpha and Delta VOC RBDs, but not the Omicron BA.1 RBDs _{Male men} Cells, but almost none of these individuals had B specifically reacted with Omacron BA.1 RBD _{Male men} Cells (FIG. 18, H). B specifically recognizing Omicron BA.1 and Alpha RBD, or Omicron BA.1 and Delta RBD, was also not detected _{Male men} And (3) cells.

In addition, RBD-specific B was identified in all individuals _{Male men} Two other subpopulations of cells. One subpopulation was characterized by binding to MN908947, alpha and Omicron ba.1, but not to Delta RBD. The other population showed binding to MN908947 and Alpha, but not to omacron ba.1 or Delta RBD (fig. 18, h). Sequence alignment identified L452R as the unique RBD mutation unique to Delta that was not shared by the other 3 variant RBDs (fig. 18, top i). Similarly, the only RBD site that has been found to be conserved in MN908947 and Alpha and altered in Delta and Omicron BA.1 is T478K (FIG. 18, bottom of I). Known as L452R andboth T478K changes were associated with escape of the neutralizing antibody response induced by the vaccine. Notably, B is not detected in all combined subgroups _{Male men} Cells, in the combinatorial subgroups, multiple sequence alignments were unable to identify unique epitopes in RBD sequences that met boolean selection criteria (e.g., MN908947 or MN908947 only and Omicron ba.1, without Alpha, delta). These findings indicate B for RBD _{Male men} The cellular response is driven specifically by the previous BNT162b2 vaccination and is not substantially diverted to a new RBD epitope mutated in the Omacron variant after infection.

SUMMARY

SARS-CoV-2 Omicron is a partial immune escape variant that has an unprecedented number of amino acid changes in the S protein at the neutralizing antibody binding site, distinguishing the variant from previously reported variants. Recent studies of neutralizing antibody localization and molecular modeling strongly supported the functional relevance of these changes, and the importance of the changes was demonstrated by observation that duplicate vaccinated individuals did not detect neutralizing activity against SARS-CoV-2 Omicron.

The findings presented herein show that an Omicron ba.1 breakthrough infection of vaccinated individuals not only enhances the neutralizing activity against Omicron ba.1 and B _{Male men} Cells, but also broadly enhance immunity against various VOCs, and also provide insight into how to achieve broad immunity.

The data presented herein indicate that initial exposure in the MN908947 strain S protein may affect B _{Male men} Cell formation and novel B to a more unique epitope against the Omacron BA.1 variant _{Male men} The formation of cellular responses has a significant impact. Similar observations were reported in vaccinated individuals who underwent breakthrough infection with Delta variants (K.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.). Such asIn this example, it was demonstrated that Omicron ba.1 breakthrough infection largely broadened the broad range of S protein and RBD epitopes to conservation, rather than including a large number of strictly Omicron-specific B _{Male men} B of cells _{Male men} Cell banks.

Thus, similar to the third booster vaccination, omacron ba.1 breakthrough infections in double vaccinated individuals expand pre-existing B _{Male men} A cell pool. However, there is a clear difference in the pattern of immune responses elicited by homologous vaccine boosters compared to omacron ba.1 breakthrough infection. Although the B memory cell response focused on conserved epitopes, the Omicron ba.1 breakthrough infection resulted in a more pronounced increase in antibody neutralization titers against Omicron ba.1, as well as a pronounced cross-neutralization of both the prototype and the novel SARS CoV-2 variants. These effects are particularly pronounced in individuals vaccinated with two vaccines.

Without wishing to be bound by theory, three findings may be directed to potential complementary and synergistic mechanisms leading to these results:

first, S protein-specific B _{Male men} The overall cell increases. Double vaccinated individuals at the omacron ba.1 recovery phase have a higher frequency of B than triple vaccinated individuals _{Male men} Cells and higher neutralizing antibody titers against all VOCs. This breakthrough infection elicited a stronger neutralizing antibody response than the 3 rd vaccine dose in the double vaccinated individuals, which was not obvious in previous studies describing breakthrough infection of other variants (Evans et al Science Translational Medicine (2022) 14, eabn 8057) and can be explained by: neutralization of omacron ba.1 variants was weaker at the beginning of infection, potentially resulting in greater or prolonged antigen exposure of the immune system in altered S protein.

Second, specificity for RBD B _{Male men} The bias of the cellular response is stronger. Omicron BA.1 breakthrough infection contributes to RBD-specific B _{Male men} Cell ratio recognizes B of S protein-specific epitope other than RBD _{Male men} Cells are more significantly enhanced in proportion. Thus, with vaccinated individuals not infected with Omicron Compared to individuals infected with Omicron ba.1, individuals have a significantly higher ratio of RBD/S protein-specific B _{Male men} And (3) cells. RBD is a key domain of the S protein that binds to SARS-CoV-2 receptor ACE2 and has multiple neutralizing antibody binding sites in a region unaffected by the Omicron BA.1 change, e.g., position L452. More focusing of the immune response on this domain may contribute to B _{Male men} The cells produced neutralizing antibodies against the unchanged RBD epitope in Omicron ba.1.

Third, broadly neutralizing antibodies were introduced. Most of the serum from Omicron ba.1 recovery phase, but not from vaccinated individuals not infected with Omicron, was found to strongly neutralize SARS-CoV-1. This may indicate that Omicron ba.1 infection in vaccinated individuals stimulated B formation of neutralizing antibodies against spike protein epitopes conserved in the SARS-CoV-1 and SARS-CoV-2 families _{Male men} And (3) cells. Broadly neutralizing antibodies are reported to be present in individuals vaccinated with BNT162b2 with SARS-CoV-1 infection. Such pansabia virus immune response is believed to be an immune response triggered by neutralizing antibodies to highly conserved S protein domains. The greater antigenic distance of the Omicron ba.1 spike protein relative to other SARS-Cov-2 strains may help target conserved sub-dominant neutralizing epitopes, as recently described as being located in the C-terminal portion of the spike protein.

Taken together, these results indicate that although previous vaccinations may have a significant impact on immune responses, preformed pools of B memory cells can be re-pooled and quantitatively reconstituted by exposure to heterologous S proteins to allow neutralization of variants escaping previously established neutralizing antibody responses.

Finally, while the data is based on samples from individuals exposed to omacron ba.1S protein due to infection, the findings presented herein support that vaccines that adapt to omacron ba.1S protein can similarly reconstruct B memory cell banks and thus may be more beneficial than the current series of expanded boosters of MN908947-Hu-1 spike-based vaccines.

Materials and methods

Participant recruitment and sample collection

Pass-through from uninfected SARS-CoV-2 OmicronBNT162b2 double vaccination (BNT 162b 2) ² ) And triple vaccination (BNT 162b 2) ³ ) Providing informed consent to individuals of the cohort as part of their participation in the clinical trial (phase 1/2 trial BNT162-01[ NCT 04380701)]Phase 2 Rolling test BNT162-14[ NCT 0494949490 ]]Or as BNT162-17[ NCT05004181 ]]Part of the test).

As part of a study plan to recruit patients who underwent Omicron ba.1 breakthrough infection following covd-19 vaccination, groups of two-and triple-vaccinations from SARS-CoV-2 Omicron ba.1 recovery (BNT 162b2, respectively) were recruited by a university affiliated hospital (University Hospital, goethe University Frankfurt) of frankfurd university ² + Omi and BNT162b2 ³ Group + Omi) and vaccinated with other approved covd-19 vaccines or mixed regimens of individuals with subsequent omacron ba.1 breakthrough infections to provide blood samples and clinical data for the study. Omicron ba.1 strain infection was confirmed by variant specific PCR or sequencing and participants did not develop symptoms at the time of blood collection.

The sampling time points are provided in fig. 15.

Serum was isolated by centrifugation at 2000x g for 10 minutes and the serum was cryopreserved until use. Lithium Heparin (Li-Heparin) blood samples were isolated by density gradient centrifugation using Ficoll-Paque PLUS (cytova) and then the blood samples were cryopreserved until use.

VSV-SARS-CoV-2S variant pseudovirus production

Recombinant replication defective Vesicular Stomatitis Virus (VSV) vectors encoding Green Fluorescent Protein (GFP) and luciferase instead of VSV-glycoprotein (VSV-G) were pseudotyped with SARS-CoV-1 spike (S) (UniProt Ref: P59594) and SARS-CoV-2S, according to published pseudotyping protocols (M. Berger Rensch, G. Zimmer, A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-specific type I interson. PloS one.6, e25858 (2011), doi:10.1371/joumal. Fine. 0025858): MN908947 reference strain (NCBI Ref: 43740568); alpha variants (mutations: DELTA69/70, DELTA144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H); beta variants (mutation: L18F, D80A, D G, DELTA242-244, R246I, K417N, E484K, N501Y, D G, A701V); delta variants (mutation: T19R, G142D, E G, DELTA/158, K417N, L452R, T478K, D52614G, P681R, D950N); the Omicron BA.1 variant (mutation: A67V, DELTA69/70, T95I, G142D, DELTA143-145, DELTA211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E496K, E498K, E501K, E505 547K, E614K, E655 to 679K, E681K, E764K, E796K, E856 954K, E5297 969 5297F); or OmicronBA.2 variants (mutations: T19I, DELTA24-26, A27S, G142D, V213G, G339 38395 371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484 493R, Q498R, N Y, Y505Y, Y614 655Y, Y679Y, Y681Y, Y764Y, Y796Y, Y954 5297 969K).

Briefly, SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmid with liposomes LTX (Life Technologies) was transfected with a kit supplemented with 10% heat-inactivated fetal bovine serum (FBS [ Sigma-Aldrich ] according to the manufacturer's instructions]) Is GlutaMAX of (A) ^TM HEK293T/17 monolayer cells cultured in Dunaliella modified Medium (DMEM) (referred to as Medium) of (Gibco)CRL-11268 ^TM ). After 24 hours, the VSVDELTAG vector was supplemented with VSV-G. At 37℃with 7.5% CO ₂ After 2 hours of incubation, cells were washed twice with Phosphate Buffered Saline (PBS) before adding medium supplemented with anti-VSV-G antibody (clone 8G5 F11,Kerafast Inc) to neutralize residual VSV-G supplemented input virus. The medium containing the VSV-SARS-CoV-2-S pseudotype was harvested 20 hours after incubation, passed through a 0.2 μm filter (Nalgene) and stored at-80 ℃. Vero 76 cells (++) cultured in medium>CRL-1587 ^TM ) The pseudovirus batch was titrated up. From the previous at Muik et alThe relative luciferase activity units induced by a limited volume of MN908947 spike pseudovirus reference batch corresponding to an infectious titer of 200 Transduction Units (TU)/mL described in human (Muik et al, "Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elited human sera.science (New York, N.Y.) 371, 1152-1153 (2021), doi:10.1126/science.abg 6105") was used as a comparator. The input volume of SARS-CoV-2 variant pseudovirus batches was calculated to normalize the infection titer based on the relative luciferase activity units of the reference.

Pseudovirus neutralization assay

Vero 76 cells were seeded at 40,000 cells/well in 96-well white flat bottom plates (Thermo Scientific) with medium and at 37 ℃ with 7.5% co 4 hours prior to assay ₂ Culturing. Each serum was serially diluted 2-fold in medium with either a 1:5 (double and triple BNT162b2 vaccinations without Omicron infection; dilution ranging from 1:5 to 1:5, 120) or 1:30 (double and triple BNT162b2 vaccinations after subsequent Omicron breakthrough infections; dilution ranging from 1:30 to 1: 30,720). The VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in medium to obtain 200 in the assay TU. Serum dilutions were mixed with pseudovirus (n=2 technical replicates/serum/pseudovirus) at 1:1 for 30 min at room temperature before addition to Vero 76 monolayer cells and use of 7.5% co at 37 °c ₂ Incubate for 24 hours. The supernatant was removed and the cells lysed with luciferase reagent (Promega). At the position ofLuminescence was recorded on a 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. The results are expressed as Geometric Mean Titer (GMT) of the replicates. If no neutralization is observed, the detection limit [ LOD ] is recorded ]Half of any titer value.

Living SARS-CoV-2 neutralization assay

Passing micro at vismedi s.r.1. of cassianaQuantitative neutralization assay SARS-CoV-2 virus neutralization titers were determined based on cytopathic effect (CPE). Briefly, heat-inactivated serum samples from participants were serially diluted 1:2 (starting at 1:10) and incubated at 37 ℃ for 1 hour with each of 100 tcid50 to allow any antigen-specific antibodies to bind to the virus: live MN 908947-like strain 2019-nCOV/ITALY-INMI1 of SARS-CoV-2 virus (Gene Bank: MT 066156); the Beta virus strain human nCoV19 oleate/England ex-SA/HCM002/2021 (mutation: D80A, D215G, DELTA242-244, K417N, E484K, N501Y, D614G, A V); sequence verified Delta strains isolated from nasopharyngeal swabs (mutation: T19R, G142D, E156G, DELTA/158, L452R, T478K, D614G, P681R, R682Q, D950N); or Omicron ba.1 strain hCoV-19/Belgium/rega-20174/2021 (mutation: A67V, DELTA69/70, T95I, G142D, DELTA143-145, DELTA211, L212I, ins214EPE G339D, S371 79373P, S375F, K417N, N440K, G446S, S477N, T478K, E484K, E493K, E496K, E498K, E501K, E505K, E547K, E614 655K, E679K, E681K, E764K, E796K, E856K, E954K, E969K, E981F). The 2019-nCOV/ITALY-INMI1 strain S protein is identical in sequence to wild-type SARS-CoV-2S (MN 908947-Hu-1 isolate). Vero E6% CRL-1586 ^TM ) Monolayer cells were incubated with serum/virus mixtures in 96-well plates and incubated for 3 days (2019-nCOV/ITALY-INMI 1 strain) or 4 days (Beta, delta, and Omicron BA.1 variant strains) to allow infection with non-neutralized virus. Plates were observed under an inverted light microscope and the wells were rated as positive for SARS-CoV-2 infection (i.e., showing CPE) or negative for SARS-CoV-2 infection (i.e., cells were viable without CPE). Neutralization titers were determined as the reciprocal of the highest serum dilution that protected more than 50% of the cells from CPE and were recorded as GMT of replicates. If no neutralization is observed, a 5-fold record is made (for detection limit [ LOD]Half) of the total titer value.

Detection and characterization of SARS-CoV-2 specific B cells by flow cytometry

The recombinant biotinylated SARS-CoV-2 spike (Acro Biosystems: MN908947-SPN-C82E9, alpha-SPN-C82E5, delta-SPN-C82Ec, omicron-SPN-C82 Ee) and RBD (Acro Biosystems: MN908947-SPD-B28E9, alpha-SPD-C82E6, delta-SPD-C82Ed, omicron-SPD-C82E 4) proteins were used to detect spike/RBD specific B cells. Recombinant spike and RBD proteins were tetramerized with fluorescent labeled streptavidin (BioLegend, BD Biosciences) at a 4:1 molar ratio in the dark at 4℃for 1 hour. After this, the sample was spin-precipitated at 4 ℃ for 10 minutes to remove the final precipitate.

In flow cytometry analysis, PBMCs were thawed and 5x10 for each sample ⁶ Individual cells were seeded into 96-well U-shaped bottom plates. Blocking cells for Fc-receptor binding (human BD Fc Block ^TM BD Biosciences) and the cells were saturated with free Biotin (D-Biotin, invitrogen, 1. Mu.M) in running buffer (DPBS (Gibco) supplemented with 2% FB S (Sigma), 2mM EDTA (Sigma-Aldrich)) at 4℃for 20 min. The cells were washed and labeled with BCR bait tetramer in dark at 4 ℃ for 1 hour (2 μg/ml for spike and 0,25 μg/ml for RBD protein) in running buffer supplemented with free Biotin (D-Biotin, invitrogen,2 μg/m 1). Cells were washed with running buffer and washed with viability (fixed viability dye eFluor) in running buffer supplemented with Brilliant Stain Buffer Plus (BD Biosciences, according to manufacturer's instructions) ^TM 780, ebioscience) and surface markers (CD 3-clone: UCHT1 (BD Biosciences), CD 4-clone: SK3 (BD Biosciences), CD185 (CXCR 5) -clone: RF882 (BioLegend), CD279 (PD-1) -clone: EH12.1 (BD Biosciences), CD278 (ICOS) -clone: C398.4A (BioLegend), CD 19-clone: SJ25C (BD Biosciences), CD 20-clone: 2H7 (BD Biosciences), CD 21-clone: b-ly4 (BD Biosciences), CD 27-clone: l128 (BD Biosciences), CD 38-clone: HIT2 (BD Biosciences), CD11 c-clone: S-HCL-3 (BD Biosciences), CD 138-clone: MI15 (BD Biosciences), igG-clone: g18-145 (BD Biosciences), igM-clone: g20-127 (BD Biosciences), igD-clone: IA6-2 (BD Biosciences), CD 14-clone: (BD Biosciences, bleed channel), CD 16-clone: 3G8 (BD Biosciences, bleed channel)) the cells were stained at 4 ℃ for 20 min. The samples were washed and fixed with BDTM stabilization fixative (BD Biosciences, according to manufacturer's instructions), after which data collection was performed 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.).

Fragmentation and doublet are distinguished via FSC/SSC. Dead cells and monolayer cells (CD 14, CD 16-viability/bleeder channel) were then excluded. Analysis of CD 19-positive B cells for IgD and CD27 expression, thereby discriminating the initial B cells as IgD with Boolean 'set-up NOT' function ⁺ And (3) cells. Within non-primary B cells, differentiation of plasmablasts (CD 38 ^{High height} CD20 ^{Low and low} ) And B memory cells (B) _{Male men} CD38 ^Integer/low CD20 ^{High height} ). Pair B _{Male men} Cell analysis B cell decoy binding. SARS-CoV-2 spike reactivity was assessed by gating each spike/RBD variant tested via mapping from CD20 signals. Bait door covers the whole B _{Male men} On cells and for four bait channels are shown as NxN plots.

Statistical analysis

The aggregation statistical method used to analyze antibody titers was a geometric mean, and the ratio of titers for SARS-CoV-2VOC to MN908947 titers was the geometric mean and the corresponding 95% confidence interval. The use of geometric averages allows for an abnormal distribution of antibody titers across several orders of magnitude. Friedman test with Dunn correction for multiple comparisons was used for paired symbol rank test with a set of geometric mean neutralizing antibody titers under a common control group. Flow cytometry frequencies were analyzed with FlowJo software (version 10.7.1) and tables derived therefrom. The statistical analysis of the cumulative B memory cell frequency is the mean and standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism software version 9.

Example 8: vaccine-induced antibody response encoding SARS-CoV-2S protein from Omicron variant

To test the efficacy of an RNA vaccine encoding SARS-CoV-2S protein comprising one or more mutations specific for omacron variants, subjects previously administered a primary therapy comprising two doses of 30 μg of RNA encoding SARS-CoV-2S protein from MN908947 strain (e.g., BNT162b 2) and a booster therapy comprising one dose of 30 μg of RNA encoding SARS-CoV-2S protein from MN908947 strain (i.e., MN908947 specific booster, e.g., BNT162b 2) were administered another booster dose comprising: (i) 30 μg of RNA encoding SARS-CoV-2S protein from the MN908947 strain (e.g., BNT162b 2), or (ii) 30 μg of RNA encoding SARS-CoV-2S protein comprising a mutation unique to the Omicron ba.1 variant (i.e., omicron ba.1 specific enhancer, e.g., RNA encoding SARS-CoV-2S protein comprising amino acid sequence SEQ ID NO:49 and/or nucleotide sequences SEQ ID NO:50 and/or 51) (the dose administered as part of the second enhancer therapy is referred to in the figures as the "fourth agent"). Serum was collected from the subject at the time of and one month after administration of the second booster therapy.

Neutralizing antibody titers were determined using fluorescence focus reduction neutralization assay ("FFRNT"). Suitable FFRNT assays are known in the art and include, for example, those 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 are incorporated herein by reference in their entirety. Other 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. 20A, subjects administered a second booster therapy comprising a dose of RNA encoding SARS-CoV-2S protein comprising a mutation unique to the Omicron ba.1 variant exhibited a significant increase in neutralizing antibody concentration against the Omicron ba.1 variant compared to subjects administered a second booster therapy comprising a dose of RNA encoding SARS-CoV-2S protein of the MN908947 strain. Specifically, subjects administered omacron ba.1-specific enhancer exhibited 1.79-fold higher GMRs and 2.31-fold higher GMFR than those observed in subjects administered the fourth dose of RNA encoding SARS-CoV-2S protein from the MN908947 strain. The excellent immune response elicited by Omicron ba.1 specific boosters against Omicron ba.1 variants was further increased in subjects previously infected with SARS-CoV-2 (as determined by antigen assays) or currently infected with SARS-CoV-2 (as determined by PCR). Referring to fig. 20B, it shows: compared to the results observed in a population of subjects including a prior and/or current infected subject and administered an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain, the population of subjects including a prior and/or current infected subject exhibited 2.94-fold higher GMR and 1.97-fold higher GMFR ratios.

Pseudovirus neutralization assays were also performed using pseudoviruses comprising the SARS-CoV-2S protein of the MN908947 strain and using the same serum samples discussed above. Subjects administered with RNA encoding SARS-CoV-2S protein from the MN908947 strain (e.g., BNT162b 2) exhibited a neutralizing antibody titer similar to that observed in subjects administered with Omicron ba.1-specific enhancer, demonstrating at least a similar effect of both vaccines in their ability to elicit an antibody response against the MN908947 strain. Referring to fig. 20C, it shows: GMR and GMFR observed in subjects administered MN 908947-specific potentiators (e.g., BNT162b 2) were similar to the values observed in subjects administered Omicron ba.1-specific potentiators (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 with the omacron ba.1-specific enhancer exhibited an improved immune response compared to subjects administered with the MN908947 strain-specific enhancer. Referring to fig. 20D, it is shown that GMR of subjects administered omacron ba.1 specific enhancer is about 1.4 times greater than GMR of subjects administered MN908947 specific enhancer.

Subjects administered omacron ba.1-specific boosters also exhibited excellent immune responses against Delta variants in pseudovirus neutralization assays. Referring to fig. 20E, it shows: GMFR in subjects administered omacron ba.1-specific enhancer was about 1.20 times higher than that observed in subjects administered MN 908947-specific enhancer. The excellent immune response elicited by Omicron ba.1-specific potentiators against Delta variants is further increased in serum from subjects previously and/or currently infected with SARS-CoV-2. See fig. 20F.

Example 9: immunogenicity studies of vaccines encoding the S protein of SARS-CoV-2 variants in unvaccinated subjects

To test the immunogenicity of various variant-specific vaccines in non-vaccinated subjects, non-vaccinated mice were immunized twice with each of the following: (a) physiological saline (negative control); (b) An RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain; (c) An RNA vaccine encoding SARS-CoV-2S protein having a mutation unique to the Omicron BA.1 variant (Omi); (d) An RNA vaccine encoding SARS-CoV-2S protein having a mutation specific for a Delta variant (Delta); (e) A bivalent vaccine comprising an RNA encoding a SARS-CoV-2S protein from the MN908947 strain and a SARS-CoV-2S protein comprising a mutation specific for the Omicron ba.1 variant (b2+ Omi); and (f) a bivalent vaccine comprising an RNA encoding a SARS-CoV-2S protein having a mutation specific for the Delta variant and an RNA encoding a SARS-CoV-2S protein having a mutation specific for the Omacron BA.1 variant (Delta+ Omi). The immunogenicity of RNA vaccines was investigated by focusing on the antibody immune response.

Serum was obtained 7 days after immunization and analyzed using a pseudovirus neutralization assay (e.g., the assay described in example 2) using a pseudovirus comprising the SARS-CoV-2S protein from the MN908947 strain, the SARS-CoV-2S protein comprising a mutation unique to the Beta variant, the SARS-CoV-2S protein comprising a mutation unique to the Delta variant, or the SARS-CoV-2S protein comprising a mutation unique to the Omicron ba.1 variant. As shown in fig. 21, the bivalent vaccine was found to elicit the broadest immune response in the unvaccinated mice.

Example 10: vaccines encoding SARS-CoV-2S protein comprising one or more mutations unique to Beta variants induce an antibody response in subjects previously administered an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain

To test the efficacy of an RNA vaccine encoding SARS-CoV-2S protein comprising one or more mutations specific for a Beta variant, subjects who were previously administered a primary therapy and in which the two doses each were 30 μg of RNA encoding SARS-CoV-2S protein from the MN908947 strain (BNT 162b2 (SEQ ID NO: 20) in this example) were administered two booster doses each comprising 30 μg of RNA encoding SARS-CoV-2S protein comprising one or more mutations specific for a Beta variant (hereinafter referred to as Beta-specific vaccine). In this example, construct RBP020.11 was administered as a Beta-specific vaccine. Although in the present example, two booster doses are administered about 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 apart (e.g., according to exemplary dosing regimens as described herein).

Serum was collected from the subjects one month prior to administration of BNT162b2, one month after administration of two primary doses of BNT162b2, one month after administration of the first dose of Beta-specific vaccine, and one month after administration of the second dose of Beta-specific vaccine. Neutralizing antibody titers against the SARS-CoV-2S protein comprising the MN908947 strain or the SARS-CoV-2S protein comprising one or more mutations specific for Beta variants were measured using a pseudovirus neutralization assay (the results are shown in figure 22). The subject exhibited an increase in neutralizing antibody titers against both the MN908947 strain and the Beta variant of SARS-CoV-2 following administration of the third and fourth dose of Beta-specific vaccine.

Example 11: vaccines encoding SARS-CoV-2S protein comprising one or more mutations unique to Beta variants elicit an antibody response in unvaccinated subjects

To test the efficacy of an RNA vaccine encoding SARS-CoV-2S protein comprising one or more mutations specific for a Beta variant in a non-vaccinated subject, two doses of 30 μg each of the RNA encoding SARS-CoV-2S protein comprising one or more mutations specific for a Beta variant (RBP 020.11 in this example) were administered to a subject who had not been previously administered a SARS-CoV-2 vaccine and did not show signs of SARS-CoV-2 infection previously or currently (e.g., as assessed by antibody testing and/or PCR testing). Serum was collected one month after administration of the second dose and the neutralizing antibody titers were measured using a virus neutralization assay in which the virus particles used contained the SARS-CoV-2S protein from the MN908947 strain or the SARS-CoV-2S protein with one or more mutations specific for the Beta variant. Tables 15 and 16 below show the neutralization assay results for Beta variants (neutralization assay results for MN908947 strain are not shown). As shown in the table, it was found that an RNA vaccine encoding SARS-CoV-2S protein having a mutation specific for the Beta variant induced a significantly stronger antibody response against the Beta variant compared to the non-vaccinated subjects administered two doses of an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain (BNT 162b2 in this example).

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Example 12: eliciting antibody responses and reactogenicity in monovalent, bivalent and high dose BNT162b2 or Omicron ba.1 specific vaccines in participants over 55 years old.

To test the efficacy and safety of (i) higher doses of an RNA vaccine (e.g., as described herein), (ii) an RNA vaccine encoding SARS-CoV-2S protein having one or more mutations specific for an Omicron ba.1 variant (Omicron ba.1-specific vaccine) and (iii) a subject comprising an RNA encoding SARS-CoV-2S protein from an MN908947 variant and an RNA encoding SARS-CoV-2S protein having one or more mutations specific for an Omicron ba.1 variant, one of several booster doses (e.g., as described herein) was administered to a subject who had previously been administered an RNA vaccine encoding SARS-CoV-2S protein of at least one dose of an MN908947 strain. Specifically, a fourth dose is administered to a subject to whom two doses of 30ug of an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain (BNT 162b2 in this example) and a third dose of 30ug of an RNA vaccine encoding SARS-CoV-2S protein from the MN908947 strain (BNT 162b2 in this example) have been administered, the fourth dose comprising:

(a) 30ug of RNA encoding SARS-CoV-2S protein from MN908947 strain,

(b) 60ug of RNA encoding SARS-CoV-2S protein from MN908947 strain,

(c) 30ug of Omicron BA.1 specific vaccine,

(d) 60ug of Omicron BA.1 specific vaccine,

e) 30ug of bivalent RNA vaccine (Omacron BA.1-adapted bivalent vaccine) comprising 15ug of RNA encoding SARS-CoV-2S protein from MN908947 strain and 15ug of RNA encoding SARS-CoV-2S protein comprising a mutation specific for the Omacron BA.1 variant, or

(f) A 60ug bivalent RNA vaccine (omacron ba.1-adapted bivalent vaccine) comprising 30ug of RNA encoding the SARS-CoV-2S protein from the MN908947 strain and 30ug of RNA encoding the SARS-CoV-2S protein comprising a mutation specific for the omacron ba.1 variant.

