Coronavirus vaccine

Abstract

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

Description

Attorney Docket No.: 2013237-1270 CORONAVIRUS VACCINE Background

[001] In December 2019, a pneumonia outbreak of unknown cause occurred in Wuhan, China and it becameclear that a novel coronavirus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) was the underlying cause. The genetic sequence of SARS-CoV-2 became available to the WHO and public (MN908947.3) and the virus was categorized into the betacoronavirus subfamily. By sequence analysis, the phylogenetic tree revealed a closer relationship to severe acute respiratory syndrome (SARS) virus isolates than to another coronavirus infecting humans, namely the Middle East respiratory syndrome (MERS) virus.

[002] SARS-CoV-2 infections and the resulting disease COVID-19 have since spread globally, affecting agrowing number of countries. On 11 March 2020 the WHO characterized the COVID-19 outbreak as a pandemic. The ongoing pandemic remains a significant challenge to public health and economic stability worldwide.

[003] The presentation of COVID-19 is generally with cough and fever, with chest radiography showingground-glass opacities or patchy shadowing. However, many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission. For symptomatic subjects, progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multi-organ failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea / vomiting or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss of taste) may be the sole presenting symptom in approximately 3% of individuals who have COVID-19.

[004] Since the initial emergence of SARS-CoV-2, the virus has continued to evolve, giving rise to thousands ofnew variants. Many of these variants have an increased growth rate and / or an increased ability to evade immune responses as compared to the first strain of SARS-CoV-2 identified. As a result, there have been successive worldwide waves of different variants. Initial SARS-CoV-2 vaccines have shown a diminished efficacy in regard to the new SARS-CoV-2 variants, although they still provide beneficial health effects, including, e.g., a decreased risk of hospitalization and severe disease. Summary

[005] The present disclosure provides new technologies (e.g., compositions and methods) for inducing animmune response against a coronavirus (e.g., SARS-CoV-2).

[006] In some embodiments, the technologies described in the present disclosure can provide significantadvantages as compared to first generation SARS-CoV-2 vaccines (e.g., vaccines that deliver a full length S protein, such as, e.g., LNP-formulated RNA that encodes a full length S protein of a SARS-CoV-2 virus). These advantages can include, e.g., an improved response. An improved immune response can comprise, e.g., increased antibody titers and / or increased neutralizing antibody titers against a SARS-CoV-2 virus as compared to compositions that deliver a full length S protein of a coronavirus. In some embodiments, an improved immune response can include a broader cross neutralization response (i.e., higher neutralizing titers against a range of SARS-CoV-2 viruses) as compared to a composition that delivers a full length S protein. In some embodiments, an improved immune response can include a more robust immune response (e.g., an immune response that results in antibody titers that remain elevated for a longer period of time following vaccination as compared to compositions that deliver a full length S protein of a coronavirus).12410924v1 Page 1 of 696

[0007] Among other things, the present disclosure provides an insight that the S2 domain of the S protein of a coronavirus comprises a number of conserved neutralizing epitopes. Without wishing to be bound by theory, the conserved nature of these epitopes means that induction of an immune response can result in an immune response that is more broadly cross-neutralizing and / or more durable as compared to compositions that deliver a full length S protein of a coronavirus. The present disclosure, among other things, provides certain insights into the design of S2 antigens that result in an improved immune response.

[0008] Among other things, the present disclosure also provides compositions that deliver both an RBD and an S2 polypeptide of a coronavirus protein or one or more fragments of an S2 polypeptide. Among other things, the present disclosure provides a novel antigenic polypeptide design that comprises an S2 domain or one or more fragments thereof and an RBD of a coronavirus. In some embodiments, an RBD and an S2 polypeptide are delivered by an RNA comprising a nucleotide sequence that encodes a polypeptide comprising an RBD and an S2 polypeptide. In some embodiments, the present disclosure provides an immunogenic fragment of an S2 domain and an RBD of a coronavirus.

[0009] In some embodiments, the compositions provided herein deliver an immunogenic fragment of the S2 polypeptide. In particular, in some embodiments, compositions described herein deliver a fragment of the S2 comprising the stem helix and / or fusion peptide of an S2 polypeptide. Without wishing to be bound by theory, the present disclosure provides the insight that the stem helix and the fusion peptide of an S2 polypeptide are both highly conserved and also comprise neutralizing epitopes. Thus, by inducing an immune response against a stem helix and / or a fusion peptide, an improved immune response can be generated, including, e.g., improved antibody titers, neutralizing antibody titers, and / or cross-neutralization as compared to RNA encoding a full length S protein. In some embodiments, a polypeptide comprising an immunogenic fragment of the S2 polypeptide is also comprises an RBD of a coronavirus S protein.

[0010] Among other things, the present disclosure provides insights into the design of polypeptides that can provide for improved immune responses. These insights include, e.g., domains to attach to antigenic regions (e.g., secretory signal peptides, transmembrane doamins, and multimerization domains), and design of polypeptides that result in improved immune responses.

[0011] Methods and agents described herein are, in particular, useful for the prevention or treatment of coronavirus infection. Administration of RNA disclosed herein to a subject can protect the subject against coronavirus infection. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject, i.e., vaccine RNA encoding vaccine antigen. Administering to the subject RNA encoding vaccine antigen may provide (following expression of the RNA by appropriate target cells) vaccine antigen for inducing an immune response against vaccine antigen (and disease-associated antigen) in the subject.

[0012] Also disclosed herein are compositions that can produce an improved immune response (e.g., an immune response having broader cross-neutralization activity, higher neutralization titers, and / or which is less susceptible to immune escape), e.g., as compared to previously approved vaccines (e.g., previously approved RNA or protein vaccines), vaccines that deliver antigens of a Wuhan S protein, and / or vaccines that deliver antigens of other SARS-CoV-2 variants.

[0013] In some embodiments, the concentration of RNA in a composition described herein (e.g., a pharmaceutical RNA preparation) is about 0.1 mg / ml. In some embodiments, about 30 ug of RNA is administered by administering about 200 uL of RNA preparation.

[0014] In some embodiments, RNA in a pharmaceutical RNA preparation is diluted prior to administration (e.g., diluted to a concentration of about 0.05 mg / ml). In some embodiments, administration volumes are between about 200 pl and about 300 pl. In some embodiments, RNA in a pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, and about 10% sucrose.

[0015] In some embodiments, RNA in a pharmaceutical RNA preparation is present at a concentration of about 0.1 mg / ml, and is formulated in about 10 mM Tris buffer, and about 10% sucrose, and a dose of about 10 μg of RNA is administered by (i) diluting the pharmaceutical RNA preparation about 1:1 and (ii) administering about 200 pl of the diluted pharmaceutical RNA preparation. In some embodiments, RNA in a pharmaceutical RNA preparation is present at a concentration of about 0.1 mg / ml, and is formulated in about 10 mM Tris buffer, and about 10% sucrose and a dose of RNA of about 10 μg is administered by (i) diluting the pharmaceutical RNA preparation about 1:5.75 and (ii) administering about 200 pl of diluted pharmaceutical RNA preparation.

[0016] The present disclosure generally embraces immunotherapeutic treatment of a subject comprising administration of RNA, e.g., vaccine RNA, encoding an amino acid sequence, e.g., a vaccine antigen, comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, i.e., an antigenic peptide or protein. Thus, vaccine antigen comprises an epitope of SARS-CoV-2 S protein for inducing an immune response against coronavirus S protein, in particular SARS-CoV-2 S protein, in the subject. RNA encoding vaccine antigen is administered to provide (following expression of the polynucleotide by appropriate target cells) antigen for induction, i.e., stimulation, priming and / or expansion, of an immune response, e.g., antibodies and / or immune effector cells, which is targeted to target antigen (coronavirus S protein, in particular SARS-CoV-2 S protein) or a procession product thereof. In one embodiment, the immune response which is to be induced according to the present disclosure is a B cell- mediated immune response, i.e., an antibody-mediated immune response. Additionally or alternatively, in one embodiment, the immune response which is to be induced according to the present disclosure is a T cell- mediated immune response. In one embodiment, the immune response is an anti-coronavirus, in particular anti- SARS-CoV-2 immune response.

[0017] In some embodiments, compositions (e.g., vaccines) described herein comprise as an active principle single-stranded RNA that may be translated into protein upon entering cells of a recipient. In addition to wildtype or codon-optimized sequences encoding an antigen sequence, RNA may contain one or more structural elements optimized for maximal efficacy with respect to stability and translational efficiency (e.g., 5' cap, 5' UTR, 3' UTR, poly(A)-tail, and combinations thereof). In one embodiment, RNA contains all of these elements. In one embodiment, a capl structure may be utilized as specific capping structure at the 5'-end of an RNA drug substance. In one embodiment, beta-S-ARCA(Dl) (m27,2'-OGppSpG) or m27,3'-OGppp(ml2'-O)ApG may be utilized as specific capping structure at the 5'-end of an RNA drug substance. As 5'-UTR sequence, the 5'-UTR sequence of the human alpha-globin mRNA, optionally with an optimized 'Kozak sequence' to increase translational efficiency (e.g., SEQ ID NO: 12) may be used. As 3’-UTR sequence, a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) (e.g., SEQ ID NO: 13) placed between the coding sequence and the poly(A)-tail to assure higher maximum protein levels and prolonged persistence of the mRNA may be used. These features were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017 / 060314, herein incorporated by reference). Alternatively, the 3’- UTR may be two re-iterated 3'-UTRs of the human beta-globin mRNA. Additionally or alternatively, in some embodiments, a poly(A)-tail may comprise a length of at least 100 adenosine residues (including, e.g., at least110 adenosine residues, at least 120 adenosine residues, 130 adenosine residues, or longer). In some embodiments, a poly(A)-tai I may comprise a length of about 100 to about 150 adenosine residues. In some embodiments a poly(A)-tail may comprise an interrupted poly(A)-tail . For example, in some such embodiments, a poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues (e.g., SEQ ID NO: 14) may be used. This poly(A)-tail sequence was designed to enhance RNA stability and translational efficiency.

[0018] Furthermore, in some embodiments, a nucleotide sequence encoding a secretory signal peptide (sec) may be fused to antigen-encoding regions of an RNA, preferably in some embodiments in a way that the sec is translated as an N terminal tag. In one embodiment, sec corresponds to the secretory signal peptide of a SARS- CoV-2 S protein (e.g., of a Wuhan strain). In some embodiments, sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins, may be used as GS / Linkers between sec and an antigen.

[0019] In some embodiments, RNA described herein may be complexed with proteins and / or lipids, preferably lipids, to generate RNA-particles for administration. If a combination of different RNAs is used, RNAs may be complexed together or complexed separately with proteins and / or lipids to generate RNA-particles for administration.

[0020] In one aspect, the present disclosure relates to a composition or medical preparation comprising RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

[0021] In one embodiment, an immunogenic fragment of a coronavirus S protein (e.g., the SARS-CoV-2 S protein) comprises the SI subunit of the coronavirus S protein (e.g., SARS-CoV-2 S protein), or the receptor binding domain (RBD) of the SI subunit of the coronavirus S protein (e.g., SARS-CoV-2 S protein). In one embodiment, an immunogenic fragment of a coronavirus S protein (e.g., SARS-CoV-2 S protein) comprises an S2 subunit of the coronavirus S protein (e.g., SARS-CoV-2 S protein). In one embodiment, an immunogenic fragment of a coronavirus S protein (e.g., SARS-CoV-2 S protein) comprises a stem helix and / or fusion peptide of a coronavirus S protein (e.g., SARS-CoV-2 S protein).

[0022] In one embodiment, an amino acid sequence comprising a coronavirus (e.g., SARS-CoV-2) S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus (e.g., SARS-CoV-2) S protein or the immunogenic variant thereof is able to form a multimeric complex, in particular a trimeric complex. To this end, an amino acid sequence comprising a coronavirus (e.g., SARS-CoV-2) S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus (e.g., SARS-CoV-2) S protein or the immunogenic variant thereof may comprise a domain allowing the formation of a multimeric complex. In some embodiments a multimeric complex is a trimeric complex. In some embodiments, a multimeric complex comprises more than 3 polypeptides. In some embodiments, the compositions described herein can result in the delivery of a multimeric (e.g., trimeric) complex of the amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus (e.g., SARS-CoV-2) S protein or the immunogenic variant thereof. In one embodiment, the domain allowing formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein, e.g., SARS-CoV-2 S protein trimerization domain. In one embodiment, trimerization is achieved by addition of a trimerization domain, e.g., a T4-fibritin- derived "foldon" trimerization domain (e.g., SEQ ID NO: 10), in particular if the amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 Sprotein or the immunogenic variant thereof corresponds to a portion of a SARS-CoV-2 S protein that does not comprise the SARS-CoV-2 S protein trimerization domain.

[0023] In one embodiment, the amino acid sequence comprising a coronavirus (e.g., SARS-CoV-2) S protein, an immunogenic variant thereof, or an immunogenic fragment of the coronavirus (e.g., SARS-CoV-2) S protein or the immunogenic variant thereof is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.Those skilled in the art will appreciate that codon optimization involves choosing between or among alternative codons encoding the same amino acid residue. Codon optimization typically includes consideration of codon(s) preferred by a particular host in which a sequence is to be expressed. In accordance with the present disclosure, in many embodiments, a preferred host is a human. In some embodiments, a preferred host may be a domestic animal. Alternatively or additionally, in some embodiments, selection between or among possible codons encoding the same amino acid may consider one or more other features such as, for example, overall G / C content (as noted above) and / or similarity to a particular reference. For example, in some embodiments of the present disclosure, a provided coding sequence that encodes a SARS-CoV-2 S protein or immunogenic variant thereof that differs in amino acid sequence from that encoded by a BNT162b2 construct described herein utilizes a codon, in at least one position of such difference, that preserves greater similarity to the BNT162b2 construct sequence relative to at least one alternative codon encoding the same amino acid at such position of difference.

[0024] In one embodiment, an RNA is a modified RNA, in particular a stabilized mRNA. In one embodiment, an RNA comprises a modified nucleoside in place of at least one uridine. In one embodiment, an RNA comprises a modified nucleoside in place of each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).

[0025] In one embodiment, RNA comprises a modified nucleoside in place of uridine.

[0026] In one embodiment, the modified nucleoside is selected from pseudouridine (ip), Nl-methyl- pseudouridine (mlip), and 5-methyl-uridine (m5U).

[0027] In one embodiment, RNA comprises a 5' cap.

[0028] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

[0029] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

[0030] SEQ ID NO: 13:CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTA TGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGC TTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCC AGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 13).

[0031] In some embodiments, an RNA disclosed herein comprises a 3' UTR having 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% identity to a 3' UTR with the sequence of CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGU CCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAG CUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 601).

[0032] In some embodiments, an RNA disclosed herein comprises a 3' UTR with the sequence of CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGU CCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAG CUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 601).

[0033] In some embodiments, an RNA disclosed herein comprises a 3' UTR having 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% identity to a 3' UTR with the sequence of CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGU CCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAG CUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC (SEQ ID NO: 602).

[0034] In some embodiments, an RNA disclosed herein comprises a 3' UTR with the sequence of CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGU CCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAG CUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC (SEQ ID NO: 602)

[0035] In some embodiments, a 3'UTR is an FI element as described in W02017 / 060314, which is herein incorporated by reference in its entirety.

[0036] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a poly-A sequence.

[0037] In one embodiment, the poly-A sequence comprises at least 100 nucleotides.

[0038] In one embodiment, the poly-A sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 14.

[0039] In one embodiment, RNA is formulated or is to be formulated as a liquid, a solid, or a combination thereof.

[0040] In one embodiment, RNA is formulated or is to be formulated for injection.

[0041] In one embodiment, RNA is formulated or is to be formulated for intramuscular administration.

[0042] In one embodiment, RNA is formulated or is to be formulated as particles.

[0043] In one embodiment, particles are lipid nanoparticles (LNP) or lipoplex (LPX) particles.

[0044] In one embodiment, LNPs comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, l,2-Distearoyl-sn-glycero-3- phosphocholine, and cholesterol.

[0045] In one embodiment, RNA lipoplex particles are obtainable by mixing RNA with liposomes. In one embodiment, RNA lipoplex particles are obtainable by mixing RNA with lipids.

[0046] In one embodiment, RNA is formulated or is to be formulated as colloid. In one embodiment, RNA is formulated or is to be formulated as particles, forming the dispersed phase of a colloid. In one embodiment, 50% or more, 75% or more, or 85% or more of RNA is present in the dispersed phase. In one embodiment, RNA is formulated or is to be formulated as particles comprising RNA and lipids. In one embodiment, particles are formed by exposing RNA, dissolved in an aqueous phase, with lipids, dissolved in an organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, particles are formed by exposing RNA, dissolved in an aqueous phase, with lipids, dispersed in an aqueous phase. In one embodiment, the lipids dispersed in an aqueous phase form liposomes.

[0047] In one embodiment, RNA is mRNA or saRNA.

[0048] In one embodiment, a composition or medical preparation is a pharmaceutical composition.

[0049] In one embodiment, a composition or medical preparation is a vaccine.

[0050] In one embodiment, a pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0051] In one embodiment, a composition or medical preparation is a kit.

[0052] In one embodiment, RNA and optionally the particle forming components are in separate vials.

[0053] In one embodiment, a kit further comprises instructions for use of a composition or medical preparation for inducing an immune response against coronavirus in a subject.

[0054] In one aspect, the present disclosure relates to a composition or medical preparation described herein for pharmaceutical use.

[0055] In one embodiment, a pharmaceutical use comprises inducing an immune response against coronavirus in a subject.

[0056] In one embodiment, a pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus infection.

[0057] In one embodiment, a composition or medical preparation described herein is for administration to a human.

[0058] In one embodiment, the coronavirus is a betacoronavirus.

[0059] In one embodiment, the coronavirus is a sarbecovirus.

[0060] In one embodiment, the coronavirus is SARS-CoV-2.

[0061] In one aspect, the present disclosure relates to a method of inducing an immune response against coronavirus in a subject comprising administering to the subject a composition comprising RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

[0062] In one embodiment, an immunogenic fragment of the SARS-CoV-2 S protein comprises the SI subunit of the SARS-CoV-2 S protein, or the receptor binding domain (RBD) of the SI subunit of the SARS-CoV-2 S protein.

[0063] In one embodiment, an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is able to form a multimeric complex, in particular a trimeric complex. To this end, an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS- CoV-2 S protein or the immunogenic variant thereof may comprise a domain allowing the formation of a multimeric complex, in particular a trimeric complex of the amino acid sequence comprising a SARS-CoV-2 Sprotein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof. In one embodiment, the domain allowing the formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein.

[0064] In one embodiment, an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0065] In one embodiment, an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a secretory signal peptide.

[0066] In one embodiment, a secretory signal peptide is fused, preferably N-terminally, to a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof.

[0067] In one embodiment,(i) the RNA encoding the secretory signal peptide comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and / or(ii) the secretory signal peptide comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

[0068] In one embodiment, a composition described herein comprises an RNA molecule comprising a nucleotide sequence encoding a SARS-CoV-2 S protein comprising one or more mutations characteristic of an XBB.1.5, XBB.1.16, BA.4 / 5, XBB, XBB.l, XBB.2.3, XBB.2.3.2, BQ.1.1, KP.2, JN.l, or XEC Omicron variant or sublineages (i.e., descendants) thereof.

[0069] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB.1.5 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0070] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of a BA.4 / 5 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N,R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0071] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of a BA.2.75 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0072] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of a BA.2.75.2 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and D1199N, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0073] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of a BA.4.6 / BF.7 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N658S, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0074] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0075] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB.l variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0076] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB.l.16 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A145, H146Q, E180V, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N,T478R, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0077] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB.2.3 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, P521S, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0078] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of an XBB.2.3.2 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, G184V, V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, P521S, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0079] In one embodiment, a composition described herein comprises a nucleotide sequence that encodes a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing comprising one or more mutations characteristic of a BQ.1.1 variant, wherein the one or more mutations are selected from the group consisting of T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, wherein the mutations are indicated relative to SEQ ID NO: 1.

[0080] In one embodiment, a SARS-CoV-2 S protein, a variant thereof, or a truncated fragment of either of the foregoing encoded by an RNA described herein comprises one or more substitutions that stable a prefusionconfirmation. In one embodiment, the one or more substitutions that stabilize a prefusion confirmation are proline substitutions at positions corresponding to K986 and V987 of SEQ ID NO: 1.

[0081] In one embodiment, an RNA molecule comprises at least at least one modified uridine in place of a uridine. In one embodiment, an RNA molecule comprises a modified uridine in place of each uridine.

[0082] In one embodiment, an RNA molecule comprises a 5' cap. In some embodiments, the 5' cap comprises (e.g., consists of) m27,3'-OGppp(ml2'-O)ApG.

[0083] In one embodiment, an RNA molecule comprises a 5' untranslated region (UTR). In one embodiment, the 5' UTR comprises a human alpha-globin 5'-UTR. In one embodiment, the human alpha-globin 5' untranslated region (UTR) comprises SEQ ID NO: 12.

[0084] In one embodiment, an RNA molecule comprises a 3' UTR. In one embodiment, the 3' UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA. In one embodiment, the 3' UTR comprises SEQ ID NO: 13. In one embodiment, the 3' UTR comprises SEQ ID NO: 601. In one embodiment, the 3' UTR comprises SEQ ID NO: 602.

[0085] In one embodiment, an RNA molecule comprises a poly-A sequence of at least 100 A nucleotides. In one embodiment, the poly-A sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence. In some embodiments, the poly-A sequence comprises SEQ ID NO: 14.

[0086] In one embodiment, an RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein, wherein the sequence is codon-optimized (e.g., codon-optimized for expression in human cells) and / or which has a G / C content that is increased compared to a wild type coding sequence.

[0087] In some embodiments, an RNA molecule is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the LNP comprises molar ratios of 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0088] In some embodiments, a composition described herein is formulated as a liquid, a solid, or a combination thereof.

[0089] In some embodiments, a composition described herein is formulated for injection. In some embodiments, a composition described herein is formulated for intramuscular administration.

[0090] In some embodiments, an RNA described herein is mRNA.

[0091] In some embodiments, an RNA described herein is saRNA.

[0092] In some embodiments, a composition described herein is a pharmaceutical composition. In some embodiments, a pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients. In some embodiments, a composition described herein is a vaccine.

[0093] In some embodiments, the present disclosure provides a method of eliciting an immune response in a subject, where the method comprising administering a composition described herein. In some embodiments, the immune response is elicited against an Omicron variant of SARS-CoV-2. In some embodiments, the immune response is elicited against a Wuhan strain, an Omicron variant, a Beta variant, an Alpha variant, and a Delta variant of SARS-CoV-2.

[0094] In some embodiments, a composition described herein is administered to a subject that has not been previously infected with and / or has not been previously administered a SARS-CoV-2 vaccine.

[0095] In some embodiments, a composition described herein is adminsitered to a subject that has previously been infected with SARS-CoV-2 and / or has been previously administered a SARS-CoV-2 vaccine.

[0096] In some embodiments, a composition described herein is adminsitered to a subject who has previously received a vaccine that delivers a SARS-CoV-2 S protein of a Wuhan strain (e.g., a full length protein or an antigenic fragment thereof).

[0097] In some embodiments, a composition described herein is adminsitered to subject who has previously been administered RNA encoding a SARS-CoV-2 S protein of a Wuhan strain (e.g., a full length protein or an antigenic fragment thereof).

[0098] In some embodiments, a composition described herein is adminsitered to subject who has previously been administered two or more doses of RNA encoding a SARS-CoV-2 S protein of a Wuhan strain.

[0099] In some embodiments, a composition described herein is adminsitered to subject who has previously been administered:(i) two or more doses of RNA encoding a SARS-CoV-2 S protein of a Wuhan strain (e.g., BNT162b2); and(ii) one or more doses of a bivalent vaccine comprising:(a) a first RNA comprising a nucleotide sequence encoding a SARS-CoV-2 S protein of a Wuhan strain; and(b) a second RNA comprising a nucleoside sequence encoding a SARS-CoV-2 S protein of an Omicron BA.4 / 5 variant.

