SARS-COV-2 immunogenic compositions

Abstract

Disclosed herein are compositions comprising protein antigens and RNA encoding the same (e.g., compositions comprising protein antigens and RNA encoding antigens) that can be used to induce an immune response against SARS-CoV-2. Also disclosed herein are immunogenic compositions and medical preparations comprising the same, and methods of making and using the same. In some embodiments, the technologies provided herein can result in an improved immune response as compared to current SARS-COV-2 vaccines.

Description

SARS-COV-2 IMMUNOGENIC COMPOSITIONS Background

[0001] SARS-CoV-2 first emerged in 2019, and quickly spread around the world, resulting in millions of deaths. The development and authorization of the first-generation SARS-CoV-2 vaccines in less than 12 months was a landmark scientific achievement that helped save countless lives. Since these initial vaccines were developed, the SARS-CoV-2 disease landscape has progressed, with researchers now understanding more about the SARS-CoV-2 virus, disease progression, and immune responses elicited by infection and vaccines. In parallel, SARS-CoV-2 has continued to evolve, with thousands of variants having arisen around the world, many with increased growth rates and immune escape potential as compared to initial SARS-CoV-2 strains. Summary

[0002] The present disclosure, among other things, describes new RNA technologies (e.g., compositions and methods) that can be used to induce an immune response against SARS-CoV-2. In some embodiments, the immune response induced by technologies provided herein is improved as compared to the first generation of approved SARS-CoV-2 vaccines (e.g., vaccines that deliver a full-length SARS-CoV-2 S protein, including, e.g., LNP- formulated RNA vaccines that encode a full-length, prefusion stabilized S protein, such as Comirnaty from Pfizer and SpikeVax from Moderna). The improved immune response provided by the technologies described herein can include, e.g., increased neutralization antibody titers against a SARS-CoV-2 variant of concerned and / or increased antibody titers against a wider array of SARS-CoV-2 variants. In some embodiments, technologies provided herein are more potent than first generation vaccines (e.g., can induce higher titers of neutralizing antibodies at a given concentration of RNA and / or similar titers of neutralizing antibodies at a lower dose of RNA). In some embodiments, RNA provided herein is more stable (e.g., more thermostable) as compared to first generation SARS-CoV-2 vaccines, which can be valuable, e.g., for increasing the shelf-life of pharmaceutical compositions, and / or removing the need to store and transport pharmaceutical compositions at ultra-cold temperatures.

[0003] Technologies provided herein include, among others, immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of manufacturing immunogenic compositions. In some embodiments, an immunogenic composition delivers a SARS-CoV-2 antigen (e.g., comprises a SARS-CoV-2 antigen or a nucleic acid encoding a SARS-CoV-2 antigen). In some embodiments, an immunogenic composition delivers an immunogenic portion of a SARS-CoV-2 S protein, including, e.g., an RBD, NTD or a truncated S1 subdomain of a SARS-CoV-2 virus, or a variant of any of the foregoing. In some embodiments, technologies provided herein can provide an improved immune response (e.g., higher neutralization antibody titers, increased naïve B cell activation, and / or higher titers of antibodies recognizing an epitope unique to a variant of concern, as compared, e.g., to a composition that delivers a full-length SARS-CoV-2 S protein.

[0004] Among other things, the present disclosure provides certain insights into the design of SARS-CoV-2 antigens that can result in significant improvements as compared to current vaccines (e.g., vaccines delivering a full- length SARS-CoV-2 S protein). These advantages include, e.g., increased antigen expression, increased antibody titers (in some embodiments, including increased neutralization titers), increased efficacy, increased stability (e.g., increased thermostability), improved cross-neutralization, and / or enhanced durability of antibody responses ascompared to current SARS-CoV-2 vaccines (e.g., as compared to first generation SARS-CoV-2 RNA vaccines that deliver a full length Spike protein (e.g., BNT162b2 (Comirnaty) and mRNA1273 (SpikeVax))).

[0005] Among other things, the present disclosure provides insights in regard to antigenic regions of the SARS- CoV-2 S protein that provide an improved immune response as compared to the full-length S protein (e.g., increased neutralization titers and / or more durable immune responses). Among other things, the present disclosure provides the insight that a truncated S1 subdomain that comprises an endogenous sequence connecting the NTD and RBD regions can induce an improved immune response as compared to a full-length S protein or a polypeptide comprising an NTD and RBD connected via a heterologous flexible linker.

[0006] Also provided herein are improved polypeptide designs for delivery via RNA, including the identification of preferred domains (e.g., secretory signal peptides and transmembrane regions) to link to SARS-CoV-2 antigens and configurations thereof that result in significant improvement in antigen expression and immune responses. These improved designs include, e.g., improved secretory signal peptides, transmembrane regions, multimerization domains, GS linkers, nucleotide sequences, and combinations and configurations thereof, each of which individually provides significant improvements as compared to existing SARS-CoV-2 vaccines, and which can be combined to result in RNA with significantly increased antigen expression and / or immunogenicity.

[0007] In some embodiments, compositions described herein can be used to induce a significantly improved immune response as compared to a reference composition. In some embodiments, the reference composition comprises an RNA encoding a full-length SARS-CoV-2 S protein, optionally comprising one or more mutations that stabilize the prefusion confirmation.

[0008] In some embodiments, the improved immune response includes an improved B cell response. In some embodiments, an improved B cell immune response comprises inducing an increased number of B cells that can recognize a SARS-CoV-2 S protein. In some embodiments, an improved B cell immune response comprises an increased number of B cells that can recognize the RBD region of a SARS-CoV-2 S protein. In some embodiments, an improved B cell immune response comprises an increased number of B cells that can recognize the NTD region of a SARS-CoV-2 S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by at least 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, ,90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by an amount within a range having a lower bound of 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, ,90%, or 100%, and an upper bound of 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is increased by at least about 200%.

[0009] In some embodiments, an improved immune response includes increased titers of antibodies that can neutralize a SARS-CoV-2 virus. In some embodiments, titers of neutralizing antibodies are increased by at least 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, titers of neutralizing antibodies are increased by an amount within a range having a lower bound of10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, and an upper bound of 150%, 200%, 250%, 300%, 350%, or 400% as compared to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, titers of neutralizing antibodies are increased by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, titers of neutralizing antibodies are increased by at least about 200%. In some embodiments, titers of neutralizing antibodies are increased by at least about 400%.

[0010] In some embodiments, technologies provided herein can provide a dose sparing effect (i.e., require a lower amount of RNA required to produce a given immune response). For example, as demonstrated in the Examples of the present disclosure, in some embodiments, a composition described herein can provide a dose sparing effect of at least about 2-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 3-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 4-fold. In some embodiments, a composition described herein can provide a dose sparing effect of at least about 5-fold.

[0011] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an amino acid sequence that is at least 80% identical to that of a reference SARS-CoV-2 S protein (or an amino acid sequence of the corresponding portion of a reference SARS-CoV-2 S protein).

[0012] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant does not comprise an S2 domain.

[0013] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of an S1 domain, a truncated S1 subdomain, or a receptor binding domain (RBD), or a variant of any of the foregoing.

[0014] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the RBD.

[0015] In some embodiments, an RNA encodes a SARS-CoV-2 antigen comprising one or more mutations of a SARS-CoV-2 variant. In some embodiments, a SARS-CoV-2 variant is a variant of concern or is predicted to become a variant of concern (e.g., by a health organization, including, e.g., WHO or the CDC). In some embodiments, a SARS-CoV-2 variant is rapidly growing in a region and / or exhibits increased immune escape potential as compared to currently prevalent SARS-CoV-2 viruses. In some embodiments, an RNA encodes a SARS-CoV-2 antigen that comprises one or more mutations of a variant that a health organization has recommended providing seasonally updated vaccines against.

[0016] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has a high immune escape potential (e.g., a variant of concern).

[0017] In some embodiments, a SARS-CoV-2 variant has been determined to have a high immune escape potential using an in vitro assay (e.g., a viral neutralization assay), in silico analysis (e.g., sequence analysis and / or molecular dynamic simulations), and / or based on infection rates and / or growth rates.

[0018] In some embodiments, a SARS-CoV-2 variant with a high immune escape potential is an Omicron variant.

[0019] In some embodiments, a SARS-CoV-2 variant is an XBB variant (e.g., an XBB.1 or XBB.1.5 variant), a BQ.1 variant, a BA.2.86 variant, a JN.1 variant, a KP.2 variant, or a descendant of any of the foregoing.

[0020] In some embodiments, one or more mutations associated with -26, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0021] In some embodiments, one or more mutations associated with an XBB.1.5 RBD are G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, and Y505H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0022] In some embodiments, one or more mutations associated with an XBB.1.5 S1 domain -26, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0023] In some embodiments, one or more mutations associated -26, A27S, V83A, G142D, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0024] In some embodiments, one or more mutations associated with an XBB.1.5 S1 -26, A27S, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0025] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of a SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3.

[0026] In some embodiments, an RNA comprises a nucleotide sequence that encodes an immunogenic portion of the SARS-CoV-2 S protein variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 5.

[0027] In some embodiments, a variant polypeptide comprises a secretion signal. In some embodiments, a secretion signal is a homologous secretion signal. In some embodiments, a secretion signal is a heterologous secretion signal.

[0028] In some embodiments, a secretion signal is present in the N-terminal portion of a polypeptide (e.g., at the N-terminus).

[0029] In some embodiments, a secretion signal is a SARS-CoV-2 S protein secretion signal, a gD2 secretion signal, a gD1 secretion signal, a gB1 secretion signal, a gI2 secretion signal, a gE2 secretion signal, an Eboz secretion signal, or an HLA-DR secretion signal.

[0030] In some embodiments, a SARS-CoV-2 S protein secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 15.

[0031] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

[0032] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

[0033] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

[0034] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13.

[0035] In some embodiments, a gD1 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12.

[0036] In some embodiments, a gB1 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

[0037] In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

[0038] In some embodiments, a gI2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

[0039] In some embodiments, a gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

[0040] In some embodiments, an EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

[0041] In some embodiments, an HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0042] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain.

[0043] In some embodiments a multimerization domain is in the C-terminal region of a SARS-COV-2 variant protein or an immunogenic portion thereof (e.g., at the C-terminus).

[0044] In some embodiments, a multimerization domain is a fibritin trimerization domain.

[0045] In some embodiments, a fibritin trimerization domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.

[0046] In some embodiments, a fibritin trimerization domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0047] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a transmembrane (TM) domain.

[0048] In some embodiments, a TM domain is a homologous TM domain.

[0049] In some embodiments, a TM domain is a heterologous TM domain.

[0050] In some embodiments, a TM domain is present in the C-terminal portion of a polypeptide (e.g., at the C-terminus).

[0051] In some embodiments, a SARS-CoV-2 S protein variant (or immunogenic portion thereof) comprises a multimerization domain and a TM domain in the C-terminal portion of the polypeptide, wherein the TM domain is C- terminal to the multimerization domain (e.g., the TM domain is at the C-terminus of the variant polypeptide and themultimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and / or connected to the TM domain via a GS linker)).

[0052] In some embodiments, a TM domain is a SARS-CoV-2 S protein TM domain, or an influenza TM domain.

[0053] In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

[0054] In some embodiments, a SARS-CoV-2 TM domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

[0055] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 120.

[0056] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises a sequence that is at least 80% identical to SEQ ID NO: 130.

[0057] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 135.

[0058] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 145.

[0059] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 150.

[0060] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in mammalian subjects.

[0061] In some embodiments, a nucleotide sequence that encodes a SARS-CoV-2 S protein variant (or immunogenic portion thereof) is codon-optimized for expression in human subjects.

[0062] In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 S protein variant (or immunogenic portion thereof) has an enriched G / C content relative to wild-type sequence.

[0063] In some embodiments, G / C content is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%.

[0064] In some embodiments, an RNA comprises a heterologous 3’ UTR or 5’UTR.

[0065] In some embodiments, a heterologous 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR.

[0066] In some embodiments, a heterologous 3’ UTR comprises or consists of a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

[0067] In some embodiments, an RNA comprises a poly(A) sequence.

[0068] In some embodiments, a poly(A) sequence has a length of about 100-150 nucleotides.

[0069] In some embodiments, a poly(A) sequence is a disrupted poly(A) sequence.

[0070] In some embodiments, an RNA comprises a 5' cap.

[0071] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 122 or 124.

[0072] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

[0073] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

[0074] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

[0075] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0076] In some embodiments, an RNA is unmodified RNA.

[0077] In some embodiments, an RNA comprises one or more modified nucleotides.

[0078] In some embodiments, a modified nucleotide is pseudouridine (e.g., N1-methyl-pseudouridine).

[0079] In some embodiments, an RNA comprises a modified nucleotide in place of each uridine.

[0080] In some embodiments an RNA is a self-amplifying RNA or trans-amplifying RNA.

[0081] In some embodiments, a composition an RNA described herein, wherein the RNA is fully or partially encapsulated within lipid nanoparticles (LNP), polyplexes (PLX), lipidated polyplexes (LPLX), oligo- or poly-saccharide particles, or liposomes. In some embodiments, an RNA is fully or partially encapsulated within an LNP. In some embodiments, an LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol and a lipid conjugate.

[0082] In some embodiments, an LNP comprises from about 40 to about 50 mol percent of the cationically ionizable lipid; from about 5 to about 15 mol percent of the neutral lipid; from about 35 to about 45 mol percent of the sterol; and from about 1 to about 10 mol percent of the PEG-lipid.

[0083] In some embodiments, the present disclosure provides a method of inducing an immune response, comprising administering an RNA described herein, or a composition described herein.

[0084] In some embodiments, an immune response is induced in a subject who has previously been administered one or more doses of one or more vaccines that deliver a reference SARS-CoV-2 S protein.

[0085] In some embodiments, an immune response comprises a naïve B cell immune response.

[0086] In some embodiments, an immune response comprises a reduced memory B cell immune response or an immune response that does not comprise a memory B cell immune response.

[0087] In some embodiments, a SARS-CoV-2 S protein variant or immunogenic portion thereof comprises a sequence that corresponds to an immunogenic portion of a reference SARS-CoV-2 S protein.

[0088] In some embodiments, an RNA or composition is administered to a subject previously exposed to a SARS-CoV-2 antigen (e.g., via vaccination or prior infection, where prior infection was determined by, e.g., a positive PCR and / or antigen diagnostic assay result. In some embodiments, a reference SARS-CoV-2 S protein or an immunogenic portion thereof is from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant jurisdiction.

[0089] In certain embodiments, antigens described herein can be engineered to incorporate sequences and / or mutations from two or more SARS-COV-2 variants (e.g., epitopes from RBDs, S proteins, and / or S1 domains from two or more SARS-CoV-2 variants). For example, in some such embodiments, mutations of a one or more SARS- CoV-2 variants can be introduced in conserved epitopes of a variant SARS-CoV-2 S protein, or an immunogenic portion thereof (e.g., an S1 domain or an RBD). Such engineering can be useful, e.g., to eliminate additional B cell epitopes (e.g., conserved B cell epitopes). Exemplary approaches for introducing mutations or sequences from two or more SARS-CoV-2 variants are described, e.g., in the US provisional application titled “Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses”, filed February 24, 2023, and having U.S. ProvisionalApplication No.63 / 448,215. Said application describes, among other things, technologies directed to in-silico design of custom, engineered, antigens (e.g., including engineered versions of SARS-CoV 2 variant proteins and portions thereof) for reducing an extent to which a memory immune response is triggered.

[0090] In some embodiments, an RNA comprises a nucleotide sequence that encodes a polypeptide, wherein the polypeptide comprises encoding an immunogenic fragment of a SARS-CoV-2 S protein. In some embodiments, an immunogenic fragment comprises a Receptor Binding Domain (RBD) of a SARS-CoV-2 S protein. In some embodiments, an RBD of a SARS-CoV-2 S protein comprises an amino acid sequence from the region corresponding to 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. In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an N-Terminal Domain (NTD). In some embodiments, an NTD of a SARS-CoV-2 S protein 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. In some embodiments, an immunogenic fragment of a SARS-COV-2 S protein comprises an S1 domain of a SARS-CoV-2 S protein, or an immunogenic fragment thereof. In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an RBD and an NTD of a SARS-CoV-2 S protein. In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises a truncated S1 subdomain or a variant thereof. In some embodiments, an RBD is at the C-terminus of the truncated S1 subdomain or the variant thereof (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). In some embodiments, a truncated S1 subdomain or variant thereof comprises amino acids 14- 528 of SEQ ID NO: 1, amino acids 17-528 of SEQ ID NO: 1, amino acids 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.

[0091] In some embodiments, an RNA comprises a nucleotide sequence that encodes a polypeptide comprising 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. In some embodiments, a secretory signal peptide is a secretory signal peptide of a SARS-CoV-2 S protein. In some embodiments, a secretory signal peptide is a heterologous secretory signal peptide. In some embodiments, a heterologous secretory signal peptide is a secretory signal peptide of a viral protein that is not a SARS-CoV-2 S protein.

[0092] In some embodiments, a polypeptide comprises a secretory signal peptide (e.g., a secretory signal peptide of a viral protein). In some embodiments, a polypeptide comprises a secretory signal peptide that comprises (i) an amino acid sequence that is listed in Table 2, Table XXXI, or Figure 15 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 or Table XXXI, and / or (ii) wherein the RNA comprises a nucleotide sequence that is listed in Table 3 or Table XXXI or Figure 15 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 or Table XXXI sequence that is listed in Table 3 and / or Table XXXI and / or Figure 15.

[0093] 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 MCRGLSAVLILLVSLSAQLHVVVG (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 MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 22)).

[0094] 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 ID NO: 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).

[0095] 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 MGGAAARLGAVILFVVIVGLHGVRG (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 MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12).

[0096] In some embodiments, a polypeptide comprises an SP32-GBD_HHV1K secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of MHQGAPSWGRRWFVVWALLGLTLGVLVASAAP (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 MHQGAPSWGRRWFVVWALLGLTLGVLVASAAP (SEQ ID NO: 38).

[0097] 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 MARGAGLVFFVGVWVVSCLA (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 MARGAGLVFFVGVWVVSCLA (SEQ ID NO: 366).

[0098] In some embodiments, a polypeptide comprises a SARS-CoV-2 secretory signal peptide (e.g., a secretory signal peptide comprising the amino acid sequence of one of the SARS-CoV-2 secretory signal peptides provided 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 one or more of the amino acid sequence of the SARS-CoV-2 secretory signal peptide provided in Table 2.

[0099] In some embodiments, a polypeptide comprises a transmembrane domain. In some embodiments, a transmembrane region further comprises an amino acid sequence that is adjacent to a membrane in its endogenous protein. In some embodiments, a transmembrane region comprises a transmembrane domain and a membrane adjacent region from the same protein.

[0100] In some embodiments, a polypeptide comprises a transmembrane domain. In some embodiments, a transmembrane domain is from a viral membrane protein. In some embodiments, a transmembrane domain is a SARS-CoV-2 S protein transmembrane domain. In some embodiments, a transmembrane domain is obtained from a viral protein that is not a SARS-CoV-2 S protein.

[0101] In some embodiments, a transmembrane domain is a SARS-CoV-2 S protein transmembrane domain (e.g., wherein the transmembrane domain comprises an amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC, 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 EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC).

[0102] In some embodiments, a transmembrane domain is a heterologous transmembrane domain.

[0103] In some embodiments, a heterologous transmembrane domain is a transmembrane domain of a viral protein that is not a SARS-CoV-2 S protein.

[0104] In some embodiments, a transmembrane domain is capable of inducing multimerization (e.g., trimerization).

[0105] In some embodiments, a transmembrane domain comprises a transmembrane domain that is listed in Table 4 (e.g., comprising an amino acid sequence provided in Table 4 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 listed in Table 4) and / or wherein the transmembrane domain is encoded by a nucleotide sequence that is listed in Table 5 or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence listed in Table 5).

