IMMUNOGENIC COMPOSITIONS AGAINST SARS-COV-2

Abstract

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

Description

Title of the invention: IMMUNOGENIC COMPOSITIONS AGAINST SARS-COV-2 Field of invention

[0001] The present disclosure relates to the field of RNA technologies (e.g., compositions and methods) that can be used to induce an immune response against SARS-CoV-2. Context

[0002] SARS-CoV-2 first emerged in 2019 and rapidly spread worldwide, resulting in millions of deaths. The development and approval of first-generation SARS-CoV-2 vaccines in less than 12 months was a major scientific breakthrough that helped save countless lives. Since these initial vaccines were developed, the SARS-CoV-2 disease landscape has advanced, with researchers now better understanding the SARS-CoV-2 virus, disease progression, and the immune responses elicited by infection and vaccines. At the same time, SARS-CoV-2 has continued to evolve, with thousands of variants emerging worldwide, many of which exhibit increased growth rates and immune escape potential compared to the initial SARS-CoV-2 strains. Summary

[0003] The present invention relates, among other things, to novel 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 the technologies provided herein is enhanced 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, for example, RNA vaccines formulated in LNPs that encode a prefusion-stabilized full-length S protein, such as Pfizer's Comimaty and Moderna's SpikeVax). The enhanced immune response provided by the technologies described herein may include, for example, increased neutralizing antibody titers against a SARS-CoV-2 variant and / or increased antibody titers against a broader range of SARS-CoV-2 variants.In some embodiments, the technologies provided herein are more potent than first-generation vaccines (e.g., they may 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, the RNA provided herein is . more stable (e.g., more thermostable) compared to first-generation SARS-CoV-2 vaccines, which may be useful, for example, in increasing the shelf life of pharmaceutical compositions, and / or eliminating the need to store and transport pharmaceutical compositions at ultra-cold temperatures.

[0004] The technologies provided herein include, among others, immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of preparing 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, comprising, for example, a truncated RBD, NTD, or S1 subdomain of a virus against SARS-CoV-2, or a variant of any of the foregoing.In some embodiments, the technologies provided herein may provide an enhanced immune response (e.g., higher neutralizing antibody titers, increased activation of naive B cells, and / or higher titers of antibodies recognizing a unique epitope) to a variant of concern, compared to, for example, a composition that delivers a full-length SARS-CoV-2 S protein.

[0005] The present invention provides, among other things, certain insights into the design of SARS-CoV-2 antigens that may result in significant improvements over current vaccines (e.g., vaccines delivering a full-length SARS-CoV-2 S protein). These advantages include, for example, 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 improved durability of antibody responses compared to current SARS-CoV-2 vaccines (e.g., compared to first-generation SARS-CoV-2 RNA vaccines that deliver a full-length spike protein (e.g., the BNT162b2 vaccine (Comimaty) and the mRNA1273 vaccine (SpikeVax)).

[0006] The present invention provides, among other things, indications of antigenic regions of the SARS-CoV-2 S protein that provide an enhanced immune response compared to the full-length S protein (e.g., increased neutralization titers and / or longer-lasting immune responses). The present invention provides, among other things, the indication that a truncated S1 subdomain that includes an endogenous sequence connecting the NTD and RBD regions can induce a response enhanced immune response compared to a full-length S protein or a polypeptide comprising an NTD and an RBD connected via a heterologous flexible linker.

[0007] Also provided are improved designs of polypeptides for delivery via RNA, including the identification of preferred domains (e.g., secretory signal peptides and transmembrane regions) for binding to SARS-CoV-2 antigens and configurations thereof that result in significantly enhanced antigen expression and immune responses. These improved designs include, for example, improved secretory signal peptides, transmembrane regions, multimerization domains, GS linkers, nucleotide sequences, and combinations and configurations thereof, each of which individually provides significant improvements over existing SARS-CoV-2 vaccines, and which can be combined to produce RNA with significantly enhanced antigen expression and / or immunogenicity.

[0008] In some embodiments, the compositions described herein may be used to induce a significantly enhanced immune response compared to a reference composition. In some embodiments, the reference composition comprises RNA encoding a full-length SARS-CoV-2 S protein, optionally comprising one or more mutations that stabilize prefusion confirmation.

[0009] In some embodiments, the enhanced immune response comprises an enhanced B cell response. In some embodiments, an enhanced 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 enhanced 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 enhanced 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% relative 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 limit of 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and an upper limit of 150%, 200%, 250%, 300%, 350%, or 400% relative to the number of B cells induced by an RNA composition delivering a . complete 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 1,000%, from about 10% to about 600%, from about 10% to about 500%, from 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%.

[0010] In some embodiments, an enhanced immune response includes increased titers of antibodies that can neutralize a virus against SARS-CoV-2. In some embodiments, the neutralizing antibody titers are increased by at least 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% relative to the number of B cells induced by an RNA composition delivering a full-length S protein. In some embodiments, neutralizing antibody titers are increased by an amount within a range having a lower limit of 10%, 20%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, and an upper limit of 150%, 200%, 250%, 300%, 350% or 400% relative to the number of B cells induced by an RNA composition delivering a full-length S protein.In some embodiments, neutralizing antibody titers 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, neutralizing antibody titers are increased by at least about 200%. In some embodiments, neutralizing antibody titers are increased by at least about 400%.

[0011] In some embodiments, the technologies provided herein may provide a dose-sparing effect (i.e., require a lesser amount of RNA than is necessary to produce a given immune response). For example, as demonstrated in the examples herein, in some embodiments, a composition described herein may provide a dose-sparing effect of at least about 2-fold. In some embodiments, a composition described herein may provide a dose-sparing effect of at least about 3-fold. In some embodiments, a composition described herein may provide a dose-sparing effect of at least about 4-fold. In some embodiments, a composition described herein may provide a dose-sparing effect of at least about 5-fold.

[0012] In some embodiments, a SARS-CoV-2 S protein variant (or an 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 part of a reference SARS-CoV-2 S protein).

[0013] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant does not comprise an S2 domain.

[0014] 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.

[0015] In some embodiments, an immunogenic portion of a SARS-CoV-2 S protein variant comprises or consists of the RBD.

[0016] 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, for example, the WHO or the CDC (Centers for Disease Control and Prevention)). In some embodiments, a SARS-CoV-2 variant is rapidly expanding in a region and / or has increased immune escape potential 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 against which a health organization has recommended providing seasonally updated vaccines.

[0017] In some embodiments, a SARS-CoV-2 S protein variant (or an immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant that has high immune escape potential (e.g., a variant of concern).

[0018] In some embodiments, a SARS-CoV-2 variant has been determined to have 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 dynamics simulations), and / or based on infection rates and / or growth rates.

[0019] In some embodiments, a SARS-CoV-2 variant with high immune escape potential is an Omicron variant.

[0020] 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.

[0021] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, 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 (sequence identification number): 1.

[0022] In some embodiments, one or more mutations associated with an RBD of XBB.1.5 are G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y and Y505H, wherein 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 with an XBB.1.5 SI domain are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, and P681H, wherein the positions of the mutation(s) are indicated relative to SEQ ID NO: 1.

[0024] In some embodiments, one or more mutations associated with an XBB.1.5 variant are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, wherein the positions of the mutation(s) are indicated with respect to SEQ ID NO: 1.

[0025] In some embodiments, one or more mutations associated with an SI XBB.1.5 are T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, where the positions of the one or more mutations are indicated relative to SEQ ID NO: 1.

[0026] 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.

[0027] 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: 5.

[0028] 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.

[0029] In some embodiments, a secretion signal is present in the N-terminal portion of a polypeptide (e.g., at the N-terminus).

[0030] 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.

[0031] 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.

[0032] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 9.

[0033] In some embodiments, a SARS-CoV-2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 16.

[0034] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 8.

[0035] In some embodiments, a gD2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 13

[0036] In some embodiments, a gDl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 12

[0037] In some embodiments, a gBl secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 37.

[0038] In some embodiments, a gC2 polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 35.

[0039] In some embodiments, a gl2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

[0040] In some embodiments, a gE2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 38.

[0041] In some embodiments, an EboZ secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 39.

[0042] In some embodiments, an HLA-DR secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 40.

[0043] In some embodiments, a variant of the SARS-CoV-2 S protein (or an immunogenic portion thereof) comprises a multimerization domain.

[0044] In some embodiments, a multimerization domain is located in the C-terminal region of a SARS-COV-2 variant protein or an immunogenic portion thereof (e.g., at the C terminus).

[0045] In some embodiments, a multimerization domain is a fibritin trimerization domain.

[0046] In some embodiments, a fibritin trimerization domain comprises a sequence that is at least 80% identical to SEQ ID NO: 95.

[0047] In some embodiments, a fibritin trimerization domain comprises a sequence that is at least 80% identical to SEQ ID NO: 96.

[0048] In some embodiments, a variant of the SARS-CoV-2 S protein (or an immunogenic portion thereof) comprises a transmembrane (TM) domain.

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

[0050] In some embodiments, a TM domain is a heterologous TM domain.

[0051] In some embodiments, a TM domain is present in the C- portion terminal of a polypeptide (e.g., at the C-terminus).

[0052] In some embodiments, a variant of the SARS-CoV-2 S protein (or an 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 the multimerization 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)).

[0053] In some embodiments, a TM domain is a SARS-CoV-2 S protein TM domain or an influenza TM domain.

[0054] In some embodiments, a TM domain of SARS-CoV-2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 89.

[0055] In some embodiments, a TM domain of SARS-CoV-2 comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 90.

[0056] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 120.

[0057] 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.

[0058] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 135.

[0059] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 145.

[0060] In some embodiments, an RNA comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 150.

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

[0062] In some embodiments, a nucleotide sequence that encodes a variant of the SARS-CoV-2 S protein (or an immunogenic portion thereof) is codon-optimized for expression in human subjects.

[0063] In some embodiments, a nucleotide sequence encoding a variant of the SARS-CoV-2 S protein (or an immunogenic portion thereof) has an enriched G / C content relative to the wild-type sequence.

[0064] In some embodiments, the 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%.

[0065] In some embodiments, an RNA comprises a heterologous 3' UTR or 5' UTR.

[0066] In some embodiments, a heterologous 5' UTR comprises or consists of a modified human alpha-globin 5'-UTR.

[0067] In some embodiments, a heterologous 3' UTR comprises or consists of a first sequence from the amino terminal enhancer of split messenger RNA (AES) and a second sequence from mitochondrially encoded 12S ribosomal RNA.

[0068] In some embodiments, an RNA comprises a poly(A) sequence.

[0069] In some embodiments, a poly(A) sequence has a length of about 100 to 150 nucleotides.

[0070] In some embodiments, a poly(A) sequence is a disrupted poly(A) sequence.

[0071] In some embodiments, an RNA comprises a 5' cap.

[0072] In some embodiments, an RNA comprises a sequence that is at less than 80% identical to SEQ ID NO: 122 or 124.

[0073] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 131 or 133.

[0074] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 136 or 138.

[0075] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 146 or 148.

[0076] In some embodiments, an RNA comprises a sequence that is at least 80% identical to SEQ ID NO: 151 or 153.

[0077] In some embodiments, an RNA is an unmodified RNA.

[0078] In some embodiments, an RNA comprises one or more nucleotides modified.

[0079] In some embodiments, a modified nucleotide is pseudouridine (e.g., Nl-methyl-pseudouridine).

[0080] In some embodiments, an RNA comprises a modified nucleotide in place of each uridine.

[0081] In some embodiments, an RNA is a self-amplifying RNA or a trans-amplifying RNA.

[0082] In some embodiments, a composition contains an RNA described herein, wherein the RNA is fully or partially encapsulated in lipid nanoparticles (LNPs), polyplexes (PLXs), lipidated polyplexes (LPLXs), oligo- or polysaccharide particles, or liposomes. In some embodiments, an RNA is fully or partially encapsulated in an LNP. In some embodiments, an LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a lipid conjugate.

[0083] In some embodiments, an LNP comprises from about 40 to about 50 mole percent of the cationically ionizable lipid; from about 5 to about 15 mole percent of the neutral lipid; from about 35 to about 45 mole percent of the sterol; and from about 1 to about 10 mole percent of the PEG lipid.

[0084] In some embodiments, the present invention provides a method of inducing an immune response, comprising administering an RNA described herein, or a composition described herein.

[0085] In some embodiments, an immune response is induced in a subject who has previously received one or more doses of one or more vaccines delivering a reference SARS-CoV-2 S protein.

[0086] In some embodiments, an immune response comprises a naive B cell immune response.

[0087] 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.

[0088] In some embodiments, a variant of the SARS-CoV-2 S protein or an immunogenic portion thereof comprises a sequence that corresponds to an immunogenic portion of a reference SARS-CoV-2 S protein.

[0089] In some embodiments, an RNA or composition is administered to a subject previously exposed to a SARS-CoV-2 antigen (e.g., by vaccination or by previous infection, where previous infection was determined, for example, by a positive PCR and / or antigen diagnostic test result).

[0090] In some embodiments, a reference SARS-CoV-2 S protein or immunogenic portion thereof is derived from a SARS-CoV-2 strain or variant that was previously prevalent or is currently prevalent in a relevant territory.

[0091] In some embodiments, the antigens described herein may 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 SI domains from two or more SARS-CoV-2 variants). For example, in some of these embodiments, mutations from one or more SARS-CoV-2 variants may be introduced into conserved epitopes of a S protein of the SARS-CoV-2 variant, or an immunogenic portion thereof (e.g., an SI domain or an RBD). Such engineering may be useful, for example, to remove 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, for example, in the U.S. Provisional Application titled “Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses,” filed on February 24, 2023, and assigned U.S. Provisional Application No. 63 / 448,215. Said application describes, among other things, technologies for the in silico design of customized and engineered antigens (e.g., comprising modified versions of SARS-CoV 2 variant proteins and parts thereof) to reduce the degree to which a memory immune response is triggered.

[0092] 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 327-528 of SEQ ID NO: 1, 330-528 of SEQ ID NO: 1, amino acids 327-528 of SEQ ID NO: 1, or amino acids 330-541 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 elements 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 (e.g., wherein the RBD comprises amino acids 327-528 of SEQ ID NO: 1, 330-528 of SEQ ID NO: 1, amino acids 327-528 of SEQ ID NO: 1, or amino acids 330-541 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 these from an S protein of a SARS-CoV-2 variant.

[0093] 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.

[0094] 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.

[0095] 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 or Table XXXI, or in [Fig. 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 in [Fig. 15] or an 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 to a sequence of Table XXXI listed in Table 3 and / or Table XXXI and / or in [Fig. 15].

[0096] In some embodiments, a polypeptide comprises a secretory signal peptide SP24-Q7PUJ5_ANOGA (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)).

[0097] In some embodiments, a polypeptide comprises a secretory signal peptide SP24-SP18-HEMA_CVBM (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).

[0098] In some embodiments, a polypeptide comprises a secretory signal peptide SP25-GD_HHV1K (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).

[0099] In some embodiments, a polypeptide comprises a secretion signal peptide SP32-GBD_HHV1K (e.g., a secretion signal peptide comprising the amino acid sequence of MHQGAPSWGRRWFVVWALLGLT LGVLVASAAP (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).

[0100] In some embodiments, a polypeptide comprises a secretory signal peptide SP20-A7U881_HHV2 (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).

[0101] 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 sequences of the SARS-CoV-2 secretory signal peptide provided in Table 2.

[0102] In some embodiments, a polypeptide comprises a transmembrane domain. In some embodiments, a transmembrane region also 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.