[0100] In some embodiments, a composition described herein is administered to a subject who has previously been administered one or more doses of a bivalent vaccine comprising(1) a first RNA that comprises a nucleotide sequence of that is at least 90% identical to that set forth in SEQ ID NO: 20 (e.g., at least 95% identical, at least 96% identical, at least 97% identical, at least 99% identical, or 100% identical), and(2) a second RNA that comprises a nucleotide sequence that is at least 90% identical to that set forth in SEQ ID NO: 72 (e.g., at least 95% identical, at least 96% identical, at least 97% identical, at least 99% identical, or 100% identical).[OO1O1] In some embodiments, a composition described herein comprises about 3 μg to about 30 μg of RNA (e.g., about 3 pg, about 10 pg, or about 30 μg of RNA).

[0102] In some embodiments, a composition described herein comprises about 3 μg of RNA, and is administered to a subject who is about 6 months to less than about 5 years old.

[0103] In some embodiments, a composition described herein comprises about 10 μg of RNA, and is administered to a subject who is about 5 years old to less than about 12 years old.

[0104] In some embodiments, a composition described herein comprises about 30 μg of RNA, and is administered to a subject who is about 12 years or older.

[0105] In some embodiments, a composition described herein is co-administered with one or more vaccines against a non-SARS-CoV-2 disease (e.g., a non-SARS-CoV-2 respiratory disease). In some embodiments, the one or more vaccines against a non-SARS-CoV-2 disease comprises an influenza vaccine, an RSV vaccine, or a combination thereof.

[0106] In some embodiments, the present disclosure provides a kit comprising a composition described herein. In some embodiments the kit further comprises instructions for use of the composition to induce an immune response against coronavirus in a subject.

[0107] In some embodiments, a composition described herein is for pharmaceutical use. In some embodiments, the pharmaceutical use comprises inducing an immune response against coronavirus (e.g., a betacoronavirus, a sarbecovirus, or a SARS-CoV-2 virus) in a subject. In some embodiments, an immune response is induced in a human. In some embodiments, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus (e.g., a betacoronavirus, a sarbecovirus, or a SARS-CoV-2 virus) infection.

[0108] In some embodiments, a composition described herein is for administration to a human.

[0109] In some embodiments, a composition described herein is for pharmaceutical use. In some embodiments, the pharmaceutical use comprises inducing an immune response against coronavirus in a subject. In some embodiments, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a coronavirus (e.g., a betacoronavirus, a sarbecovirus, or a SARS-CoV-2 virus) infection.

[0110] In some embodiments, described herein is the use of a composition described herein for the manufacture of a medicament for inducing an immune response against coronavirus in a subject. In some embodiments, the medicament is for therapeutic or prophylactic treatment of a coronavirus (e.g., a betacoronavirus, a sarbecovirus, or a SARS-CoV-2 virus) infection.

[0111] In some embodiments, a composition comprises an RNA molecule comprising:(i) Nl-methyl-pseudouridine in place of each uridine; and(ii) a 5' cap that comprises m27,3'-OGppp(ml2'-O)ApG; wherein the RNA molecule is encapsulated in a lipid nanoparticle (LNP); and wherein the LNP comprises molar ratios of 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0112] In some embodiments, a composition described herein comprises about 10 mM Tris buffer and about 10% sucrose.

[0113] In some embodiment, a composition comprises at least one unit dose of LNP-encapsulated RNA molecules, optionally wherein the unit dose comprises the RNA molecule in an amount of about 30 pg, or wherein the unit dose comprises the RNA molecule in an amount of about 10 pg, or wherein the unit dose comprises the RNA molecule in an amount of about 3 pg.

[0114] In some embodiments, a composition described herein is formulated as a multi-dose formulation in a vial.

[0115] In some embodiments, a composition described herein is for use in a method of inducing an immune response against coronavirus in a subject, said method comprising administering to a subject the composition. In some embodiments: the subject is 12 years or older, and the composition comprises 30 μg of the RNA molecule, or the subject is 5 years to less than 12 years old, and the composition comprises 10 pg of the RNA molecule, or the subject is 6 months to less than 5 years old, and the composition comprises 3 pg of the RNA molecule.

[0116] In some embodiments, a composition described herein is administered in a volume of about 200 pL to 300 μL.

[0117] In some embodiments, a composition described herein is adminsitered to a subject previously administered one or more doses of a SARS-CoV-2 vaccine, preferably wherein the subject was previously administered a complete dosing regimen of a SARS-CoV-2 vaccine.

[0118] In some embodiments, a composition described herein is adminsitered to a subject previously administered a first dose and a second dose of BNT162b2, wherein the first dose and the second dose were administered about 21 days apart, and / or wherein the subject was previously administered as a booster dose a bivalent vaccine that delivers (i) a SARS-CoV-2 S protein of an Omicron BA.4 / 5 variant and (ii) a SARS-CoV-2 S protein of a Wuhan strain.

[0119] In some embodiments, a composition described herein is co-administered with one or more vaccines against a non-SARS-CoV-2 disease, preferably wherein the one or more vaccines comprises an RSV vaccine, an influenza vaccine, or a combination thereof.

[0120] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

[0121] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

[0122] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 601, or a nucleotidesequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 601.

[0123] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 602, 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: 602.

[0124] In one embodiment, RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprises a poly-A sequence.

[0125] In one embodiment, a poly-A sequence comprises at least 100 nucleotides.

[0126] In one embodiment, a poly-A sequence comprises or consists of the nucleotide sequence of SEQ IDNO: 14.

[0127] In one embodiment, RNA is formulated as a liquid, a solid, or a combination thereof.

[0128] In one embodiment, RNA is formulated as particles.

[0129] In one embodiment, the particles are lipid nanoparticles (LNP) or lipoplex (LPX) particles.

[0130] In one embodiment, LNPs comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2- hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, l,2-Distearoyl-sn-glycero-3- phosphocholine, and cholesterol.

[0131] In one embodiment, RNA lipoplex particles are obtainable by mixing RNA with liposomes. In one embodiment, RNA lipoplex particles are obtainable by mixing RNA with lipids.

[0132] In one embodiment, RNA is formulated as colloid. In one embodiment, RNA is formulated as particles, forming the dispersed phase of a colloid. In one embodiment, 50% or more, 75% or more, or 85% or more of RNA are present in the dispersed phase. In one embodiment, RNA is formulated as particles comprising RNA and lipids. In one embodiment, particles are formed by exposing RNA, dissolved in an aqueous phase, with lipids, dissolved in an organic phase. In one embodiment, the organic phase comprises ethanol. In one embodiment, particles are formed by exposing RNA, dissolved in an aqueous phase, with lipids, dispersed in an aqueous phase. In one embodiment, the lipids dispersed in an aqueous phase form liposomes.

[0133] In one embodiment, a method disclosed herein is a method for vaccination against coronavirus.

[0134] In one embodiment, a method disclosed herein is a method for therapeutic or prophylactic treatment of a coronavirus infection.

[0135] In one embodiment, a subject is a human.

[0136] In one embodiment, the coronavirus is a betacoronavirus.

[0137] In one embodiment, the coronavirus is a sarbecovirus.

[0138] In one embodiment, the coronavirus is SARS-CoV-2.

[0139] In one embodiment of methods described herein, a composition described herein is administered to a subject.

[0140] In one aspect, the present disclosure relates to a composition or medical preparation described herein for use in a method described herein.

[0141] Among other things, the present disclosure teaches that a composition comprising a lipid nanoparticle encapsulated RNA encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV- 2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein) can achieve detectable antibody titer againstan epitope in serum within 7 days after administration to a population of adult human subjects according to a regimen that includes administration of at least one dose of the composition. Moreover, the present disclosure teaches persistence of such antibody titer. In some embodiments, such antibody titer is increased when a modified mRNA is used, as compared with titer achieved with a corresponding unmodified mRNA.

[0142] In some embodiments, a provided regimen includes at least one dose. In some embodiments, a provided regimen includes a first dose and at least one subsequent dose. In some embodiments, the first dose is the same amount as at least one subsequent dose. In some embodiments, the first dose is the same amount as all subsequent doses. In some embodiments, the first dose is a different amount as at least one subsequent dose. In some embodiments, the first dose is a different amount than all subsequent doses. In some embodiments, a provided regimen comprises two doses. In some embodiments, a provided regimen consists of two doses.

[0143] In particular embodiments, an immunogenic composition is formulated as a single-dose in a container, e.g., a vial. In some embodiments, an immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in the vial is equal in volume. In some embodiments, a first dose is a different volume than a subsequent dose.

[0144] A "stable" multi-dose formulation exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunologic response when administered to a subject.

[0145] In some embodiments, a multi-dose formulation remains stable for a specified 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 usages, when contained within a multi-dose container. In some embodiments, multi-dose formulations remain stable with 2-20 inoculations / insertions.

[0146] In some embodiments, administration of a composition comprising a lipid nanoparticle encapsulated RNA (e.g., in some embodiments mRNA) encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein), e.g., according to a regimen as described herein, may result in lymphopenia in some subjects (e.g., in all subjects, in most subjects, in about 50% or fewer, in about 40% or fewer, in about 40% or fewer, in about 25% or fewer, in about 20% or fewer, in about 15% or fewer, in about 10% or fewer, in about 5% or fewer, etc). Among other things, the present disclosure teaches that such lymphopenia can resolve over time. For example, in some embodiments, lymphopenia resolves within about 14, about 10, about 9, about 8, about 7 days or less. In some embodiments, lymphopenia is Grade 3, Grade 2, or less.

[0147] Thus, among other things, the present disclosure provides compositions comprising a lipid nanoparticle encapsulated RNA (e.g., in some embodiments mRNA) encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein) that are characterized, when administered to a relevant population of adults, to display certain characteristics (e.g., achieve certain effects) as described herein. In some embodiments, provided compositions may have been prepared, stored, transported, characterized, and / or used under conditions where temperature does not exceed a particular threshold. Alternatively or additionally, in some embodiments, provided compositions may have been protected from light (e.g., from certain wavelengths) during some or all of their preparation, storage, transport,characterization, and / or use. In some embodiments, one or more features of provided compositions (e.g., RNA stability, as may be assessed, for example, by one or more of size, presence of particular moiety or modification, etc; lipid nanoparticle stability or aggregation, pH, etc) may be or have been assessed at one or more points during preparation, storage, transport, and / or use prior to administration.

[0148] Among other things, the present disclosure documents that certain provided compositions in which nucleotides within an RNA (e.g., in some embodiments mRNA) are not modified (e.g., are naturally-occurring A, U, C, G), and / or provided methods relating to such compositions, are characterized (e.g., when administered to a relevant population, which may in some embodiments be or comprise an adult population), by an intrinsic adjuvant effect. In some embodiments, such a composition and / or method can induce an antibody and / or a T cell response. In some embodiments, such a composition and / or method can induce a higher T cell response, as compared to conventional vaccines (e.g., non-RNA vaccines such as protein vaccines).

[0149] Alternatively or additionally, the present disclosure documents that provided compositions (e.g., compositions comprising a lipid nanoparticle encapsulated RNA encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein)) in which nucleotides within an RNA are modified, and / or provided methods relating to such compositions, are characterized (e.g., when administered to a relevant population, which may in some embodiments be or comprise an adult population), by absence of an intrinsic adjuvant effect, or by a reduced intrinsic adjuvant effect as compared with an otherwise comparable composition (or method) with unmodified results. Alternatively or additionally, in some embodiments, such compositions (or methods) are characterized in that they (e.g., when administered to a relevant population, which may in some embodiments be or comprise an adult population) 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 relevant population, which may in some embodiments be or comprise an adult population) induce a higher CD4+ T cell response than that observed with an alternative vaccine format (e.g., a peptide vaccine). In some embodiments involving modified nucleotides, such modified nucleotides may be present, for example, in a 3' UTR sequence, an antigen-encoding sequence, and / or a 5'UTR sequence. In some embodiments, modified nucleotides are or include one or more modified uracil residues and / or one or more modified cytosine residues.

[0150] Among other things, the present disclosure documents that provided (e.g., compositions comprising a lipid nanoparticle encapsulated RNA encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein)) and / or methods are characterized by (e.g., when administered to a relevant population, which may in some embodiments be or comprise an adult population) sustained expression of an encoded polypeptide (e.g., of a SARS-CoV-2-encoded protein [such as an S protein] or portion thereof, which portion, in some embodiments, may be or comprise an epitope thereof). For example, in some embodiments, such compositions and / or methods are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such human and, in some embodiments, such expression persists for a period of time that is at least at least 36 hours or longer, including, e.g., 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 longer.

[0151] Those skilled in the art, reading the present disclosure, will appreciate that it describes various RNA constructs (e.g., in some embodiments mRNA constructs) encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein)). Such person of ordinary skill, reading the present disclosure, will particularly appreciate that it describes various RNA constructs(e.g., in some embodiments mRNA constructs) encoding at least a portion of a SARS-CoV-2 S protein, for example at least an RBD portion of a SARS-CoV-2 S protein. Still further, such a person of ordinary skill, reading the present disclosure, will appreciate that it describes particular characteristics and / or advantages of RNA constructs (e.g., in some embodiments mRNA constructs) encoding at least a portion (e.g., that is or comprises an epitope) of a SARS-CoV-2-encoded polypeptide (e.g., of a SARS-CoV-2-encoded S protein). In some embodiments, an RNA construct (e.g., in some embodiments, an mRNA construct) may encode at least one domain of a SARS-CoV-2 encoded polypeptide (e.g., one or more domains of a SARS-CoV-2 encoded polypeptide as described in WO 2021 / 159040, including, e.g., an N-terminal domain (NTD) of a SARS-CoV-2 Spike protein, a receptor binding domain (RBD) of a SARS- CoV-2 Spike protein, Heptapeptide repeat sequence 1 (HR1) of a SARS-CoV-2 Spike protein, Heptapeptide repeat sequence 2 (HR1) of a SARS-CoV-2 Spike protein, and / or combinations thereof). Among other things, the present disclosure particularly documents surprising and useful characteristics and / or advantages of certain RNA constructs (e.g., in some embodiments mRNA constructs) encoding a SARS-CoV-2 RBD portion and, in some embodiments, not encoding a full length SARS-CoV-2 S protein. Without wishing to be bound by any particular theory, the present disclosure suggests that provided RNA constructs (e.g., in some embodiments mRNA constructs) that encode less than a full-length SARS-CoV-2 S protein, and particularly those that encode at least an RBD portion of such SARS-CoV-2 S protein may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine), and / or for achieving immunological effects as described herein (e.g., generation of SARS-CoV-2 neutralizing antibodies, and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)).

[0152] In some embodiments, the present disclosure provides an RNA (e.g., mRNA) comprising an open reading frame encoding a polypeptide that comprises a receptor-binding portion of a SARS-CoV-2 S protein, which RNA is suitable for intracellular expression of the polypeptide. In some embodiments, such an encoded polypeptide does not comprise the complete S protein. In some embodiments, an encoded polypeptide comprises the receptor binding domain (RBD), for example, as shown in SEQ ID NO: 5. In some embodiments, the encoded polypeptide comprises the peptide according to SEQ ID NO: 29 or 31. In some embodiments, such an RNA (e.g., mRNA) may be complexed by a (poly)cationic polymer, polyplex(es), protein(s) or peptide(s). In some embodiments, such an RNA may be formulated in a lipid nanoparticle (e.g., ones described herein). In some embodiments, such an RNA (e.g., mRNA) may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine), and / or for achieving immunological effects as described herein (e.g., generation of SARS-CoV-2 neutralizing antibodies, and / or T cell responses (e.g., CD4+ and / or CD8+ T cell responses)). In some embodiments, such an RNA (e.g., mRNA) may be useful for vaccinating humans (including, e.g., humans known to have been exposed and / or infected by SARS-CoV-2, and / or humans not known to have been exposed to SARS-CoV-2).

[0153] Those skilled in the art, reading the present disclosure, will further appreciate that it describes various mRNA constructs comprising a nucleic acid sequence that encodes a full-length SARS-CoV-2 Spike protein (e.g., including embodiments in which such encoded SARS-CoV-2 Spike protein may comprise at least one or more amino acid substitutions, e.g., proline substitutions as described herein, and / or embodiments in which the mRNA sequence is codon-optimized e.g., for mammalian, e.g., human, subjects). In some embodiments, such a full-length SARS-CoV-2 Spike protein may have an amino acid sequence that is or comprises that set forth in SEQ ID NO: 7. Still further, such a person of ordinary skill, reading the present disclosure, will appreciate, among other things, that it describes particular characteristics and / or advantages of certain mRNA constructs comprising a nucleic acid sequence that encodes a full-length SARS-CoV-2 Spike protein. Without wishing to be bound by anyparticular theory, the present disclosure suggests that provided mRNA constructs that encode a full-length SARS- CoV-2 S protein may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine) in particular subject population (e.g., particular age populations). For example, in some embodiments, such an mRNA composition may be particularly useful in younger (e.g., less than 25 years old, 20 years old, 18 years old, 15 years, 10 years old, or lower) subjects; alternatively or additionally, in some embodiments, such an mRNA composition may be particularly useful in elderly subjects (e.g., over 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, or higher). In particular embodiments, an immunogenic composition comprising such an mRNA construct provided herein exhibits a minimal to modest increase (e.g., no more than 30% increase, no more than 20% increase, or no more than 10% increase, or lower) in dose level and / or dose number-dependent systemic reactogenicity (e.g., fever, fatigue, headache, chills, diarrhea, muscle pain, and / or joint pain, etc.) and / or local tolerability (e.g., pain, redness, and / or swelling, etc.), at least in some subjects (e.g., in some subject age groups); in some embodiments, such reactogenicity and / or local tolerability is observed particularly, in in younger age group (e.g., less than 25 years old, 20 years old, 18 years old or lower) subjects, and / or in older (e.g., elderly) age group (e.g., 65-85 years old). In some embodiments, provided mRNA constructs that encode a full-length SARS-CoV-2 S protein may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine) for inducing SARS-CoV-2 neutralizing antibody response level in a population of subjects that are at high risk for severe diseases associated with SARS-CoV-2 infection (e.g., an elderly population, for example, 65-85 year-old group). In some embodiments, a person of ordinary skill, reading the present disclosure, will appreciate, among other things, that provided mRNA constructs that encode a full-length SARS-CoV-2 S protein, which exhibit a favorable reactogenicity profile (e.g., as described herein) in younger and elderly age populations, may be particularly useful and / or effective for use as or in an immunogenic composition (e.g., a vaccine) for achieving immunological effects as described herein (e.g., generation 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 suggests that provided mRNA constructs that encode a full-length SARS-CoV-2 S protein may be particularly effective to protect against SARS-CoV-2 infection, as characterized by earlier clearance of SARS-CoV-2 viral RNA in non-human mammalian subjects (e.g., rhesus macaques) that were immunized with immunogenic compositions comprising such mRNA constructs and subsequently challenged by SARS-CoV-2 strain. In some embodiments, such earlier clearance of SARS-CoV-2 viral RNA may be observed in the nose of non-human mammalian subjects (e.g., rhesus macaques) that were immunized with immunogenic compositions comprising such mRNA constructs and subsequently challenged by SARS-CoV-2 strain.

[0154] In some embodiments, the present disclosure provides an RNA (e.g., mRNA) comprising an open reading frame encoding a full-length SARS-CoV-2 S protein (e.g., a full-length SARS-CoV-2 S protein with one or more amino acid substitutions), which RNA is suitable for intracellular expression of the polypeptide. In some embodiments, the encoded polypeptide comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, such an RNA (e.g., mRNA) may be complexed by a (poly)cationic polymer, polyplex(es), protein(s) or peptide(s). In some embodiments, such an RNA may be formulated in a lipid nanoparticle (e.g., ones described herein).

[0155] In some embodiments, an immunogenic composition provided herein may comprise a plurality of (e.g., at least two or more, including, e.g., 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 variants thereof. In some such embodiments, such a plurality of immunoreactive epitopes may be encoded by a pluralityof RNAs (e.g., mRNAs). In some such embodiments, such a plurality of immunoreactive epitopes may be encoded by a single RNA (e.g., mRNA). In some embodiments, nucleic acid sequences encoding a plurality of immunoreactive epitopes may be separated from each other in a single RNA (e.g., mRNA) by a linker (e.g., a peptide linker in some embodiments). Without wishing to be bound by any particular theory, in some embodiments, provided polyepitope immunogenic compositions (including, e.g., those that encode a full-length SARS-CoV-2 spike protein) may be particularly useful, when considering the genetic diversity of SARS-CoV-2 variants, to provide protection against numerous viral variants and / or may offer a greater opportunity for development of a diverse and / or otherwise robust (e.g., persistent, e.g., detectable about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days after administration of one or more doses) neutralizing antibody and / or T cell response, and in particular a particularly robust THl-type T cell (e.g., CD4+ and / or CD8+ T cell) response.

[0156] In some embodiments, the present disclosure documents that provided compositions and / or methods are characterized by (e.g., when administered to a relevant population, which may in some embodiments be or comprise an adult population) in that they achieve one or more particular therapeutic outcomes (e.g., effective immune responses as described herein and / or detectable expression of encoded SARS- CoV-2 S protein or an immunogenic fragment thereof) with a single administration; in some such embodiments, an outcome may be assessed, for example, as compared to that observed in absence of RNA vaccines (e.g., mRNA vaccines) described herein. In some embodiments, a particular outcome may be achieved at a lower dose than required for one or more alternative strategies.

[0157] In some embodiments, the present 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 that comprises a receptor-binding portion of a SARs-CoV-2 S protein, and wherein the isolated mRNA polynucleotide is formulated in at least one lipid nanoparticle. For example, in some embodiments, such a lipid nanoparticle may comprise a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid (e.g., neutral lipid), 25-55% sterol or steroid, and 0.5-15% polymer- conjugated lipid (e.g., PEG-modified lipid). In some embodiments, a sterol or steroid included in a lipid nanoparticle may be or comprise cholesterol. In some embodiments, a neutral lipid may be or comprise 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, a polymer-conjugated lipid may be or comprise PEG2000 DMG. In some embodiments, such an immunogenic composition may comprise a total lipid content of about 1 mg to 10 mg, or 3 mg to 8 mg, or 4 mg to 6 mg. In some embodiments, such an immunogenic composition may comprise a total lipid content of about 5 mg / mL -15 mg / mL or 7.5 mg / mL- 12.5 mg / mL or 9-11 mg / mL. In some embodiments, such an isolated mRNA polynucleotide is provided in an effective amount to induce an immune response in a subject administered at least one dose of the immunogenic composition. In some embodiments, a polypeptide encoded by a provided isolated mRNA polynucleotide does not comprise the complete S protein. In some embodiments, such an isolated mRNA polynucleotide provided in an immunogenic composition is not self-replicating RNA.

[0158] In some embodiments, an immune response may comprise generation of a binding antibody titer against SARS-CoV-2 protein (including, e.g., a stabilized prefusion spike trimer in some embodiments) or a fragment thereof. In some embodiments, an immune response may comprise generation of a binding antibody titer against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some embodiments, a provided immunogenic composition has been established to achieve a detectable binding antibody titer after administration of a first dose, with seroconversion in at least 70% (including, e.g., at least 80%, at least 90%, atleast 95% and up to 100%) of a population of subjects receiving such a provided immunogenic composition, for example, by about 2 weeks.

[0159] In some embodiments, an immune response may comprise generation of a neutralizing antibody titer against SARS-CoV-2 protein (including, e.g., a stabilized prefusion spike trimer in some embodiments) or a fragment thereof. In some embodiments, an immune response may comprise generation of a neutralizing antibody titer against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In some embodiments, a provided immunogenic composition has been established to achieve a neutralizing antibody titer in an appropriate system (e.g., in a human infected with SARS-CoV-2 and / or a population thereof, and / or in a model system therefor). For example, in some embodiments, such neutralizing antibody titer may have been demonstrated in one or more of a population of humans, a non-human primate model (e.g., rhesus macaques), and / or a mouse model.