[0106] In some embodiments, a polypeptide comprises, C-terminally adjacent to the transmembrane domain, a sequence that is endogenously C-terminal to the transmembrane domain and membrane adjacent in its native protein.

[0107] In some embodiments, a sequence that is endogenously C-terminally to the transmembrane domain and adjacent to the plasma membrane is MTSCCSCLKGCCSCGSCC, 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 MTSCCSCLKGCCSCGSCC.

[0108] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 90( Q W W W G G V V CC SCCSC GCCSCGSCC), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90.

[0109] In some embodiments, a polypeptide comprises a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin trimerization domain). In some embodiments, a polypeptide does not comprise a soluble multimerization domain (e.g., a trimerization domain, e.g., a T4 fibritin trimerization domain). In some embodiments, a polypeptide comprises a transmembrane domain that can induce multimerization (e.g., trimerization), and the polypeptide lacks a soluble peptide domain.

[0110] In some embodiments, a polypeptide comprises: (1) a fragment of an S1 polypeptide (e.g., amino acids 1-528 of SEQ ID NO: 1 or a corresponding region of a SARS-CoV-2 variant); (2) a secretory signal peptide (e.g., a SP24-Q7PUJ5_ANOGA, SP24-SP18-HEMA_CVBM, SP25- GD_HHV1K, SP32-GD_HHV1K, or SP20-A7U881_HHV2 secretory signal peptide described herein); and (3) a transmembrane domain (e.g., a viral transmembrane domain, a transmembrane domain of a coronavirus S protein, a transmembrane domain of an influenza virus HA protein, or a transmembrane domain of a SARS-CoV-2 S protein).

[0111] In some embodiments, the N-terminal to C-terminal orientation of the polypeptide is: (secretory signal peptide)-(fragment of an S1 polypeptide)-(transmembrane domain). In some embodiments, (1) the secretory signal peptide and the fragment of an S1 polypeptide; and / or (2) the fragment of an S1 polypeptide and the transmembrane domain are connected to one another by a linker (e.g., an artificial linker, a flexible linker, a flexible linker comprising a GS sequence). In some embodiments, a GS sequence comprises a sequence provided in Table 5. In some embodiments, a GS sequence comprises at least 5, at least 10, at least 15, or at least 20 amino acids (e.g., G or S amino acids). In some embodiments, a GS sequence comprises (G4S)1, (GRS)2, (G4S)3, or (G4S)4sequence. In some embodiments, a fragment of an S1 polypeptide and a transmembrane domain are connected via a flexible linker that comprises 10-20 amino acids (e.g., about 15 amino acids).

[0112] In some embodiments, a fragment of an S protein is adapted to a SARS-CoV-2 strain or variant (e.g., an Omicron variant, XBB.1.5 variant, JN.1 variant, KP.2 variant, XEC variant, and / or any variant described in the present disclosure).

[0113] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 156 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: 156; (ii) the nucleotide sequence of SEQ ID NO: 158 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: 158; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 155, 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: 155.

[0114] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 161 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: 161; (ii) the nucleotide sequence of SEQ ID NO: 163 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: 163; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 160, 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: 160.

[0115] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 166 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: 166; (ii) the nucleotide sequence of SEQ ID NO: 168 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: [168; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 165, 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: 165.

[0116] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 171 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: 171; (ii) the nucleotide sequence of SEQ ID NO: 173 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: 173; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 170, 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: 170.

[0117] In some embodiments, an RNA comprises (i) 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: 176; (ii) the nucleotide sequence of SEQ ID NO: 178 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: 178; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 175, 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: 175.

[0118] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 181 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical toSEQ ID NO: 181; (ii) the nucleotide sequence of 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; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 180, 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: 180.

[0119] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 186 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: 186; (ii) the nucleotide sequence of 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; 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.

[0120] In some embodiments, an RNA comprises (i) the nucleotide sequence of 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; (ii) the nucleotide sequence of SEQ ID NO: 193 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: 193; 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.

[0121] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 196 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: 196; (ii) the nucleotide sequence of SEQ ID NO: 198 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: 198; 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.

[0122] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 201 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: 201; (ii) the nucleotide sequence of SEQ ID NO: 203 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: 203; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 200, 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: 200.

[0123] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 211 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: 211; (ii) the nucleotide sequence of SEQ ID NO: 213 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: 213; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 210, 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: 210.

[0124] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 221 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: 221; (ii) the nucleotide sequence of SEQ ID NO: 223 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: 223; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 220, 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: 220.

[0125] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 226 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: 226; (ii) the nucleotide sequence of SEQ ID NO: 228 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: 228; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 225, 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: 225.

[0126] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 231 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: 231; (ii) the nucleotide sequence of SEQ ID NO: 233 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: 233; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 230, 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: 230.

[0127] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 236 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: 236; (ii) the nucleotide sequence of SEQ ID NO: 238 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: 238; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 235, 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: 235.

[0128] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 241 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: 241; (ii) the nucleotide sequence of SEQ ID NO: 243 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: 243; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 240, 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: 240.

[0129] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 246 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: 246; (ii) the nucleotide sequence of SEQ ID NO: 248 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: 248; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 245, or a sequence that is atleast 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 245.

[0130] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 251 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: 251; (ii) the nucleotide sequence of SEQ ID NO: 253 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: 253; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 250, 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: 250.

[0131] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 256 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: 256; (ii) the nucleotide sequence of SEQ ID NO: 258 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: 258; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 255, 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: 255.

[0132] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 261 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: 261; (ii) the nucleotide sequence of SEQ ID NO: 263 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: 263; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 260, 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: 260.

[0133] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 266 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: 266; (ii) the nucleotide sequence of SEQ ID NO: 268 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: 268; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 265, 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: 265.

[0134] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 271 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: 271; (ii) the nucleotide sequence of SEQ ID NO: 273 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: 273; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 270, 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: 270.

[0135] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 276 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: 276; (ii) the nucleotide sequence of SEQ ID NO: 278 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: 278; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 275, 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: 275.

[0136] In some embodiments, an RNA comprises comprising (i) the nucleotide sequence of SEQ ID NO: 281 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: 281; (ii) the nucleotide sequence of SEQ ID NO: 283 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: 283; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 280, 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: 280.

[0137] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 286 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: 286; (ii) the nucleotide sequence of SEQ ID NO: 288 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: 288; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 285, 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: 285.

[0138] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 291 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: 291; (ii) the nucleotide sequence of SEQ ID NO: 293 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:293; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 290, 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: 290.

[0139] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 296 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: 296; (ii) the nucleotide sequence of SEQ ID NO: 298 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: 298; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 295, 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: 295.

[0140] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 301 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: 301; (ii) the nucleotide sequence of SEQ ID NO: 303 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: 303; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 300, 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: 300.

[0141] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 306 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: 306; (ii) the nucleotide sequence of SEQ ID NO: 308 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: 308; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 305, 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: 305.

[0142] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 311 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: 311; (ii) the nucleotide sequence of SEQ ID NO: 313 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: 313; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 310, 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: 310.

[0143] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 321 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: 321; (ii) the nucleotide sequence of SEQ ID NO: 323 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: 323; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 320, 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: 320.

[0144] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 343 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: 343 (ii) the nucleotide sequence of SEQ ID NO: 345 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: 345; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 342; 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: 342.

[0145] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 348 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: 348 (ii) the nucleotide sequence of SEQ ID NO: 350 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: 350; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 347; 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: 347.

[0146] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 353 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: 353 (ii) the nucleotide sequence of SEQ ID NO: 355 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: 355; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 352; or a sequence that is atleast 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 352.

[0147] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 358 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: 358 (ii) the nucleotide sequence of SEQ ID NO: 360 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: 360; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 357; 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: 357.

[0148] 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.

[0149] In some embodiments, (i) a 5’ cap comprises a Cap1 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) an RNA comprises a combination of any one of (i)-(iv).

[0150] In some embodiments, a 5’ cap comprises a Cap1 structure, and the Cap1 structure comprises m7(3’OMeG)(5')ppp(5')(2'OMeA1)pG2, wherein A1 is position +1 of the RNA, and G2 is position +2 of the RNA.

[0151] In some embodiments, a cap proximal sequence comprises A1and G2of the Cap1 structure, and a sequence comprising: A3N4N5at positions +3, +4 and +5 respectively of the RNA, wherein N4and N5are each independently selected from A, G, C, and U.

[0152] 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. In some embodiments, a 5’-UTR sequence comprises SEQ ID NO: 112, 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: 112. In some embodiments, a 3’-UTR sequence comprises SEQ ID NO: 118, 647, or 648 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: 118, 647, or 648.

[0153] In some embodiments, an interrupted polyA tail sequence comprises SEQ ID NO: 114, 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: 114.

[0154] 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.

[0155] In some embodiments, an RNA is saRNA, self-amplifying RNA, trans-amplifying RNA (taRNA), or mRNA.

[0156] In some embodiments, an RNA is unmodified RNA or an RNA comprises one or modified uridines in place of one or more uridines.

[0157] In some embodiments, an RNA comprises a single modified uridine in place of each uridine.

[0158] In some embodiments, a modified uridine is N1-methyl-pseudouridine.

[0159] In some embodiments, a nucleotide sequence encoding a 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.

[0160] Among other things, provided herein is a composition comprising an RNA described herein.

[0161] In some embodiments, a composition comprises an RNA formulated in a nanoparticle. In some embodiments, a nanoparticle is a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle. In some embodiments, a nanoparticle is a lipid nanoparticle. In some embodiments, a lipid nanoparticle comprises a cationically ionizable lipid, a sterol, a neutral lipid, and a polymer-conjugated lipid. In some embodiments, a polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, an RNA is In some embodiments, a nanoparticle has an average diameter of about 50-150 nm. In some embodiments, a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose. In some embodiments, a composition comprises an aqueous buffered solution, optionally wherein the aqueous buffered solution comprises one or more of Tris base, Tris HCl, NaCl, KCl, Na2HPO4, and KH2PO4. In some embodiments, a composition comprises about 10 mM Tris buffer and about 10% sucrose.

[0162] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an RNA described herein (e.g., in a particular formulation, a lipid formulation, a lipoplex formulation, or a lipid nanoparticle formulation). In some embodiments, a pharmaceutical composition comprises an RNA described herein or a composition described herein and one or more pharmaceutically acceptable salts.

[0163] 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.

[0164] In some embodiments, a pharmaceutical composition formulated to provide a dose of about 100 μg or less (e.g., about 90 μg or less) of total RNA. In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 90 μg, about 60 μg, about 30 μg, about 25 μg, about 20 μg, about 10 μg, about 6 μg, about 5 μg, or about 3 μg of total RNA.

[0165] In some embodiments, provided herein is a method comprising administering an RNA, a composition, or a pharmaceutical composition provided herein.

[0166] In some embodiments: (i) a subject is 12 years or older, and a method comprises administering 30 μg of the RNA, (ii) a subject is 5 years to less than 12 years old, and a method comprises administering 10 μg of the RNA, or (iii) a subject is 6 months to less than 5 years old, and a method comprises administering 3 μg of the RNA.

[0167] In some embodiments, a method comprises administering a composition described herein in a volume of about 200 μL to about 300 μL.

[0168] In some embodiments, a subject has not previously been administered a SARS-CoV-2 vaccine and / or a subject has not previously been determined to have been infected with SARS-CoV-2 (e.g., as determined using a PCR or antigen diagnostic assay).

[0169] In some embodiments, a method comprises administering a single dose of the RNA, composition, or pharmaceutical composition to a subject.

[0170] In some embodiments, a method comprises administering two or more doses of an RNA, composition, or pharmaceutical composition described herein to a subject, optionally wherein the two doses are administered about 21 days apart.

[0171] In some embodiments, an RNA, composition, or pharmaceutical composition described herein is administered three times to a subject, optionally wherein the first and the second dose are administered about 21 days apart, and the third dose is administered about 28 days after the second dose.

[0172] In some embodiments, a method described herein comprises administering a further dose of an RNA, composition, or pharmaceutical composition described herein, at least about 2 months after administering a first dose of an RNA, composition, or pharmaceutical composition described herein (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).

[0173] In some embodiments, a subject has previously been exposed to a SARS-CoV-2 antigen (e.g., by vaccination and / or by infection).

[0174] In some embodiments, a subject has previously been administered one or more doses of a SARS-CoV-2 vaccine.

[0175] In some embodiments, a subject has previously been administered a complete dosing regimen of a SARS-CoV-2 vaccine.

[0176] In some embodiments, a subject has previously been 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.1 variant).

[0177] In some embodiments, a method described herein 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.

[0178] In some embodiments, a method results in induction of an immune response against SARS-CoV-2 in the subject. In some embodiments, an immune response comprises a B-cell response. In some embodiments, a B cell response comprises production of antibodies directed against one or more SARS-CoV-2 viruses. 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.

[0179] In some embodiments, a method described herein 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.

[0180] In some embodiments, the RNA, compositions, or pharmaceutical compositions described herein can be used to inducing an immune response in a subject.

[0181] In some embodiments, the RNA, compositions, or pharmaceutical compositions described herein can be used for the manufacture of a medicament for inducing an immune response in a subject.

[0182] In some embodiments, a medicament is formulated to be administered to the subject in accordance with a method described herein.

[0183] In some embodiments a method provided herein is a method of inducing an immune response to a coronavirus in a subject, and wherein the method comprises administering an RNA or a pharmaceutical composition provided herein. In some embodiments, a method described herein induces an immune response against a SARS- CoV-2 virus.

[0184] In some embodiments, provided herein is a method of manufacturing an RNA, comprising in vitro transcribing an RNA provided herein.

[0185] In some embodiments, provided herein is DNA (e.g., linear DNA or a plasmid DNA) encoding an RNA provided herein. In some embodiments, provided herein is a polypeptide encoded by an RNA provided herein.

[0186] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89.

[0187] In some embodiments, RNA described herein comprises (i) the nucleotide sequence of SEQ ID NO: 328 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: 328 (ii) the nucleotide sequence of SEQ ID NO: 330 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: 330; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 327; 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: 327.

[0188] In some embodiments, a fragment of an S protein comprises one or more mutations of a SARS-CoV-2 strain or variant (e.g., one or more mutations in the S protein of a variant described herein, e.g., one or more mutations associated with a variant listed in Table 1). In some embodiments, a fragment of an S protein comprises one or more mutations associated with a JN.1, KP.2, or XEC variant, or a descendent thereof. A skilled artisan will understand that introducing one or more mutations that are associated with a SARS-CoV-2 variant into a fragment of an S protein (e.g., a truncated S1 subdomain) will not substantially affect the general antigen properties of the fragment of the S protein, including, e.g., the ability of the fragment to induce a strong B cell response and high titers of neutralizing antibodies.

[0189] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, 64, or more) of the --R408S, K417N, N440K, V445H, G Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or M1229I relative to SEQ ID NO: 1.

[0190] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, or more of the follow -26,-G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W,L4

[0191] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following --70, V12V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0192] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, -26, A27S, S50L, -L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T,S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K,

[0193] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following --V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0194] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41 -26,- G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, 1.

[0195] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following --V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or E1150D relative to SEQ ID NO: 1.

[0196] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, or more of the following mutations: ins16MPLF -26, A27S, S50L,- S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K,

[0197] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following --V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or E1150D relative to SEQ ID NO: 1.

[0198] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, -26, A27S, S50L, -, V203G, L206F, H245N, A264D, I332V, G339H, K356T,S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K,

[0199] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following --V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, or P1143L relative to SEQ ID NO: 1.

[0200] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, -26, A27S, - R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, ID NO: 1.

[0201] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, - 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, or N969K relative to SEQ ID NO: 1.

[0202] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or more) of the following -26, A27S, V83A, G142D S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0203] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, 64, 65, 66, or 67 or --70, V127F, G142D,, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L.

[0204] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, or more of the following list of mutations: ins16MPLF, --H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, and Y505H.

[0205] In some embodiments, a fragment of a GS sequence comprises (G4S)1, (GRS)2, (G4S)3, or (G4S)4sequence.

[0206] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89.

[0207] In some embodiments, an S1 polypeptide comprises: (a) amino acids 1-528 of SEQ ID NO: 1, or a corresponding region of any of the foregoing from the S protein of a SARS-CoV-2 variant; (b) QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNL REFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRS LRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPK, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto; and / or) QCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLRE FVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNE NGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYN FAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRK SKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPK, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0208] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising: (i) a truncated S1 subdomain of a SARS-CoV-2 S protein or a variant thereof; (ii) a heterologous secretory signal peptide; and (iii) a homologous transmembrane domain, wherein the N-terminal to C-terminal orientation of the truncated S1 subdomain, heterologous secretory signal peptide, and homologous transmembrane domain is (secretory signal peptide)-(truncated S1 subdomain)- (transmembrane domain).

[0209] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising an NTD, RBD, and / or truncated S1 subdomain, or a variant of any of the foregoing, and wherein the polypeptide further comprises one or more T cell epitopes from a SARS-CoV-2 protein that is not an S protein. In some embodiments, a polypeptide comprises one or more T cell epitopes from a SARS-CoV-2 nucleocapsid (N) protein, NS9b protein, membrane (M) protein, ORF1ab protein, ORF3a protein, ORF9b protein, or NSP1-4, or any combination thereof. In some embodiments, a polypeptide comprises: (i) one or more T cell epitopes from an N protein and one or more T cell epitopes from an NS9b protein; (ii) one or more T cell epitopes from an N protein and one or more T cell epitopes from an M protein; (iii) one or more T cell epitopes from an N protein, one or more T cell epitopes from an M protein; and one or more T cell epitopes from an NS9b protein; (iv) one or more T cell epitopes from an M protein, one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP3, one or more T cell epitopes from NSP1, and one or more T cell epitopes from an N protein; (v) one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP1, one or more T cell epitopes from NSP3, one or more T cell epitopes from an N protein, one or more T cell epitopes from NSP4, and one or more T cell epitopes from an M protein; (vi) one or more T cell epitopes from an N protein, one or more T cell epitopes from NSP1, one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP3, and one or more T cell epitopes from NSP4. In some embodiments, an RNA encodes a polypeptide comprising: (a) a truncated S1 subdomain, or a variant thereof, and a SARS-CoV-2 transmembrane domain; (b) a truncated S1 subdomain, or a variant thereof, and an HSV-1 gD secretory signal peptide; (c) a truncated S1 subdomain or a variant thereof, a transmembrane domain, and an HSV-1 gD secretory signal peptide;(d) a truncated S1 subdomain or a variant thereof, a SARS-CoV-2 S protein transmembrane domain, and a secretory signal peptide; or (e) a truncated S1 subdomain or a variant thereof, a SARS-CoV-2 S protein transmembrane domain, and an HSV-1 gD secretory signal peptide.

[0210] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising: (a) a truncated S1 subdomain comprising (i) amino acids 20-528 of SEQ ID NO: 1 (including, e.g., amino acids 1-528, 14-528, 17-528, and 50-528), or a sequence of a corresponding region of an S protein of a SARS- CoV-2 variant or (ii) amino acids 20-541 of SEQ ID NO: 1 (including, e.g., amino acids 1-541, 14-541, 17-541, and 20-541), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or a variant of either of the foregoing; (b) a secretory signal peptide comprising an amino acid sequence of MGGAAARLGAVILFVVIVGLHGVRG, 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 MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12); and / or (c) a transmembrane domain comprising an amino acid sequence of SEQ ID NO: 90( Q ), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90.

[0211] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a transmembrane, a truncated S1 subdomain or variant thereof, and a transmembrane domain, wherein the N-terminal to C-terminal orientation of the polypeptide is (secretory signal peptide)-(truncated S1 subdomain)-(transmembrane domain), and the truncated S1 subdomain or variant thereof and transmembrane are connected via a sequence that comprises a GS linker, optionally wherein the GS linker comprises about 10-20 residues (e.g., about 15 or about 20 residues).