[0103] In some embodiments, a polypeptide comprises a transmembrane domain. In some embodiments, a transmembrane domain is derived from a viral membrane protein. In some embodiments, a transmembrane domain is a transmembrane domain of a SARS-CoV-2 S protein. In some embodiments, a transmembrane domain is obtained from a viral protein that is not a SARS-CoV-2 S protein.

[0104] In some embodiments, a transmembrane domain is a transmembrane domain of the SARS-CoV-2 S protein (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).

[0105] In some embodiments, a transmembrane domain is a heterologous transmembrane domain.

[0106] In some embodiments, a heterologous transmembrane domain is a transmembrane domain of a viral protein that is not a SARS-CoV-2 S protein.

[0107] In some embodiments, a transmembrane domain is capable of inducing multimerization (e.g., trimerization).

[0108] 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).

[0109] 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 adjacent to the membrane in its native protein.

[0110] In some embodiments, a sequence that is endogenously C-terminal 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.

[0111] In some embodiments, 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.

[0112] 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.

[0113] In some embodiments, a polypeptide comprises:

[0114] (1) a fragment of an S1 polypeptide (e.g., amino acids 1 to 528 of SEQ ID NO: 1 or a corresponding region of a SARS-CoV-2 variant);

[0115] (2) a secretory signal peptide (e.g., a secretory signal peptide SP24- Q7PUJ5_ANOGA, SP24-SP18-HEMA_CVBM, SP25-GD_HHV1K, SP32-GD_HHV1K or SP20-A7U881_HHV2 described herein); and

[0116] (3) a transmembrane domain (e.g., a transmembrane domain viral, 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).

[0117] 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 each other by a linker (e.g., an artificial linker, a flexible linker, a flexible linker comprising a GS sequence). In 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 a (G4S)1, (GRS)2, (G4S)3, or (G4S)4 sequence. In some embodiments, a fragment of an S1 polypeptide and a transmembrane domain are connected via a flexible linker that comprises 10 to 20 amino acids (e.g., about 15 amino acids).

[0118] In some embodiments, a fragment of an S protein is adapted to a strain or variant of SARS-CoV-2 (e.g., an Omicron variant, an XBB.1.5 variant, a JN.1 variant, a KP.2 variant, an XEC variant and / or any variant described herein).

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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 to SEQ 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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 sequence nucleotide 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.

[0135] 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 at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 245.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] In some embodiments, an RNA comprises (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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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 at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 352.

[0153] 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.

[0154] 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.

[0155] In some embodiments,

[0156] (i) a 5' cap comprises a Cap 1 structure;

[0157] (ii) a 5'-UTR sequence comprises a modified 5'-UTR of human alpha-globin;

[0158] (iii) a 3'-UTR sequence comprises a first split messenger RNA amino terminal enhancer (AES) sequence and a second mitochondrially encoded 12S ribosomal RNA sequence;

[0159] (iv) a polyA sequence comprises at least 100 A nucleotides; or

[0160] (v) an RNA comprises a combination of any of (i) to (iv).

[0161] In some embodiments, a 5' cap comprises a Cap-1 structure, and the Cap-1 structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeAl)pG2, wherein Al is position +1 of the RNA, and G2 is position +2 of the RNA.

[0162] In some embodiments, a cap proximal sequence comprises A1 and G2 of the Cap-1 structure, and a sequence comprising: A3N4N5 at positions +3, +4 and +5 respectively of the RNA, wherein N4 and N5 are each independently selected from A, G, C and U.

[0163] 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, the 30 adenine nucleotides and the 70 adenine nucleotides being 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.

[0164] 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.

[0165] 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.

[0166] In certain embodiments, an RNA is an oRNA, a self-amplifying RNA, a trans-amplifying RNA (taRNA) or an mRNA.

[0167] In some embodiments, an RNA is an unmodified RNA or an RNA comprises one or more modified uridines in place of one or more uridines.

[0168] In some embodiments, an RNA comprises a single modified uridine in place of each uridine.

[0169] In some embodiments, a modified uridine is Nl-methyl-pseudouridine.

[0170] In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 S protein is encoded by a codon-optimized sequence (e.g., codon-optimized for expression in human cells) and / or has a G / C content that is increased relative to a wild-type coding sequence.

[0171] Among other things, the invention relates to a composition comprising an RNA described in the present invention.

[0172] In some embodiments, a composition comprises 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 encapsulated in a lipid nanoparticle (LNP), preferably wherein the LNP comprises molar ratios of 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.In some embodiments, a nanoparticle has an average diameter of about 50 to 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, wherein the aqueous buffered solution optionally 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.

[0173] In some embodiments, the present invention 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.

[0174] 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 pre-filled syringe.

[0175] In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 100 pg or less (e.g., about 90 pg or less) of total RNA. In some embodiments, a pharmaceutical composition is formulated to provide a dose of about 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg, or about 3 pg of total RNA.

[0176] In some embodiments, the invention provides a method comprising administering an RNA, composition, or pharmaceutical composition provided herein.

[0177] In some embodiments:

[0178] (i) a subject is 12 years of age or older, and a method comprises administering 30 pg of RNA,

[0179] (ii) a subject is aged 5 years to less than 12 years, and a method comprises administering 10 pg of RNA, or

[0180] (iii) a subject is aged 6 months to less than 5 years, and a method comprises administering 3 pg of RNA.

[0181] In some embodiments, a method comprises administering a composition described herein in a volume of about 200 µl to about 300 µl.

[0182] In some embodiments, a subject has not previously received a SARS-CoV-2 vaccine and / or a subject has previously been determined not to have been infected with SARS-CoV-2 (e.g., as determined using a PCR test or an antigen diagnostic test).

[0183] In some embodiments, a method comprises administering a single dose of the RNA, composition, or pharmaceutical composition to a subject.

[0184] 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.

[0185] In some embodiments, an RNA, composition, or pharmaceutical composition described herein is administered three times to a subject, optionally wherein the first and second doses are administered about 21 days apart, and the third dose is administered about 28 days after the second dose.

[0186] In some embodiments, a method described herein comprises administering an additional 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 to 12 months, 2 to 10 months, 2 to 8 months, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months).

[0187] In some embodiments, a subject has previously been exposed to a SARS-CoV-2 antigen (e.g., by vaccination and / or infection).

[0188] In some embodiments, a subject has previously received one or more doses of a SARS-CoV-2 vaccine.

[0189] In some embodiments, a subject has previously received a complete dosage regimen of a SARS-CoV-2 vaccine.

[0190] In some embodiments, a subject has previously received 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 RNA formulated into LNPs encoding for a SARS-CoV-2 S protein), wherein the first dose and the second dose were administered approximately 21 days apart, and optionally wherein the subject previously received as a booster dose a monovalent or bivalent vaccine that delivers a SARS-CoV-2 S protein of one or more variants (e.g., (i) an S protein of a Wuhan strain and an S protein of an Omicron BA.4 / 5 strain, (ii) an S protein of an XBB.1.5 variant, (iii) an S protein of a KP.2 variant, and / or (iv) an S protein of a JN.l variant).

[0191] In some embodiments, a method described herein comprises administering one or more vaccines against a disease unrelated to SARS-CoV-2, optionally wherein the one or more vaccines comprise an RSV vaccine, an influenza vaccine, or a combination thereof.

[0192] In some embodiments, a method results in inducing 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 the 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 a CD8+ T cell response.

[0193] In some embodiments, a method described herein is a method of preventing or reducing the risk of becoming infected with a SARS-CoV-2 virus and / or treating a SARS-CoV-2 infection.

[0194] In certain embodiments, the RNA, compositions, or pharmaceutical compositions described herein may be used to induce an immune response in a subject.

[0195] In certain embodiments, the RNA, compositions, or pharmaceutical compositions described herein may be used for the manufacture of a medicament for inducing an immune response in a subject.

[0196] In some embodiments, a medicament is formulated for administration to the subject in accordance with a method described herein.

[0197] 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 pharmaceutical composition provided herein. In some embodiments, a method described herein induces an immune response against a SARS-CoV-2 virus.

[0198] In some embodiments, the invention provides a method of making an RNA, comprising in vitro transcription of an RNA provided herein.

[0199] In some embodiments, the invention provides a DNA (e.g., a linear DNA or a plasmid DNA) encoding an RNA provided herein. In some embodiments, the invention provides a polypeptide encoded by an RNA provided herein.

[0200] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCK FDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 89.

[0201] In some embodiments, an 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.

[0202] 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 descendant thereof.

[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 or more) of the following mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or M1229I with respect to SEQ ID NO: 1.

[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, or more) of the following mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0205] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0206] 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: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H compared to SEQ ID NO: 1.

[0207] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L relative to SEQ ID NO: 1.

[0208] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0209] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or El 150D with respect to SEQ ID NO: 1.

[0210] 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: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H compared to SEQ ID NO: 1.

[0211] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or El 150D with respect to SEQ ID NO: 1.

[0212] 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: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, or Y505H compared to SEQ ID NO: 1.

[0213] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, 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, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, VI104L, or PI 143L with respect to SEQ ID NO: 1.

[0214] 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, 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, A483, E484K, F486P, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0215] 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 the following mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H or N969K with respect to SEQ ID NO: 1.

[0216] 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 mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, or Y505H relative to SEQ ID NO: 1.

[0217] 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 more) of the following mutations: inslôMPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, 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, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L.

[0218] 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 mutations: inslôMPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, 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, A483, E484K, F486P, Q498R, N501Y, and Y505H.

[0219] In some embodiments, a fragment of a GS sequence comprises a (G4S)1, (GRS)2, (G4S)3, or (G4S)4 sequence.

[0220] In some embodiments, a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCK FDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 89.

[0221] In some embodiments, an SI polypeptide comprises:

[0222] (a) amino acids 1 to 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;

[0223] (b) QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFF SNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDS KTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIGRDF PQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAADYYV GYLQP RTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSN FRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVL YNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIA DYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDIST EIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPA TVCGPK, or a sequence that is 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) QCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLF LPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIF GTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFR VYSSANNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFKIYSKHTP INLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTA GAAAYYVGYLQPR TFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEK GIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNC VADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAP GQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKP FERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLS FELLHAPATVCGPK, or a sequence that is 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. ;

[0224] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising:

[0225] (i) a truncated SI subdomain of a SARS-CoV-2 S protein or a variant thereof this one;

[0226] (ii) a heterologous secretory signal peptide; and

[0227] (iii) a homologous transmembrane domain,

[0228] wherein the N-terminal to C-terminal orientation of the truncated SI subdomain, the heterologous secretory signal peptide and the transmembrane domain homolog is (secretory signal peptide)-(truncated SI subdomain)-(transmembrane domain).

[0229] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a truncated NTD, RBD, and / or S1 subdomain, or a variant of any of the foregoing, and wherein the polypeptide also 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, an NS9b protein, a membrane (M) protein, an ORFlab protein, an ORF3a protein, an ORF9b protein, or an NSP1-4, or any combination thereof. In some embodiments, a polypeptide comprises:

[0230] (i) one or more T cell epitopes from an N protein and one or several T cell epitopes from an NS9b protein;

[0231] (ii) one or more T cell epitopes from an N protein and one or more T cell epitopes from an M protein;

[0232] (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;

[0233] (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;

[0234] (v) one or more T cell epitopes from an NSP2 protein, one or multiple T cell epitopes from an NSP1 protein, one or more T cell epitopes from an NSP3 protein, one or more T cell epitopes from an N protein, one or more T cell epitopes from an NSP4 protein, and one or more T cell epitopes from an M protein;

[0235] (vi) one or more T cell epitopes from an N protein, one or more T cell epitopes from an NSP1 protein, one or more T cell epitopes from an NSP2 protein, one or more T cell epitopes from an NSP3 protein, and one or more T cell epitopes from NSP4.

[0236] In some embodiments, an RNA encodes a polypeptide comprising:

[0237] (a) a truncated SI subdomain, or a variant thereof, and a domain transmembrane of SARS-CoV-2;

[0238] (b) a truncated SI subdomain, or a variant thereof, and a signal peptide secretory HSV-1 gD;

[0239] (c) a truncated SI subdomain, or a variant thereof, a domain transmembrane, and a secretory signal peptide HSV-1 gD;

[0240] (d) a truncated SI subdomain or a variant thereof, a domain transmembrane S protein of SARS-CoV-2 and a secretory signal peptide; or

[0241] (e) a truncated SI subdomain, or a variant thereof, of a SARS-CoV-2 S protein CoV-2, and a secretory signal peptide HSV-1 gD.

[0242] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising:

[0243] (a) a truncated SI subdomain comprising (i) amino acids 20 to 528 of SEQ ID NO: 1 (comprising, for example, amino acids 1 to 528, 14 to 528, 17 to 528 and 50 to 528), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or (ii) amino acids 20 to 541 of SEQ ID NO: 1 (comprising, for example, amino acids 1 to 541, 14 to 541, 17 to 541 and 20 to 541), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or a variant of any of the foregoing;

[0244] (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

[0245] (c) a transmembrane domain comprising an amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCC SCGSCC), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 90.

[0246] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a transmembrane, a truncated S1 subdomain or a 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 a variant thereof and the transmembrane are connected via a sequence that comprises a GS linker, optionally wherein the GS linker comprises about 10 to 20 residues (e.g., about 15 or about 20 residues).

[0247] 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.

[0248] In some embodiments, an RNA comprises a nucleotide sequence encoding

[0249] (a) a truncated S1 subdomain, or a variant thereof and a domain transmembrane of SARS-CoV-2;

[0250] (b) a truncated SI subdomain, or a variant thereof and a signal peptide secretory HSV-1 gD;

[0251] (c) a truncated SI subdomain, or a variant thereof, a domain transmembrane, and a secretory signal peptide HSV-1 gD.

[0252] (d) a truncated SI subdomain or a variant thereof, a domain transmembrane protein of SARS-CoV-2, and a secretory signal peptide; or

[0253] (e) a truncated SI subdomain, or a variant thereof, of a SARS- CoV-2, and a secretory signal peptide HSV-1 gD.

[0254] In some embodiments, an RNA comprising a nucleotide sequence encoding a polypeptide comprising:

[0255] (a) a truncated SI subdomain comprising (i) amino acids 20 to 528 of SEQ ID NO: 1 (comprising, for example, amino acids 1 to 528, 14 to 528, 17 to 528 and 50 to 528), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or (ii) amino acids 20 to 541 of SEQ ID NO: 1 (comprising, for example, amino acids 1 to 541, 14 to 541, 17 to 541 and 20 to 541), or a sequence of a corresponding region of an S protein of a SARS-CoV-2 variant or a variant of any of the foregoing;

[0256] (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 any of the foregoing; and / or

[0257] (c) a transmembrane domain comprises an amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSC GSCC), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 90.

[0258] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising a truncated S1 subdomain or a variant thereof, and a transmembrane domain, and a secretory signal peptide, 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 a variant thereof and the transmembrane are connected via a sequence that includes a GS linker, optionally wherein the GS linker comprises about 10 to 20 residues (e.g., about 15 or about 20 residues). Brief description of Fig.s

[0259] [Fig. 1]. Novel spike antigen designs - S1 and RBD subdomain vaccines. The mutation density in novel SARS-CoV-2 variants of concern (e.g., XBB) is highest in the S1 fragment and especially in the RBD. Therefore, in some embodiments, omission of the highly conserved S2 fragment may result in more efficient priming (e.g., by removing conserved epitopes that may activate BMEM cells and / or prevent activation of naive B cells). Shown are some exemplary antigen designs, including (1) an RBD of a VOC attached to a trimerization domain (e.g., an RBD of XBB. 1.5 attached to a T4 fibritin trimerization domain), (2) an S1 domain of a 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 a 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 SI domain of a VOC attached to a trimerization domain and a transmembrane domain (comprising a transmembrane domain (e.g., an SI 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, while constructs (3) and (4) are TM-anchored.