[0160] In some embodiments, a neutralizing antibody titer is a titer that is (e.g., that has been established to be) sufficient to reduce viral infection of B cells relative to that observed for an appropriate control (e.g., an unvaccinated control subject, 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, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0161] In some embodiments, a neutralizing antibody titer is a titer that is (e.g., that has been established to be) sufficient to reduce the rate of asymptomatic viral infection relative to that observed for an appropriate control (e.g., an unvaccinated control subject, 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, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, such reduction can be characterized by assessment of SARS- CoV-2 N protein serology. Significant protection against asymptomatic infection was also confirmed by real life observations (see also: Dagan N. et al., N Engl J Med. 2021, doi: 10.1056 / NEJMoa2101765. Epub ahead of print. PMID: 33626250)

[0162] In some embodiments, a neutralizing antibody titer is a titer that is (e.g., that has been established to be) sufficient to reduce or block fusion of virus with epithelial cells and / or B cells of a vaccinated subject relative to that observed for an appropriate control (e.g., an unvaccinated control subject, 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, such reduction is of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0163] In some embodiments, induction of a neutralizing antibody titer may be characterized by an elevation in the number of B cells, which in some embodiments may include plasma cells, class-switched IgGl- and IgG2-positive B cells, and / or germinal center B cells. In some embodiments, a provided immunogenic composition has been established to achieve such an elevation in the number of B cells in an appropriate system (e.g., in a human infected with SARS-CoV-2 and / or a population thereof, and / or in a model system therefor). For example, in some embodiments, such an elevation in the number of B cells may have been demonstrated in one or more of a population of humans, a non-human primate model (e.g., rhesus macaques), and / or a mouse model. In some embodiments, such an elevation in the number of B cells may have been demonstrated in draining lymph nodes and / or spleen of a mouse model after (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, after) immunization of such a mouse model with a provided immunogenic composition.

[0164] In some embodiments, induction of a neutralizing antibody titer may be characterized by a reduction in the number of circulating B cells in blood. In some embodiments, a provided immunogenic composition has been established to achieve such a reduction in the number of circulating B cells in blood of an appropriate system (e.g., in a human infected with SARS-CoV-2 and / or a population thereof, and / or in a model system therefor). For example, in some embodiments, such a reduction in the number of circulating B cells in blood may have been demonstrated in one or more of a population of humans, a non-human primate model (e.g., rhesus macaques), and / or a mouse model. In some embodiments, such a reduction in the number of circulating B cells in blood may have been demonstrated in a mouse model after (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, after) immunization of such a mouse model with a provided immunogenic composition. Without wishing to be bound by theory, a reduction in circulating B cells in blood may be due to B cell homing to lymphoid compartments.

[0165] In some embodiments, an immune response induced by a provided immunogenic composition may comprise an elevation in the number of T cells. In some embodiments, such an elevation in the number of T cells may include an elevation in the number of T follicular helper (TFH) cells, which in some embodiments may comprise one or more subsets with ICOS upregulation. One of skilled in the art will understand that proliferation of TFH in germinal centres is integral for generation of an adaptive B-cell response, and also that in humans, TFH occurring in the circulation after vaccination is typically correlated with a high frequency of antigen-specific antibodies. In some embodiments, a provided immunogenic composition has been established to achieve such an elevation in the number of T cells (e.g., TFH cells) in an appropriate system (e.g., in a human infected with SARS- CoV-2 and / or a population thereof, and / or in a model system therefor). For example, in some embodiments, such an elevation in the number of T cells (e.g., TFH cells) may have been demonstrated in one or more of a population of humans, a non-human primate model (e.g., rhesus macaques), and / or a mouse model. In some embodiments, such an elevation in the number of T cells (e.g., e.g., TFH cells) may have been demonstrated in draining lymph nodes, spleen, and / or blood of a mouse model after (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, after) immunization of such a mouse model with a provided immunogenic composition.

[0166] In some embodiments, a protective response against SARS-CoV-2 induced by a provided immunogenic composition has been established in an appropriate model system for SARS-CoV-2. For example, in some embodiments, such a protective response may have been demonstrated in an animal model, e.g., a non- human primate model (e.g., rhesus macaques) and / or a mouse model. In some embodiments, a non-human primate (e.g., rhesus macaque) or a population thereof that has / have received at least one immunization with a provided immunogenic composition is / are challenged with SARS-CoV-2, e.g., through intranasal and / or intratracheal route. In some embodiments, such a challenge may be performed several weeks (e.g., 5-10 weeks) after at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition. In some embodiments, such a challenge may be performed when a detectable level of a SARS-CoV- 2 neutralizing titer (e.g., antibody response to SARS-CoV-2 spike protein and / or a fragment thereof, including, e.g., but not limited to a stabilized prefusion spike trimer, S-2P, and / or antibody response to receptor-binding portion of SARS-CoV-2) is achieved in non-human primate(s) (e.g., rhesus macaque(s)) that has received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition. In some embodiments, a protective response is characterized by absence of or reduction in detectable viral RNA in bronchoalveolar lavage (BAL) and / or nasal swabs of challenged non-human primate(s) (e.g., rhesus macaque(s)).In some embodiments, immunogenic compositions described herein may have been characterized in that a larger percent of challenged animals, for example, non-human primates in a population (e.g., rhesus macaques), that have received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition display absence of detectable RNA in their BAL and / or nasal swab, as compared to a population of non-immunized animals, for example, non-human primates (e.g., rhesus macaques). In some embodiments, immunogenic compositions described herein may have been characterized in that challenged animals, for example, non-human in a population (e.g., rhesus macaques), that have received at least one immunization (including, e.g., at least two immunizations) with a provided immunogenic composition may show clearance of viral RNA in 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., as compared to a population of non-immunized animals, for example, non-human primates (e.g., rhesus macaques).

[0167] In some embodiments, immunogenic compositions described herein when administered to subjects in need thereof do not substantially increase the risk of vaccine-associated enhanced respiratory disease. In some embodiments, such vaccine-associated enhanced respiratory disease may be associated with antibody-dependent enhancement of replication and / or with vaccine antigens that induced antibodies with poor neutralizing activity and Th2-biased responses. In some embodiments, immunogenic compositions described herein when administered to subjects in need thereof do not substantially increase the risk of antibody-dependent enhancement of replication.

[0168] In some embodiments, a single dose of an RNA composition (e.g., mRNA formulated in lipid nanoparticles) can induce a therapeutic antibody response in less than 10 days of vaccination. In some embodiments, such a therapeutic antibody response may be characterized in that when such an RNA vaccine can induce production of about 10-100 ug / mL IgG measured at 10 days after vaccination at a dose of 0.1 to 10 ug or 0.2- 5 ug in an animal model. In some embodiments, such a therapeutic antibody response may be characterized in that such an RNA vaccine induces about 100-1000 ug / mL IgG measured at 20 days of vaccination at a dose of 0.1 to 10 ug or 0.2- 5 ug in an animal model. In some embodiments, a single dose may induce a pseudovirusneutralization titer, as measured in an animal model, of 10-200 pVN50 titer 15 days after vaccination. In some embodiments, a single dose may induce a pseudovirus-neutralization titer, as measured in an animal model, of 50-500 pVN50 titer 15 days after vaccination.

[0169] In some embodiments, a single dose of an RNA composition (e.g., mRNA composition) can expand antigen-specific CD8 and / or CD4 T cell response by at least at 50% or more (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more), as compared to that observed in absence of such an RNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain). In some embodiments, a single dose of an RNA composition can expand antigenspecific CD8 and / or CD4 T cell response by at least at 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), as compared to that observed in absence of such an RNA construct encoding a SARS-COV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain).

[0170] In some embodiments, a regimen (e.g., a single dose of an mRNA composition) can expand T cells that exhibit a Thl phenotype (e.g., as characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5) by at least at 50% or more (including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more), as compared to that observed in 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 someembodiments, a regimen (e.g., a single dose of an mRNA composition) can expand T cells that exhibit a Thl phenotype (e.g., as characterized by expression of IFN-gamma, IL-2, IL-4, and / or IL-5), for example by at least at 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), as compared to that observed in 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, a T-cell phenotype may be or comprise a Thl-dominant cytokine profile (e.g., as characterized by INF-gamma positive and / or IL-2 positive), and / or no by or biologically insignificant IL-4 secretion.

[0171] In some embodiments, a regimen as described herein (e.g., one or more doses of an mRNA composition) induces and / or achieves production of RBD-specific CD4+ T cells. Among other things, the present disclosure documents that mRNA compositions encoding an RBD-containing portion of a SARS-CoV-2 spike protein (e.g., and not encoding a 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 an mRNA composition described herein (e.g., by an mRNA composition that encodings an RBD- containing-portion of a SARS-CoV-2 spike protein and, in some embodiments not encoding a full-length SARS- CoV-2 spike protein) demonstrate a Thl-dominant cytokine profile (e.g., as characterized by INF-gamma positive and / or IL-2 positive), and / or by no or biologically insignificant IL-4 secretion.

[0172] In some embodiments, characterization of CD4+ and / or CD8+ T cell responses (e.g., described herein) in subjects receiving RNA compositions (e.g., as described herein) may be performed using ex vivo assays using PBMCs collected from the subjects.

[0173] In some embodiments, immunogenicity of RNA (e.g., mRNA) compositions described herein may be assessed by one of or more of the following serological immunongenicity assays: detection of IgG, IgM, and / or IgA to SARS-CoV-2 S protein present in blood samples of a subject receiving a provided RNA composition, and / or neutralization assays using SARS-CoV-2 pseudovirus and / or a wild-type SARS-CoV-2 virus.

[0174] In some embodiments, an RNA composition (e.g., as described herein) provide a relatively low adverse effect (e.g., Grade 1-Grade 2 pain, redness and / or swelling) within 7 days after vaccinations at a dose of 10 ug - 100 ug or 1 ug-50 ug. In some embodiments, RNA compositions (e.g., as described herein) provide a relatively low observation of systemic events (e.g., Grade 1-Grade 2 fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, medication, and combinations thereof ) within 7 days after vaccinations at a dose of 10 ug - 100 ug.

[0175] In some embodiments, RNA (e.g., mRNA) compositions are characterized in that when administered to subjects at 10-100 ug dose or 1 ug-50 ug, IgG directed to a SARS-CoV2 immunogenic protein or fragment thereof (e.g., spike protein and / or receptor binding domain) may be produced at a level of 100-100,000 U / mL or 500-50,000 U / mL 21 days after vaccination.

[0176] In some embodiments, an RNA (e.g., mRNA) encodes a natively-folded trimeric receptor binding protein of SARS-CoV-2. In some embodiments, an RNA (e.g., mRNA) encodes a variant of such receptor binding protein such that the encoded variant binds to ACE2 at a Kd of 10 pM or lower, including, e.g., at a Kd of 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, or lower. In some embodiments, an RNA (e.g., mRNA) encodes a variant of such receptor binding protein such that the encoded variant binds to ACE2 at a Kd of 5 pM. In some embodiments, an RNA (e.g., mRNA) encodes a trimeric receptor binding portion of SARS-CoV-2 that comprises an ACE2 receptor binding site. In some embodiments, an RNA (e.g., mRNA) comprises a coding sequence for a receptor-binding portion of SARS-CoV-2 and a trimerization domain (e.g., a natural trimerization domain (foldon) of T4 fibritin)such that the coding sequence directs expression of a trimeric protein that has an ACE2 receptor binding site and binds ACE2. In some embodiments, an RNA (e.g., mRNA) encodes a trimeric receptor binding portion of SARS- CoV-2 or a variant thereof such that its Kd is smaller than that for a monomeric receptor-binding domain (RBD) of SARS-CoV-2. For example, in some embodiments, an 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 1000-fold, etc.) smaller than that for a RBD of SARS-CoV-2.

[0177] In some embodiments, a trimer receptor binding portion of SARS-CoV-2 encoded by an RNA (e.g., as described herein) may be determined to have a size of about 3-4 angstroms when it is complexed with ACE2 and B0AT1 neutral amino acid transporter in a closed conformation, as characterized by electron cryomicroscopy (cryoEM). In some embodiments, geometric mean SARS-CoV-2 neutralizing titer that characterizes and / or is achieved by an RNA composition or method 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 higher, that of a COVID-19 convalescent human panel (e.g., a panel of sera from COVID-19 convalescing humans obtained 20-40 days after the onset of symptoms and at least 14 days after the start of asymptomatic convalescence.

[0178] In some embodiments, RNA compositions as provided herein may be characterized in that subjects who have been treated with such compositions (e.g., with at least one dose, at least two doses, etc) may show reduced and / or more transient presence of viral RNA in relevant site(s) (e.g., nose and / or lungs, etc, and / or any other tissue susceptible to infection) as compared with an appropriate control (e.g., an established expected level for a comparable subject or population not having been so treated and having been exposed to virus under reasonably comparable exposure conditions)

[0179] In some embodiments, the RBD antigen expressed by an mRNA construct (e.g., as described herein) can be modified by addition of a T4-fibritin-derived "foldon" trimerization domain, for example, to increase its immunogenicity.

[0180] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) may appear and / or peak at Day 2 after vaccination. In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that certain local reactions (e.g., pain, redness, and / or swelling, etc.) and / or systemic events (e.g., fever, fatigue, headache, etc.) may resolve by Day 7 after vaccination.

[0181] In some embodiments, RNA compositions (e.g., mRNA) and / or methods described herein are characterized in that no Grade 1 or greater change in routine clinical laboratory values or laboratory abnormalities are observed in subjects receiving RNA compositions (e.g., as described herein). Examples of such clinical laboratory assays may include lymphocyte count, hematological changes, etc.

[0182] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that by 21 days after a first dose (e.g., 10-100 ug inclusive or 1 ug-50 ug inclusive), geometric mean concentrations (GMCs) of IgG directed to a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof (e.g., RBD) may reach 200-3000 units / mL or 500-3000 units / mL or 500-2000 units / mL, compared to 602 units / mL for a panel of COVID-19 convalescent human sera. In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that by 7 days after a second dose (e.g., 10-30 ug inclusive; or 1 ug-50 ug inclusive), geometric mean concentrations (GMCs) of IgG directed to a SARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g., RBD) may increase by at least 8-fold or higher, including, e.g., 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 higher. In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that by 7 days after a second dose (e.g., 10-30 ug inclusive; or 1 ug-50 ug inclusive), geometric mean concentrations (GMCs) of IgG directed to a SARS-CoV-2 S polypeptide or an immunogenic fragment thereof (e.g., RBD) may increase to 1500 units / mL to 40,000 units / mL or 4000 units / mL to 40,000 units / mL. In some embodiments, antibody concentrations described herein can persist to at least 20 days or longer, including, e.g., at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, after a first dose, or at least 10 days or longer, including, e.g., at least 15 days, at least 20 days, at least 25 days, or longer, after a second dose. In some embodiments, antibody concentrations can persist to 35 days after a first dose, or at least 14 days after a second dose.

[0183] In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that when measured at 7 days after a second dose (e.g., 1-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptide or an 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, as compared to antibody concentrations observed in a panel of COVID-19 convalescent human serum. In many embodiments, geometric mean concentration (GMC) of IgG described herein is GMCs of RBD-binding IgG.

[0184] In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that when measured at 7 days after a second dose (e.g., 10-50 ug inclusive), GMC of IgG directed to a SARS-CoV-2 S polypeptide or an 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), as compared to antibody concentrations observed in a panel of COVID-19 convalescent human serum, In many embodiments, geometric mean concentration (GMC) of IgG described herein is GMCs of RBD-binding IgG.

[0185] In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that when measured at 21 days after a second dose, GMC of IgG directed to a SARS-CoV-2 S polypeptide or an 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), as compared to antibody concentrations observed in a panel of COVID-19 convalescent human serum, In many embodiments, geometric mean concentration (GMC) of IgG described herein is GMCs of RBD-binding IgG.

[0186] In some embodiments, 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 SARS-CoV-2 neutralizing geometric mean titers (GMTs) is observed 21 days after a first dose. In some embodiments, RNA (e.g., mRNA) compositions described herein are characterized in that a substantially greater serum neutralizing GMTs are achieved 7 days after subjects receive a second dose (e.g., 10 pg-30 μg inclusive), reaching 150-300, compared to 94 for a COVID-19 convalescent serum panel.

[0187] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that 7 days after administration of the second dose, the protective efficacy is at least 60%, e.g., at least 70%, at least 80%, at least 90, or at least 95%. In one embodiment, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that 7 days after administration of the second dose, the protective efficacy is at least 70%. In one embodiment, RNA (e.g., mRNA) compositions and / or methodsdescribed herein are characterized in that 7 days after administration of the second dose, the protective efficacy is at least 80%. In one embodiment, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that 7 days after administration of the second dose, the protective efficacy is at least 90%. In one embodiment, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that 7 days after administration of the second dose, the protective efficacy is at least 95%.

[0188] In some embodiments, an RNA composition provided herein is characterized in that it induces an immune response against SARS-CoV-2 after at least 7 days after a dose (e.g., after a second dose). In some embodiments, an RNA composition provided herein is characterized in that it induces an immune response against SARS-CoV-2 in less than 14 days after a dose (e.g., after a second dose). In some embodiments, an RNA composition provided herein is characterized in that it induces an immune response against SARS-CoV-2 after at least 7 days after a vaccination regimen. In some embodiments, a vaccination regimen comprises a first dose and a second dose. In some embodiments, a first dose and a second dose are administered by at least 21 days apart. In some such embodiments, an immune response against SARS-CoV-2 is induced at least after 28 days after a first dose.

[0189] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that geometric mean concentration (GMCs) of antibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g., RBD), as measured in serum from subjects receiving RNA (e.g., mRNA) compositions of the present disclosure (e.g., at a dose of 10-30 ug inclusive), is substantially higher than in a convalescent serum panel (e.g., as described herein). In some embodiments where a subject may receive a second dose (e.g., 21 days after 1 first dose), geometric mean concentration (GMCs) of antibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g., RBD), as measured in serum from the subject, may be 8.0-fold to 50-fold higher than a convalescent serum panel GMC. In some embodiments where a subject may receive a second dose (e.g., 21 days after 1 first dose), geometric mean concentration (GMCs) of antibodies directed to a SARS-CoV-2 spike polypeptide or an immunogenic fragment thereof (e.g., RBD), as measured in serum from the subject, may be at least 8.0-fold or higher, including, e.g., 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 a convalescent serum panel GMC.

[0190] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that the SARS-CoV-2 neutralizing geometric mean titer, as measured at 28 days after a first dose or 7 days after a second dose, may be at least 1.5-fold or higher (including, e.g., at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold or higher), as compared to a neutralizing GMT of a convalescent serum panel.

[0191] In some embodiments, a regimen administered to a subject may be or comprise a single dose. In some embodiments, a regimen administered to a subject may comprise a plurality of doses (e.g., at least two doses, at least three doses, or more). In some embodiments, a regimen administered to a subject may comprise a first dose and a second dose, which are given at least 2 weeks apart, at least 3 weeks apart, at least 4 weeks apart, or more. In some embodiments, such doses may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more apart. In some embodiments, doses may be administered days apart, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days apart. In some embodiments, doses may be administered about 1 to about 3 weeks apart, or about 1 to about 4 weeks apart, or about 1 to about 5 weeks apart, or about 1 to about6 weeks apart, or about 1 to more than 6 weeks apart. In some embodiments, doses may be separated by a period of about 7 to about 60 days, such as for example about 14 to about 48 days, etc. In some embodiments, a minimum number of days between doses may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In some embodiments, a maximum number of days between doses may be about 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, TJ, 26, 25, 24, 23, 22, 21, or fewer. In some embodiments, doses may be about 21 to about 28 days apart. In some embodiments, doses may be about 19 to about 42 days apart. In some embodiments, doses may be about 7 to about 28 days apart. In some embodiments, doses may be about 14 to about 24 days. In some embodiments, doses may be about 21 to about 42 days.

[0192] In some embodiments, particularly for compositions established to achieve elevated antibody and / or T-cell titres for a period of time longer than about 3 weeks - e.g., in some embodiments, a provided composition is established to achieve elevated antibody and / or T-cell titres (e.g., specific for a relevant portion of a SARS-CoV- 2 spike protein) for a period of time longer than about 3 weeks; in some such embodiments, a dosing regimen may involve only a single dose, or may involve two or more doses, which may, in some embodiments, be separated from one another by a period of time that is longer than about 21 days or three weeks. For example, in some such embodiments, such 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 wees, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks or more, 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 more, or in some embodiments about a year or more.

[0193] In some embodiments, a first dose and a second dose (and / or other subsequent dose) may be administered by intramuscular injection. In some embodiments, a first dose and a second dose may be administered in the deltoid muscle. In some embodiments, a first dose and a 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., 0.3 mL each) 21 days part. In some embodiments, each dose is about 30 ug. In some embodiments, each dose may be higher than 30 ug, e.g., about 40 ug, about 50 ug, about 60 ug. In some embodiments, each dose may be lower than 30 ug, e.g., about 20 ug, about 10 ug, about 5 ug, etc. In some embodiments, each dose is about 3 ug or lower, e.g., about 1 ug. In some such embodiments, an RNA (e.g., mRNA) composition described herein is administered to subjects of age 16 or older (including, e.g., 16-85 years). In some such embodiments, an RNA composition (e.g., mRNA) described herein is administered to subjects of age 18-55. In some such embodiments, an RNA composition (e.g., mRNA) described herein is administered to subjects of age 56-85. In some embodiments, an RNA (e.g., mRNA) composition described herein is administered (e.g., by intramuscular injection) as a single dose.

[0194] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that RBD-specific IgG (e.g., polyclonal response) induced by such RNA compositions and / or methods exhibit a higher binding affinity to RBD, as compared to a reference human monoclonal antibody with SARS-CoV-2 RBD-binding affinity (e.g., CR3022 as described in J. ter Meulen et al., PLOS Med. 3, e237 (2006).)

[0195] In particular embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that sera of vaccinated subjects display neutralizing activity against SARs-CoV-2 spike variant "B.l.1.28".

[0196] In particular embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that sera of vaccinated subjects display neutralizing activity against SARs-CoV-2 spike variant "B.l.1.248".

[0197] In particular embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that sera of vaccinated subjects display neutralizing activity against SARs-CoV-2 spike variant "501.V2".

[0198] In particular embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that sera of vaccinated subjects display neutralizing activity against SARs-CoV-2 spike variant "B.l.1.248".

[0199] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein are characterized in that sera of vaccinated subjects display neutralizing activity against SARs-CoV-2 spike variant of the Omicron (B.l.1.529) variant. Omicron (B.l.1.529) variant is a variant of SARS-CoV-2 which was detected in South Africa. Multiple Omicron variants or sublineages have arisen, including e.g., the BA.l, BA.2, BA.2.12.1, BA.3, BA.4, BA.5, BA.2.75, XBB, XBB.l, XBB.1.5, XBB.1.16, XBB.2.3, and XBB.2.3.2 sublineages. As used herein, unless otherwise specified, "Omicron variant" refers to the first disclosed Omicron variant (BA.l) or any variant thereof that has since arisen (e.g., Omicron variants described herein).

[0200] SARs-CoV-2 spike proteins encoded by RNA described herein may or may not include a D614G mutation as compared to SEQ ID NO: 1.

[0201] In some embodiments, SARS-CoV-2 spike proteins encoded by RNA described herein comprise a mutation in a furin cleavage site (e.g., in some embodiments residues 682-685 of SEQ ID NO: 1). In some embodiments, SARS-CoV-2 spike proteins encoded by RNA described herein comprise a mutation in the furin cleavage site that prevents cleavage by a furin protease (e.g., a human furin protease). In some embodiments, a SARS-CoV-2 protein described herein comprises a furin mutation disclosed in WO2021163365 or WO2021243122 (e.g., a GSAS mutation), the contents of both of which are incorporated by reference herein in their entirety.

[0202] In some embodiments, RNA (e.g., mRNA) compositions and / or methods described herein can provide protection against SARS-CoV-2 and / or decrease severity of SARS-CoV-2 infection in at least 50% of subjects receiving such RNA compositions and / or methods.