[0212] In some embodiments, an RNA comprises (i) the nucleotide sequence of SEQ ID NO: 650 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: 650; (ii) the nucleotide sequence of SEQ ID NO: 652 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: 652; and / or (iii) comprises a nucleotide sequence that encodes a polypeptide comprising SEQ ID NO: 649, 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: 649.

[0213] In some embodiments, an RNA comprises a nucleotide sequence encoding (a) a truncated S1 subdomain or a variant thereof and a SARS-CoV-2 transmembrane domain; (b) a truncated S1 subdomain or a variant thereof and an HSV-1 gD secretory signal peptide; (c) a truncated S1 subdomain or a variant thereof, a transmembrane domain, and an HSV-1 gD secretory signal peptide; (d) a truncated S1 subdomain or a variant thereof, a SARS-CoV-2 S protein transmembrane domain, and a secretory signal peptide; or (e) a truncated S1 subdomain or a variant thereof, a SARS-CoV-2 S protein transmembrane domain, and an HSV-1 gD secretory signal peptide.

[0214] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising: (a) a truncated S1 subdomain comprising (i) amino acids 20-528 of SEQ ID NO: 1 (including, e.g., amino acids 1-528, 14-528, 17-528, and 50-528), or a sequence of a corresponding region of an S protein of a SARS- CoV-2 variant or (ii) amino acids 20-541 of SEQ ID NO: 1 (including, e.g., amino acids 1-541, 14-541, 17-541, and 20-541), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or a variant of either of the foregoing; (b) a secretory signal peptide comprising an amino acid sequence of MGGAAARLGAVILFVVIVGLHGVRG, 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 MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12) or a variant of either for the foregoing; and / or (c) a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC), or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90.

[0215] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a truncated S1 subdomain or a variant thereof, a transmembrane domain, and a secretory signal peptide, where the N- terminal to C-terminal orientation of the polypeptide is (secretory signal peptide)-(truncated S1 subdomain)- (transmembrane domain), and the truncated S1 subdomain or variant thereof and transmembrane are connected via a sequence that comprises a GS linker, optionally wherein the GS linker comprises about 10-20 residues (e.g., about 15 or about 20 residues).

[0216] In some embodiments, a subject is 12 years or older, and a method comprises administering about 20 μg of an RNA. In some embodiments, a subject is 12 years or older, and a method comprises administering about 10 μg of an RNA. In some embodiments, a subject is 12 years or older, and a method comprises administering about 5 μg of an RNA.

[0217] In some embodiments, a subject is 5 years to less than 12 years old, and a method comprises administering about 6.6 μg of an RNA. In some embodiments, a subject is 5 years to less than 12 years old, and a method comprises administering about 3.3 μg (e.g., 3 μg) of an RNA. In some embodiments, a subject is 5 years to less than 12 years old, and a method comprises administering about 1.6 μg (e.g., 1.5 μg) of an RNA.

[0218] In some embodiments, a subject is 6 months to less than 5 years old, and a method comprises administering about 2 μg of an RNA. In some embodiments, a subject is 6 months to less than 5 years old, and a method comprises administering about 1 μg of an RNA. In some embodiments, a subject is 6 months to less than 5 years old, and a method comprises administering about 0.5 μg of an RNA. Brief description of the Figures

[0219] Fig.1. Novel Spike antigen designs – S1 and RBD-subdomain vaccines. Mutation density in new SARS-CoV-2 variants of concern (e.g., XBB) is highest in the S1-fragment and especially in the RBD. Hence, in some embodiments, omitting the highly conserved S2 fragment can result in more efficient priming (e.g., by removing conserved epitopes that can activate BMEMcells and / or prevent activation of naïve B cells). Shown are certain exemplary antigen designs, including (1) an RBD of an VOC attached to a trimerization domain (e.g., an RBDof XBB.1.5 attached to a T4 fibritin trimerization domain), (2) an S1 domain of an VOC attached to a trimerization domain (e.g., an S1 of XBB.1.5 attached to a T4 fibritin trimerization domain), (3) an RBD of an VOC attached to a trimerization domain and a transmembrane(TM) domain (e.g., an RBD of XBB.1.5 attached to a T4 fibritin trimerization domain and a TM domain of a SARS-CoV-2 S protein), and (4) an S1 domain of an VOC attached to a trimerization domain and a transmembrane domain (e.g., an S1 of XBB.1.5 attached to a T4 fibritin trimerization domain and a TM domain of a SARS-CoV-2 S protein)). Constructs (1) and (2) are soluble and secreted, whereas constructs (3) and (4) are TM-anchored.

[0220] Fig.2. Exemplary Immunogenecity Study in Vaccine-Experienced Mice. Mice are administered two doses of BNT162b2 (encoding an S protein of a Wuhan strain), or a composition comprising a first RNA that encodes a SARS-CoV-2 S protein of a Wuhan strain and a second RNA encoding a full length S protein of an Omicron BA.4 / 5 variant (Bivalent b2 + BA.4 / 5), followed by a third dose of a candidate vaccine. Third doses include RNA encoding full length Spike protein of a Wuhan strain (BNT162b2); RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal and a timerization domain (RBD (XBB.1.5)); RNA encoding an S1 domain of an XBB.1.5 S protein comprising a timerization domain (S1 (XBB.1.5)); RNA encoding an S1 domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (S1-TM (XBB.1.5)); and RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal, a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5)). Yellow-filled cells indicate days on which sera sample are collected, gray-filled cells indicate days on which vaccines are administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0221] Fig.3. Exemplary Protocol for Characterizing Immune Cell (Including B Cell and T Cell) Responses. Spleen sample, lymph nodes are collected and analyzed as shown in the Figure. Figure also summarizes analysis of blood samples collected throughout a study (including collection of viral neutralizing titers (pVNTs) and virus binding antibody titers (ELISA), which can be performed in parallel. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0222] Fig.4. Exemplary Immunogenecity Study in Vaccine-Naive mice. Mice are administered two doses of RNA encoding (i) a full length Spike protein of a Wuhan strain (BNT162b2); (ii) RNA encoding a full length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); (iii) RNA encoding a full length S protein of an XBB.1.5 variant and comprising a 19 amino acid C-terminal truncation (BNT162b2 (XBB.1.5) Cd19); (iv) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19) and a timerization domain (RBD (XBB.1.5) (SP19)); (v) RNA encoding an S1 domain of an XBB.1.5 S protein comprising a trimerization domain (S1 (XBB.1.5)); (vi) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19), a timerization domain, and a transmembrane domain (RBD-TM (XBB.1.5) (SP19)); (vii) RNA encoding an S1 domain of an XBB.1.5 S protein comprising a timerization domain and a transmembrane domain (S1-TM (XBB.1.5); and (viii) RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP16) and a timerization domain (RBD (XBB.1.5) (SP16)). Yellow-filled cells indicate days on which sera sample are collected, gray-filled cells indicate days on which vaccines are administered, and green-filled cells indicate days on which mice are sacrificed and final samples collected. The exemplary protocol can be used to characterize immune responses induced by compositions described herein.

[0223] Fig.5. Exemplary Protocol for Characterizing Immune Cell (Including B Cell and T Cell) Responses. Spleen samples can be collected and analyzed as shown in the figure to characterize immune cell responses induced by compositions described herein (including, e.g., T cell and B cell immune responses).

[0224] Fig.6. Exemplary polypeptides comprising an RBD or an NTD. “SP“ stands for secretory peptide, “F“ stands for a fibritin trimerization domain, “TM“ stands for a transmembrane domain, and “CT“ stands for a C-terminal sequence derived from a SARS-CoV-2 S protein.

[0225] Fig.7. Exemplary Polypeptides Comprising an RBD and a “T-string“ (comprising one or more T-cell epitopes of a SARS-CoV-2 antigen). “SP“, “TM“, “F“, and “CT“ refer to the same regions as in Fig. 6. A “T string“ refers to a polypeptide comprising one or more T cell epitopes or antigens (e.g., as described herein).

[0226] Fig.8. Exemplary “Dumbbell“ Constructs. (A) Shows graphics summarizing certain “dumbbell“ designs that are described herein (i.e., a polypeptide comprising one or more NTD polypeptides and / or one or more RBD polypeptides, and a multimerization domain such that multiple polypeptides associate in solution, forming a multimer of polypeptides, each polypeptide comprising two or more RBD polypeptides, two or more NTD polypeptides, and / or one or more RBD polypeptides and one or more NTD polypeptides). (B) Depicts a model of the structure of one such dumbbell construct, comprising three polypeptides, each comprising two RBD polypeptides and a T4 fibritin trimerization domain, such that an oligomer comprising 6 RBD polypeptides is formed in solution.

[0227] Fig.9. Exemplary Library for Characterizing Effect of N-terminal Secretory Signals on Antigen Expression.

[0228] Fig.10. Exemplary Experimental Protocol for Identifying Vaccine Constructs with Improved Expression. Shown is an exemplary in vitro protocol for testing extracellular expression of vaccine candidates in vitro. A cell line (HEK293 in the Fig.10) is transfected with a plasmid encoding a vaccine candidate. Cells are incubated with ACE2 or an antibody that binds RBD, followed by a 2oantibody, and then screened by flow cytometry to measure cell surface expression of RBD.

[0229] Fig.11. Initial in vitro expression data. Shown is flow cytometry data obtained using BNT162b3, which comprises an RBD of an XBB.1.5 SARS-CoV-2 variant, and a secretory signal of a SARS-CoV-2 S protein (aa 1- 19) and a transmembrane domain. The amount of transfected plasmid was varied so as to identify a dynamic range (concentration of plasmid at which to transfect cells).

[0230] Fig.12. Effect of N-terminal Secretory Signal on In Vitro Expression of Vaccine Candidates. HEK293 were transfected with a library of vaccine candidates, comprising various N-terminal secretory signals (design shown in Fig.9, specific sequences tested include those shown in Table XXXI of the present disclosure). (A) Shows background flourescent signal (transfected cells incubated with 2oantibody but not 1oantibody). (B) Shows intial results from a library of candidates. Signfiicant background signal was observed but could be addressed by subtracting background signal. As shown in (B), all signal peptides tested resulted in significantly increased cell surface expression of RBD as compared to a SARS-CoV-2 S protein secretory signal (aa 1-19).

[0231] Fig.13. Further Data Characterizing the Effect of Secreotry Signals on Cell Surface Expression of Antigens. A repeat of the experimental protocol shown in Fig. 10 was performed to confirm the results shown in Fig. 12. (A) Shows background signal was observed, after incubation with 2oantibody only. (B) Shows results from screening for expression. Once again, all signal peptides tested resulted in significantly higher surface expression of antigen as compared to the SARS-CoV-2 spike signal peptide (aa 1-19).

[0232] Fig.14. Further Data Characterizing the Effect of Secretory Signals on Cell Surface Expression of Antigens (Anti-RBD Antibody + Human 2oAntibody). A repeat of the experimental protocol shown in Fig. 10 was performed using a 1oantibody that binds the RBD and human 2oantibodies. (A) Shows background signal (cell not incubated with 1oantibody_ As shown, reduced background fluorescence was observed relative to mouse 2oantibody and ACE2 labelling. (B) Shows RBD signal. Once again, all signal peptides tested resulted in signifcantly higher surface expression of RBD as compared to the SARS-CoV-2 spike signal peptide (aa 1- 19).

[0233] Fig.15. Top N-terminal Secretory Signals. Shown are the five N-terminal secretory signals that resulted in the highest expression of RBD.

[0234] Fig.16. (A), (B), and (C) Show Results from Figs.12, 13, and 14, Respectively for the Top 5 Secretory Signals.

[0235] Fig.17. Effect of N-terminal Secretory Signals on Expression of RBD and Truncated S1 Subdomain Polypeptides. (A) Shows in vitro expression data from 5 different secretory signal peptides attached to a polypeptide comprising a SARS-CoV-2 RBD. Red arrow indicates improved expression of the top-performing candidate (RNA003) as compared to a polypeptide comprising an RBD linked to the SARS-CoV-2 S protein secretory signal peptide (RNA040). As shown in (A), optimizing the secretory signal peptide resulted in a dose sparing effect of approximately 16-fold for polypeptides comprising an RBD (i.e., 16-fold less RNA was required to produce the same level of in vitro expression). (B) Shows in vitro expression data from the top 5 performing secretory signal peptides when attached to a fragment of the S1 subdomain of the SARS-CoV-2 S protein (comprising a sequence corresponding to amino acids 1-528 of SEQ ID NO: 1, with the N-terminal secretory signal peptide replaced in constructs comprising a heterologous secretory signal peptide). RNA035 comprises the native SARS-CoV-2 secretory peptide. As shown in (B), optimizing the secretory signal peptide was also found to improve expression of the fragment of the S1 subdomain, resulting in an about 4-fold dose-sparing effect.

[0236] Fig.18. Omitting a Fibritin Trimerization Domain Improves Expression and Does Not Reduce Antigenicity of a Truncated S1 Subdomain. (A) Shows results from an in vitro experiment in which a cell line was transfected with nucleic acid encoding a construct with (RNA008) and without (RNA017) a fibritin trimerization domain. As shown in the Fig.18(A), omission of the fibritin trimerization domain was found to significantly improve antigen expression. (B) Shows results from a mouse experiment in which mice were administered RNA encoding a polypeptide with (RNA008) and without (RNA017) a fibritin trimerization domain. As shown in the figure, omission of the trimerization domain did not interfere with the immune response.

[0237] Fig.19. Screening Transmembrane Domains for Effect on Antigen Expression. (A) Shows results from an experiment in which the same antigen (corresponding to amino acids 1-528 of the S protein where, in some embodiments, the N-terminal secretory signal peptide was replaced with a heterologous secretory signal peptide) was fused to different transmembrane domains. As shown in Fig. 19(A), each of the RNAs tested resulted in antigen expression, and little variation in the amount of expression was observed. (B) Provides expression results from the top 5 transmembrane domains in terms of expression identified in initial experiments. (C) and (D) provide results from a further confirmatory experiment, characterizing the same constructs plus a few newly identified constructs. (D) shows the results from the top candidates tested in the further confirmatory experiment. Once again, it was concluded that the top performers provided about the same level of expression as those comprising the transmembrane domain of a SARS-CoV-2 S protein.

[0238] Fig.20. Exemplary Experimental Protocol for Testing the Immunogenicity of Different Fragments of the S1 Subdomain in Mice. Shown is an exemplary experimental protocol for testing the immunogenicity of different fragments of the S protein in mice. Orange cells indicate days on which serum samples were collected, gray cells indicate days on which a vaccine was administered, and green cells indicate the final day of the experiment, on which mice were euthanized and final samples collected.

[0239] Fig.21. A Fragment of the S1 Subdomain Induces a Strong Immune Response in Mice. (A), (B), (C), (D), and (E) show neutralization titers (pVNT50) 7, 14, 21, 28, and 35 days thereafter, respectively; (F) shows a line graph of neutralization titers (pVNT50) for constructs across vaccination days. As shown in (C), a statistically significant difference was observed at day 21 between (i) mice administered RNA encoding a fragment of the S1 subdomain (RNA035) or RNA encoding an NTD and RBD connected via a GS linker (RNA038), and (ii) RNA encoding a full-length S protein.

[0240] Fig.22. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-Cov-2 Antigens in Mice. Yellow highlighted cells indicate days on which serum samples were collected. Gray cells indicate days on which the indicated compositions were administered. Green cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0241] Fig.23. Mouse Immunogenicity Data Generated Using Improved SARS-Cov-2 Vaccines.(A), (B), (C), (D), and (E) show neutralization titers (pVNT50) 7, 15, 21, 28, and 34 days after vaccination,respectively, in an experiment performed in accordance with the protocol shown in Fig.22. (F) provides a summary of the neutralization titers that were collected at different time points.

[0242] Fig.24. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-Cov-2 RNA Compositions in Mice. Yellow cells indicate days on which serum samples were collected. Gray cells indicate days on which the indicated compositions were administered. Green cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0243] Fig.25. Mouse Immunogenicity Data Generated Using Improved SARS-Cov-2 Vaccines. (A), (B), (C), and (D) show neutralization titers (pVNT50) 7, 14, 21, and 28 days after vaccination, respectively, in an experiment performed in accordance with the protocol shown in Fig.24. (E) provides a summary of the neutralization titers that were collected at different time points.

[0244] Fig.26. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-Cov-2 Antigens in Mice. Yellow highlighted cells indicate days on which serum samples were collected. Gray cells indicate days on which the indicated compositions were administered. Green cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0245] Fig.27. Mouse Immunogenicity Data Generated Using Improved SARS-Cov-2 Vaccines. (A), (B), (C), and (D) show neutralization titers (pVNT50) 7, 14, 21, and 28 days after vaccination, in an experiment performed in accordance with the protocol shown in Fig.26. (E) provides a summary of the neutralization titers that were collected at different time points. (F) and (G) provide a summary of the same data summarized in (E), along with neutralization titers from a repeated experiment, shown neutralizing titers for the original BNT162b2 construct (RNA041) and the optimized construct (RNA017).

[0246] Fig.28. Summary of Effects of Optimizing Signal Peptide, Multimerization Domain, Linker Domain, and Transmembrane Domain on Antigen Expression. (A)-(D) show the effects of optimizing the indicated domains on polypeptide expression (in the figure, a fragment of the S1 polypeptide comprising a sequencecorresponding to amino acids 1-528). Aside from the indicated domain (signal peptide, multimerization domain, linker, transmembrane domain), all other portions of the encoded polypeptide were identical in each of the panels. Red arrow indicates improvement in expression relative to a reference. In (D), “reference” refers to a polypeptide comprising the transmembrane domain of the SARS-CoV-2 S protein. (E) Provides expression domains from polypeptides (a fragment of the S1 domain comprising a sequence corresponding to amino acids 1-528 of SEQ ID NO: 1) comprising a combination of different optimized domains. As indicated in the figure, the optimized construct improved expression approximately 5- to 10-fold as compared to the original BNT162b2 product (encoding a full-length S protein), and approximately 2-fold as compared to other optimized constructs.

[0247] Fig.29. Correlation of Surface Expression (In Vitro) and Elicited nAb Titers (In Vivo). Provided is an analysis showing the correlation between in vitro expression measurements and neutralizing antibody titers observed in mice (neutralization titers measured 28 days after administering a first dose of a composition to vaccine-immune mice). As shown in the figure, in vitro expression data showed a strong correlation with mouse neutralization titers, indicating that in vitro expression data was a good predictor for in vivo immunogenicity.

[0248] Fig.30. Exemplary Protocol for Investigating Immunogenicity of Improved SARS-Cov-2 Antigens in Mice. Yellow highlighted cells indicate days on which serum samples were collected. Gray cells indicate days on which the indicated compositions were administered. Green cells indicate the final day of the experiment, during which mice with euthanized and final samples were collected.

[0249] Fig.31. Mouse Immunogenicity Data Generated Using Improved SARS-Cov-2 Vaccines. (A), (B), (C), and (D) show neutralization titers (pVNT50) 7, 14, 21, and 28 days after vaccination, in an experiment performed in accordance with the protocol shown in Fig.30. (E) provides a summary of the neutralization titers that were collected at different time points.

[0250] Fig.32. Mouse Immunogenicity Data Confirms Dose-Sparing Effect Provided by Vaccines Described Herein. Vaccine naïve mice were administered 0.4, 0.1, or 0.025 μg of LNP-formulated RNA. 7, 14, 21, 28, and 35 days after administration, serum samples were collected and neutralization titers against an XBB.1.5- adapted pseudovirus were measured. Results are shown in Fig.32(A)-(F). As shown in the figure, a clear dose sparing effect was observed by days 21, 28, and 35 for constructs described herein as compared to vaccines encoding a full-length SARS-CoV-2 S protein.