[0260] [Fig.2]. Example of immunogenicity study in mice that have already been vaccinated. Mice receive two doses of BNT162b2 (encoding an S protein of a Wuhan strain), or a composition comprising a first RNA encoding 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 vaccine candidate. The third doses include RNA encoding the 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 timing domain (RBD (XBB.1.5)); RNA encoding an S1 domain of an S protein XBB.1.5 comprising a timing domain (S1 (XBB. 1.5)); and RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal, a timing domain, and a transmembrane domain (RBD-TM (XBB.1.5)). Yellow-filled cells indicate days when serum samples were collected, gray-filled cells indicate days when vaccines were administered, and green-filled cells indicate days when mice were sacrificed and final samples were collected. The exemplary protocol can be used to characterize immune responses induced by the compositions described herein.

[0261] [Fig.3]. Example protocol for characterizing immune cell responses (including B cells and T cells). A spleen sample, lymph nodes are collected and analyzed as shown in the figure. The figure also summarizes the analysis of blood samples collected throughout a study (including the collection of virus neutralization titers (from pVNTs) and virus binding antibody titers (ELISA), which can be performed in parallel). The example protocol can be used to characterize immune responses induced by the compositions described herein.

[0262] [Fig.4]. Example of immunogenicity study in vaccinated naive mice. Mice are administered two doses of RNA encoding (i) a full-length spike protein of a Wuhan strain (BNT162b2); (ii) an RNA encoding a full-length S protein of an XBB.1.5 variant (BNT162b2 (XBB.1.5)); (iii) an RNA encoding a full-length S protein of an XBB.1.5 variant and comprising a C-terminal truncation of 19 amino acids (BNT162b2 (XBB.1.5) Cdl9); (iv) an RNA encoding an RBD of an XBB.1.5 S protein comprising a secretory signal (SP19) and a timing domain (RBD (XBB.1.5) (SP19)); (v) an RNA encoding an SI domain of an XBB.1.5 S protein comprising a trimerization domain (SI (XBB.1.5)); (vi) an RNA encoding an RBD of an S protein XBB.1.5 comprising a secretory signal (SP19), a timing domain and a transmembrane domain (RBD-TM (XBB.1.5) (SP19)); (vii) an RNA encoding an S1 domain of an S protein XBB.1.5 comprising a timing domain and a transmembrane domain (S1-TM (XBB.1.5); and (viii) an RNA encoding an RBD of an S protein XBB.1.5 comprising a secretory signal (SP 16) and a timing domain (RBD (XBB.1.5) (SP16)). Yellow-filled cells indicate days when serum samples are collected, gray-filled cells indicate days when vaccines are administered, and green-filled cells indicate days when mice are sacrificed and final samples are collected. The exemplary protocol can be used to characterize immune responses induced by the compositions described herein.

[0263] [Fig.5]. Example protocol for characterizing immune cell responses (including B cells and T cells). Spleen samples may be collected and analyzed as shown in the figure to characterize immune cell responses induced by the compositions described herein (including, for example, T cell and B cell immune responses).

[0264] [Fig.6]. Examples of polypeptides comprising an RBD or an NTD. “SP” stands for secretory peptide, “F” stands for a fibritin domain, “TM” stands for a transmembrane domain, and “CT” stands for a C-terminal sequence derived from a SARS-CoV-2 S protein.

[0265] [Fig.7]. Examples of polypeptides comprising an RBD polypeptide and a " “T chain” (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 chain” refers to a polypeptide comprising one or more T cell epitopes or antigens (e.g., as described herein).

[0266] [Fig.8]. Examples of “dumbbell” constructions. (A) represents graphs 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 polypeptide multimer, 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) illustrates a model of the structure of such a 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.

[0267] [Fig.9]. Example of a library to characterize the effect of secretory signals N-terminals on antigen expression.

[0268] [Fig. 10]. Example of an experimental protocol for the identification of vaccine constructs with enhanced expression. An example of an in vitro protocol for testing the extracellular expression of vaccine candidates in vitro is presented. A cell line (HEK293 in [Fig. 10]) is transfected with a plasmid encoding a vaccine candidate. The cells are incubated with ACE2 or an antibody that binds to the RBD, followed by a 2e antibody, and then screened by flow cytometry to measure cell surface expression of the RBD.

[0269] [Fig. 11]. Initial in vitro expression data. Flow cytometry data presented obtained using BNT162b3, which comprises an RBD from a SARS-CoV-2 XBB.1.5 variant, and a secretory signal from a SARS-CoV-2 S protein (aa 1-19) and a transmembrane domain are shown. The amount of plasmid transfected was varied to identify a dynamic range (plasmid concentration at which to transfect cells).

[0270] Fig. 12. Effect of N-terminal secretory signal on in vitro expression of vaccine candidates. HEK293s were transfected with a library of vaccine candidates, including various N-terminal secretory signals (design shown in [Fig.9], specific sequences tested include those shown in Table XXXI of the present invention). (A) shows background fluorescent signal (transfected cells incubated with antibody 2, but not with antibody 1). (B) shows initial results from a candidate library. Significant background signal was observed, but could not be corrected by subtracting the background signal. As shown in Figure (B), all signal peptides tested resulted in a significant increase in cell surface expression of the RBD compared to the secretory signal of the SARS-CoV-2 S protein (aa 1-19).

[0271] Fig. 13. Additional data characterizing the effect of secretory signals on cell surface expression of antigens. A repeat of the experimental protocol presented in [Fig. 10] was performed to confirm the results presented in Fig. 12. Once again, highly variable background fluorescence was observed, but this could be corrected by subtracting the background fluorescence when interpreting the data. Once again, all signal peptides tested resulted in significantly higher surface expression of the antigen than the SARS-CoV-2 spike signal peptide (aa 1-19).

[0272] Fig. 14. Supplementary data characterizing the effect of secretory signals on cell surface antigen expression (anti-RBD antibody + human antibody 2). A repeat of the experimental protocol presented in [Fig. 10] was performed using antibody 1 that binds RBD and human antibodies 2. Reduced background fluorescence was observed compared to mouse antibody 2 and ACE2 labeling. Again, background fluorescence was subtracted when interpreting the data. Once again, all signal peptides tested resulted in significantly higher RBD surface expression compared to the SARS-CoV-2 spike signal peptide (aa 1-19).

[0273] [Fig. 15]. Best N-terminal secretory signals. Shown are the five N-terminal secretory signals that resulted in the highest RBD expression.

[0274] Fig. 16. (A), (B) and (C) show the results of Figs. 12, 13 and 14, respectively for the 5 main secretory signals.

[0275] [Fig. 17]. Effect of N-terminal secretory signals on the expression of RBD polypeptides and the truncated SI subdomain. (A) Shows in vitro expression data of 5 different secretory signal peptides attached to a polypeptide comprising a SARS-CoV-2 RBD. The red arrow indicates enhanced expression of the best-performing candidate (ARN003) compared to a polypeptide comprising an RBD linked to the secretory signal peptide of the protein S of SARS-CoV-2 (ARN040). As shown in (A), optimization of 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 expression in vitro). (B) Shows in vitro expression data of the 5 best-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 to 528 of SEQ ID NO: 1, with the N-terminal secretory signal peptide replaced in constructs comprising a heterologous secretory signal peptide). ARN035 includes the native SARS-CoV-2 secretory peptide. As shown in Figure (B), optimization of the secretory signal peptide was also found to enhance the expression of the SI subdomain fragment, resulting in a dose-sparing effect of approximately 4-fold.

[0276] Fig. 18. Omission of a fibritin trimerization domain enhances expression and does not reduce the antigenicity of a truncated SI subdomain. (A) Shows the results of an in vitro experiment in which a cell line was transfected with a nucleic acid encoding a construct with a fibritin trimerization domain (RNA008) and without a fibritin trimerization domain (RNA017). As shown in Fig. 18(A), omission of the fibritin trimerization domain was found to significantly enhance antigen expression. (B) Shows the results of a mouse experiment in which mice were given RNA encoding a polypeptide with a fibritin trimerization domain (RNA008) and without a fibritin trimerization domain (RNA017). As shown in the figure, omission of the trimerization domain did not interfere with the immune response.

[0277] Fig. 19. Screening of transmembrane domains to determine their effect on antigen expression. (A) shows the results of an experiment in which the same antigen (corresponding to amino acids 1 to 528 of the S protein) where, in some embodiments, the N-terminal secretory signal peptide was replaced by 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 the expression results of the top 5 transmembrane domains in terms of expression identified in the initial experiments. (C) and (D) provide the results of an additional confirmatory experiment, characterizing the same constructs as well as a few newly identified constructs.(D) shows the results of the best candidates tested in the additional confirmation experiment. Once again, it was concluded that the domains . The best performing ones showed roughly the same level of expression as those constituting the transmembrane domain of a SARS-CoV-2 S protein.

[0278] [Fig.20]. Example of experimental protocol for testing immunogenicity of different fragments of the S subdomain in mice. The example shown shows an experimental protocol for testing the immunogenicity of different fragments of the S protein in mice. Orange cells indicate the days when serum samples were collected, gray cells indicate the days when a vaccine was administered, and green cells indicate the last day of the experiment, when the mice were euthanized and the last samples were collected.

[0279] Fig. 21. A fragment of the SI subdomain induces a strong immune response in mice. (A), (B), (C), (D), and (E) show the neutralization titers (pVNT50) 7, 14, 21, 28, and 35 days after vaccination, respectively; (F) shows a line graph of the neutralization titers (pVNT50) for the constructs over the days of vaccination. As shown in (C), a statistically significant difference was observed at day 21 between (i) mice receiving RNA encoding a fragment of the SI subdomain (RNA035) or RNA encoding an NTD and an RBD connected via a GS linker (RNA038), and (ii) RNA encoding a full-length S protein.

[0280] [Fig.22]. Example of a protocol for studying the immunogenicity of antigens of the Enhanced SARS-CoV-2 in mice. Cells highlighted in yellow indicate the days serum samples were collected. Gray cells indicate the days the indicated compositions were administered. Green cells indicate the last day of the experiment, on which euthanized mice and final samples were collected.

[0281] Fig. 23. Mouse immunogenicity data generated using improved SARS-CoV-2 vaccines. (A), (B), (C), (D), and (E) show the neutralization titers (pVNT50) 7, 15, 21, 28, and 34 days after vaccination, respectively, in an experiment performed according to the protocol presented in [Fig. 22]. (F) provides a summary of the neutralization titers that were collected at different time points.

[0282] [Fig.24]. Example of a protocol for studying the immunogenicity of compositions of enhanced RNA against SARS-CoV-2 in mice. Yellow cells indicate the days on which serum samples were collected. Gray cells indicate the days on which the indicated compositions were administered. Green cells indicate the last day of the experiment, on which the euthanized mice and the final samples were collected.

[0283] 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 post-vaccination, respectively, in an experiment performed according to the protocol presented in [Fig.24]. (E) provides a summary of the neutralization titers that were collected at different times.

[0284] [Fig.26]. Example of a protocol for studying the immunogenicity of antigens of the Enhanced SARS-CoV-2 in mice. Cells highlighted in yellow indicate the days serum samples were collected. Gray cells indicate the days the indicated compositions were administered. Green cells indicate the last day of the experiment, on which euthanized mice and final samples were collected.

[0285] 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 according to the protocol presented 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), as well as neutralization titers from a replicate experiment, showing neutralizing titers for the original BNT162b2 construct (ARN041) and the optimized construct (ARN017).

[0286] Fig. 28. Summary of the effects of optimizing the signal peptide, multimerization domain, linker domain, and transmembrane domain on antigen expression. (A) to (D) show the effects of optimizing the indicated domains on polypeptide expression (in the figure, a fragment of the S1 polypeptide comprising a sequence corresponding to amino acids 1 to 528). Except for the indicated domain (signal peptide, multimerization domain, linker, transmembrane domain), all other parts of the encoded polypeptide were identical in each of the panels. The red arrow indicates an improvement in expression compared 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 to 528 of SEQ ID NO: 1) comprising a combination of different optimized domains. As shown in the figure, the optimized construct improved expression by approximately 5- to 10-fold compared to the original BNT162b2 product (encoding a full-length S protein), and by approximately 2-fold compared to the other optimized constructs.

[0287] [Fig.29]. Correlation of surface expression (in vitro) and nAb titers induced (in vivo). An analysis is provided showing the correlation between in vitro expression measurements and neutralizing antibody titers observed in mice (neutralizing titers measured 28 days after administration of a first dose of a composition to vaccine-immunized mice). As shown in the figure, the in vitro expression data showed a strong correlation with mouse neutralization titers, indicating that the in vitro expression data were a good predictor of in vivo immunogenicity.

[0288] [Fig.30]. Example of a protocol for studying the immunogenicity of antigens of Enhanced SARS-CoV-2 in mice. Cells highlighted in yellow indicate the days serum samples were collected. Gray cells indicate the days the indicated compositions were administered. Green cells indicate the last day of the experiment, on which euthanized mice and final samples were collected.

[0289] Fig. 31. Mouse immunogenicity data generated using improved SARS-CoV-2 vaccines. (A), (B), (C), and (D) show the neutralization titers (pVNT50) 7, 14, 21, and 28 days after vaccination, in an experiment performed according to the protocol presented in [Fig. 30]. (E) provides a summary of the neutralization titers that were collected at different time points.

[0290] Fig. 32. Immunogenicity data in mice confirm the dose-sparing effect provided by the vaccines described here. Vaccine-naïve mice received 0.4, 0.1, or 0.025 pg of RNA formulated in LNPs. At 7, 14, 21, 28, and 35 days post-administration, serum samples were collected, and neutralization titers against an XBB.1.5-adapted pseudovirus were measured. The results are shown in Fig. 32(A) to (F). As shown in the figure, a clear dose-sparing effect was observed at days 21, 28, and 35 for the constructs described here compared to vaccines encoding a full-length SARS-CoV-2 S protein.

[0291] Fig. 33. The SARS-CoV-2 vaccines described here induce higher neutralization titers and broader cross-neutralization in vaccine-naïve mice compared to RNA encoding the full-length S protein. Vaccine-naïve mice received a single dose of the indicated RNAs. Serum samples were collected 7, 14, 21, 28, and 35 days after RNA administration. The 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 other SARS-CoV-2 strains and variants (Wuhan, Omicron BA.1, and Omicron BA.4 / 5), in a serum sample collected 35 days after RNA administration.As shown in each of Figures 33(A) to (F), the RNA described here resulted in a considerable increase in neutralization titers against a corresponding SARS-CoV-2 variant compared to RNA encoding a full-length S protein (about 10-fold higher titers at day 35). Cross-neutralization titers were also considerably increased, with neutralization titers of . BA.4 / 5 being approximately 10 times higher for the RNA described here compared to the RNA encoding the full-length S protein.

[0292] Fig. 34. The RNA described here results in enhanced in vitro expression of the SARS-CoV-2 XBB.1.5 and KP.2 antigens. Cell surface expression was measured in RNA-transfected cells using the fluorescently labeled ACE2 protein. (A) and (B) compare the antigen expression of an RNA encoding a truncated S1 subdomain (RNA017) and an RNA encoding a full-length S protein (RNA041), where the truncated S1 subdomain and the full-length S protein are each adapted to the SARS-CoV-2 XBB.1.5 variant. (E) and (F) compare the antigenic expression of an RNA encoding a polypeptide comprising a truncated SI subdomain of a SARS-CoV-2 KP.2 S protein (RNA042) with RNA encoding a full-length KP.2 S protein (RNA044). In each experiment, RNA encoding a truncated SI subdomain was found to enhance antigen expression compared to RNA encoding a full-length S protein.