In this example, for the fourth dose, the RNA encoding the SARS-CoV-2S protein from the MN908947 variant is BNT162b2 and the RNA encoding the SARS-CoV-2S protein having the mutation unique to the omacron ba.1 variant comprises the amino acid sequence of SEQ ID NO: 51.

Serum samples were taken at dose 4 and 7 days after administration and tested for neutralizing antibody titers against viral particles containing SARS-CoV-2S protein from the MN908947 strain or SARS-CoV-2S protein containing mutations specific for Delta variants or omacron ba.1 variants.

Neutralizing antibody titers were determined using fluorescence focus reduction neutralization assay (FFRNT). Suitable FFRNT assays are known in the art as discussed in example 8. The neutralization reaction is shown in fig. 23.

As shown in fig. 23 (a), subjects administered the fourth dose 30ug Omicron BA.1-specific vaccine exhibited an increase in neutralizing antibodies against the omacron ba.1 variant compared to the fourth dose 30ug bnt162b 2. Administration of 60ug of RNA increased neutralization against BNT162b2 and Omicron ba.1 specific vaccine, wherein 60ug of Omicron ba.1 specific vaccine showed a stronger immune response against Omicron ba.1 variant. As shown in fig. 23 (B), similar effects were observed in populations including subjects previously or currently infected with SARS-CoV-2 (e.g., as determined by antibody and PCR assays, respectively).

FIG. 23 (C-D) provides data of neutralization responses of MN908947 strain against SARS-CoV-2 in a population of subjects not including subjects previously or presently 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 Delta variants in a population of subjects (FIG. 23 (E)) that excludes subjects previously or currently infected with SARS-CoV-2 and a population of subjects (FIG. 23 (F)) that include these subjects.

Fig. 23 (G) shows the neutralization response with subjects administered with dose 4, 30ug bnt162b 2. As can be seen from the table, the omacron ba.1 specific vaccine elicited a strong response against the omacron ba.1 variant, and at least comparable to the response of BNT162b2 against the other variants. The bivalent vaccine (omacron ba.1 adaptive bivalent vaccine) produced a strong immune response against each SARS-CoV-2 variant tested at doses of 30ug and 60 ug.

The reactivity of the fourth dose tested in the patient was also monitored within 7 days after administration of dose 4. Fig. 24 (a) shows the local immune response observed in the different groups of subjects as indicated. As can be seen in the figures, the 60ug dose of omacron-specific vaccine and bivalent vaccine was found to be more likely to produce pain at the injection site than was observed with the other tested booster doses; however, pain was rated as mild or moderate for both doses. Redness and swelling responses were lower and comparable at each dose tested.

Fig. 24 (B) shows the systemic immune response observed in the subjects of the different groups as indicated. For each dose, the systemic response (e.g. by fever, fatigue, headache, chill, vomiting, diarrhea, muscle pain or joint pain) was similar, whereas fatigue tended to be higher at the 60ug dose.

The immune response and reactogenicity of omacron ba.1 adaptive vaccine (monovalent and bivalent vaccine as described in the examples) as booster doses was also demonstrated in phase 2/3 trials of more than 1,000 participants aged 56 and older. The Geometric Mean Titer (GMT) and Geometric Mean Ratio (GMR) of each of the tested doses compared to the subjects administered the 4 th dose 30ug bnt162b2 are shown in table 23 below.

Table 23: neutralization in subjects administered 30ug or 60ug of monovalent or bivalent Omicron ba.1 specific vaccine

The Omicron ba.1 adaptive vaccine administered as booster doses (monovalent or bivalent; and 30ug or 60 ug) elicited a higher neutralizing antibody response against Omicron ba.1 when compared to the neutralizing antibody response induced by BNT162b2 (encoding SARS-CoV-2S protein from MN908947 strain). The pre-specified simple dominance criterion is measured by the ratio of the neutralization geometric mean titer (GMR) with 95% confidence interval lower limit > 1. The pre-specified superdominance criterion is measured by the ratio of the neutralization geometric mean titer (GMR) with a 95% confidence interval lower limit > 1.5. Based on these criteria, each of the tested doses showed better than 30ug of BNT162b2, and administration of 30ug or 60ug of BNT162b2 OMI showed superior performance compared to 30ug of BNT162b 2.

The serum response rates are summarized in table 24 below. As shown in the table below, each of the doses tested was found to be no less than BNT162b2 30ug.

Table 24: serum reactivity of Omicron BA.1

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¹ Non-bad efficiency criteria: the 95% confidence interval has a lower limit of > -5%

After collecting the data summarized in table 24, data was continued to be collected on the subjects administered the omacron monovalent vaccine. Further data are summarized in table 25 below and confirm the findings shown in table 24.

Table 25: further data on Omicron ba.1 serum reactivity

Abbreviations: nt50=50% neutralization titer; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

Note that: serum responses were defined as achieving a 4-fold increase from baseline (prior to the first study vaccination). If the baseline measurement is below LLOQ, then a post-vaccination measurement of 4×LLOQ is considered a serum response.

Note that: included in the analysis were subjects who had no serological or virological evidence of past SARS-CoV-2 infection (prior to blood sample collection 1 month after the first study vaccination) (i.e., were negative for N-binding antibodies [ serum ] at the time of the first study vaccination and 1 month visit after the first study vaccination, were negative for NAAT [ nasal swabs ] at the time of the first study vaccination visit and any unplanned visit prior to blood sample collection 1 month after the first study vaccination) and had no history of covd-19.

a.N = number of participants with valid and determined assay results at pre-vaccination time point and given sampling time point for a given assay.

This value is the denominator of the percentage calculation.

b.n = number of participants with serum response to a given assay at a given sampling time point.

c. Accurate double sided CI based on Clopper and Pearson methods.

d. The ratio difference is expressed as a percentage (monovalent Omicron ba.1[30mcg ] -comimary [30mcg ]).

e. The double sided CI based on the mietinien and nurrminen methods of the ratio differences are expressed as percentages.

The change in titer for each dose is shown in figure 25 before the administration of the 4 th booster dose and 1 month after the administration of the 4 th booster dose. As shown in fig. 25, one month after administration, the boost dose (30 μg and 60 μg) of Omicron ba.1-adapted monovalent vaccine increased the neutralization Geometric Mean Titer (GMT) against Omicron ba.1.13.5 and 19.6 fold higher than the pre-boost level, while the boost dose (30 μg and 60 μg) of Omicron ba.1-adapted bivalent vaccine conferred a 9.1 and 10.9 fold increase against Omicron ba.1-neutralized GMT. The boost dose (30 μg) of Omicron ba.1-adapted bivalent vaccine induced neutralization titers were about 3 times lower than the neutralization titers induced for ba.1. Of the participants who received one or the other omacron ba.1-adapted vaccine, both omacron ba.1-adapted vaccines (e.g., monovalent and bivalent vaccine) were well-tolerated and exhibited good safety and tolerability characteristics similar to BNT162b2 (encoding SARS-CoV-2S protein from MN908947 strain).

In addition, in SARS-CoV-2 live virus neutralization assays tested on sera from participants who received an Omicron-adapted vaccine (e.g., monovalent or bivalent vaccine as described in this example) at and above 56 years old, the sera also neutralized Omicron BA.4/BA.5, titres 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 Omacron subfsystem with further changes in SARS-CoV-2S protein continue to appear, where European disease prevention and control center (ECDC) treated BA.4 and BA.5 as VOCs at day 12 of 5.2022 (European disease prevention and control center, epidemiological update: SARS-CoV-2 Omacron subfsystem BAA and BA.5 (2022) (available at https:// www.ecdc.europa.eu/en/new-events/epidemic-update-sarscov-2-Omicron-sub-linear-ba 4-and-ba 5)).

This example 13 is an extension of example 7 in which serum samples taken from ba.1-breakthrough cases as described in example 7 were further analyzed for neutralizing activity against Omicron ba.4 and ba.5 variants.

As described in example 7, 50% pseudovirus neutralization of Beta and Delta VOCs was found in duplicate vaccinated individuals not infected with Omicron (pVN ₅₀ ) Geometric Mean Titers (GMT) were reduced compared to the MN908947 strain, whereas neutralization of omacron sub-lines ba.1 and ba.2 was barely detectable. In this example, FIG. 26 (a) shows that neutralization titers of BA.4/5 were also barely detectable in double vaccinated BA.1-breakthrough patients.

As described in example 7, the omacron-uninfected triple vaccinated individuals were directed against all VOCs testedExhibits a significantly higher pVN than the double vaccinated individual ₅₀ GMT. Potent neutralization of Alpha, beta and Delta variants was observed, whereas neutralization by Omicron ba.1 and ba.2 was reduced compared to MN908947 (GMT 160 and 211 vs 398). As shown in fig. 26 (a) of the present example, neutralization by omacron ba.4/5 was further reduced (GMT 74) in the patients with triple vaccination without omacron, corresponding to a titer 5-fold lower than that of the MN908947 strain.

As shown in 26 (b), the Omacron BA.1 breakthrough infection was found to have only a slight potentiation on BA.4/5 neutralization. pVN against Omacron BA.4/5 in double vaccinated patients ₅₀ GMTs were significantly lower than those for MN908947 (GMT 135 vs 740). Similar patterns were observed in ba.1 recovery serum from triple vaccinated individuals and control serum. As shown in example 7, BA.1 recovery serum showed a high pVN against the previous SARS-CoV-2 VOCs, including Beta (1182), omicron BA.1 (1029) and Omicron BA.2 (836) ₅₀ GMT, which is close to the titer referenced (1182) for MN 908947. In contrast, as shown in fig. 26 (b), neutralization of ba.4/5 was significantly reduced, pVN, in triple vaccinated individuals with ba.1 breakthrough infection compared to the MN908947 strain ₅₀ GMT was 197, 6-fold lower than for MN908947 strain.

Notably, in all groups, the neutralization titers for BA.4/5 were closer to the low levels observed for phylogenetically more distant SARS-CoV-1 pseudoviruses than those observed for MN 908947. Comparison of SARS-CoV-2 VOC normalized to MN908947 and SARS-CoV-1 pVN ₅₀ Ratio of GMT (fig. 26 (c)), it is notable that breakthrough infection by omacron ba.1 did not result in more effective cross-neutralization of omacron ba.4/5 in double-vaccinated and triple-vaccinated individuals. Taken together, these data demonstrate that omacron ba.1 breakthrough infection in vaccinated individuals mediates broad neutralization activity against ba.1, ba.2 and several previous SARS-CoV-2 variants, but not against ba.4/5.

As shown in fig. 27, similar results were found in patients previously administered a non-BNT 162b2 vaccine and with ba.1 breakthrough infection.

As described in example 7, it was found that Omacron BA.1 breakthrough infection in BNT162B2 vaccinated individuals resulted in strong neutralization activity against Omacron BA.1, BA.2 and the previous SARS-CoV-2 VOC, mainly by amplifying B against an epitope widely shared between different SARS-CoV-2 strains _MEM And (3) cells. These data demonstrate that B of the vaccination print _MEM The cell pool was sufficiently plastic to remodel by exposure to the heterologous SARS-CoV-2S protein. Although B recognizes shared epitopes _MEM Selective expansion of cells allows for efficient neutralization of most variants of the immunity established prior to escape, but obtaining altered variants at sites conserved so far may have enhanced sensitivity to escape. Neutralization activity against Omicron ba.4/5 pseudovirus (which has additional changes in RBD L452R and F486V) significantly reduced the mechanisms supporting escape of immunity by losing some of the remaining conserved epitopes.

Discussion of

Unexpectedly, and contrary to the results observed in example 7, neutralization by omacron sublines ba.4and ba.5 was not enhanced in ba.1-breakthrough patients, but rather titers comparable to those for phylogenetically more distant SARS-CoV-1. While this example focused on individuals vaccinated with BNT162b2 mRNA vaccine, similar observations have recently been reported in individuals with CoronaVac (a whole inactivated virus vaccine developed by Sinovac Biotech), suggesting that Omacron BA.4/5 may bypass BA.1 infection-mediated humoral immune boosting (Y.Cao et al BA.2.12.1, BA.4and BA.5 escape antibodies elicited by Omicron infection, bioRxiv: the preprint server for biology (2022)).

The present disclosure provides a thorough understanding of how immunity against multiple variants, the causes of which omacron ba.4 and ba.5 sublines can partially escape neutralization, is achieved in ba.1 breakthrough cases, and provides vaccination protocols and techniques to enhance protection across coronavirus strains and lineages, including in particular across omacron lineages (e.g., including ba.4 and/or ba.5). Without wishing to be bound by a particular theory, the present disclosure suggests that initial exposure to MN908947 toxinThe strain S protein can shape the formation of BMEM cells and is directed against novel B recognizing epitopes unique to Omicron BA.1 variants _MEM The cellular response produces a blot.

The Omicron ba.1 breakthrough infection in bnt162B2 vaccinated individuals amplified predominantly broad B against the conserved SARS-CoV-2S protein and RBD epitope _MEM Cell banks other than those that induce stringent Omicron ba.1 specificity B _MEM And (3) cells. The Omicron ba.1 breakthrough infection resulted in a more pronounced increase in antibody neutralization titers against Omicron, as well as a potent cross-neutralization of many SARS CoV-2 variants, compared to the immune response elicited by homologous vaccine boosting.

As described in example 7, one possible explanation for the broad neutralization caused by ba.1 breakthrough infection is the induction of broad neutralizing antibodies. The serum from Omicron ba.1-convalescence vaccinated individuals was found to neutralize SARS-CoV-2 Omicron ba.4/5 and SARS-CoV-1 to a much lower extent than the previous SARS-CoV-2 VOCs (including ba.1 and ba.2). This finding suggests that omacron ba.1 infection in vaccinated individuals stimulated BMEM cells that produced neutralizing antibodies to the S protein epitope conserved in the SARS-CoV-2 variant (up to and including omacron ba.2), but largely lost in ba.4/5 and largely not shared by SARS-CoV-1.

The greater antigen distance of the Omicron BA.1S protein from the early SARS-CoV-2 strain may facilitate the targeting of the epitope in the masking site as described recently (Li, ringing, et al, "Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants," Nature communications.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-eitic 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 S b.5) or in the membrane proximal S glycoprotein subunit designated S2 (Pinto be expressed as" 3.13 ").

As described in example 7, individuals infected with Omicron BA.1 appeared to have significantly higher RBD/S protein-specific B compared to vaccinated, uninfected Omicron individuals _MEM Cell ratio. Omicron BA.1 carries multiple S protein changes (such as del69/70 and del 143-145) in the critical neutralizing antibody binding sites of NTD, which significantly reduce the targeting surface of memory B cell responses in this region. Although Omicron ba.1 RBD has multiple changes, some neutralizing antibody binding sites are unaffected (20). B (B) _MEM The neutralization of ba.1 and ba.2 variants can be helped by the expansion of cells that produce neutralizing antibodies against RBD epitopes that are unchanged in omacron ba.1, as indicated in this example. Importantly, the strong neutralization of Omicron ba.1 and ba.2 should not mask the following facts: neutralization B in Omicron ba.1 recovery phase vaccinated individuals _MEM The immune response is driven by a small number of epitopes. The significantly reduced neutralizing activity against Omicron ba.4/5 pseudovirus (which has additional changes in RBD L452R and F486V) demonstrates a mechanism to evade immunity by losing some of the remaining conserved epitopes. Meanwhile, other sublines with L452 alterations (e.g., ba.2.12.1) were reported to evade humoral immunity triggered by ba.1 breakthrough infection (y.cao et al, cited above).

The present disclosure suggests that immunization at the early stage of Omicron ba.1 infection in vaccinated individuals may be based on recognition of conserved epitopes and narrowly focused on a small number of neutralization sites that are not altered in Omicron ba.1 and ba.2. Such narrow immune responses are at high risk, i.e. during the evolution of Omicron, those small amounts of epitopes may be lost due to further changes obtained and may lead to immune escape as experienced in the case of the sub-lines ba.2.12.1, ba.4 and ba.5 (y.cao et al, cited and k.khan et al, omicron sub-linear ba.4/ba.5 escape ba.1 infection elicited neutralizing immunity (202)2)). Importantly, omacron ba.1 breakthrough infection did not appear to reduce the overall profile of (MN 908947) S glycoprotein-specific memory B cells, as memory B cells that did not recognize omacron ba.1S were still detectable in blood with similar frequency. MN908947 specific (non-Omicron BA.1 reactivity) B _MEM Cells were consistently detected in Omicron ba.1 breakthrough infected individuals at levels similar to those in duplicate/triple vaccinated individuals not infected with Omicron. Without wishing to be bound by a particular theory, the present disclosure indicates that these findings may reflect the identification of BB sharing epitopes by selective amplification _MEM Cell, total B _MEM An increase in cell banks.

This embodiment provides, inter alia, the following insight: for subjects who have been infected or administered at least one dose (including, for example, at least two, at least three doses) of vaccine (e.g., but not limited to, protein-based vaccine or RNA-based vaccine such as BNT162b2, moderna mRNA-1273) adapted to the MN908947 strain, it may be more beneficial to receive at least one dose of vaccine (e.g., protein-or RNA-based vaccine) adapted to a strain other than omacron ba.1. In some embodiments, the vaccine adapted to a strain other than omacron ba.1 may be or comprise a vaccine adapted to omacron ba.4 and/or omacron ba.5. This embodiment also provides, among other things, the following insight: i.e., a non-vaccinated subject that is not previously infected with SARS-CoV-2, may wish to administer a combination of vaccines that includes at least one dose of a vaccine that is adapted to a strain of MN908947 (e.g., an RNA vaccine, such as BNT162b2 in some embodiments) and at least one dose of a vaccine that is adapted to a strain other than omicroba.1. In some embodiments, such vaccines in combination can be administered at different times, e.g., in some embodiments, as initial doses and/or booster doses administered at predetermined intervals of time periods (e.g., according to certain dosing regimens as described herein). In some embodiments, such vaccines in combination may be administered as a single multivalent vaccine.

Materials and methods

Serum samples, neutralization assays and all other experiments described in this example were performed as in example 7. Pseudotyped a ba.4/5 VSV-SARS-CoV-2S variant pseudovirus generation a recombinant replication defective Vesicular Stomatitis Virus (VSV) vector encoding a Green Fluorescent Protein (GFP) and a luciferase other than VSV-glycoprotein (VSV-G), comprising a SARS-CoV-2S protein comprising the following mutations relative to the MN908947 strain: T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D38408S, K417N, N K, L452R, S477N, T478K, E484A, F486V, Q498R, N Y, Y H, D655Y, N679K, P681 764K, P796K, P954 5297 969K.

Example 14. Omicron BA.2 breakthrough infection of vaccinated individuals induced extensive cross-neutralization against Omicron BA.1, BA.2 and other VOCs (including BA.4 and BA.5).

This example shows that ba.2 Omicron breakthrough infection in individuals vaccinated with BNT162b2 surprisingly drives superior cross variant neutralization, including improved production of neutralizing antibodies against ba.4/5 Omicron variants, compared to ba.1 breakthrough infection in individuals vaccinated with BN162b2 triple. Thus, the present disclosure demonstrates, among other things, the feasibility of defining immunocompetent classes of coronavirus strains and/or sequences (e.g., spike protein sequences).

In some embodiments of the improved coronavirus vaccination strategies provided by the present disclosure, the subject is exposed to each of at least two different such synergistic categories. In some embodiments, the subject is infected, has been infected or becomes infected with a virus of a first class and receives at least one dose of a vaccine of a second class characterized by a synergistic effect with the immunity of the first class. Alternatively or additionally, in some embodiments, the subject receives or has received such first and second vaccine doses of the first and second categories. In some embodiments, different classes of vaccines can be administered separately (e.g., at different time points and/or to different sites on a subject). In some embodiments, different classes of vaccines can be administered together (e.g., substantially simultaneously and/or to about or exactly the same site and/or in a single composition).

This example shows that ba.2 Omicron breakthrough infection in individuals vaccinated with BNT162b2 unexpectedly drives superior cross-variant neutralization, including improved production of neutralizing antibodies to ba.4/5 Omicron variants, compared to neutralization of Omicron ba.1 breakthrough infection in vaccinated individuals (relative to other SARS-CoV-2 variants (including, e.g., MN908947-Hu-1 strain, alpha variant, beta variant, delta variant, omicron ba.1, omicron ba.2, and Omicron ba.2.11.2). Thus, in some embodiments, the present disclosure demonstrates, among other things, that SARS-CoV-2 strains and/or variants can be grouped into at least two different categories such that subjects exposed to SARS-CoV-2 strains and/or variants from each of the two different categories can benefit from immune synergistic protection conferred by the two different categories. In some embodiments, the first class of SARS-CoV-2 strains/variants comprises: wuhan-Hu-1 strain, alpha variants, beta variants, delta variants, omicron ba.1 and sub variants derived from the above strains and/or variants; while the second category includes Omicron ba.2, omicron ba.2.12.1, omicron ba.4/ba.5 and sub-variants derived from the above strains and/or variants. Thus, in some embodiments, the present disclosure provides, inter alia, the following insights: that is, a combination of at least one dose (including, e.g., at least 1, at least 2, at least 3, at least 4, or more doses) of a first vaccine (e.g., an mRNA vaccine encoding a spike protein polypeptide as described herein) comprising or delivering a SARS-CoV-2 spike protein polypeptide having a sequence unique to a first class 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 doses) of a second vaccine (e.g., an mRNA vaccine encoding a spike protein polypeptide as described herein) comprising or delivering a SARS-CoV-2 spike protein polypeptide having a sequence unique to a second class as described above) can synergistically provide excellent cross variant neutralization, including enhanced production of neutralizing antibodies to ba.4/5 Omicron variants.

In some embodiments, the disclosure specifically teaches the unexpected efficacy of administering at least one dose of a vaccine comprising or delivering a SARS-CoV-2 spike protein polypeptide having a sequence specific for a ba.2 Omicron variant (e.g., an mRNA vaccine encoding a spike protein polypeptide as described herein) to a subject who has received at least one (e.g., 2, 3, or more) doses of a vaccine comprising or delivering a SARS-CoV-2 spike protein polypeptide having a sequence specific for a MN908947-hu-1 strain (e.g., a vaccine encoding a spike protein polypeptide as described herein).

Background

The occurrence of SARS-CoV-2 Omicron concern Variants (VOCs) at month 11 of 2021 (reference 1) can be considered a turning point of the covd-19 pandemic due to its ability to basically escape the previously established immunity. Omicron ba.1, which replaces Delta as the dominant circulating VOC within weeks, has obtained significant changes in the Receptor Binding Domain (RBD) and N-terminal domain (NTD) (reference 2). These changes resulted in the loss of many epitopes recognized by neutralizing antibodies (references 3-4), and severe impairment of humoral immunity induced by ancestral MN908947 strain-based vaccines or exposure to ancestral strains or previous variants (references 5-7). Ba.1 is then replaced with a ba.2 variant, which in turn generates a further subline. Ba.4 and ba.5, which are derived from ba.2, are currently dominant variants in many countries around the world, and many studies have shown that there are significant changes in their antigenic properties compared to ba.2 and especially compared to ba.1 (references 8-9). Since BA.4 and BA.5 share the same S glycoprotein sequence, they are referred to herein as BA.4/5. Although many amino acid changes in RBD were shared between omacron sublines, changes within NTD of the ba.2 derived sublines (including ba.4/5) were mostly different from those found in ba.1 (fig. 33).

The vast majority of people worldwide have been immunized with MN908947 strain adaptive vaccines (including, for example, mRNA vaccines such as BNT162b2 and mRNA-1273 (ref 10), which thus essentially model the SARS-CoV-2 population immunity, however, the emergence of the immune escape variant Omicron ba.1 results in a dramatic increase in breakthrough infection in vaccinated individuals.

Some findings

To determine if ba.2 breakthrough infection will refocus immunity against omacron ba.2 and sub-lines of ba.2 origin (such as ba.4/5), the size and breadth of neutralizing antibody responses in samples from individuals who received mRNA vaccine for the ba.2 lineage between march 2022 and march during which time the ba.2 lineage was dominant in germany (all vax+ Omi ba.2) were studied (BNT 162b 2/mRNA-1273) and subsequently experienced SARS-CoV-2 breakthrough infection. Such findings are of great significance to ongoing vaccine design efforts, as suppressing the covd-19 pandemic requires the development of durable and broad enough immunity to provide protection against current and future SARS-CoV-2 variants.

Two reference groups were generated from the data previously disclosed in Quandt et al (reference 12), including (i) individuals vaccinated with BNT162b2 triple vaccine (BNT 162b 2) who did not precede or breakthrough SARS-CoV-2 infection at the time of sample collection ³ ) And (ii) individuals vaccinated with mRNA vaccine triplets with subsequent breakthrough infections during Omicron ba.1 predominance (all vax+ Omi ba.1).

After triple vaccination with mRNA-based COVID-19 vaccine (BNT 162b2, mRNA-1273 or heterologous regimen comprising both vaccines; all Vax+ Omi BA.1, all Vax+ Omi BA.2), breakthrough infection of SARS-CoV-2 Omicron BA.1 and BA.2 occurred in the median of about 4 months or 3 weeks, respectively (FIG. 29). Immune serum for BNT162b2 used to characterize serum neutralization activity ³ The groups were collected at 28 days median post vaccination, 43 days post ba vaccination, 43 days median post ba.1 breakthrough for all vax+ Omi ba.1 groups, and 39 days median post ba.2 breakthrough infection for all vax+ Omi ba.2 groups. The median ages of the groups were similar (32-38 years). For this study, the ba.2.12.1 neutralization data was from group BNT162b ³ And all Vax+OmBA.1 serum samples 。

To evaluate the neutralizing activity of immune serum, a pseudovirus neutralization assay (pnnt) as described, for example, in references 13, 14 was used. Pseudoviruses carrying the S glycoprotein of SARS-CoV-2MN908947, alpha, beta, delta, omicron BA.1, BA.2, BA.2.12.1 and the newly emerging Omicron sub-lines BA.4 and BA.5 were used to evaluate the extent of neutralization. Since BA.4 and BA.5 share the same S glycoprotein sequence, including the critical alterations L452R and F486V, they are referred to herein as BA.4/5. In addition, SARS-CoV (herein referred to as SARS-CoV-1; ref.15) was assayed to detect potential ubiquity virus neutralization activity.

As previously reported in reference 12, 50% pseudovirus neutralization of omacron BA.1 and BA.2 against immune serum from triple vaccinated individuals not infected with SARS-CoV-2 (pVN) ₅₀ ) Geometric Mean Titers (GMT) were significantly reduced compared to the MN908947 strain (GMT 160 and 221 pair 398). Neutralization activity was even further reduced for ba.2.12.1 and ba.4/5 (GMT 111 and 74), titers for ba.4/5 were 5.4-fold lower than for MN908947 strain (fig. 30 (a)).

Compared to triple vaccinated immune serum not infected with SARS-CoV-2, the Omacron BA.2 breakthrough infection significantly increased pVN against BA.2 and BA.2.12.1 ₅₀ GMT, such that neutralization of ba.2 after breakthrough infection was comparable to the MN908947 strain (fig. 31 (B-C)). Similarly, ba.1 breakthrough incubation was effective for neutralizing activity against ba.1 (fig. 30 (B), fig. 31 (a). Importantly, although pVN against ba.4/5 in ba.2 convalescence serum ₅₀ GMT was lower than for the MN908947 strain (GMT 391 pair 922, i.e. 2.4 fold reduction), but this reduction was still lower than BNT162b2 in uninfected omicon ³ The latter serum showed a 5.4-fold decrease in ba.4/5 neutralization activity observed in the group (fig. 31 (C)). In contrast, pVN against BA.4/5 and MN908947 after breakthrough BA.1 infection ₅₀ GMT was 266 and 1327, respectively (i.e., 5-fold reduction; fig. 30 (B)). Thus, the Omicron ba.1 breakthrough infection of triple vaccinated individuals did not result in more effective cross-neutralization of Omicron ba.4/5 compared to the non-Omicron infected individuals of triple vaccinated individuals. Neutralization titers and needles for BA.4/5 in both groupsThe low levels observed for MN908947 were closer to those observed for phylogenetically more distant SARS-CoV-1 (figure 30). Notably, pVN against the MN908947 strain following a ba.1 breakthrough infection ₅₀ GMT was slightly higher than those observed for ba.2 breakthrough infection (GMT 1327 pair 922), which is not wishing to be bound by a particular theory, which may be associated with a longer interval between third vaccination and infection (22 days for ba.1 versus 127.5 days for ba.2) (fig. 31).