[0251] Fig.33. SARS-Cov-2 Vaccines Described Herein Induce Higher Neutralization Titers and Broader Cross Neutralization in Vaccine Naïve Mice As Compared To RNA Encoding Full Length S Protein. Vaccine naïve mice were administered a single dose of the indicated RNAs. Serum samples were collected 7, 14, 21, 28, and 35 days after administration of RNA. Results are shown in (A), (B), (C), (D), and (E). (F) provides a summary of the neutralization titers collected at each time point. (G) shows neutralization titers against further SARS-CoV-2 strains and variants (Wuhan, Omicron BA.1, and Omicron BA.4 / 5), in sera sample collected 35 days after administration of RNA. As shown in each of Figs.33(A)-(F), RNA described herein resulted in greatly increased neutralization titers against a matched SARS-CoV-2 variant as compared to RNA encoding a full-length S protein (titers ~10-fold higher by day 35). Cross-neutralization titers were also greatly increased, with neutralization titers of BA.4 / 5 being ~10-fold higher for RNA described herein as compared to RNA encoding full length S protein.

[0252] Fig.34. RNA Described Herein Results in Improved In Vitro Expression Of XBB.1.5 and KP.2 SARS-Cov-2 Antigens. Cell surface expression was measured in cells transfected with RNA using fluorescently labeled ACE2 protein. (A) and (B) Compare antigen expression for an RNA encoding a truncated S1 subdomain(RNA017) and an RNA encoding a full-length S protein (RNA041), where the truncated S1 subdomain and full-length S protein are each adapted to the XBB.1.5 SARS-CoV-2 variant. (C) and (D) Compare antigen expression of an RNA encoding a polypeptide comprising a truncated S1 subdomain of a KP.2 SARS-CoV-2 S protein (RNA042) to RNA encoding a full length KP.2 S protein (RNA044). In each experiment, RNA encoding a truncated S1 subdomain was found to improve antigen expression as compared to RNA encoding a full-length S protein.

[0253] Fig.35. Phenotypic Characterization of Antigen-Specific B Cells. (A) Provides an illustration showing the B cell maturation process and the various intermediary states a B cell transitions through as it transforms from a naïve cell to a plasma cell. Also indicated are cell surface markers that are characteristic of the different B cell differentiation stages. (B) Lists the different mixtures of fluorescently labeled bait proteins that were used in the experiment described in Example 14 to phenotypically characterize antigen-specific B cells obtained from mice administered a composition described herein. (C) Lists flow antibodies from the B-cell flow panel that was used in the experiment described in Example 14. FLS is an abbreviation for “Full Length Spike,” and BC is an abbreviation for B Cell.

[0254] Fig.36. An Example of a Protocol for Phenotypically Screening B cells. Cells are screened to identify single cell lymphocytes. Single cell lymphocytes are probed for cell surface expression of CD19 and CD20 to identify B cells, which can be screened for a variety of markers of interest and binding to various bait proteins. CD19+ / CD20+ B cells are screened for staining Spike-antigen bait positive. Antigen-specific B cells are then analyzed for cell surface expression of CD38 (activation marker), CD95 (Germinal Center marker), and CD273 / 80 (markers for memory B cells) and CD138 (a marker of plasma cells) for phenotypic characterization though a combinatorial / Boolean gating approach.

[0255] Fig.37. Representative Plots Showing Antigen-specificity of B Cells Obtained from Mice Administered Compositions Described Herein. (A)-(C) show exemplary plots of CD19+ / CD20+ cells probed for binding to full length S protein, RBD, and NTD respectively. Each plot characterizes B cells obtained from a single mouse. As shown in (C), an RNA encoding a truncated S1 polypeptide (RNA017) was found to induce surprising high numbers of B cells capable of binding the NTD. Without wishing to be bound by theory, the improved immune response to NTD may result from increased accessibility of the NTD in the truncated S1 subdomain as compared to a full-length S protein.

[0256] Fig.38. Summary of Phenotypic Characteristics of B Cells Obtained from Mice Administered Compositions Described Herein. (A)-(C) Show the percent of CD19+ B cells that were found to bind full length S protein, RBD, and NTD, respectively. As shown, RNA017 was found to produce significantly higher numbers of B cells that bind the full-length S protein, RBD, and NTD at each concentration of RNA administered. For both the NTD and the RBD, B cell responses were ~4x higher for RNA encoding a truncated S1 subdomain (RNA017) as compared to those induced by RNA encoding a full-length S protein (RNA041) for each concentration of RNA tested. The B cell response induced by 0.1 μg of RNA encoding a truncated S1 subdomain was comparable to that induced by 0.4 μg of RNA encoding a full-length S protein, representing a ~4-fold dose sparing effect.

[0257] Fig.39. RNA Compositions Described Herein Induce a Prolonged Germinal Center Response. B cells found to bind (A) a full-length S protein, (B) an RBD, or (C) an NTD were probed for cell surface expression of germinal center markers. As shown, when administered at 0.4 μg, an RNA encoding a truncated S1 subdomain (RNA017) produced a higher proportion of B cells showing a germinal center phenotype as compared to mice administered the same amount of RNA encoding a full-length S protein (RNA041). This observation suggeststhat RNA encoding a truncated S1 subdomain produces a germinal center reaction that is extended as compared to RNA encoding a full-length S protein and is consistent with observations in other experiments described herein, in which neutralization titers induced by a truncated S1 subdomain continued to increase for a longer period of time as compared to RNA encoding a full-length S protein.

[0258] Fig.40. Neutralizing Titers Induced in Vaccine-Naïve Mice Administered Two Doses (~21 days apart) of RNA Compositions Described. (A)-(B) Show geometric mean titers (GMTs) of antibodies that neutralize a KP.2 SARS-CoV-2 virus 7 and 14 days after administering a first dose of the indicated composition to vaccine naïve mice. (C)-(E) Show SARS-CoV-2 KP.2 neutralization titers 7, 14, and 21 days after administering a second dose of the indicated composition (28, 35, and 42 days after administering the first dose). As shown, by day 14, RNA encoding a truncated S1 subdomain (RNA042) began to show increased neutralization titers as compared to RNA encoding a full-length SARS-CoV-2 S protein (RNA044), and by days 28-42, had produced neutralization titers that were over 2-fold higher than those induced by the same amount of RNA encoding a full-length S protein.

[0259] Fig.41. Neutralizing Titers Induced in Vaccine-Naïve Mice Administered Two Doses (~28 days apart) of RNA Compositions Described. (A)-(D) Show geometric mean titers (GMTs) of antibodies that neutralize a KP.2 SARS-CoV-2 virus 7, 14, 21, and 28 days after administering a first dose of the indicated composition to vaccine naïve mice. (E) Shows SARS-CoV-2 KP.2 neutralization titers 7 days after administering a second dose of the indicated composition (35 days after administering the first dose). (F) Is a time plot, summarizing the data shown in (A)-(E). The data again shows that RNA encoding a truncated S1 subdomain (RNA042) begins to show increased neutralization titers as compared to RNA encoding a full-length SARS-CoV-2 S protein by day 14, which increases to be >4x those induced by RNA encoding a full-length SARS-CoV-2 S protein by day 21 and ~8x higher by day 28. 35 days after the first dose, and 7 days after the second dose, titers had increased to be ~3x those induced by an RNA encoding a full-length S protein. Titers induced by a 0.1 μg dose of the RNA encoding a truncated S1 subdomain were also found to be comparable to those induced by a 0.5 μg dose of RNA encoding a full-length S protein at each time point, indicating a ~5-fold dose-sparing effect.

[0260] Fig.42. Neutralization Titers Induced in Vaccine-Experienced Mice Administered RNA Compositions Described Herein. (A)-(C) Show results from an experiment in which vaccine experienced mice were administered an RNA encoding a full-length S protein or RNA encoding a truncated S1. Prior to administering a candidate RNA, each mouse was first administered 2 doses of RNA encoding an S protein of a SARS-CoV-2 Wuhan strain (on days 0 and 21) and one dose of a bivalent composition comprising an RNA encoding an S protein of a SARS-CoV-2 Wuhan strain and an RNA encoding an S protein of a BA.4 / 5 Omicron SARS-CoV-2 variant (on day 49). 133 days after administering the first vaccine (d133) candidate RNA were administered. (A), (B), and (C) show neutralization titers 0 days (d133 of the experiment), 7 days (d140 of the experiment), and 14 days (d147 of the experiment) after administering the indicated candidate. (D) Provides Geometric Mean Fold Increases (GMFI) of neutralizing antibody titers at d147 as compared to mice administered carrier solution. As shown in (C), by d147, absolute titers of neutralization antibodies induced by RNA encoding a truncated S1 subdomain were ~2-fold higher than those induced by RNA encoding a full-length S protein. As shown in (D), GMFI of neutralizing antibody titers were ~3-fold higher for the RNA encoding a truncated S1 subdomain as compared to RNA encoding a full length, prefusion-stabilized S protein. Certain Definitions

[0261] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0262] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0263] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0264] Agent: As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and / or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0265] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0266] Antibody agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments,an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRs1, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and / or 3 and light chain CDRs 1, 2, and / or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domainantibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., poly-ethylene glycol, etc.)).

[0267] Antigen: Those skilled in the art, reading the present specification, will appreciate that the term “antigen” refers to a molecule that is recognized by the immune system, e.g., in particular embodiments, the adaptive immune system, such that it elicits an antigen-specific immune response. In some embodiments, an antigen-specific immune response may be or comprise generation of antibodies and / or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide that comprises at least one epitope against which an immune response can be generated. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. In one embodiments, an antigen or a processed product thereof such as a T-cell antigen is bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a processed product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a parasitic antigen. In accordance with the present disclosure, in some embodiments, an antigen may be delivered by RNA molecules as described herein. In some embodiments, a peptide or polypeptide 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 or polypeptide antigen can be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen can be greater than 100 amino acids. In some embodiments, an antigen is recognized by an immune effector cell. In some embodiments, an antigen, if recognized by an immune effector cell, is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and / or expansion of the immune effector cell carrying an antigen receptor recognizing the antigen. In the context of the embodiments of the present disclosure, in some embodiments, an antigen can be presented or present on the surface of a cell, e.g., 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.

[0268] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In someembodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0269] Binding: Those skilled in the art, reading the present specification, will appreciate that the term “binding” typically refers to a non-covalent association between or among entities or moieties. In some embodiments, binding data are expressed in terms of “IC50”. As is understood in the art, IC50is the concentration of an assessed agent in a binding assay at which 50% inhibition of binding of reference agent known to bind the relevant binding partner is observed. In some embodiments, assays are run under conditions in which (e.g., limiting binding target and reference concentrations), IC50values approximate KDvalues. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31:813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. For example, can be based on its IC50, relative to the IC50of a reference standard peptide. Binding can also be determined using other assay systems including those using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol.2:443 (1990); Hill et al., J. Immunol.147:189 (1991); del Guercio et al., J. Immunol.154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol 21:2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem.268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med.180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol.149:1896 (1992)).

[0270] Cap: As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as “m7G.” In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art). Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a single- stranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.

[0271] Cell-mediated immunity: “Cell-mediated immunity,” “cellular immunity,” “cellular immune response,” or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC. A cellular responserelates to immune effector cells, in particular to T cells or T lymphocytes which act as either “helpers” or “killers.” The helper T cells (also termed CD4+T cells or CD4 T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+T cells, CD8 T cells, or CTLs) kill diseased cells such as virus-infected cells, preventing the production of more diseased cells.

[0272] Co-administration: As used herein, the term “co-administration” refers to use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent. The combined use of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent may be combined in one pharmaceutically acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0273] Codon-optimized: As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon- optimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0274] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0275] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity isrequired in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0276] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, fASTA, gGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0277] As those skilled in the art are aware, sequence alignment strategies enable consideration, for example, of “gaps” in sequences, and / or of “repeated” residues. Moreover, those skilled in the art understand that in some cases, it is not possible to unambiguously determine the exact location of a sequence change relative to a reference sequence. For example, when a reference sequence includes a stretch of two or more contiguous identical residues, and a changed sequence has one fewer of the residues, it is not possible to assign a particular singular residue in the reference sequence as the one that was deleted, as deletion of any one of the identical contiguous residues would generate the same changed sequence. Those skilled in the art therefore appreciate the convention of arbitrarily assigning one of the reference residue positions as the deleted residue. To give a specific example, SEQ ID NO:1 is a polypeptide sequence in which two adjacent Y residues are present at positions 144 and 145. If one of these amino acid residues is deleted, a person of skill in the art will not be able to determine whether amino acid 144 or 145 has been deleted in the changed sequence. They will understand, however, that either deletion describes the same polypeptide sequence, and therefore will be able to unambiguously determine the sequence of a polypeptide described as having a deletion at position 144 or 145 of SEQ ID NO: 1 (i.e., they will understand that a polypeptidedescribed as having a deletion at a position corresponding to position 144 of SEQ ID NO: 1 and a polypeptide described as having a deletion at a position corresponding to position 145 of SEQ ID NO: 1 have the same amino acid sequence).

[0278] Derived: In the context of an amino acid sequence (peptide or polypeptide) “derived from” a designated amino acid sequence (peptide or polypeptide), refers to a structural analogue of a designated amino acid sequence. In some embodiments, an 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.

[0279] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0280] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0281] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule (e.g., an mRNA) encodes a polypeptide if transcription and translation of RNA (e.g., mRNA) corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the RNA (e.g., mRNA) sequence of such a target antigen. In some embodiments, a coding region of an RNA molecule encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0282] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences thatare not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0283] Epitope: As used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous fragment of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0284] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0285] Five prime untranslated region: As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of an RNA (e.g., mRNA) molecule between a transcription start site and a start codon of a coding region of an RNA. In some embodiments, “5’ UTR” refers to a sequence of an RNA (e.g., mRNA) molecule that begins at a transcription start site and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA molecule, e.g., in its natural context.

[0286] Fragment: The term “fragment” as used herein in the context of a nucleic acid sequence (e.g., RNA sequence) or an amino acid sequence may typically be a fragment of a reference sequence. In some embodiments, a reference sequence is a full-length sequence of e.g., a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, refers to a sequence that is identical to a corresponding stretch within a reference sequence. In some embodiments, a fragment comprises a continuous stretch of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the total length of a reference sequence from which the fragment is derived. In some embodiments, the term “fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, e.g., a sequence which represents the amino acid sequence shortened at the N-terminus and / or C-terminus. In some embodiments, a fragment of an amino acid sequence 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.

[0287] Homology: As used herein, the term “homology” or “homolog” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptidemolecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0288] Humoral immunity: As used herein, the term “humoral immunity” or “humoral immune response” refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0289] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0290] Immunologically equivalent: The term “immunologically equivalent” means that an 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 immunologicaleffects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, in some embodiments, the term “immunologically equivalent” is used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

[0291] In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope of an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0292] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may be “increased” relative to that obtained with a comparable reference pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine). Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value 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 higher, as compared to a comparable reference.

[0293] Ionizable: The term “ionizable” refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid ma ha e a Ka within a range of about 5 to about 7.

[0294] Isolated: The term “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.

[0295] Lipid: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties orgroups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0296] RNA lipid nanoparticle: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s). In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z- average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0297] Lipidoid: As used herein, a “lipidoid” refers to a lipid-like molecule. In some embodiments, a lipoid is an amphiphilic molecule with one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.

[0298] Nanoparticle: As used herein, the term “nanoparticle” refers to a particle having an average size suitable for parenteral administration. In some embodiments, a nanoparticle has a longest dimension (e.g., a diameter) of less than 1,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) of less than 100 nm. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 μm and about 500 nm, or between about 1 nm and 1,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticle may be substantially spherical so that its longest dimension may be its diameter. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health.

[0299] Naturally occurring: The term “naturally occurring” as used herein refers to an entity that 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.

[0300] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0301] Nucleic acid particle: A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may comprise at least one cationic orcationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. In some embodiments, a nucleic acid particle is a lipid nanoparticle. In some embodiments, a nucleic acid particle is a lipoplex particle.

[0302] Nucleic acid / Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double- stranded fragments. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.

[0303] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0304] Patient: As used herein, the term “patient” refers to any organism who is suffering or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non- human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has beendiagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes a HSV infection. In some embodiments, a patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or susceptible to a HSV infection.

[0305] PEG-conjugated lipid: The term “PEG-conjugated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0306] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, or intravenous injection as, for example, a sterile solution or suspension formulation.

[0307] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to inhibition of the course of the disease. In some embodiments, such inhibition may comprise slowing down the progress of a disease and / or interrupting or reversing the progress of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease or a condition. An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend, for example, on a disease or condition to be treated, the severity of such a disease or condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0308] Poly(A) sequence: As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3’-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0309] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural aminoacids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0310] Prevent: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder and / or condition and / or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0311] Recombinant: The term “recombinant” in the context of the present disclosure means “made through genetic engineering”. In some embodiments, a “recombinant” entity such as a recombinant nucleic acid in the context of the present disclosure is not naturally occurring.

[0312] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by thoseskilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0313] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded fragments. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0314] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0315] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0316] RNA lipoplex particle: As used herein, the term “RNA lipoplex particle” refers to a complex comprising liposomes, in particular cationic liposomes, and RNA molecules. Without wishing to bound by a particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. In some embodiments, positively charged liposomes may comprise a cationic lipid, such as in some embodiments DOTMA, and additional lipids, such as in some embodiments DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0317] Selective or specific: The term “selective” or “specific”, when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0318] Stable: As used herein, the term “stable” in the context of the present disclosure refers to a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as a whole and / or components thereof meeting or exceeding pre-determined acceptance criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the integrity of RNA molecules being maintained at least above 90% or more. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to at least 90% or more (including, e.g., at least 95%, at least 96%, at least 97%, or more) of RNA molecules being maintained to be encapsulated within lipid nanoparticles. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to a formulation that remains capable of eliciting a desired immunologic response when administered to a subject. In some embodiments, a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) remains stable for a specified period of time under certain conditions.

[0319] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., a HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0320] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0321] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have beendiagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0322] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0323] Therapy: The term “therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0324] Three prime untranslated region: As used herein, the terms “three prime untranslated region” or “3' UTR” refer to a sequence of an RNA (e.g., mRNA) molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.

[0325] Threshold level (e.g., acceptance criteria): As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and / or classify the results of a measurement, for example, the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g., a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0326] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition.

[0327] Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and / or after exposure to a disease-associated agent, and in certain embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent.

[0328] Vaccine: As used herein, the term “vaccine” refers to a composition that induces an immune response upon administration to a subject. In some embodiments, an induced immune response provides protective immunity.

[0329] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0330] Vector: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In some embodiments, known techniques may be used, for example, for generation or manipulation of recombinant DNA, for oligonucleotide synthesis, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.

[0331] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. Detailed Description

[0332] In some embodiments, the present provides technologies (e.g., compositions, pharmaceutical compositions, immunogenic compositions, vaccines, and methods) that can be used to induce an immune response against SARS-CoV-2. In some embodiments, technologies provided in the present disclosure can be used to mitigate immune imprinting effects and / or induce a stronger de novo immune response (e.g., as compared to other vaccination approaches).SARS-CoV-2 Overview

[0333] SARS-CoV-2 Spike (S) protein can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. S1 of SARS-CoV-2 comprises a receptor-binding domain (RBD), which mediates virus entry into host cells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0334] The presentation of COVID-19 is generally with cough and fever, with chest radiography showing ground-glass opacities or patchy shadowing. However, many patients present without fever or radiographic changes, and infections may be asymptomatic which is relevant to controlling transmission. For symptomatic subjects, progression of disease may lead to acute respiratory distress syndrome requiring ventilation and subsequent multi- organ failure and death. Common symptoms in hospitalized patients (in order of highest to lowest frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea / vomiting or diarrhoea, headache, weakness, and rhinorrhoea. Anosmia (loss of smell) or ageusia (loss of taste) may be the sole presenting symptom in approximately 3% of individuals who have COVID-19.