[0203] In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 18-55. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 56-85. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include older subjects (e.g., over age 60, 65, 70, 75, 80, 85, etc, for example subjects of age 65-85). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 18-85. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 18 or younger. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 12 or younger. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include subjects of age 10 or younger. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include adolescent populations (e.g., individuals approximately 12 to approximately 17 years of age). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include pediatric populations (e.g., as described herein). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include infants (e.g., less than 1 year old). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein do not include infants (e.g., less than 1 year) whose mothers have received such RNA (e.g., mRNA) compositions described herein during pregnancy. Without wishing to be bound by any particular theory, a rat study has suggested that a SARS-CoV-2 neutralizing antibody response induced in femalerats given such RNA compositions during pregnancy can pass onto fetuses. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein include infants (e.g., less than 1 year) whose mothers did not receive such RNA compositions described herein during pregnancy. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include pregnant women; in some embodiments, infants whose mothers were vaccinated during pregnancy (e.g., who received at least one dose, or alternatively only who received both doses), are not vaccinated during the first 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) post-birth. Alternatively or additionally, in some embodiments, infants whose mothers were vaccinated during pregnancy (e.g., who received at least one dose, or alternatively only who received both doses), receive reduced vaccination (e.g., lower doses and / or smaller numbers of administrations - e.g., boosters - and / or lower total exposure over a given period of time) after birth, for example during the first 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) post-birth or may need reduced vaccination (e.g., lower doses and / or smaller numbers of administrations - e.g., boosters - over a given period of time), In some embodiments, compositions as provided herein are administered to populations that do not include pregnant women.

[0204] In some particular embodiments, compositions as provided herein are administered to pregnant women according to a regimen that includes a first dose administered 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, compositions as provided herein are administered to pregnant women according to a regimen that includes a first dose administered before about 34 weeks of gestation (e.g., before about 30, 31, 32, 33, 34, 35, 36, 37, 38 weeks of gestation). In some embodiments, compositions as provided herein are administered to pregnant women according to a regimen that includes a first dose administered after about 24 weeks (e.g., after about 27 weeks of gestation, e.g., between about 24 weeks and 34 weeks, or between about 27 weeks and 34 weeks) of gestation and a second dose administered about 21 days later; in some embodiments both doses are administered prior to delivery. Without wishing to be bound by any particular theory, it is proposed that such a regimen (e.g., involving administration of a first dose after about 24 weeks, or 27 weeks of gestation and optionally before about 34 weeks of gestation), and optionally a second dose within about 21 days, ideally before delivery, may have certain advantages in terms of safety (e.g., reduced risk of premature delivery or of fetal morbidity or mortality) and / or efficacy (e.g., carryover vaccination imparted to the infant) relative to alternative dosing regimens (e.g., dosing at any time during pregnancy, refraining from dosing during pregnancy, and / or dosing later in pregnancy for example so that only one dose is administered during gestation. In some embodiments, infants born of mothers vaccinated during pregnancy, e.g., according to a particular regimen as described herein, may not need further vaccination, or may need reduced vaccination (e.g., lower doses and / or smaller numbers of administrations - e.g., boosters and / or lower overall exposure over a given period of time), for a period of time (e.g., as noted herein) after birth.

[0205] In some embodiments, compositions as provided herein are administered to populations in which women are advised against becoming pregnant for a period of time after receipt of the vaccine (e.g., after receipt of a first dose of the vaccine, after receipt of a final dose of the 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 more, 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 more.

[0206] In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include one or more populations with one or more particularly high risk conditions or history, e.g., as noted herein. For example, in some embodiments, populations to be treated with RNA compositions described herein may include subjects whose profession and / or environmental exposure may dramatically increase their risk of getting SARS-CoV-2 infection (including, e.g., but not limited to mass transportation, prisoners, grocery store workers, residents in long-term care facilities, butchers or other meat processing workers, healthcare workers, and / or first responders, e.g., emergency responders). In particular embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include healthcare workers and / or first responders, e.g., emergency responders. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include those with a history of smoking or vaping (e.g., within 6 months, 12 months or more, including a history of chronic smoking or vaping). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include certain ethnic groups that have been determined to be more susceptible to SARS-CoV-2 infection.

[0207] In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include certain populations with a blood type that may have been determined to more susceptible to SARS-CoV-2 infection. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include immunocompromised subjects (e.g., those with HIV / AIDS; cancer patients (e.g., receiving antitumor treatment); patients who are taking certain immunosuppressive drugs (e.g., transplant patients, cancer patients, etc.); autoimmune diseases or other physiological conditions expected to warrant immunosuppressive therapy (e.g., within 3 months, within 6 months, or more); and those with inherited diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency)). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include those with an infectious disease. For example, in some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include those infected with human immunodeficiency virus (HIV) and / or a hepatitis virus (e.g., HBV, HCV). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include those with underlying medical conditions. Examples of such underlying medical conditions may include, but are not limited to hypertension, cardiovascular disease, diabetes, chronic respiratory disease, e.g., chronic pulmonary disease, asthma, etc., cancer, and other chronic diseases such as, e.g., lupus, rheumatoid arthritis, chronic liver diseases, chronic kidney diseases (e.g., Stage 3 or worse such as in some embodiments as characterized by a glomerular filtration rate (GFR) of less than 60 mL / min / 1.73m2). In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include overweight or obese subjects, e.g., specifically including those with a body mass index (BMI) above about 30 kg / m2. In some embodiments, populations to be treated with RNA (e.g., mRNA) compositions described herein may include subjects who have prior diagnosis of COVID-19 or evidence of current or prior SARS-CoV-2 infection, e.g., based on serology or nasal swab. In some embodiments, populations to be treated include white and / or non-Hispanic / non-Latino.

[0208] In some embodiments, certain RNA (e.g., mRNA) compositions described herein may be selected for administration to Asian populations (e.g., Chinese populations), or in particular embodiments to older Asian populations (e.g., 60 years old or over, e.g., 60-85 or 65-85 years old).

[0209] In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to and / or assessed in subject(s) who have been determined not to show evidence of prior infection, and / or of present infection, before administration; in some embodiments, evidence of prior infection and / or of presentinfection, may be or include evidence of intact virus, or any viral nucleic acid, protein, lipid etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and / or tissue), and / or evidence of a subject's immune response to the same. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to and / or assessed in subject(s) who have been determined to show evidence of prior infection, and / or of present infection, before administration; in some embodiments, evidence of prior infection and / or of present infection, may be or include evidence of intact virus, or any viral nucleic acid, protein, lipid etc. present in the subject (e.g., in a biological sample thereof, such as blood, cells, mucus, and / or tissue), and / or evidence of a subject's immune response to the same. In some embodiments, a subject is considered to have a prior infection based on having a positive N-binding antibody test result or positive nucleic acid amplification test (NAAT) result on the day of Dose 1.

[0210] In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject who has been informed of a risk of side effects that may include one or more of, for example: chills, fever, headache, injection site pain, muscle pain, tiredness; in some embodiments, an RNA (e.g., mRNA) composition is administered to a subject who has been invited to notify a healthcare provider if one or more such side effects occurs, is experienced as more than mild or moderate, persists for a period of more than a day or a few days, or if any serious or unexpected event is experienced that the subject reasonably considers may be associated with receipt of the composition. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject who has been invited to notify a healthcare provider of particular medical conditions which may include, for example, one or more of allergies, bleeding disorder or taking a blood thinner medication, breastfeeding, fever, immunocompromised state or taking medication that affects the immune system, pregnancy or plan to become pregnant, etc. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject who has been invited to notify a healthcare provider of having received another COVID-19 vaccine. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject not having one of the following medical conditions: experiencing febrile illness, receiving immunosuppressant therapy, receiving anticoagulant therapy, suffering from a bleeding disorder (e.g., one that would contraindicate intramuscular injection), or pregnancy and / or breastfeeding / lactation. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject not having received another COVID-19 vaccine. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject who has not had an allergic reaction to any component of the RNA (e.g., mRNA) composition. Examples of such allergic reaction may include, but are not limited to difficulty breathing, swelling of fact and / or throat, fast heartbeat, rash, dizziness and / or weakness. In some embodiments, an RNA (e.g., mRNA) composition as provided herein is administered to a subject who received a first dose and did not have an allergic reaction (e.g., as described herein) to the first dose. In some embodiments where allergic reaction occurs in subject(s) after receiving a dose of an RNA (e.g., mRNA) composition as provided herein, such subject(s) may be administered one or more interventions such as treatment to manage and / or reduce symptom(s) of such allergic reactions, for example, fever-reducing and / or anti-inflammatory agents.

[0211] In some embodiments, a subject who has received at least one dose of an RNA (e.g., mRNA) composition as provided herein is informed of avoiding being exposed to a coronavirus (e.g., SARS-CoV-2) unless and until several days (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.) have passed since administration of a second dose. For example, a subject who has received at least one dose of an RNA (e.g., mRNA) composition as provided herein is informed of taking precautionary measures against SARS-CoV-2 infection (e.g., remaining socially distant, wearing masks,frequent hand-washing, etc.) unless and until several days (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.) have passed since administration of a second dose. Accordingly, in some embodiments, methods of administering an RNA (e.g., mRNA) composition as provided herein comprise administering a second dose of such an RNA (e.g., mRNA) composition as provided herein to a subject who received a first dose and took precautionary measures to avoid being exposed to a coronavirus (e.g., SARS-CoV-2).

[0212] In some embodiments, RNA (e.g., mRNA) compositions described herein may be delivered to a draining lymph node of a subject in need thereof, for example, for vaccine priming. In some embodiments, such delivery may be performed by intramuscular administration of a provided RNA (e.g., mRNA) composition.

[0213] In some embodiments, different particular RNA (e.g., mRNA) compositions may be administered to different subject population(s); alternatively or additionally, in some embodiments, different dosing regimens may be administered to different subject populations. For example, in some embodiments, RNA (e.g., mRNA) compositions administered to particular subject population(s) may be characterized by one or more particular effects (e.g., incidence and / or degree of effect) in those subject populations. In some embodiments, such effect(s) may be or comprise, for example titer and / or persistence of neutralizing antibodies and / or T cells (e.g., THl-type T cells such as CD4+ and / or CD8+ T cells), protection against challenge (e.g., via injection and / or nasal exposure, etc), incidence, severity, and / or persistence of side effects (e.g., reactogenicity), etc.

[0214] In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to reduce COVID-19 incidence per 1000 person-years, e.g., based on a laboratory test such as nucleic acid amplification test (NAAT). In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to reduce COVID-19 incidence per 1000 person-years based on a laboratory test such as nucleic acid amplification test (NAAT) in subjects receiving at least one dose of a provided RNA (e.g., mRNA) composition with no serological or virological evidence (e.g., up to 7 days after receipt of the last dose) of past SARS-CoV-2 infection. In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to reduce confirmed severe COVID-19 incidence per 1000 person-years. In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to reduce confirmed severe COVID-19 incidence per 1000 person-years in subjects receiving at least one dose of a provided RNA (e.g., mRNA) composition with no serological or virological evidence of past SARS-CoV-2 infection.

[0215] In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to produce neutralizing antibodies directed to a SARS-CoV-2 spike polypeptide and / or an immunogenic fragment thereof (e.g., RBD) as measured in serum from a subject that achieves or exceeds a reference level (e.g., a reference level determined based on human SARS-CoV-2 infection / COVID-19 convalescent sera) for a period of time and / or induction of cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, e.g., 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 an immunogenic fragment thereof (e.g., RBD) for a period of time. 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) may be presented on a MHC class I allele that is present in at least 50% of subjects in aleast 80%, at least 90%, or more; in some such HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA-HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA- B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0216] In some embodiments, efficacy is assessed as COVID-19 incidence per 1000 person-years in individuals without serological or virological evidence of past SARS-CoV-2 infection before and during vaccination regimen; alternatively or additionally, in some embodiments, efficacy is assessed as COVID-19 incidence per 1000 person-years in subjects with and without evidence of past SARS-CoV-2 infection before and during vaccination regimen. In some such embodiments, such incidence is of COVID-19 cases confirmed within a specific time period after the final vaccination dose (e.g., a first dose in a single-dose regimen; a second dose in a two-dose regimen, etc); in some embodiments, such time period may be within (i.e., up to and including 7 days) 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). In some embodiments, such time period may be within 7 days or within 14 days or within 21 days or within 28 days. In some embodiments, such time period may be within 7 days. In some embodiments, such time period may be within 14 days.

[0217] In some embodiments (e.g., in some embodiments of assessing efficacy), a subject is determined to have experienced COVID-19 infection if one or more of the following is established: detection of SARS-CoV-2 nucleic acid in a sample from the subject, detection of antibodies that specifically recognize SARS-CoV-2 (e.g., a SARS-Co-V-2 spike protein), one or more symptoms of COVID-19 infection, and combinations thereof. In some such embodiments, detection of SARS-CoV-2 nucleic acid may involve, for example, NAAT testing on a mid- turbinatae swap sample. In some such embodiments, detection of relevant antibodies may involve serological testing of a blood sample or portion thereof. In some such embodiments, symptoms of COVID-19 infection may be or include: fever, new or increased cough, new or increased shortness of breath, chills, new or increased muscle pain, new loss of taste or smell, sore throat, diarrhea, vomiting and combinations thereof. In some such embodiments, symptoms of COVID-19 infection may be or include: fever, new or increased cough, new or increased shortness of breath, chills, new or increased muscle pain, new loss of taste or smell, sore throat, diarrhea, vomiting, fatigue, headache, nasal congestion or runny nose, nausea, and combinations thereof. In some such embodiments, a subject is determined to have experienced COVID-19 infection if such subject both has experienced one such symptom and also has received a positive test for SARS-CoV-2 nucleic acid or antibodies, or both. In some such embodiments, a subject is determined to have experienced COVID-19 infection if such subject both has experienced one such symptom and also has received a positive test for SARS-CoV-2 nucleic acid. In some such embodiments, a subject is determined to have experienced COVID-19 infection if such subject both has experienced one such symptom and also has received a positive test for SARS-CoV-2 antibodies.

[0218] In some embodiments (e.g., in some embodiments of assessing efficacy), a subject is determined to have experienced severe COVID-19 infection if such subject has experienced one or more of: clinical signs at rest indicative or severe systemic illness (e.g., one or more of respiratory rate at greater than or equal to 30 breaths per minute, heart rate at or above 125 beats per minute, SpO2 less than or equal to 93% on room air at sea level or a PaO2 / FiO2 below 300 m Hg), respiratory failure (e.g., one or more of needing high-flow oxygen, noninvasive ventilation, mechanical ventilation, ECMO), evidence of shock (systolic blood pressure below 90 mm Hg, diastolic blood pressure below 60mm Hg, requiring vasopressors), significant acute renal, hepatic, or neurologic dysfunction, admission to an intensive care unit, death, and combinations thereof.

[0219] In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to reduce the percentage of subjects reporting at least one of the following: (i) one or more local reactions (e.g., as described herein) for up to 7 days following each dose; (ii) one or more systemic events for up to 7 days following each dose; (iii) adverse events (e.g., as described herein) from a first dose to 1 month after the last dose; and / or (iv) serious adverse events (e.g., as described herein) from a first dose to 6 months after the last dose.

[0220] In some embodiments, one or more subjects who have received an RNA (e.g., mRNA) composition as described herein may be monitored (e.g., for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more, 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 more, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) to assess, for example, presence of an immune response to component(s) of the administered composition, evidence of exposure to and / or immune response to SARS-CoV-2 or another coronavirus, evidence of any adverse event, etc. In some embodiments, monitoring may be via tele-visit.Alternatively or additionally, in some embodiments, monitoring may be in-person.

[0221] In some embodiments, a treatment effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized by (i) a SARS-CoV-2 anti-Sl binding antibody level above a predetermined threshold; (ii) a SARS-CoV-2 anti-RBD binding antibody level above a pre-determined threshold; and / or (iii) a SARS-CoV-2 serum neutralizing titer above a threshold level, e.g., at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 months after completion of vaccination. In some embodiments, anti-Sl binding antibody and / or anti-RBD binding antibody levels and / or serum neutralizing titers may be characterized by geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold-rise (GMFR).

[0222] In some embodiments, a treatment effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized in that percentage of treated subjects showing a SARS-CoV-2 serum neutralizing titer above a pre-determined threshold, e.g., at baseline, 1 month, 3 months, 6 months, 9 months, 12 months, 18 months, and / or 24 months after completion of vaccination, is higher than the percentage of nontreated subjects showing a SARS-CoV-2 serum neutralizing titer above such a pre-determined threshold (e.g., as described herein). In some embodiments, a serum neutralizing titer may be characterized by geometric mean concentration (GMC), geometric mean titer (GMT), or geometric mean fold-rise (GMFR).

[0223] In some embodiments, a treatment effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized by detection of SARS-CoV-2 NVA-specific binding antibody.

[0224] In some embodiments, a treatment effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized by SARS-CoV-2 detection by nucleic acid amplification test.

[0225] In some embodiments, a treatment effect conferred by one or more RNA (e.g., mRNA) compositions described herein may be characterized by induction of cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, e.g., 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 an immunogenic fragment thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine- induced T cells (e.g., CD8+ T cells) may be presented on a MHC class I allele that is present in at least 50% of subjects in a population, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more; in some such embodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA- A*0201. In some embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV;HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA- B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0226] In some embodiments, primary vaccine efficacy (VE) of one or more RNA (e.g., mRNA) compositions described herein may be established when there is sufficient evidence (posterior probability) that either primary VE1 or both primary VE1 and primary VE2 are >30% or higher (including, e.g., 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 higher), wherein primary VE is defined as primary VE = 100 x (1 - IRR); and IRR is calculated as the ratio of COVID-19 illness rate in the vaccine group to the corresponding illness rate in the placebo group. Primary VE1 represents VE for prophylactic RNA (e.g., mRNA) compositions described herein against confirmed COVID-19 in participants without evidence of infection before vaccination, and primary VE2 represents VE for prophylactic RNA (e.g., mRNA) compositions described herein against confirmed COVID-19 in all participants after vaccination. In some embodiments, primary VE1 and VE2 can be evaluated sequentially to control the overall type I error of 2.5% (hierarchical testing). In some embodiments where one or more RNA (e.g., mRNA) compositions described herein are demonstrated to achieve primary VE endponts as discussed above, secondary VE endpoints (e.g., confirmed severe COVID-19 in participants without evidence of infection before vaccination and confirmed severe COVID-19 in all participants) can be evaluated sequentially, e.g., by the same method used for the primary VE endpoint evaluation (hierarchical testing) as discussed above. In some embodiments, evaluation 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) randomized in a 1: 1 ratio to the vaccine or placebo group, e.g., based on the following assumptions: (i) 1.0% illness rate per year in the placebo group, and (ii) 20% of the participants being non-evaluable or having serological evidence of prior infection with SARS- CoV-2, potentially making them immune to further infection.

[0227] In some embodiments, one or more RNA (e.g., mRNA) compositions described herein may be administered according to a regimen established to achieve maintenance and / or continued enhancement of an immune response. For example, in some embodiments, an administration regimen may include a first dose optionally followed by one or more subsequent doses; in some embodiments, need for, timing of, and / or magnitude of any such subsequent dose(s) may be selected to maintain, enhance, and / or modify one or more immune responses or features thereof. In some embodiments, number, timing, and / or amount(s) of dose(s) have been established to be effective when administered to a relevant population. In some embodiments, number, timing and / or amount(s) of dose(s) may be adjusted for an individual subject; for example, in some embodiments, one or more features of an immune response in an individual subject may be assessed at least once (and optionally more than once, for example multiple times, typically spaced apart, often at pre-selected intervals) after receipt of a first dose. For example, presence of antibodies, B cells, and / or T cells (e.g., CD4+ and / or CD8+ T cells), and / or of cytokines secreted thereby and / or identity of and / or extent of responses to particular antigen(s) and / or epitope(s) may be assessed. In some embodiments, need for, timing of, and / or amount of a subsequent dose may be determined in light of such assessments.

[0228] As noted hereinabove, in some embodiments, one or more subjects who have received an RNA (e.g., mRNA) composition as described herein may be monitored (e.g., for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more, 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 more, including for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more) from receipt of any particular dose to assess, for example, presence of an immune response to component(s) of the administered composition, evidence ofexposure to and / or immune response to SARS-CoV-2 or another coronavirus, evidence of any adverse event, etc, including to perform assessment of one or more of presence of antibodies, B cells, and / or T cells (e.g., CD4+ and / or CD8+ T cells), and / or of cytokines secreted thereby and / or identity of and / or extent of responses to particular antigen(s) and / or epitope(s) may be assessed. Administration of a composition as described herein may be in accordance with a regimen that includes one or more such monitoring steps.

[0229] For example, in some embodiments, need for, timing of, and / or amount of a second dose relative to a first dose (and / or of a subsequent dose relative to a prior dose) is assessed, determined, and / or selected such that administration of such second (or subsequent) dose achieves amplification or modification of an immune response (e.g., as described herein) observed after the first (or other prior) dose. In some embodiments, such amplification of an immune response (e.g., ones 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 higher, as compared to the level of an immune response observed after the first dose. In some embodiments, such amplification of an 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 higher, as compared to the level of an immune response observed after the first dose.

[0230] In some embodiments, need for, timing of, and / or amount of a second (or subsequent) dose relative to a first (or other prior) dose is assessed, determined, and / or selected such that administration of the later dose extends the durability of an immune response (e.g., as described herein) observed after the earlier dose; in some such embodiments, the durability 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 longer. In some embodiments, an immune response observed after the first dose may be characterized by production of neutralizing antibodies directed to a SARS-CoV-2 spike polypeptide and / or an immunogenic fragment thereof (e.g., RBD) as measured in serum from a subject and / or induction of cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, e.g., 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 an immunogenic fragment thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells) may be presented on a MHC class I allele that is present in at least 50% of subjects in a population, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more; in some such embodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA- A*0201. In some embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV;HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA- B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0231] In some embodiments, need for, timing of, and / or amount of a second dose relative to a first dose (or other subsequent dose relative to a prior dose) is assessed, determined and / or selected such that administration of such second (or subsequent) dose maintains or exceeds a reference level of an immune response; in some such embodiments, the reference level is determined based on human SARS-CoV-2 infection / COVID-19 convalescent sera and / or PBMC samples drawn from subjects (e.g., at least a period of time such as at least 14 days or longer, 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 longer, after PCR-confirmed diagnosis when the subjects were asymptomatic. In some embodiments, an immune response may be characterized by production of neutralizing antibodies directed to a SARS-CoV-2 spike polypeptide and / or an immunogenicfragment thereof (e.g., RBD) as measured in serum from a subject and / or induction of cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, e.g., 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 an immunogenic fragment thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine-induced T cells (e.g., CD8+ T cells) may be presented on a MHC class I allele that is present in at least 50% of subjects in a population, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more; in some such embodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402, HLA- B*3501, HLA-B*4401, or HLA-A*0201. In some embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA-B*3501 IPFAMQMAY; or HU\-B*3501 LPFNDGVYF.

[0232] In some embodiments, determination of need for, timing of, and / or amount of a second (or subsequent) dose may include one or more steps of assessing, after (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or longer after) a first (or other prior) dose, presence and / or expression levels of neutralizing antibodies directed to a SARS-CoV-2 spike polypeptide and / or an immunogenic fragment thereof (e.g., RBD) as measured in serum from a subject and / or induction of cell-mediated immune response (e.g., a T cell response against SARS-CoV-2), including, e.g., 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 an immunogenic fragment thereof (e.g., RBD). In some embodiments, one or more epitopes recognized by vaccine- induced T cells (e.g., CD8+ T cells) may be presented on a MHC class I allele that is present in at least 50% of subjects in a population, including, e.g., at least 60%, at least 70%, at least 80%, at least 90%, or more; in some such embodiments, the MHC class I allele may be HLA-B*0702, HLA-A*2402, HLA-B*3501, HLA-B*4401, or HLA- A*0201. In some embodiments, an epitope may comprise HLA-A*0201 YLQPRTFLL; HLA-A*0201 RLQSLQTYV; HLA-A*2402 QYIKWPWYI; HLA-A*2402 NYNYLYRLF; HLA-A*2402 KWPWYIWLGF; HLA-B*3501 QPTESIVRF; HLA- B*3501 IPFAMQMAY; or HLA-B*3501 LPFNDGVYF.

[0233] In some embodiments, a kit as provided herein may comprise a real-time monitoring logging device, which, for example in some embodiments, is capable of providing shipment temperatures, shipment time and / or location.