Separate analyses were performed, including only individuals triple vaccinated with BNT162b2 (with ba.2 or ba.1 breakthrough infection, or uninfected Omicron). In these assays, similar observations were made regarding BA.4/5 neutralization activity: pVN against BA.4/5 in BA.2 convalescence serum ₅₀ GMT was 2.4-fold lower than for MN908947 strain, whereas the decrease was 6-fold after ba.1 breakthrough infection (fig. 31). Although the relative neutralization of ba.2 and ba.2.12.1 was comparable in ba.2 and ba.1 convalescence sera, the neutralization activity for these variants was slightly higher than that observed in serum not infected with Omicron.

Immune serum from triple vaccinated uninfected omacron individuals has broad neutralization activity against ancestral SARS-CoV-2 VOC. The neutralization activity against Beta was slightly higher in serum at recovery of BA.1, whereas the neutralization activity of Alpha and Delta was not affected by either BA.1 or BA.2 breakthrough infection (FIG. 31 (C)).

Taken together, these data demonstrate that Omicron ba.2 breakthrough infection in vaccinated individuals mediates broad neutralization activity against ba.1, ba.2, ba.2.12.1 and several ancestral SARS-CoV-2 variants. Furthermore, the neutralization activity against ba.4/5 was lower than that of the MN908947 reference, but to a greater extent than in ba.1 recovery serum.

Recent studies have demonstrated that omacron ba.1 breakthrough infection in individuals vaccinated with mRNA vaccine (BNT 162b2 or mRNA-1273) not only enhances serum neutralization titers against ancestral MN908947 strain, but also against VOCs including ba.2 (references 8, 11, 12). This effect was observed in triple vaccinated individuals, but was particularly pronounced in double vaccinated individuals, whose serum contained little or no neutralizing activity against ba.2. However, the breakthrough infection with BA.1 did not induce strong neutralization activity against BA.4/5, which is currently the dominant VOC worldwide. Without wishing to be bound by a particular theory, this immune escape is due to the amplification and/or recall of a preexisting neutralizing antibody reaction that recognizes epitopes not present in the omacron sublines ba.2.12.1, ba.4 and ba.5.

This embodiment provides, inter alia, the following insight: ba.2 breakthrough infections trigger recall reactions that mediate enhanced neutralization of sub-lines of ba.2 origin (including ba.4/5), suggesting that higher S protein sequence similarity between ba.2, ba.2.12.1 and ba.4/5 drives more efficient cross neutralization compared to breakthrough infections with more distant ba.1 variants. Despite the importance of vaccination with currently approved MN 908947-derived vaccines (such as BNT162b 2) that provide effective protection against severe disease caused by current VOCs (including omacron ba.1 and ba.2), the findings of the present invention directed to the broad cross-neutralizing activity of current VOCs (including ba.4/5) after ba.2 breakthrough infection, in particular, provide the following insight: i.e., a vaccine that is adapted to the MN908947 strain sequence or variant sequences from the same immune-related class (e.g., alpha strain, beta strain, delta strain, omicron ba.1) as described above in combination with a vaccine that is adapted to the ba.2 variant sequence or variant sequences from the same immune-related class (e.g., omicron ba.2.12.1, omicron ba.4/ba.5) as described above may provide enhanced cross-neutralizing activity against variants from two different classes. In some embodiments, the evidence provided by this example supports the implementation of a licensing program modeled on seasonal influenza vaccine that uses up-to-date epidemiological data to select a strain of covd-19 vaccine. In some embodiments, the evidence further provided by this example supports the establishment of rapid plant selection for seasonal updates of the covd-19 vaccine, similar to the selection process implemented by the World Health Organization (WHO) global influenza monitoring and response system (GISRS), and/or protocols for accelerated approval pathways based on alternative immunogenic endpoints.

Neutralization titers from subjects vaccinated against SARS-CoV-2 and having ba.1 or ba.2 breakthrough infections are shown in fig. 31 (a) and (B), respectively, and GMR for both groups of subjects is shown in fig. 31 (C). As shown in fig. 31 (a) and (B), serum from subjects previously vaccinated against SARS-CoV-2 and having breakthrough infections of ba.1 or ba.2 was found to have significant neutralization titers against pseudoviruses comprising SARS-CoV-2S protein, alpha variant, beta variant, delta variant and Omicron ba.1 variant of the MN908947 strain. As previously described, the neutralization titers for ba.2 were slightly lower in serum from ba.1 breakthrough patients (GMT 875 for ba.2 versus 1327 for MN908947 strain) and lower for ba.2.12.1 and ba.4/5 (GMT 584 and 266, respectively, versus 1327 for MN908947 strain). Ba.2 breakthrough patients showed similar neutralization responses to ba.1 breakthrough patients for SARS-CoV-2MN908947 strain, alpha variant, beta variant and Delta variant. The neutralization response for omacron ba.1 was slightly higher in ba.1 breakthrough patients than in ba.2 breakthrough patients (GMR 0.76 vs. 0.60), while the neutralization titer for omacron ba.2 was higher in ba.2 breakthrough patients than in ba.1 breakthrough patients (GMR 0.94 vs. 0.66). Unexpectedly, however, the neutralization response to ba.4/5 was significantly higher in ba.2 breakthrough patients (GMR 0.39 in ba.2 breakthrough patients versus 0.2 in ba.1 breakthrough subjects). Thus, the present disclosure thus demonstrates that ba.2 breakthrough infection can elicit a broader immune response than ba.1 breakthrough infection in subjects vaccinated against SARS-CoV-2, and teaches that administration of a booster vaccine comprising RNA encoding an S protein comprising mutations unique to ba.2 omacron variants can achieve surprising and unexpected benefits.

Further, the present disclosure provides the following insight: that is, given the similarity between the S protein sequences of the ba.2 and ba.4/5 variants, combining a vaccination dose comprising or delivering the ba.4 and/or ba.5 variant spike sequences with a vaccination dose comprising or delivering the MN908947 spike sequence may also achieve particularly broad immunization (i.e., synergistic immunization as described herein).

In some embodiments, these findings indicate that coronavirus strain and/or variant sequences (e.g., synergistic class of SARS-CoV-2 strain and/or variant sequences) can be defined, for example, in some embodiments based on shared amino acid changes in the S glycoprotein of the coronavirus strain and/or variant sequences. For example, while many amino acid changes in RBD of S protein are shared between omacron sublines (e.g., ba.1, ba.2, ba.2.12.1 and ba.4/5), changes within NTD of sublines of ba.2 and ba.2 origin (including ba.4/5) are mostly different from those found in ba.1. Thus, in some embodiments, the synergistic class of coronavirus strains 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 within the NTD of the S protein. For example, in some embodiments in which two SARS-CoV-2 strains 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 the S protein, both SARS-CoV-2 strains and variant sequences can be grouped into the same class. In some embodiments where two SARS-CoV-2 strains and/or variant sequences share no more than 50% (including no more than 45%, no more than 40%, no more than 30% or less) of the amino acid mutations present in the NTD of the S protein, both SARS-CoV-2 strains and variant sequences can be grouped into different classes. The findings of the present invention provide, inter alia, the following insight: even exposure of a subject (e.g., by infection and/or vaccination) to at least two antigens with different synergy categories (e.g., as shown in the table below) can produce a more potent immune response (e.g., expand cross-neutralization profiles for different variants and/or produce an immune response that is less prone to immune escape).

Class I	Class II
		MN908947 strain	Omicron BA.2
Alpha variants	Omicron BA.2.11.2
		Beta variants	Omicron BA.4
Delta variants	Omicron BA.5
		Omicron BA.1	Subgenera derived from any of the above
Subgenera derived from any of the above

For example, in some embodiments, a combination of vaccines can be administered to a non-vaccinated subject that is not previously infected, at least two of which are each adapted for SARS-CoV-2 strain having a different synergy class (e.g., as described herein). In some embodiments, such vaccines in combination can be administered at different times, e.g., in some embodiments, as a first dose and a second dose administered at predetermined intervals of time periods (e.g., according to certain dosing regimens as described herein). In some embodiments, such vaccines in combination may be administered as multivalent vaccines. In some embodiments, a subject infected or vaccinated with one class of SARS-CoV-2 strain may be administered a vaccine (e.g., as described herein) that is tailored to the different classes of SARS-CoV-2 strain. In some embodiments, such vaccines may be polypeptide-based or RNA-based vaccines.

While the findings of the present invention are based on retrospective analysis of samples from different studies, using relatively small sample volumes and groups that are not completely consistent in terms of immune spacing and demographic characteristics (such as age and sex of individuals), the findings of the present invention provide useful insights into vaccine design and vaccination strategies to improve cross-neutralization against a broader spectrum of SARS-COV-2 variants.

In some embodiments, the vaccine can comprise a polypeptide (e.g., a non-native polypeptide, such as a chimeric polypeptide) comprising one or more mutations that are characteristic of one or more different SARS-CoV-2 variants; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine can comprise a polypeptide (e.g., a non-native polypeptide, such as a chimeric polypeptide) comprising one or more mutations that are characteristic of a first SARS-CoV-2 variant and one or more mutations that are characteristic of a second SARS-CoV-2 variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the first SARS-CoV-2 variant may be a SARS-CoV-2 strain/variant from class I of the above table, and the second SARS-CoV-2 variant may be a SARS-CoV-2 strain/variant from class II of the above table. For example, in some embodiments, a vaccine can comprise a polypeptide comprising an RBD comprising one or more mutations unique to a first SARS-CoV-2 variant and an NTD comprising one or more mutations unique to a second SARS-CoV-2 variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments).

In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to a ba.1omicron variant and NTD comprising one or more mutations unique to a second SARS-CoV-2 variant (which is not a ba.1omicron variant); or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise or encode a polypeptide comprising an NTD comprising one or more mutations unique to a ba.1 omacron variant and an RBD comprising one or more mutations unique to a second SARS-CoV-2 variant (which is not a ba.1 omacron variant). In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to the ba.1omicron variant and NTD comprising one or more mutations unique to the ba.2omicron variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising an RBD comprising one or more mutations unique to the ba.1Omicron variant and an NTD comprising one or more mutations unique to the ba.4/5Omicron variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments).

In some embodiments, the vaccine may comprise a polypeptide comprising an NTD comprising one or more mutations unique to a ba.1 omacron variant and an RBD comprising one or more mutations unique to a second SARS-CoV-2 variant (which is not a ba.1 omacron variant); or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to a ba.1omicron variant and NTD comprising one or more mutations unique to a second SARS-CoV-2 variant (which is not a ba.1omicron variant); or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising an NTD comprising one or more mutations unique to the ba.1omicron variant and an RBD comprising one or more mutations unique to the ba.2omicron variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising one or more mutations unique to the NTD of the ba.1Omicron variant and an RBD comprising one or more mutations unique to the ba.4/5Omicron variant; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments).

In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to the ba.1omicron variant and NTD of MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to the ba.2omicron variant and NTD of MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising RBD comprising one or more mutations unique to the ba.4/5Omicron variant and NTD of MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments).

In some embodiments, the vaccine may comprise a polypeptide comprising an NTD comprising one or more mutations unique to the ba.1omicron variant and an RBD of the MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising an NTD comprising one or more mutations unique to the ba.2omicron variant and an RBD of the MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments). In some embodiments, the vaccine may comprise a polypeptide comprising an NTD comprising one or more mutations unique to the ba.4/5Omicron variant and an RBD of the MN908947S protein; or a nucleic acid encoding the polypeptide (e.g., RNA in some embodiments).

Materials and methods

Participant recruitment and sample collection

Triple BNT162b2 vaccination from uninfected SARS-CoV-2 (BNT 162b2 ³ ) Individuals in the cohort provided informed consent as part of their participation in phase 2 trial BNT162-17 (NCT 05004181). Individuals with omacron ba.1 or ba.2 breakthrough infections (all vax+ Omi ba.1 and all vax+ Omi ba.2 groups) were triple vaccinated, e.g., using one or more doses of BNT162b2, modema mRNA-1273, astraZeneca ChAdOx1-S recombinant vaccine, or combinations thereof, and recruited to provide blood samples and clinical data for study. Omicron infection was confirmed by variant-specific PCR between month 11 of 2021 and month 1 of 2022 (all vax+ Omi ba.1) or between month 3 of 2022 and month 5 of 2022 (when sublines ba.1 and ba.2 are dominant, respectively) (reference 24). Some participants in the studyThe infection (e.g., of at least 7 participants) was further characterized by genomic sequencing, and genomic sequencing confirmed omacron ba.1 or ba.2 infection.

Participants had no symptoms at the time of blood collection. Table 26 is a summary of characteristics of vaccinated individuals analyzed for neutralizing antibody responses. All participants had no recorded history of SARS-CoV-2 infection prior to vaccination.

Table 26

N/A: inapplicable; n/a, unavailable; d, dosage; yrs, the year old; n, number.

* SARS-CoV-2PCR assay # negative at recruitment, no sign of previous SARS-CoV-2 infection (based on symptoms/signs of COVID-19 and SARS-CoV-2PCR assay)

As part of a government vaccination program, participants received an initial 2 dose series of BNT162b2 vaccine, and the interval between doses was not recorded

Omicron ba.1 infection was confirmed at recruitment to the study

Serum was isolated by centrifugation of the withdrawn blood at 2000x g for 10 minutes and stored frozen until use.

VSV-SARS-CoV-2S variant pseudovirus production

The recombinant replication defective Vesicular Stomatitis Virus (VSV) vector encoding Green Fluorescent Protein (GFP) and luciferase other than VSV-glycoprotein (VSV-G) was pseudotyped according to the published pseudotyping protocol (reference 49) with SARS-CoV-1S glycoprotein (UniProt reference: P59594) and with SARS-CoV-2S glycoprotein derived from: MN reference strain (NCBI reference: alpha variants (changes: Δ69/70, Δ144, N501 614 681 716 982 1118H), beta variants (changes: L18 80G, delta242-244, R246 484 501 614V), delta variants (changes: T19 142G, delta157/158, K417 452 478 681, delta69/70, T95 142D, delta143-145, delta211, L212 EPE, G339 371 373 417 440, delta47478 493 496 498 501 505 547 614 655, delta856 954 969F), omicron BA.2 variants (changes: T19I, delta24-26, A27 142 213 371 373 376, whereby DeltaEvaporation 67, delta67, 679, 684, 9594 969K), omicron BA.2.12.1 variants (changes: T19I, delta24-26, A27, 371, 37375, 440, 4935, 493, 4968, 499, and 499, whereby Delta67, and/or Omicron BA.47, whereby the Omicron BA.2.12.1 variants (changes: T19I, delta24-26, A27, delta371, deltawear on 373, deltawear, delta7, deltawear, delta7, delta1, delta7, delta1, stro, delta1, delta7, delta1, delta7, deltaStro,.

A graphical representation of the SARS-CoV-2S glycoprotein alteration is shown in FIG. 62, and a separate alignment of the S glycoprotein alterations in the Omicon subline is shown in FIG. 60. Briefly, a kit supplemented with 10% heat-inactivated fetal bovine serum (FBS [ Sigma-Aldrich]) Dulbecco's modified Eagle's medium; DMEM) plus GlutaMAX ^TM HEK293T/17 monolayer [. Sup.CRL-11268 ^TM ) SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmid verified by Sanger sequencing according to the manufacturer' S instructions plus Lipofectamine LTX (Life technology)Genies) transfection. At 24 hours, VSV-G supplemented VSV.DELTA.G vector. After incubation at 37 ℃ and 7.5% co2 for 2 hours, the cells were washed twice with Phosphate Buffered Saline (PBS) and then medium supplemented with anti-VSV-G antibody (clone 8G5F11,Kerafast Inc) was added to neutralize residual VSV-G supplemented input virus. The medium containing the VSV-SARS-CoV-2-S pseudotype was harvested 20 hours after inoculation, passed through a 0.2 μm filter (Nalgene) and stored at-80 ℃. Vero 76 cells in culture medium were used (-)>CRL-1587 ^TM ) To titrate each batch of pseudoviruses. The relative luciferase units induced by a defined volume of MN908947S glycoprotein pseudovirus reference lot corresponding to an infectious titer of 200 Transduction Units (TUs) per milliliter, previously described in Muik et al, 2021, were used as a control. The input volumes of each batch of SARS-CoV-2 variant pseudoviruses were calculated to normalize the infection titer based on relative luciferase units relative to the reference.

Pseudovirus neutralization assay

Vero76 cells were seeded at 40,000 cells/well in medium in 96-well white flat bottom plates (Thermo Scientific) 4 hours prior to assay and cultured at 37 ℃ and 7.5% co 2. Serum from each individual was serially diluted 2-fold in medium at a first dilution of 1:5 (triple BNT162b2 vaccination without Omicron; dilution range 1:5 to 1:5, 120) or 1:30 (triple vaccination after subsequent Omicron BA.1 or BA.2 breakthrough infection; dilution range 1:30 to 1:30, 720). In the case of SARS-CoV-1 pseudovirus assay, the serum of all individuals is initially diluted 1:5 (dilution range 1:5 to 1:5, 120). The VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in the medium to obtain 200 TUs in the assay. Serum dilutions were mixed with pseudoviruses (n=2 technical replicates per pseudovirus per serum) for 30 min at room temperature, then added to Vero76 cell monolayers and incubated at 37 ℃ and 7.5% co2 for 24 hours. The supernatant was removed and the supernatant was washed with luciferase reagent (Promega) lyse cells. At the position ofLuminescence was recorded on a 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. The results are expressed in terms of Geometric Mean Titres (GMT) from replicates. If no neutralization is observed, the limit of detection [ LOD ] is reported ]Half of any titer value.

Statistical analysis

The statistical polymerization method used to analyze antibody titers was a geometric mean and for the ratio of SARS-CoV-2VOC titers to MN908947 titers was a geometric mean and corresponding 95% confidence interval. The use of geometric averages accounts for the non-normal distribution of antibody titers across several orders of magnitude. Paired symbol rank test (Friedman test) was performed on group geometric mean neutralizing antibody titers under a common control group using Friedman test (Dunn's correction) for multiple comparisons. All statistical analyses were performed using GraphPad Prism software version 9.

Reference cited in example 14

1.WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution(TAG-VE)，Classification of Omicron(B.1.1.529)：SARS-CoV-2 Variant of Concern(2021).

2.WHO Headquarters(HQ)，WHO Health Emergencies Programme，Enhancing response to Omicron SARS-CoV-2 variant：Technical brief and priority actions for Member States(2022).

3.M.Hoffmann et al.，The Omicron variant is highly resistant against antibody-mediated neutralization.Cell.185，447-456.e11(2022)，doi：10.1016/i.cell.2021.12.032.

4.W.Dejnirattisai et al.，SARS-CoV-2 Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.Cell.185，467-484.e15(2022)，doi：10.1016/j.cell.2021.12.046.

5.V.Servellita et al.，Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants.Cell.185，1539-1548.e5(2022)，doi：10.1016/j.cell.2022.03.019.

6.C.Kurhade et al.，Neutralization of Omicron BA.1，BA.2，and BA.3 SARS-CoV-2 by 3doses 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 COVlD-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-2Omicron by BNT162b2mRNA 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-2lineage B.1.1.7pseudovirus by BNT162b2vaccine-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-1Survivors.The New England journal of medicine.385，1401-1406(2021)，doi：10.1056/NEJMoa2108453.

Example 15: further renewal of immune response elicited by vaccines encoding SARS-CoV-2S protein from Omicron variants

Following the experiment described in example 8, further subjects were included in a clinical trial that investigated an RNA vaccine encoding SARS-CoV-2S protein comprising one or more mutations unique to the ba.1 omacron variant. In this example, 30ug of RNA encoding SARS-CoV-2S protein of MN908947 strain (BNT 162b2 in this example) or 30ug of RNA encoding SARS-CoV-2S protein having one or more mutations unique to the Omicron variant (BNT 162b2OMI in this example) encoding SARS-CoV-2S protein having mutations unique to the BA.1Omicron variant, comprising amino acids SEQ ID NO:50 and 51, and SEQ ID NO: 49) was administered to a subject (18 to 55 years with or without evidence of prior infection).

In a primary immunogenicity analysis of participants without signs of previous infection, BNT162b2OMI (n=132) elicited an excellent neutralizing antibody response against ba.1omicron SARS-CoV-2 virus compared to BNT162b2 (n=141). BNT162b2OMI GMT for BA.1Omicron is 1929 (CI: 1632, 2281), compared to BNT162b2GMT of 1100 (CI: 932, 1297); GMT ratio 1.75 (95% CI:1.39, 2.22).

In contrast to BNT162b2, BNT162b2OMI elicited a similar neutralizing antibody response against the MN908947 strain of SARS-CoV-2. BNT162b2OMI GMT was 11997 (CI: 10554, 13638) in contrast to BNT162b2GMT being 12009 (CI: 10744, 13425).

The data shows that omacron monovalent vaccine as booster vaccination (dose 4) improves neutralizing antibody response against ba.1 omacron compared to RNA vaccine encoding S protein of MN908947 strain, and does not adversely affect neutralizing antibody response against MN908947 strain of SARS-CoV-2.

EXAMPLE 16 immune Effect of VOC vaccination

This example describes the immune impact of administering a BNT162b2 vaccine encoding spike proteins from certain variants ("VOCs") of interest. In particular, this example describes the immune effect of administering a "booster dose" to a subject (in this example, a mouse) who has received two doses (i.e., according to an established model immunization regimen) of a "raw" BNT162b2 vaccine (i.e., encoding MN908947 spike protein, as described herein).

Fig. 34 presents an immunization protocol used in this example. Specifically, BALB/c mice were immunized twice with BNT162b2 (1 ug per dose) and then at a later time point with BNT162b2/VOC (1 ug per dose). Immunization occurred up to 3 or 4 times. Animals were bled periodically to analyze antibody immune responses by ELISA and pseudovirus neutralization assays. At the end of the experiment, animals were euthanized and the spleen was analyzed for T cell responses.

Reinforcement was performed with: (a) raw BNT162b2 ("BNT 162b 2"); (b) BNT162b2OMI BA.1 ("OMI BA.1"); (c) BNT162b2OMI BA.4/5 ("OMI BA.4/5"); (d) BNT162b2+OMI BA.1 (0.5 g each); (e) BNT162b2+OMI BA.4/5 (0.5 ug each); (f) OMI BA.1+OMI BA.4/5 (0.5 ug each); and (g) BNT162b2+OMI BA.1+OMI BA.4/5 (0.33 ug each).

Omicron variants ba.4 and ba.5 were first reported in the circulation at month 1 of 2022 and have been dominant variants by month 6 of 2022. Both lineages contain the amino acid substitutions F486V and R493Q. Preliminary studies showed significant changes in the antigenic properties of ba.4 and ba.5 compared to ba.1 and ba.2, especially compared to ba.1. In addition, as an increasing trend in the proportion of ba.5 variants was observed at a particular site (e.g., portugal), the number of cases of covd-19 and the test positivity increased. The present disclosure suggests that ba.4/5 (which are considered together in this example in view of their common spike protein mutations) may represent escaping VOCs. The present disclosure demonstrates particular benefits of a dosing regimen (e.g., as described herein and specifically as exemplified in the present examples) comprising one or more doses of a vaccine comprising or delivering (e.g., by expression of administered RNA) a spike protein containing the relevant ba.4/5 sequence (e.g., amino acid substitution).

Figures 35 and 36 present Geometric Mean Titers (GMT) relative to the baseline (measured on day 104, before boosting) of the various SARS-CoV-2 strains as indicated. It can be seen that the baseline immunity of the different mouse groups was comparable. Specifically, the group GMT for each pseudovirus is always within the same range between groups; no more than about 2-fold difference was observed. Consistent with observations in the human population as described above, the neutralising GMT for the MN908947 strain was quite high compared to the neutralising GMT for VOCs (GMT up to 3, 044). In general, the order of GMT is MN908947 > BA.1' BA.2 > BA.2.12.1 > BA.4/5.

Figure 37 shows a baseline (measured on day 104, before boosting) cross-neutralization analysis and demonstrates that baseline immune groups are comparable in cross-neutralization capacity. Specifically, at baseline, the calculated variant/MN 908947 reference GMT ratio indicated that cross-neutralization capacity was quite similar between groups (only one outlier was observed in BNT162b2 monovalent group re.ba.1 neutralization). Again, consistent with observations of the human population, ba.1 = ba.2 > ba.2.12.1 > ba.4/5.

Figures 38-40 present data obtained 7 days after boost and record the significant effectiveness of ba.4/5, and in particular monovalent ba.4/5, in achieving a significant geometric mean fold increase in GMT (figures 38 and 39) and effective cross-neutralization (figure 40). It can be seen that BNT162b2 booster immunization produced a comparable titer increase (3.9-7.1 fold) for all VOCs, while monovalent BA.1 and BA.4/5 boosters produced a fairly strong increase in homologous VOC titers (16.8 fold for BA.1 and 67.3 fold for BA.4/5).

The monovalent ba.4/5 booster was most effective in driving an increase in titre in the pseudovirions tested. Divalent enhancers show a similar but reduced trend compared to monovalent VOC enhancers; among the divalent boosters, the b2+BA.4/5 combination is most effective in driving broad cross-neutralization. Trivalent booster (b2+ba.1+ba.4/5) is superior to divalent booster and provides intermediate immunity between divalent b2+ba.4/5 and monovalent ba.4/5 booster.

Fig. 40 presents, inter alia, a calculated variant/MN 908947 reference GMT ratio, which demonstrates:

(i) BNT162b2 potentiators give relatively poor cross-neutralization, especially of BA.2 and its progeny (BA.2.12.1, BA.4/5)

(ii) The BA.1 enhancer produced excellent cross-neutralization of BA.1, but neutralization of BA.2.12.1, BA.4/5 was still relatively poor

(iii) The BA.4/5 enhancer produced balanced pan Omicron neutralization with very encouraging neutralization against BA.2, BA.2.12.1 and BA.4/5

Divalent enhancers show a similar but reduced trend compared to monovalent VOC enhancers; among the divalent boosters, the b2+BA.4/5 combination is most effective in driving broad cross-neutralization; trivalent reinforcing agents (b2+BA.1+BA.4/5) induce cross-neutralization comparable to the BA.1/BA.4/5 reinforcing agents.

Two RNA molecules may also be administered to the mice, wherein the ratio of the two RNA molecules is not 1:1. For example, a bivalent vaccine comprising BNT162b2 and BA.4/5 in a 1:2 ratio may be administered to mice (e.g., by administering 0.33ug of BNT162b2 and 0.66ug of BA.4/5). The mice may also be administered a bivalent vaccine comprising BNT162b2 and BA.4/5 in a 1:3 ratio (e.g., by administering 0.25ug of BNT162b2 and 0.75ug of BA.4/5). Such compositions may be administered to unvaccinated mice or to mice previously vaccinated with BNT162b2 (e.g., two doses of BNT162b2 previously administered with 1 ug).

The present specification demonstrates significant efficacy of ba.4/5 immunization (and particularly BNT162b2+ ba.4/5 immunization, e.g., with the sequences provided herein).

Furthermore, the present specification demonstrates the efficacy of ba.4/5 immunization in monovalent, divalent and trivalent forms and records unexpected efficacy of monovalent ba.4/5.

The present disclosure demonstrates in particular the significant effectiveness of one or more ba.4/5 doses administered to a subject who has been previously immunized (e.g., using a MN908947 vaccine, such as using at least (or just) two doses of a MN908947 vaccine). Without wishing to be bound by a particular theory, the present disclosure teaches that the immunological features of omicron ba.4/5 spike may make it particularly useful or effective for immunizing subjects, including subjects that have been immunized (e.g., by prior administration of one or more vaccine doses and/or by prior infection) with the MN908947 strain (and/or with one or more strains associated with the MN908947 strain), including specifically by vaccination with one or more doses (e.g., 1, 2, 3, 4, or more doses) of the original BNT162b 2.

Example 17: omicron ba.2 breakthrough infections enhance cross neutralization of ba.2.12.1 and ba.4/ba.5

This example 17 is an extension of example 14 and describes an experiment to analyze the neutralizing activity of serum samples taken from ba.1-and ba.2-breakthrough cases against omacron ba.4 and ba.5 variants. In addition to confirming the results described in example 14, this example 17 also provides a further characterization of the antibody responses elicited by ba.1 and ba.2 breakthrough infections, and provides insight as to which aspects may contribute to an increased neutralization of the ba.4/5Omicron variant as observed in ba.2 breakthrough infections compared to ba.1 breakthrough infections. In particular, this example demonstrates that ba.2 breakthrough infection can induce higher titers of neutralizing antibodies that bind to the N-terminal domain (NTD) of the SARS-CoV-2S protein (e.g., ba.4/5SARS-CoV-2Omicron variant), which can result in increased neutralization of the ba.4/5Omicron variant.