[0335] All ages may present with the disease, but notably case fatality rates (CFR) are elevated in persons >60 years of age. Comorbidities are also associated with increased CFR, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers are overrepresented among COVID-19 patients due to occupational exposure to infected patients.

[0336] In most situations, a molecular test is used to detect SARS-CoV-2 and confirm infection. The reverse transcription polymerase chain reaction (RT-PCR) test methods targeting SARS-CoV-2 viral RNA is one method for diagnosing suspected cases of COVID-19. Samples to be tested are collected from the nose and / or throat with a swab. SARS-CoV-2 Variants

[0337] Since the initial discovery of SARS-CoV-2, a number of variants have arisen around the world. The emergence of these novel circulating variants of SARS-CoV-2 has raised significant concerns about geographic and temporal efficacy of vaccine interventions. The emergence of Omicron (B.1.1.529) variants, which comprise a number of mutations in the S protein, has been of particular concern. As used herein, a SARS-CoV-2 variant refers to a SARS-CoV-2 virus that has acquired one or more mutations that differentiate it from the Wuhan strain of SARS- CoV-2 that first emerged in 2019. Variants can be identified by virologists and / or health organizations using an appropriate classification system, including, e.g., the Pango or NextClade classification systems, examples of which are described herein.

[0338] 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: https: / / www.gisaid.org, 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.

[0339] The Omicron BA.1 variant was first reported to WHO on 24 November 24, 2021, and was detected in South Africa. Omicron and its sublineages have had a major impact on the epidemiological landscape of the COVID- 19 pandemic since their initial emergence (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE):Classification of Omicron (B.1.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Enhancing Response to Omicron SARS-CoV-2 variant: Technical brief and priority actions for Member States (2022)). Significant alterations in the spike (S) glycoprotein of the first Omicron variant BA.1 resulted in the loss of many neutralizing antibody epitopes (M. Hoffmann et al., “The Omicron variant is highly resistant against antibody mediated neutralization: Implications for control of the COVID-19 pandemic”, Cell 185, 447–456.e11 (2022)) and rendered BA.1 capable of partially escaping previously established SARS-CoV-2 wild-type strain (Wuhan-Hu-1)-based immunity (V. Servellita, et al., “Neutralizing immunity in vaccine breakthrough infections from the SARS-CoV-2 Omicron and Delta variants”, Cell 185, 1539–1548.e5 (2022); Y. Cao et al., “Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies”, Nature 602, 657–663 (2022)).

[0340] As a result, breakthrough infection of vaccinated individuals with Omicron is more common than with previous Variants of Concern (VOCs). While Omicron BA.1 was displaced by the BA.2 variant in many countries around the globe, other variants such as BA.1.1 and BA.3 temporarily and / or locally gained momentum but did not become globally dominant (S. Xia et al., “Origin, virological features, immune evasion and intervention of SARS-CoV- 2 Omicron sublineages. Signal Transduct. Target. Ther.7, 241 (2022); H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022).). Omicron BA.2.12.1 subsequently displaced BA.2 to become dominant in the United States, whereas BA.4 and BA.5 displaced BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID-19 country overview -Country overview report: Week 312022 (2022); J. Hadfield et al., “Nextstrain: Real- time tracking of pathogen evolution,” Bioinformatics 34, 4121–4123 (2018)). Currently, Omicron BA.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https: / / covid.cdc.gov / coviddata-tracker (2022)).

[0341] Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, among which some are shared between all Omicron VOCs while others are specific to one or more Omicron sublineages. Antigenically, BA.2.12.1 exhibits high similarity with BA.2 but not BA.1, whereas BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more so from BA.1, in line with their genealogy (A. Z. Mykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct,” Sci. –145, L212I, or ins214EPE in the S glycoprotein N-terminal domain and G446S or G496S in the receptor binding domain (RBD). Amino acid changes T376A, D405N, and R408S in the RBD are in turn common to BA.2 and its descendants but not found in BA.1. In addition, some alterations are specific for individual BA.2-descendant VOCs, including L452Q for BA.2.12.1 or L452R and F486V for BA.4 and BA.5 (BA.4 and BA.5 encode for the same S sequence). Most of these shared and VOC-specific alterations were shown to play an important role in immune escape from monoclonal antibodies and polyclonal sera raised against the wild-type S glycoprotein. In particular, the BA.4 / BA.5- specific alterations are strongly implicated in immune escape of these VOCs (P. Wang et al., “Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130–135 (2021); Q. Wang et al., “Antibody evasion by SARS- CoV-2 Omicron subvariants BA.2.12.1, BA.4, & BA.5. Nature 608, 603–608 (2022)).SARS-CoV-2 Variant Adaptions

[0342] In some embodiments, an antigen utilized as described herein is or comprises a fragment or domain of a viral polypeptide, or an antigenic fragment thereof. In some embodiments, an antigen utilized as described herein is a membrane-tethered antigen (e.g., an antigenic fragment thereof fused with a membrane-associating moiety, such as for example, a transmembrane moiety). In some embodiments, a provided pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) comprises or delivers antigen sequences that are or comprise one or more antibody epitopes and / or one or more CD4 T cell and / or CD8 T cell epitopes.

[0343] In some embodiments, an antigen utilized as described herein includes one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked; alternatively or additionally, in some embodiments, a terminally truncated antigen is utilized, and / or one or more mutations associated with a viral variant (e.g., a SARS-CoV-2 variant of concern) is present in the antigen.

[0344] In some embodiments, an antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element).

[0345] In some embodiments, an antigen utilized as described herein includes a membrane association element (e.g., a homologous membrane association element), such as a transmembrane domain.

[0346] In some embodiments, an antigen utilized as described herein includes a secretion signal (e.g., a homologous secretion signal).

[0347] In some embodiments, utilized sequences may comprise one or more mutations associated with a viral variant (e.g., a variant that prevalent and / or that is predicted to be highly immune escaping). In some embodiments, utilized sequences comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by WHO). In some embodiments, utilized sequences comprise one or more mutations associated with a viral variant that has been determined to be or has been predicted to be highly immune escaping (e.g., highly immune escaping relative to an immune response developed in subjects administered a previously approved vaccine and / or a previously prevalent viral variant).

[0348] Among other things, described herein are certain SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, a SARS-CoV-2 antigen comprise immunogenic portions of a full- length SARS-CoV-2 polypeptide (e.g., an S1 domain of a SARS-COV-2 S protein, a truncated S1 subdomain, and / or an RBD of a SARS-CoV-2 S protein) or a variant thereof. In some embodiments, such antigens are delivered as protein antigens to induce an immunogenic response. In some embodiments, such antigens are delivered using RNA (e.g., modRNA encoding an S1 domain, truncated S1 subdomain and / or RBD of a SARS-CoV-2 S protein and formulated in LNP particles) to induce an immunogenic response.

[0349] As used herein, a full-length SARS-CoV-2 S protein comprising a “Wild-Type” or “Wuhan” sequence has a sequence corresponding to that of the first detected SARS-CoV-2 strain, consisting of 1273 amino acids and having an amino acid seq ence according to SEQ ID NO: 1:CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS

[0350] Unless otherwise indicated, position numberings in a SARS-CoV-2 S protein given herein are in relation to the amino acid sequence of SEQ ID NO: 1. One of skill in the art reading the present disclosure will understand and be able to determine corresponding positions in a SARS-CoV-2 S protein variant sequence from locations of positions provided relative to the amino acid sequence of SEQ ID NO: 1 (i.e., a person of skill in the art provided positions relative to SEQ ID NO: 1, or another variant, will be able to determine corresponding positions in the S protein sequence of another SARS-CoV-2 variant or a fragment thereof). One of skill in the art will also understand that a fragment of a SARS-CoV-2 S protein that comprises one or more mutations of a variant, comprises only those mutations that fall within the fragment region. For example, if a truncated S1 subdomain comprises an amino acid sequence corresponding to amino acids 20-528 of SEQ ID NO: 1 and comprises one or more mutations of a SARS- CoV-2 variant, one of skill in the art will understand that the truncated S1 subdomain comprises only those mutations located within the corresponding region of the SARS-CoV-2 variant.

[0351] In some embodiments, a fragment of an S protein (e.g., a truncated S1 domain, including, e.g., a sequence comprising amino acids 20-528 of SEQ ID NO: 1, 17-528 of SEQ ID NO: 1, 14-528 of SEQ ID NO: 1, 20- 541 of SEQ ID NO: 1, 17-528 of SEQID NO: 1, or 14-528 of SEQ ID NO: 1) includes 1 or more mutations associated with a SARS-CoV-2 variant (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 25, 36 or more (e.g., all)), to the extent they are present in the corresponding fragment of the S protein of the SARS-CoV-2 variant.

[0352] In specific embodiments, a spike (S) protein described herein or a fragment thereof can be modified in such a way that the prototypical prefusion conformation is stabilized. Certain mutations that stabilize a prefusion confirmation are known in the art, e.g., as disclosed in WO 2021243122 A2 and Hsieh, Ching-Lin, et al. ("Structure- based design of prefusion-stabilized SARS-CoV-2 spikes," Science 369.6510 (2020): 1501-1505), the contents of each which are incorporated by reference herein in their entirety. In some embodiments, a SARS-CoV-2 S protein may be stabilized by introducing one or more proline mutations. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to residues 986 and / or 987 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at one or more positions corresponding to residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, and 942 of SEQ ID NO: 1. In some embodiments, a SARS-CoV-2 S protein comprises a proline substitution at positions corresponding to each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO: 1.

[0353] In some embodiments, stabilization of the prototypical prefusion conformation of a SARS-CoV-2 S protein may be obtained by introducing two consecutive proline substitutions at residues 986 and 987. Specifically, spike (S) protein stabilized protein variants are obtained in a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline. In one embodiment, aSARS-CoV-2 S protein variant wherein the prototypical prefusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 2:(SEQ ID NO: 2)

[0354] Those skilled in the art are aware of various SARS-COV-2 Spike variants, and / or resources that document them. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences and, in particular, modifications thereof compared to wildtype SARS-CoV-2 S protein amino acid sequence, e.g., as compared to SEQ ID NO: 1, are useful herein.

[0355] B.1.1.7 ("Variant of Concern 202012 / 01" (VOC-202012 / 01)

[0356] B.1.1.7 (“alpha variant”) is a SARS-CoV-2 variant that was first detected in October 2020 in the United Kingdom from a sample taken the previous month, and quickly began to spread by mid-December. It is correlated with a significant increase in the rate of COVID-19 infection; this increase is thought to be at least partly due to a change of N501Y inside the spike glycoprotein's receptor-binding domain, which is needed for binding to ACE2 in human cells. B.1.1.7 is defined by 23 mutations: 13 non-synonymous mutations, 4 deletions, and 6 synonymous mutations (i.e., there are 17 mutations that change proteins and six that do not). Spike protein changes in B.1.1.7 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0357] B.1.351 (501.V2)

[0358] B.1.351 lineage ( “Beta variant”), colloquially known as South African COVID-19 variant, has increased transmissibility relative to the original Wuhan strain. The B.1.351 variant is defined by multiple spike protein changes including: L18F, D80A, D215G, deletion 242-244, R246I, K417N, E484K, N501Y, D614G and A701V. There are three mutations of particular interest in the spike region of the B.1.351 genome: K417N, E484K, N501Y.

[0359] B.1.1.298 (Cluster 5)

[0360] B.1.1.298 was discovered in North Jutland, Denmark, and is believed to have been spread from minks to humans via mink farms. Several different mutations in the spike protein of the virus have been confirmed. The specific mutations include deletion 69–70, Y453F, D614G, I692V, M1229I, and optionally S1147L.

[0361] P.1 (B.1.1.248)

[0362] Lineage B.1.1.248 (the “gamma variant”), known as the Brazil(ian) variant, is one of the variants of SARS-CoV-2 which has been named P.1 lineage. P.1 has a number of S-protein modifications (L18F, T20N, P26S,D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F) and is similar in certain key RBD positions (K417, E484, N501) to variant B.1.351 from South Africa.

[0363] B.1.427 / B.1.429 (CAL.20C)

[0364] Lineage B.1.427 / B.1.429 (the “epsilon variant”), also known as CAL.20C, is defined by the following modifications in the S-protein: S13I, W152C, L452R, and D614G, of which the L452R modification is of particular concern. CDC has listed B.1.427 / B.1.429 as a "variant of concern".

[0365] B.1.525

[0366] B.1.525 ( “eta variant”) carries the same E484K modification as found in the P.1, and B.1.351 variants, and also carri D614G, Q677H and F888L.

[0367] B.1.526

[0368] B.1.526 ( “iota variant”) was detected as an emerging lineage of viral isolates in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

[0369] B.1.1.529

[0370] B.1.529 (“Omicron variant”) was first detected in South Africa in November 2021. Omicron multiplies around 70 times faster than Delta variants, and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, a number of Omicron sublineages have arisen. Listed below are the current Omicron variants of concern, along with certain characteristic mutations associated with the S protein of each. The S protein of BA.4 and BA.5 have the same set of characteristic mutations, which is why the below table has a single row for “BA.4 or BA.5”, and why the present disclosure refers to a “BA.4 / 5” S protein in some embodiments. Similarly, the S proteins of the BA.4.6 and BF.7 Omicron variants have the same set of characteristic mutations, which is why the below table has a single row for “BA.4.6 or BF.7”).

[0371] The JN.1 variant emerged in August 2023, in Luxemburg. It is a descendant of the BA.2.86 variant. BA.2.86 initially drew the attention of health authorities because it had a large number of S protein mutations as compared to previous variants (~30 more than other variants circulating at the time). BA.2.86 never came to dominate circulating SARS-CoV-2 variants, however. Unlike BA.2.86, the JN.1 variant (and descendants thereof) has the ability to transmit efficiently between humans, an ability that is thought to be due to the acquisition of an L455S mutation in the S protein (position shown relative to SEQ ID NO: 1). The JN.1 rapidly came to dominate SARS-CoV-2 variants, increasing from less than 5% in November 2023, to 60% of cases by January 2024. Since the initial emergence of the JN.1 variant, descendants have continued to be identified, including the JN.1.2, JN.1.6, JN.1.7, KP.2, KP.3, and XEC variants, that have acquired further mutations relative to JN.1, and which are thought to further increase the infectivity and / or transmissibility of SARS-CoV-2 variants.

[0372] Since the emergence of JN.1, descendants of JN.1 have arisen and quickly supplanted the JN.1 variant. These JN.1 descendants include “SLip” variants (including, e.g., JN.1.16), which include L455S and F456L mutations; and “FLiRT" variants (including, e.g., KS.1.1, KP.2), which include the mutations associated with SLip variants and an additional R346T mutation. FLuQE variants (e.g., KP.3.3), in turn, are descendants of the FLiRT variants, and include the same mutations plus an additional Q493E mutation. The 455 position has also continued to be a mutation hot spot, with “FLip” including mutations L455F and F456L. In some embodiments, an S protein or fragment thereof comprises one or more mutations associated with each of the Slip, FLiRT, and / or Flip variants.

[0373] The XEC variant is a hybrid of the KS.1.1 and KP.3.3 variants. A description of the emergence of the JN.1 variant and descendants thereof, is provided, e.g., in E. Topol, “Are We FLiRTing With A New Covid Wave?,” April 18, 2024, accessible at erictopol.substack.com / p / are-we-flirting-with-a-new-covid; and Sankaran, V. “New Covid XEC variant starting to spread in Europe – what we know,” September 4, Independent, accessible at www.independent.co.uk / news / science / covid-variant-xec-europe-symptoms-b2613485.html.

[0374] Table 1: Omicron Variants of Concern and Characteristic mutationsBF.7 S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A,R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N,N969K, V1104L, P1143L

[0375] In some embodiments, SARS-CoV-2 S proteins described herein comprise one or more mutations (including, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) characteristic of a certain Omicron variant (e.g., one or more mutations of an Omicron variant listed in Table 1, e.g., each of the mutations associated with a given XBB, JN.1, KP.2, or XEC variant in the above Table 1).

[0376] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., an RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1), or a variant thereof, wherein the fragment of the S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, 64, or more) mutations associated with a SARS-CoV-2 variant (e.g., one or more mutations associated with a SARS-CoV- 2 variant listed in Table 1).

[0377] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., an RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1), or a variant thereof, wherein the fragment of the S protein comprises at least 5% (e.g., at least 5%, 10%, 155, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the mutations associated with a given SARS-CoV-2 variant in the S protein fragment region. For example, in some embodiments, a construct comprises at least 5 % of the mutations associated a SARS-CoV-2 variant in the region corresponding to amino acids 1-528 of the S protein.

[0378] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., an RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1), wherein the fragment of the S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, 64, or more) mutations associated with a SARS-CoV-2 variant.

[0379] In some embodiments, RNA described herein encodes an immunogenic fragment of a SARS-CoV-2 S protein or a variant thereof comprising one or more mutations characteristic of a JN.1, JN.1.2, JN.1.6, KP.2, KP.3, XEC and / or JN.1.7 variant (e.g., one or more mutations described herein). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 455 of SEQ ID NO: 1 (e.g., L455S). In some embodiments, the one or more mutations include mutations at a position corresponding to position 455 of SEQ ID NO: 1 (e.g., L455F). In some embodiments, the one or more mutations include a mutation at a position corresponding position 456 of SEQ ID NO: 1 (e.g., F456L). In some embodiments, the one or more mutations include mutations at positions corresponding positions 455 and 456 of SEQ ID NO: 1 (e.g., F456L and L455F). In some embodiments, the one or mutations include a mutation at a position corresponding to position 346 of SEQ ID NO: 1 (e.g., R346T). In some embodiments, the one or more mutations include a mutation at a positioncorresponding to position 1104 of SEQ ID NO: 1 (e.g., V1104L). In some embodiments, the one or more mutations include mutations at positions corresponding to positions 346 and 1104 of SEQ ID NO: 1 (e.g., R346T and V1104L).

[0380] In some embodiments, one or more mutations characteristic of a KP.2 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, --213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L, where mutations are indicated relative to SEQ ID NO: 1. In some embodiments, one or mutations characteristic of a KP.2 variant include R346T and V1104L, where positions are indicated relative to SEQ ID NO: 1. In some embodiments, one or mutations characteristic of a KP.2 variant include R346T, F456L, and / or V1104L, where positions are indicated relative to SEQ ID NO: 1.

[0381] In some embodiments, one or more mutations characteristic of a KP.3 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, - D405N, R408S E484K, F486P, Q493E, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L, or any combination thereof. In some embodiments one or more mutations characteristic of a KP.3 variant include F456L, Q493E, and / or V1104L, wherein positions are indicated relative to SEQ ID NO: 1.

[0382] In some embodiments, one or more mutations characteristic of a XEC variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, --70, V127F,S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L, where mutations are indicated relative to SEQ ID NO: 1. In some embodiments one or more mutations characteristic of a XEC variant include T22N, F59S, F456L, Q493E, and / or V1104L, wherein positions are indicated relative to SEQ ID NO: 1.

[0383] In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, 64, 65, or 66 or more ofthe following list of mutations: - - , F157S,11, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456LN460K, S477N, T478K, N481K,S939F, Q954H, N969K, P1143L, or M1229I. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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 or more of the following list ofmutations: - - , 11, L212I,V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L Q498R, N501Y, Y505H.

[0384] In some embodiments, one or more mutations characteristic of a JN.1 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61,62, 63 or more of (e.g., all of) ins16MPLF, - -D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or any combination thereof, where mutations are indicated relative to SEQ ID NO: 1. In someembodiments, one or mor -26, A27S,- K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L, relative to SEQ ID NO: 1. In some embodiments, one or mutations characteristic of a JN.1 variant include L455S.