[0293] Fig. 35. Phenotypic characterization of antigen-specific B cells. (A) Provides an illustration showing the process of B cell maturation and the various intermediate states that a B cell passes through as it transitions from a naive cell to a plasma cell. Also shown are the cell surface markers characteristic of the various stages of B cell differentiation. (B) Lists the various 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 the flow antibodies of the B cell flow panel that was used in the experiment described in Example 14. FLS is the abbreviation for "Full Length Spike" and BC is the abbreviation for B Cell.

[0294] [Fig.36]. An example of a B cell phenotypic screening protocol. The cells are screened to identify single-cell lymphocytes. Single-cell lymphocytes are probed for cell surface expression of CD 19 and CD20 to identify B cells, which can be screened for a variety of markers of interest and binding to various bait proteins. CD 19+ / CD20+ B cells are screened for positive staining for the spike antigen bait. Antigen-specific B cells are then analyzed for cell surface expression of CD38 (activation marker), CD95 (germinal center marker), and CD273 / 80 (memory B cell markers), and CD138 (a plasma cell marker) for phenotypic characterization via a combinatorial / Boolean approach.

[0295] Fig. 37. Representative plots showing the antigen specificity of B cells obtained from mice administered the compositions described herein. (A) to (C) show exemplary plots of CD19+ / CD20+ cells probed for binding to the full-length S protein, the RBD, and the 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 a surprisingly high number of B cells capable of binding the NTD. While not intended to be bound by theory, the enhanced immune response to the NTD may result from increased accessibility of the NTD in the truncated S1 subdomain compared to a full-length S protein.

[0296] [Fig.38]. Summary of phenotypic characteristics of B cells obtained from mice administered the compositions described herein. (A) to (C) show the percentage of CD 19+ B cells that were found to bind to full-length S protein, RBD, and NTD, respectively. As shown, FARN017 was found to produce significantly higher numbers of B cells that bind full-length S protein, RBD, and NTD at each RNA concentration administered. For both NTD and RBD, B cell responses were approximately 4-fold higher for RNA encoding a truncated S1 subdomain (ARN017) compared to those induced by RNA encoding a full-length S protein (ARN041) for each RNA concentration tested.The B cell response induced by 0.1 pg of RNA encoding a truncated S1 subdomain was comparable to that induced by 0.4 pg of RNA encoding a full-length S protein, representing a dose-sparing effect of approximately 4-fold.

[0297] [Fig.39]. The RNA compositions described here induce a prolonged germinal center response. B cells that 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 pg, an RNA encoding a truncated S1 subdomain (RNA017) produces a higher proportion of B cells with a germinal center phenotype compared to mice given the same amount of RNA encoding a full-length S protein (RNA041).This observation suggests that RNA encoding a truncated S1 subdomain produces a germinal center response that is extensive compared to RNA encoding a full-length S protein, and is consistent with observations in other experiments described here, in which neutralizing titers induced by a truncated S1 subdomain continued to increase for a longer period compared to RNA encoding a full-length S protein.

[0298] Fig. 40. Neutralizing titers induced in vaccine-naive mice receiving two doses (approximately 21 days apart) of the described RNA compositions. (A) and (B) show the geometric mean titers (GMT) of antibodies that neutralize a SARS-CoV-2 KP.2 virus 7 and 14 days after administration of a first dose of the indicated composition to vaccine-naïve mice. (C) to (E) Show the SARS-CoV-2 KP.2 neutralizing titers 7, 14, and 21 days after administration of a second dose of the indicated composition (28, 35, and 42 days after administration of the first dose). As shown, at day 14, RNA encoding a truncated SI subdomain (RNA042) began to show increased neutralizing titers compared to RNA encoding a full-length SARS-CoV-2 S protein (RNA044), and by days 28 to 42, had produced neutralizing titers that were more than 2-fold higher than those induced by the same amount of RNA encoding a full-length S protein.

[0299] Fig. 41. Neutralizing titers induced in vaccine-naïve mice given two doses (approximately 28 days apart) of the described RNA compositions. (A) to (D) show the geometric mean titers (GMTs) of antibodies that neutralize a SARS-CoV-2 KP.2 virus 7, 14, 21, and 28 days after administration of a first dose of the indicated composition to vaccine-naïve mice. (E) Shows the SARS-CoV-2 KP.2 neutralizing titers 7 days after administration of a second dose of the indicated composition (35 days after administration of the first dose). (F) is a time plot summarizing the data shown in (A) to (E).The data again show that RNA encoding a truncated SI subdomain (RNA042) begins to show increased neutralizing titers compared to RNA encoding a full-length SARS-CoV-2 S protein at day 14, which increases to be >4-fold those induced by RNA encoding a full-length SARS-CoV-2 S protein at day 21 and ~8-fold higher by day 28. By 35 days after the first dose and 7 days after the second dose, titers had increased to be ~3-fold those induced by RNA encoding a full-length S protein. Titers induced by a 0.1 pg dose of RNA encoding a truncated SI subdomain were also found to be comparable to those induced by a 0.5 pg dose of RNA encoding a full-length S protein at each time point, indicating a dose-sparing effect of approximately 5-fold.

[0300] Fig. 42. Neutralization titers induced in previously vaccinated mice administered RNA compositions described herein. (A) to (C) show the results of an experiment in which vaccinated mice received RNA encoding a full-length S protein or RNA encoding a truncated S1 subdomain. Before administering a candidate RNA, each mouse first received 2 doses of RNA encoding an S protein from a Wuhan strain of SARS-CoV-2 (on days 0 and 21) and one dose of a bivalent composition comprising RNA encoding an S protein from a Wuhan strain of SARS-CoV-2 and RNA encoding an S protein from a BA.4 / 5 Omicron variant of SARS-CoV-2 (on day 49). 133 days later administration of the first vaccine (d 133), candidate RNAs were administered. (A), (B), and (C) show neutralizing titers 0 days (d 133 of the experiment), 7 days (d 140 of the experiment), and 14 days (d 147 of the experiment) after administration of the indicated candidate. (D) provides geometric mean increases (GMFI) in neutralizing antibody titers at day 147 compared to mice administered carrier solution. As shown in (C), at day 147, absolute neutralizing antibody titers induced by RNA encoding a truncated S1 subdomain were approximately 2-fold higher than those induced by RNA encoding a full-length S protein. As shown in (D), the GMFI of neutralizing antibody titers was approximately 3-fold higher for RNA encoding a truncated S1 subdomain than for RNA encoding a prefusion-stabilized full-length S protein. Some definitions

[0301] In general, the terminology used herein is consistent with its meaning as understood in the art, unless otherwise clearly indicated. Explicit definitions of certain terms are provided below; the meaning of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from the context.

[0302] In order that the present invention may be more easily understood, certain terms are first defined below. Additional definitions for the following and other terms are set forth throughout the description.

[0303] About: The term "about," when used herein in reference to a value, refers to a value that is analogous, in context, to the referenced value. In general, one skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by the term "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values ​​that are 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 referenced value.

[0304] 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 characteristic 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, a polypeptide, a nucleic acid, a saccharide, a lipid, a 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 embodiment, the term "agent" may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymer moiety. In some embodiments, the term may refer to a compound, molecule, or entity that is free or substantially free of any polymer or polymer moiety.

[0305] 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, for example, by 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-naturally occurring 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.“Non-standard amino acid” refers to any amino acid, other than standard amino acids, whether 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, may contain a structural modification relative to 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) relative to the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid relative to a polypeptide 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, relative to a polypeptide containing an otherwise identical unmodified amino acid. As is clear from the 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.

[0306] 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 comprises sufficient immunoglobulin structural elements 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 that 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 and 5 amino acid substitutions relative to the reference CDR.In some embodiments, an included CDR is substantially identical to a reference CDR in that it has 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 has 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 in the included CDR is deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, an included CDR is substantially identical to a reference CDR in that at least amino acids 1-5 in the included CDR are deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that at least one amino acid in the included CDR is substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR.In some embodiments, an included CDR is substantially identical to a reference CDR in that at least amino acids 1 to 5 in the included CDR are deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of 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 is present in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as corresponding to CDRs 1, 2, and 3 of an antibody variable domain; in . In some of these embodiments, an antibody agent is present in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together comprise structural elements recognized by those skilled in the art as corresponding 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 that is homologous or broadly 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 comprise one or more constant region sequences that are characteristic of a particular organism, such as a camel, a human, a mouse, a primate, a rabbit, a rat; in many embodiments, an antibody agent may comprise one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may comprise one or more sequence elements that would be recognized by those 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-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modula ImmunoPharmaceuticals (“SMIP™”); single-chain or tandem diabodies (TandAb®); VHHs; Anticalins®; Nanocorps® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; . TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, an antibody may lack a covalent modification (e.g., glycan attachment) that it would have if naturally produced. In some embodiments, an antibody may contain a covalent modification (e.g., glycan attachment, charge useful (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.) or another pendant group (e.g., polyethylene glycol, etc.)).

[0307] Antigen: Those skilled in the art, upon reading the present description, will recognize that the term "antigen" refers to a molecule that is recognized by the immune system, for example, 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 include the generation of antibodies and / or antigen-specific T cells. In some embodiments, an antigen is a peptide or polypeptide that includes 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 embodiment, 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 can react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a parasite antigen. In accordance with the present invention, in some embodiments, an antigen can be delivered by RNA molecules as described herein. In some embodiments, a peptide or polypeptide antigen can be 2 to 100 amino acids in length, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length.In some embodiments, a peptide or polypeptide antigen may be greater than 50 amino acids. In some embodiments, a peptide or polypeptide antigen may 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 capable of inducing, in the presence of appropriate co-stimulatory signals, stimulation, priming, and / or expansion of the immune effector cell bearing an antigen receptor recognizing the antigen. In the context of embodiments of the present invention, in some embodiments, an antigen may be presented or present on the surface of a cell, for example, an antigen-presenting cell.In one embodiment, an antigen is presented by a diseased cell such as a cell infected with a virus. In one embodiment, an antigen receptor is a TCR that binds to an epitope of an antigen. presented in the context of the 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.

[0308] Associated: Two events or entities are "associated" with each other, 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., a polypeptide, a genetic signature, a metabolite, a microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level, and / or form correlates with the incidence, susceptibility, severity, stage, etc. of the disease, disorder, or condition (e.g., in a relevant population). In some embodiments, two or more entities are physically "associated" with each other if they interact, directly or indirectly, such that they are and / or remain in physical proximity to each other.In some embodiments, two or more entities that are physically associated with each other are covalently bonded to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other but are non-covalently associated, e.g., by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0309] Binding: Those skilled in the art, upon reading this disclosure, will recognize that the term "binding" generally refers to a non-covalent association between or among entities or moieties. In some embodiments, the binding data is expressed in terms of "IC50." As understood in the art, the IC50 is the concentration of an agent being evaluated in a binding assay at which a 50% inhibition of the binding of the reference agent known to bind to the relevant binding partner is observed. In some embodiments, the assays are performed under conditions in which (e.g., by limiting the target binding and reference concentrations) the IC50 values ​​approximate the KD values.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 as 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 may be expressed relative to binding by a reference standard peptide. For example, it may be based on its IC50 relative to the IC50 of 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 solution-phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)); and measurement of MHC class I 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)).

[0310] Cap: As used herein, the term "cap" refers to a structure comprising or consisting essentially of a nucleoside 5'-triphosphate that is generally 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 5' RNA cap, including, for example, 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 has the ability to stabilize RNA if attached thereto, including, for example, but not limited to, anti-reverse cap analogs (ARCA) known in the art.Those skilled in the art will appreciate that methods for attaching 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, for example, but not limited to, the vaccinia capping enzyme system or the Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA may be obtained by in vitro transcription (IVT) of a single-stranded DNA template in the presence of a dinucleotide or trinucleotide cap analog.

[0311] Cell-mediated immunity: “Cell-mediated immunity”, “cellular immunity”, “cellular immune response” or similar terms are intended to include a cellular response directed toward cells characterized by the expression of an antigen, in particular characterized by the presentation of an antigen with an MHC class I or class II. A cellular response involves immune effector cells, in particular T cells or T lymphocytes that act either as “helpers” or “killers”. Helper T cells (also called CD4+ T cells or CD4 T cells) play a central role in regulating the immune response and killer cells (also called 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.

[0312] Co-administration: As used herein, the term "co-administration" refers to the use of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein and an additional therapeutic agent. The combined use of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein and an additional therapeutic agent may be performed simultaneously or separately (e.g., sequentially in any order). In some embodiments, a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein and an additional therapeutic agent may be combined in a 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 considered to fall within the definition of "co-administration" or "combination," provided that a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein and an additional therapeutic agent are delivered or administered at times sufficiently close in time that there is at least some temporal overlap in the biological effect(s) generated by each on a target cell or treated subject.

[0313] Codon-Optimized: As used herein, the term "codon-optimized" refers to the modification 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. In the context of the present invention, in some embodiments, the coding regions are codon-optimized for expression optimal 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 comprise increasing the guanosine / cytosine (G / C) content of a coding region of the RNA described herein relative 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 altered from the amino acid sequence.

[0314] Combination Therapy: As used herein, the term "combination therapy" refers to 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, these regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before the administration of any dose of a second regimen); in some embodiments, these agents are administered in overlapping regimens. In some embodiments, "administering" a combination therapy may involve administering one or more agents or modality(ies) to a subject receiving the other agent(s) or modality(ies) of 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 fractions thereof, may be administered together in a combined composition.

[0315] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., which may not be identical to each other, but are sufficiently similar to permit comparison with each other such that a person skilled in the art will appreciate that inferences can reasonably be drawn based on the observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical characteristics and one or a small number of varied characteristics. One skilled in the art will understand, in this context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable.For example, a person skilled in the art will appreciate that sets of circumstances, individuals or populations are comparable. to each other when they are characterized by a sufficient number and type of substantially identical characteristics to justify a reasonable conclusion that differences in results obtained or phenomena observed in or with different sets of circumstances, individuals, or populations are caused by or indicate variation in those characteristics which are varied.

[0316] 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 refer to the position / identity of a structural element in one compound or composition relative to another compound or composition (e.g., to a suitable 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 a suitable reference polymer.For example, those skilled in the art will appreciate that, for simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a related reference polypeptide, such that an amino acid "matching" a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those skilled in the art will readily appreciate how to identify "matching" amino acids.For example, those skilled in the art will be familiar with 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 may be used, for example, to identify "matching" residues in polypeptides and / or nucleic acids in accordance with the present invention. Those skilled in the art will also appreciate that, in some instances, the term "matching" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., a suitable reference event or entity).To give just one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein of another organism to indicate, in some embodiments, that it plays a similar role or performs a similar function and / or that it exhibits a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0317] As is known to those skilled in the art, sequence alignment strategies allow for the consideration of, for example, "gaps" in sequences and / or "repeated" residues. Moreover, the skilled person in the art understands 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, where a reference sequence has a segment of two or more contiguous identical residues, and a modified sequence has one less residue, it is not possible to assign a particular singular residue in the reference sequence as the one that has been deleted, since deletion of any of the contiguous identical residues would generate the same modified sequence. The skilled person will therefore be sensitive to 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 skilled in the art will not be able to determine whether amino acid 144 or 145 has been deleted in the modified sequence. They will, however, understand that either deletion describes the same polypeptide sequence and will therefore 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 polypeptide described 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).

[0318] 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 analog of a designated amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially 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, one skilled in the art will understand that antigens suitable for use herein may be modified such that they vary in sequence from the naturally occurring or native sequences from which they are derived, while retaining the desirable activity of the native sequences.

[0319] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or has been selected by human hand; (ii) which is produced by a process requiring human intervention; and / or (iii) which is distinct from natural substances and other known agents.