[0234] In some embodiments, an RNA (e.g., mRNA) composition as described herein may be shipped, stored, and / or utilized, in a container (such as a vial or syringe), e.g., a glass container (such as a glass vial or syringe), which, in some embodiments, may be a single-dose container or a multi-dose container (e.g., may be arranged and constructed to hold, and / or in some embodiments may hold, a single dose, or multiple doses of a product for administration). In some embodiments, a multi-dose container (such as a multi-dose vial or syringe) may be arranged and constructed 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, a singledose or multi-dose container (such as a single-dose or multi-dose vial or syringe) may be arranged and constructed to hold and / or may hold a volume or amount greater than the indicated number of doses, e.g., in order to permit some loss in transfer and / or administration. In some embodiments, an RNA (e.g., mRNA) composition as described herein may be shipped, stored, and / or utilized, in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a single-dose or multi-dose preservative-free glass vial or syringe). In some embodiments, an RNA (e.g., mRNA) composition as described herein may be shipped, stored, and / or utilized, in a preservative-free glass container (e.g., a preservative-free glass vial or syringe, e.g., a singledose or multi-dose preservative-free glass vial or syringe) that contains a frozen liquid, e.g., in someembodiments 0.45 ml of frozen liquid (e.g., including 5 doses). In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a vial or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature below room temperature, at or below 4 °C, at or below 0 °C, at or below -20 °C, at or below -60 °C, at or below -70 °C, at or below -80 °C , at or below -90 °C, etc. In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature between -80°C and -60°C and in some embodiments protected from light. In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature below about 25oC, and in some embodiments protected from light. In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature below about 5oC (e.g., below about 4oC), and in some embodiments protected from light. In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature below about -20oC, and in some embodiments protected from light. In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a viral or syringe) in which it is disposed, is shipped, stored, and / or utilized may be maintained at a temperature above about -60oC (e.g., in some embodiments at or above about -20oC, and in some embodiments at or above about 4-5oC, in either case optionally below about 25oC), and in some embodiments protected from light, or otherwise without affirmative steps (e.g., cooling measures) taken to achieve a storage temperature materially below about -20oC.

[0235] In some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a vial or syringe) in which it is disposed is shipped, stored, and / or utilized together with and / or in the context of a thermally protective material or container and / or of a temperature adjusting material. For example, in some embodiments, an RNA (e.g., mRNA) composition as described herein and / or a container (e.g., a vial or syringe) in which it is disposed is shipped, stored, and / or utilized together with ice and / or dry ice and / or with an insulating material. In some particular embodiments, a container (e.g., a vial or syringe) in which an RNA (e.g., mRNA) composition is disposed is positioned in a tray or other retaining device and is further contacted with (or otherwise in the presence of) temperature adjusting (e.g., ice and / or dry ice) material and / or insulating material. In some embodiments, multiple containers (e.g., multiple vials or syringes such as single use or multi-use vials or syringes as described herein) in which a provided RNA (e.g., mRNA) composition is disposed are co-localized (e.g., in a common tray, rack, box, etc.) and packaged with (or otherwise in the presence of) temperature adjusting (e.g., ice and / or dry ice) material and / or insulating material. To give but one example, in some embodiments, multiple containers (e.g., multiple vials or syringes such as single use or multi-use vials or syringes as described herein) in which an RNA (e.g., mRNA) composition is disposed are positioned in a common tray or rack, and multiple such trays or racks are stacked in a carton that is surrounded by a temperature adjusting material (e.g., dry ice) in a thermal (e.g., insulated) shipper. In some embodiments, temperature adjusting material is replenished periodically (e.g., within 24 hours of arrival at a site, 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, re-entry into a thermal shipper should be infrequent, and desirably should not occur more than twice a day. In some embodiments, a thermal shipper is reclosed within 5, 4, 3, 2, or 1 minute, or less, of having been opened. In some embodiments, a provided RNA (e.g., mRNA) composition that has been stored within a thermal shipper for a period of time, optionally within aparticular temperature range remains useful. For example, in some embodiments, if a thermal shipper as described herein containing a provided RNA (e.g., mRNA) composition is or has been maintained (e.g., stored) at a temperature within a range of about 15 °C to about 25 °C, the RNA (e.g., mRNA) composition may be used for up to 10 days; that is, in some embodiments, a provided RNA (e.g., mRNA) composition that has been maintained within a thermal shipper, which thermal shipper is at a temperature within a range of about 15 °C to about 25 °C, for a period of not more than 10 days is administered to a subject. Alternatively or additionally, in some embodiments, if a provided RNA (e.g., mRNA) composition is or has been maintained (e.g., stored) within a thermal shipper, which thermal shipper has been maintained (e.g., stored) at a temperature within a range of about 15 °C to about 25 °C, it may be used for up to 10 days; that is, in some embodiments, a provided RNA (e.g., mRNA) composition that has been maintained within a thermal shipper, which thermal shipper has been maintained at a temperature within a range of about 15 °C to about 25 °C for a period of not more than 10 days is administered to a subject.

[0236] In some embodiments, a syringe is a prefilled syringe. In some embodiments, a prefilled syringe is a glass or plastic syringe. Suitable syringes include e.g., those described in Makwana, Sagar, et al. "Prefilled syringes: an innovation in parenteral packaging." International journal of pharmaceutical investigation 1.4 (2011): 200; Sacha, Gregory, J. Aaron Rogers, and Reagan L. Miller. "Pre-filled syringes: a review of the history, manufacturing and challenges." Pharmaceutical development and technology 20.1 (2015): 1-11; and Borse, Sandip Arun, Ashish Prakash Gorle, and Kuldip Patil. "Prefilled syringe: A review of injectable dosage form delivery system." Int. J. Pharm. Sci. Res 11 (2020): 167-174, the contents of each of which is incorporated by reference herein in their entirety.

[0237] In some embodiments, a composition described herein is administered a volume of about 0.1 to 5.0 ml. In some embodiments, a composition described herein is administered a volume of about 0.1 to 1.0 ml. In some embodiments, a composition described herein is administered a volume of about 0.1 to 0.50 ml. In some embodiments, a composition described herein is administered in a volume of about 100 pL to about 300 μL. In some embodiments, a composition described herein is administered in a volume of about 50 pL to about 300 μL. In some embodiments, a composition described herein is administered in a volume of about 50 pL to about 200 pL. In some embodiments, a composition described herein is administered in a volume of about 100 pL to about 300μL. In some embodiments, a composition described herein is administered in a volume of about 200 pL to about 300μL. In some embodiments, a composition described herein is administered in a volume of about 50 pL to about 500 μL. In some embodiments, a composition described herein is administered in a volume of about 50 pL, about 100 pL, about 150 pL, about 200 pL, about 250 pL, about 300 pL, about 350 pL, about 400 pL, about 450 pL, or about 500 μL.

[0238] In some embodiments, a provided RNA (e.g., mRNA) composition is shipped and / or stored in a frozen state. In some embodiments, a provided RNA (e.g., mRNA composition is shipped and / or stored as a frozen suspension, which in some embodiments does not contain preservative. In some embodiments, a frozen RNA (e.g., mRNA) composition is thawed. In some embodiments, a thawed RNA (e.g., mRNA) composition (e.g., a suspension) may contain white to off-white opaque amorphous particles. In some embodiments, a thawed RNA (e.g., mRNA) composition may be used for up to a small number (e.g., 1, 2, 3, 4, 5, or 6) of days after thawing if maintained (e.g., stored) at a temperature at or below room temperature (e.g., below about 30 °C, 25 °C, 20 °C, 15 °C, 10 °C, 8 °C, 4 °C, etc). In some embodiments, a thawed RNA (e.g., mRNA) composition may be used after being stored (e.g., for such small number of days) at a temperature between about 2 °C and about 8 °C; alternatively or additionally, a thawed RNA (e.g., mRNA) composition may be used within a small number (e.g., 1,2, 3, 4, 5, 6) of hours after thawing at room temperature. Thus, in some embodiments, a provided RNA (e.g., mRNA) composition that has been thawed and maintained at a temperature at or below room temperature, and in some embodiments between about 2 °C and about 8 °C, for not more than 6, 5, 4, 3, 2, or 1 days is administered to a subject. Alternatively or additionally, in some embodiments, a provided RNA (e.g., mRNA) composition that has been thawed and maintained at room temperature for not more than 6, 5, 4, 3, 2, or 1 hours is administered to a subject. In some embodiments, a provided RNA (e.g., mRNA) composition is shipped and / or stored in a concentrated state. In some embodiments, such a concentrated composition is diluted prior to administration. In some embodiments, a diluted composition is administered within a period of about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s) post-dilution; in some embodiments, such administration is within 6 hours post-dilution. Thus, in some embodiments, diluted preparation of a provided RNA (e.g., mRNA) composition is administered to a subject within 6 hours post-dilution (e.g., as described herein after having been maintained at an appropriate temperature, e.g., at a temperature below room temperature, at or below 4 °C, at or below 0 °C, at or below -20 °C, at or below -60 °C, at or below -70 °C, at or below - 80 °C, etc, and typically at or above about 2 °C, for example between about 2 °C and about 8 °C or between about 2 °C and about 25 °C). In some embodiments, unused composition is discarded within several hours (e.g., about 10, about 9, about 8, about 7, about 6, about 5 or fewer hours) after dilution; in some embodiments, unused composition is discarded within 6 hours of dilution.

[0239] In some embodiments, an RNA (e.g., mRNA) composition that is stored, shipped or utilized (e.g., a frozen composition, a liquid concentrated composition, a diluted liquid composition, etc.) may have been maintained at a temperature materially above -60oC for a period of time of 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 composition may have been maintained at a temperature at or above about -20oC for such period of time, and / or at a temperature up to or about 4-5oC for such period of time, and / or may have been maintained at a temperature above about 4-5oC, and optionally about 25oC for a period of time up that is less than two (2) months and / or optionally up to about one (1) month. In some embodiments, such composition may not have been stored, shipped or utilized (or otherwise exposed to) a temperature materially above about 4-5oC, and in particular not at or near a temperature of about 25oC for a period of time as long as about 2 weeks, or in some embodiments 1 week. In some embodiments, such composition may not have been stored, shipped or utilized (or otherwise exposed to) a temperature materially above about -20oC, and in particular not at or near a temperature of about 4-5oC for a period of time as long as 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 as long as about 8 weeks or 6 weeks or materially more than about 2 months or, in some embodiments, 3 months or, in some embodiments 4 months.

[0240] In some embodiments, an RNA (e.g., mRNA) composition that is stored, shipped or utilized (e.g., a frozen composition, a liquid concentrated composition, a diluted liquid composition, etc.) may be protected from light. In some embodiments, one or more steps may be taken to reduce or minimize exposure to light for such compositions (e.g., which may be disposed within a container such as a vial or a syringe). In some embodiments, exposure to direct sunlight and / or to ultraviolent light is avoided. In some embodiments, a diluted solution may be handled and / or utilized under normal room light conditions (e.g., without particular steps taken to minimize or reduce exposure to room light). It should be understood that strict adherence to aseptic techniques is desirable during handling (e.g., diluting and / or administration) of an RNA (e.g., mRNA) composition as described herein. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered (e.g., is not injected) intravenously. In some embodiments, an RNA (e.g., mRNA) composition as described herein is notadministered (e.g., is not injected) intradermally. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered (e.g., is not injected) subcutaneously. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered (e.g., is not injected) any of intravenously, intradermally, or subcutaneously. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered to a subject with a known hypersensitivity to any ingredient thereof. In some embodiments, a subject to whom an RNA (e.g., mRNA) composition has been administered is monitored for one or more signs of anaphylaxis. In some embodiments, a subject to whom an RNA (e.g., mRNA) composition is administered had previously received at least one dose of a different vaccine for SARS-CoV-2; in some embodiments, a subject to whom an RNA (e.g., mRNA) composition is administered had not previously received a different vaccine for SARS- CoV-2. In some embodiments, a subject's temperature is taken promptly prior to administration of an RNA (e.g., mRNA) composition (e.g., shortly before or after thawing, dilution, and / or administration of such composition); in some embodiments, if such subject is determined to be febrile, administration is delayed or canceled. In some embodiments, an RNA (e.g., mRNA) composition as described herein is not administered to a subject who is receiving anticoagulant therapy or is suffering from or susceptible to a bleeding disorder or condition that would contraindicate intramuscular injection. In some embodiments, an RNA (e.g., mRNA) composition as described herein is administered by a healthcare professional who has communicated with the subject receiving the composition information relating to side effects and risks. In some embodiments, an RNA (e.g., mRNA) composition as described herein is administered by a healthcare professional who has agreed to submit an adverse event report for any serious adverse events, which may include for example one or more of death, development of a disability or congenital anomaly / birth defect (e.g., in a child of the subject), in-patient hospitalization (including prolongation of an existing hospitalization), a life-threatening event, a medical or surgical intervention to prevent death, a persistent or significant or substantial disruption of the ability to conduct normal life functions; or another important medical event that may jeopardize the individual and may require medical or surgical intervention (treatment) to prevent one of the other outcomes.

[0241] In some embodiments, provided RNA compositions are administered to a population of individuals under 18 years of age, or under 17 years of age, or under 16 years of age, or under 15 years of age, or under 14 years of age, or under 13 years of age, for example according to a regimen established to have a rate of incidence for one or more of the local reaction events indicated below that does not exceed the rate of incidence indicated below: pain at the injection site (75% after a first dose and / or a second dose, and / or a lower incidence after a second dose, e.g., 65% after a second dose); redness at the injection site (less than 5% after a first dose and / or a second dose); and / or swelling at the injection site (less than 5% after a first dose and / or a second dose).

[0242] In some embodiments, provided RNA compositions are administered to a population of individuals under 18 years of age, or under 17 years of age, or under 16 years of age, or under 15 years of age, or under 14 years of age, or under 13 years of age, for example according to a regimen established to have a rate of incidence for one or more of the systemic reaction events indicated below that does not exceed the rate of incidence indicated below: fatigue (55% after a first dose and / or a second dose); headache (50% after a first dose and / or a second dose); muscle pain (40% after a first dose and / or a second dose); chills (40% after a first dose and / or a second dose);joint pain (20% after a first dose and / or a second dose); fever (25% after a first dose and / or a second dose); vomiting (10% after a first dose and / or a second dose); and / or diarrhea (10% after a first dose and / or a second dose).

[0243] In some embodiments, medication that alleviates one or more symptoms of one or more local reaction and / or systemic reaction events (e.g., described herein) are administered to individuals under 18 years of age, or under 17 years of age, or under 16 years of age, or under 15 years of age, or under 14 years of age, or under 13 years of age who have been administered with provided RNA compositions and have experienced one or more of the local and / or systemic reaction events (e.g., described herein). In some embodiments, antipyretic and / or pain medication can be administered to such individuals.

[0244] Among other things, the present disclosure describes an RNA comprising a nucleotide sequence encoding a polypeptide comprising an S2 domain, or a fragment thereoof.

[0245] In some embodiments, an S2 domain comprises amino acids 686-1213 of SEQ ID NO: 1, 686-1211 of SEQ ID NO: 1, amino acids 687-1206 of SEQ ID NO: 1, or amino acids 687-1211 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant, optionally wherein the S2 domain compries one or more mutations that stabilize the S2 domain (e.g., stabilize the prefusion conformation of the S2 domain).

[0246] In some embodiments, an S2 domain comprises: an amino acid sequence of:SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAV EQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYH LMSFPQSAPHGWFLHVTYVPAQEKN FTTAPAICH DG KAH FPREGVFVSNGTHWFVTQRN FYEP QIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWP, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto; an amino acid sequence of:SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAV EQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYH LMSFPQSAPHGWFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRN FYEP QIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIK, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto; an amino acid sequence of:VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVE QDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT VLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASA LGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS ECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIK, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto. an amino acid sequence of:VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVE QDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT VLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASA LGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS ECVLGQSKRVDFCGKGYH LM SFPQSAPHG WFLHVTYVPAQEKN FTTAPAICH DGKAH FPREGVFVSNGTHWFVTQRN FYEPQ IITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKY, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto.

[0247] In some embodiments, an S2 domain or a fragment thereof comprises a stem helix and / or a fusion peptide of an S2 domain.

[0248] In some embodiments, a fusion peptide comprises a sequence of PSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto, and the stem helix comprises a sequence of LQPELDSFKEELDKYFKNHTSPDV, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto.

[0249] In some embodiments, an S2 domain comprises one or more stabilizing mutations.

[0250] In some embodiments, one or more stabilizing mutations comprise one or more mutations that can stabilize the prefusion confirmation of an S protein.

[0251] In some embodiments, one or more stabilizing mutations include one or more Pro substitutions.

[0252] In some embodiments, an S2 domain comprises one or more of the following mutations relative toSEQ ID NO: 1, or corresponding mutation(s) in an S2 domain of an S protein of a SARS-CoV-2 variant:(a) K986P and V987P;(b) A892P, A899P, A942P, or combinations thereof, optionally in combination with K986P and V987P;(c) F817P, A892P, A899P, A942P, or combinations thereof, optionally in combination with K986P and V987P; or(d) any combination of (a)-(c).

[0253] In some embodiments, an S2 domain comprises one or more mutations that can result in the formation of a disulfide bond.

[0254] In some embodiments, an S2 domain comprises one of the following combinations of mutations relative to SEQ ID NO: 1, or a corresponding combination of mutations in an S2 domain of a SARS-CoV-2 variant:(a) V707C and T883C;(b) I770C and A1015C;(c) V826C and A1015C;(d) V826C and L948C;(e) F970C and G999C;(f) S735C and T859C; or(g) any combination of (a)-(e).

[0255] In some embodiments, an S2 domain comprises one or more of the following combinations of mutations relative to SEQ ID NO: 1, or a corresponding combination of mutations in an S protein of a SARS-CoV-2 variant:(a) V707C, T883C, F970C, and G999C;(b) S735C, T859C, I770C, and A1015C;(c) S735C, T859C, V826C, and L948C;(d) I770C, A1015C, V826C, and L948C;(e) S735C, I770C, A1015C, V826C, and L948C; or(f) any combination of (a)-(e).

[0256] In some embodiments, an S2 domain comprises one or more Pro substitions and one or more mutations that can lead to the formation of a disulfide bond.

[0257] In some embodiments, an S2 domain comprises one of the following combinations of mutations relative to SEQ ID NO: 1, or a corresonding combination of mutations of an S2 domain of a SARS-CoV-2 variant:A892P, A899P, 941, K986P, V987P, G999C, and I770C;A892P, A899P, A942P, V987P, V707C, T883C, optionally wherein the S2 domain compises one or more Gly substitutions at positions corresponding to amino acids 814 and 815 of SEQ ID NO: 1;A892P, A899P, A942P, V987P, V707C, T883C, F970C, G999C, optionally wherein the S2 domain compises one or more Gly substitutions at positions corresponding to amino acids 814 and 815 of SEQ ID NO: 1.

[0258] In some embodiments, an the S2 domain comprises one or more mutations that can prevent or result in the reduction of cleavage at the S2' cleavage site.

[0259] In some embodiments, an S2 domain comprises one or more mutations that can remove the S2' protease cleavage site.

[0260] In some embodiments, an S2 domain comprises one or more mutations at positions corresponding to amino acids 814 and / or 815 of SEQ ID NO: 1, optionally wherein the one or more mutations are a Gly, Ser, or Ala substition (e.g., a Gly substition).

[0261] In some embodiments, an S2 domain comprises Gly substitutions at positions corresponding to amino acids 814 and 815 of SEQ ID NO: 1.

[0262] In some embodiments, an S2 domain comprises one or more Pro substitutions that can stabilize the S2 domain and one or more mutations that disrupt the S2' protease cleavage site.

[0263] In some embodiments, an S2 domain comprises one or more of the following mutations relative to SEQ ID NO: 1: A892P, A899P, A942P, K986P, V987P, or any combination thereof, or corresponding mutations in a SARS-Cov-2 variant, and a Gly substituion at positions 814 and / or 815 of SEQ ID NO: 1.

[0264] In some embodiments, an S2 domain comprises one or more substitutions of a hydrophillic amino acid for a hydrophobic amino acid, wherein the one or more hydrophobic amino acids are at positions that are solvent explosed in the S2 domain.

[0265] In some embodiments, an S2 domain comprises a substitution of a hydrophillic amino acid at one or more positions corresponding to amino acid 855, 861, 864, 976, or 984 of SEQ ID NO: 1, or any combination thereof.

[0266] In some embodiments, an S2 domain comprises one or more of the following mutations relative to SEQ ID NO: 1 F855S, L861E, L864D, V976D, and L984Q, or any combination thereof, or corresponding mutations in an S2 domain of a SARS-CoV-2 variant.

[0267] In some embodiments, an S2 domain comprises an amino acid substitution at one or more of the amino acids at positions 901, 1020, 1058, or any combination thereof relative to SEQ ID NO: 1, or corresponding substitutions in an S2 domain of a SARS-CoV-2 variant.

[0268] In some embodiments, an S2 domain comprises one or more of the following mutations relative to SEQ ID NO: 1: Q901M, A1020Q, H1058Y, or any combination thereof, or corresponding mutations in an S2 domain of a SARS-CoV-2 variant.

[0269] In some embodiments, an S2 domain comprises:(a) one or more of the following mutations: S375C, T859C, Q901M, A1020Q, H1058Y, or any combination thereof, or corresponding mutations in an S protein of a SARS-CoV-2 variant, wherein mutations are shown relative to SEQ ID NO: 1, Pro substitutions at positions corresponding to amino acids K986P and / or V987P of SEQ ID NO: 1, and optionally a Gly substition at one or both of the positions corresponding to amino acids 814 and 815 of SEQ ID NO: 1;(b) one or more of the following mutations: S735C, T859C, Q901M, A1020Q, H1058Y, I770C, A1015C, V826C, L948C, or any combination thereof, or corresponding mutations in an S protein of a SARS-CoV-2 variant, and Pro substiutions at positions corresponding to amino acids K986P and V987P of SEQ ID NO: 1, wherein mutations are shown relative to SEQ ID NO: 1, and optionally a Gly substition at one or both of the positions corresponding to amino acids 814 and 815 of SEQ ID NO: 1;(c) one or more of the following mutations: S735C, T859C, Q901M, A1020Q, H1058Y, I770C, A1015C, V826C, L948C, or any combination thereof, or corresponding mutations in an S protein of a SARS-CoV-2 variant, wherein mutations are shown relative to SEQ ID NO: 1; or(d) any combination of (a)-(c).

[0270] In some embodiments, an S2 domain comprises one or more subsitions of a hydrophobic residue for a hydrophillic residue at one or more positions that are solvent buried in the context of the full length S protein, but which are solvent exposed in the absense of the SI domain.

[0271] In some embodiments, one or more hydrophic residues include F855S, L861E, L864D, V976D, and L984Q, where mutations are indicated relative to SEQ ID NO: 1, or corresponding mutations in an S2 domain of a SARS-CoV-2 variant.

[0272] In some embodiments, an S2 domain comprises one or more of the following mutations: L861E, L864D, V976D, and L984Q, a Pro substitution at one or more positions corresponding to amino acids F817P, A892P, A899P, A942P, or combinations thereof of SEQ ID NO: 1, optionally in combination with a Pro substitution at one or both positions corresponding to amino acids K986P and V987P of SEQ ID NO: 1; or where mutations are indicated relative to SEQ ID NO: 1, or corresponding mutations in an S2 domain of a SARS-CoV-2 variant.

[0273] In some embodiments, a polypeptide comprises a multimerization domain, optionally wherein the multimerization domain is a T4 fibritin domain.

[0274] In some embodiments, a multimerization domain is at the C-terminus of the polypeptide.

[0275] In some embodiments, a polypeptide does not comprise a transmembrane domain.

[0276] In some embodiments, a polypeptide comprises a transmembrane domain, optionally where the transmembrane domain is a SARS-CoV-2 transmembrane domain (e.g., a transmembrane domain comprising an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC, or a variant thereof, including e.g., an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC, or a variant thereof).

[0277] In some embodiments, a polypeptide comprises a multimerization domain, and wherein the polypeptide does not comprise a transmembrane domain.

[0278] In some embodiments, a polypeptide comprises a multimerization domain and a transmembrane domain, optionally wherein the multimerization domain is C-terminal to the S2 domain and the transmembrane domain is C-terminal to the trimerization domain.