[0385] In some embodiments, a fragment of an S protein (e.g., an RBD or a truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with a JN.2 variant. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following list of mutations: -- 11, L212I, V213G,L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, or more of the following list of mutations: -26, A27S, -11, L212I, V213G, L216F, H245N, A264D, I332V, G339H,R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, and Y505H.

[0386] In some embodiments, one or more mutations characteristic of a JN.1.2 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, --D405N F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and M1229I, where mutations are indicated relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a JN.1.2 variant include M1229I, where position is indicated relative to SEQ ID NO: 1.

[0387] In some embodiments, one or more mutations characteristic of a JN.1.6 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, --R403K, D405N, R408S, K417N, N4 E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L, where mutations are indicated relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a JN.1.6 variant include R346T.

[0388] In some embodiments, one or more mutations characteristic of a JN.1.7 variant include one or more of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 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, 61, -26, A27S, - F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and E1150D, where mutations are indicated relative to SEQ ID NO: 1.

[0389] In some embodiments, a fragment of an S protein (e.g., an RBD or a truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with a JN.6 variant. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following list of mutations: --L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and E1150D. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more of the following list of mutations: -26, A27S, - K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S,N .

[0390] In some embodiments, a fragment of an S protein (e.g., an RBD or a truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with a JN.6 variant. In someembodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, 61, 62, 63, or more of the following list of mutations: --L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 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, or more of the following list of mutations: - - G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, and Y505H.

[0391] In some embodiments, a fragment of an S protein (e.g., an RBD or a truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with an XBB.1.5 variant. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or more) of - 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, N969K. In some embodiments, a fragment of an S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or more) of the following list of - S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, and Y505H.

[0392] In some embodiments, an S protein encoded by an RNA molecule comprises a majority of mutations associated with a KP.2, KP.3, or XEC variant and one or more additional mutations. In some embodiments an S protein comprises 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, 61, 62, 63 or more of (e.g., all of) mutations associated with a KP.2, KP.3, or XEC variant (e.g., mutations provided herein) and one or more additional mutations. In some embodiments, an S protein comprises a majority of mutations associated with a JN.1 variant (e.g., as described herein) and one or more additional mutations. In some embodiments, an S protein comprises a majority of mutations associated with a JN.1 variant (e.g., as described herein) and one or more additional mutations associated with a descendent of a JN.1 variant (e.g., a descendant described herein). In some embodiments, an S protein comprises a majority of mutations associated with a JN.1 variant (e.g., as described herein) and one or more additional mutations associated with increased spread of a descendent of a JN.1 variant (e.g., a descendant described herein).

[0393] In some embodiments, an S protein comprises a majority of mutations associated with a JN.1 variant (e.g., as described herein) and: (a) a mutation at a position corresponding to position 346 of SEQ ID NO: 1 (e.g., R346T); (b) a mutation at a position corresponding to position 456 of SEQ ID NO: 1 (e.g., F456L);(c) mutations at positions corresponding to positions 455 and 456 of SEQ ID NO: 1 (e.g., L455S and F456L); (d) a mutation at a position corresponding to position 1104 of SEQ ID NO: 1 (e.g., V1104L).

[0394] In some embodiments, an S protein comprises a majority of mutations associated with a KP.2 and / or JN.1 variant and one or more additional mutations. In some embodiments, an S protein comprises a majority of mutations associated with a KP.2 and / or JN.1 variant and a mutation at a position corresponding to position 493 of SEQ ID NO: 1 (e.g., Q493E).

[0395] In some embodiments, an S protein of a XEC variant includes a majority of mutations associated with a KP.2, KP.3 and / or JN.1 variant and one or more additional mutations. In some embodiments, an S protein comprises a majority of mutations associated with a KP.2, KP.3 and / or JN.1 variant and a mutation at a position corresponding to position 22 of SEQ ID NO: 1 (e.g., T22N). In some embodiments, an S protein comprises a majority of mutations associated with a KP.2, KP.3 and / or JN.1 variant and a mutation at a position corresponding to position 592 of SEQ ID NO: 1 (e.g., F592).

[0396] As used herein, ever change in an amino acid is counted as a single mutation. For example, ins16MPLF would be counted as 4 mutations. Immunogenic Portions of SARS-CoV-2 S Protein

[0397] As noted elsewhere in the present disclosure, in some embodiments, compositions described herein deliver an immunogenic portion of a full-length SARS-CoV-2 S protein. As used herein, “immunogenic portion of a full-length SARS-CoV-2 S protein” is synonymous with an “immunogenic fragment of a full length SARS-CoV-2 S protein).

[0398] An immunogenic portion of a SARS-CoV-2 S protein lacks certain features that are present in the full- length polypeptide. In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein lacks regions of the S protein other than the RBD, the NTD, or the NTD and RBD (where the secretory signal peptide present in the NTD is optionally replaced with a heterologous secretory signal peptide). For example, in some embodiments, an immunogenic portion of a SARS-CoV-2 S protein lacks a full S2 domain. In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein lacks the entire S2 domain. In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein lacks a full S2 domain, but comprises certain sequences that can improve immunogenicity and / or stability of an immunogenic portion (e.g., in some embodiments, an immunogenic portion lacks a full S2 domain but retains a TM sequence and optionally a sequence that is endogenously C-terminally adjacent to the TM sequence in a SARS-CoV-2 S protein).

[0399] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein comprises an N-terminal domain (NTD) of the S protein. In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein comprises a receptor binding domain (RBD) of the S protein. In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein comprises an S1 domain of the S protein or a truncated S1 subdomain or a variant thereof.

[0400] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein comprises an RBD and an NTD and omits other features of the S1 domain, optionally wherein the endogenous secretory signal peptide in the NTD is replaced with a heterologous secretory signal peptide.

[0401] SARS-CoV-2 S proteins are well characterized, and a person of skill in the art will be able to determine which portions of an S protein sequence correspond to immunogenic portions discussed herein (e.g., which portions of an S protein sequence correspond to the NTD, the RBD, the S1, and the S2 domains).

[0402] In some embodiments, an RBD comprises a portion of SEQ ID NO: 1 corresponding to the amino acid sequence between (i) about amino acid 317 to about amino acid 330 (inclusive) of SEQ ID NO: 1, and (ii) about amino acid 528 and about amino acid 541 (inclusive) of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, the N-terminal residue of an RBD corresponds to about position 317 in SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS-CoV-2 variant. In some embodiments, the N- terminal residue of an RBD corresponds to about position 327 in SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS-CoV-2 variant. In some embodiments, the N-terminal residue of an RBD corresponds to about position 330 of SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an RBD corresponds to about position 528 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an RBD corresponds to about position 541 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments the N-terminal residue of an RBD corresponds to an amino acid between amino acids 317 and 330 (inclusive) of SEQ ID NO: 1 and the C-terminal residue of an RBD corresponds to an amino acid between amino acids 528 and 541 (inclusive) of SEQ ID NO: 1. In some embodiments, an RBD of a SARS-CoV-2 S protein comprises residues 327 to 528 of SEQ ID NO: 1, residues 330 to 528 of SEQ ID NO: 1, residues 330 to 541 of SEQ ID NO: 1, or residues 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.

[0403] In some embodiments, an RBD of SARS-CoV-2 comprises the amino acid sequence:VRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQ TGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTY GVGHQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 3), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0404] In some embodiments, an RBD of SARS-CoV-2 comprises the amino acid sequence: PNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNI ADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGH QPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 325), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0405] TGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN GVGYQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 4), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0406] In some embodiments, an RBD of SARS-CoV-2 comprises the amino acid sequence: VRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQ TGNIADYNYKLPDDFTGCVIAWNSNKLDSK(SEQ ID NO: 326), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0407] In some embodiments, an immunogenic fragment of a SARS-CoV-2 S protein comprises an N-terminal domain (“NTD”) polypeptide (i.e., a polypeptide that includes a spike protein NTD polypeptide, an immunogenic fragment thereof, or a variant thereof, e.g., as described herein). In some embodiments, an NTD lacks an endogenous SARS-CoV-2 secretory signal peptide, and a polypeptide instead comprises a heterologous secretory signal peptide (i.e., a secretory signal peptide that is not from a SARS-CoV-2 S protein). As used herein, an NTD encompasses both domains comprising a SARS-CoV-2 S protein secretory signal peptide, and domains in which the SARS-CoV-2 S protein secretory signal peptide has been replaced by a heterologous secretory signal peptide.

[0408] In some embodiments, an NTD comprises a portion of SEQ ID NO: 1 corresponding to the amino acid sequence between about amino acid 1 and about amino acid 20 of SEQ ID NO: 1 and about amino acid 302 to 318 (inclusive) of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, the N-terminal residue of an RBD corresponds to about position 317 in SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS-CoV-2 variant. In some embodiments, the N-terminal residue of an NTD corresponds to about position 1 in SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS- CoV-2 variant. In some embodiments, the N-terminal residue of an NTD corresponds to about position 14 of SEQ ID NO: 1, or a corresponding amino acid in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an NTD corresponds to about position 17 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an NTD corresponds to about position 20 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments, the C- terminal residue of an NTD corresponds to about position 302 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an NTD corresponds to about position 303 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments, the C-terminal residue of an NTD corresponds to about position 318 of SEQ ID NO: 1, or a corresponding residue in an S protein of a SARS-CoV-2 variant. In some embodiments the N-terminal residue of an NTD corresponds to an amino acid between amino acids 1 and 20 (inclusive) of SEQ ID NO: 1 and the C-terminal residue of an NTD corresponds to an amino acid between amino acids 302-318 (inclusive) of SEQ ID NO: 1. In some embodiments, an NTD of a SARS-CoV-2 S protein comprises residues 1 to 302 of SEQ ID NO: 1, residues 1 to 302 of SEQ ID NO: 1, residues 1 to 303 of SEQ ID NO: 1, residues 14 to 318 of SEQ ID NO: 1, residues 14 to 302 of SEQ ID NO: 1, residues 14 to 302 of SEQ ID NO: 1, residues 14 to 303 of SEQ ID NO: 1, residues 14 to 318 of SEQ ID NO: 1, residues 17 to 302 of SEQ ID NO: 1, residues 17 to 302 of SEQ ID NO: 1, residues 17 to 303 of SEQ ID NO: 1, residues 17 to 318 of SEQ ID NO: 1, residues 20 to 302 of SEQ ID NO: 1, residues 20 to 302 of SEQ ID NO: 1, residues 20 to 303 of SEQ ID NO: 1, or residues 20 to 318 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant.

[0409] In some embodiments, an NTD comprises a sequence corresponding to amino acids 14-209, 14-303, 20-318 or 20-302 of SEQ ID NO: 1, variants thereof, or immunogenic fragments or variants thereof.

[0410] In some embodiments, an RNA comprising a nucleotide sequence that encodes a polypeptide comprising an NTD polypeptide can be administered in combination with an RNA comprising a nucleotide sequence that encodes a polypeptide comprising an RBD polypeptide (e.g., as described herein). For example, in some embodiments, one or more RNAs encoding a polypeptide comprising an RBD polypeptide can be administered in combination with one or more RNAs encoding a polypeptide comprising an NTD polypeptide, wherein the one ormore RNAs encoding a polypeptide comprising an RBD polypeptide and the one or more RNAs encoding a polypeptide comprising an NTD polypeptide can be formulated in the same or separate nanoparticles.

[0411] In some embodiments, an NTD polypeptide comprises a sequence provided in Table LXIV, below, a variant thereof, or an immunogenic fragment thereof. In some embodiments, an RNA construct encodes a polypeptide comprising an NTD polypeptide that is depicted in Figure 6. Table LXIV: Exemplary NTD sequences( Q : )

[0412] In some embodiments, an NTD of SARS-CoV-2 comprises SEQ ID NO: 643, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0413] In some embodiments, an NTD of SARS-CoV-2 comprises SEQ ID NO: 644, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0414] In some embodiments, an NTD of SARS-CoV-2 comprises SEQ ID NO: 645, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0415] In some embodiments, an NTD of SARS-CoV-2 comprises SEQ ID NO: 646, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0416] In some embodiments, an S1 domain of a SARS-CoV-2 S protein comprises amino acids 1 to 678 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an S1 domain of a SARS-CoV-2 S protein comprises amino acids 1 to 683 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an S1 domain of a SARS-CoV-2 S protein comprises amino acids 1 to 685 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant.

[0417] In some embodiments, an S1 domain of a SARS-CoV-2 S protein comprises the amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVY FASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKEGNFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGY LQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRI SNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPS GNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVP VAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 5), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0418] In some embodiments, an S1 domain of a SARS-CoV-2 S protein comprises the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAY YVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWN RKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKK STNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVN CTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 6), or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0419] In some embodiments, an immunogenic portion or fragment of a SARS-CoV-2 S protein comprises a truncated S1 subdomain or a variant thereof. As used herein, a “truncated S1 subdomain” refers to a polypeptide comprising the NTD and the RBD of the S1 domain, where the NTD and RBD are connected via an endogenous linker, and wherein at least one amino acid C-terminal to the RBD in the S1 domain has been deleted. In some embodiments, a truncated S1 subdomain comprises an RBD at its C-terminus (e.g., an RBD as described herein). In some embodiments, a truncated S1 subdomain comprises an NTD and an RBD, wherein the RBD is at the C-terminus of the truncated S1 subdomain, and wherein the endogenous secretory signal peptide in the NTD is optionally replaced with a heterologous secretory signal peptide.

[0420] In some embodiments, a truncated S1 subdomain comprises or consists of amino acids 20 to 528 of SEQ ID NO: 1 (e.g., amino acids 14 to 528 or 17 to 528 of SEQ ID NO: 1), or a corresponding region in an S protein in a SARS-CoV-2 variant, or a variant of either of the foregoing. In some embodiments, a truncated S1 subdomain comprises or consists of amino acids 20 to 541 (e.g., amino acids 14 to 541 or 17 to 541) of SEQ ID NO: 1, or a corresponding region in a SARS-CoV-2 variant, or a variant of either of the foregoing.

[0421] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFND GVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAY

[0422] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence:( Q ), q , , , , 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0423] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence:at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0424] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence:YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN F, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0425] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acidWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0426] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG WIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLR EFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0427] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: TTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIF GTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFV FKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNS ASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0428] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: TTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIF GTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFV FKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNS ASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRK SNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF, or a sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0429] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLR E Y Lleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0430] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence:80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0431] In some embodiments, an S2 domain of a SARS-CoV-2 S protein comprises amino acids 679 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an S2 domain of a SARS-CoV-2 S protein comprises amino acids 684 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant. In some embodiments, an S2 domain of a SARS-CoV-2 S protein comprises amino acids 686 to 1273 of SEQ ID NO: 1, or a corresponding region in an S protein of a SARS-CoV-2 variant.

[0432] In some embodiments, compositions described herein deliver an immunogenic portion of an S protein of a SARS-CoV-2 variant. In some embodiments, the variant is a variant of concern (e.g., a variant that has been predicted to and / or has been shown to spread rapidly in a relevant jurisdiction, e.g., as identified by certain public health agencies, e.g., the Center for Disease Control and Prevention (CDC), Public Health England and the COVID-19 Genomics UK Consortium for the UK, the Canadian COVID Genomics Network (CanCOGeN), and / or the World Health Organization (WHO)). In some embodiments, a variant has been predicted to have a highly likelihood of becoming a variant of concern (e.g., using sequence-based algorithms that predict the ability of a variant to escape previously developed immune responses and / or measure the “fitness” of a given variant, such as described, e.g., in WO2022 / 235847 and WO2022 / 235853, the contents of each of which are incorporated by reference herein in their entirety). In some embodiments, a variant is a SARS-CoV-2 variant described herein, or a descendent thereof.

[0433] In some embodiments, an RBD comprises mutations associated with a variant described herein. A person of skill in the art will be able to identify which portions of a given variant correspond to immunogenic portions described herein.

[0434] In some embodiments, a polypeptide comprises two or more SARS-CoV-2 subdomains (e.g., two or more S1 domains, truncated S1 subdomains, or RBDs or variants thereof). In some embodiments, a polypeptide comprises two or more receptor binding domains linked in tandem, e.g., as described in Dai, Lianpan, et al. "A universal design of betacoronavirus vaccines against COVID-19, MERS, and SARS," Cell 182.3 (2020): 722-733, and Han, Yuxuan, et al. "mRNA vaccines expressing homo-prototype / Omicron and hetero-chimeric RBD-dimers against SARS-CoV-2," Cell Research 32.11 (2022): 1022-1025, the contents of each of which are incorporated by referenceherein in their entirety. In some embodiments, the two or more subdomains are from the same SARS-CoV-2 variant (e.g., a variant described herein). In some embodiments, at least two of the two or more subdomains are from different SARS-CoV-2 variants (e.g., from different variants of concern, different Omicron variants, an Omicron variant and a non-Omicron variant, or a Wuhan strain and an Omicron variant). Secretory Signals

[0435] In some embodiments, an antigen construct described herein includes a secretory signal, e.g., that is functional in mammalian cells. In some embodiments, a utilized secretory signal is a heterologous secretory signal (i.e., heterologous relative to a SARS-CoV-2 antigen present in the same polypeptide). In some embodiments, a utilized secretory signal is a homologous secretory signal (i.e., a secretory signal that is naturally found in the same protein as an antigen in the same polypeptide, including e.g., the N-terminal 16 or 19 amino acids of a SARS-CoV-2 S protein when attached to an antigen of a SARS-CoV-2 S protein). In some embodiments, a heterologous secretory signal comprises or consists of a non-human secretory signal. In some embodiments, a heterologous secretory signal comprises or consists of a viral secretory signal. In some embodiments, a viral secretory signal comprises or consists of an HSV secretory signal (e.g., an HSV-1 or HSV-2 secretory signal). As used, herein, reference to a “secretory signal” in the context of a polypeptide, is synonymous with a “secretory signal peptide.”

[0436] In some embodiments, a secretory signal comprises or consists of an Ebola virus secretory signal. In some embodiments, an Ebola virus secretory signal comprises or consists of an Ebola virus spike glycoprotein (SGP) secretory signal.

[0437] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.

[0438] In many embodiments, a secretory signal is positioned at the N-terminus of a SARS-CoV-2 antigen construct as described herein. In some embodiments, a secretory signal preferably allows transport of the SARS-CoV- 2 antigen construct with which it is associated into a defined cellular compartment, preferably a cell surface, endoplasmic reticulum (ER) or endosomal-lysosomal compartment.

[0439] In some embodiments, a secretory signal is selected from an S1S2 signal peptide (e.g., aa 1-16 or 1- 19), an immunoglobulin secretory signal (e.g., aa 1-22), an HSV-1 gD signal peptide (MGGAAARLGAVILFVVIVGLHGVRSKY; SEQ ID NO: 7), an HSV-2 gD signal peptide (MGRLTSGVGTAALLVVAVGLRVVCA; SEQ ID NO: 8); a human SPARC signal peptide, a human insulin isoform 1 signal, a human albumin signal peptide, etc. Those skilled in the art will be aware of other secretory signal such as, for example, as disclosed in WO2017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1-1115 and 1728, or fragments variants thereof).

[0440] In some embodiments, a SARS-CoV-2 antigen construct described herein does not comprise a secretory signal.

[0441] In certain embodiments, a signal peptide is an IgG signal peptide, such as an IgG kappa signal peptide.

[0442] In some embodiments, a SARS-CoV-2 secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal.

[0443] In some embodiments, a string construct sequence encodes an antigen that may comprise or otherwise be linked to a signal sequence (e.g., secretory signal), such as those listed in Table 2 or at least a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a secretory signal such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 9), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.

[0444] In some embodiments, a secretory signal is selected from a gI signal peptide. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGLVVR (SEQ ID NO: 10), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGL (SEQ ID NO: 11), or at least a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.