As demonstrated in the previous examples, individuals previously administered with RNA encoding the SARS-CoV-2S protein from the MN908947 strain and subsequently developed Omicron ba.1 breakthrough infections have strong serum neutralization activity against Omicron ba.1, ba.2 and previous SARS-CoV-2 variants of interest (VOCs), but lower serum neutralization activity against highly infectious Omicron sub-lines ba.4 and ba.5 that replaced the previous variants. Since the latter subline was derived from omacron ba.2, serum neutralization activity was analyzed for the covd-19 mRNA vaccine triple immunized individuals who underwent ba.2 breakthrough infection. This example demonstrates that the sera of these individuals have broad neutralization activity against the previous VOC and all omacron sublines tested, including ba.2-derived variants ba.2.12.1, ba.4/ba.5 (confirming the results of example 14 above). Furthermore, using antibody depletion, this example shows that neutralization of ba.2 recovery stage sera to ba.2 and ba.4/ba.5 sublines is driven largely by antibodies targeting the N-terminal domain (NTD) of the spike glycoprotein, whereas neutralization of omacron ba.1 recovery stage sera to them is entirely dependent on antibodies targeting the Receptor Binding Domain (RBD). These findings indicate that exposure to omacron ba.2 elicits a significant NTD-specific recall response in vaccinated individuals compared to ba.1 spike glycoprotein and thereby enhances neutralization of the ba.4/ba.5 subline. Given the current epidemiology and rapid ongoing evolution of the ba.2 derived sublines such as ba.4/ba.5, these findings are highly relevant for the development of Omicron-adapted vaccines.

Introduction to the invention

The occurrence of SARS-CoV-2Omicron variants of interest (VOCs) at month 11 of 2021 (reference 1) can be considered a turning point for the pandemic of COVID-19. Omicron ba.1 significantly altered in spike (S) glycoprotein Receptor Binding Domain (RBD) and N-terminal domain (NTD) relative to S protein from MN908947 strain, partially escaped previously established immunity (reference 2).

The loss of many epitopes (references 3, 4) reduced the susceptibility to neutralizing antibodies induced by wild-type strain (MN 908947-Hu-1) S glycoprotein-based vaccines or by pre-infection strains (references 5-7), requiring a third vaccine dose to establish complete immunity (references 8-10). Omicron ba.1 is replaced by a ba.2 variant, which in turn is replaced by its offspring ba.2.12.1, ba.4 and ba.5, which are now dominant in many areas (references 11-14).

Antigenically, ba.2.12.1 shows a high similarity to ba.2 instead of ba.1, whereas ba.4 and ba.5 are significantly different from ba.2 and even more significantly different from ba.1, consistent with their pedigree (references 15 and 16). Although some amino acid changes in RBD are shared between all omacron sublines, L452Q changes are found only in ba.2.12.1 and are the only residues of RBD that distinguish their RBD from ba.2 variants. L452R and F486V changes were BA.4/BA.5 specific, while S371F, T376A, D N and R408S were shared by BA.2 and its progeny BA.2.12.1 and BA.4/BA.5, but not by BA.1 (FIG. 33). These amino acid exchanges are associated with further escape of neutralizing antibodies induced from the vaccine and therapeutic antibody drugs targeting wild-type S glycoprotein (references 6, 15, 17-20). The NTD of ba.2 and its progeny is antigenically closer to the wild-type strain and lacks several amino acid changes, insertions and deletions that occur in ba.1 (fig. 33). For example, Δ143-145, L212I or ins214EPE, which confers resistance to a panel of NTG-directed monoclonal antibodies against wild-type S glycoprotein, to BA.1 variants were not found in BA.2 and offspring (references 21, 22).

As demonstrated in the previous examples, omacron ba.1 breakthrough infection of BNT162b2 vaccinated individuals enhanced broad neutralization activity against omacron ba.1, ba.2 and previous VOCs at levels similar to those observed for SARS-CoV-2 wild type. Ba.1 breakthrough infection in triple BNT162B2 vaccinated individuals induced a potent recall response, mainly by expansion of memory B cells against epitopes shared extensively in variants, rather than B cells specific only for ba.1. Neutralization by omacron sublines ba.4 and ba.5 increased to a lesser extent in ba.1 breakthrough patients compared to ba.1 variants, and the geometric mean titers were comparable to those for phylogenetically more distant SARS-CoV-1.

Given that Omicron ba.2 is more closely related to ba.4/ba.5 than to ba.1, it was evaluated whether ba.2 breakthrough infections would shift cross-neutralizing activity more towards these more recent Omicron sublines than ba.1 breakthrough infections. Serum samples from three different groups of individuals vaccinated with triple mRNA covd-19 vaccine, i.e. individuals without history of SARS-CoV-2 infection, and individuals experiencing breakthrough infections with ba.1 or ba.2 were compared for neutralization of the different Omicron sublines. In addition, the contribution of serum antibodies targeting the S glycoprotein RBD versus NTD to Omicron subline neutralization was also characterized. The data thus generated increases the current knowledge of the omacron immune escape mechanism and the impact of immunity on variant cross-neutralization and thereby helps guide further vaccine development.

Results

Group and sampling

The present study investigatedSerum samples from three groups: BNT162b2 triple vaccinated individuals not infected with SARS-CoV-2 at the time of sampling (BNT 162b2 ³ N=18), individuals vaccinated with three doses of mRNA covd-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) and subsequently with Omicron breakthrough infection when ba.1 is dominant (mRNA-Vax ³ +ba.1, n=14) or triple mRNA vaccinated individuals with breakthrough infections in ba.2 predominance (mRNA-Vax ³ +ba.2, n=13). For convalescence groups, relevant intervals between critical events, such as the most recent vaccination and infection, are provided in fig. 41. Serum was derived from biological sample collection of BNT162b2 vaccine trials and non-intervention studies of vaccinated patients who underwent omacron breakthrough infection were explored.

Omicron BA.2 breakthrough infection in triple mRNA vaccinated individuals induces extensive neutralization of VOCs including Omicron BA.4/BA.5

In a well characterized pseudovirus neutralization assay (pVNT) (reference 24, 25) 50% pseudovirus neutralization was determined by using pseudoviruses with S glycoprotein of SARS-CoV-2 wild-type strain or Alpha, beta, delta, omicron BA.1, BA.2 and BA.2 derived sublines BA.2.12.1, BA.4 and BA.5 (pVN) ₅₀ ) Geometric Mean Titer (GMT) to test neutralizing activity of immune serum. Since BA.4 and BA.5 share the same S glycoprotein sequence, in this example they are referred to as BA.4/5 in the context of pVNT. In addition, SARS-CoV (herein referred to as SARS-CoV-1) was assayed to detect potential ubiquity virus neutralization activity (ref.26). As an orthogonal assay system, a live SARS-CoV 2 neutralization assay (VNT) was also used which analyzes the neutralizing effect of antibodies present throughout the assay period during multicycle replication of real viruses (wild-type strain of SARS-CoV-2 and VOCs including ba.4, except Omicron ba.2.12.1).

In the pVNT, sera from all three groups were potent in neutralizing wild-type strains, alpha, beta, delta VOC and Omicron BA.1 and BA.2 lineages, with neutralization activity being more pronounced in breakthrough infected individuals, especially in the BA.1 breakthrough infected group (mRNA-Vax3+BA.1). However, with wild typeIn contrast, triple-vaccinated individuals with BNT162b2 (BNT 162b 2) ³ ) And mRNA-Vax ³ The serum neutralization activity of individuals +ba.1 was significantly reduced against ba.2.12.1 (p < 0.05), and even more significantly against ba.4/5 (p < 0.001; > 5-fold compared to wild-type strain) (fig. 42 (a)). In contrast, serum from individuals vaccinated with triple mRNA with omacron ba.2 breakthrough infection (mRNA-vax3+ba.2) neutralized ba.2.12.1 pseudoviruses as strongly as the wild-type strain. Neutralization of ba.4/5 was approximately similar to that of ba.2.12.1 and the reduction relative to the wild type strain was significant (p < 0.05) but less significant (about 2.5 fold) compared to the two other groups.

In order to compare the groups with respect to neutralization breadth regardless of the size of the antibody titer, VOC pVN was used ₅₀ GMT was normalized to MN908947 strain. The ratio shows that it is identical to mRNA-Vax ³ +BA.1 and BNT162b2 ³ Serum (GMT ratio of 0.18 to 0.17) was compared to the BA.4/5 cross-neutralization on mRNA-Vax ³ The +ba.2 is significantly stronger (GMT ratio 0.38) (fig. 42 (B)). Similarly, mRNA-Vax ³ Cross-neutralization of Omicron BA.2.12.1 by +BA.2 serum (GMT ratio 0.52) was stronger than mRNA-Vax ³ +ba.1 serum (GMT ratio 0.43), and even stronger than BNT162b2 ³ Serum (GMT ratio 0.26).

Separate analysis of individuals vaccinated with BNT162b2 in only these three groups demonstrated that the BA.2 breakthrough infection was associated with considerable cross-neutralization of BA.4/5 (0.42 ratio of BA.4/5 to wild-type GMT), whereas pVN against BA.4/5 was post-BA.1 breakthrough infection ₅₀ GMT was about 6-fold lower than those for wild type (i.e., GMT ratio 0.17) (fig. 45 ((a) - (C)). Ba.1 and ba.2 convalescence sera were superior to sera from individuals vaccinated with BNT162b2 that were not infected with SARS-CoV-2 in cross-neutralization of ba.2 and ba.2.12.1.

The VOC neutralization titer provided by the authentic live SARS-CoV-2 virus neutralization assay was highly correlated with the neutralization titer from the pnnt assay (fig. 46), and confirmed the principal findings in fig. 42. In this assay, BNT162b2 compared to that for the wild type ³ Targeting Omi in serum50% virus neutralization of cron BA.2 (VN ₅₀ ) GMT was strongly reduced (p < 0.0001) whereas sera from both recovery groups showed strong neutralizing activity, VN ₅₀ GMT was comparable to that for the wild-type strain (fig. 43 (a)). And BNT162b2 ³ And mRNA-Vax ³ The decrease in neutralizing activity against omacron ba.4 was less pronounced in the focal ba.2 recovery phase group compared to the +ba.1 group (VN ₅₀ GMT was about 2.5-fold lower compared to about 15-fold and about 5-fold lower for wild-type strains, respectively).

Consistent with the pVNT data, VN was calculated for the wild-type strain by calculating VOC VN ₅₀ Size independent analysis of GMT ratios showed that the samples were not dependent on mRNA-Vax ³ +ba.1 group (GMT ratio 0.20) and BNT162b2 ³ (GMT ratio 0.07) in comparison, BA.4 cross-neutralization was performed on mRNA-Vax ³ Stronger in the +ba.2 group (GMT ratio 0.39) (fig. 43 (B)) and similar in the subgroup of BNT162B2 triple vaccinated individuals (fig. 45 (D) - (F)).

Taken together, these data demonstrate that Omicron ba.2 breakthrough infection of vaccinated individuals is associated with broad neutralization activity against all tested Omicron sublines and the previous SARS-CoV-2 VOCs. In particular, these data indicate that ba.2 breakthrough infection is more effective in refocusing the neutralizing antibody response towards the ba.4/ba.5s glycoprotein (about 2-fold higher cross neutralization) than ba.1 breakthrough infection.

Neutralization of omacron ba.2 and ba.4/5 by serum of triple mRNA vaccinated ba.2 convalescence individuals is largely mediated by antibodies targeting NTD.

To profile the effect of serum antibodies binding to RBD or NTD of S glycoprotein on neutralizing SARS-CoV-2 wild type, omacron ba.1, ba.2 and ba.4/5, these antibody fractions were well depleted from the serum of the three groups, respectively (each n=6, fig. 47 (a)). Depletion was performed using SARS-CoV-2 wild-type strain S glycoprotein RBD and NTD baits, as VOC breakthrough infection has been demonstrated to mainly trigger recall responses that recognize epitopes conserved in known VOCs (references 10, 23 and 27).

Depletion experiments removed > 97% of all RBD binding antibodies and > 74% of all NTD binding antibodies(fig. 47 (B)). The depleted serum was then tested in a pVNT assay. In sera from all groups RBD antibody depletion strongly reduced neutralizing activity against wild-type strains, whereas after depletion of NTD binding antibodies, neutralizing activity was largely retained (> 80% of residual activity) (fig. 44 (a)). Neutralization by Omicron ba.1 was completely abolished after RBD-binding antibody depletion and was substantially unaffected by NTD-binding antibody depletion. For the neutralization of BA.2, RBD antibody depletion almost completely abrogated mRNA-Vax ³ Neutralization activity of +ba.1 serum (about 2% residual neutralization activity). BNT162b2 ³ And in particular mRNA-Vax ³ The decrease in neutralization titer of +ba.2 serum was less severe, about 12% and about 24% of residual neutralization activity, respectively. In contrast, depletion of NTD-binding antibodies was associated with BNT162b2 ³ And mRNA-Vax ³ The neutralizing activity of +BA.1 serum had no significant effect (about 91% and about 99% of the unconsumed control, respectively), whereas mRNA-Vax ³ The neutralization activity of +ba.2 serum was reduced to about 50%. A similar pattern was observed for BA.4/5 neutralization after RBD antibody depletion, mRNA-Vax ³ Neutralization activity of +ba.1 serum was strongly reduced (about 3% residual activity), in contrast to BNT162b2 ³ And mRNA-Vax ³ The neutralization activity of +ba.2 serum was less severely reduced (about 20% and about 26% residual activity, respectively). Depletion of NTD-binding antibodies has a greater effect on BA.4/5 neutralization than BA.2, where BNT162b2 ³ And mRNA-Vax ³ The residual neutralization activity of +BA.1 serum was about 70% and about 90%, respectively, again mRNA-Vax ³ The effect of +ba.2 serum was strongest (about 48% of the unconsumed control).

As an orthogonal approach, the neutralizing activity of sera from vaccinated individuals of those 3 groups against pseudoviruses containing engineered hybrid S glycoproteins consisting of Omicron ba.1 n-terminus (including NTD (amino acids 1-338)) and ba.4/5C-terminus (including RBD) was assessed.

From BNT162b2 ³ pVN against Omicron BA.1-BA.4/5 hybrid pseudovirus in the serum of (E) ₅₀ GMT is slightly lower than that of ba.4/5 pseudovirus (1.86 times) and is only slightly affected (< 1) during the ba.1 recovery period5-fold decrease) (fig. 44 (B)). In contrast, in ba.2 recovery serum, titers against hybrid pseudoviruses were much lower than those against ba.4/5 pseudoviruses (GMT decrease > 3-fold) (fig. 44 (B)), suggesting that the massive neutralization activity was attributable to the NTD epitope shared between omacron ba.2 and ba.4/5.

Overall, the data obtained in both experiments indicate that RBD binding antibody neutralization provides the major contribution in all these VOCs. Furthermore, exposure to BA.1 (which is significantly different in its NTD from the previous VOC; FIG. 33) enhanced the vaccine-induced recall reaction of neutralizing antibodies that predominantly bind RBD, whereas exposure to BA.2S glycoprotein (with NTD more closely related to the previous VOC) can build up on the basis of existing memory and elicit a massive recall of antibodies targeting NTD, which in turn greatly contributed to the neutralization of BA.2 and BA.4/5.

Discussion of

Recent studies have demonstrated that in individuals vaccinated with the mRNA vaccine BNT162b2 or mRNA-1273 or an inactivated viral vaccine, omacron ba.1 breakthrough infection enhances serum neutralization titers against VOCs including ba.2 (references 10, 15, 23), but not against ba.2.12.1 or ba.4/ba.5. Immune escape has been attributed to the enhancement of preexisting neutralizing antibody responses that recognize epitopes shared between SARS-CoV-2 wild-type strain and omacron ba.1, but partially deleted in ba.2.12.1, ba.4 and ba.5 due to alterations in key residues including L452Q/L452R and F486V (reference 15).

In this example, ba.2 breakthrough infections are associated with broad neutralization activities, including ba.2 and its progeny ba.2.12.1, ba.4 and ba.5. These findings indicate that higher sequence similarity of ba.2 to ba.2.12.1 and ba.4/5 drives more efficient cross-neutralization in S glycoprotein RBD as well as NTD than breakthrough infections with antigenically more distant ba.1 variants. In particular, whereas breakthrough infection of the heterologous SARS-CoV-2 strain mainly amplified memory B cell repertoires against conserved S glycoprotein epitopes (references 10, 23), ba.1 breakthrough infection may not trigger strong recall of NTD-specific memory B cells due to the large number of changes within ba.1NTD (fig. 33). The data obtained in the antibody depletion and hybrid pseudovirus experiments described herein indicate that NTD-binding antibodies have a great contribution to neutralizing activity against omacron ba.4/5 in triple vaccinated ba.2 convalescence sera, whereas in ba.1 convalescence sera neutralizing activity is largely dependent on RBD-binding antibodies. This finding is consistent with the observation that NTD-binding antibodies isolated from individuals with ba.2 breakthrough infections do not neutralize ba.1 (reference 29). Together, these important findings extend our knowledge of how to shape the immune pattern in a population with current wild-type strain-based vaccines for vaccination and boosting and breakthrough infections of various VOCs, and provide important information for further vaccine development and adaptation in response to current and emerging VOCs.

Despite the importance of vaccination with currently approved wild-type strain-based vaccines such as BNT162b2, which provide effective protection against severe diseases caused by current VOCs including omacron ba.1 and ba.2 (references 30 and 31), the findings of the present invention underscore that it is also important to guide vaccine adaptation programs considering rapidly evolving epidemiological situations and emerging SARS-CoV-2 variants. For example, while efficacy of vaccines adapted to the S glycoprotein sequence of strain ba.1 is currently being investigated in clinical trials, the data of the present invention demonstrate that further benefits can be obtained from vaccines adapted to the sequence of ba.2 or offspring.

Materials and methods

Study design, participant recruitment and sample collection

The aim of this study was to investigate the effect of omacron ba.2 breakthrough infection on the cross variant neutralising capacity of human serum. The immune response in triple mRNA (BNT 162b 2/mRNA-1273) vaccinated and with the confirmed subsequent SARS-CoV-2 breakthrough infected individual (2022, 3 months to 5 months; mRNA-Vax3+BA.2) over a period of time in which the Omicron BA.2 lineage is dominant was compared with the immune response in triple mRNA vaccinated and with the confirmed subsequent SARS-CoV-2 breakthrough infected individual (BNT 162b 23) over a period of time in which the Omicron BA.1 lineage is dominant (2021, 11 months to 2022, 1 month; mRNA-Vax3+BA.1) (references 1 and 2) and with the individual vaccinated with triple BNT162b2 not infected with SARS-CoV-2 (nucleocapsid seronegative) at the time of sample collection. Serum neutralization capacity was characterized using pseudovirus and live SARS-CoV-2 neutralization assays. The data for the reference group BNT162b23 and mRNA-Vax3+BA.1 were previously disclosed (reference 10), except for the newly generated BA.2.12.1 neutralization data. Cross-neutralization of variants in a smaller subgroup after depleting neutralizing antibodies targeting wild-type S glycoprotein NTD or RBD is further characterized.

From BNT162b2 ³ Individuals in the cohort provided informed consent as part of their participation in phase 2 trial BNT162-17 (NCT 05004181). From mRNA-Vax ³ + Omi BA.1 and mRNA-Vax ³ Participants in the +BA.2 group were recruited from university of Frankfursingard university (Goethe University Frankfurt) as non-intervention studies (protocol approved by the university medical ethics Committee [ No.: 2021-560) that had undergone Omicron breakthrough infection after vaccination with the COVID-19 vaccine]) A portion of the exploratory patient. Omicron ba.1 infection was confirmed by variant specific PCR. Infection of 4 ba.1 recovery participants in this study was further characterized by genomic sequencing. In all 4 cases, genomic sequencing confirmed Omicron ba.1 infection.

Demographic and clinical data for all participants are provided as well as sampling time points (fig. 41). All participants had no recorded history of SARS-CoV-2 infection prior to vaccination. Participants had no symptoms at the time of blood collection.

VSV-SARS-CoV-2S variant pseudovirus production

The recombinant replication defective Vesicular Stomatitis Virus (VSV) vector encoding Green Fluorescent Protein (GFP) and luciferase other than VSV-glycoprotein (VSV-G) was pseudotyped according to the published pseudotyping protocol (reference 3) with SARS-CoV-1S glycoprotein (UniProt reference: P59594) and SARS-CoV-2S glycoprotein derived from: MN-Hu-1 reference strain (NCBI reference: alpha variants (changes: Δ69/70, Δ144, N501 614 681 716 982 1118H), beta variants (changes: L18 80 215G, delta242-244, R246 484 501 614V), delta variants (changes: T19 142G, delta157/158, K417 452 478 614 681 950N), omicron BA.1 variants (changes: A67V, delta69/70, T95 142D, delta143-145, delta211, L212 EPE, G339 371 373 417 440, delta478 493 496 498 501 505 547 614-655 856 954 969F), omicron BA.2 variants (changes: T19I, delta24-26, A27 142 213 371 373 376, etc.) changes: A27 142, delta339-373, A39, delta67, A27, taking on, delta67, A27, delta67, A27, deltawear, delta67, delta35, delta1, delta35, delta7, E, delta7, delta95, E, delta95, E, delta95, E, delta95, E, E, delta95, E, E, delta95, deltal, delta95, deltal, deltal, Δ143-145, Δ211, L212I, ins EPE, G339D, S371F, S373P, S375 376A, D405N, R408 79417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y H, D614 655Y, N359K, P681H, N764K, D796Y, Q954H, N969K. A graphical representation of the SARS-CoV-2S glycoprotein alteration is shown in FIG. 48, and a separate alignment of the S glycoprotein alterations in the Omicon subline is shown in FIG. 33.

Briefly, a kit supplemented with 10% heat-inactivated fetal bovine serum (FBS [ Sigma-Aldrich]) Du's modified Issat (DMEM) plus Glutamax ^TM HEK293T/17 monolayer [. Sup.CRL-11268 ^TM ) SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmid plus Lipofectamine LTX (Life technologies) transfection was verified by Sanger sequencing according to the manufacturer' S instructions. 24 hours after transfection, cells were infected with VSV-G-supplemented VSV.DELTA.G vector at a multiplicity of infection (MOI) of 3. At 37℃and 7.5% CO ₂ After incubation for 2 hours, the cells were washed twice with Phosphate Buffered Saline (PBS) and then medium supplemented with anti-VSV-G antibodies (clone 8G5F11,Kerafast Inc) was added to neutralize residual VSV-G supplemented input virus. The medium containing the VSV-SARS-CoV-2-S pseudotype was harvested 20 hours after inoculation, passed through a 0.2 μm filter (Nalgene) and stored at-80 ℃. Vero 76 cells in culture medium were used (-)>CRL-1587 ^TM ) To titrate each batch of pseudoviruses. The relative luciferase units induced by a defined volume of SARS-CoV-2 wild-type strain S glycoprotein pseudovirus reference batch corresponding to an infectious titer of 200 Transduction Units (TU) per milliliter, previously described in Muik et al, 2021 (reference 4), were used as a control. The input volumes of each batch of SARS-CoV-2 variant pseudoviruses were calculated to normalize the infection titer based on relative luciferase units relative to the reference.

Pseudovirus neutralization assay

Vero 76 cells were seeded at 40,000 cells/well in 96-well white flat bottom plate (Thermo Scientific) medium 4 hours prior to assay and incubated at 37℃and 7.5% CO ₂ Culturing was performed under the following conditions. Serum from each individual was serially diluted 2-fold in medium at a first dilution of 1:5 (triple BNT162b2 vaccination without SARS-CoV-2; dilution range 1:5 to 1:5, 120) or 1:30 (subsequent triple vaccination after breakthrough infection by Omicron BA.1 or BA.2; dilution range 1:30 to 1:30, 720). Pseudo-viralization in SARS-CoV-1In the case of the assay, the serum of all individuals was initially diluted 1:5 (dilution range 1:5 to 1:5, 120). The VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in the medium to obtain 200 TUs in the assay. Serum dilutions were mixed with pseudoviruses (n=2 technical replicates per pseudovirus per serum) 1:1 for 30 min at room temperature, then added to Vero 76 cell monolayers and incubated at 37 ℃ and 7.5% co ₂ Incubate for 24 hours. The supernatant was removed and the cells were lysed with luciferase reagent (Promega). At the position ofLuminescence was recorded on a 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. For depletion studies, resolution was increased with respect to neutralization titers in order to distinguish less than 2-fold differences in individual serum levels. Neutralization titers were determined by generating a 4 parameter logistic (4 PL) fit of percent neutralization at each serial serum dilution. 50% pseudovirus neutralization (pVN) ₅₀ ) Titres are reported as the reciprocal interpolation of dilutions resulting in 50% reduction in luminescence. The results of all pseudo-virus neutralization experiments are expressed in terms of Geometric Mean Titer (GMT) of the replicates. If no neutralization is observed, the limit of detection [ LOD ] is reported]Half of any titer value. The SARS-CoV-2 wild-type strain and Alpha, beta, delta, BA.1, BA.4/5VOC of the non-SARS-CoV-2 infected BNT162b2 triple vaccine inoculation group and triple vaccinated BA.1 recovery period group were reported in Quandt et al (ref.10) before neutralization of GMT by SARS-CoV-1 pseudovirus. Only BA.2.12.1 neutralization data were newly generated from serum samples in this study.

Living SARS-CoV-2 neutralization assay

SARS-CoV-2 virus neutralization titers were determined by a micro-neutralization assay based on cytopathic effect (CPE) in vismedi s.r.1. Briefly, heat-inactivated serum samples from individuals were serially diluted 1:2 (starting from 1:10; n=2 technical replicates per virus) and incubated at 37℃with a 100TCID50 live wild-type strain of SARS-CoV-2 virus 2019-nCOV/ITALY-INMI1 (GenBank:MT 066156), alpha virus strain nCoV19 isolate/england/MIG 457/2020 (change: Δ69/70, Δ144, N501Y, A570D, D614G, P681H, T716I, S A, D1118H), beta virus strain nCoV19 isolate/england ex-SA/HCM002/2021 (change: D80A, D215G, Δ242-244, K417N, E484K, N501Y, D614G, A V), sequence verified Delta strain isolated from nasopharyngeal swabs (change: T19R, G142D, E G, Δ157/158, L452R, T478K, D614G, P681R, R682Q, D950N), omicron BA.1 strain hCoV-19/Belgium/rega-20174/2021 (change: a67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I, ins EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N Y, Y H, T547K, D614 35614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q95959595959595959595 954H, N969K, L981F), sequence verified Omicron ba.2 strain (change: T19I, Δ24-26, a27S, V213G, G339D, S371F, S373P, S375 376A, D405N, R408S, K417N, S477N, T478K, E484A, Q493R, Q5483R, Q501Y, Y505H, D614G, H655Y, N679 681H, R682W, N764K, D796Y, Q954H, N969K) or sequence verified Omicron ba.4 strain (change: V3G, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D N, R408 6767 417N, N440K, L452K, S477N, T478K, E484A, F486V, Q498R, N Y, Y614Y, Y655Y, Y679 681 764Y, Y796 5297 954 5299K) to allow any antigen-specific antibodies to bind to viruses. A graphical representation of S glycoprotein changes is shown in figure 48. The sequence of the S glycoprotein of 2019-nCOV/Italian-INMI 1 strain is identical to that of wild-type SARS-CoV-2S (MN 908947-Hu-1 isolate). Vero E6% CRL-1586 ^TM ) Cell monolayers were inoculated with serum/virus mixtures in 96-well plates and incubated for 3 days (2019-nCOV/Italian-INMI 1 strain) or 4 days (Alpha, beta, delta, omicron BA.1, BA.2 and BA.4 variant strains) to allow non-neutralizing viral infection. The plates were observed under an inverted light microscope and wells were scored as positive for SARS-CoV-2 infection (i.e., CPE was shown) or negative for SARS-CoV-2 infectionSex (i.e. cell survival without CPE).

Neutralization titers were determined as the reciprocal of the highest serum dilution that protected more than 50% of the cells from CPE and reported as GMT for the repeated experiments. If no neutralization is observed, an arbitrary titer value of 5[ half LOD ] is reported.