[0320] Dosage Regimen: Those skilled in the art will appreciate that the term "dosage regimen" may be used to refer to a set of unit doses (generally more than one) that are administered individually to a subject, generally separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosage regimen, which may involve one or more doses. In some embodiments, a dosage regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, the individual doses are separated from each other by a period of the same duration; in some embodiments, a dosage regimen comprises a plurality of doses and at least two different periods separating the individual doses. In some embodiments, all doses in a dosage regimen are of the same unit dose amount.In some embodiments, different doses within a dosage regimen are of different amounts. In some embodiments, a dosage regimen includes 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 dosage regimen includes 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 dosage regimen is correlated with a desired or beneficial outcome when administered to a relevant population (i.e., is a therapeutic dosage regimen).

[0321] Encode: As used herein, the term "encode" or "coding" refers to sequence information in a first molecule that guides the 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 may encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule may encode a polypeptide (e.g., by a translation process). Thus, a gene, cDNA, or RNA molecule (e.g., mRNA) encodes a polypeptide if transcription and translation of the 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 whose nucleotide sequence is identical to the RNA (e.g., mRNA) sequence of such a target antigen. In . 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 can be used as a template for transcription of a gene or cDNA.

[0322] Modified: In general, the term "modified" refers to having been manipulated by human hands. For example, a polynucleotide is considered to be "modified" when two or more sequences that are not linked together in that order in nature are manipulated by human hands to be directly linked to each other in the modified polynucleotide and / or when a particular residue in a polynucleotide is not present in nature and / or is caused, by human action, to be linked to an entity or moiety with which it is not linked in nature.

[0323] Epitope: As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin-binding component (e.g., an antibody or a receptor). 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, these chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of these chemical atoms are groups physically separated from each other when the antigen adopts an alternative conformation (e.g., is linearized).Accordingly, in some embodiments, an epitope of an antigen may comprise 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 be from about 5 to about 30 amino acids in length, or from about 10 to about 25 amino acids in length, or from about 5 to about 15 amino acids in length, or from about 5 to 12 amino acids in length, or from about 6 to about 9 amino acids in length.

[0324] 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 may be a transcript. In some embodiments, a gene product may 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.

[0325] 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 (typically AUG) of a coding region of an RNA molecule, e.g., in its natural context.

[0326] Fragment: The term "fragment" as used herein in the context of a nucleic acid sequence (e.g., an RNA sequence) or an amino acid sequence may generally be a fragment of a reference sequence. In some embodiments, a reference sequence is a full-length sequence, e.g., a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment generally refers to a sequence identical to a corresponding strand in a reference sequence. In some embodiments, a fragment comprises a continuous strand of nucleotides or amino acid residues that corresponds to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the full length of a reference sequence from which the fragment is derived.In some embodiments, the term "fragment", in reference to an amino acid sequence (peptide or polypeptide), relates to a portion of an amino acid sequence, for example a sequence that represents the amino acid sequence shortened at the N-terminus and / or the 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.

[0327] 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 polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered "homologous" to each other 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 each other 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 to those skilled in the art, certain amino acids are generally classified as being similar to each other as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “nonpolar” side chains. The substitution of one amino acid for another of the same type can often be considered a “homologous” substitution.

[0328] Humoral Immunity: As used herein, the term "humoral immunity" or "humoral immune response" refers to antibody production and accompanying accessory processes, 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-mediated promotion of pathogen phagocytosis and clearance.

[0329] 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 "substantially identical" to each other 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.Calculating the percent identity of two nucleic acid or polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison (e.g., gaps may be introduced into one or both of a first and second sequence for optimal alignment and non-identical sequences may be ignored for comparison). In some embodiments, the length of a sequence aligned for comparison 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 the corresponding positions are then compared.When a position in the first sequence is occupied by the same residue (e.g., a nucleotide or an 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 must be introduced for optimal alignment of the two sequences. Comparing the sequences and determining the percent identity between two sequences can be performed 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 performed with the ALIGN program use a residue table of weight PAM120, 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 a NWSgapdna.CMP matrix.

[0330] Immunologically equivalent: The term "immunologically equivalent" means that an immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or substantially the same immunological properties and / or exerts the same or substantially the same immunological effects, for example with respect to the type of immunological effect. In the context of the present invention, in certain embodiments, the term "immunologically equivalent" is used with respect to the immunological effects or properties of the 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 response having a reaction specificity with the reference amino acid sequence.

[0331] In one embodiment, an antigen receptor is an antibody or B cell receptor that binds to an epitope of an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

[0332] Augmented, induced, or reduced: As used herein, these terms or grammatically comparable comparative terms indicate values ​​relative to a comparable reference measurement. For example, in some embodiments, an evaluated value obtained with a provided pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) may be "augmented" relative to that obtained with a comparable reference pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). Alternatively or additionally, in some embodiments, an assessed value obtained in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., before or after an event; or presence or absence of an event such as administration of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as described herein, or in a different, comparable subject (e.g., in a comparable subject who differs from the subject of interest by prior exposure to a condition, e.g., absence of administration of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as described herein).In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of sufficient prevalence and / or magnitude to achieve statistical significance). One skilled in the art will be aware, in a given context, of or readily able to determine 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 refer to a reduction in the level of an evaluated value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or more, relative to a comparable reference. In some embodiments, the term "reduced" or equivalent terms refer to complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.In some embodiments, the term "augmented" or "induced" refers to an increase in the level of an evaluated value of 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 more, relative to a comparable reference.

[0333] 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 functional group or atom thereof carries 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 primarily neutral at physiological pH values, e.g., in some embodiments about 7.0 to 7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa ranging from about 5 to about 7.

[0334] Isolated: The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or 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 may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.

[0335] Lipid: As used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules that include one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules including both hydrophobic moieties and hydrophilic moieties are also generally referred to as amphiphiles.

[0336] RNA Lipid Nanoparticle: As used herein, the term "nanoparticle “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and one or more RNA molecules. 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., a PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein may have an average size (e.g., a 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 invention, the RNA lipid nanoparticles may 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 can be prepared by mixing lipids with RNA molecules described herein.

[0337] 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 invention, the term lipid is considered to encompass lipidoids.

[0338] 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. e.g., a diameter) less than 1,000 nanometers (nm). In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) less than 300 nm. In some embodiments, a nanoparticle may be characterized by a longest dimension (e.g., a diameter) less than 100 nanometers (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 pm 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 less than about 1000 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 greater than about 1 nm.In many embodiments, a nanoparticle may be substantially spherical such 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.

[0339] 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 present in an organism (including viruses) that can be isolated from a natural source and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0340] Neutralization: As used herein, the term "neutralization" refers to an event in which binding agents such as antibodies bind to a biologically active site of a virus such as a receptor binding protein, thereby inhibiting parasitic infection of cells. In some embodiments, the term "neutralization" refers to an event in which the binding agents eliminate or significantly reduce the ability to infect cells.

[0341] Nucleic Acid Particle: A "nucleic acid particle" may be used to deliver nucleic acid to a target site of interest (e.g., a cell, tissue, organ, etc.). A nucleic acid particle may comprise at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and a nucleic acid. In some embodiments, a nucleic acid particle is a lipid nanoparticle. In some embodiments, a nucleic acid particle is a lipoplex nanoparticle.

[0342] Nucleic Acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments embodiment, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises a 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-stranded and double-stranded fragments. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester bonds. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester bonds.For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite bonds 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, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues.In some embodiments, a non-naturally occurring residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 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, bases methylated, intercalated bases and combinations thereof). In some embodiments, a non-naturally occurring residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose) relative to those of the naturally occurring residues.In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or a polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, by enzymatic synthesis (e.g., by complementary template-based polymerization, e.g., in vivo or in vitro), by reproduction in a cell or recombinant system, or by chemical synthesis. In some embodiments, a nucleic acid has a length of 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, 10000, 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 in length.

[0343] Nucleotide: As used herein, the term "nucleotide" refers to its recognized meaning in the art. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0344] Patient: As used herein, the term "patient" refers to any organism suffering from or at risk of suffering from a disease, 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 is at risk of suffering from one or more diseases, disorders, or conditions. In some embodiments, a patient is exhibiting one or more symptoms of a disease, disorder, or condition. In some embodiments, a patient has been diagnosed with one or more diseases, disorders, or conditions. In some embodiments, a disease, disorder, or condition that may be treated by the provided technologies is or includes an HSV infection.In some embodiments, a patient is receiving or has received a certain therapy to diagnose and / or treat a disease, disorder, or condition. In some embodiments, a patient is a patient suffering from or at risk of suffering from an HSV infection.

[0345] PEG-conjugated lipid: The term “PEG-conjugated lipid” refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0346] Pharmaceutical Composition: As used herein, the term "pharmaceutical composition" refers to an active agent, formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a therapeutic regimen that has a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be specially formulated for parenteral administration, e.g., by subcutaneous, intramuscular, or intravenous injection, such as, for example, a sterile solution or suspension formulation.

[0347] Pharmaceutically Effective Amount: The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount that achieves a desired reaction or effect, alone or in combination with other doses. In the case of the treatment of a particular disease, a desired reaction in some embodiments relates to the inhibition of the progression of the disease. In some embodiments, such inhibition may include slowing the progression of a disease and / or halting or reversing the progression of the disease. In some embodiments, a desired reaction in a treatment of a disease may be or include the delaying or preventing the onset of a disease or condition.An effective amount of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) described herein will depend on, for example, a disease or condition to be treated, the severity of such disease or condition, individual patient parameters, including, for example, age, physiological status, height and weight, duration of treatment, type of accompanying therapy (if present), 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 these parameters. In the event that a response in a patient is insufficient with an initial dose, higher doses (or actually higher doses achieved by a different, more localized route of administration) may be used.

[0348] 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 that is generally located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled 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. The RNAs described herein may 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.

[0349] 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 is found in nature. In some embodiments, a polypeptide has an amino acid sequence that is not found in nature. In some embodiments, a polypeptide has an amino acid sequence that is modified in that it is designed and / or produced by human action. In some embodiments, A polypeptide may comprise or consist of naturally occurring amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only naturally occurring amino acids or only unnatural amino acids. 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 comprise one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the N terminus of the polypeptide, at the C terminus of the polypeptide, or any combination thereof.In some embodiments, such pendant groups or modifications include acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic and / or may include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share the relevant activity or structure and may therefore be considered members of the same polypeptide class or family.For each of these classes, the present disclosure provides and / or those skilled in the art will likely know exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, these exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family has 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 has an overall degree of homology or sequence 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 comprises at least one region (e.g., a conserved region which may in some embodiments be or comprise a characteristic sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region generally encompasses at least 3 to 4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one strand 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.

[0350] 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 delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of a disease, disorder, or condition has been delayed for a predefined period of time.

[0351] Recombinant: The term "recombinant" in the context of the present invention means "made by genetic engineering." In some embodiments, a "recombinant" entity such as a recombinant nucleic acid in the context of the present invention is not naturally occurring.

[0352] Reference: As used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to 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 test or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally incorporated into a tangible medium. Generally, as would be understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being evaluated.A person skilled in the art will recognize when sufficient similarities are present to justify recourse to a particular possible reference or witness and / or comparison with them.

[0353] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a ribonucleotide polymer. In some embodiments, an RNA is single-stranded. In some embodiments, an RNA is double-stranded. In some embodiments, an RNA comprises both single-stranded and double-stranded fragments. strand and double strand. In some embodiments, an RNA may comprise a backbone structure as described in the definition of "Nucleic acid / Polynucleotide" above. An RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, an RNA is an mRNA. In some embodiments where an RNA is an mRNA, an RNA generally comprises at its 3' end a poly(A) region. In some embodiments where an RNA is an mRNA, an RNA generally comprises at its 5' end a cap structure recognized in the art, e.g., for recognition and attachment of an mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0354] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses both unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides comprise the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may comprise 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 pair with an expanded repertoire of partners, or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) internucleoside linkage modifications, including modification or replacement of phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

[0355] Risk: As will be understood from the context, the "risk" of a disease, disorder, and / or condition refers to the probability that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, the risk is expressed as a percentage. In some embodiments, the risk is 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, the 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 has a known risk of the disease, disorder, condition, and / or event. In some embodiments, a reference sample or group of reference samples are derived from individuals comparable to a particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater. In some embodiments, the risk may reflect one or more genetic attributes, for example, that may predispose an individual to developing (or not developing) a particular disease, disorder, and / or condition. In some embodiments, the risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0356] RNA Lipoplex Particle: As used herein, the term "RNA lipoplex particle" refers to a complex comprising liposomes, particularly cationic liposomes, and RNA molecules. While not intended to be limited to any particular theory, electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and spontaneous formation of RNA lipoplex particles. In some embodiments, the positively charged liposomes may comprise a cationic lipid, such as in some DOTMA embodiments, and additional lipids, such as in some DOPE embodiments. In one embodiment, an RNA lipoplex particle is a nanoparticle.

[0357] Selective or Specific: The term "selective" or "specific," when used herein in reference to an agent having 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 preferentially binds to that target in the presence of one or more competing alternative targets. In many embodiments, the specific interaction is dependent on the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors).In some embodiments, the specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, the specificity is evaluated relative to that of a reference specific binding moiety.

[0358] Stable: As used herein, the term "stable" in the context of the present invention refers to a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as a whole and / or components thereof meeting or exceeding predetermined acceptance criteria. For example, in some embodiments, a composition stable pharmaceutical composition (e.g., an immunogenic composition, e.g., a 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., an immunogenic composition, e.g., a vaccine) refers to the integrity of the RNA molecules being maintained at least above 90% or more. In some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) refers to at least 90% or more (including, for example, at least 95%, at least 96%, at least 97%, or more) of the RNA molecules being maintained to be encapsulated in lipid nanoparticles.In some embodiments, a stable pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) refers to a formulation that remains capable of eliciting a desired immunological response when administered to a subject. In some embodiments, a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) remains stable for a specified period of time under certain conditions.

[0359] Subject: As used herein, the term "subject" refers to an organism to be administered a composition described herein, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic animals, 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., HSV infection). In some embodiments, a subject is likely to be suffering from a disease, disorder, or condition (e.g., HSV infection). In some embodiments, a subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition (e.g., HSV infection).In some embodiments, a subject exhibits one or more non-specific symptoms of a disease, disorder, or condition (e.g., HSV infection). In some embodiments, a subject exhibits no symptoms or characteristics of a disease, disorder, or condition (e.g., HSV infection). In some embodiments, a subject is a person exhibiting one or more characteristics of susceptibility to or risk for a disease, disorder, or condition (e.g., HSV infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual receiving and / or having received a diagnosis and / or therapy.

[0360] Suffering from: A person who “suffers” from a disease, disorder and / or condition has been diagnosed with and / or exhibits one or more symptoms of a disease, disorder and / or condition.

[0361] Likely to suffer from: A person who is "likely to suffer from" a disease, disorder, and / or condition is a person who is at a higher risk of developing the disease, disorder, and / or condition than a person in the general public. In some embodiments, an individual who is likely to suffer from a disease, disorder, and / or condition may not have been diagnosed as having the disease, disorder, and / or condition. In some embodiments, an individual who is likely to suffer from a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is likely to suffer from 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.

[0362] Synthetic: As used herein, the term "synthetic" refers to an entity that is artificial, or is manufactured with human intervention, or results from synthesis rather than being naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, for example, in some embodiments by solid-phase synthesis. In some embodiments, the term "synthetic" refers to an entity manufactured outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) that is produced by in vitro transcription using a template.