[0445] In some embodiments, an antigen comprises an affinity label (e.g., a short sequence that can bind to an affinity reagent, and which can be useful for, e.g., affinity purification of a protein). Suitable affinity labels are known in the art, and include e.g., a His tag (e.g., HHHHHHHH) and a StrepTag® (e.g., WSHPQFEK). In some embodiments, an affinity label can be included at the N-terminus of an antigen (e.g., adjacent to a secretory signal (to which it is optionally connected via a fleixible linker)).

[0446] In some embodiments, a secretory signal is one listed in Table 2 and / or Table 3, or a secretory signal having 1, 2, 3, 4, or 5 amino acid differences relative thereto (optionally, with corresponding changes in a corresponding nucleotide sequence provide in Table 3). In some embodiments, a secretory signal is selected from those included in the Table 2 below and / or those encoded by the sequences in Table 3 below.

[0447] In some embodiments, an RNA (e.g., mRNA) encodes a polypeptide comprising amino acids 20 to 292 of SEQ ID NO: 145 (or variants thereof) and a secretory signal from Table 2.Table 2: Exemplary secretory signalsBovine herpesvirus 1.1 (strain P8-2) MQGPTLAVLGALLAVAVS 370CD45 SP MTMYLWLKLLAFGFAFLDTEVFVTG 396 TGGACUGCAUGGAGUGAGAAGCAAGUACsignalF) GGCCUCCAUGGGGUCCGCGGC(straintrain A / Tern / AustTransmembrane Regions

[0448] In some embodiments, an RNA described herein encodes a membrane association element (e.g., a homologous or heterologous membrane association element). In some embodiments, a SARS-CoV-2 antigen construct as described herein includes a transmembrane region. In some embodiments, a utilized transmembrane domain is a heterologous transmembrane domain (i.e., heterologous relative to a SARS-CoV-2 antigen in the same polypeptide as the transmembrane doamin). In some embodiments, a utilized transmembrane domain is a homologous transmembrane domain (i.e., a transmembrane domain that is naturally found in the same protein as an antigen in the same polypeptide as the transmembrane domain, including e.g., a transmembrane domain of a SARS- CoV-2 S protein when attached to an antigen of a SARS-CoV-2 S protein). In some embodiments, a heterologous transmembrane domain comprises or consists of a non-human transmembrane domain. In some embodiments, a heterologous transmembrane domain comprises or consists of a viral transmembrane domain.

[0449] In some embodiments, a transmembrane region is located at the N-terminus of a SARS-CoV-2 construct (e.g., N-terminal to an immunogenic portion of a SARS-CoV-2 S protein and / or C-terminal to a secretory signal peptide). In some embodiments, a transmembrane region is located at the C-terminus of a SARS-CoV-2 construct (e.g., C-terminal to an immunogenic portion of a SARS-CoV-2 S protein, optionally, wherein, C-terminally adjacent tothe transmembrane domain, is an endogenous, membrane-adjacent sequence (e.g.., MTSCCSCLKGCCSCGSCC in the case of the SARS-CoV-2 transmembrane domain)). In some embodiments, a transmembrane region is not located at the N-terminus or C-terminus of a SARS-CoV-2 construct (i.e., is an internal sequence).

[0450] A coding sequence of a transmembrane element (in some embodiments a transmembrane domain) is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences of sequences (e.g., sequences encoding polypeptide(s)) with which it is to be linked.

[0451] In some embodiments, a transmembrane region comprises or is a transmembrane domain of a SARS- CoV-2 S protein or a transmembrane domain of a SARS-CoV-2 S protein and a C-terminal, membrane adjacent sequence (where the membrane adjacent sequence optionally has a C-terminal truncation (e.g., a 19 amino acid or 37 amino acid C-terminal truncation).

[0452] Transmembrane regions are known in the art, any of which can be utilized in a SARS-CoV-2 construct described herein. In some embodiments, a transmembrane region comprises or is a transmembrane domain of a SARS-CoV-2 S protein, a transmembrane domain of a SARS-CoV-2 S protein with a C-terminal truncation (e.g., a 19 amino acid C-terminal truncation). In some embodiments, a transmembrane domain is a heterologous transmembrane domain. As used herein, “heterologous” refers to an amino acid sequence or nucleotide sequence that is from a different species than the species of a disease specific antigen (e.g., viral antigen) that is within the same polypeptide or nucleotide sequence. For a polypeptide comprising a SARS-CoV-2 antigen (e.g., a truncated S1 subdomain) a heterologous sequence would be a sequence of non-SARS-CoV-2 origin. In some embodiments, a heterologous sequence is a viral sequence. In some embodiments, a heterologous transmembrane domain is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.

[0453] In some embodiments, a transmembrane domain is a heterologous transmembrane domain. In some embodiments, a heterologous transmembrane domain is a transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.

[0454] In some embodiments, a heterologous transmembrane region does not comprise a hemagglutinin transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a non-human transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of a viral transmembrane region. In some embodiments, a heterologous transmembrane region comprises or consists of an HSV transmembrane region, e.g., an HSV-1 or HSV-2 transmembrane region. In some embodiments, an HSV transmembrane region comprises or consists of an HSV gD transmembrane region, e.g., comprising or consisting of an amino acid sequence of GLIAGAVGGSLLAALVICGIVYWMRRHTQKAPKRIRLPHIR (SEQ ID NO: 90).

[0455] In some embodiments, a heterologous transmembrane region comprises or consists of a human transmembrane region. In some embodiments, a human transmembrane region comprises or consists of a human decay accelerating factor glycosylphosphatidylinositol (hDAF-GPI) anchor region. In some embodiments, an hDAF- GPI anchor region comprises or consists of an amino acid sequence of PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 92).

[0456] In some embodiments, a utilized transmembrane region is a heterologous transmembrane region.

[0457] In some embodiments, a construct described herein does not comprise a transmembrane region.

[0458] In some embodiments, a transmembrane domain includes a sequence that is membrane proximal and C-terminal to a membrane inserted portion of a transmembrane in its native protein. In some embodiments, a membrane proximal, C-terminal sequence is endogenously C-terminal to the transmembrane domain. In some embodiments, a construct described herein comprising a transmembrane domain comprises a membrane proximal, C-terminal sequence of a SARS-CoV-2 S protein (e.g., MTSCCSCLKGCCSCGSCC), e.g., C-terminal to the transmembrane domain.

[0459] In some embodiments, a transmembrane domain can induce multimerization (e.g., trimerization) and does not include a soluble trimerization domain (e.g., a T4 fibritin trimerization domain).

[0460] Among other things, the present disclosure provides an insight that a soluble multimerization domain (e.g., a multimerization domain that does not comprise a region inserted in the membrane) may not be required to induce an immune response to an immunogenic fragment of a SARS-CoV-2 S protein that is sufficient to provide protection from SARS-CoV-2 infection and / or COVID disease if a transmembrane region is included that can drive multimerization (e.g., trimerization) on its own. The present disclosure specifically provides an insight that not including a soluble multimerization (e.g., trimerization) can provide for certain advantages as compared to inclusion of a soluble multimerization domain. For example, omitting a multimerization domain can allow for a shorter RNA (e.g., by about 100 bp if a T4 fibritin trimerization domain is omitted), and in some embodiments such shorter RNA may show increased thermostability and / or potency (as measured by the immune response induced by a given mass of RNA) as compared with an appropriate reference RNA (e.g., an RNA that is otherwise identical except that it comprises a soluble multimerization domain). Without wishing to be being bound by theory, the present disclosure also provides the insight that a subject can mount an immune response against a solvent-exposed multimerization domain (i.e., against the multimerization domain itself, in addition to or instead of a provided antigen region). Thus, by using multimerization domains that are not solvent-exposed or which have reduced accessibility (e.g., a transmembrane domain), an immune response against a multimerization domain can be reduced and / or eliminated. The present disclosure also provides the surprising discovery that not including a T4 fibritin trimerization domain can increase expression in some embodiments, while not reducing immunogenicity. Furthermore, the present disclosure also demonstrates that excluding a T4 fibritin trimerization domain can improve polypeptide expression.

[0461] Transmembrane regions that can drive trimerization include, e.g., those described in Fu, Qingshan, and James J. Chou. "A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike," Journal of the American Chemical Society 143.23 (2021): 8543-8546, the contents of which are hereby incorporated by reference in their entirety. Examples of such transmembrane regions also include a SARS-CoV-2 transmembrane domain (e.g., a polypeptide comprising: a sequence corresponding to residues 1207 to 1236 of SEQ ID NO: 1; EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC; or fragments or variants thereof); a PIV5-F transmembrane region (e.g., a polypeptide comprising: a sequence corresponding to residues 480-517 of the PIV5-F polypeptide; a sequence corresponding to residues 485-517 of the PIV5-F polypeptide; ATTTSVLSIIAIALGSLGLILIILLSVVVWKTIVVA; or VLSIIAIALGSLGLILIILLSVVV; or a variant or fragment thereof); or a HeV-F transmembrane region (e.g., a polypeptide comprising a sequence corresponding to residues 479-526 of a HeV-F polypeptide; residues 484-521 of a HeV-F polypeptide; VLSIIAIALGSLGLILIILLSVVV; or ISMLSMIILYVLSIAALCIGLITFISFVIVEKK; variants thereof; or fragments thereof).

[0462] In some embodiments, adjacent to a transmembrane domain, is a C-terminal, membrane proximal region of a SARS-CoV-2 S protein (e.g., a polypeptide comprising a sequence that corresponds to amino acids 1237- 1245 of SEQ ID NO: 1; or MTSCCSCLKGCCSCGSCC or a corresponding region thereof of a SARS-CoV-2 variant). In some embodiments, a polypeptide comprises a short stretch of a membrane proximal sequence of the SARS-CoV-2 S protein (e.g., an amino acid sequence corresponding to residues 1209-1217 of SEQ ID NO: 1), C-terminal to (e.g., immediately C-terminal to, or connected by a linker to) a transmembrane domain (e.g., a transmembrane domain of a SARS-CoV-2 S protein). In some embodiments, a transmembrane region provided herein can induce a higher proportion of trimers as compared to an appropriate reference polypeptide (e.g., as compared to a polypeptide comprising a transmembrane region that cannot induce trimerization and / or a polypeptide described in Fu, Qingshan, and James J. Chou. "A trimeric hydrophobic zipper mediates the intramembrane assembly of SARS-CoV-2 spike," Journal of the American Chemical Society 143.23 (2021): 8543-8546), examples of which include SARS-CoV-2 transmembrane regions comprising mutations at one or more of I1221, I1225, L1229, and L1233 (e.g., I1225Y (e.g., EQYIKWPWYIWLGFIAGLYAIVMVTIMLCC) or L1229Y); and a VSV-G transmembrane region.

[0463] In some embodiments, a transmembrane domain is a VSV-G transmembrane domain. In some embodiments, a VSV-G transmembrane domain can provide the benefit of inducing formation of viral-like particles (VLP) when attached to immunogenic polypeptides, which can further improve immunogenicity of a delivered construct. In some embodiments, a VSV-G transmembrane domain optionally includes a short membrane proximal sequence of VSV-G. In some embodiments a VSV-G transmembrane domain include a short membrane proximal sequence (e.g., a signal portion of a membrane proximal active domain), include a polypeptide comprising: IELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK or FEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCI, variants thereof, or fragments thereof. In some embodiments, a polypeptide comprises a VSV-G transmembrane domain, optionally further comprising a soluble multimerization (e.g., a trimerization) domain. A VSV-G transmembrane domain comprising a short membrane-proximal sequence provides the advantage of being able to induce formation of a viral- like particle (VLP), which can increase valency and induce a stronger immune response against any antigen it is linked to. In some embodiments, a VSV-G polypeptide is at the C-terminus of a polypeptide (wherein, if the VSV-G polypeptide comprises a membrane-proximal sequence, the membrane-proximal sequence is optionally C-terminal to the VSV-G transmembrane region).

[0464] In some embodiments, a polypeptide comprises an endosomal sorting complex required for transport (ESCRT)- and ALG-2-interacting protein X (ALIX) binding region (collectively referred to as EABR), e.g., as described in Hoffmann, Magnus AG, et al. "ESCRT recruitment to SARS-CoV-2 spike induces virus-like particles that improve mRNA vaccines." Cell 186.11 (2023): 2380-2391, the contents of which are incorporated by reference herein in their entirety. In some embodiments, an EABR sequence comprises FNSSINNIHEMEIQLKDALEKNQQWLVYDQQREVYVKGLLAKIFELEKKTETAAHSLP. In some embodiments, a polypeptide comprising an EABR sequence also comprises an EPM sequence (e.g., ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSSPY). In some embodiments, an EABR sequence comprises LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ. In some embodiments, a polypeptide comprising an EABR sequence comprises a transmembrane domain (e.g., a transmembrane of a SARS- CoV-2 S protein (e.g., as described herein, optionally with a C-terminal proximal sequence). An exemplarypolypeptide comprising a SARS-CoV-2 antigen and an EABR sequence is shown in Figure 25, and two exemplary polypeptide sequences are provided in Table IV.

[0465] Exemplary RNA encoding polypeptides comprising transmembrane region that are capable of inducing trimerization, and which lack a soluble trimerization domain are provided in Tables V, VI, VII-X, and XII of the present disclosure. Exemplary RNA which encode polypeptides that comprise transmembrane region which cannot induce trimerization (or which result in a proportionally lower amount of trimers as compared to the constructs provided in Tables V, VI, VII-X, and XII of the present disclosure, e.g., about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 1%, or less trimer), are shown in Tables VII and XI of the present disclosure.

[0466] In some embodiments, a transmembrane region is one listed in Table 4 or a transmembrane region having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a transmembrane region is selected from those included in the Table 4 below, optionally encoded by a corresponding nucleotide sequence provided in Table 5 below. Table 4: Exemplary transmembrane regions (amino acid sequences)453 Vaccinia Virus H3L (279-305) LISFFGLFDINVIGLIVILFIMFMLIF7 n uenza S V SS V VS G S Table 5: Exemplary nucleotide sequences encoding transmembrane regionsUGUAGCUGCCUGAAGGGCUGUUGUAGCUGUGGCAGCUGCUGC Multimerization Regions

[0467] In some embodiments, a SARS-CoV-2 construct as described herein includes one or more multimerization regions (e.g., a heterologous multimerization region).

[0468] In some embodiments, a heterologous multimerization region comprises a dimerization, trimerization or tetramerization region.

[0469] In some embodiments, a multimerization region is one described in WO2017 / 081082, which is incorporated herein by reference in its entirety (e.g., SEQ ID NOs: 1116-1167, or fragments or variants thereof).Exemplary trimerization and tetramerization regions include, but are not limited to, engineered leucine zippers, fibritin trimerization domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.

[0470] In some embodiments, a provided SARS-CoV-2 construct described herein is able to form a trimeric complex. For example, a provided SARS-CoV-2 construct may comprise a multimerization region allowing formation of a multimeric complex, such as for example a trimeric complex of a SARS-CoV-2 construct described herein. In some embodiments, a multimerization region allowing formation of a multimeric complex comprises a trimerization region, for example, a trimerization region described herein. In some embodiments, a SARS-CoV-2 construct includes a T4-fibritin-derived “foldon” trimerization region (also referred to herein as T4 fibritin trimerization domain or fibritin domain for short), for example, to increase its immunogenicity. In some embodiments, a SARS-CoV-2 construct includes a multimerization region comprising or consisting of the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 95). In some embodiments, a SARS-CoV-2 construct includes a multimerization region comprising or consisting of the amino acid sequence(SEQ ID NO: 98).

[0471] In some embodiments, a multimerization domain is a soluble multimerization domain (i.e., does not comprise a transmembrane domain). In some embodiments, a transmembrane region is capable of inducing multimerization (e.g., trimerization). In some embodiments, a polypeptide described herein comprises a transmembrane domain that is capable of inducing multimerization (e.g., trimerization), and lacks a soluble trimerization domain. In some embodiments, a polypeptide described herein comprises a transmembrane region that is capable of inducing multimerization and also comprises a soluble trimerization domain. Inclusion of a multimerization domain can provide certain immunogenic fragments as compared to the same polypeptide lacking a multimerization domain. Such advantages include, e.g., increased structural stability of an antigen, an antigen with a structure that more closely matches the structure of an endogenous antigen, and / or improved induction of an immune response.

[0472] Among other things, the present disclosure provides approaches for producing higher order oligomers (e.g., oligomers comprising more than 3 SARS-CoV-2 antigens). In some embodiments, polypeptides described in here can induce highly multimeric oligomers (e.g., oligomers approximately 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 or more SARS-CoV-2 antigens), generate an improved immune response (e.g., as compared to a composition comprising the same amount of RNA (in terms of mass or moles)) encoding a polypeptide producing a monomer or an oligomer comprising fewer SARS-CoV-2 antigens), and / or can display antigenic polypeptides in a confirmation that is preferred for generating an immune response.

[0473] In some embodiments, an RNA can encode a polypeptide comprise two or more antigenic regions (e.g., two or more RBDs, three or more RBDs, four or more RBDs, or five or more RBDs), e.g., as described in Gao et al., the contents of which are incorporated by reference herein in their entirety.

[0474] In some embodiments, an RNA can encode two or more antigenic regions (e.g., can encode a polypeptide comprising two or more RBD polypeptides), and further comprise a multimerization domain (e.g., a T4 fibritin trimerization domain). For example, in some embodiments, a polypeptide provides two or more (e.g., 2, 3, or 4) antigen regions (e.g., RBD polypeptides) and a multimerization (e.g., trimerization) domain. In some embodiments, a polypeptide provided herein comprises two RBD polypeptides and a trimerization domain, such that an oligomer comprising six RBD polypeptides (a “trimer of dimers”, where each “dimer” refers to a polypeptide comprising two RBD polypeptides) can be formed. In some embodiments, an RNA encodes a polypeptide depicted in Figure 8.

[0475] In some embodiments, a multimerization can induce highly multivalent oligomers (e.g., oligomers comprising 10 or more antigenic polypeptides).

[0476] In some embodiments, a multimerization domain can induce multimerization of 10, 15, 20, 24, 25, 30, 35, 40, 45, 50, 55, 60, or more antigenic polypeptides.

[0477] In some embodiments, multimerization domains are selected so as to reduce an immune response generated against the multimerization domain itself. For example, in some embodiments, a relatively small multimerization domain is used (e.g., a multimerization domain comprising about 100 amino acids or less and / or a multimerization domain that comprises less than 20% of the total amino acids in a polypeptide). Additionally or alternatively, in some embodiments, a multimerization domain is used that forms an oligomer structure in which (i) the multimerization domain is not freely accessible in solution, and / or has reduced solvent accessibility as compared to a relevant comparator (e.g., other multimerization domains commonly used in commercial vaccines, e.g., a T4 fibritin trimerization domain); and / or (ii) the majority of antigen is freely accessible in solution and / or forms the majority of the solvent exposed surface area of an oligomer (e.g., 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more).

[0478] nanoscaffolded spike-RBD vaccine provides protection against SARS-CoV-2 with minimal anti-scaffold response," Vaccines 9.5 (2021): 431; Joyce, M. Gordon, et al. "SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity," Cell reports 37.12 (2021); Joyce, M. Gordon, et al. "A SARS-CoV-2 ferritin nanoparticle vaccine elicits protective immune responses in nonhuman primates," Science translational medicine 14.632 (2021): eabi5735; Wang, Chong, et al. "Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice." Antiviral research 140 (2017): 55-61; Johnston, Sara C., et al. "A SARS-CoV-2 spike ferritin nanoparticle vaccine is protective and promotes a strong immunological response in the cynomolgus macaque coronavirus disease 2019 (COVID-19) model," Vaccines 10.5 (2022): 717; Zhang, Baoshan, et al. "A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone," Scientific reports 10.1 (2020): 18149; Malhi, Harman, et al. "Immunization with a self-assembling nanoparticle vaccine displaying EBV gH / gL protects humanized mice against lethal viral challenge." Cell Reports Medicine 3.6 (2022); and WO2022043449A1, the contents of each of which is incorporated by reference herein in their entirety.