Depleting RBD or NTD binding antibodies in human serum

SARS-CoV-2 wild-type strain S glycoprotein RBD and NTD-coupled magnetic beads (Acro Biosystems, catalog nos. MBS-K002 and MBS-K019; 40. Mu.g RBD/mg beads and 38. Mu.g NTD/mg beads, respectively) were prepared according to the manufacturer' S instructions. The beads were resuspended in ultrapure water at 1mg beads/mL and the beads were collected using a magnet and washed with PBS. The beads were resuspended in serum to obtain 20 μg RBD or NTD baits per 100 μl serum. By adding 0.5mg biotin saturated MyOne per 100. Mu.L serum ^TM Streptavidin T1Dynabeads ^TM T1Dynabeads ^TM (thermo fisher, catalog No. 65601), each serum was subjected to simulated depletion (non-depleted control). The beads were incubated with human serum for 1 hour under gentle rotation. A magnet was administered to separate the bead-bound antibodies from the depleted supernatant. The cross-neutralization capacity of both depleted and non-depleted serum was analyzed using a pseudovirus neutralization assay. The depletion efficacy of both RBD and NTD binding antibodies was determined by multiplex electrochemiluminescence immunoassay (Meso Scale Discovery, V-Plex SARS-CoV-2 group 1 kit, catalog number K15359U-2).

Statistical analysis

The statistical polymerization method used to analyze antibody titers was a geometric mean and for the ratio of SARS-CoV-2VOC titers to wild-type strain titers was a geometric mean and a corresponding 95% confidence interval. The use of geometric averages accounts for the non-normal distribution of antibody titers across several orders of magnitude. Paired symbol rank test was performed on group geometric mean neutralizing antibody titers under a common control group using friedemann assay with dunn correction for multiple comparisons. The Spearman correlation was used to evaluate monotonic relationships between the non-normal distribution datasets. All statistical analyses were performed using GraphPad Prism software version 9.

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28.P.Arora et al.，Augmented neutralisation resistance of emerging omicron subvariants BA.2.12.1，BA.4，and BA.5.Lancet Infect Dis，(2022).

29.E.Andreano et al.，Anatomy of Omicron BA.1and BA.2neutralizing antibodies in

COVID-19mRNA vaccinees.Nat Commun 13，3375(2022).

30.N.Andrews et al.，Covid-19Vaccine Effectiveness against the Omicron(B.1.1.529)Variant.N Engl J Med 386，1532-1546(2022).

31.S.Y.Tartof et al.，Immunocompromise and durability of BNT162b2vaccine against severe outcomes due to omicron and delta variants.Lancet Respir Med 10，e61-e62(2022).

Example 18: exposure to ba.4/ba.5 spike glycoprotein drives pan Omicron neutralization in vaccinated humans and mice

This example is an extension of examples 7, 13, 16 and 17 and describes (i) immune response data from human subjects previously vaccinated against SARS-CoV-2 and having had a ba.4/5 breakthrough infection (in this example, human subjects who had previously been administered three doses of an RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 variant), and (ii) further mouse data showing immune responses induced by RNAs encoding SARS-CoV-2S proteins comprising mutations specific for certain omacron variants.

Abstract

Due to the large number of amino acid changes within the spike (S) glycoprotein, the SARS-CoV-2Omicron variant and its sublines showed significant viral escape of neutralizing antibodies. As demonstrated in the previous examples, breakthrough infections of individuals vaccinated with omacron progeny ba.1 and ba.2 have been associated with different cross-neutralizing activity patterns against SARS-CoV-2 variants of interest (VOCs), most notably against the most recently occurring ba.2 offspring omacron ba.4 and ba.5. Here, the effect of Omacron BA.4/BA.5S glycoprotein exposure on the size and breadth of the neutralizing antibody response was studied in vaccinated humans and mice. Immune sera from individuals vaccinated with triple mRNA with Omicron ba.4/ba.5 breakthrough infection showed broad and potent neutralizing activity against Omicron ba.1, ba.2, ba.2.12.1 and ba.4/ba.5. Following primary immunization based on the SARS-CoV-2 wild-type (MN 908947) strain, administration of BA.4/BA.5-adapted mRNA boost vaccine to mice was associated with similarly extensive neutralization activity. Immunization of non-vaccinated mice with bivalent mRNA vaccine encoding ba.4/ba.5-adapted and wild-type S glycoprotein immunogen was further shown to induce strong and broad neutralization activity against Omicron and non-Omicron vocs. These findings support the conclusion that omacron ba.4/ba.5 adaptive vaccines (e.g., RNAs encoding SARS-CoV-2S proteins comprising mutations unique to these variants) can improve the extent of neutralization against current and emerging VOCs (especially improved compared to ba.1 adaptive vaccines) when administered as monovalent or bivalent booster agents. These findings also indicate that the ba.4/ba.5 vaccine when administered in bivalent form has the ability to provide protection to subjects who do not have preexisting immunity to SARS-CoV-2 (e.g., to unexposed subjects).

Introduction to the invention

Since the advent of month 11 of 2021, SARS-CoV-2Omicron concerns Variants (VOCs) have had a significant impact on epidemiological situations of the COVID-19 pandemic (references 1-2). Significant changes in the spike (S) glycoprotein of the original omacron variant ba.1 resulted in the loss of many neutralizing antibody epitopes (reference 3) and enabled ba.1 to partially escape the previously established immunity based on the SARS-CoV-2 wild-type strain (MN 908947-hu-1) (references 4-6). Thus, omicron breakthrough infections are more common in vaccinated individuals than in previous VOCs. Although Omicron ba.1 is replaced by ba.2 variants in many countries around the world, other variants such as ba.1.1 and ba.3 are temporary and/or in the local landscape are strong, but are not dominant worldwide (references 7-9). Omicron ba.2.12.1 substituted ba.2 during which time ba.4 and ba.5 were dominant in europe, african parts and asia/pacific parts substituted ba.2 (references 8, 10-12). Currently, VOCs Omacron BA.4 and BA.5 predominate in most parts of the world, including the United states, where they ultimately replace BA.2.12.1 (reference 13).

The omacron lineage VOCs have acquired a number of changes (amino acid exchanges, insertions or deletions) in the S glycoprotein, some of which are shared among all omacron VOCs, while others are specific for one or more sublines (see figure 33). Antigenically, ba.2.12.1 shows a high similarity to ba.2 instead of ba.1, whereas ba.4 and ba.5 are significantly different from ba.2 and even more significantly different from ba.1, consistent with their pedigree (reference 14). The main differences between BA.1 and the remaining Omicon VOCs include Δ143-145 in the N-terminal domain of S glycoprotein, L212I or ins214EPE and G446S or G496S in the Receptor Binding Domain (RBD). The amino acid changes T376A, D N and R408S in RBD are in turn common to ba.2 and its progeny, but not in ba.1. In addition, some changes are specific to certain ba.2-progeny VOCs, including L452Q for ba.2.12.1 or L452R and F486V for ba.4 and ba.5. Most of these shared and subline-specific changes appear to play an important role in immune escape against monoclonal antibodies and polyclonal serum raised against wild-type S glycoprotein (reference 15).

As described in the previous examples, omicron ba.1 or ba.2 breakthrough infections of individuals immunized with mRNA vaccines or inactivated virus vaccines have been associated with potent neutralizing activity against Omicron ba.1, ba.2 and previous VOCs (see also references 16-21). The considerable enhancement of Omicron ba.2.12.1 and ba.4/ba.5 neutralization relative to the serum of vaccinators not infected with SARS-CoV-2 was only evident in ba.2 breakthrough cases, with titers against ba.4/ba.5 being lower than those against the remaining VOCs (see also references 19-21). The neutralizing activity against ba.1 was shown to be enhanced compared to the wild-type booster before administration of Omicron ba.1 adaptation booster to mice after the initial immunization based on the wild-type strain (see previous example 16 and reference 22). Preliminary analysis of phase 2 clinical trials also showed that serum from individuals vaccinated with omacron ba.1-adapted mRNA vaccine as dose 4, which were not infected with SARS-CoV-2, showed a significantly increased neutralization response against ba.1 compared to serum from individuals boosted with prototype mRNA vaccine (reference 23). However, the ba.1 adaptation enhancer was not associated with increased ba.4/ba.5 cross-neutralization, as the response to ba.4/ba.5 was approximately 3-fold and 5-fold lower than that to ba.1 and prototype strains, respectively, in serum from ba.1 and prototype-enhanced individuals.

Given the current predominance of highly infectious VOCs Omicron BA.4 and BA.5 in most regions of the world, this example aims to investigate whether exposure to Omicron BA.4/BA.5S glycoprotein would trigger a more broadly neutralizing antibody response to the relevant Omicron subline. Immune serum neutralization from vaccinated individuals with omacron ba.4/ba.5 breakthrough infection was tested for breadth of neutralizing activity against omacron VOC in serum from mice that received omacron ba.4/ba.5 adaptive booster vaccine after primary immunization with BNT162b 2. In addition, the breadth of neutralizing activity in serum from mice immunized with omacron ba.4/ba.5-adapted vaccine but not previously exposed to wild-type S glycoprotein was evaluated. The data described in this example provides further insight into the mechanism of omacron immune escape and the effect of immunity on variant cross-neutralization, and is therefore useful for guiding the selection of vaccination strategies.

Results

Study design, group and sampling

The effect of omacron ba.4/ba.5 breakthrough infection in individuals vaccinated with three doses of mRNA covd-19 vaccine (BNT 162B2/mRNA-1273 homologous or heterologous regimen) (fig. 49 (a)) and the effect of omacron ba.4/ba.5 adaptive booster vaccination of mice prior to BNT162B2 immunization on the extent of neutralization activity in immune serum (fig. 49 (B)) was studied. Furthermore, the effect of primary immunization with Omicron ba.4/ba.5 adaptive vaccine was studied in uninfected mice (i.e., mice not previously exposed to SARS-CoV-2S protein, see fig. 49 (C)).

For breakthrough infection studies, individuals vaccinated from triple mRNA and subsequently undergoing Omacron BA.4 or BA.5 breakthrough infection (mRNA-Vax ³ +ba.4/ba.5, n=17, fig. 53) were collected. Three groups are included for reference: with Omicron BA.2 (mRNA-Vax) ³ +BA.2, n=19) or BA.1 (mRNA-Vax ³ +ba.2, n=14) breakthrough infected triple mRNA vaccinated individuals, and BNT162b2 triple vaccinated individuals not infected with SARS-CoV-2 at the time of sampling (BNT 162b2 ³ N=18, fig. 53). Serum was derived from the study described previously in examples 7, 13 and 17.

Omicron BA.4/BA.5 breakthrough infections in triple mRNA vaccinated individuals lead to pan Omicron neutralizing activity

50% pseudovirus neutralization was determined in a well-characterized pseudovirus neutralization assay (pVNT) (reference 24-25) by using pseudoviruses with S glycoprotein of SARS-CoV-2 wild-type strain or Omicron BA.1, BA.2 and BA.2 derived sub-lines BA.2.12.1, BA.4 and BA.5 (pVN) ₅₀ ) Geometric Mean Titer (GMT) to test neutralizing activity of immune serum. Since the S glycoprotein sequences of BA.4 and BA.5 are identical, BA.4/5 was used in the context of pVNT. In addition, SARS-CoV (herein referred to as SARS-CoV-1) was assayed to detect potential ubiquity virus neutralization activity (ref.26). As an orthogonal assay system, a live SARS-CoV-2 neutralization assay (VNT) was also used which analyzes the neutralization of immune serum present throughout the assay period during multicycle replication of real viruses (SARS-CoV-2 wild-type strain and Omicron VOCs ba.1, ba.2 and ba.4).

In the pVNT assay, the gene from the Omicron BA.4/BA.5 breakthrough infection group (mRNA-Vax ³ +BA.4/BA.5) serum neutralization was strongWild-type strains and all omacron VOCs tested were presented (fig. 50 (a)). pVN against Omicron BA.2 and BA.2.12.1 pseudoviruses ₅₀ GMT is within 2-fold range of GMT for wild-type strain (GMT 613 for omacron versus GMT 1085 for wild-type). Neutralization by BA.1 and BA.4/5 (GMT 500-521) was substantially similar to that by BA.2 and was significantly reduced (p < 0.05) relative to the wild type strain, but also in the range of about 2-fold. GMT against SARS-CoV-1 was significantly lower (p < 0.0001; 50 times lower than wild type).

In order to target the gene with Omicron BA.1 or BA.2 breakthrough infection (mRNA-Vax ³ +BA.1 and mRNA-Vax ³ +BA.2) mRNA-Vax of the reference group ³ +BA.4/BA.5 individuals vaccinated with triple BNT162b2 not infected with SARS-CoV-2 (BNT 162b 2) ³ ) For comparison, VOC pVN was used ₅₀ GMT was normalized to wild-type strains to allow evaluation of neutralization luminosity, irrespective of the size of antibody titers, which unexpectedly differed between individuals with breakthrough infected triple vaccination and those without infection (references 16 and 21). Although BNT162b2 ³ Serum mediated massive cross-neutralization of Omicron ba.1 and ba.2, but breakthrough infections of Omicron ba.1 and ba.2 correlated with higher cross-neutralization of the corresponding homologous strains (fig. 50 (B)). With mRNA-Max ³ Cross-neutralization of BA.2.12.1 and especially BA.4/5 in mRNA-Vax compared to +BA.4/BA.5 (GMT ratios of 0.57 and 0.48) ³ The +ba.1 group (GMT ratios of 0.43 and 0.18, respectively) was less effective. In mRNA-Vax ³ In the +ba.2 group (GMT ratios of 0.53 and 0.37, respectively), cross neutralization of ba.2.12.1 and ba.4/5 was less reduced. Surprisingly, in the reciprocal case, cross-neutralization of omacron ba.1 and ba.2 in mRNA-Vax ³ The +BA.4/BA.5 group (GMT ratios of 0.46 and 0.57, respectively) remained at a relatively high level. Thus, the BA.4/BA.5 breakthrough infection resulted in the most effective cross-neutralization among all tested VOCs (GMT ratio. Gtoreq.0.46) in all groups evaluated.

The true live SARS-CoV-2 virus neutralization assay largely confirms the major pVNT assay findings shown in FIGS. 50 (A) - (B). Omicron BA.4/BA.5 breakthrough in serum against50% virus neutralization of BA.2 and BA.4 (VN ₅₀ ) GMT was comparable to (i.e., in the 1.5-fold range) against wild-type strains (fig. 50 (C)). The reduction in neutralization by BA.1 was significant (p < 0.01), but in the 2.5-fold range. GMT normalized to wild strain shows mRNA-Vax ³ Strong cross-neutralization of Omicron BA.1, BA.2 and BA.4 by +BA.4/BA.5 serum (GMT ratio.gtoreq.0.40) in mRNA-Vax ³ +BA.1 (GMT ratio 0.20) and mRNA-Vax ³ In +ba.2 (GMT ratio 0.39) serum, ba.4 cross-neutralization was quite inefficient (fig. 50 (D)) (references 16 and 21). Thus, findings in the pnnt and VNT assay systems indicate that omacron ba.4/ba.5 breakthrough infections are associated with broad neutralization activity against all tested omacron sublines.

Boosting with Omicron ba.4/ba.5s glycoprotein-adapted mRNA vaccine boost pan Omicron neutralization was driven in BNT162b2 double vaccinated mice

The enhanced extent of neutralization observed following omacron ba.4/ba.5 breakthrough infection suggests that variant adaptive vaccines based on omacron ba.4/5S glycoprotein sequences are able to elicit recall responses with broader cross-neutralization compared to vaccines based on omacron ba.1. To test this hypothesis, a booster study was performed in BNT162B2 pre-immunized mice (fig. 49 (B)). Mice were administered an initial series of two BNT162b2 immunizations on days 0 and 21, and a third dose of BNT162b2 (1 μg) or a variant adaptive vaccine derived from BNT162b2 encoding Omicron ba.1 or ba.4/ba.5s glycoprotein on day 104 (fig. 54). The adaptive vaccine was used as a monovalent vaccine (1. Mu.g) encoding Omicron BA.1 or BA.4/5S glycoprotein or as a bivalent vaccine comprising BNT162b2 and Omicron BA.1 or BA.4/5S glycoprotein adaptive vaccine (0.5. Mu.g each). Equivalent RNA purity and integrity of BNT162B2 and omacron-adapted vaccines and in vitro antigen expression were confirmed (fig. 55 (a) - (B)).

Neutralization titers against pseudoviruses expressing wild-type strains, omacron ba.1, ba.2, ba.2.12.1 or ba.4/5S glycoproteins were determined in the pnnt assay using serum drawn before the booster (day 104, before D3) and at days 7, 21 and 35 (D7D 3, D21D3 and D35D 3) after the booster. The live SARS-CoV-2 neutralization assay was used as an orthogonal assay system to confirm the observed pseudovirus neutralization activity after D21D3 and D35D3 boosting.

Mice were assessed for baseline immunity by measuring SARS-CoV-2 pseudovirus neutralizing activity in serum withdrawn prior to D3. pVN specific to groups of various reinforcing agents ₅₀ GMT was comparable, i.e. within a range of 3-fold difference (fig. 56). pVN against omacron ba.1 and ba.2 compared to wild type strain ₅₀ GMT was 3 to 11 times lower (GMT ratio +.0.32, fig. 56 (B)). GMT for ba.2.12.1 and ba.4/5 was 10 to 25 times lower than for wild type (GMT ratio +.0.10).

At D7D3, the neutralizing GMT increased significantly between groups and for all tested variants (fig. 57), peak titers were reached at D21D3 (fig. 51). In serum from BNT162b 2-boosted mice, strong neutralizing activity against the wild-type strain was observed, while pVN against the omacron variant ₅₀ GMT is significantly lower (fig. 51 (a)). The Omicron ba.1 enhancer resulted in considerable neutralization of ba.1 and wild type strains, while pVN for the remaining VOCs ₅₀ GMT is significantly lower. In particular, GMT for ba.4/5 was reduced 13-fold compared to wild type. In contrast, administration of the BA.4/5 enhancer resulted in broad neutralization activity against all Omacron variants, pVN ₅₀ GMT was comparable (in the 1.5-fold range) to that for wild-type strains. Serum from mice receiving the BNT162b2/BA.1 bivalent booster had a height pVN against the wild-type strain ₅₀ GMT, and potent neutralization to Omicron ba.1, whereas GMT against ba.2 and its progeny strains was slightly lower. The BNT162b 2/Omicon BA.4/5 bivalent enhancer produced a high titre against the wild type strain, comparable to the BNT162b2 monovalent enhancer. Omicron neutralization was approximately equivalent (in the 2-fold range) in all sublines, pVN ₅₀ GMT was slightly lower than for the wild type strain.

To quantify the potentiating effect of the third dose of vaccine variant on the neutralization of individual VOCs, pVN detected at D21D3 was evaluated ₅₀ Fold change in GMT relative to baseline GMT measured prior to administration of the third dose. BNT162b2 considerably increased the neutralization of all variants tested (pVN) ₅₀ GMT 6 to 10 times higher than baseline), whereas detectionThe most pronounced effect on neutralization of homologous VOCs by the ba.1 enhancer (26 fold increase) (fig. 51 (B)). The ba.4/5 enhancer strongly enhances neutralization activity (> 120-fold) against ba.2.12.1 and ba.4/5, and also has a significant effect on ba.1 and ba.2 neutralization (34-fold and 37-fold increases, respectively). BNT162b2/BA.1 and BNT162b2/BA.4/5 bivalent vaccines showed similar enhancement patterns to the BA.1 and BA.4/5 monovalent vaccines, respectively, although neutralization was less focused on the increase of homologous VOCs.

To compare groups with respect to neutralization breadth regardless of size, VOC pVN was calculated ₅₀ GMT was normalized to wild-type strain. The GMT ratio showed that the Omicron BA.4/5 plus vaccine mediated a broad Omicron neutralization (0.65 or more for all variants tested) (FIG. 51 (C)). In contrast, BA.1 booster vaccines favor neutralization of BA.1 (GMT ratio 0.77), whereas the ratio is significantly lower for BA.2 (0.39) and especially for BA.2.12.1 and BA.4/5 (. Ltoreq.0.16). The bivalent BNT162b2/BA.4/5 vaccine also mediated broad neutralization activity, with enhanced cross-neutralization of BA.2, BA.2.12.1 and BA.4/5 compared to the BNT162b2/BA.1 bivalent vaccine, although the GMT ratio was lower (between 0.27 and 0.46) compared to the BA.4/5 monovalent vaccine.

Again, the true live SARS-CoV-2 virus neutralization assay largely confirmed the major pnnt assay findings shown in fig. 51 (a) - (C). Serum from mice administered with ba.4/5 booster dose neutralized all tested variants strongly (fig. 51 (D)), confirming the ability of this approach to mediate generalized omacron neutralization (GMT ratio ∈0.39) (fig. 51 (E)). Although neutralization of Omicron variants in serum from mice boosted with BNT162b2/ba.4/5 bivalent vaccine was lower compared to ba.4/5 monovalent vaccine, both ba.4/5 containing vaccines showed stronger cross-neutralization of Omicron variants compared to BNT162b2/ba.1 vaccine and even stronger compared to monovalent ba.1 vaccine and BNT162b 2.

Although the absolute titre was slightly lower at D7D3 compared to D21D3, the omacron ba.4/5 vaccine mediated similar neutralization activity for all variants (fig. 57). BNT162b2/BA.4/5 bivalent enhancer mediated a considerable extent of neutralization (GMT ratio. Gtoreq.0.35), whereas the sera of all other enhancer groups showed a considerably lower cross-neutralization to BA.2, BA.2.12.1 (and BA.4/5). Similarly, subsequent analysis of D35D3 serum showed that neutralization activity against omacron variants was significantly enhanced by ba.4/5 vaccine booster compared to baseline, resulting in pan omacron neutralization (fig. 58 (a) - (C)). The divalent BNT162b2/BA.4/5 booster also showed extensive neutralization in all variants tested, whereas the BA.1 and BNT162b2/BA.1 vaccines showed lower cross-neutralization capacity, especially against BA.2.12.1 and BA.4/5. Similarly, in the VNT assay, sera from both groups boosted with the vaccine containing ba.4/5 showed significantly stronger cross-neutralization of all tested Omicron variants compared to the vaccine containing ba.1 and BNT162b2 (fig. 58 (D) - (E)).

These mouse booster results are consistent with those observed in ba.4/5 breakthrough infected humans as described above, and further demonstrate that the use of a booster of Omicron ba.4/5S glycoprotein adaptive vaccine can elicit superior broad spectrum Omicron neutralizing activity over ba.1S glycoprotein based boosters after primary immunization with wild type strain based vaccines.

Boosting with Omicron ba.4/ba.5s glycoprotein-adapted mRNA vaccine boost driving pan Omicron neutralization in previously unvaccinated mice

Next, experiments were performed to better understand the neutralizing capacity of serum samples after immunization with omacron-adapted vaccine in mice without pre-existing immune response against SARS-CoV-2. Uninfected mice were immunized twice with either BNT162b2, BA.1 or BA.4/5S glycoprotein-adapted monovalent vaccine or bivalent BNT162b2/BA.4/5 vaccine on day 0 and day 21 (FIG. 49 (C)). Neutralization titers against pseudoviruses expressing wild-type strain S glycoprotein, previous VOCAlpha or Delta or Omicron ba.1, ba.2, ba.2.12.1 or ba.4/5 were determined in the pnnt assay using serum withdrawn 14 days after the second immunization (D14D 2). In serum from BNT162b2 immunized mice, strong neutralizing activity against wild type strains was observed. In these sera, potent neutralizing activity against Alpha and Delta (in the 4-fold range of wild type) was also detected, while pVN against omacron variants ₅₀ GMT is remarkable (14 to 37 times)Lower than the wild type (fig. 52). Immunization with omacron ba.1 monovalent vaccine resulted in a high degree of neutralization of ba.1. In these sera, potent neutralization was also detected on omacron ba.2 and ba.2.12.1 (in the 3-fold range of ba.1), while pVN against wild-type strain and remaining VOCs ₅₀ GMT was significantly (7 to 32 times) lower than ba.1. Immunization with Omicron BA.4/5 monovalent vaccine resulted in a high degree of neutralization of BA.4/5. In these sera, potent neutralization (in the 2.5-fold range of BA.4/5) was also detected on omacron BA.2 and BA.2.12.1, while pVN was directed against the wild-type strain and the remaining VOCs ₅₀ GMT was significantly (14 to > 42 times) lower than ba.4/5. Immunization with BNT162b2/BA.4/5 bivalent vaccine resulted in high neutralization activity against BA.4/5. Potent neutralizing activity (in the 6-fold range of ba.4/5) against wild-type strains and all remaining VOCs was also detected in these sera compared to other vaccines.

These results indicate that in uninfected animals (e.g., animals that were not previously dosed for SARS-CoV-2 and/or were not infected with SARS-CoV-2), monovalent vaccines can induce highly neutralizing antibody responses primarily in a variant-specific manner, but may lose efficacy when tested against more distant variants. In contrast, bivalent vaccines can elicit strong and broad neutralizing antibody responses in uninfected animals.

Discussion of

In this example, the ba.4/ba.5 breakthrough infection of triple mRNA vaccinated individuals was associated with potent neutralization of all current or previous dominant Omicron subvariants, i.e., pan Omicron neutralization was observed in ba.4/5 breakthrough patients. These findings are consistent with recent reports showing strong cross-neutralization of omacron ba.1, ba.2 and Beta and Delta in serum from individuals vaccinated with BNT162b2 or adenovirus-based vaccines and subsequently ba.4 breakthrough infected (reference 27). Consistent with those observed in humans, pan Omicron neutralization activity was also observed in serum of mice receiving Omicron ba.4/5 booster vaccine after primary immunization with BNT162b2, whereas Omicron ba.1 booster induced significantly reduced neutralizing antibody titers against ba.4/ba.5. The divalent BNT162b2/BA.4/5 boost elicited extensive Omacron neutralization, although not as pronounced as the BA.4/5 monovalent boosters. Taken together, these findings further understand how breakthrough infections or vaccine boosters in monovalent or bivalent form that are adapted to Omicron VOCs can shape immunity and demonstrate that exposure to Omicron ba.4/5S glycoprotein can confer enhanced protection against currently circulating and potentially future Omicron sub-line VOCs. Immunization of non-infected mice with the BNT162b2/BA.4/5 bivalent vaccine was found to elicit a strong neutralizing antibody response against wild-type strains as well as Omicron and non-Omicron VOCs, suggesting that this bivalent approach may provide broad protection to previously non-vaccinated individuals not infected with SARS-CoV-2 (e.g., pediatric patients), and thus may be particularly suitable for these individuals.

Although currently approved vaccines based on wild-type strain of SARS-CoV-2, such as BNT162b2, have proven effective in protecting against severe disease (references 28-30), prevention of transmission remains a significant challenge due to the continued emergence of new variants antigenically distant from the wild-type strain (references 16-18, 20). The data described in this example demonstrate that monovalent or bivalent ba.4/ba.5s glycoprotein fitness enhanced vaccines (e.g., the monovalent or bivalent vaccines described herein) can confer higher benefits against highly prevalent ba.4 and ba.5 vocs than vaccines based on the previously dominant omacron subline (e.g., ba.1). Given their predominance in many parts of the world and their high transmissibility (see, for example, references 8, 10, 11 and 13), it is possible that new variants with further growth advantages will be generated from omacron ba.4 or ba.5, which retain some or all of the sensitivity to ba.4/ba.5 adaptive vaccines. Thus boosting pre-existing immunity with an adaptive vaccine based on omacron ba.4/5S glycoprotein might represent a suitable strategy to cope with current pandemic situations, while closely monitoring viral evolution and epidemiological situations still helps to guide potential further vaccine adaptation in response to emerging threats.

Materials and methods

Human study design, participant recruitment and sample collection

The purpose of the study described in this example was to investigate Omicron BA.4/BA.5 breakthroughEffects of infection on the neutralizing capacity of cross variants of human serum. Neutralization activity was assessed in immune serum from individuals vaccinated with triple mRNA (BNT 162b 2/mRNA-1273) and with confirmed subsequent SARS-CoV-2 breakthrough infections that occurred in germany over a period of time that predominates in the Omicron ba.4/ba.5 lineage (2022, late 6 to late 7; or by genomic sequencing (mRNA-Vax ³ The +BA.4/5 variant confirmed (BA.4 or BA.5) (FIG. 53). Neutralization activity was associated with a period of time from triple mRNA vaccination with confirmed predominance over the Omacron BA.2 lineage (3 months to 5 months in 2022; mRNA-Vax) ³ +ba.2), a period of time that predominates in the omacron ba.1 lineage of germany (11 months 2021 to 1 middle 2022; mRNA-Vax ³ +BA.1) (reference 1, 2) and triple BNT162b2 vaccinated individuals not infected with SARS-CoV-2 (nucleocapsid seronegative) at the time of sample collection (BNT 162b 2) ³ ) The neutralizing activity in the immune serum of (a) was compared. Serum neutralization capacity was characterized using pseudovirus and live SARS-CoV-2 neutralization assays. Reference group mRNA-Vax ³ +BA.2、mRNA-Vax ³ +BA.1 and BNT162b2 ³ Is disclosed (references 16, 21).