[0279] In some embodiments, a multimerization domain is a trimerization domain (e.g., a fibritin domain, including, e.g., a peptide having an amino acid sequence of GYIPEAPRDGQAYVRKDGEWVLLSTFL, or a variant thereof) and / or wherein the tramsmembrane domain is a transmembrane domain of a SARS-CoV-2 S protein (e.g., a transmembrane domain comprising an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC, or a variant thereof, including e.g., an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC, or a variant thereof).

[0280] In some embodiments, a polypeptide comprises a secretory signal peptide (e.g., a secretory signal peptide of a viral protein), optionally wherein the secretory signal peptide is at the N-terminus of the polypeptide.

[0281] In some embodiments, a secretory signal peptide is a secretory signal peptide of a SARS-CoV-2 S protein (e.g., wherein the secretory signal peptide comprises amino acids 1-19 of SEQ ID NO: 1, or a an amino acid sequence of a correseponding region of an S protein of a SARS-CoV-2 variant).

[0282] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 178.

[0283] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 179.

[0284] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 180.

[0285] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 181.

[0286] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 182.

[0287] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 183.

[0288] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 184.

[0289] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 185.

[0290] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 186.

[0291] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 187.

[0292] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 192.

[0293] In some embodiments, an RNA comprises a nuceltide sequence encoding polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 193.

[0294] In some embodiments, an RNA comprises a nuceltide sequence encoding polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 194.

[0295] In some embodiments, an RNA comprises a nuceltide sequence encoding a polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 195.

[0296] In some embodiments, an RNA comprises a nuceltide sequence encoding a polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 196.

[0297] In some embodiments, an RNA comprises a nuceltide sequence encoding a polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 197.

[0298] In some embodiments, an RNA comprises a nuceltide sequence encoding a polypeptide comprises an amino acid sequence of SEQ ID NO: 178, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 198.

[0299] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 446 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 446; (ii) the nucleotide sequence of SEQ ID NO: 447 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 447; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 183, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 183.

[0300] In some embodiments, an RNA comprises RNA comprises (i) the nucleotide sequence of SEQ ID NO: 448 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 448; (ii) the nucleotide sequence of SEQ ID NO: 449 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 449; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 184, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 184.

[0301] In some embodiments, an RNA comprises RNA comprises (i) the nucleotide sequence of SEQ ID NO: 450 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 450; (ii) the nucleotide sequence of SEQ ID NO: 451 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQID NO: 451; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 185, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 185.

[0302] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 452 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 452; (ii) the nucleotide sequence of SEQ ID NO: 453 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 453; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 442, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 442.

[0303] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 455 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 455; (ii) the nucleotide sequence of SEQ ID NO: 456 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 456; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 443, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 443.

[0304] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 457 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 457; (ii) the nucleotide sequence of SEQ ID NO: 458 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 458; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 195, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 195.

[0305] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 459 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 459; (ii) the nucleotide sequence of SEQ ID NO: 460 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 460; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 197, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 197.

[0306] Among other things, the present disclosure comprising an RNA comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an S2 domain or an immunogenic fragment thereof, and a Receptor Binding Domain of a SARS-CoV-2 S protein.

[0307] In some embodiments, an S2 domain and an RBD are connected by a non-endogenous sequence.

[0308] In some embodiments, an S2 domain is N-terminal or C-terminal to the RBD.

[0309] In some embodiments, a polypeptide does not comprise an NTD.

[0310] In some embodiments, an RBD is directly linked to the S2 domain.

[0311] In some embodiments, an RBD is connected to the S2 domain via a linker sequence.

[0312] In some embodiments, a linker sequence is a flexible linker sequence, a helical linker sequence, or a rigid linker sequence.

[0313] In some embodimetns, a linker sequence comprises a protease recognition site.

[0314] In some embodiments, a linker sequence comprises a furin recognition site (e.g., an amino acid sequence of AGNRVRRSVG, or an amino acid with 1, 2, 3, 4, or more mutations thereto).

[0315] In some embodiments, an RNA encodes a polypeptide comprising one or more multimerization domains (e.g., two or mmore multimerization domains).

[0316] In some embodiments, a multimerization domain (e.g., a fibritin domain is attached to each of the S2 polyeptpide and the RBD, and the RBD and the S2 domain are connected via a linker that comprises a protease cleavage site, and the polypeptide is configured such that, upon cleavage of the furin cleavage site, a fibritin domain is attached to each of the S2 domain and the RBD.

[0317] In some embodiments, an RNA encodes a polypeptide comprising a transmembrane domain, an RBD, and an S2 domain (e.g., a transmembrane domain comprising EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC, or a variant thereof, including, e.g., EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC), and the polypeptide is configured such that, upon cleavage, one of the RBD and the S2 domain is membrane bound and the other is soluble. In some embodiments, the polypeptide is configured such that the RBD is membrane bound and the S2 domain is soluble. In some embodiments, the S2 is N-terminal to the RBD. In some embodiments, a transmembrane domain is C-terminal to the RBD. In some embodiments, a multimerization domain is attached to each of the S2 and the RBD, wherein each of the fibritin domains is C-terminal to the S2 and the RBD.

[0318] In some embodiments, a polypeptide comprises a fibritin domain directly linked to the C-terminus of the S2 domain, a fibritin domain is directly linked to the C-terminus of the RBD, the S2 is N-terminal to the RBD, a transmembrane domain is linked to the C-terminus of the fibritin domain that is at the C-terminus of the RBD, and wherein a linker sequence is located between the fibritin domain that is attached to the C-terminus of the S2 domain, and the RBD.

[0319] In some embodiments, an RBD comprises amino acids 327 to 528 of SEQ ID NO: 1, 330 to 528 of SEQ ID NO: 1, amino acids 327 to 528 of SEQ ID NO: 1, or amino acids 330 to 541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of a SARS-CoV-2 variant.

[0320] In some embodiments, an S2 domain comprises amino acid 686-1213 of SEQ ID NO: 1, 686-1211 of SEQ ID NO: 1, amino acids 687-1206 of SEQ ID NO: 1, or amino acids 687-1211 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant, optionally wherein the S2 domain comprises one or more mutations that stabilize the S2 domain.

[0321] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 462 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 462; (ii) the nucleotide sequence of SEQ ID NO: 463 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 463; and / or (Hi) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 188, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 188.

[0322] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 469 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 469; (ii) the nucleotide sequence of SEQ ID NO: 470 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 470; and / or (Hi) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 444, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 444.

[0323] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 471 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 471; (ii) the nucleotide sequence of SEQ ID NO: 472 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 472; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 445, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 445.

[0324] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, where the polypeptide comprises:

[0325] (i) an RBD, and

[0326] (ii) a stem helix and a fusion peptide of an S2 domain.

[0327] In some embodiments, a stem helix and a fusion peptide are connected via a non-endogenous peptide sequence.

[0328] In some embodiments, a stem helix and a fusion peptide are directly linked to one another and / or a stem helix and a fusion peptide are connected via a sequence that comprises a flexible linker.

[0329] In some embodiments, an RBD is connected to a stem helix and / or a fusion peptide via a non- endogenous sequence.

[0330] In some embodiments, an RBD is directly linked to the stem helix and / or the fusion peptide.

[0331] In some embodiments, an RBD is linked to a stem helix and / or a fusion peptide via a flexible linker.

[0332] In some embodiments, an RBD is N-terminal to the stem helix and the fusion peptide.

[0333] In some embodiments, an RBD is C-Terminal to the stem helix and the fusion peptide.

[0334] In some embodiments, the N-terminal to C-terminal orientation of the polypeptide is: (RBD)-(SH) -(FP), (SH)-(FP)-(RBD), (FP)-(SH)-(RBD), or (RBD)-(FP)-(SH) optionally wherein there is one or more linkers (e.g., a flexible linker) and / or domains (e.g., transmembrane domain or multimerization domains) between one or more of the RBD, stem helix, and fusion peptide.

[0335] In some embodiments, a stem helix and a fusion peptide are directly linked to one another, and wherein an RBD is N-terminal or C-terminal to the stem helix-fusion peptide region.

[0336] In some embodiments, an RNA encodes a polypetpide having an N-terminal to C-terminal orientation of:

[0337] (fusion peptide)-(stem helix)-(RBD)-(transmembrane domain); or

[0338] (RBD-(fusion peptide)-(stem helix)-(transmembrane domain).

[0339] In some embodiments, a stem helix, fusion peptide, and RBD are connected via a non-endogenous sequence

[0340] In some embodiments, a fusion peptide comprises a sequence of PSKPSKRSFIEDLLFNKVFLADAGFIKQYGDCLGD, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto, and the stem helix comprises a sequence of LQPELDSFKEELDKYFKNHTSPDV, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto.

[0341] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 464 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 464; (ii) the nucleotide sequence of SEQ ID NO: 465 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 465;and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 190, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 190.

[0342] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 466 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 466; (ii) the nucleotide sequence of SEQ ID NO: 467 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 467; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 191, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 191.

[0343] In some embodiments, a polypeptide comprises a secretory signal peptide (e.g., a secretory signal peptide of a viral protein), optionally wherein the secretory signal peptide is at the N-terminus of the polypeptide.

[0344] In some embodiments, a secretory signal peptide is a secretory signal peptide of a SARS-CoV-2 S protein (e.g., wherein the secretory signal peptide comprises an amino acid sequence of MFVFLVLLPLVSSQCVNLT, or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant).

[0345] In some embodiments, a polypeptide comrpises one or more multimerization domains.

[0346] In some embodiments, a multimerization domain is a |3-Annulus peptide, a ferritin domain, a fibritin domain, or a lumazine synthase multimerization domain.

[0347] In some embodiments, a fJ-Annulus peptide comprises SEQ ID NO: 201, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 201.

[0348] In some embodiments, a ferritin domain comprises:

[0349] SEQ ID NO: 202, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 202; or

[0350] SEQ ID NO: 203, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 203.

[0351] In some embodiments, a fibritin domain comprises:

[0352] SEQ ID NO: 199, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 199; or

[0353] SEQ ID NO: 200, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 200.

[0354] In some embodiments, a lumazine synthase multimerization domain comprises SEQ ID NO: 204, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 204.

[0355] Among other things, the present disclosure describes an RNA comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an antigenic fragment of an S protein, or a variant thereof, and a multimerization domain.

[0356] In some embodiments, an RNA encodes an antigenic fragment of a coronavirus S protein and a multimerization domain, where:

[0357] the antigenic fragment of the coronavirus S protein comprises an RBD and wherein the multimerization domain comprises a fJ-Annulus peptide;

[0358] the antigenic fragment of the coronavirus S protein comprises an RBD and wherein the multimerization domain comprises a ferritin domain;

[0359] the antigenic fragment of the coronavirus S protein comprises an RBD and wherein the multimerization domain comprises a lumazine synthase multimerization domain;

[0360] the antigenic fragment of the coronavirus S protein comprises a truncated SI domain and wherein the multimerization domain comprises a p-Annulus peptide;

[0361] the antigenic fragment of the coronavirus S protein comprises a truncated SI domain and wherein the multimerization domain comprises a ferritin domain;

[0362] the antigenic fragment of the coronavirus S protein comprises a truncated SI domain and wherein the multimerization domain comprises a lumazine synthase multimerization domain;

[0363] the antigenic fragment of the coronavirus S protein comprises an S2 domain and wherein the multimerization domain comprises a p-Annulus peptide;

[0364] the antigenic fragment of the coronavirus S protein comprises an S2 domain and wherein the multimerization domain comprises a ferritin domain; or

[0365] the antigenic fragment of the coronavirus S protein comprises an S2 domain and wherein the multimerization domain comprises a lumazine synthase multimerization domain.

[0366] In some embodiments, an antigenic fragment of a coronavirus S protein and a multimerization domain are connected to one another via a linker.

[0367] In some embodimetns, a linker is a flexible linker, a rigid linker, or a helical linker.

[0368] In some embodiments, a linker is a flexible linker and comprises a GS linker (e.g., wherein the GS linker 5, 10, 15, or 20 amino acids in length), optioanlly wherein the GS linker comprises a sequence of (G4S)1, (G4S)2, (G4S)3, or (G4S)4.

[0369] In some embodiments, an RBD comprises amino acids 327 to 528 of SEQ ID NO: 1, 330 to 528 of SEQ ID NO: 1, amino acids 327 to 528 of SEQ ID NO: 1, or amino acids 330 to 541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of a SARS-CoV-2 variant.

[0370] In some embodimetns, an antigenic fragment of a SARS-CoV-2 S protein comprises an RBD and an NTD of a SARS-CoV-2 S protein.

[0371] In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises a truncated SI domain.

[0372] In some embodiments, an RBD is at the C-terminus of the truncated SI domain (e.g., wherein the RBD comprises amino acids 327 to 528 of SEQ ID NO: 1, 330 to 528 of SEQ ID NO: 1, amino acids 327 to 528 of SEQ ID NO: 1, or amino acids 330 to 541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of a SARS-CoV-2 variant).

[0373] In some embodiments, a truncated SI domain comprises amino acids 14-528 of SEQ ID NO: 1, amino acids 17-528 of SEQ ID NO: 20-528 of SEQ ID NO: 1, amino acids 14-541 of SEQ ID NO: 1, amino acids 17-541 of SEQ ID NO: 1, or amino acids 20-541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing from an S protein of a SARS-CoV-2 variant.

[0374] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 205, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 205.

[0375] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 206, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 206.

[0376] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 207, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 207.

[0377] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 208, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 208.

[0378] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 209, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 209.

[0379] In some embodiments, an RNA encodes a polypeptide comprising SEQ ID NO: 210, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 210.

[0380] In some embodiments, an RNA comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 211, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 211;

[0381] In some embodiments, an RNA comprises a nucleotide sequence of SEQ ID NO: 212, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 212

[0382] In some embodiments, an RNA comprises a nucleotide sequence of SEQ ID NO: 212, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 214.

[0383] Among other things, the present disclosure describes, an RNA comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an immunogenic fragment of a SARS-CoV-2 S protein, and one or more domains that can induce formation of a viral-like particle (VLP) when the RNA is transfected into a cell.

[0384] In some embodiments, one or more domains that can induce formation of a VLP comprise an endosomal sorting complex required for transport (ESCRT)- and ALG-2-interacting protein X (ALIX) binding region (collectively referred to as EABR).

[0385] In some embodiments, an EABR sequence comprises an amino acid sequence of FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP, or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP.

[0386] In some embodiments, an EABR sequence comprises an amino acid sequence of LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ.

[0387] In some embodiments, a polypeptide comprising an EABR sequence further comprises a transmembrane domain.

[0388] In some embodiments, a transmembrane domain is a heterologous transmembrane domain or a homologous transmembrane domain.

[0389] In some embodiments, a transmembrane domain is or comprises a SARS-CoV-2 S protein transmembrane domain.

[0390] In some embodimetns, a transmembrane domain comprises an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC.

[0391] In some embodiments, an EABR and a transmembrane domain are C-terminal to a SARS-CoV-2 S protein fragment.

[0392] In some embodimetns, an EABR sequence is C-terminal to a transmembrane domain.

[0393] In some embodiments, a transmembrane domain and a EABR sequence are directly linked to one another, or a transmembrane domain and a EABR sequence are connected via a flexible linker. In some embodiments, the flexible linker connecting a transmembrane domain and an EABR sequence is a GS linker comprising about 5, about 10, about 15, or about 20 amino acids.

[0394] In some embodiments, a polypeptide comprises a peptide having an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCGSGSGSFNSSINNIHEMEIQLKDALEKNQQWLVYDQ QREVYVKGLLAKIFELEKKTETAAHSLP (SEQ ID NO: 441), or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical thereto,

[0395] optionally wherein the peptide having an amino acid sequence of SEQ ID NO: 441 or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical thereto is at the C-terminus of the polypeptide.

[0396] In some embodiments, a polypeptide comprising an EABR further comprises an EPM sequence.

[0397] In some embodiments, an EPM sequence comprises ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSSPY, or sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, identical to ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSSPY.

[0398] In some embodiments, one or more domains that can induce formation of a VLP comprise a VSV-G transmembrane domain.

[0399] In some embodiments, a VSV-G transmembrane domain comprises an amino acid sequence of KLKHTKKRQIYTDIEMNRLGK, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to KLKHTKKRQIYTDIEMNRLGK.

[0400] In some embodiments, a VSV-G transmembrane domain comprises a sequence that is endogenously proximal to the membrane in the VSV-G protein.

[0401] In some embodiments, a sequence that is endogenously proximal to the membrane in the VSV-G protein comprises:

[0402] an amino acid sequence of IELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to IELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI;

[0403] an amino acid sequence of FFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to FFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI;

[0404] an amino acid sequence of QDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to QDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI; or

[0405] an amino acid sequence of FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIor an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI.

[0406] In some embodiments, a VSV-G membrane proximal region comprises:

[0407] an amino acid sequence ofIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to IELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK;

[0408] an amino acid sequence ofFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to FFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK;

[0409] an amino acid sequence ofQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to QDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK; or

[0410] an amino acid sequence of FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNR LGKor an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNR LG.

[0411] In some embodiments, a VSV-G transmembrane domain is at the C-terminus of a polypeptide.

[0412] In some embodiments, an immunogenic fragment of an S protein and a VSV-G transmembrane domain are directly linked to one another or are linked via a linker sequence.

[0413] In some embodiments, a linker sequence is a GS linker sequence, and optionally comprises about 5- 20 amino acids.

[0414] In some embodiments, a GS linker sequence comprises about 5 to about 10 amino acids.

[0415] In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an RBD, a truncated SI subdomain, and / or an S2 domain, optionally wherein the S2 domain comprises one or more mutations that increase stability.

[0416] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide that the comprises an amino acid sequence of SEQ ID NO: 411, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 411;

[0417] the RNA comprises a nucleotide sequence of SEQ ID NO: 412, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 412; and / or

[0418] the RNA comprises a nucleotide sequence of SEQ ID NO: 414, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 414.

[0419] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0420] the polyepptide comprises an amino acid sequence of SEQ ID NO: 406, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 406;

[0421] the RNA comprises a nucleotide sequence of SEQ ID NO: 407, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 407; and / or

[0422] the RNA comprises a nucleotide sequence of SEQ ID NO: 409, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 409.

[0423] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0424] the polyepptide comprises an amino acid sequence of SEQ ID NO: 416, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 416;

[0425] the RNA comprises a nucleotide sequence of SEQ ID NO: 417, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 417; and / or

[0426] the RNA comprises a nucleotide sequence of SEQ ID NO: 419, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 419.

[0427] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0428] the polyepptide comprises an amino acid sequence of SEQ ID NO: 421, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 421;

[0429] the RNA comprises a nucleotide sequence of SEQ ID NO: 422, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 422; and / or

[0430] the RNA comprises a nucleotide sequence of SEQ ID NO: 424, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 424.

[0431] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0432] the polypeptide comprises an amino acid sequence of SEQ ID NO: 426, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 426;

[0433] the RNA comprises a nucleotide sequence of SEQ ID NO: 427, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 427; and / or

[0434] the RNA comprises a nucleotide sequence of SEQ ID NO: 428, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 428.

[0435] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0436] the polyepptide comprises an amino acid sequence of SEQ ID NO: 431, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 431;

[0437] the RNA comprises a nucleotide sequence of SEQ ID NO: 432, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 432; and / or

[0438] the RNA comprises a nucleotide sequence of SEQ ID NO: 434, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 434.

[0439] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide, wherein:

[0440] the polyepptide comprises an amino acid sequence of SEQ ID NO: 436, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 436;

[0441] the RNA comprises a nucleotide sequence of SEQ ID NO: 437, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 437; and / or

[0442] the RNA comprises a nucleotide sequence of SEQ ID NO: 438, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 438.

[0443] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 475 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 475; (ii) the nucleotide sequence of SEQ ID NO: 176 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 476; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 474, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 474.

[0444] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 480 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 480; (ii) the nucleotide sequence of SEQ ID NO: 482 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 482; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 479, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 479.

[0445] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 484 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 484; (ii) the nucleotide sequence of SEQ ID NO: 486 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 486; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 483, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 483.

[0446] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 489 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 489; (ii) the nucleotide sequence of SEQ ID NO: 491 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 491; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 488, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 488.

[0447] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 494 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 494; (ii) the nucleotide sequence of SEQ ID NO: 496 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 496; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 493, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 493.

[0448] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 499 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 499; (ii) the nucleotide sequence of SEQ ID NO: 501 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 501; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 498, or asequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 498.

[0449] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 504 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 504; (ii) the nucleotide sequence of SEQ ID NO: 506 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 506; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 503, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 503.

[0450] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 509 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 509; (ii) the nucleotide sequence of SEQ ID NO: 511 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 511; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 508, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 508.

[0451] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 514 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 514; (ii) the nucleotide sequence of SEQ ID NO: 516 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 516; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 513, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 513.

[0452] In some embodiments, one or more domains that can induce formation of a VLP comprise a VP40 TSG101 peptide and / or a p6 Alix peptide.

[0453] In some embodiemnts, a VP40 TSG101 peptide comprises an amino acid sequence of VILPTAPPEYMEA, or an amino acid sequence having 1, 2, 3, 4, or 5 modifications relative to VILPTAPPEYMEA.

[0454] In some embodiments, a p6 Alix peptide comprises:

[0455] an amino acid sequence of DKELYPLTSLRSLFGN, or an amino acid sequence having 1, 2, 3, 4, or 5 modifications relative to DKELYPLTSLRSLFGN; or

[0456] an amino acid sequence of TQNLYPDLSEIKKEYNVKEKDQVEDLNLDSLWE, or an amino acid sequence having 1, 2, 3, 4, or 5 modifications relative to TQNLYPDLSEIKKEYNVKEKDQVEDLNLDSLWE.

[0457] In some embodiments, a p6 Alix peptide and the VP40 TSG101 peptide are directly linked to one anotheer, or wherein the p6 Alix peptide and the VP40 TSG101 peptide are connected via a linker. In some embodiments, the linker is a flexible linker, and optionally comprises a GS linker (e.g., wherein the GS linker is about 5 to about 20 amino acids in length, including, e.g., about 5, about 10, about 15, or 20 amino acids in length).

[0458] In some embodiments, a polypeptide comprises an amino acid sequence of VILPTAPPEYMEAGSGSGSDKELYPLTSLRSLFGN, or or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to VILPTAPPEYMEAGSGSGSDKELYPLTSLRSLFGN.

[0459] In some embodiments, one or more domains that can induce formation of a VLP comprise an amino acid sequence of PTAPPEYGSGSGSLYPLTSLRSLGSGSGSPTAPGSGSGSLYPDLNLDSLGSGSGSPSAP, or or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to PTAPPEYGSGSGSLYPLTSLRSLGSGSGSPTAPGSGSGSLYPDLNLDSLGSGSGSPSAP.

[0460] In some embodiments, one or more domains that can induce formation of a VLP comprise an amino acid seuqence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCGSGSGS VILPTAPPEYMEAGSGSGSDKELYPLTSLRSLFGN or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0461] In some embodiments, one or more domains that can induce formation of a VLP comprise an amino acid seuqence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCGSGSGSVILPTAPPEYMEAGSGSGSTQNLYPDLSEIK KEYNVKEKDQVEDLNLDSLWE or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0462] In some embodiments, one or more domains that can induce formation of a VLP comprise an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCGSGSGSPTAPPEYGSGSGSLYPLTSLRSLGSGSGSPT APGSGSGSLYPDLNLDSLGSGSGSPSAP or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0463] In some embodiments, a VP40 TSG101 peptide and / or p6 Alix peptide are C-terminal to a transmembrane domain.

[0464] In some embodiments, a transmembrane doamin and a VP40 TSG101 peptide and / or a p6 Alix peptide are C-terminal to an immunogenic fragment of a SARS-CoV-2 S protein.

[0465] In some embodiments, a VP40 TSG101 peptide and / or p6 Alix peptide are at the C-terminus of a polypeptide.

[0466] In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an RBD, a truncated SI subdomain, and / or an S2 domain, optionally wherein the S2 domain comprises one or more mutations that increase stability.

[0467] In some embodiments, an RBD comprises amino acids 327 to 528 of SEQ ID NO: 1, 330 to 528 of SEQ ID NO: 1, amino acids 327 to 528 of SEQ ID NO: 1, or amino acids 330 to 541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of a SARS-CoV-2 variant.