[0363] Therapy: The term "therapy" refers to the administration or delivery of an agent or intervention that has a therapeutic effect and / or causes a desired biological and / or pharmacological effect (e.g., has been shown to be statistically likely to have such an effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to slow, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation therapy, phototherapy) that can be performed to slow, alleviate, inhibit, present, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0364] 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 after 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 some embodiments, the 3' UTR does not begin immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context.

[0365] Threshold Level (e.g., Acceptance Criteria): As used herein, the term "threshold level" refers to a level used as a reference to obtain information about and / or classify the results of a measurement, e.g., the results of a measurement obtained in a test. For example, in some embodiments, a threshold level means a measured value in a test that defines the dividing line between two subsets of a population (e.g., a lot that meets quality control criteria versus a lot that does not meet quality control criteria). Thus, a value equal to or greater than the threshold level defines one subset of the population, and a value less than the threshold level defines the other subset of the population. A threshold level may be determined based on one or more control samples or on a population of control samples.A threshold level may be determined before, concurrently with, or after the measurement of interest. In some embodiments, a threshold level may be a range of values.

[0366] Treat: As used herein, the term "treat," "treatment," or "treating" refers to any method used to slow, ameliorate, alleviate, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the occurrence of one or more symptoms or characteristics of a disease, disorder, and / or condition, partially or completely. The treatment may be administered to a subject who is not exhibiting signs of a disease, disorder, and / or condition. In some embodiments, the treatment may be administered to a subject who is exhibiting only early signs of the disease, disorder, and / or condition, for example, for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and / or condition. disorder and / or condition. In some embodiments, the treatment may be administered to a subject at a later stage of the disease, disorder and / or condition.

[0367] Vaccination: As used herein, the term "vaccination" refers to administering a composition intended to generate an immune response, e.g., to a disease-associated (e.g., disease-causing) agent. In some embodiments, the vaccination may be administered before, during, and / or after exposure to a disease-associated agent, and in some embodiments, before, during, and / or shortly after exposure to the agent. In some embodiments, the vaccination comprises multiple, appropriately spaced administrations of a vaccine composition. In some embodiments, the vaccination generates an immune response to an infectious agent.

[0368] 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.

[0369] 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 exhibits significant structural identity with a reference molecule, but differs structurally from the reference molecule, e.g., in the presence or absence or in one or more chemical moieties relative to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be readily recognized by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements.A variant, by definition, is a distinct molecule that shares one or more of these 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 due to one or more differences in the amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., that are attached to the backbone of the polypeptide or nucleic acid). In some embodiments, a variant polypeptide or nucleic acid has overall sequence identity with a reference polypeptide or nucleic acid that is . of 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 exhibits a reduced level of one or more biological activities relative to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference, except for a small number of sequence changes at particular positions. Generally, less 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, relative 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 1 substituted residues relative to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) 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 no 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, relative to the reference.In some embodiments, a variant polypeptide or nucleic acid comprises less 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 generally less than about 5, about 4, about 3, or about 2 additions or deletions relative to the reference. In some embodiments, a reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

[0370] 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 can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some 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 mammalian episomal 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 are thereby replicated along with the host genome.In addition, certain vectors are capable of directing the expression of the genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." In certain embodiments, known techniques may be used, for example, for the generation or manipulation of recombinant DNA, for the synthesis of oligonucleotides, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The foregoing techniques and procedures may generally be performed according to standard methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this disclosure. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which is incorporated herein by reference for any purpose.

[0371] All literature and similar documentation 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 documentation, are expressly incorporated by reference in their entirety. In the event that any one or more of the incorporated literature and similar documents deviate from or conflict with this application, including, but not limited to, defined terms, use of terms, techniques described, or the like, this application shall control. The section headings used herein are for organizational purposes only and shall not be construed as limiting in any manner the subject matter disclosed. Detailed Description

[0372] In some embodiments, the present invention 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, the technologies provided herein can be used to mitigate immune imprinting effects and / or induce a stronger de novo immune response (e.g., compared to other vaccination approaches). Presentation of SARS-CoV-2

[0373] The SARS-CoV-2 spike (S) protein can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The SARS-CoV-2 S2 includes a receptor binding domain (RBD), which facilitates viral entry into host cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0374] The presentation of COVID-19 typically presents with cough and fever, with a chest radiograph showing ground-glass opacities or focal shadows. However, many patients do not present with fever or radiographic changes, and infections may be asymptomatic, which is important for controlling transmission. In symptomatic individuals, disease progression may lead to acute respiratory distress syndrome requiring ventilation and subsequent multiorgan failure or even death. Common symptoms in hospitalized patients (in order of decreasing frequency) include fever, dry cough, shortness of breath, fatigue, myalgias, nausea / vomiting or diarrhea, headache, weakness, and rhinorrhea.Anosmia (loss of smell) or ageusia (loss of taste) may be the only symptom present in about 3% of people with COVID-19.

[0375] The disease can affect all age groups, but case fatality rates (CFRs) are particularly high in people over 60 years of age. Comorbidities are also associated with an increased case fatality rate, including cardiovascular disease, diabetes, hypertension, and chronic respiratory disease. Healthcare workers are overrepresented among COVID-19 patients due to their occupational exposure to infected patients.

[0376] In most situations, a molecular test is used to detect SARS-CoV-2 and confirm infection. Reverse transcription polymerase chain reaction (RT-PCR) testing methods targeting SARS-CoV-2 viral RNA are a diagnostic method for suspected cases of COVID-19. Samples to be tested are taken from the nose and / or throat using a swab. SARS-CoV-2 variants

[0377] Since the initial discovery of SARS-CoV-2, a number of variants have emerged worldwide. The emergence of these new circulating SARS-CoV-2 variants has raised significant concerns about the geographic and temporal effectiveness of vaccine interventions. Of particular concern is the emergence of Omicron (B. 1.1.529) variants, which include a number of mutations in the S protein. 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 may be identified by virologists and / or healthcare organizations using an appropriate classification system, including the Pango or NextClade classification systems, examples of which are described herein.

[0378] In some embodiments, the present invention refers to a SARS-CoV-2 variant that is prevalent and / or rapidly spreading in a relevant territory. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided in the GIS AID 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 described herein.

[0379] The Omicron BA. 1 variant was first reported to WHO on 24 November 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): Omicron Classification (B.1.1.259): SARS-CoV-2 Variant of Concern (2021); WHO Headquarters (HQ), WHO Health Emergencies Programme, Strengthening the Response to the SARS-CoV-2 Omicron Variant: Technical Brief and Priority Actions for Member States (2022)). Significant alterations in the spike (S) glycoprotein of the first Omicron BA.l variant 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.ell (2022)) and made BA.l capable of partially evading previously established SARS-CoV-2 wild-type (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)).

[0380] As a result, post-vaccination infection of individuals vaccinated with Omicron is more frequent than with previous variants of concern (VOC). While the Omicron variant BA.1 has been supplanted by the BA.2 variant in many countries worldwide, other variants such as BA.1.1 and BA.3 have temporarily and / or locally gained momentum, but have not become globally dominant (S. Xia et al., “Origin, virological features, immune evasion and intervention of SARS-CoV-2 Omicron sublineages.” Signal Transduct. Ther. 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's BA.2.12.1 subsequently supplanted BA.2 to become dominant in the United States, while BA.4 and BA.5 supplanted BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages showcase distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022); European Centre for Disease Prevention and Control, Weekly COVID-19 country overview -Country overview report: Week 31 2022 (2022); J. Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,” Bioinformatics 34, 4121-4123 (2018)). Currently, Omicron BA.5 is dominant globally, including in the United States (Centers for Disease Control and Prevention). COVID Data Tracker. Atlanta, GA: U.S. Department of Health and Human Services, CDC; August 12, 2022. https: / / covid.cdc.gov / coviddata-tracker (2022)).

[0381] Omicron has acquired numerous alterations (amino acid exchanges, insertions, or deletions) in the S glycoprotein, some of which are shared by all Omicron VOCs while others are specific to one or more Omicron sublineages. Antigenically, BA.2.12.1 shows high similarity to BA.2 but not to BA.l, while BA.4 and BA.5 differ considerably from their ancestor BA.2 and even more from BA.l, consistent with their genealogy (AZ Mykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.l and BA.2 are antigenically distinct,” Sci. Immunol. 7, eabq4450 (2022).). The main differences between BA.l and other Omicron VOCs include the A143-145, L212I or ins214EPE in the N-terminal domain of the S-glycoprotein and the G446S or G496S in the receptor binding domain (RBD).The amino acid changes T376A, D405N, and R408S in the RBD are in turn common to BA.2 and its descendants, but are not found in BA.L. In addition, some alterations are specific to 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 the same S sequence). Most of these shared, VOC-specific alterations have been shown to play important roles in the immune escape of monoclonal antibodies. and polyclonal sera directed against wild-type S glycoprotein. In particular, BA.4 / BA.5-specific alterations are strongly implicated in the 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)). Adaptations of SARS-CoV-2 variants

[0382] In some embodiments, an antigen used as described herein is or comprises a fragment or domain of a viral polypeptide, or an antigenic fragment thereof. In some embodiments, an antigen used as described herein is a membrane-attached 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., an immunogenic composition, e.g., a vaccine) comprises or delivers antigen sequences that are or comprise one or more antibody epitopes and / or one or more CD4 T cell epitopes and / or CD8 T cell epitopes.

[0383] In some embodiments, an antigen used as described herein comprises one or more variant sequences relative to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is deleted or blocked; alternatively or additionally, in some embodiments, a terminally truncated antigen is used, and / or one or more mutations associated with a viral variant (e.g., a SARS-CoV-2 variant of concern) are present in the antigen.

[0384] In some embodiments, an antigen used as described herein comprises a multimerization element (e.g., a heterologous multimerization element).

[0385] In some embodiments, an antigen used as described herein comprises a membrane associating element (e.g., a homologous membrane associating element), such as a transmembrane domain.

[0386] In some embodiments, an antigen used as described herein comprises a secretion signal (e.g., a homologous secretion signal).

[0387] In some embodiments, the sequences used may comprise one or more mutations associated with a viral variant (e.g., a variant that is prevalent and / or is believed to be highly immune evasive). In some embodiments, the sequences used comprise one or more mutations associated with a variant of concern (e.g., a variant of concern identified by the WHO). In some embodiments, the sequences used comprise one or more mutations associated with a viral variant that has been determined to be or was predicted to be highly immune evasive (e.g., highly immune evasive compared to an immune response developed in subjects who received a previously approved vaccine and / or a previously prevalent viral variant).

[0388] The present invention relates to, among other things, certain SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, a SARS-CoV-2 antigen comprises 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, these antigens are delivered as protein antigens to induce an immunogenic response. In some embodiments, such antigens are delivered using RNA (e.g., a modRNA encoding an S1 domain, a truncated S1 subdomain, and / or an RBD of a SARS-CoV-2 S protein and formulated into LNP particles) to induce an immunogenic response.

[0389] 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 1,273 amino acids and having an amino acid sequence according to SEQ ID NO: 1:

[0390] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHST QDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWM ESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSF TVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKR ISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKS NLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV VVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECD IPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAI PTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTD EMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE NQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSN FGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQ EKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGIN ASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGL IAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO : 1)

[0391] Unless otherwise indicated, the position numberings in a SARS-CoV-2 S protein given herein are in relation to the amino acid sequence of SEQ ID NO: 1. A person skilled in the art reading the present invention will understand and be able to determine corresponding positions in a SARS-CoV-2 S protein variant sequence from the locations of the provided positions relative to the amino acid sequence of SEQ ID NO: 1 (i.e., a person skilled in the art having 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 fragment thereof). A person skilled in the art will also understand that a fragment of a SARS-CoV-2 S protein that includes one or more mutations of a variant, includes only those mutations that lie within the region of the fragment.For example, if a truncated SI subdomain comprises an amino acid sequence corresponding to amino acids 20 to 528 of SEQ ID NO: 1 and comprises one or more mutations of a SARS-CoV-2 variant, a person skilled in the art will understand that the truncated SI subdomain comprises only the mutations located in the corresponding region of the SARS-CoV-2 variant.

[0392] In specific embodiments, a spike (S) protein described herein or a fragment thereof may be modified such that the prototypical prefusion conformation is stabilized. Certain mutations that stabilize a prefusion confirmation are known in the art, for example, as described 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 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 S protein of . SARS-CoV-2 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.

[0393] In some embodiments, stabilization of the prototypical prefusion conformation of a SARS-CoV-2 S protein may be achieved by introducing two consecutive proline substitutions at residues 986 and 987. More specifically, the spike (S) protein-stabilized protein variants are achieved such that the amino acid residue at position 986 is exchanged with a proline and the amino acid residue at position 987 is also exchanged with a proline. In one embodiment, a SARS-CoV-2 S protein variant in which the prototypical prefusion conformation is stabilized comprises the amino acid sequence shown in SEQ ID NO: 2:

[0394] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHST QDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIR GWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWM ESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIY SKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSS SGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSF TVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKR ISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKS NLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV VVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLP FQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLY QDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECD IPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAI PTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRS FIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTD EMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE NQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQ EKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFV SGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGIN ASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGL IAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0395] (SEQID NO: 2)

[0396] Those skilled in the art are aware of various SARS-CoV-2 spike variants and / or resources documenting them. For example, the following strains, their SARS-CoV-2 S protein amino acid sequences, and, in particular, their modifications relative to the wild-type SARS-CoV-2 S protein amino acid sequence, e.g., relative to SEQ ID NO: 1, are useful herein.

[0397] B.1.1.7 (“Variant of concern 202012 / 01” (VOC-202012 / 01)

[0398] B.1.1.7 (“alpha variant”) is a variant of SARS-CoV-2 that has been detected for the first identified in October 2020 in the UK from a sample collected the previous month and rapidly began to spread in mid-December. It correlates with a significant increase in the COVID-19 infection rate; this increase is thought to be at least partly due to a change in N501Y within the receptor-binding domain of the spike glycoprotein, which is required 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 modify proteins and six that do not). The spike protein modifications in B. 1.1.7 include deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0399] B.1.351 (501.V2)

[0400] The B.1.351 lineage (“beta variant”), colloquially known as the South African COVID-19 variant, exhibits increased transmissibility compared to the original Wuhan strain. The B.1.351 variant is defined by multiple changes in the spike protein, 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.

[0401] Bl1.298 (Cluster 5)

[0402] The B. 1.1.298 virus was discovered in North Jutland, Denmark, and is thought to have been transmitted from mink to humans via mink farms. Several different mutations in the spike protein of the virus have been confirmed. Specific mutations include deletions 69-70, Y453F, D614G, I692V, Ml2291, and possibly SI 147L.

[0403] Pl (B. 1.1.248)

[0404] Lineage B. 1.1.248 (the “gamma variant”), known as the Brazilian or Brazil variant, is one of the SARS-CoV-2 variants that has been named the PL lineage. Pl exhibits a number of changes in the S protein (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, VI176F) and resembles in some key positions of the RBD (K417, E484, N501) the B.1.351 variant from South Africa.

[0405] B.1.427 / B.1.429 (CAL.20C)

[0406] The B.1.427 / B.1.429 lineage (the “epsilon variant”), also known as CAL.20C, is defined by the following changes in the S protein: S131, W152C, L452R, and D614G, of which the L452R change is of particular concern. The CDC has classified B.1.427 / B.1.429 as a “variant of concern.”

[0407] B. 1.525

[0408] B. 1.525 (“eta variant”) carries the same E484K modification found in variants Pl and B. 1.351, and also carries the same AH69 / AV70 deletion found in B.1.1.7 and B.1.1.298. It also carries modifications D614G, Q677H, and F888L.