From mRNA-Vax ³ +Omi BA.4/5、mRNA-Vax ³ + Omi BA.2 and mRNA-Vax ³ Participants in the +BA.1 group were recruited from university of Frankfursingard university (Goethe University Frankfurt) as non-intervention studies (protocol approved by the university medical ethics Committee [ number: 2021-560) that had undergone Omicron breakthrough infection after vaccination with the COVID-19 vaccine]) A portion of the exploratory patient. From BNT162b2 ³ Individuals in the cohort provided informed consent as part of their participation in phase 2 trial BNT162-17 (NCT 05004181).

Infection of 5 BA.4/5 and 4 BA.1 recovery phase participants in this study was confirmed by genomic sequencing (ref 16).

All participants had no recorded history of SARS-CoV-2 infection prior to vaccination. Participants had no symptoms at the time of blood collection.

In vitro transcription of RNA and lipid-nanoparticle (LNP) formulations

BNT162b2 vaccine was designed in the context of S sequences with pre-fusion conformational stable K986P and V987P mutations from SARS-CoV-2 isolate MN908947-Hu-1 (GenBank: MN 908947.3). The Omicron ba.1 and Omicron ba.4/5 vaccine candidates were designed based on BNT162b2 comprising sequence changes as shown in figure 54. RNA production and formulation is performed as described elsewhere (see, e.g., reference 32, the contents of which are incorporated herein by reference in their entirety). Briefly, DNA templates are cloned into plasmid vectors with backbone sequence elements (T7 promoter, 5 'and 3' utr, poly (a) tail of 100 nucleotides) interrupted by linkers (a 30LA70, 10 nucleotides) to improve RNA stability and translation efficiency, amplified by PCR and purified (see, e.g., references 33, 34). RNA was transcribed in vitro by T7RNA polymerase in the presence of trinucleotide cap1 analog ((m 27,3 '-O) Gpp (m 2' -O) ApG; triLink) and with N1-methyl pseudouridine-5 '-triphosphate (m 1 ψTP; thermo Fisher Scientific) instead of uridine-5' -triphosphate (UTP) (ref.35). RNA was purified using magnetic particles (reference 36) and RNA integrity was assessed by microfluidic capillary electrophoresis (Agilent 2100 bioanalyzer), and all three RNAs showed a single spike, yielding comparable and higher purity and integrity (fig. 55 (a)). In addition, the concentration, pH, osmotic pressure, endotoxin level and bioburden of the solution were measured.

An ionizable lipid ((4-hydroxybutyl) azetidinediyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), two structural lipids (1, 2-distearoyl-sn-glycero-3-phosphorylcholine [ DSPC ] and cholesterol) and one pegylated lipid (2- [ (polyethylene glycol) -2000] -N, N-tetracosanamide) were used for the formulation of RNA. After transfer into an aqueous buffer system by dialysis, the LNP composition was analyzed to ensure, but not limited to, high RNA integrity and encapsulation efficacy and particle sizes of less than 100 nm. The vaccine candidates were stored at a concentration of 0.5mg/mL at-70 ℃ to-80 ℃ until the point of use.

In vitro expression of RNA and vaccine

Using ribojet according to manufacturer's instructions ^TM mRNA transfection kits (Merck Millipore) HEK293T cells were transfected with 0.15. Mu.g BNT162b2 or Omicon-adapted vaccine (lipid-nanoparticle formulated) or with vaccine RNA and incubated for 18 hours. Transfected HEK293T cells were stained with an fixable vital dye (eBioscience) and incubated with mFc-labeled recombinant human ACE-2 (Sino Biological). A second donkey anti-mouse antibody conjugated to AF647 was used to detect surface expression. Cells were fixed (fixed buffer, biolegend) prior to flow cytometry analysis using a FACSCelesta flow cytometer (BD Biosciences, BD FACSDiva software version 8.0.1) and FlowJo software version 10.6.2 (FlowJo, BD Biosciences).

Mouse study

All mouse studies were performed at BioNTech SE and protocols were approved by local authorities (local welfare committee) and were performed according to the recommendations of the european laboratory animal science association. Study execution and containment were in compliance with German animal welfare and Command 2010/63/EU. Mice were kept under separate ventilation cages for a 12 hour light/dark cycle, controlled environmental conditions (22 ℃ ±2 ℃,45% to 65% relative humidity) and specific pathogen free conditions. Food and water are provided at will. Only mice with good health condition were selected for the test procedure.

For immunization, female BALB/c mice (Janvier) (9-21 weeks old) were randomly assigned to groups. BNT162b2 and Omacron-based vaccine candidates were diluted in 0.9% NaCl and 1. Mu.g of vaccine candidate was injected into gastrocnemius muscle under isoflurane anesthesia at a volume of 20. Mu.l. For the mouse booster study, mice were immunized twice with BNT162b2 (day 0 and day 21). A third immunization with BNT162b2 and omacron-based vaccine candidate was performed on day 104 after study initiation, and mice were bled shortly before the third immunization and as indicated in fig. 49. For uninfected mice studies, animals were immunized with BNT162b2 and omacron-based vaccine candidates on days 0 and 21. Blood was drawn 14 days after the second immunization. Peripheral blood was collected from facial veins without anesthesia. Final bleeds were performed under isoflurane anesthesia from the retroorbital venous plexus. For serogenesis, blood is centrifuged at 16,000 g for 5 minutes and serum is immediately used for downstream assays or stored at-20 ℃ until the point of use.

VSV-SARS-CoV-2S variant pseudovirus production

According to the disclosed pseudotyping scheme (e.g., as described in reference 3, the contents of which are incorporated herein by reference in their entirety), recombinant replication defective Vesicular Stomatitis Virus (VSV) vectors encoding Green Fluorescent Protein (GFP) and luciferase, but not VSV-glycoprotein (VSV-G) are pseudotyped with SARS-CoV-1S glycoprotein (Unit Prot reference: P59594) and with SARS-CoV-2S glycoprotein derived from: wild-type strain (MN-Hu-1, ncbi reference: alpha variants (changes: Δ69/70, Δ144, N501 614 681 716 982 1118H), delta variants (changes: T19 142G, Δ157/158, K417 452 478 681N), omicron BA.1 variants (changes: A67V, Δ69/70, T95 142D, Δ143-145, Δ211, L212 EPE, G339 371 373 440 477N, 1478 484 493 496 498 501 505 547 614 679 681K 764,954 969F), omicron BA.2 variants (changes: T19I, Δ24-26, A27 142 213 339 373 376 478 484 493 501, changing: A67V, Δ69/70, T95 142D, Δ143-145, Δ211, L212 EPE, G339 371 375 440 477 493 498K), omicron BA.47 679 704 7694 969K, omicron 4/37A 7 484 499, and Omicron BA.45 484 493 498K), omicron BA.47 484 37, changing: A19I, Δ24-26, A27 142, A27, 339 37, and Omicron 7, changing: 484, A7, 484, 484.12.1 variants (change: A27, FIG. 39, FIG. 7, etc. 484, and Omicron 7/7, changing: 484.40 7 to be a 7, so that they are changed: A27, and A27.7, and a change, thereby to change to thereby to.7.7.7.7.7.7.7, to.7, to change to.7 to,. A graphical representation of the SARS-CoV-2S glycoprotein alteration is shown in FIG. 32, and a separate alignment of the S glycoprotein alterations in the Omicon subline is shown in FIG. 33.

Briefly, a kit supplemented with 10% heat-inactivated fetal bovine serum (FBS [ Sigma-Aldrich]) Du's modified Issat (DMEM) plus Glutamax ^TM HEK293T/17 monolayer [. Sup.CRL-11268 ^TM ) SARS-CoV-1 or variant-specific SARS-CoV-2S expression plasmid plus Lipofectamine LTX (Life technologies) transfection was verified by Sanger sequencing according to the manufacturer' S instructions. 24 hours after transfection, cells were infected with VSV-G-supplemented VSV.DELTA.G vector at a multiplicity of infection (MOI) of 3. At 37℃and 7.5% CO ₂ After incubation for 2 hours, the cells were washed twice with Phosphate Buffered Saline (PBS) and then medium supplemented with anti-VSV-G antibodies (clone 8G5F11,Kerafast Inc) was added to neutralize residual VSV-G supplemented input virus. The medium containing the VSV-SARS-CoV-2-S pseudotype was harvested 20 hours after inoculation, passed through a 0.2 μm filter (Nalgene) and stored at-80 ℃. Vero 76 cells in culture medium were used (-)>CRL-1587 ^TM ) To titrate each batch of pseudoviruses. The relative luciferase units induced by a defined volume of SARS-CoV-2 wild-type strain S glycoprotein pseudovirus reference batch corresponding to an infectious titer of 200 Transduction Units (TU) per milliliter, previously described in Muik et al, 2021 (ref.31), were used as controls. The input volumes of each batch of SARS-CoV-2 variant pseudoviruses were calculated to normalize the infection titer based on relative luciferase units relative to the reference.

Pseudovirus neutralization assay

Vero 76 cells were seeded at 40,000 cells/well in 96-well white flat bottom plate (Thermo Scientific) medium 4 hours prior to assay and at 37℃and 7.5% CO ₂ Culturing was performed under the following conditions. Human and mouse serum samples were studied at 1:5 to 1:30, 720 (human serum), 1:40 to 1:102, 400 (mouse serum; initial dilution 1:40 [ D3 pre-f ]]，1∶200[d7D3]) 1:100 [ D21D3, D35D3 ]]) Dilutions within the range were performed in medium and in uninfected environment (mouse serum; monovalent vaccination groups starting dilutions 1:120 to 1:15, 360, and bivalent vaccination groups starting dilutions 1:100) 2-fold serial dilutions. The VSV-SARS-CoV-2-S/VSV-SARS-CoV-1-S particles were diluted in the medium to obtain 200 TUs in the assay. Serum dilutions were mixed with pseudoviruses (n=2 technical replicates per pseudovirus per serum) 1:1 for 30 min at room temperature, then added to Vero 76 cell monolayers and incubated at 37 ℃ and 7.5% co ₂ Incubate for 24 hours. The supernatant was removed and the cells were lysed with luciferase reagent (Promega). At the position ofLuminescence was recorded on a 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. The results of all pseudo-virus neutralization experiments are expressed in terms of Geometric Mean Titer (GMT) of the replicates. If no neutralization is observed, the limit of detection [ LOD ] is reported ]Half of any titer value. A table of neutralization titers in human serum is provided.

Living SARS-CoV-2 neutralization assay

SARS-CoV-2 virus neutralization titers were determined by a micro-neutralization assay based on cytopathic effect (CPE) in vismedi s.r.1. Briefly, human and mouse serum samples were serially diluted 1:2 (n=2 technical replicates per virus per serum; starting from 1:10 for human samples and 1:100 for murine samples [ after boosting, day 125)]Or 1:50 [ after boosting, day 139 ]]Start) and at 37 ℃ with 100TCID ₅₀ Live wild-type strain of SARS-CoV-2 virus 2019-nCOV/Italy-INMI 1 (GenBank: MT 066156), omicron BA.1 strain hCoV-19/Belgium/rega-20174/2021 (changes: A67V, Δ69/70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G)9D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547 614 79614G, H79655Y, N679K, P681H, N764K, D796Y, N856 954H, N969K, L981F), sequence verified Omicron ba.2 strain (change: T19I, Δ24-26, a27S, V213G, G339D, S371F, S373P, S375 376A, D405N, R408S, K417N, S477N, T478K, E484A, Q493R, Q5483R, Q501Y, Y505H, D614G, H655Y, N679 681H, R682W, N764K, D796Y, Q954H, N969K) or sequence verified Omicron ba.4 strain (change: V3G, T19I, Δ24-26, a27S, Δ69/70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D N, R408 6767 417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N Y, Y614Y, Y655Y, Y679 681 764Y, Y796 5297 954 5299K) to allow any antigen-specific antibodies to bind to viruses. A graphical representation of S glycoprotein changes is shown in figure 48. The sequence of the S glycoprotein of 2019-nCOV/Italian-INMI 1 strain is identical to that of wild-type SARS-CoV-2S (MN 908947-Hu-1 isolate). Vero E6% CRL-1586 ^TM ) Cell monolayers were inoculated with serum/virus mixtures in 96-well plates and incubated for 3 days (2019-nCOV/Italian-INMI 1 strain) or 4 days (Omicron BA.1, BA.2 and BA.4 variant strains) to allow non-neutralising viral infection. The plates were observed under an inverted light microscope and wells were scored as positive for SARS-CoV-2 infection (i.e., CPE was shown) or negative for SARS-CoV-2 infection (i.e., cells survived without CPE). Neutralization titers were determined as the reciprocal of the highest serum dilution that protected more than 50% of the cells from CPE and reported as GMT for the repeated experiments. If no neutralization is observed, half of the 5[ LOD ] is reported]Any titer value of (a).

Statistical analysis

Reference to example 18

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Example 19: effects of BA.4/BA.5 breakthrough infection on Omicron immune response

This example 19 is an extension of examples 14, 17 and 18 and describes experiments to analyze the neutralization activity of serum samples taken from ba.1-, ba.2-and ba.4/5-breakthrough cases against omacron ba.4, ba.5, ba.4.6, bf.7 and ba.2.75 variants. In addition to confirming the results described in the previous examples, this example 19 also provides further characterization of antibody responses induced by BA.1-, BA.2-and BA.4/5-breakthrough infections in relation to the BA.4.6, BF.7 and BA.2.75 variants. This example further demonstrates that: ba.4/5 breakthrough infections can produce a broader neutralization response than ba.1-and ba.2-breakthrough infections, induce a better neutralization titer against ba.4/5 and ba.4.6/bf.7 variants than ba.1-and ba.2-breakthrough infections, and are comparable to neutralization titers against ba.2.75 virus.

The continued evolution of SARS-CoV-2Omicron variants has led to the emergence of numerous sublines with different neutralizing antibody escape patterns. The recently occurring sub-lines ba.4.6, bf.7 and ba.2.75 have attracted attention because of their slow but steadily increasing popularity in many areas. This example investigated the neutralizing activity against these Omicron sub-lines and the currently dominant ba.4/ba.5 in the immune serum of individuals who later developed triple vaccination with covd-19 mRNA of the ba.1, ba.2 or ba.4/ba.5sars-CoV-2 variants. Serum from individuals vaccinated with triple or quadruple mRNA that did not infect SARS-CoV-2 was also tested. Serum from the recovery phases of BA.1, BA.2 and BA.4/BA.5 was approximately comparable in terms of its ability to cross-neutralize BA.2.75 relative to the SARS-CoV-2 wild-type strain. The strongest cross-neutralization of omacron ba.4.6/bf.7 was detected in ba.4/ba.5 convalescence serum. These findings indicate that recent breakthrough infections of the omacron subline may confer at least partial protection from emerging subline infections, and that further evidence is provided to support the efficacy of ba.4/5-specific vaccines (in particular, it is further demonstrated that ba.4/5-specific vaccines may provide a broad immune response against omacron variants).

SARS-CoV-2 omicon concerns Variants (VOCs) containing more than 30 amino acid changes in their spike (S) glycoprotein, which mediate partial escape of previously established immunity. Thus, breakthrough infections by omacron have been more frequent between vaccinated populations than previously cycled VOCs. Sub-lines ba.1, ba.2, ba.4 and ba.5 have continuously dominated in the case of pandemic since the first occurrence of omacron at 11 in 2021. Although ba.5 has been the globally dominant subline since 2022, virus evolution continues to produce new sublines with additional amino acid changes in their S glycoproteins. Many countries in each continent report a slow but steadily increasing popularity of offspring from the previous omacron subline. These offspring include omicon ba.2.75, ba.4.6 and bf.7, from ba.2, ba.4 and ba.5, respectively.

Omicron ba.2.75 contains five amino acid changes within the N-terminal domain (NTD) of the S protein, which makes it different from previous Omicron variants of interest (VOCs), including its parent subline ba.2 (fig. 60). In addition, ba.2.75 has three changes in its Receptor Binding Domain (RBD) that are not present in ba.2, with G446S shared with ba.1. The most prevalent omacron strains ba.4.6 and bf.7 have the same S glycoprotein sequence that shows a high similarity to their corresponding parent lineages ba.4 and ba.5, which also share their S glycoprotein sequences. A single R346T change within RBD distinguishes BA.4.6/BF.7 from BA.4/BA.5 and eliminates therapeutics Neutralization by monoclonal antibody (mAb) of cygavitab Wei Shankang (reference 1). Combination of Sigavirtuzumab and tixagevimab (Evusheld) ^TM ) There was a high clinical relevance to pre-exposure covd-19 prophylaxis in immunocompromised patients, and the combination was expected to be ineffective against Omicron ba.4.6 and bf.7 given the lack of neutralizing activity against Omicron ba.4/ba.5 and its progeny for tibevacizumab Sha Gewei (references 1 and 2).

Primary SARS-CoV-2 immunity in many parts of the world is based on infection with the original wild-type virus or on a wild-type strain of vaccine such as mRNA vaccine BNT162b2 and mRNA-1273. The immune response is typically further developed by breakthrough of an Omicron subline virus or recently authorized Omicron adaptive vaccine booster that infects antigenically distinct. An important question is whether breakthrough infections and Omicron-adaptive vaccine boosters will elicit a massive neutralizing antibody response against the recently occurring and currently powerful Omicron subline.

This example investigated the neutralizing activity against Omicron ba.4.6/bf.7 and ba.2.75 of immune serum isolated from five groups of individuals who received three or four doses of mRNA covd-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen), with or without subsequent Omicron subline variant breakthrough infections. Individuals with triple vaccinated BNT162b2 (BNT 162b 2) ³ The method comprises the steps of carrying out a first treatment on the surface of the n=18), elderly individuals with non-infected SARS-CoV-2 vaccinated with a quadruple of BNT162b2 (BNT 162b 2) ⁴ The method comprises the steps of carrying out a first treatment on the surface of the n=15) and triple mRNA vaccination and underwent breakthrough infections Omicron ba.1 (mRNA-Vax) ³ +BA.1；n＝14)、BA.2(mRNA-Vax ³ +BA.2, n=19) or BA.4/BA.5 (mRNA-Vax) ³ +ba.4/ba.5, n=17) (fig. 61). Given that local health authorities recommend vaccination of elderly individuals with a fourth dose of vaccine and that omacron breakthrough infections are highly frequent compared to previous variants, the study design includes groups considered to represent a large part of the european and north american population. In a well characterized pseudovirus neutralization assay (pVNT) (reference 3-5) by using S-glycoprotein having SARS-CoV-2 wild-type strain or Omicron BA.4/BA.5 (the S glycoprotein sequences of BA.4 and BA.5 are identical), BA.4.6/BF.7 (BA.4.6 and BF.7)White sequence identical) or the S glycoprotein of ba.2.75 determines 50% pseudovirus neutralization (pVN) ₅₀ ) Serum neutralization activity was tested by Geometric Mean Titer (GMT).

At BNT162b2 ³ 、BNT162b2 ⁴ And mRNA-Vax ³ pVN to Omicron BA.4/BA.5 in the +BA.1 group ₅₀ The titre was significantly lower than for the wild-type strain (GMT reduced 5-to 6-fold) (fig. 59 (a)). In mRNA-Vax ³ +BA.2 and mRNA-Vax ³ The reduction in ba.4/ba.5 neutralization in the +ba.4/ba.5 group was also statistically significant but less pronounced than in the other groups (2-to 3-fold lower GMT compared to wild type). In mRNA-Vax ³ In +BA.2, pVN against Omicron BA.4.6/BF.7 compared to BA.4/BA.5 ₅₀ The titre was further significantly reduced (GMT 239 vs 386; P < 0.05). Also in BNT162b2 ⁴ The reduction in neutralization of BA.4.6/BF.7 relative to BA.4/BA.5 was observed to be quite substantial but not statistically significant (GMT 55 vs 121). In all other groups, omicron ba.4.6/bf.7gmt was largely comparable to GMT for Omicron ba.4/ba.5 and mRNA-Vax ³ Titers in +BA.4/BA.5 were the most potent (GMT 443). In mRNA-Vax ³ In +BA.4/BA.5, the titer against BA.2.75 was reduced 1.8-fold compared to BA.4/BA.5 (GMT 295 vs 521), whereas in mRNA-Vax ³ In +ba.1, the neutralized GMT for ba.2.75 was even increased by a factor of 2 compared to the neutralized GMT for ba.4/ba.5 (GMT 525 vs 263). Titers for ba.2.75 were comparable to titers for ba.4/5 in the other groups.

To allow assessment of neutralization breadth regardless of the size of antibody titers, omacron subline pVN ₅₀ GMT was normalized to wild-type strain. At BNT162b2 ³ And BNT162b2 ⁴ The GMT ratios of all omacron subvariant pseudoviruses in the group were similar (ratio of all pseudoviruses ∈0.22, fig. 59 (B)), indicating that the fourth dose of BNT162B2 did not improve cross-neutralization of the tested subvariants, although the antibody titres were slightly increased overall. And BNT162b2 ³ In contrast, in the case of mRNA-Vax ³ Cross-neutralization of BA.4/BA.5 and BA.4.6/BF.7 in serum of +BA.2 was significantly stronger (p < 0.05) (for BA.4/BA.5, GMT)Ratio 0.37 vs 0.17; and 0.23 vs 0.12 for ba.4.6/bf.7). In mRNA-Vax ³ For BA.4/BA.5 pseudoviruses in the +BA.4/BA.5 group (GMT ratio 0.48, p < 0.01, comparison BNT162b 2) ³ ) And BA.4.6/BF.7 pseudoviruses (0.41, p < 0.0001) the GMT ratio was even higher. Cross-neutralization of Omicron BA.2.75 pseudoviruses was approximately comparable in most groups, only in mRNA-Vax ³ +BA.1 with BNT162b2 ³ A significant (p < 0.05) increase was observed.

Neutralization activity against omacron ba.4.6/bf.7 was found to be further reduced compared to ba.4/ba.5 in sera of vaccinated individuals and ba.2 recovery phase, indicating that: the R346T change mediates further escape of neutralizing antibodies in polyclonal serum. The convergent evolution of RBD at this site in Omicron ba.4.6, bf.7 and other strains that are currently less prevalent (reference 1) indicates that the resulting immune escape may confer considerable growth advantages. The findings summarized here indicate that omacron ba.4/ba.5 breaks through the infection refocusing neutralizing antibody reaction to partially restore ba.4.6/bf.7 neutralization. As demonstrated in example 18, a similar pattern of cross-neutralization of the Omicron lineage was also observed in ba.4/ba.5 breakthrough infected humans and mice boosted with Omicron ba.4/5 adaptive vaccine. Thus, such findings provide evidence that omacron ba.4/5 adaptive vaccine boosters can also elicit a relevant neutralizing antibody response against ba.4.6/bf.7 in humans.

The observations that the cross-neutralization of Omicron ba.2.75 is approximately equivalent to the cross-neutralization of ba.4/5 are consistent with previous reports (references 2, 6 and 7). Without wishing to be bound by a particular theory, this observation suggests that the growth advantage of ba.2.75 over ba.5 may be related to factors other than immune evasion. For example, in some embodiments, minor differences in susceptibility of omacron ba.2.75 and ba.4/ba.5 to ba.1 and ba.4/ba.5 convalescence serum neutralization may be directed to amino acid changes that have a potential environmental dependent effect in immune evasion.

The results of the study summarized in this example focused on neutralization of the new omacron sub-lines ba.4.6/bf.7 and ba.2.75, as these sub-lines may replace ba.5 in the future according to their current trajectories. The findings associated with Omicron breakthrough infection with enhanced cross-neutralization of the new sublines are consistent with the data provided in the previous examples, which show enhanced neutralization breadth, including against early Omicron sublines and previous SARS-CoV-2 VOCs. Together, these findings show that omacron ba.4/ba.5 breakthrough infection is associated with the most extensive neutralization for all variants, including omacron sublines, providing evidence that vaccination with RNA encoding SARS-CoV-2S protein containing mutations unique to ba.4/5 variants can produce a broad neutralization response.

Materials and methods

Study design, participant recruitment and sample collection

The aim of this study was to investigate the cross-neutralization activity of five different sets of sera against Omicron sub-lines ba.4.6/bf.7 and ba.2.75 compared to SARS-CoV-2 wild type and Omicron ba.4/ba.5. Neutralization activity was assessed in immune sera from: (i) Triple BNT162b2 vaccinated young adult individuals not infected with SARS-CoV-2 (BNT 162b 2) ³ ) The method comprises the steps of carrying out a first treatment on the surface of the (ii) Quadruple BNT162b2 vaccinated elderly individuals not infected with SARS-CoV-2 (BNT 162b 2) ⁴ ) And triple mRNA (BNT 162b 2/mRNA-1273) vaccinated individuals, wherein the confirmed subsequent SARS-CoV-2 breakthrough infection occurred (iii) within a period of time that the omacron ba.1 lineage predominated (2021, 11 months to 2022, 1 middle ten days; mRNA-Vax ³ +ba.1), (iv) over a period of time in which the omacron ba.2 lineage predominates (3 months to 5 months of 2022; mRNA-Vax ³ +ba.2) or (v) germany (2022, late 6 to late 7 in 6; mRNA-Vax ³ +ba.4/5). Serum neutralization capacity was characterized using a pseudovirus neutralization assay. Group BNT162b2 ³ 、mRNA-Vax ³ +BA.1、mRNA-Vax ³ +BA.2、mRNA-Vax ³ SARS-CoV-2 wild-type +BA.4/5 and Omicron BA.4/BA.5 neutralization data have been previously disclosed.

From mRNA-Vax ³ +Omi BA.1、mRNA-Vax ³ + Omi BA.2 and mRNA-Vax ³ Participants in the +BA.4/5 cohort were recruited from university of Frankfurd university (Goethe University Frankfurt)As a non-intervention study (protocol approved by the university medical institute of ethics Committee [ number: 2021-560) that has undergone Omicron breakthrough infection after being vaccinated with a COVID-19 vaccine]) A portion of the exploratory patient. From BNT162b2 ³ And BNT162b2 ⁴ Individuals in the cohort provided informed consent as part of their participation in phase 2 trial BNT162-17 (NCT 05004181) and BNT162-16 sub-study F (NCT 04955626), respectively. All participants had no recorded history of SARS-CoV-2 infection prior to vaccination. Participants had no symptoms at the time of blood collection.

The recombinant replication defective Vesicular Stomatitis Virus (VSV) vector encoding Green Fluorescent Protein (GFP) and luciferase but not VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-1S glycoprotein (UniProt reference: P59594) or with SARS-CoV-2S glycoprotein derived from the following according to the published pseudotyping protocol (Berger Rentsch, marianne, and Gert Zimmer. "A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-specific type I interson." PloS one 6.10 (2011): e 25858): wild-type strain (MN-Hu-1, ncbi reference: omicron ba.4/ba.5 variant (change: T19I, Δ24-26, a27S, Δ69/70, G142 213 339 371 373 375 376 405 417 440 477 478 484 486 498 501 505 614 679 681 764 796 954 969K), omacron ba.4.6/bf.7 variants (changes: T19I, delta24-26, A27S, delta69/70, G142 213 339 346 373 375 376 405 408 417 440 477 478 484 486 498 501 505 614 679 681 764 796 954 969K) and Omicron BA.2.75 variants (changes: T19I, delta24-26, A27 142 147 152 157 210 257 339 373 375 376 408 417 440 446 477 478 498 501 505 614 67 484 501 614 67 655 689 684 794 969K). A graphical representation of the SARS-CoV-2S glycoprotein alteration is shown in FIG. 62, and a separate alignment of the S glycoprotein alterations in the Omicron VOC is shown in FIG. 60.