[0479] In some embodiments, an RNA (e.g., mRNA) construct described herein comprises a nucleotide sequence that encodes a multimerization domain listed in Table 14.

[0480] Table 14: Exemplary Multimerization Domains

[0481] In some embodiments, a multimerization domain comprises a ferritin domain (e.g., as described in Table II). In some embodiments a multimerization domain comprises a ferritin domain that has been modified to reduced autoimmunity (e.g., as described in Kanekiyo, Masaru, et al. "Rational design of an Epstein-Barr virus vaccine targeting the receptor-binding site," Cell 162.5 (2015): 1090-1100, the contents of which are incorporated by reference herein in their entirety). Linkers

[0482] In some embodiments, a SARS-CoV-2 construct described herein includes one or more linkers. In some embodiments, a linker is or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, or more amino acids. In some embodiments, a linker is or comprises no more than about 30, 25, 20, 15, 10 or fewer amino acids. In some embodiments, a linker comprises 5 to 30 amino acids. In some embodiments, a linker comprises 5 to 20 amino acids. In some embodiments, a linker comprises 10 to 20 amino acids. In some embodiments, a linker comprises about 5 amino acids. In some embodiments, a linker comprises about 10 amino acids. In some embodiments, a linker comprises about 15 amino acids. In some embodiments, a linker comprises about 20 amino acids.

[0483] In some embodiments, a linker is a flexible linker (e.g., a linker comprising one or more gly residues and one or more ser residues). In some embodiments, a linker is a rigid linker. In some embodiments, a linker is a helical linker. In some embodiments, a linker comprises a protease recognition site and can be cleaved by a protease. In some embodiments, said protease is one that is expressed on the surface of a human cell. One example of such a protease is a furin protease.

[0484] In some embodiments, a linker is a flexible linker. As used herein, a flexible linker in the context of a polypeptide refers to an amino acid sequence that connects two protein regions while allowing for movement andinteraction between the two protein regions. Flexible linkers often comprise small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which allow for linker flexibility.

[0485] A linker can include any amino acid sequence and is not limited to any particular amino acids. In some embodiments, a linker comprises one or more glycine (G) amino acids and / or one or more serine (S) amino acids. In some embodiments, a linker includes amino acids selected based on a cleavage predictor to generate highly- cleavable linkers.

[0486] In some embodiments, a linker described herein is a rigid linker. In some embodiments, a rigid linker comprises one or more Pro residues. In some embodiments, a rigid linker comprises one or more repeats of a PA motif, optionally where the PA motif is repeated to a reach a total length of 10-40 amino acids, 10-30 amino acids, 10-20 amino acids, 25-30 amino acids, about 5 amino acids, about 10 amino acids, or about 20 amino acids.

[0487] In some embodiments, a linker described herein is a helical linker. In some embodiments, a helical linker comprises an amino acid sequence of Aor AKA, or sequences comprising 1, 2, 3, 4, or 5 amino acid modifications thereto.

[0488] In some embodiments, a linker is or comprises S-G4-S-G4-S (SEQ ID NO: 99). In some embodiments, a linker is or comprises GSPGSGSGS (SEQ ID NO: 100). In some embodiments, a linker is or comprises GGSGGGGSGG (SEQ ID NO: 101). In some embodiments, a linker is or comprises GSGSGS (SEQ ID NO: 102). In some embodiments, a linker is one presented in Table 5. In some embodiments, a linker is or comprises a sequence as set forth in WO2017 / 081082, which is incorporated herein by reference in its entirety (see SEQ ID NOs: 1509-1565, or a fragment or variant thereof).

[0489] In some embodiments, a SARS-CoV-2 construct described herein comprises a linker between a C- terminal region or fragment thereof and a transmembrane region.

[0490] Exemplary linkers are provided in the following Table 5: Table 5: Exemplary linkersGSGGS 476RNA Encoding T Cell Epitopes

[0491] In some embodiments, an RNA (e.g., mRNA) construct encodes a polypeptide comprising (i) an RBD, truncated S1 domain, and / or an NTD, or a variant thereof, and (ii) one or more T-cell epitopes of a SARS-CoV-2 virus (e.g., as depicted in Figure 7).

[0492] In some embodiments, the one or more T-cell epitopes are derived from a SARS-CoV-2 protein that is not a SARS-CoV-2 S protein. In some embodiments, the one or more T-cell epitopes are derived from one or more SARS-CoV-2 proteins that are not a SARS-CoV-2 S protein and which have previously been shown to be conserved and / or comprise a number of T cell epitopes.

[0493] In some embodiments, a polypeptide comprises one or more peptides (e.g., peptides comprising less than 50, 40, 30, 20, 10, or about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids) that have previously been shown to comprise a T cell epitope.

[0494] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and one or more of a SARS-CoV-2 nucleocapsid (N) protein, NS9b protein, membrane (M) protein, ORF1ab protein, ORF3a protein, ORF9b protein, or NSP1-4, or any combination thereof, or a fragment thereof that has previously been shown to include a number of T cell epitopes.

[0495] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant of thereof and a SARS-CoV-2 nucleocapsid (N) protein or a region thereof that contains a number of T cell epitopes.

[0496] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 NS9b protein or a region thereof that contains a number of T cell epitopes.

[0497] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 membrane (M) protein or a region thereof that contains a number of T cell epitopes.

[0498] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 ORF1ab protein or a region thereof that contains a number of T cell epitopes.

[0499] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 ORF3a protein or a region thereof that contains a number of T cell epitopes.

[0500] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 ORF9b protein or a region thereof that contains a number of T cell epitopes.

[0501] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and a SARS-CoV-2 NSP1-4 protein or a region thereof that contains a number of T cell epitopes.

[0502] In some embodiments, a polypeptide comprises an NTD, RBD, and / or truncated S1 subdomain or a variant thereof and: (i) an N protein, one or more regions thereof, and / or one or more T cell epitopes thereof and an NS9b protein, one or more regions thereof, and / or one or more T cell epitopes thereof; (ii) an N protein, one or more regions thereof, and / or one or more T cell epitopes thereof and an M protein, one or more regions thereof, and / or one or more T cell epitopes thereof; (iii) an N protein, an M protein, and an NS9b protein, or one or more regions, and / or one or more T cell epitopes of any of the foregoing; (iv) an M protein, NSP2, NSP3, NSP1, and an N protein, or one or more regions, and / or one or more T cell epitopes of any of the foregoing; (v) NSP2, NSP1, NSP3, N protein, NSP4, and an M protein, or one or more regions, and / or one or more T cell epitopes of any of the foregoing; (vi) an N protein, NSP1, NSP2, NSP3, and NSP4, or one or more regions, and / or one or more T cell epitopes of any of the foregoing.

[0503] In some embodiments, the one or more regions of a SARS-CoV-2 protein that is not a SARS-CoV-2 protein and comprises a number of T cell epitopes, and / or the one or more peptides comprising one or more T-cell epitopes are one of those described in WO2021188969 or WO2023049272A1, the entire contents of both of which are incorporated by reference herein in their entirety. In some embodiments, an RNA (e.g., mRNA) encodes a polypeptide comprising (i) an RBD, NTD, and / or a truncated S1 subdomain (e.g., a polypeptide described herein, with or without any transmembrane domain or C-terminal domain that may be present), and (ii) a polypeptide described in WO2021188969 (e.g., a polypeptide comprising any one of SEQ ID NOs: RS C1p1full, RS C2p1full, RS C3p1full, RS C4p1full, RS C5p1full, RS C5p2, RS C5p2full, RS C6p1full, RS C6p2full, RS C6p2, RS C7p1full, RS C7p2, RS C8p1full, RS C8p2, or RS C8p2full of WO2021 / 188969).

[0504] Exemplary designs for polypeptides comprising an RBD polypeptide attached to additional T-cell antigens are shown in Figure 7. Without wishing to be bound by theory, an RNA (e.g., mRNA) construct encoding a polypeptide that comprises (i) an RBD and / or NTD, and (ii) one or more additional T-cell epitopes can provide certain advantages as compared to a composition that comprises the RBD and / or NTD and the one or more additional T-cell epitopes in separate RNA (e.g., mRNA) constructs. In particular, providing a single RNA (e.g., mRNA) can reduce thenumber of RNA (e.g., mRNA) constructs that need to be administered to a subject, which reduces the dose of RNA to be administered, which can (i) reduce unwanted side effects, (ii) allow for a higher dose of antigen (i.e., the number of polypeptides comprising an RBD and / or NTD and one or more additional T-cell epitopes delivered to a subject is higher), and / or (iii) allow for the inclusion of additional RNA (e.g., mRNA) constructs in a composition (e.g., RNA (e.g., mRNA) constructs encoding antigens of additional diseases (e.g., non-SARS-CoV-2 diseases, respiratory disease, influenza, RSV, etc.)). Polyribonucleotides

[0505] In many embodiments, provided pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) deliver antigens as described herein by delivering a nucleic acid construct, e.g., in many embodiments, an RNA that encodes one or more antigens as described herein and is expressed in the subject upon administration of the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine).

[0506] Among other things, the present disclosure encompasses the recognition that administration of nucleic acid, and particularly of RNA to achieve delivery (e.g., by expression) of encoded antigen can provide a variety of benefits relative to other strategies for immunizing against an SARS-CoV-2 infection.

[0507] Among other things, the present disclosure provides an insight that RNA may be particularly useful and / or effective as an active agent in pharmaceutical compositions (e.g., immunogenic compositions, e.g., SARS- CoV-2 vaccines) for a variety of reasons including specifically that RNA can have intrinsic adjuvanticity. As noted herein, ability to induce very high antibody titers to SARS-CoV-2 proteins, e.g., particularly SARS-CoV-2 antigens associated with a variant of concern with high immune escape potential.

[0508] Still further, experience with SARS-CoV-2 vaccines has demonstrated that RNA actives, can also elicit significant and diverse T cell responses which, particularly when combined with strong antibody response, represents a combination of immune characteristics thought to potentially maximize the probability of protection. Exemplary Polyribonucleotides Features

[0509] Polyribonucleotides described herein encode one or more SARS-CoV-2 constructs described herein. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence...

Claims

CLAIMS What is claimed is:

1. A ribonucleic acid (RNA) comprising a nucleotide sequence encoding a polypeptide comprising: (i) a truncated S1 subdomain of a SARS-CoV-2 Spike (S) protein or a variant thereof; (ii) a heterologous secretory signal peptide; and (iii) a homologous transmembrane domain, optionally wherein the N-terminal to C-terminal orientation of the polypeptide is (secretory signal peptide)- (truncated S1 subdomain)-(transmembrane domain).

2. The RNA of claim 1, wherein the truncated S1 domain comprises amino acids 20-528 of SEQ ID NO: 1, amino acids 14-528 of SEQ ID NO: 1, amino acids 17-528 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.

3. The RNA of claim 1 or 2, wherein the secretory signal peptide comprises: (i) an amino acid sequence that is at least 70% identical to MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 22); (ii) an amino acid sequence of that is at least 70% identical to MFLLLRFVLVSCIIGSLG (SEQ ID NO: 391); (iii) an amino acid sequence that is at least 70% identical to MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12); (iv) an amino acid sequence that is at least 70% identical to MHQGAPSWGRRWFVVWALLGLTLGVLVASAAP (SEQ ID NO: 38); or (v) an amino acid sequence that is at least 70% identical to MARGAGLVFFVGVWVVSCLA (SEQ ID NO: 366).

4. The RNA of any one of claims 1-3, wherein the polypeptide comprises: (i) an amino acid sequence that I at least 70% identical to SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC); or (ii) an amino acid sequence that is at least 70% identical to SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT).

5. The RNA of any one of claims 1-4, wherein the polypeptide does not comprise a soluble multimerization domain, optionally wherein the soluble multimerization domain is a trimerization domain, further optionally wherein the multimerization domain is a T4 fibritin trimerization domain.

6. The RNA of any one of claims 1-5, wherein the truncated S1 subdomain or variant thereof and the transmembrane domain are connected to one another by a flexible linker.

7. The RNA of claim 6, wherein the flexible linker comprises a (G4S)2, (G4S)3, or (G4S)4sequence.

8. The RNA of any one of claims 1-7, wherein the RNA comprises (i) a sequence that is at least 70% identical to SEQ ID NO: 236; (ii) a nucleotide sequence that is at least 70% identical to SEQ ID NO: 238; and / or (iii) a nucleotide sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

235.

9. The RNA of any one of claims 1-7, wherein the RNA comprises (i) a sequence that is at least 70%identical to SEQ ID NO: 328 (ii) a sequence that is at least 70%, identical to SEQ ID NO: 330; and / or (iii) a nucleotide sequence that encodes a polypeptide that is at least 70% identical to SEQ ID NO:

327.

10. The RNA of any one of claims 1-9, wherein the truncated S1 subdomain or variant thereof comprises one or more amino acid mutations of a SARS-CoV-2 variant, optionally wherein the truncated S1 domain or variant thereof comprises one or more mutations associated with a variant listed in Table 1 (such as JN.1, KP.2, or XEC variant, or a descendent thereof).

11. The RNA of any one of claims 1-10, wherein the polypeptide further comprises one or more T cell epitopes from a SARS-CoV-2 protein that is not an S protein, optionally wherein the polypeptide comprises one or more T cell epitopes from a SARS-CoV-2 nucleocapsid (N) protein, NS9b protein, membrane (M) protein, ORF1ab protein, ORF3a protein, ORF9b protein, or NSP1-4, or any combination thereof.

12. The RNA of claim 11, wherein the polypeptide comprises: (i) one or more T cell epitopes from an N protein and one or more T cell epitopes from an NS9b protein; (ii) one or more T cell epitopes from an N protein and one or more T cell epitopes from an M protein; (iii) one or more T cell epitopes from an N protein, one or more T cell epitopes from an M protein; and one or more T cell epitopes from an NS9b protein; (iv) one or more T cell epitopes from an M protein, one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP3, one or more T cell epitopes from NSP1, and one or more T cell epitopes from an N protein; (v) one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP1, one or more T cell epitopes from NSP3, one or more T cell epitopes from an N protein, one or more T cell epitopes from NSP4, and one or more T cell epitopes from an M protein; (vi) one or more T cell epitopes from an N protein, one or more T cell epitopes from NSP1, one or more T cell epitopes from NSP2, one or more T cell epitopes from NSP3, and one or more T cell epitopes from NSP4.

13. 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; optionally wherein(i) the 5’ cap comprises a Cap1 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).

14. The RNA of claim 13, wherein the 5’ cap comprising a Cap1 structure, and the Cap1 structure comprises m7(3’OMeG)(5')ppp(5')(2'OMeA1)pG2, wherein A1 is position +1 of the RNA, and G2 is position +2 of the RNA, optionally wherein the cap proximal sequence comprises A1and G2of the Cap1 structure, and a sequence comprising: A3N4N5at positions +3, +4 and +5 respectively of the RNA, wherein N4and N5are each independently selected from A, G, C, and U.

15. The RNA of claim 13 or 14, wherein the 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.

16. The RNA of any one of claims 13-15, wherein the 5’-UTR sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 112 or 113; and / or wherein the 3’-UTR sequence comprises a sequence that is at least 70%, identical to SEQ ID NO: 118, 647, or 648; and / or wherein the interrupted polyA tail sequence comprises a sequence that is at least 70% identical to SEQ ID NO:

114.

17. The RNA of any one of claims 13-16, wherein the sequence at the 5‘ end of the 3’UTR sequence is CUCGAG or GGAUCCGAU.

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

19. The RNA of any one of the preceding claims, wherein the RNA is unmodified RNA or wherein the RNA comprises one or modified uridines in place of one or more uridines, optionally wherein the RNA comprises a single modified uridine in place of each uridine, further optionally wherein the modified uridine is N1-methyl- pseudouridine.

20. The RNA of any one of the preceding claims, wherein the nucleotide sequence encoding the SARS- CoV-2 S protein is encoded by a sequence that is codon-optimized and / or which has a G / C content that is increased compared to a wild type coding sequence.

21. A composition comprising an RNA of any one of claims 1-20, optionally wherein the RNA is formulated in a nanoparticle, further optionally wherein the nanoparticle is a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome, or a polysaccharide nanoparticle.

22. The composition of claim 21, wherein the RNA is fully or partially encapsulated in the nanoparticle.

23. The composition of claim 21 or 22, further comprising a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose and / or an aqueous buffered solution, optionally wherein the aqueous buffered solution comprises one or more of Tris base, Tris HCl, NaCl, KCl, Na2HPO4, and KH2PO4, further optionally wherein the aqueous buffered solution comprises about 10 mM Tris buffer and about 10% sucrose.

24. A pharmaceutical composition comprising (i) an RNA of any one of claims 1-20 or a composition of any one of claims 21-23 and (ii) a pharmaceutically acceptable excipient.

25. The pharmaceutical composition of claim 24, 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.

26. The pharmaceutical composition of claim 24 or 25, formulated to provide a dose of about 100 μg or less of total RNA, about 90 μg, about 60 μg, about 30 μg, about 25 μg, about 20 μg, about 10 μg, about 6 μg, about 5 μg, or about 3 μg of total RNA.

27. A method comprising administering an RNA of any one of claims 1-20, a composition of any one of claims 21-23, or a pharmaceutical composition of any one of claims 24-26 to a subject.

28. The method of claim 27, wherein: (i) the subject is 12 years or older, and the method comprises administering about 20 μg, about 10 μg, or about 5 μg of the RNA, (ii) the subject is 5 years to less than 12 years old, and the method comprises administering about 6.6 μg, about 3.3 μg, or about 1.6 μg of the RNA, or (iii) the subject is 6 months to less than 5 years old, and the method comprises administering about about 2 μg, about 1 μg, or about 0.5 μg of the RNA.

29. The method of claim 27 or 28, wherein the composition is administered in a volume of about 200 μL to about 300 μL.

30. The method of any one of claims 27-29, wherein the method comprises administering a single dose of the RNA, composition, or pharmaceutical composition to the subject.

31. The method of any one of claims 27-29, wherein the method comprises administering two or moredoses of the RNA, composition, or pharmaceutical composition to the subject, optionally wherein the two doses are administered about 21 days apart.

32. The method of any one of claims 27-29, wherein RNA, composition, or pharmaceutical composition is administered three times to the subject, optionally wherein the first and the second dose are administered about 21 days apart, and the third dose is administered about 28 days after the second dose.

33. The method of any one of claims 27-32, further comprising 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.

34. The method of any one of claims 27-33, wherein the method results in induction of an immune response against SARS-CoV-2 in the subject, optionally wherein the immune response comprises production of antibodies directed against one or more SARS-CoV-2 viruses and / or a T cell response.

35. The method of any one of claims 27-34, 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, increase the change of experiencing an asymptomatic SARS-CoV-2 infection, and / or treating a SARS-CoV-2 infection.

36. An RNA of any one of claims 1-20, a composition of any one of claims 21-23, or a pharmaceutical composition of any one of claims 24-26, for use in inducing an immune response in a subject, optionally wherein the use comprises performing steps in accordance with the method of any one of claims 27-35.

37. Use of an RNA of any one of claims 1-20, a composition of any one of claims 21-23, or a pharmaceutical composition of any one of claims 24-26, for the manufacture of a medicament for inducing an immune response in a subject, optionally wherein the medicament is formulated to be administered to the subject in accordance with the method of any one of claims 27-35.

38. A method of manufacturing an RNA, comprising in vitro transcribing the RNA of any one of claims 1-27.

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