[0468] In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an N- Terminal Domain (NTD).

[0469] In some embodiments, an NTD comprises amino acids 14-209, 14-303, 20-318, or 20-302 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant.

[0470] In some embodiments, an immunogenic fragment comprises an SI domain of a SARS-CoV-2 S protein, or an immunogenic fragment thereof.

[0471] In some embodimetns, an immunogenic fragment of a SARS-CoV-2 S protein comprises an RBD and an NTD of a SARS-CoV-2 S protein.

[0472] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 362, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NO: 362.

[0473] In some embodiments, an RNA encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 362, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NO: 363.

[0474] In some embodiments, an RNA encodes a polypeptide a polypeptide comprising an amino acid sequence of SEQ ID NO: 364, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical SEQ ID NO: 364.

[0475]

[0476] In some embodiments, one or more domains that can induce formation of a VLP comprise a portion of a capsid protein of Porcine circovirus 2 (PCV-2).

[0477] In some embodiments, a portion of a capsid protein of Porcine circovirus 2 (PCV-2): comprises an amino acid sequence ofMTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNIDDFVPP GGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLD STIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPPLKP, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to MTYPRRRYRRRRHRPRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTVKATTVRTPSWAVDMMRFNIDDFVPP GGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVNYSSRHTIPQPFSYHSRYFTPKPVLD STIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPPLKP; and / or is encoded by a nucleotide sequence ofAUGACGUAUCCAAGGAGGCGUUACCGCAGAAGAAGACACCGCCCCCGCAGCCAUCUUGGCCAGAUCCUCCGCCGCCGCCC CUGGCUCGUCCACCCCCGCCACCGCUACCGUUGGAGAAGGAAAAAUGGCAUCUUCAACACCCGCCUCUCCCGCACCUUCG GAUAUACUGUCAAGGCUACCACAGUCAGAACGCCCUCCUGGGCGGUGGACAUGAUGAGAUUUAAUAUUGACGACUUUGU UCCCCCGGGAGGGGGGACCAACAAAAUCUCUAUACCCUUUGAAUACUACAGAAUAAGAAAGGUUAAGGUUGAAUUCUGGC CCUGCUCCCCCAUCACCCAGGGUGAUAGGGGAGUGGGCUCCACUGCUGUUAUUCUAGAUGAUAACUUUGUAACAAAGGC CACAGCCCUAACCUAUGACCCAUAUGUAAACUACUCCUCCCGCCAUACAAUCCCCCAACCCUUCUCCUACCACUCCCGUUA CUUCACACCCAAACCUGUUCUUGACUCCACUAUUGAUUACUUCCAACCAAAUAACAAAAGGACUCAGCUUUGGCUGAGGC UACAAACCUCUAGAAAUGUGGACCACGUAGGCCUCGGCACUGCGUUCGAAAACAGUAUAUACGACCAGGACUACAAUAUC CGUGUAACCAUGUAUGUACAAUUCAGAGAAUUUAAUCUUAAAGACCCCCCACUUAAACCC, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical toAUGACGUAUCCAAGGAGGCGUUACCGCAGAAGAAGACACCGCCCCCGCAGCCAUCUUGGCCAGAUCCUCCGCCGCCGCC CCUGGCUCGUCCACCCCCGCCACCGCUACCGUUGGAGAAGGAAAAAUGGCAUCUUCAACACCCGCCUCUCCCGCACCUUC GGAUAUACUGUCAAGGCUACCACAGUCAGAACGCCCUCCUGGGCGGUGGACAUGAUGAGAUUUAAUAUUGACGACUUUG UUCCCCCGGGAGGGGGGACCAACAAAAUCUCUAUACCCUUUGAAUACUACAGAAUAAGAAAGGUUAAGGUUGAAUUCUGG CCCUGCUCCCCCAUCACCCAGGGUGAUAGGGGAGUGGGCUCCACUGCUGUUAUUCUAGAUGAUAACUUUGUAACAAAGG CCACAGCCCUAACCUAUGACCCAUAUGUAAACUACUCCUCCCGCCAUACAAUCCCCCAACCCUUCUCCUACCACUCCCGUU ACUUCACACCCAAACCUGUUCUUGACUCCACUAUUGAUUACUUCCAACCAAAUAACAAAAGGACUCAGCUUUGGCUGAGG CUACAAACCUCUAGAAAUGUGGACCACGUAGGCCUCGGCACUGCGUUCGAAAACAGUAUAUACGACCAGGACUACAAUAU CCGUGUAACCAUGUAUGUACAAUUCAGAGAAUUUAAUCUUAAAGACCCCCCACUUAAACCC.

[0478] In some embodiments, a portion of a capsid protein of PCV-2: comprises an amino acid sequence ofTPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSFAVILDDNFVTKATALTYDPYVNYSSR HTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPP,or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to TPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVNYSSR HTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPP; and / or is encoded by a nucleotide sequence of ACGCCCUCCUGGGCGGUGGACAUGAUGAGAUUUAAUAUUGACGACUUUGUUCCCCCGGGAGGGGGGACCAACAAAAUCU CUAUACCCUUUGAAUACUACAGAAUAAGAAAGGUUAAGGUUGAAUUCUGGCCCUGCUCCCCCAUCACCCAGGGUGAUAGG GGAGUGGGCUCCACUGCUGUUAUUCUAGAUGAUAACUUUGUAACAAAGGCCACAGCCCUAACCUAUGACCCAUAUGUAAA CUACUCCUCCCGCCAUACAAUCCCCCAACCCUUCUCCUACCACUCCCGUUACUUCACACCCAAACCUGUUCUUGACUCCAC UAUUGAUUACUUCCAACCAAAUAACAAAAGGACUCAGCUUUGGCUGAGGCUACAAACCUCUAGAAAUGUGGACCACGUAG GCCUCGGCACUGCGUUCGAAAACAGUAUAUACGACCAGGACUACAAUAUCCGUGUAACCAUGUAUGUACAAUUCAGAGAA UUUAAUCUUAAAGACCCCCCACUU, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical ACGCCCUCCUGGGCGGUGGACAUGAUGAGAUUUAAUAUUGACGACUUUGUUCCCCCGGGAGGGGGGACCAACAAAAUCU CUAUACCCUUUGAAUACUACAGAAUAAGAAAGGUUAAGGUUGAAUUCUGGCCCUGCUCCCCCAUCACCCAGGGUGAUAGG GGAGUGGGCUCCACUGCUGUUAUUCUAGAUGAUAACUUUGUAACAAAGGCCACAGCCCUAACCUAUGACCCAUAUGUAAA CUACUCCUCCCGCCAUACAAUCCCCCAACCCUUCUCCUACCACUCCCGUUACUUCACACCCAAACCUGUUCUUGACUCCAC UAUUGAUUACUUCCAACCAAAUAACAAAAGGACUCAGCUUUGGCUGAGGCUACAAACCUCUAGAAAUGUGGACCACGUAG GCCUCGGCACUGCGUUCGAAAACAGUAUAUACGACCAGGACUACAAUAUCCGUGUAACCAUGUAUGUACAAUUCAGAGAA UUUAAUCUUAAAGACCCCCCACUU.

[0479] In some embodiments, a portion of a capsid protein of PCV-2 comprises an amino acid sequence of TPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVDYSSR HTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPPL, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to TPSWAVDMMRFNIDDFVPPGGGTNKISIPFEYYRIRKVKVEFWPCSPITQGDRGVGSTAVILDDNFVTKATALTYDPYVDYSSR HTIPQPFSYHSRYFTPKPVLDSTIDYFQPNNKRTQLWLRLQTSRNVDHVGLGTAFENSIYDQDYNIRVTMYVQFREFNLKDPPL.

[0480] In some embodiments, a portion of a PCV-2 capside protein is at the C-terminus of a polypeptide.

[0481] In some embodiments, a polypeptide comprises a secretory signal peptide that comprises (i) an amino acid sequence that is listed in Table 2 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence that is listed in Table 2, and / or (ii) wherein the RNA comprises a nucleotide sequence that is listed in Table 3 or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to an amino acid sequence that is listed in Table 2.

[0482] In some embodiments, a polypeptide comprises an SP24-Q7PUJ5_ANOGA secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of MCRGLSAVLILLVSLSAQLHVWG (SEQ ID NO: 22) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to MCRGLSAVLILLVSLSAQLHVWG (SEQ ID NO: 22)).

[0483] In some embodiments, a polypeptide comprises an SP24-SP18-HEMA_CVBM secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MFLLLRFVLVSCIIGSLG (SEQ IDNO: 391) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to MFLLLRFVLVSCIIGSLG (SEQ ID NO: 391).

[0484] In some embodiments, a polypeptide comprises an SP25-GD_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising an amino acid sequence of MGGAAARLGAVILFWIVGLHGVRG (SEQ ID NO: 12) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to MGGAAARLGAVILFWIVGLHGVRG (SEQ ID NO: 12).

[0485] In some embodiments, a polypeptide comprises an SP32-GB_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MHQGAPSWGRRWFWWALLGLTLGVLVASAAP (SEQ ID NO: 38) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to MHQGAPSWGRRWFWWALLGLTLGVLVASAAP (SEQ ID NO: 38).

[0486] In some embodiments, a polypeptide comprises an SP20-A7U881_HHV2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MARGAGLVFFVGVWWSCLA (SEQ ID NO: 366) or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to MARGAGLVFFVGVWWSCLA (SEQ ID NO: 366).

[0487] In some embodiments, an RNA comprises a 5' cap, a cap proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence.

[0488] In some embodiments,(i) a 5' cap comprises a Capl structure;(ii) a 5'-UTR sequence comprises a modified human alpha-globin 5'-UTR;(iii) a 3'-UTR sequence comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA;(iv) a polyA sequence comprises at least 100 A nucleotides; or(v) a combination of any one of (i)-(iv).

[0489] In some embodiments, a 5' cap comprises a Capl structure, and the Capl structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeAl)pG2, wherein Al is position +1 of the RNA, and G2 is position +2 of the RNA.

[0490] In some embodiments, a cap proximal sequence comprises Al and G2 of the Capl structure, and a sequence comprising: A3N4N5 at positions +3, +4 and +5 respectively of the RNA, wherein N4 and N5 are each independently selected from A, G, C, and U.

[0491] In some embodiments, a polyA sequence comprises an interrupted sequence of A nucleotides, optionally wherein the interrupted 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.

[0492] In some embodiments, a 5'-UTR sequence comprises SEQ ID NO: 12, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 12.

[0493] In some embodiments, a 3'-UTR sequence comprises SEQ ID NO: 13, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 13. In some embodiments, a 3'-UTR sequence comprises SEQ ID NO: 601, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 601. In some embodiments, a 3'-UTR sequence comprises SEQ ID NO: 602, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 602.

[0494] In some embodiments, an interrupted polyA tail sequence comprises SEQ ID NO: 14, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 14.

[0495] In some embodiments, a sequence at the 5' end of the 3'UTR sequence (e.g., the sequence immediately adjacent to a sequence encoding an antigenic polypeptide) is CUCGAG or GGAUCCGAU.

[0496] In some embodiments, an RNA is saRNA, self-amplifying RNA, trans-amplifying RNA (taRNA), or mRNA.

[0497] In some embodiments, an RNA is unmodified RNA or wherein the RNA comprises one or modified uridines in place of one or more uridines.

[0498] In some embodiments, an RNA comprises a single modified uridine in place of each uridine.

[0499] In some embodiments, a modified uridine is Nl-methyl-pseudouridine.

[0500] In some embodiments, a nucleotide sequence encoding the SARS-CoV-2 S protein is encoded by a sequence that is codon-optimized (e.g., codon-optimized for expression in human cells) and / or which has a G / C content that is increased compared to a wild type coding sequence.

[0501] In some embodiments, an RNA is formulated in a nanoparticle.

[0502] In some embodiments, a nanoparticle is a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle.

[0503] In some embodiments, a nanoparticle is a lipid nanoparticle.

[0504] In some embodiments, a lipid nanoparticle comprises a cationically ionizable lipid, a sterol, a neutral lipid, and a polymer-conjugated lipid.

[0505] In some embodiments, a polymer-conjugated lipid comprises a PEG-conjugated lipid.

[0506] In some embodiments, an RNA is encapsulated in a lipid nanoparticle (LNP), preferably wherein the LNP comprises molar ratios of 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0507] In some embodiments, nanoparticles have an average diameter of about 50-150 nm.

[0508] In some embodiments, a composition comprises a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose.

[0509] In some embodiments, a composition comprises an aqueous buffered solution, optionally wherein the aqueous buffered solution comprises one or more of Tris base, Tris HCI, NaCI, KCI, Na2HPO4, and KH2PO4.

[0510] In some embodiments, a compostion comprises about 10 mM Tris buffer and about 10% sucrose.

[0511] Among other things, the present disclosure describes a pharmaceutical composition comprising (i) an RNA or composition described herein and (ii) a pharmaceutically acceptable excipient.

[0512] In some embodiments, a pharmaceutical composition is formulated as a multi-dose formulation in a vial, a single-dose formulation in a vial, or a prefilled syringe.

[0513] In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 100 μg or less (e.g., about 90 μg or less) of total RNA.

[0514] In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg, or about 3 μg of total RNA.

[0515] Among other things, the present disclosure describes a method comprising administering an RNA, composition, or pharmaceutical composition described herein to a subject.

[0516] In some embodiments, if a subject is 12 years or older, a method comprises administering 30 μg of the RNA. In some embodiments, if a subject is 5 years to less than 12 years old, a method comprises administering 10 μg of the RNA. In some embodiments, if a subject is 6 months to less than 5 years old, amethod comprises administering 3 μg of the RNA. In some embodiments, a composition described herein is administered in a volume of about 200 pL to about 300 μL.

[0517] In some embodiments, a subject has not previously been administered a SARS-CoV-2 vaccine and / or wherein the subject has not previously been determined to have been infected with SARS-CoV-2 (e.g., as determined using a PCR or antigen test).

[0518] In some embodiments, a method comprises administering a single dose of RNA, composition, or pharmaceutical composition to a subject.

[0519] In some embodiments, a method comprises administering two or more doses of the RNA, composition, or pharmaceutical composition to a subject, optionally wherein the two doses are administered about 21 days apart.

[0520] In some embodiments, RNA, composition, or pharmaceutical composition is administered three times to a subject, optionally wherein a first and a second dose are administered about 21 days apart, and a third dose is administered about 28 days after the second dose.

[0521] In some embodiments, a method comprises administering a further dose of the RNA, composition, or pharmaceutical composition, at least about 2 months after the first dose of the RNA, composition, or pharmaceutical composition (e.g., 2-12 months, 2-10 months, 2-8 months, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months).

[0522] In some embodiments, a subject has previously been exposed to a SARS-CoV-2 antigen (e.g., by vaccination and / or by infection).

[0523] In some embodiments, a subject was previously administered one or more doses of a SARS-CoV-2 vaccine.

[0524] In some embodimetns, a subject was previously administered a complete dosing regimen of a SARS-CoV-2 vaccine.

[0525] In some embodiments, a subject was previously administered a first dose and a second dose of a vaccine that delivers a full length SARS-CoV-2 S protein (e.g., a composition comprising LNP-formulated RNA encoding a SARS-CoV-2 S protein), wherein the first dose and the second dose were administered about 21 days apart, and optionally wherein the subject was previously administered as a booster dose a monovalent or bivalent vaccine that delivers a SARS-CoV-2 S protein of one or more variants (e.g., (i) an S protein of a Wuhan strain and an S protein of an Omicron BA.4 / 5 strain, (ii) an S protein of an XBB.1.5 variant, (iii) an S protein of a KP.2 variant, and / or (iv) an S protein of a JN.l variant).

[0526] In some embodiments, a method comprises administering one or more vaccines against a non- SARS-CoV-2 disease, optionally wherein the one or more vaccines comprises an RSV vaccine, an influenza vaccine, or a combination thereof.

[0527] In some embodiments, method described herein can induce an immune response against SARS- CoV-2 in the subject.

[0528] In some embodiments, an immune response comprises a B-cell response.

[0529] In some embodiments, a B cell response comprises production of antibodies directed against one or more SARS-CoV-2 viruses.

[0530] In some embodiments, an immune response comprises a T cell response, optionally wherein the T- cell response comprises a CD4+ T cell response and / or CD8+ T cell response.

[0531] In some embodiments, a method is a method of preventing or reducing the chances of being infected with a SARS-CoV-2 virus and / or treating a SARS-CoV-2 infection.

[0532] In some embodiments, an RNA, composition, or pharmaceutical composition described herein can be used to induce an immune response in a subject. In some embodiments, an immune response is induced usign a method described herein.

[0533] In some embodiments, an RNA, composition, or pharmaceutical composition described herein can be used for the manufacture of a medicament for inducing an immune response in a subject. In some embodiments, a medicament is formulated to be administered to a subject in accordance with a method described herein.

[0534] Among other things, the present disclosure provides a method of manufacturign RNA, wherein the method comprises in vitro transcribing an RNA described herein.

[0535] In some embodiments, a ribonucleic acid (RNA) comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises:(i) a receptor binding domain (RBD) of a coronavirus Spike (S) protein; and(ii) an S2 domain of a coronavirus S protein or one or more fragments thereof; wherein the RBD and the S2 domain or the one or more fragments thereof are directly adjacent to one another or are connected via a non-endogenous sequence.Brief description of the drawings

[0536] Fig. 1. Schematic Overview of the S Protein Organization of the SARS-CoV-2 S Protein.The sequence within the SI subunit in the native SARS-CoV-2 S protein comprises a signal sequence (SS) and the receptor binding domain (RBD) which is a subunit within the S protein that is relevant for binding to the human cellular receptor ACE2. The S2 subunit in the native SARS-CoV-2 S protein contains the S2 protease cleavage site (529 followed by a fusion peptide (FP) for membrane fusion, heptad repeats (HR1 and HR2) with a central helix (CH) domain, the transmembrane domain (TM) and a cytoplasmic tail (CT).

[0537] Fig. 2. Schematics of an Exemplary Dosing Regimen.

[0538] Fig. 3. Sequences of RBDs of SARS-COV-2 Wuhan strain and Variants Thereof. Variantspecific amino acid alterations are indicated in bold red font, with the original Wuhan amino acid highlighted in bold blue font.

[0539] Fig. 4. Exemplary Polypeptides Comprising an S2 Polypeptide, an RBD, an NTD, and / or Fragments of an S2 Polypeptide. (A) Depects exemplary designs of polypeptides comprising a secretory signal peptide C'SP), a Receptor Binding Domain (RBD), an S2 domain, a transmembrane domain ("TM), and a C- terminal sequence. Lightening bolts are used to indicate linkers that comprise a protease cleavage site (e.g., a human furin protease) (e.g., a sequence described herein). (B) Depects Exemplary construct designs for polypeptides comprising an S2 domain, optionally with one or more mutations that stabilize a prefusion confirmation of the S2 polypeptide (indicated with in the figure) and a fibritin trimerization domain ("F"). (B) Also depicts exemplary polyepptides comprising a stem helix and fusion peptide of an S2 domain and an S2 and RBD, optionally with one or more muations that stabilize a prefusion confirmation fo the S2 domain and a prease cleavage site.

[0540] Fig. 5. Exemplary Polypeptides Comprising a SARS-Cov-2 Antigen That are Capable of Producing Viral Like Particles (VLPs). "C" stands for a capsid protein of Porcine circovirus 2. "SP" refers to a secretory signal peptide.

[0541] Fig. 6. Exemplary Polypeptide Comprising a SARS-Cov-2 Antigen and Components For Forming a VLP (EABR and EPM).

[0542] Fig. 7. In Vitro Expression of Soluble S2 Polypeptides. (A) and (B) show intracellular S2 polypeptide signal in cells (% positive cells and mean flource intensity (MFI) receptively) transfected with an RNA encoding a full length S protein (Construct 1) and cells transfected with various RNAs described herein that encode a polypeptide comprising an S2 polypeptide with one or more stabilizing mutations, and which lack a transmembrane domain (Constructs 2-8). Signal was assessed using a fluorescently labeled antibody that binds the S2 polypeptide region. As shown, each of the constructs tested produced detectable S2 signal. Further information regarding the characterized constructs is provided in Example 5.

[0543] Fig. 8. In Vitro Stem Helix (SH) Signal Produced by RNA Encoding Soluble S2Polypeptides. (A)-(C) Show intracellular Stem Helix (SH) signal measured in cells transfected with RNA encoding a full length S protein (Construct 1) and various RNAs described herein that encode a polypeptide comprising an S2 polypeptide with one or more stabilizing mutations and which lack a transmembrane domain (Constructs 2-7). SH signal was measured using three different antibodies, each of which had previously been shown to bind to the SH region and also to be capable of neutralizing a SARS-CoV-2 virus (SH nAbl-3). As shown in each of (A)-(C), RNA encoding an S2 polypeptide was shown to result in increased SH signal for each construct tested. Further information regarding the characterized constructs is provided in Example 5.

[0544] Fig. 9. In Vitro Fusion Peptide (FP) Signal Produced by RNA Encoding Soluble S2 Polypeptides. (A)-(C) Show intracellular Fusion Peptide (FP) signal observed for an RNA encoding a full length S protein (Construct 1), and various RNAs described herein that encode a polypeptide comprising an S2 polypeptide with one or more stabilizing mutations and which lack a transmembrane domain (Constructs 2-7). Cells were transfected with one of the indicated RNA molecules, and intracellular FP signal was measured using three different antibodies that had previously been shown to bind to the FP region and to be capable of neutralizing a SARS-CoV-2 virus (FP nAbl-3). As shown, low FP signal was observed for an RNA encoding a full length S protein. Certain RNA molecules described herein, in contrast, displayed strong FP signal. Further information regarding the characterized constructs is provided in Example 5.

[0545] Fig. 10. Exemplary RBD-S2 and RBD-SH-FP Constructs. Shown are exemplary designs for polypeptides encoded by RNA described herein. TM is an abbreviation for transmembrane domain.

[0546] Fig. 11. RBD Signal Produced by RNA Encoding an S2 Polypeptide or SH and FP Peptides, and Optionally Linked to an RBD. Cells were transfected with RNA encoding (1) a full length S protein (Construct 1), (2) RNA encoding an S2 polypeptide (Construct 2), (3) RNA encoding a polypeptide comprising an RBD, a transmembrane domain, and a fibritin domain, and lacking regions from the S2 polypeptide (Construct 3), (4) RNA encoding an S2 polypeptide linked to an RBD (Constructs 4-6), or (5) RNA encoding a polypeptide comprising an RBD linked to an SH and FP region of the S2 polypeptide (Constructs 7 and 8). Shown is cell surface RBD signal, as determined using a flouresently labeled ACE2 protein. As shown, certain RNA molecules described herein can result in significantly increased cell surface expression of RBD as compared to RNA encoding a full length S protein. Further information regarding the characterized constructs is provided in Example 6.

[0547] Fig. 12. In Vitro S2 Signal Produced by RNA Encoding an S2 Polypeptide or SH and FP Peptides, and Optionally Linked to an RBD. Cells were transfected with RNA encoding (1) a full length S protein (Construct 1), (2) RNA encoding an S2 polypeptide (Construct 2), (3) RNA encoding a polypeptide comprising an RBD, a transmembrane domain, and a fibritin domain, and lacking regions from the S2 polypeptide (Construct 3), (4) RNA encoding an S2 polypeptide linked to an RBD (Constructs 4-6), or (5) RNA encoding a polypeptide comprising an RBD linked to an SH and FP region of the S2 polypeptide (Constructs 7 and 8). Intracellular (shown in (A)) and cell surface (shown in (B)) S2 signal was then measured using an antibody thatbinds the S2 polypeptide. As shown, RNA described herein induced higher intracellular S2 signal as compared to RNA encoding a full length S protein. An RNA encoding an RBD and SH and FP regions of the S protein (Construct 8) was also shown to induce higher cell surface S protein signal as compared to RNA encoding a full length S protein. Further information regarding the characterized constructs is provided in Example 6.