Briefly, a kit supplemented with 10% heat-inactivated fetal bovine serum (FBS [ Sigma-Aldrich]) Du's modified Issat (DMEM) plus Glutamax ^TM HEK293T/17 monolayer [. Sup.CRL-11268 ^TM ) Variant-specific SARS-CoV-2S expression plasmid plus Lipofectamine LTX (Life Technologies) transfection was verified by Sanger sequencing according to the manufacturer' S instructions. 24 hours after transfection, cells were infected with VSV-G-supplemented VSV.DELTA.G vector at a multiplicity of infection (MOI) of 3. At 37℃and 7.5% CO ₂ After incubation for 2 hours, the cells were washed twice with Phosphate Buffered Saline (PBS) and then medium supplemented with anti-VSV-G antibodies (clone 8G5F11,Kerafast Inc) was added to neutralize residual VSV-G supplemented input virus. The medium containing the VSV-SARS-CoV-2-S pseudotype was harvested 20 hours after inoculation, passed through a 0.2 μm filter (Nalgene) and stored at-80 ℃. Vero 76 cells in culture medium were used (-)>CRL-1587 ^TM ) To titrate each batch of pseudoviruses. The relative luciferase units induced by a defined volume of SARS-CoV-2 wild-type strain S glycoprotein pseudovirus reference batch corresponding to an infectious titer of 200 Transduction Units (TU) per milliliter was used as a control, which was previously described in Muik et al, 2021 (Muik, alexander, et al, "Neutralization of SARS-CoV-2lineage B.1.1.7pseudovirus by BNT 162b2vaccine-eisite human serum." Science 371.6534 (2021): 1152-1153). The input volumes of each batch of SARS-CoV-2 variant pseudoviruses were calculated to normalize the infection titer based on relative luciferase units relative to the reference.

Pseudovirus neutralization assay

Vero was run 4 hours prior to assay76 cells were seeded at 40,000 cells/well in 96 well white flat bottom plate (Thermo Scientific) medium at 37℃and 7.5% CO ₂ Culturing was performed under the following conditions. Human serum samples were serially diluted 2-fold in culture medium in the range of 1:10 to 1: 10,240. VSV-SARS-CoV-2-S particles were diluted in medium to obtain 200 TUs in the assay. Serum dilutions were mixed with pseudoviruses (n=2 technical replicates per pseudovirus per serum) 1:1 for 30 min at room temperature, then added to Vero 76 cell monolayers and incubated at 37 ℃ and 7.5% co ₂ Incubate for 24 hours. The supernatant was removed and the cells were lysed with luciferase reagent (Promega). At the position ofLuminescence was recorded on a 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. The results of all pseudo-virus neutralization experiments are expressed in terms of Geometric Mean Titer (GMT) of the replicates. If no neutralization is observed, the limit of detection [ LOD ] is reported]Half of any titer value.

Statistical analysis

The statistical polymerization method used to analyze antibody titers was a geometric mean and for the ratio of SARS-CoV-2VOC titers to wild-type strain titers was a geometric mean and a corresponding 95% confidence interval. The use of geometric averages accounts for the non-normal distribution of antibody titers across several orders of magnitude. Paired symbol rank test was performed on group geometric mean neutralizing antibody titers under a common control group using friedemann assay with dunn correction for multiple comparisons. Unpaired symbol rank test (unpaired signed-rank test) was performed on the group GMT ratios using Kruskal-wales test (Kruskal-walis test) which makes dane corrections for multiple comparisons. All statistical analyses were performed using GraphPad Prism software version 9.

Reference cited in example 19

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Example 20: it was confirmed that booster doses including Omicron ba.4/5-specific vaccine could lead to clinical trial results of a strong immune response against Omicron ba.4/5 variant

The present example provides clinical trial data to confirm that: ba.4/5-specific vaccines (e.g., a bivalent RNA vaccine as described herein comprising a first RNA encoding the SARS-CoV-2S protein of the MN908947 strain and a second RNA encoding the SARS-CoV-2S protein comprising one or more mutations unique to ba.4/5 omacron variants) can result in a strong immune response in a subject. Specifically, the present embodiment provides data to demonstrate that: such an RNA vaccine may result in an immune response (e.g., neutralizing antibody titer) against the ba.4/5 variant that is higher than that caused by an RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 strain. These results confirm the insight provided by the ba.4/5-breakthrough infection data and the mouse experimental data described in the previous examples regarding the benefits of administering ba.4/5-specific vaccines (e.g., the vaccines described herein).

The immune response data provided by this example is from a phase 2/3 clinical trial in which 30- μg of a booster dose of Omicron ba.4/ba.5-adaptive bivalent covd-19 vaccine (comprising 15 μg of RNA encoding the full length SARS-CoV-2S protein of MN908947 variant), e.g., in some embodiments RNA comprising a nucleotide sequence at least 95% or more (including up to 100%) identical to SEQ ID No. 20 (e.g., in some embodiments BNT162b 2), and 15 μg of RNA encoding the full length SARS-CoV-2S protein comprising one or more mutations unique to Omicron ba.4/ba.5) are administered to 18-55 year old individuals (n=38) and those over 55 years old (n=36) who have previously received three doses of an RNA vaccine encoding the SARS-CoV-2S protein of MN908947 strain, e.g., in some embodiments, with a nucleotide sequence at least 95% or more (including up to 100%) identical to SEQ ID No. 20 (e.g., in some embodiments, BNT162b 2). A 30- μg booster dose (as a fourth dose) of an RNA vaccine encoding SARS-CoV-2S protein of the MN908947 strain was administered to a group of participants (n=40) over 55 years old as a comparator. In this example, a subject that has been administered a bivalent vaccine as described herein receives on average 11 months before administration of the bivalent vaccine its last dose of the vaccine encoding the SARS-CoV-2S protein of the MN908947 strain, and a subject that has been administered an RNA vaccine encoding the SARS-CoV-2 protein of the MN908947 strain, such as BNT162b2 (as the fourth dose), receives on average its third dose 6 months before administration of the bivalent vaccine. Despite this difference, the pre-boost antibody titers in the two groups were similar. Both groups included subjects who had previously had evidence of SARS-CoV-2 infection and subjects who had previously had no evidence of SARS-CoV-2 infection.

A significantly higher increase in Omicron ba.4/ba.5-neutralizing antibody titers was observed between subjects administered Omicron ba.4/ba.5-specific bivalent vaccine compared to pre-booster levels. For individuals 18 to 55 years of age administered bivalent vaccine, the Geometric Mean Titer (GMT) against omacron ba.4/ba.5 was about 600 (e.g., 606), which is 9.5 times higher than the pre-booster level (95% ci:6.7, 13.6). For individuals over 55 years of age administered a ba.4/5 bivalent vaccine, GMT was about 900 (e.g., 896), which was 13.2 times higher than pre-booster levels (95% ci:8.0, 21.6). In contrast, participants over 55 years old who received 30- μg booster doses of the RNA vaccine encoding SARS-CoV-2S protein of the MN908947 strain observed a lower neutralizing antibody response to omacron ba.4/ba.5 measured 1 month after the booster. For these participants, GMT was about 230 (e.g., 236), indicating 2.9 times higher than the pre-booster level (95% ci:2.1, 3.9). The safety profile observed for bivalent vaccines in clinical trials is favourable and consistent with BNT162b 2.

In addition, when the population of subjects with and without prior signs of SARS-CoV-2 infection was examined, an increase in neutralizing antibodies to Omicron BA.4/BA.5 was observed in both populations following booster doses of bivalent vaccine, demonstrating that: the ba.4/5-bivalent vaccine improves protection in subjects receiving such vaccine, regardless of the previous infection status.

Example 21: neutralization of Omacron BA.4/5, BA.4.6, BA.2.75.2, BQ.1.1 and XBB.1 with bivalent BA.4/5 vaccine

This example provides further data to demonstrate that: a bivalent RNA vaccine (e.g., a bivalent vaccine as described herein) comprising RNA encoding the SARS-CoV-2S protein of the MN908947 strain and RNA encoding the SARS-CoV-2S protein comprising one or more mutations unique to the ba.4/5 Omicron variant may provide an improved immune response compared to a monovalent RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 strain. For example, in some embodiments, such improved immune responses include increased neutralization titers against one or more, including two or more, three or more, four or more, five or more, six or more, SARS-CoV-2 variants of interest. In some embodiments, such improved immune responses include increased neutralization titers against one or more, including two or more, three or more, four or more, five or more, six or more Omicron-focused variants. In some embodiments, the improved immune response comprises: neutralization titers were increased for a number of omacron-focused variants compared to that observed for the monovalent RNA vaccine encoding SARS-CoV-2S protein of the MN908947 strain.

In particular, the present example provides data to demonstrate that: such bivalent vaccines can elicit an improved immune response (e.g., in some embodiments a higher neutralization response) against the sublines derived from ba.5 (e.g., ba.4.6, bq.1.1, and xbb.1) and the sublines derived from ba.2 (e.g., ba.2.75.2) when administered at the fourth dose of booster as compared to the monovalent RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 strain. The present example also provides data to demonstrate that: a subject having a history of infection with SARS-COV-2 (e.g., a subject having a prior or current infection with SARS-COV-2) may develop a higher immune response (e.g., a higher neutralization titer) after administration of a fourth booster dose (e.g., a monovalent or bivalent vaccine) relative to a subject that had not been infected with SARS-COV-2. This example demonstrates that: regardless of the history of SARS-CoV-2 infection, the BA.4/5 bivalent vaccine can elicit an improved immune response (e.g., an improved neutralization response) as compared to a monovalent RNA vaccine encoding the SARS-CoV-2S protein of the MN908947 strain. The present example also provides data to demonstrate that: the immune response provided by the ba.4/5 bivalent vaccine against certain omacron sublines (e.g., omacron sublines tested herein) was improved to a greater extent in subjects not previously infected with SARS-CoV-2 than in subjects previously infected with SARS-CoV-2 relative to the MN908947 monovalent vaccine.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants have evolved worldwide into a number of sub-lines since the advent of month 11 in 2021. To alleviate ongoing omicon pandemic, the U.S. FDA mandates emergency use of bivalent ba.4/5-vaccine at 9 months 2022. The us FDA-approved bivalent vaccine contains two mrnas: a SARS-CoV-2S protein encoding the original (MN 908947) SARS-CoV-2 spike protein (BNT 162b 2) and another encoding comprising a mutation unique to the omacron ba.4/5 variant (in some embodiments comprising the amino acid sequence of SEQ ID NO:69 and in some embodiments comprising the amino acid sequence of SEQ ID NO: 72). Since U.S. FDA approval, new Omacron BA.2-and BA.5-derived sublines (e.g., BA.4.6, BA.2.75.2, BQ.1.1 and XBB.1) have emerged and have become popularhttps://covid.cdc.gov/covid-data-tracker/#variant- proportions). Although early epidemiological data indicate no increase in disease severity, these novel sublines have accumulated additional spike mutations that may further evade vaccine-and/or infection-induced antibody neutralization (references 2-4).

This example describes data from a clinical trial in which neutralization titers against omacron sublines ba.4/5, ba.4.6, ba.2.75.2, bq.1.1 and xbb.1 were measured in serum collected from subjects administered with a fourth dose (a) 30- μg booster dose of omacron ba.4/ba.5-adaptive bivalent covd-19 vaccine comprising in some embodiments 15 μg of a vaccine comprising a peptide sequence identical to SEQ ID NO:20 and 15 μg of RNA encoding a full length SARS-CoV-2S protein comprising one or more mutations unique to the omacron ba.4/ba.5 variant and comprising a nucleotide sequence that is at least 95% (including and up to 100%) identical to SEQ ID NO:72 RNA of a nucleotide sequence of at least 95% (including and up to 100%) identical. In this example, a test subject is administered a composition comprising (i) 15 μg of a polypeptide comprising SEQ ID NO:20 and (ii) 15 μg of RNA comprising the sequence of SEQ ID NO:72 sequence of RNA Omicron BA.4/BA.5-adaptive bivalent COVID-19 vaccine.

To a 55 year old participant who had previously received 3 doses of a vaccine that delivered SARS-CoV-2S protein of the MN908947 strain (in this example, the subject had previously received 3 30- μg doses of BNT162b 2) a fourth booster dose comprising 30- μg monovalent BNT162b2 at about 6.6 months after the third dose or a fourth booster dose comprising 30- μg bivalent vaccine (15- μg BNT162b2 plus 15- μg ba.4/5) at about 11 months after the third dose. Serum was collected on the day of administration of the fourth dose (pre-serum sample) and 1 month after administration of the fourth dose (1 MPD4 serum sample). Screening all participants for signs of previous and current SARS-CoV-2 infection using viral nucleocapsid antibodies and RT-PCR assays; the two vaccine groups in the neutralization assay were evenly distributed among people with or without signs of infection. In the neutralization assay, the complete spike gene from omacron ba.4/5 (ba.4 and ba.5 encoding the same spike sequence), ba.2.75.2, bq.1.1 or xbb.1 WAs cloned into the backbone of the fluorescent reporter USA-WA1/2020 SARS-CoV-2 (a strain isolated in month 1 2020, see reference 5). The resulting wild-type- (WT), BA.4/5-, BA.2.75.2-, BQ.1.1-and XBB.1-spike mNG USA-WA1/2020 were used to measure 50% fluorescence focal reduction neutralization titers (FFRNT 50) of each collected serum sample.

For all participants (including subjects with and without signs of SARS-CoV-2 infection), the fourth dose of monovalent BNT162b2 vaccine was found to result in 3.0 fold, 2.9 fold, 2.3 fold, 2.1 fold, 1.9 fold, and 1.5 fold geometric mean neutralization titer fold increase (GMFR) for MN908947 strain, ba.4/5, ba.4.6, ba.2.75.2, bq.1.1, and xbb.1, respectively; bivalent vaccines were found to result in 5.8-fold, 13.0-fold, 11.1-fold, 6.7-fold, 8.7-fold, and 4.8-fold GMFR (fig. 63 (a)). BNT162b2 resulted in 4.4-fold, 3.0-fold, 2.6-fold, 2.1-fold, 1.5-fold and 1.3-fold GMFR, respectively, for individuals without a history of SARS-CoV-2 infection; the bivalent vaccine resulted in 9.9-fold, 25.9-fold, 21.2-fold, 8.6-fold, 13.0-fold, and 4.6-fold GMFR (fig. 63 (B)). BNT162b2 resulted in 2.0-fold, 2.8-fold, 2.1-fold, 2.2-fold, and 1.8-fold GMFR, respectively, for individuals previously infected with SARS-CoV-2; bivalent vaccines resulted in 3.5-fold, 6.7-fold, 6.2-fold, 5.3-fold, 6.3-fold, and 4.9-fold GMFR (fig. 63C). Although the interval between the third and fourth doses was different, the neutralization titers before the fourth dose in the monovalent and bivalent vaccine groups were similar in all participants and in the infection-free group.

The present embodiment provides at least three findings. First, the bivalent ba.4/5 vaccine continuously elicited higher neutralization against the subline derived from ba.5 (ba.4.6, bq.1.1 and xbb.1) and subline derived from ba.2 (ba.2.75.2) than the monovalent RNA vaccine encoding the SARS-CoV-2S protein when administered with the fourth dose of booster. Second, after the fourth dose of booster, individuals who previously or currently have evidence of SARS-CoV-2 infection exhibit a higher neutralization titer than those without infection. An improvement in neutralization by bivalent vaccine was observed regardless of the history of SARS-CoV-2 infection. Again, for each omacron subline tested, the difference between BNT162b2-GMFR and bivalent-GMFR was greater for previously uninfected sera than for previously infected sera.

Between all omacron sub-lines, ba.2.75.2, bq.1.1 and xbb.1 exhibited the greatest escape from vaccine-primed neutralization; however, the neutralization titer after the bivalent enhancer was several times higher than that after the monovalent RNA encoding SARS-CoV-2S protein of MN908947 strain. These data demonstrate that: bivalent vaccines are more immunogenic against the circulating omacron subfsystem and underscores the importance of monitoring real world effectiveness.

Method

Cells

Vero E6 from American type culture Collection (American Type Culture Collection; ATCC, bethesda, md.)CRL-1586) and Vero E6 cells expressing TMPRSS2 from SEKISUI XenoTech, LLC at 37℃and 5% CO2Maintained in high glucose Du's modified Itemia medium (DMEM) containing 10% fetal bovine serum (FBS; hyClone Laboratories, south Logan, UT) and 1% penicillin/streptomycin. Media and antibiotics were purchased from Thermo Fisher Scientific (Waltham, MA). Cell lines were tested negative for mycoplasma.

Human serum

In the past 55 years old participants who received three 30- μg BNT162b2 doses, serum samples were taken just prior to the use of the fourth dose booster of monovalent original 30- μg BNT162b2 or 30- μg bivalent BA.4/BA.5 vaccine (15 μg primordial plus 15 μg BA.4/BA.5) and 1 month after the use of the booster. The institutional review board of each study center participating in the study approves the protocol and informed consent. The study was conducted following all the guidelines of the international coordination committee for good clinical practice (International Council for Harmonisation Good Clinical Practice) and the ethical principles of the declaration of helsinki (Declaration of Helsinki). For BNT162b2 and bivalent BA.4/5 vaccine, the median time between the third and fourth dose was 6.3 months and 11.3 months, respectively. A subset of the serum of the participants selected for the neutralization test (approximately 40 per vaccine group) was evenly distributed among the individuals informed by either test with and without signs of infection. Human serum was heat-inactivated at 56 ℃ for 30 minutes after the neutralization test.

Recombinant Omicron subline-FP SARS CoV-2 virus

Recombinant omacron sublines ba.4/5-, ba.4.6-, ba.2.75.2-, bq.1.1-and xbb.1-spike Fluorescent Protein (FP) SARS-CoV-2s were constructed by engineering the complete spike gene from the indicated variants into infectious cDNA clones of mNG USA-WA1/2020 and have been previously reported (references 6-10). Viruses were rescued after 2-3 days post electroporation and served as P0 stocks. The P0 stock was further passaged once on Vero E6 cells to produce P1 stock. Spike genes from all P1 stock viruses were sequenced to ensure that no unwanted mutations were present. The infectious titer of P1 virus was quantified against Vero E6 cells using a fluorescent focus assay. The P1 virus was used in the neutralization assay.

Fluorescence focus reduction neutralization assay (FFRNT)

Neutralization titers of human serum were measured by FFRNT using USA-WA1/2020-, BA.4/5, BA.4.6-, BA.2.75.2-, BQ.1.1-, and XBB.1-spike FP SARS-CoV-2 s. All sera were tested sequentially, first USA-WA1/2020 and BA.4/5, followed by the remaining Omacron sub-lines. Details of FFRNT protocols have been previously reported (see references 6 and 10-13). Briefly, vero E6 cells were seeded in 96-well plates (Greiner Bio-one) at 2.5X104 cells per well ^TM ) Is a kind of medium. Cells were incubated overnight. On the next day, each serum was serially diluted 2-fold in medium at a first dilution of 1:20 (final dilution range 1:20 to 1:20, 480). The diluted serum was incubated with 100-150 FFU of FP SARS-CoV-2 for 1 hour at 37℃after which the serum virus mixture was loaded onto a pre-inoculated monolayer of Vero E6 cells in a 96-well plate. After 1 hour of infection, the inoculum was removed and 100 μl of cover medium (supplemented with 0.8% methylcellulose) was added to each well. After incubating the plates at 37 ℃ for 16 hours, raw images of FP foci were acquired using cyatiiontm 7 (BioTek) equipped with a wide field of view 2.5×fl Zeiss (Zeiss) objective and set up using gene 5 software (GFP [469, 525]Threshold 4000, object selection size 50-1000 μm). Foci in each well were counted and normalized to the non-serum treated control group to calculate relative infectivity. FFRNT50 values were defined as the minimum serum dilution that inhibited > 50% fluorescence focus. The neutralization titers of each serum were determined in duplicate assays and geometric mean was taken. All attempts at replication were successful. Data was initially drawn in GraphPad Prism 9 software and assembled in Adobe Illustrator.

The references cited in example 21 (each incorporated by reference in its entirety herein)

1.Cele S，Jackson L，Khoury DS，et al.Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization.Nature 2022；602(7898)：654-6.

2.Kurhade C，Zou J，Xia H，et al.Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by 3 doses of BNT162b2 vaccine or BA.1 infection.Emerg Microbes Infect 2022：1-18.

3.Liu Z，VanBlargan LA，Bloyet LM，et al.Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization.Cell Host Microbe 2021；29(3)：477-88 e4.

4.Davis-Gardner ME，Lai L，Wali B，et al.mRNA bivalent booster 1 enhances neutralization against BA.2.75.2 and BQ.1.1.bioRxiv 2022：https://doi.org/10.1101/2022.10.31.514636.

5.Muruato AE，Fontes-Garfias CR，Ren P，et al.A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation.Nat Commun 2020；11(1)：4059.

6.Kurhade C，Zou J，Xia H，et al.Low neutralization of SARS-CoV-2 Omicron BA.2.75.2，BQ.1.1，and XBB.1 by 4 doses of parental mRNA vaccine or a BA.5-bivalent booster.bioRxiv 2022：2022.10.31.514580.

7.Xie X，Muruato A，Lokugamage KG，et al.An Infectious cDNA Clone of SARS-CoV-2.Cell host&microbe 2020：S1931-3128(20)30231-6.

8.Xie X，Lokugamage KG，Zhang X，et al.Engineering SARS-CoV-2 using a reverse genetic system.Nature Protocols 2021；16(3)：1761-84.

9.Muruato AE，Fontes-Garfias CR，Ren P，et al.A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation.Nat Commun 2020；11(1)：4059.

10.Xie X，Zou J，Liu M，Ren P，Shi PY.Neutralization of SARS-CoV-2 Omicron sublineages by 4 doses of mRNA vaccine.bioRxiv 2022.

11.Zou J，Xie X，Liu M，Shi PY，Ren P.Neutralization Titers in Vaccinated Patients with SARS-CoV-2 Delta Breakthrough Infections.mBio 2022；13(4)：e0199622.

12.Zou J，Xia H，Xie X，et al.Neutralization against Omicron SARS-CoV-2 from previous non-Omicron infection.Nat Commun 2022；13(1)：852.

13.Kurhade C，Zou J，Xia H，et al.Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by 3 doses of BNT162b2 vaccine or BA.1 infection.Emerg Microbes Infect 2022：1-18.

Example 22: vaccine-induced and convalescent immune serum has unique cross-neutralization of omacron sub-lineages.

Blood samples were drawn from the following individuals: individuals vaccinated with (i) triple BNT162b2 or (ii) quadruple BNT162b2 with non-infected SARS-CoV-2, and individuals with breakthrough infections with three doses of mRNA COVID-19 vaccine (BNT 162b2/mRNA-1273 homologous or heterologous regimen) with (iii) Omicron BA.1, (iv) Omicron BA.2 or (v) Omicron BA.4/BA.5. Breakthrough infections occurred at the time when the respective variant of interest was dominant (ba.1:2021, 11-2021, 1-2021, ba.2:2022, 3-5, ba.4/5:2022, 6-middle 7-middle) and/or were confirmed as variants by genomic sequencing. Neutralization assay using pseudovirus neutralization titers were measured using pseudoviruses with ba.4/5, ba.4.6/bf.7, bq.1.1, ba.2.75, ba.2.75.2 or XBB Omicron variants of SARS-CoV-2S protein on the surface (e.g., using the methods described in the previous examples; fig. 65). 50% pseudovirus neutralization titer (pVNT) ₅₀ ) As shown in fig. 64 (a). pVNT by targeting Omacron sub-lineage pVNT50 to wild type strains ₅₀ The neutralization width was evaluated by normalization regardless of the size of the antibody titer (fig. 64 (B)).

pVN against omacron ba.4/5 in triple/quadruple vaccinated individuals without breakthrough infection ₅₀ GMT was 5 to 6 times lower than GMT for wild type strain (GMT for ba.4/5 was in the range of 69-121) (fig. 64 (a)). GMT for ba.4/5 was similarly reduced in ba.1 recovery (GMT 263, 5-fold lower than wild type), while titers for ba.4/5 were still higher in ba.2 and ba.4/ba.5 recovery queues (GMT 386 and 521, respectively; 3-fold and 2-fold lower than wild type).

In all three recovery queues, the neutralization titers against omacron ba.4.6/bf.7 and ba.2.75 were significantly higher than those vaccinated with triple/quadruple vaccine for uninfected SARS-CoV-2 (recovery GMT ranged from 239 to 525, and uninfected 55 to 139). In the recovery phase, the GMT for Omicron BA.4.6/BF.7 and BA.2.75 was largely unchanged from the GMT for Omicron BA.4/BA.5. In contrast, the pVN titers for Omicron bq.1.1, ba.2.75.2 and XBB were lower in the queue than for ba.4/5. Titers for BQ.1.1 were generally lower in vaccinated and BA.1 recovery (GMT.ltoreq.38) cohorts that were not infected with SARS-CoV-2, and higher in BA.2 and BA.4/BA.5 recovery cohorts (GMT 100 and 154, respectively). Titers for BA.2.75.2 and XBB were relatively low in each queue (GMT. Ltoreq.88 and. Ltoreq.33, respectively).

To evaluate the extent of neutralization regardless of the size of the antibody titer, omacron sub-lineage pVN was used ₅₀ pVN of GMT against wild type strains ₅₀ GMT is normalized. GMT ratio of all Omicron subvariant pseudoviruses at BNT162b2 ³ And BNT162b2 ⁴ The queue is comparable (fig. 64 (B)) indicating that the fourth dose did not improve cross-neutralization of the tested sub-lineages. In both queues, the GMT ratios of BA.4/5, BA.4.6/BF.7 and BA.2.75 were in the range of 0.09-0.22, while the GMT ratios of BQ.1.1, BA.2.75.2 and XBB were in the range of 0.05.

Cross neutralization of BA.4/BA.5 and BA.4.6/BF.7 was significantly (p < 0.05) higher in serum from individuals in the BA.2. Recovery phase than in individuals vaccinated with triple vaccination (GMT ratio of 0.37 and 0.1 for BA.4/BA.5 and 0.23 and 0.12 for BA.4.6/BF.7) and pseudovirus (GMT ratio of 0.48 and BNT162b 2) for BA.4/BA.5 in the BA.4/BA.5 recovery phase ³ This is especially true for p < 0.01) and BA.4.6/BF.7 pseudoviruses (GMT ratio of 0.41, p < 0.0001). Cross-neutralization of BA.4.6/BF.7 was also significantly stronger in individuals at the recovery phase of BA.4/BA.5 (p < 0.0001) compared to individuals vaccinated with the quadruple vaccine. Although the cross-neutralization efficiency of BQ.1.1 was lower than BA.4/5 (GMT ratio. Ltoreq.0.14) in all cohorts, cross-neutralization in individuals at recovery of BA.4/BA.5 and BA.2 was triple or quadruple vaccinated with uninfected SARS-CoV and a breakthrough of BA.1 occurred The infected queue is still significantly stronger. Cross-neutralization of ba.2.75, ba.2.75.2 and XBB pseudoviruses has a broad comparability among different queues. Together, these data demonstrate that partial neutralization of certain omacron sub-lineages is retained, especially in ba.4/ba.5 convalescence individuals, and that neutralizing antibody responses observed in vaccinated and breakthrough infected individuals appear to be less effective on sub-lineages ba.2.75.2 and XBB. These data also demonstrate that in some embodiments, vaccines comprising RNA encoding SARS-CoV-2S protein that is characteristic of XBB variants, bq.1.1 variants, or any lineages derived therefrom, can provide improved immune responses (e.g., increased neutralization titers).

Example 23: t cell epitopes and neutralizing B cell epitopes are conserved among variants of SARS-CoV-2 interest.

To estimate the non-synonymous mutation rate of T cell epitopes in S glycoprotein, epitopes of T cell reactivity confirmed in experimental assays were obtained using the immune epitope database (https:// www.iedb.org /). The database was filtered using the following criteria: organisms: SARS-COV2; antigen: a spike glycoprotein; positive determination; b cell free assay; MHC-free assay; MHC restriction type: class I; and (3) a host: homo sapiens (human). The results table was filtered by deleting the epitope "deduced from the pool of reactive overlapping peptides" and epitopes longer than 14 amino acids in length in order to limit the dataset to only the minimum identified epitopes. The experimental assays to confirm the reactivity of these epitopes are dependent on multimeric assays, ELISpot or ELISpot-like assays, T cell activation assays, etc. Epitopes of at least 27 different HLA-I alleles are reported, including HLA-A, HLA-B, and HLA-C alleles. Of the 251 unique epitope sequences obtained in this way, 244 were found in the MN908947 strain spike glycoprotein. Wherein 36 epitopes (14.8%) include the positions reported as mutated by the sequence analysis described herein.

Furthermore, conservation of neutralizing B cell epitopes was calculated by counting the number of antibody neutralizing epitopes that may be affected by the mutation. It was calculated by first mapping 719 binding epitopes observed in 332 experimentally resolved nAb structures onto the S protein using the available protein structure (as described in https:// www.biorxiv.org/content/10.1101/2021.12.24.474095v2). For each structure, the epitope of each antibody is calculated as the set of positions in contact with the antibody, two residues are considered to be in contact if the minimum Euclidean distance between the atoms of the two residues is less than 4 angstroms. Each nAb will be evaluated and if any position of the epitope of a certain nAb is mutated, it is considered that the nAb will be escaped by the variant.