[0409] B. 1.526

[0410] B. 1.526 (“iota variant”) has been detected as an emerging lineage of viral isolates in the New York City area that shares mutations with previously reported variants. The most common spike mutation sets in this lineage are L5F, T95I, D253G, E484K, D614G, and A701V.

[0411] Bl1.529

[0412] The B.1.529 virus (“Omicron variant”) was first detected in South Africa in November 2021. Omicron multiplies approximately 70 times faster than the Delta variants and has rapidly become the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, a number of Omicron sublineages have emerged. 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 has the same set of characteristic mutations, which is why the table below has a single row for “BA.4 or BA.5,” and why the present invention refers to a “BA.4 / 5” S protein in certain embodiments. Similarly, the S proteins of the BA.4.6 and BF variants.7 of Omicron have the same set of characteristic mutations, which is why the table below has only one row for the "BA.4.6 or BF.7").

[0413] The JN.l variant emerged in August 2023 in Luxembourg. It is a descendant of the BA.2.86 variant. The BA.2.86 variant initially attracted the attention of health authorities because it had a large number of S protein mutations compared to previous variants (~30 more than other variants circulating at the time). However, BA.2.86 never dominated the circulating SARS-CoV-2 variants. Unlike BA.2.86, the JN.l variant (and its descendants) has the ability to transmit efficiently between humans, an ability that is believed to be due to the acquisition of an L455S mutation in the S protein (position shown relative to SEQ ID NO: 1). JN.l rapidly became dominant among SARS-CoV-2 variants, increasing from less than 5% in November 2023 to 60% of cases in January 2024. Since the initial emergence of the JN.l variant, descendants have continued to be identified, including JN.1.2, JN.1.6, JN.1.7, KP.2, KP.3, and XEC, which have acquired additional mutations relative to JN.l, and are expected to further increase the infectivity and / or transmissibility of SARS-CoV-2 variants.

[0414] Since the emergence of JN.l, descendants of JN.l have emerged and rapidly supplanted the JN.l variant. These JN.l descendants include "SLip" variants (including, for example, JN.l.16), which include the L455S and F456L mutations; and "FLiRT" variants (including, for example, KS.1.1, KP.2), which include the mutations associated with the SLip variants and an additional R346T mutation. The FLuQE variants (for example, KP.3.3), in turn, are descendants of the FLiRT variants and include the same mutations plus an additional Q493E mutation. Position 455 has also continued to be a mutational hotspot, with "FLip" including the L455F and F456L mutations. 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.

[0415] The XEC variant is a hybrid of the KS.1.1 and KP.3.3 variants. A description of the emergence of the JN.l variant and its descendants is provided, for example, in E. Topol, “Are We FLiRTing With A New Covid Wave?”, April 18, 2024, available 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, available at www.independent.co.uk / news / science / covid-variant-xec-europe-symptoms-b2613485.html.

[0416] Table 1: Omicron variants of concern and characteristic mutations <h2 style=";text-align:left;direction:ltr">Sub-variable mutations BA.l A67V, A69-70, T95I, G142D, A143-145, A211, L212I, ins214EPE, G339D, S37IL, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F BA.2 T19I, A24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N5<h2 style=";text-align:left;direction:ltr"> 01Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969 K BA.2.12.1 T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E48 4A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N658S, N679K, P681H, N764K, D796Y, Q954H, N969K BA.4 or B T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, A.5 T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F48 6V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y , Q954H, N969K BA.2.75 T19I, A24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N44 OK, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H et N969K BA.2.75.<h2 style=";text-align:left;direction:ltr">2 T19I, A24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376A, D405N, R408S, K41 7N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K et D1199N BJ.l T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G339H, R 346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, S477N, T478K, V483A, E484A, F490V, Q493R, Q498R, N5 01Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, G798D, Q954H, N969K and S1OO3I BA.4.6 or T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, BF.7 S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E48 4A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N658S, N679K, P681H, N764K, D796Y, Q954H and N969K XBB T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G339H, R 346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q493R, Q49 8R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H et N969K XBB.l T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N , N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q49 . <h2 style=";text-align:left;direction:ltr">3R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K XBB.2 T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, D253G, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q49 3R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K XBB.1.3 T19I, A24-26, A27S, V83A, G142D, A144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, A484T, F486S, F490S, Q49 3R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K BA.2.3.20 T19I, A24-26, A27S, G142D, M153T, N164K, V213G, H245N, G257D, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444R, E4 84R N450D, L452M, N460K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H and N969K BQ.1.<h2 style=";text-align:left;direction:ltr">1 T19I, A24-26, A27S, A69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K, S47 7N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K JN.l inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L222I, V213G, L216F, H245N, A264D, I332V, G339 H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N 440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, PI 143 L JN.1.2 inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145 , F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339 H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K4 17N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478 K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D 614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969 K, P1143L JN.1.6 inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145 . F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339 . H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N 440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481 K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D6 14G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K , P1143L, E1150D. KP.2(1) inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145 , F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339 H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N 440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481 K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P6 21S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, PI 143 L, M12291 KP.2(2) inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145 , F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339 H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K4 17N, N440K, V445H, G446S, N450D, L452W, L455S, F456LN460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A57 OV, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, M1229I XEC inslôMPLF, T19I, R21T, T22N, A24-26, A27S, S5OL, A69-70, V127F, G142D , A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408 S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S 477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K , A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q9 54H, N969K, V1104L, P1143L JN.1.7 inslôMPLF, T19I, R21T, A24-26, A27S, S5OL, A69-70, V127F, G142D, A145 , F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339 H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K4 17N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N , T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A57 OV, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L XBB.1.5 T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417 N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q . 498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q95 4H, N969K

[0417] In some embodiments, the SARS-CoV-2 S proteins described herein comprise one or more mutations (including, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) characteristic of a certain Omicron variant (for example, one or more mutations of an Omicron variant listed in Table 1, for example, each of the mutations associated with an XBB, JN.1, KP.2, or XEC variant given in Table 1 above).

[0418] 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 to 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- variant 2 (e.g., one or more mutations associated with a SARS-CoV-2 variant listed in Table 1).

[0419] In some embodiments, an RNA comprises a nucleotide sequence encoding a polypeptide comprising an S protein fragment (e.g., an RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1 to 528 of SEQ ID NO: 1), or a variant thereof, wherein the S protein fragment 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 region of the S protein fragment. For example, in some embodiments, a construct comprises at least 5% of the mutations associated with a SARS-CoV-2 variant in the region corresponding to amino acids 1 to 528 of the S protein.

[0420] 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 to 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.

[0421] In some embodiments, the 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 comprise 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 comprise mutations at a position corresponding to position 455 of SEQ ID NO: 1 (e.g., L455F). In some embodiments, the one or more mutations comprise a mutation at a position corresponding to position 456 of SEQ ID NO: 1 (e.g., F456L).In some embodiments, the one or more mutations comprise mutations at positions corresponding to position 455 and 456 of SEQ ID NO: 1 (e.g., F456L and L455F). In some embodiments, the one or more mutations comprise 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 comprise a mutation at a position corresponding to position 1104 of SEQ ID NO: 1 (e.g., V1104L). In some embodiments, the one or more mutations comprise mutations at positions corresponding to position 346 and 1104 of SEQ ID NO: 1 (e.g., R346T and V1104L).

[0422] In some embodiments, one or more mutations characteristic of a KP.2 variant comprise one or more of the following mutations (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 (for example, the whole of) inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, 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, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and PI 143L, where the mutations are indicated relative to SEQ ID NO: 1.In some embodiments, one or more mutations characteristic of a KP.2 variant comprise R346T and V1104L, where the positions are indicated relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a KP.2 variant comprise R346T, F456L and / or V1104L, where the positions are indicated relative to SEQ ID NO: 1.

[0423] In some embodiments, one or more mutations characteristic of a KP.3 variant comprise one or more of the following mutations (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 more) (e.g. the entirety of) inslôMPLF, T19, A24-26, A27S, S50L, A69 / 70, V127F, G142D, A144, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, A483, 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 comprise F456L, Q493E and / or VI104L, the positions being indicated relative to SEQ ID NO: 1.

[0424] In some embodiments, one or more mutations characteristic of a XEC variant comprise one or more of the following mutations (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, 67 or more of (for example, the whole of) inslôMPLF, T19I, R21T, T22N, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, 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, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, PI 143L, where the mutations are indicated relative to SEQ ID NO: 1.In some embodiments, one or more mutations characteristic of a XEC variant comprise T22N, F59S, F456L, Q493E, and / or VI104L where the positions are indicated relative to SEQ ID NO: 1.

[0425] 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 more) from the following mutation list: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, 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, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, 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, A483, E484K, F486P, Q498R, N501Y, Y505H.

[0426] In some embodiments, one or more mutations characteristic of a JN.l variant comprise one or more of the following mutations (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 (for example, the entirety of) inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or any combination thereof, wherein the mutations are indicated relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a JN.l variant comprise inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145.F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K and P1143L, relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a JN.l variant comprise L455S.

[0427] In some embodiments, a fragment of an S protein (e.g., a truncated RBD or 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 mutations (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: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and PI 143L.In some embodiments, a fragment of an S protein comprises one or more mutations (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) from the following list of mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y and Y505H. .

[0428] In some embodiments, one or more mutations characteristic of a JN.1.2 variant comprise one or more of the following mutations (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 (for example, the whole of) inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L and M1229I, where the mutations are indicated relative to SEQ ID NO: 1.In some embodiments, one or more mutations characteristic of a JN.1.2 variant comprise M1229I where the position is indicated relative to SEQ ID NO: 1.

[0429] In some embodiments, one or more mutations characteristic of a JN.1.6 variant comprise one or more of the following mutations (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 (e.g. all) of the following mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R346T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L, or the mutations are indicated relative to SEQ ID NO: 1. In some embodiments, one or more mutations characteristic of a JN.1.6 variant comprise R346T.

[0430] In some embodiments, one or more mutations characteristic of a JN.1.7 variant comprise one or more of the following mutations (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 (e.g. the whole of): inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L and E1150D, where the mutations are indicated relative to SEQ ID NO: 1.

[0431] In some embodiments, a fragment of an S protein (e.g., a truncated RBD or 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 from the following mutation list: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145.F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H, 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 mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145. F157S, R158G, A201, L202I, V203G, L206F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, N460K, S477N, T478K, N481K, A483, E484K, F486P, Q498R, N501Y, Y505H. .

[0432] 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 mutations (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) from the list of following mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, 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, A483, E484K, F486P, Q498R, 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 mutations (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) from the following list of mutations: inslôMPLF, T19I, R21T, A24-26, A27S, S50L, A69-70, V127F, G142D, A145, F157S, R158G, A201, L202I, V203G, L206F, 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, A483, E484K, F486P, Q498R, N501Y and Y505H. .

[0433] In some embodiments, a fragment of an S protein (e.g., a truncated RBD or 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 the following list of mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, 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 mutations: T19I, A24-26, A27S, V83A, G142D, A145, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, . R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y and Y505H.

[0434] 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) the mutations associated with a KP.2, KP.3 or XEC variant (e.g., the 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 variant.l (e.g., as described herein) and one or more additional mutations associated with a descendant of a JN.l variant (e.g., a descendant described herein). In some embodiments, an S protein comprises a majority of mutations associated with a JN.l variant (e.g., as described herein) and one or more additional mutations associated with increased spread to a descendant of a JN.l variant (e.g., a descendant described herein).

[0435] In some embodiments, an S protein comprises a majority of mutations associated with a JN. 1 variant (e.g., as described herein) and:

[0436] (a) a mutation at a position corresponding to position 346 of SEQ ID NO: 1 (e.g., R346T);

[0437] (b) a mutation at a position corresponding to position 456 of SEQ ID NO: 1 (e.g., F456L);

[0438] (c) mutations at positions corresponding to positions 455 and 456 of SEQ ID NO: 1 (e.g., L455S and F456L);

[0439] (d) a mutation at a position corresponding to position 1104 of SEQ ID NO: 1 (e.g., V1104L).

[0440] In some embodiments, an S protein comprises a majority of mutations associated with a variant of KP.2 and / or JN.1 and one or more additional mutations. In some embodiments, an S protein comprises a majority of mutations associated with a variant of KP.2 and / or JN.1 and a mutation at a position corresponding to position 493 of SEQ ID NO:1 (e.g., Q493E).

[0441] In some embodiments, an S protein of an XEC variant comprises a majority of mutations associated with a KP.2, KP.3 and / or JN.l 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.l 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).

[0442] As used herein, each change in an amino acid is counted as a single mutation. For example, inslôMPLF would be counted as 4 mutations.

[0443] Immunogenic parts of the SARS-CoV-2 S protein

[0444] As noted elsewhere herein, in certain embodiments, the 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 “immunogenic fragment of a full-length SARS-CoV-2 S protein.”

[0445] An immunogenic portion of a SARS-CoV-2 S protein lacks certain features 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 both the NTD and the RBD (wherein 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-length 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 complete S2 domain, but includes certain sequences that may enhance the immunogenicity and / or stability of an immunogenic portion (e.g., in some embodiments, an immunogenic portion lacks a complete S2 domain but retains a TM sequence and optionally a sequence that is endogenously adjacent C-terminally to the TM sequence in a SARS-CoV-2 S protein).

[0446] 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.

[0447] 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 SI domain, possibly in which the endogenous secretory signal peptide in the NTD is replaced by a heterologous secretory signal peptide.

[0448] The S proteins of SARS-CoV-2 are well characterized, and one skilled in the art will be able to determine which portions of an S protein sequence correspond to the immunogenic portions described herein (e.g., which portions of an S protein sequence correspond to the NTD, RBD, S1, and S2 domains).

[0449] 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 to 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 to 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 to 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 in SEQ ID NO: 1, or to 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 in SEQ ID NO: 1, or to 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 in SEQ ID NO: 1, or to 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 327-528 of SEQ ID NO: 1, residues 330-528 of SEQ ID NO: 1, residues 330-541 of SEQ ID NO: 1, or residues 330-541 of SEQ ID NO: 1, or a corresponding region of any of the foregoing of an S protein of a SARS-CoV-2 variant.

[0450] In some embodiments, a SARS-CoV-2 RBD comprises the amino acid sequence: VRFPNITNLCPFHEVFNATTFASVYAW NRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRG NEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRL FRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQ PYRVVVLSFELLHAPATVCGPK (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.

[0451] In some embodiments, a SARS-CoV-2 RBD comprises the amino acid sequence: PNITNLCPFHEVFNATTFASVYAWNRK RISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEV SQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRK SKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYR VVVLSFELLHAPATVCGPK (SEQ ID NO: 325), or a sequence that is 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.

[0452] In some embodiments, a SARS-CoV-2 RBD comprises the amino acid sequence: VRFPNITNLCPFGEVFNATRFASVYAW NRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRG DEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRL FRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ PYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 4), or a sequence that is 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.

[0453] In some embodiments, a SARS-CoV-2 RBD comprises the amino acid sequence: VRFPNITNLCPFDEVFNATRFASVYAW NRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRG DEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSK (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.

[0454] 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 comprises 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 with a heterologous secretory signal peptide.

[0455] 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 to 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 to 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 to 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 in 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 in 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 in 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 in SEQ ID NO: 1, or to 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 and 318 (inclusive) of SEQ ID NO: 1.In some embodiments, an NTD of a SARS-CoV-2 S protein comprises residues 1-302 of SEQ ID NO: 1, residues 1-302 of SEQ ID NO: 1, residues 1-303 of SEQ ID NO: 1, residues 14-318 of SEQ ID NO: 1, residues 14-302 of SEQ ID NO: 1, residues 14-302 of SEQ ID NO: 1, residues 14-302 of SEQ ID NO: 1, residues 14-303 of SEQ ID NO: 1, residues 14-318 of SEQ ID NO: 1, residues 17-302 of SEQ ID NO: 1, residues 17-302 of SEQ ID NO: 1, residues 17-303 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 preceding regions of an S protein of a SARS-CoV-2 variant.