[0548] Fig. 13. In Vitro Stem Helix (SH) Signal Produced by RNA Encoding an S2 Polypeptide or SH and FP Peptides, and Optionally Linked to an RBD. Cells were transfected with RNA encoding (1) a full length S protein (Construct 1), (2) RNA encoding an S2 polypeptide (Construct 2), (3) RNA encoding a polypeptide comprising an RBD, a transmembrane domain, and a fibritin domain, and lacking regions from the S2 polypeptide (Construct 3), (4) RNA encoding an S2 polypeptide linked to an RBD (Constructs 4-6), or (5) RNA encoding a polypeptide comprising an RBD linked to an SH and FP region of the S2 polypeptide (Constructs 7 and 8). Intracellular (shown in (A)) and cell surface (shown in (B)) SH signal was then measured using an antibody previously shown to bind the SH region and to be capable of neutralizing a SARS-CoV-2 virus. As shown, RNA encoding an S2 polypeptide or an SH and FP peptide was shown to result in increased intracellular SH signal for each construct tested. Constructs comprising an SH and FP region linked to an RBD (Constructs 7 and 8) were also shown to produce higher cell surface SH signal as compared to RNA encoding a full length S protein. Further information regarding the characterized constructs is provided in Example 6.

[0549] Fig. 14. In Vitro Fusion Peptide (FP) Signal Produced by RNA Encoding an S2 Polypeptide or SH and FP Peptides, and Optionally Linked to an RBD. Cells were transfected with RNA encoding (1) a full length S protein (Construct 1), (2) RNA encoding an S2 polypeptide (Construct 2), (3) RNA encoding a polypeptide comprising an RBD, a transmembrane domain, and a fibritin domain, and lacking regions from the S2 polypeptide (Construct 3), (4) RNA encoding an S2 polypeptide linked to an RBD (Constructs 4-6), or (5) RNA encoding a polypeptide comprising an RBD linked to an SH and FP region of the S2 polypeptide (Constructs 7 and 8). Intracellular (shown in (A)) and cell surface (shown in (B) FP signal was then measured using an antibody previously shown to bind the FP region and to be capable of neutralizing a SARS-CoV-2 virus.As shown, RNA encoding an S2 polypeptide or an SH and FP peptide was shown to result in increased intracellular FP signal for each construct tested while low FP signal was observed for Construct 1 (encoding a full length S protein). Constructs encoding a polypeptide comprising an SH, FP, and RBD regions of a SARS-CoV-2 S protein were also shown to produce much higher cell surface FP signal. Further information regarding the characterized constructs is provided in Example 6.

[0550] Fig. 15. Exemplary Experimental Protocol for Characterizing Immune Responses inVaccine Naive Mice. Two doses of the indicated composition were administered about 21 days apart. Serum samples were collected before administering a composition and 7, 14, 21, and 34 days after administering the first dose. On day 34, final samples were collected and mice were sacrificed.

[0551] Fig. 16. Neutralization Titers Induced in Vaccine Naive Mice by RNA Encoding an S2 Polypeptide and an RBD or SH and FP Peptides and an RBD. (A)-(D) Show neutralization titers against XBB.1.5-adapted, BA.4 / 5-adapted, and Wuhan-adapted pseudoviruses measured in sera samples collected from mice administered compositions in accordance with the protocol indicated in Fig. 15. Neutralization titers 7, 14, 21, and 34 days after administering the indicated composition are shown. As demonstrated in (D), by day 34, neutralization titers induced by Construct 5 (encoding a polypeptide comprising comprising an RBD, SH, and FP of a SARS-CoV-2 S protein) were ~1.5x those induced by Construct 1 (encoding a full length S protein) for both an XBB.1.5 and BA.4 / 5-adapted pseudovirus. Neutralizing titers induced by Construct 3 (encoding a polypeptide comprising an S2 and RBD) against a Wuhan S protein, were ~2x those induced by Construct 1, demonstratingincreased cross-neutralization. "XBB.1.5" refers to neutralizing titers measured in a repetition of the pVNT assay against XBB.l.S-adpated pseudoviruses.

[0552] Fig. 17. Antibody Titers Induced in Vaccine Naive Mice by RNAs Encoding an S2 Polypeptide and an RBD or SH and FP Peptides and an RBD (ELISA Data). (A) and (B) Show antibody titers as determined using ELISA against an XBB.1.5 RBD and a Wuhan RBD, respectively, in sera samples collected at day 34 of the experiment summarized in Fig. 15. As shown, Constructs 4 and 5 (encoding an RBD linked to an SH and an FP) resulted in significantly increased antibody titers as compared to Construct 1 (encoding a full length S protein).

[0553] Fig. 18. Assessing VLP Formation and (3-Annulus Tagged Polypeptide Expression. (A) Provides a schematic summarizing the design of certain constructs described herein. "L" refers to a linker. (B) Provides the membrane proximal sequence of a VSV-G protein, and indicates the different membrane proximal sequences described therein. (C) Provides supernatant RBD signal from cells transfected with RNA encoding a SARS-CoV-2 antigen (RBD or a truncated SI domain) and a transmembrane domain (VSV-GJong, VSV-G_short, or SARS-CoV-2 transmembrane domain) or a p-Annulus multimerization domain. As expected, 4012, the one RNA encoding a polypeptide lacking a transmembrane domain, exhibited the highest supernatant signal. Each of the RNAs encoding a polypeptide comprising a VSV-G transmembrane domain also produced supernatant signal, which is consistent with VLP formation, as any VLPs formed would be expected to remain in the supernatant. VSV-GJong, in particular, may be especially effective at promoting VLP formation, given the high supernatant signal it produced.

[0554] Fig. 19. Assessing Immunogenicity of VLP Inducing RNA. (A)-(F) GMTs (geometric mean titers) against an XBB.1.5-adapted pseudovirus in serum samples collected from mice administered two doses (21 days apart) of the indicated composition.

[0555] Fig. 20. Characterizing VLP Formation In Vitro Using Polypeptides Comprising a VSV-G transmembrane domain and an RBD. RNA encoding polypeptides comprising an RBD and different VSV-G domains were transfected into cells. (A) Shows RBD signal as assessed in the supernatant and at the cell surface. (B) Shows RBD concentration measured in the supernatant of cells transfected with a certain amount of RNA. Each of the VSV-G transmembrane domains was found to produce increased supernatant signal and decreased cell surface signal as compared to an RNA encoding a SARS-CoV-2 antigen linked to a SARS-CoV-2 transmembrane domain, which is consistent with these transmembrane domains being capable of inducing VLP formation. VSV-GJong and VSV-G_mid_Q427 may be especially effective at inducing VLP formation, given the high supernatant signal and low cell surface signal produced by these constructs.

[0556] Fig. 21. Characterizing VLP Formation In Vitro Using Polypeptides Comprising a VSV-G transmembrane domain and a truncated SI Domain. RNA encoding polypeptides comprising a truncated SI domain and different VSV-G domains were transfected into cells. (A) Shows RBD signal as assessed in the supernatant and at the cell surface. (B) Shows RBD concentration measured in the supernatant of cells transfected with a certain amount of RNA. Each of the VSV-G transmembrane domains was found to produce increased supernatant signal and decreased cell surface signal as compared to an RNA encoding a SARS-CoV-2 antigen linked to a SARS-CoV-2 transmembrane domain, which is consistent with these transmembrane domains being capable of inducing VLP formation. VSV-GJong and VSV-G_mid_Q427 may be especially effective at inducing VLP formation, given the high supernatant signal and low cell surface signal produced by these constructs.

[0557] Fig. 22. Assessing Immunogenicity of VLP Inducing RNA. (A)-(F) GMTs (geometric mean titers) against an XBB.l.S-adapted pseudovirus in serum samples collected from mice administered a single dose of the indicated composition.

[0558] Fig. 23. Improved GS Linkers for VSV-G and (3-Annulus Polypeptides. (A) and (B) show cell surface (membrane-exposed) and supernatant (soluble antigen) RBD signal observed in cells transfected with various RNAs encoding a polypeptide comprising a truncated SI polypeptide and a VSV-G_mid_Q427 transmembrane domain, which are identical except for the linker sequence connecting the truncated SI domain and the transmembrane domain. As shown, a GS linker comprising 5 or 10 amino acids resulted in increased cell surface and supernatant expression as compared to a GS linker comprising 15 amino acids. (C) Characterizes the RBD signal (soluble antigen) observed in the supernatant of cell cultures transfected with RNAs encoding a polypeptide comprising a truncated SI domain and a fJ-Annulus multimerization domain, and which are identical other than the linker connecting the p-Annulus multimerization domain and the truncated SI domain. As shown, a 5 amino acid long GS linker resulted in increased soluble antigen expression as compared to a GS linker comprising 10, 15, or 20 amino acids.Detailed description

[0559] Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0560] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0561] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0562] In the following, the elements of the present disclosure will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements should be considered disclosed by this description unless the context indicates otherwise.

[0563] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure was not entitled to antedate such disclosure.Definitions

[0564] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0565] The term "about" means approximately or nearly, and in the context of a numerical value or range set forth herein in one embodiment means ± 20%, ± 10%, ± 5%, or ± 3% of the numerical value or range recited or claimed.

[0566] The terms "a" and "an" and "the" and similar reference used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-daimed element essential to the practice of the disclosure.

[0567] "Activation" or "stimulation", as used herein, refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.

[0568] The term "antigen" relates to an agent comprising an epitope against which an immune response can be generated. The term "antigen" includes, in particular, proteins and peptides. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. An antigen or a procession product thereof such as a T-cell epitope is in one embodiment bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a procession product thereof may react specifically with antibodies orT lymphocytes (T cells). In one embodiment, an antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS-CoV-2 S protein, and an epitope is derived from such antigen.

[0569] The term "viral antigen" refers to any viral component having antigenic properties, i.e. being able to provoke an immune response in an individual. The viral antigen may be coronavirus S protein, e.g., SARS-CoV-2 S protein.

[0570] "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen-specific antibodies added to the cells.

[0571] The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction withany other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0572] The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells.

[0573] Unless expressly specified otherwise, the term "comprising" is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising". It is, however, contemplated as a specific embodiment of the present disclosure that the term "comprising" encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment "comprising" is to be understood as having the meaning of "consisting of or "consisting essentially of".

[0574] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

[0575] The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0576] "Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

[0577] The term "immunologically equivalent" means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of antigens or antigen variants used forimmunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

[0578] Terms such as "increase", "enhance" or "exceed" preferably relate to an increase or enhancement by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

[0579] According to the present disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" or "polypeptide" refers to large peptides, in particular peptides having at least about 150 amino acids, but the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.

[0580] The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

[0581] Terms such as "reduce", "decrease", "inhibit" or "impair" as used herein relate to an overall reduction or the ability to cause an overall reduction, preferably of at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or even more, in the level. These terms include a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0582] A "therapeutic protein" has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term "therapeutic protein" includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

[0583] The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

[0584] By "variant" herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence.Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

[0585] By "wild type" or "WT" or "native" herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

[0586] A peptide and protein antigen which is provided to a subject according to the present disclosure by administering RNA encoding the peptide and protein antigen, i.e., a vaccine antigen, preferably results in the induction of an immune response, e.g., a humoral and / or cellular immune response in the subject being provided the peptide or protein antigen. Said immune response is preferably directed against a target antigen, in particular coronavirus S protein, in particular SARS-CoV-2 S protein. Thus, a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragment or variant is immunologically equivalent to the target antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent which results in the induction of an immune response which immune response targets the antigen, i.e. a target antigen. Thus, the vaccine antigen may correspond to or may comprise the target antigen, may correspond to or may comprise a fragment of the target antigen or may correspond to or may comprise an antigen which is homologous to the target antigen or a fragment thereof. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence being homologous to an immunogenic fragment of a target antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

[0587] The peptide and protein antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0588] The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein.

[0589] In one embodiment, vaccine antigen is recognized by an immune effector cell. Preferably, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate costimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present disclosure, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell such as a virus-infected cell. In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and / or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and / or present on T cells to an antigen presented on diseased cells results in cytolysis and / or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g. perforins and granzymes.

[0590] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0591] In some embodiments, the present disclosure refers to a SARS-CoV-2 variant that is prevalent and / or rapidly spreading in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided in the GISAID Initiative database: httg^www^s^.or2, and / or data provided by the World Health Organization WHO (e.g., as provided at https: / / www.who.int / activities / tracking-SARS-CoV-2-variants). In some embodiments, such a variant refers to a variant disclosed herein.

[0592] For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

[0593] Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and / or carboxyterminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and / or C-terminal end of the protein are also called N-terminal and / or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and / or to replacing amino acids with other ones having similar properties.Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), 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 classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0594] Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: :needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0595] "Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0596] The terms "% identical", "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FAST A, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match / Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

[0597] Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

[0598] In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% ofthe entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0599] Homologous amino acid sequences exhibit according to the present disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0600] The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

[0601] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0602] An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

[0603] As used herein, an "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions of the present disclosure or be shipped together with a container which contains the compositions. Alternatively, the instructional material maybe shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

[0604] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0605] The term "recombinant" in the context of the present disclosure means "made through genetic engineering". Preferably, a "recombinant object" such as a recombinant nucleic acid in the context of the present disclosure is not occurring naturally.

[0606] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

[0607] "Physiological pH" as used herein refers to a pH of about 7.5.

[0608] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and / or an organism of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection.Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

[0609] The term "seroconversion" includes a ≥4-fold rise from before vaccination to 1-month post Dose 2.

[0610] As used herein, the term "vaccine" refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.Coronavirus

[0611] Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+) ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNA viruses and are phylogenetically divided into four genera (a, P, y, and 3), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some huma...

Claims

Claims1. A ribonucleic acid (RNA) comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises:(i) a receptor binding domain (RBD) of a coronavirus Spike (S) protein; and(ii) an S2 domain of a coronavirus S protein or one or more fragments thereof; wherein the RBD and the S2 domain or the one or more fragments thereof are directly adjacent to one another or are connected via a non-endogenous sequence.

2. The RNA of claim 1, wherein the polypeptide comprises a stem helix and a fusion peptide of an S2 domain of a coronavirus S protein.

3. The RNA of claim 1 or 2, wherein the RBD comprises amino acids 327 to 528 of SEQ ID NO: 1, 330 to 528 of SEQ ID NO: 1, amino acids 327 to 528 of SEQ ID NO: 1, or amino acids 330 to 541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant.

4. The RNA of any one of claims 1-3, wherein the S2 domain comprises amino acid 686-1213 of SEQ ID NO: 1, 686-1211 of SEQ ID NO: 1, amino acids 687-1206 of SEQ ID NO: 1, or amino acids 687-1211 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant, wherein the S2 domain comprises one or more mutations that stabilize the S2 domain.

5. The RNA of any one of claims 1-4, wherein the S2 domain comprises:(a) an amino acid sequence that is at least 70% identical to:SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAV EQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEP QIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIKWP;(b) an amino acid sequence that is at least 70% identical to:SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAV EQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGL TVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTAS ALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEP QIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLI DLQELGKYEQYIK, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto;(c) an amino acid sequence that is at least 70% identical to:VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT VLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASA LGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS ECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQ IITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIK, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto; or(d) an amino acid sequence that is at least 70% identical to:VASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVE QDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLT VLPPLLTDEMIAQYTSALLAGTITSGV / FFGAGAALQIPFAMQMAYRFNGIGVFQNVLYENQKLIANQFNSAIGKIQDSLSSTASA LGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMS ECVLGQSKRVDFCGKGYH LM SFPQSAPHG WFLHVTYVPAQEKN FTTAPAICH DGKAH FPREGVFVSNGTHWFVTQRN FYEPQ IITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDL QELGKY, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical thereto.

6. The RNA of any one of claims 1-5, wherein the S2 domain comprises one or more mutations that can stabilize the prefusion confirmation of an S protein.

7. The RNA of claim 6, wherein the S2 domain comprises one or more of the following mutations relative to SEQ ID NO: 1, or corresponding mutation(s) in an S2 domain of an S protein of a SARS-CoV-2 variant:(a) K986P and V987P;(b) A892P, A899P, A942P, or combinations thereof, optionally in combination with K986P and V987P;(c) F817P, A892P, A899P, A942P, or combinations thereof, optionally in combination with K986P and V987P;(d) V707C and T883C;(e) I770C and A1015C;(f) V826C and A1015C;(g) V826C and L948C;(h) F970C and G999C;(i) S735C and T859C; or fl) any combination of (a)-(i).

8. The RNA of any one of claims 1-7, wherein the non-endogenous sequence comprises a flexible linker sequence.

9. The RNA of any one of claims 1-8, wherein the polypeptide does not comprise sequences of a coronavirus S protein other than that of the RBD and the S2 domain or one or more fragments thereof, and wherein:(i) the polypeptide does not comprise a SARS-CoV-2 S protein secretory signal and / or a SARS-CoV- 2 S protein transmembrane domain;(ii) the polypeptide comprises a SARS-CoV-2 S protein secretory signal and / or a SARS-CoV-2 S protein transmembrane domain.

10. The RNA of any one of claims 1-9, wherein the polypeptide comprises a stem helix (SH) and fusion peptide (FP) of an S2 domain and does not comprise sequences of other regions of the S2 domain, aside from a transmembrane domain of a SARS-CoV-2 S protein.

11. The RNA of claim 10, wherein the RBD is directly adjacent to the SH and / or the FP, or wherein the RBD is connected to the SH and / or the FP via a flexible linker sequence.

12. The RNA of any one of claims 1-11, wherein the SH and the FP are directly adjacent to one another or are connected to one another via a non-endogenous sequence comprising a flexible linker sequence.

13. The RNA of any one of embodiments 1-12, wherein the RBD is N-terminal to the SH and the FP, or the RBD is C-Terminal to the SH and the FP.

14. The RNA of any one of claims 1-13, wherein the N-terminal to C-terminal orientation of the polypeptide is: (RBD)-(FP)-(SH), or (FP)-(SH)-(RBD).

15. The RNA of claim 14, wherein the SH and the FP are directly adjacent to one another, and wherein the RBD is N-terminal or C-terminal to the SH-FP region.

16. The RNA of any one of claims 1-16, wherein the polypeptide comprises a transmembrane domain, wherein the transmembrane domain comprises an amino acid sequence that is at least 70% identical toEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT orEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC.

17. The RNA of claim 16, wherein the N-terminal to C-terminal orientation of the polypeptide is:(a) (FP)-(SH)-(RBD)-(transmembrane domain); or(b) (RBD)-(FP)-(SH)-(transmembrane domain).

18. The RNA of any one of claims 1-17, wherein the FP comprises a sequence that is at least 70% identical to PSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGD, and the SH comprises a sequence that is at least 70% identical to LQPELDSFKEELDKYFKNHTSPDV.

19. The RNA of any one of claims 1-18, wherein the polypeptide comprises a secretory signal peptide, wherein the secretory signal peptide is a homologous or a heterologous secretory signal peptide.

20. The RNA of any one of claims 1-19, wherein the polypeptide comprises a secretory signal peptide comprising:(i) an amino acid sequence that is at least 70% identical to MFVFLVLLPLVSSQCVNLT, or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant.(ii) an amino acid sequence that is at least 70% identical to MCRGLSAVLILLVSLSAQLHVWG;(Hi) an amino acid sequence that is at least 70% identical to MFLLLRFVLVSCIIGSLG;(iv) an amino acid sequence that is at least 70% identical to MGGAAARLGAVILFVVIVGLHGVRG;(v) an amino acid sequence that is at least 70% identical toMHQGAPSWGRRWFWWALLGLTLGVLVASAAP or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical thereto; or(vi) an amino acid sequence that is at least 70% identical to MARGAGLVFFVGVWVVSCLA.

21. The RNA of any one of claims 1-20, wherein the polypeptide comprises:(a) an amino acid sequence that is at least 70% identical to SEQ ID NO: 190; or(b) an amino acid sequence that is at least 70% identical to SEQ ID NO: 191.

22. The RNA of any one of claims 1-21, wherein the RBD comprises one or more mutations of a SARS-CoV-2 variant.

23. The RNA of any one of the preceding claims, wherein the RNA comprises a 5' cap, a cap proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence; wherein(i) the 5' cap comprises a Capl structure;(ii) the 5'-UTR sequence comprises a modified human alpha-globin 5'-UTR;(iii) the 3 -UTR sequence comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA;(iv) the polyA sequence comprises at least 100 A nucleotides; or(v) the RNA comprises a combination of any one of (i)-(iv).

24. The RNA of claim 23, wherein: the 5' cap comprising a Capl structure, and the Capl structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeAl)pG2, wherein Al is position +1 of the RNA, and G2 is position +2 of the RNA, wherein the cap proximal sequence comprises Ai and G2 of the Capl structure, and a sequence comprising: A3N4N5 at positions +3, +4 and +5 respectively of the RNA, wherein N< and Ns are each independently selected from A, G, C, and U; and / or the polyA sequence comprises an interrupted sequence of A nucleotides; and / or the 5'-UTR sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 112; and / or the 3'-UTR sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 113; and / or the sequence at the 5' end of the 3'UTR sequence (e.g., the sequence immediately adjacent to a sequence encoding an antigenic polypeptide) is CUCGAG or GGAUCCGAU.

25. The RNA of any one of the preceding claims, wherein the RNA is self-amplifying RNA (saRNA), transamplifying RNA (taRNA), or messenger RNA (mRNA).

26. The RNA of any one of the preceding claims, wherein the RNA is unmodified RNA or wherein the RNA comprises a single Nl-methyl-pseudouridine in place of each uridine.

27. A composition comprising an RNA of any one of claims 1-26, wherein the RNA is formulated in a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle.

28. The composition of claim 27, wherein the RNA is fully or partially encapsulated in the nanoparticle.

29. The composition of any one of claims 27-28, further comprising about 10 mM Tris buffer and about 10% sucrose.

30. A pharmaceutical composition comprising (i) an RNA of any one of claims 1-26 or a composition of any one of claims 27-29 and (ii) a pharmaceutically acceptable excipient.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition is formulated as a multi-dose formulation in a vial, a single-dose formulation in a vial, or a prefilled syringe.

32. The pharmaceutical composition of claim 30 or 31, formulated to provide a dose of about 100 pg or less of total RNA, about 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg, or about 3 μg of total RNA.

33. A method comprising administering an RNA of any one of claims 1-26, a composition of any one of claims 27-29, or a pharmaceutical composition of any one of claims 30-32 to a subject.

34. The method of claim 33, wherein:(i) the subject is 12 years or older, and the method comprises administering 30 pg of the RNA,(ii) the subject is 5 years to less than 12 years old, and the method comprises administering 10 pg of the RNA, or(iii) the subject is 6 months to less than 5 years old, and the method comprises administering 3 pg of the RNA.

35. The method of claim 33 or 34, wherein the composition is administered in a volume of about 200 pL to about 300μL.

36. The method of any one of claims 33-35, wherein the method comprises administering a single dose of the RNA, composition, or pharmaceutical composition to the subject.

37. The method of any one of claims 33-36, wherein the method comprises administering two or more dosesof the RNA, composition, or pharmaceutical composition to the subject.

38. The method of any one of claims 33-37, wherein RNA, composition, or pharmaceutical composition is administered three times to the subject.

39. The method of any one of claims 33-38, further comprising administering one or more vaccines against a non-SARS-CoV-2 disease.

40. The method of any one of claims 33-39, wherein the method results in induction of an immune response against SARS-CoV-2 in the subject.

41. The method of any one of claims 33-40, wherein the method is a method of preventing SARS-CoV-2 infection, reducing the chance of SARS-CoV-2 infection, preventing or reducing the change of deleterious symptoms associated with SARS-CoV-2 infection (which can result, e.g., in a reduced change of hospitalization), increase the change of experiencing an asymptomatic SARS-CoV-2 infection, and / or treating a SARS-CoV-2 infection.

42. An RNA of any one of claims 1-26, a composition of any one of claims 27-29, or a pharmaceutical composition of any one of claims 30-32, for use in inducing an immune response in a subject.

43. Use of an RNA of any one of claims 1-26, a composition of any one of claims 27-29, or a pharmaceutical composition of any one of claims 30-32, for the manufacture of a medicament for inducing an immune response in a subject.

44. A method of manufacturing an RNA, comprising in vitro transcribing the RNA of any one of claims 1-26.

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