Given that humoral and cell-mediated immunity together determine susceptibility to severe covd-19 disease, the degree of conservation between neutralizing B-and T-cell epitopes of S glycoprotein in omacron subfreeds was assessed. The findings presented herein indicate that more than 80% of the S glycoprotein T cell epitopes encoded by BNT162b2 are fully conserved in Omicron lineages including ba.2.75.2, bq.1.1 and XBB (fig. 66). In sharp contrast, B cell epitopes are partially conserved (. Gtoreq.50%) among the early variants Alpha, beta and Delta, but most of these epitopes are altered in the Omacron lineage (. Ltoreq.20%) especially in BA.2.752 and XBB (. Ltoreq.10%). These findings indicate that in the vaccinated population, strong T cell mediated immunity can be maintained against omacron variants including sub-lineages that evade neutralizing antibodies.

Certain sequences

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Claims

1. A composition or pharmaceutical formulation comprising RNA comprising a nucleotide sequence encoding a SARS-CoV-2S protein or an immunogenic fragment thereof comprising one or more mutations characteristic of omacron variants.

2. The composition or pharmaceutical formulation of claim 1, wherein the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

3. The composition or pharmaceutical formulation of claim 1 or 2, wherein the SARS-CoV-2S protein or immunogenic fragment thereof comprises one or more mutations characteristic of ba.4/5, ba.1, ba.2, XBB, xbb.1 or bq.1.1omicron variant or a subline thereof.

4. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA encodes a polypeptide of SEQ ID No. 69 and comprises the nucleotide sequence of SEQ ID No. 70 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 97%, at least 98%, or 99% or more) identical to SEQ ID No. 70, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

5. The immunogenic composition of claim 4, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

6. The immunogenic composition of claim 4 or 5, wherein the modified uridine is each N1-methyl-pseudouridine.

7. The immunogenic composition of any one of claims 4-6, 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.

8. The immunogenic composition of claim 7, 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.

9. The immunogenic composition of claim 7, wherein the poly a sequence comprises SEQ ID No. 14.

10. The immunogenic composition of any one of claims 4 to 10, wherein the RNA comprises SEQ ID No. 72.

11. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA encodes a 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 least 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 more) identical to SEQ ID No. 50, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

12. The immunogenic composition of claim 11, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

13. The immunogenic composition of claim 11 or 12, wherein the modified uridine is each N1-methyl-pseudouridine.

14. The immunogenic composition of any one of claims 11-13, 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.

15. The immunogenic composition of claim 14, 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.

16. The immunogenic composition of claim 14, wherein the poly a sequence comprises SEQ ID No. 14.

17. The immunogenic composition of any one of claims 13 to 14, wherein the RNA comprises SEQ ID No. 51.

18. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA encodes a 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 least 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 more) identical to SEQ ID NO:56, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

19. The immunogenic composition of claim 18, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

20. The immunogenic composition of claim 18 or 19, wherein the modified uridine is each N1-methyl-pseudouridine.

21. The immunogenic composition of any one of claims 18-20, 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.

22. The immunogenic composition of claim 21, 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.

23. The immunogenic composition of claim 22, wherein the poly a sequence comprises SEQ ID No. 14.

24. The immunogenic composition of any one of claims 18-23, wherein the RNA comprises SEQ ID NO 57.

25. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA encodes a 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 least 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 more) identical to SEQ ID NO:59, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

26. The immunogenic composition of claim 25, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

27. The immunogenic composition of claim 25 or 26, wherein the modified uridine is each N1-methyl-pseudouridine.

28. The immunogenic composition of any one of claims 25-27, 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.

29. The immunogenic composition of claim 28, 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.

30. The immunogenic composition of claim 29, wherein the poly a sequence comprises SEQ ID No. 14.

31. The immunogenic composition of any one of claims 25-30, wherein the RNA comprises SEQ ID No. 60.

32. An immunogenic composition comprising: a Lipid Nanoparticle (LNP) comprising an RNA, wherein the RNA encodes a polypeptide of SEQ ID No. 61 and comprises the nucleotide sequence of SEQ ID No. 62a 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 97%, at least 98% or 99% or more) identical to SEQ ID No. 62a, and wherein the RNA comprises:

(a) Modified uridine;

(b) A 5' cap; and is also provided with

33. The immunogenic composition of claim 32, wherein the nucleotide sequence comprises a modified uridine instead of all uridine.

34. The immunogenic composition of claim 32 or 33, wherein the modified uridine is each N1-methyl-pseudouridine.

35. The immunogenic composition of any one of claims 32-34, 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.

36. The immunogenic composition of claim 35, 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.

37. The immunogenic composition of claim 36, wherein the poly a sequence comprises SEQ ID No. 14.

38. The immunogenic composition of any one of claims 32-37, wherein the RNA comprises SEQ ID No. 63a.

39. 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 least 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 more) identical to SEQ ID NO. 9, an

The second RNA encodes the polypeptide of SEQ ID NO. 69 and comprises the nucleotide sequence of SEQ ID NO. 70 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 97%, at least 98%, or 99% or more) identical to SEQ ID NO. 70, an

Wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

wherein the first RNA and the second RNA are formulated in a Lipid Nanoparticle (LNP), wherein the LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate.

40. The immunogenic composition according to claim 39, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

41. The immunogenic composition according to claim 39, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

42. The immunogenic composition of any one of claims 39-41, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

43. The immunogenic composition of any one of claims 39-42, wherein the modified uridine are each N1-methyl-pseudouridine.

44. The immunogenic composition of any one of claims 39-43, wherein the first RNA and the second RNA each independently 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.

45. The immunogenic composition according to claim 44, 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.

46. The immunogenic composition according to claim 44, wherein the poly A sequence comprises SEQ ID NO 14.

47. An immunogenic composition comprising a first RNA and a second RNA, wherein:

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 least 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 more) identical to SEQ ID NO. 50, an

Wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

48. The immunogenic composition according to claim 47, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

49. The immunogenic composition according to claim 47, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

50. The immunogenic composition of any one of claims 47-49, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

51. The immunogenic composition of any one of claims 47-50, wherein the modified uridine is each N1-methyl-pseudouridine.

52. The immunogenic composition of any one of claims 47-51, wherein the first RNA and the second RNA each independently 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.

53. The immunogenic composition of claim 52, 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.

54. The immunogenic composition according to claim 52, wherein the poly A sequence comprises SEQ ID NO 14.

55. The immunogenic composition of any one of claims 49-54, wherein the first RNA comprises SEQ ID No. 20 and the second RNA comprises SEQ ID No. 51.

56. An immunogenic composition comprising a first RNA and a second RNA, wherein:

the first RNA comprises a nucleotide sequence encoding 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 least 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 more) identical to SEQ ID NO. 9 and

the second RNA comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO. 55, 58 or 61 and comprises the nucleotide sequence of SEQ ID NO. 56, 59 or 62a 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 97%, at least 98% or 99% or more) identical to SEQ ID NO. 56, 59 or 62a, and

Wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

57. The immunogenic composition according to claim 56, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

58. The immunogenic composition according to claim 56, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

59. The immunogenic composition of any one of claims 56-58, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

60. The immunogenic composition of any one of claims 56-59, wherein each of the modified uridine is N1-methyl-pseudouridine.

61. The immunogenic composition of any one of claims 56-60, wherein each of the first RNA and the second RNA, independently, 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.

62. The immunogenic composition of any one of claims 56-61, 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.

63. The immunogenic composition according to claim 62, wherein the poly A sequence comprises SEQ ID NO 14.

64. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 9 and the second RNA comprises SEQ ID No. 56.

65. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 9 and the second RNA comprises SEQ ID No. 59.

66. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 9 and the second RNA comprises SEQ ID No. 62a.

67. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 20 and the second RNA comprises SEQ ID No. 57.

68. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 20 and the second RNA comprises SEQ ID No. 60.

69. The immunogenic composition of any one of claims 56-63, wherein the first RNA comprises SEQ ID No. 20 and the second RNA comprises SEQ ID No. 63a.

70. 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 least 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 more) identical to SEQ ID NO. 59 and

the second RNA encodes a polypeptide of SEQ ID NO. 49, 55 or 61 and comprises the nucleotide sequence of SEQ ID NO. 50, 56 or 62a 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 97%, at least 98% or 99% or more) identical to SEQ ID NO. 50, 56 or 62a, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

71. The immunogenic composition of claim 70, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

72. The immunogenic composition of claim 70, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

73. The immunogenic composition of any one of claims 70-72, wherein each of the first RNA and the second RNA comprises a modified uridine in place of all uridine.

74. The immunogenic composition of any one of claims 70-73, wherein the modified uridine is each N1-methyl-pseudouridine.

75. The immunogenic composition of any one of claims 70-74, wherein each of the first RNA and the second RNA, independently, 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.

76. The immunogenic composition of claim 75, 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.

77. The immunogenic composition of claim 75, wherein the poly-A sequence comprises SEQ ID NO 14.

78. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 59 and the second RNA comprises SEQ ID No. 50.

79. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 59 and the second RNA comprises SEQ ID No. 56.

80. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 59 and the second RNA comprises SEQ ID No. 62a.

81. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 60 and the second RNA comprises SEQ ID No. 51.

82. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 60 and the second RNA comprises SEQ ID No. 57.

83. The immunogenic composition of any one of claims 70-77, wherein the first RNA comprises SEQ ID No. 60 and the second RNA comprises SEQ ID No. 63a.

84. 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 least 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 more) identical to SEQ ID NO. 50, an

The second RNA encodes the polypeptide of SEQ ID NO. 55 or 61 and comprises the nucleotide sequence of SEQ ID NO. 56 or 62a 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 97%, at least 98% or 99% or more) identical to SEQ ID NO. 56 or 62a, and

wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

85. The immunogenic composition of claim 84, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

86. The immunogenic composition of claim 84, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

87. The immunogenic composition of any one of claims 84-86, wherein each of the first RNA and the second RNA comprises a modified uridine instead of all uridine.

88. The immunogenic composition of any one of claims 84-87, wherein the modified uridine are each N1-methyl-pseudouridine.

89. The immunogenic composition of any one of claims 84-88, 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.

90. The immunogenic composition of claim 89, 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.

91. The immunogenic composition of claim 89, wherein the poly-a sequence comprises SEQ ID No. 14.

92. The immunogenic composition of any one of claims 84-91, wherein the first RNA comprises SEQ ID No. 50 and the second RNA comprises SEQ ID No. 56.

93. The immunogenic composition of any one of claims 84-91, wherein the first RNA comprises SEQ ID No. 50 and the second RNA comprises SEQ ID No. 62a.

94. The immunogenic composition of any one of claims 84-91, wherein the first RNA comprises SEQ ID No. 51 and the second RNA comprises SEQ ID No. 57.

95. The immunogenic composition of any one of claims 84-91, wherein the first RNA comprises SEQ ID No. 51 and the second RNA comprises SEQ ID No. 63a.

96. 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 least 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 more) identical to SEQ ID NO. 56, an

The second RNA encodes the polypeptide of SEQ ID NO. 61 and comprises the nucleotide sequence of SEQ ID NO. 62a 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 97%, at least 98% or 99% or more) identical to SEQ ID NO. 62a, an

Wherein each of the first RNA and the second RNA comprises:

(a) Modified uridine; and

(b) 5' cap, and

97. The immunogenic composition of claim 96, wherein the first RNA and the second RNA are formulated in separate lipid nanoparticles.

98. The immunogenic composition of claim 96, wherein the first RNA and the second RNA are formulated in the same lipid nanoparticle.

99. The immunogenic composition of any one of claims 96-98, wherein each of the first RNA and the second RNA comprises a modified uridine instead of all uridine.

100. The immunogenic composition of any one of claims 96-99, wherein the modified uridine are each N1-methyl-pseudouridine.

101. The immunogenic composition of any one of claims 96-100, wherein each of the first RNA and the second RNA, independently, 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.

102. The immunogenic composition of claim 101, 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.

103. The immunogenic composition of claim 101, wherein the poly a sequence comprises SEQ ID No. 14.

104. The immunogenic composition of any one of claims 96-103, wherein the first RNA comprises SEQ ID No. 57 and the second RNA comprises SEQ ID No. 63a.

105. The immunogenic composition of any one of claims 4-104 wherein the 5' -cap is or comprises m ₂ ⁷ ^,3’-O Gppp(m ₁ ^2’-O )ApG。

106. The immunogenic composition of any one of claims 4-105 wherein the LNP comprises about 40 to about 50 mole% cationic ionizable lipid, about 35 to about 45 mole% sterol, about 5 to about 15 mole% neutral lipid, and about 1 to about 10 mole% polymer-lipid conjugate.

107. The immunogenic composition of any one of claims 4-106, wherein the composition comprises a plurality of LNPs, wherein the plurality of LNPs has an average diameter of about 30nm to about 200nm or about 60nm to about 120nm (e.g., as determined by dynamic light scattering measurements).

108. A method of eliciting an immune response against SARS-CoV-2, comprising administering the immunogenic composition of any one of claims 4 to 107.

109. The method of claim 108, wherein the omacron variant against SARS-CoV-2 elicits the immune response.

110. The method of claim 108, wherein the Beta variant against SARS-CoV-2 elicits the immune response.

111. The method of claim 108, wherein the Alpha variant directed against SARS-CoV-2 elicits the immune response.

112. The method of claim 108, wherein the Delta variant against SARS-CoV-2 elicits the immune response.

113. The method of claim 108, wherein the immune response is elicited against a MN908947 strain, omacron variant, beta variant, alpha variant, and Delta variant of SARS-CoV-2.

114. A method of inducing an immune response in a subject, wherein the method comprises delivering (e.g., as a polypeptide or RNA encoding such polypeptide) an antigen of a SARS-CoV-2 virus that is not a ba.1 omacron variant of SARS-CoV-2.

115. The method of claim 114, wherein the subject has been previously infected with SARS-CoV-2 or vaccinated against SARS-CoV-2.

116. The method of claim 114 or 115, wherein the subject has previously been delivered (e.g., as a polypeptide or RNA encoding such polypeptide) an antigen of the MN908947 strain of SARS-CoV-2.

117. The method of any one of claims 114-116, wherein the subject has previously been administered RNA encoding SARS-CoV-2S protein of the MN908947 strain.

118. The method of any one of claims 114-117, wherein the subject has previously been administered two or more doses of RNA encoding SARS-CoV-2S protein of MN908947 strain.

119. The method of any one of claims 114-118, wherein the method comprises administering RNA encoding an antigen of SARS-CoV-2 virus that is not a ba.1 omacron variant.

120. The method of any one of claims 114-119, wherein the method comprises administering RNA encoding SARS-CoV-2S protein from a SARS-CoV-2 variant that is a ba.1 omacron variant.

121. The method of any one of claims 114-120, wherein the method comprises administering RNA encoding the S protein of an Omicron variant of SARS-CoV-2, wherein the Omicron variant is not a ba.1Omicron variant.

122. The method of any one of claims 114-121, wherein the method comprises administering RNA encoding the S protein of the ba.2 omacron variant of SARS-CoV-2.

123. The method of any one of claims 114-121, wherein the method comprises administering RNA encoding the S protein of ba.4 or ba.5 omacron variant of SARS-CoV-2.

124. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, or Delta variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is not an Omicron variant of a ba.1Omicron variant.

125. The method of claim 124, wherein the second RNA encodes an S protein of a ba.2omicron variant.

126. The method of claim 124, wherein the second RNA encodes an S protein of ba.4 or ba.5 omacron variant.

127. A method of inducing an immune response in a subject, wherein the method comprises administering (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 omacron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is antigenically different from the S protein encoded by the first RNA.

128. The method of claim 127, wherein said second RNA encodes a SARS-CoV-2S protein that is not an omacron variant of the ba.1 omacron variant.

129. The method of claim 127, wherein the second RNA encodes a SARS-CoV-2S protein of a ba.2omicron variant.

130. The method of claim 127, wherein the second RNA encodes a SARS-CoV-2S protein of ba.4 or ba.5 omacron variant.

131. The method of any one of claims 114-130, wherein the first RNA and the second RNA are encapsulated in separate LNPs.

132. The method of any one of claims 114-131, wherein the first RNA and the second RNA are encapsulated in the same LNP.

133. The method of any one of claims 114-132, wherein the first RNA and the second RNA are administered separately, e.g., at different injection sites.

134. A composition comprising (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1 omacron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is antigenically different from the S protein encoded by the first RNA.

135. A composition comprising (a) a first RNA encoding a SARS-CoV-2S protein of a MN908947 strain, alpha variant, beta variant, delta variant, or ba.1Omicron variant, and (b) a second RNA encoding a SARS-CoV-2S protein that is not an Omicron variant of a ba.1Omicron variant.

136. The composition of claim 135, wherein said second RNA encodes a SARS-CoV-2S protein of a ba.2 omacron variant.

137. The composition of claim 135, wherein said second RNA encodes a SARS-CoV-2S protein of ba.4 or ba.5omicron variant.

138. The composition of claim 135, wherein said first RNA encodes a SARS-CoV-2S protein of the MN908947 strain and said second RNA encodes a SARS-CoV-2S protein of the ba.2 omacron variant.

139. The composition of claim 135, wherein said first RNA encodes SARS-CoV-2S protein of the MN908947 strain and said second RNA encodes SARS-CoV-2S protein of a ba.4 or ba.5 omacron variant.

140. The composition of claim 135, wherein said first RNA encodes a SARS-CoV-2S protein of a ba.1omicron variant and said second RNA encodes a SARS-CoV-2S protein of a ba.2omicron variant.

141. The composition of claim 135, wherein said first RNA encodes a SARS-CoV-2S protein of a ba.1omicron variant and said second RNA encodes a SARS-CoV-2S protein of a ba.4 or ba.5omicron variant.

142. The composition of any one of claims 134-141, wherein said first RNA and said second RNA are administered in a form encapsulated in the same LNP.

143. The composition of any one of claims 134-141, wherein said first RNA and said second RNA are encapsulated in separate LNPs.

144. A composition or pharmaceutical preparation comprising RNA encoding an amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof.

145. The composition or pharmaceutical preparation of claim 144, wherein the immunogenic fragment of SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

146. The composition or pharmaceutical formulation of claim 144 or 145, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein said codon optimization and/or said increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

147. The composition or pharmaceutical preparation of any one of claims 144-146, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said 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 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of said SARS-CoV-2S protein or 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 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.

148. The composition or pharmaceutical preparation of any one of claims 144-147, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said 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 said SEQ ID No. 30 or 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; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said 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 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.

149. The composition or pharmaceutical preparation of any one of claims 144-148, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said immunogenic variant thereof comprises the nucleotide sequence of nucleotide 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 nucleotide 49 to 3819 of SEQ ID No. 2, 8 or 9, or a fragment of the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotide 49 to 3819 of SEQ ID No. 2, 8 or 9; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or said 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 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.

150. The composition or pharmaceutical formulation of any one of claims 144 to 149, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises a secretion signal peptide.

151. The composition or pharmaceutical formulation of claim 150, wherein the secretion signal peptide is fused to a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof, preferably an N-terminal fusion.

152. The composition or pharmaceutical preparation of claim 150 or 151, wherein

(i) The RNA encoding the secretion 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 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; and/or

(ii) The secretion 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 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.

153. The composition or pharmaceutical preparation of any one of claims 144-152, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said 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 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; and/or

(ii) The SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or said 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 said 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.

154. The composition or pharmaceutical preparation of any one of claims 144-153, wherein

(i) The RNA encoding the SARS-CoV-2S protein, an immunogenic variant thereof or an immunogenic fragment of said SARS-CoV-2S protein or said 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 said SEQ ID No. 30 or 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; and/or

(ii) The SARS-CoV-2S protein, immunogenic variant thereof or immunogenic fragment of said SARS-CoV-2S protein or 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 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.

155. The composition or pharmaceutical formulation of any one of claims 144 to 153, wherein said RNA comprises a modified nucleoside in place of uridine, particularly wherein said modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ) and 5-methyl-uridine (m 5U), particularly wherein said modified nucleoside is N1-methyl-pseudouridine (m 1 ψ).

156. The composition or pharmaceutical preparation of any one of claims 144-155, wherein the RNA comprises a 5' cap.

157. The composition or pharmaceutical formulation of any one of claims 144 to 156, wherein the RNA encoding the amino acid sequence constituting a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an 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.

158. The composition or pharmaceutical formulation of any one of claims 144 to 157, wherein the RNA encoding the amino acid sequence constituting a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an 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.

159. The composition or pharmaceutical preparation of any one of claims 144-158, wherein the RNA encoding the amino acid sequence that constitutes SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of said SARS-CoV-2S protein or an immunogenic variant thereof comprises a poly a sequence.

160. The composition or pharmaceutical formulation of claim 159, wherein the poly a sequence comprises at least 100 nucleotides.

161. The composition or pharmaceutical formulation of claim 159 or 160, wherein the poly a sequence comprises or consists of the nucleotide sequence of SEQ ID No. 14.

162. The composition or pharmaceutical formulation of any one of claims 144 to 161, wherein the RNA is formulated or to be formulated as a liquid, a solid, or a combination thereof.

163. The composition or pharmaceutical formulation of any one of claims 144 to 162, wherein the RNA is formulated or to be formulated for injection.

164. The composition or pharmaceutical formulation of any one of claims 144 to 163, wherein the RNA is formulated or to be formulated for intramuscular administration.

165. The composition or pharmaceutical formulation of any one of claims 144 to 164, wherein the RNA is formulated or to be formulated as particles.

166. The composition or pharmaceutical formulation of claim 165, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

167. The composition or pharmaceutical formulation of claim 166, wherein the LNP particles comprise a cationically ionizable lipid, a neutral lipid, a sterol, and a polymer-lipid conjugate.

168. The composition or pharmaceutical formulation of claim 167, wherein the RNA cationic lipid complex particles are obtainable by mixing the RNA with a liposome.

169. The composition or pharmaceutical formulation of any one of claims 144 to 168, wherein the RNA is mRNA or saRNA.

170. The composition or pharmaceutical preparation of any one of claims 144-169, which is a pharmaceutical composition.

171. The composition or pharmaceutical preparation of any one of claims 144-170, which is a vaccine.

172. The composition or pharmaceutical formulation of claim 170 or 171, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

173. The composition or pharmaceutical preparation of any one of claims 144 to 169, which is a kit.

174. The composition or pharmaceutical formulation of claim 173, wherein the RNA and optional particle-forming components are in separate vials.

175. The composition or pharmaceutical formulation of claim 173 or 174, further comprising instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in a subject.

176. The composition or pharmaceutical preparation of any one of claims 144-175 for pharmaceutical use.

177. The composition or pharmaceutical formulation of claim 176, wherein the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

178. The composition or pharmaceutical formulation of claim 176 or 177, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus infection.

179. The composition or pharmaceutical formulation of any one of claims 144 to 178, for administration to a human.

180. The composition or pharmaceutical preparation of any one of claims 175-179, wherein the coronavirus is a Beta coronavirus.

181. The composition or pharmaceutical preparation of any one of claims 175-180, wherein the coronavirus is sand Bei Bingdu.

182. The composition or pharmaceutical preparation of any one of claims 175-181, wherein the coronavirus is SARS-CoV-2.

183. A method of inducing an immune response against a coronavirus in a subject, comprising administering to the subject a composition comprising RNA encoding an amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or the immunogenic variant thereof.

184. The method of claim 183, wherein the immunogenic fragment of the SARS-CoV-2S protein comprises the S1 subunit of the SARS-CoV-2S protein or the Receptor Binding Domain (RBD) of the S1 subunit of the SARS-CoV-2S protein.

185. The method of any one of claims 183 or 184, wherein the amino acid sequence comprising the SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to a wild-type coding sequence, wherein the codon optimization and/or the increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.

186. The method of any one of claims 183-185, wherein

187. The method of any one of claims 183-186, wherein

188. The method of any one of claims 183-187, wherein

189. The method of any one of claims 183-188, wherein the amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises a secretion signal peptide.

190. The method of claim 189, wherein the secretion signal peptide is fused to a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof, preferably an N-terminal fusion.

191. The method of claim 189 or 190, wherein

192. The method of any one of claims 183-191, wherein

193. The method of any one of claims 183-192, wherein

194. The method of any one of claims 183-192, wherein the RNA comprises a modified nucleoside in place of uridine, particularly wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U), particularly wherein the modified nucleoside is N1-methyl-pseudouridine (m 1 ψ).

195. The method of any one of claims 183-194, wherein the RNA comprises a cap.

196. The method of any one of claims 183-195, wherein the RNA encoding the amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S 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.

197. The method of any one of claims 183-196, wherein the RNA encoding the amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S 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.

198. The method of any one of claims 183-197, wherein the RNA encoding the amino acid sequence that constitutes a SARS-CoV-2S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2S protein or an immunogenic variant thereof comprises a poly-a sequence.

199. The method of claim 198, wherein the poly-a sequence comprises at least 100 nucleotides.

200. The method of claim 198 or 199, wherein the poly-a sequence comprises or consists of the nucleotide sequence of SEQ ID No. 14.

201. The method of any one of claims 183-200, wherein the RNA is formulated as a liquid, a solid, or a combination thereof.

202. The method of any one of claims 183-201, wherein the RNA is administered by injection.

203. The method of any one of claims 183-202, wherein the RNA is administered by intramuscular administration.

204. The method of any one of claims 183-203, wherein the RNA is formulated as particles.

205. The method of claim 204, wherein the particle is a Lipid Nanoparticle (LNP) or a cationic lipid complex (LPX) particle.

206. The method of claim 205, wherein the LNP particles comprise cationically ionizable lipids, neutral lipids, sterols, and polymer-lipid conjugates.

207. The method of any one of claims 205, wherein said RNA cationic lipid complex particles are obtainable by mixing said RNA with a liposome.

208. The composition or pharmaceutical formulation of any one of claims 183 to 207, wherein the RNA is mRNA or saRNA.

209. The method of any one of claims 183-208, which is a method of vaccinating against coronavirus.

210. The method of any one of claims 183-209, which is a method for the therapeutic or prophylactic treatment of a coronavirus infection.

211. The method of any one of claims 183-210, wherein the subject is a human.

212. The method of any one of claims 183-211, wherein the coronavirus is a Beta coronavirus.

213. The method of any one of claims 183-212, wherein the coronavirus is sand Bei Bingdu.

214. The method of any one of claims 183-213, wherein the coronavirus is SARS-CoV-2.

215. The method of any one of claims 108 to 133 or 183 to 214 wherein the composition is the composition of any one of claims 1 to 107 or 134 to 182.

216. The composition or pharmaceutical preparation of any one of claims 1 to 107 or 134 to 182 for use in the method of any one of claims 108 to 133 or 183 to 215.

217. The composition or pharmaceutical formulation of any one of claims 1 to 107 or 134 to 182, which is a pharmaceutical composition.

218. The composition or pharmaceutical formulation of any one of claims 1 to 107 or 134 to 182, which is a vaccine.

219. The composition or pharmaceutical formulation of claim 217 or 218, wherein the pharmaceutical composition or vaccine further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

220. The composition or pharmaceutical formulation of any one of claims 1 to 107 or 134 to 182, which is a kit.

221. The composition or pharmaceutical formulation of claim 220, wherein the RNA and optional particle-forming components are in separate vials.

222. The composition or pharmaceutical formulation of claim 220 or 221, further comprising instructions for using the composition or pharmaceutical formulation to induce an immune response against coronavirus in a subject.

223. The composition or pharmaceutical formulation of any one of claims 1 to 107 or 134 to 182 for pharmaceutical use.

224. The composition or pharmaceutical formulation of claim 223, wherein the pharmaceutical use comprises inducing an immune response against a coronavirus in a subject.

225. The composition or pharmaceutical formulation of claim 223 or 224, wherein the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus infection.

226. A composition or pharmaceutical formulation as claimed in any one of claims 1 to 107 or 134 to 182 for use in the manufacture of a medicament.

227. The composition or pharmaceutical formulation of claim 226, wherein the medicament is for inducing an immune response against coronavirus in a subject.

228. The composition or pharmaceutical formulation of claim 226 or 227, wherein the medicament is for the therapeutic or prophylactic treatment of a coronavirus infection.

229. The composition or pharmaceutical formulation of any one of claims 1 to 107 or 134 to 182, for administration to a human.

230. The composition or pharmaceutical preparation of any one of claims 222, 224, 225, 227, or 228, wherein the coronavirus is a Beta coronavirus.

231. The composition or pharmaceutical formulation of claim 230, wherein the coronavirus is sand Bei Bingdu.

232. The composition or pharmaceutical preparation of claim 231, wherein the coronavirus is SARS-CoV-2.