[0456] 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.

[0457] In some embodiments, an RNA comprising a nucleotide sequence that encodes a polypeptide comprising an NTD polypeptide may 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 may be administered in combination with one or more RNAs encoding a polypeptide comprising an NTD polypeptide, wherein the one or more RNAs encoding a polypeptide comprising an RBD polypeptide and the one or more RNAs encoding a polypeptide comprising an NTD polypeptide may be formulated in the same nanoparticles or in separate nanoparticles.

[0458] 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 [Fig. 6]. Table LXIV#: Examples of NTD sequences <h2 style=";text-align:left;direction:ltr">NTD Wuhan NTD (language) TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKS NIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDP FLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLM DLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLP QGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGW TAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPL SETKCTLKSFTVEKGIYQTSNF (SEQ ID NO: 643) Wuhan NTD (courte) TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKS NIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDP FLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLM SEQ ID NO : 644<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">NTD (longue) Omi cron BA. 1 TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNII RGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFL DHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFS ALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALPLSETKC TLKSFTVEKGIYQTSNF (SEQ ID NO: 645) NTD d'Omicron B Al (courte) TRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNII RGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFL DHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGK QGNFKNLREFVFKNIDGYFKIYSKHTPIIFREPEDLPQGFS ALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CT (SEQ ID NO : 646)<h2 style=";text-align:left;direction:ltr">

[0460] In some embodiments, a SARS-CoV-2 NTD 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.

[0461] In some embodiments, a SARS-CoV-2 NTD 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.

[0462] In some embodiments, a SARS-CoV-2 NTD 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.

[0463] In some embodiments, a SARS-CoV-2 NTD 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.

[0464] 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.

[0465] In some embodiments, an S1 domain of a SARS-CoV- S protein 2 includes the amino acid sequence: MFVFLVLLPLVSSQCVNLITRTQSYTN SFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDN PALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCE FQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEG NFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINI TRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTIT DAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCP FHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLN DLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSN KLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYS PLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF NFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSF GGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVF QTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 5), or a sequence that is 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 to this one.

[0466] In some embodiments, an S1 domain of a SARS-CoV- S protein 2 includes the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPA YTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKR FDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIK VCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPI GINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNEN GTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNIT NLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNK CVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDIT PCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTG SNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQT (SEQ ID NO: 6), or a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, or 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.

[0467] 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 the RBD are joined via an endogenous linker, and in which 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 located at the C terminus of the truncated S1 subdomain, and the endogenous secretory signal peptide in the NTD is optionally replaced with a heterologous secretory signal peptide.

[0468] In some embodiments, a truncated S1 subdomain comprises or consists of amino acids 20-528 of SEQ ID NO: 1 (e.g., amino acids 14-528 or 17-528 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 20-541 (e.g., amino acids 14-541 or 17-541 of SEQ ID NO: 1), or a corresponding region in a SARS-CoV-2 variant, or a variant of either of the foregoing.

[0469] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFR SSVLHSTQDLFLPFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFAST EKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH KNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSY LTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYL YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPK (SEQ ID NO: 362), or a sequence that is 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.

[0470] In some embodiments, an SI subdomain of a S protein of SARS-CoV-2 includes the amino acid sequence: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFR SSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFAST EKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYH KNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNI DGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSY LTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFV IRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYL YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 363), or a sequence that is 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.

[0471] In some embodiments, a truncated SI subdomain of a S protein of SARS-CoV-2 includes the amino acid sequence: QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQ TGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFE RDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE LLHAPATVCGPK, or a sequence that is 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.

[0472] In some embodiments, a truncated SI subdomain of a S protein of SARS-CoV-2 includes the amino acid sequence: QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLP FFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRV YSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAG AAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGI YQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVA DYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQ TGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFE RDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE LLHAPATVCGPKKSTNLVKNKCVNF, or a sequence that is 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 to this one.

[0473] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTL DSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYS SANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINL VRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQ TSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADY SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERD ISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPK, or a sequence that is 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.

[0474] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFF SNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTL DSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYS SANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINL VRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAA AYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQ TSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADY SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERD ISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL HAPATVCGPKKSTNLVKNKCVNF, 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 to it.

[0475] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: TTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNV TWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSK TQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRD LPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYY VGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSN FRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP ATVCGPK, or a sequence that is 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.

[0476] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: TTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNV TWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSK TQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSAN NCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRD LPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYY VGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSN FRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIST EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP ATVCGPKKSTNLVKNKCVNF, or a sequence that is 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.

[0477] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFL PFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTT LDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYS SANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIG RDFPQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAAD YYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQT SNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYS VLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGN IADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDI STEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPK, 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 to this.

[0478] In some embodiments, a truncated S1 subdomain of a SARS-CoV-2 S protein comprises the amino acid sequence: QCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLD SKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSA NNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFKIYSKHTPINLER DLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAY YVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTS NFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSV IYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNI ADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDIS TEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHA PATVCGPK, or a sequence that is 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.

[0479] 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.

[0480] In some embodiments, the 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 and / or shown to spread rapidly in a territory concerned, for example, 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 United Kingdom, the Canadian COVID Genomics Network (CanCOGeN) and / or the World Health Organization (WHO)). In some embodiments, a variant has been predicted to have a high probability of becoming a variant of concern (e.g., using sequence-based algorithms that predict a variant's ability to evade previously developed immune responses and / or measure the "fitness" of a given variant, as described, for example, 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 descendant thereof.

[0481] In some embodiments, an RBD comprises mutations associated with a variant described herein. One skilled in the art will be able to identify which portions of a given variant correspond to the immunogenic portions described herein.

[0482] In some embodiments, a polypeptide comprises two or more subdomains of SARS-CoV-2 (e.g., two or more S1 domains, truncated S1 subdomains, or RBDs or variants thereof). In some embodiments, a polypeptide comprises two or more tandemly linked receptor binding domains, 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 reference herein 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., 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

[0483] In some embodiments, an antigen construct described herein comprises a secretory signal, for example, that is functional in mammalian cells. In some embodiments, a secretory signal used is a heterologous secretory signal (i.e., heterologous with respect to a SARS-CoV-2 antigen present in the same polypeptide). In some embodiments, a secretory signal used 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, for example, comprising 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 “secretory signal peptide.”

[0484] 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.

[0485] In some embodiments, a secretory signal is characterized by a length of about 15 to 30 amino acids.

[0486] 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 enables transport of the SARS-CoV-2 antigen construct with which it is associated into a defined cellular compartment, preferably a cell surface, an endoplasmic reticulum (ER), or an endosomal-lysosomal compartment.

[0487] 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 or HSV-2 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 know of other secretory signals such as, for example, those described in WO2017 / 081082, which is incorporated herein by reference in its entirety (for example, SEQ ID NO: 1 to 1115 and 1728, or variant fragments thereof).

[0488] In some embodiments, a SARS-CoV-2 antigen construct described herein does not include a secretory signal.

[0489] In some embodiments, a signal peptide is an IgG signal peptide, such as an IgG kappa signal peptide.

[0490] In some embodiments, a SARS-CoV-2 secretory signal comprises or consists of an HSV glycoprotein D (gD) secretory signal.

[0491] In some embodiments, a segment construct sequence encodes an antigen that may comprise or be otherwise linked to a signal sequence (e.g., a secretory signal), such as those listed in Table 2 or at least one sequence having 1, 2, 3, 4, or 5 amino acid differences therefrom. In some embodiments, a secretory signal such as MFVFLVLLPLVSSQCVNLT (SEQ ID NO: 9), or at least one sequence having 1, 2, 3, 4, or at most 5 amino acid differences therefrom is used.

[0492] In some embodiments, a secretory signal is selected from a gl signal peptide. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGLVVR (SEQ ID NO: 10), or at least one sequence having 1, 2, 3, 4 or at most 5 amino acid differences therefrom is used. In some embodiments, a secretory signal such as MPGRSLQGLAILGLWVCATGL (SEQ ID NO: 11), or at least one sequence having 1, 2, 3, 4 or at most 5 amino acid differences therefrom is used.

[0493] In some embodiments, an antigen comprises an affinity tag (e.g., a short sequence that can bind to an affinity reagent, and which may be useful, for example, for affinity purification of a protein). Suitable affinity tags are known in the art and include, for example, a His-tag (e.g., HHHHHHHH) and a StrepTag® (e.g., WSHPQFEK). In some embodiments, an affinity tag may be included at the N-terminus of an antigen (e.g., adjacent to a secretory signal (to which it is optionally connected via a flexible linker)).

[0494] In some embodiments, a secretory signal is a signal listed in Table 2 and / or Table 3, or a secretory signal having 1, 2, 3, 4 or 5 amino acid differences therefrom (optionally, with corresponding changes in a corresponding nucleotide sequence provided in Table 3). In some embodiments, a secretory signal is selected from those included in Table 2 below and / or those encoded by the sequences in Table 3 below.

[0495] In some embodiments, an RNA (e.g., an mRNA) encodes a polypeptide comprising amino acids 20 to 292 of SEQ ID NO: 145 (or variants thereof) and a secretory signal of Table 2.

[0496] Table 2: Examples of secretory signals Secretory signals Sequence (amino acid) SEQ ID NO: HSV-1 gD_SKY MGGAAARLGAVILFVVIVGLHG VRSKY 7 HSV-1 gD_RG MGGAAARLGAVILFVVIVGLHG VRG 12 HSV-2 gD SP MGRLTSGVGTAALLVVAVGLR VVCA 8 HSV-2 gD + KYA MGRLTSGVGTAALLVVAVGLR VVCAKYA 13 Csp (isolate 3D7) MMRKLAILSVSSFLFVEA 14 SARS-CoV-2-S (SP14) MFVFLVLLPLVSSQ 364 SARS-CoV-2-S (SP 16) MFVFLVLLPLVSSQCV 15 SARS-CoV-2-S (SP19)_1 MFVFLVLLPLVSSQCVNLT 9 SARS-CoV-2-S (SP19)_2 MFVFLVLLPLVSSQCVNLI 16 SARS-CoV-2-S (HIS) MFVFLVLLPLVSSQCHHHHHHH HGGSG 365 signal secretory chain lourde d'I g humaine (huSec) MDWIWRILFLVGAATGAHSQM 17 Secretory signal of HuIgGk METPAQLLFLLLLWLPDTTG 18 Secretory signal of chain IgE lou rde epsilon-1 MDWTWILFLVAAAATRVHS 19 Secretory signal of PRM of Japanese encephalite MLGSNSGQRVVFTILLLLVAPA YS 20 Secretory signal of VSV g protein MKCLLYLAFLFIGVNCA 21 Indiana vesicular stomatitis virus glycoproteins MKCLLYLAFLFIGVNC 397 TRIO MCRGLSAVLILLVSLSAQLHVV VG 22 Secretory signal 1 of human Ig heavy chain MELGLSWIFLLAILKGVQC 23 Secretory signal 2 ofhuman Ig heavy chain MELGLRWVFLVAILEGVQC 24 Human Ig heavy chain secretory signal 3 MKHLWFFLLLVAAPRWVLS 25 Human Ig heavy chain secretory signal 4 MDWTWRILFLVAAATGAHS 26 Human Ig heavy chain secretory signal 5 MDWTWRFLFVVAAATGVQS 27 Human Ig heavy chain secretory signal 6 MEFGLSWLFLVAILKGVQC 28 Human Ig heavy chain secretory signal 7 MEFGLSWVFLVALFRGVQC 29 Human Ig heavy chain secretory signal 8 MDLLHKNMKHLWFFLLLVAAP RWVLS 30 Human Ig kappa chain secretory signal 1 MDMRVPAQLLGLLLLWLSGAR C 31 Human Ig kappa chain secretory signal 2 MKYLLPTAAAGLLLLAAQPAM A 32 HSV-2 gIVVR MPGRSLQGLAILGLWVCATGL WR 10 gl of HSV-2 MPGRSLQGLAILGLWVCATGL 11 HSV-1 gD (2) MGGAAARLGAVILFVVIVGLHG VRGKY 33 HSV-2 gD + KYAL MGRLTSGVGTAALLVVAVGLR VVCAKYAL 34 HSV-2 gD + KYALA MGRLTSGVGTAALLVVAVGLR VVCAKYALA 35 HSV-2 gC signal peptide MALGRVGLAVGLWGLLWVGV VVVLANA 36 IL2 signal peptide MRMQLLLLIALSLALVTNS 37 gB of HSV-1 MHQGAPSWGRRWFVVWALLG LTLGVLVASAAP 38 gE of HSV-2MARGAGLVFFVGVWVVSCLAA APRTS 39 gE of HSV-2 (short) MARGAGLVFFVGVWVVSCLA 366 Spicule glycoprotein GP of Ebola virus MGVTGILQLPRDRFKRTSFFLW VIILFQRTFSIP 40 Spicule glycoprotein GP (2) of Ebola virus MGVTGILQLPRDRFKRTSFFLW VIILFQRTFS 367 Signal peptide 41 MDSKGSSQKGSRLLLLLVVSNL LLPQGVVG 42 Signal sequence of the Japanese encephalitis JEV MWLVSLAIVTACAGA 43 MFVFLVLLPLVSSQC 44 HSV-1 gD (KOS strain) MGGAAARLGAVILFVGVGLVHVHVHVHVHVHVG 45 of influenza A (strain A / Duck / Ireland / 113 / 1983 H 5N8) MEEIVLLFAIVSLARS 368 HA protein of influenza A virus (strain A / Turkey / Ontario / 7732 / 19 66 H5N9) MERIVIALAIISVVKG 369 Bovine virus.1 (P8-2 strain) MQGPTLAVLGALLAVAVS 370 Anopheles gambiae (Frica mosquito and malaria vector) MCRGLSAVLILLVSLSAQLHVV VG 371 Ustilago maydis P4 virus MQIINVVYSFLFAAAMLPVVHS 372 Influenza A virus hemagglutinin (A / Equine / Detroit / 1 / 1964 H7N7 strain) MNTQILILATSAFLCVRA 373 Influenza A virus hemagglutinin (A / Shearwater / Australia / 1975 H5N3 strain) MERVVLLLAMISLVKS 374 Kidney herpesvirus 1 glycoprotein G1 (Rice strain) MRPFLLRAAQLLALLALALS 375 . Influenza A virus hemagglutinin (strain A / Wilson-Smith / 1933 H INI) MKAKLLVLLYAFVATDA 376 Influenza A virus hemagglutinin (strain A / Turkey / Ireland / 1378 / 1983 H5N8) MEKIVLLFAIVSLVRS 377 Hantaan virus envelope glycoprotein (strain 76-118) MGIWKWLVMASLVWPVLT 378 Influenza A virus (strain A / Equine / New Market / 1 / 1977 H7N7) MNTQILILAISAFLCVRA 379 New Jersey vesicular stomata virus glycoprotein G (strain Ogden subtype Concan) MLSYLIFALAVSPILG 380 Vaccinia virus complement control protein (strain Weste m Reserve / WR) MKVESVTFLTLLGIGCVLS 381 Human adenovirus serotype 2 C Early glycoprotein of E3 of 18,5 kDa MRYMILGLLALAAVCSA 382 Influenza A Virus Hemagglutinin (Strain A / Gull / Maryland / 704 / 19 77 H13N6) MALNVIATLTLISVCVHA 383 Influenza A Virus Hemagglutinin (Strain A / Japan / 30 / 35 H129) MAIIYLILLFTAVRG 384 Hemagglutinin of influenza A virus (strain A / Duck / Ukraine / 1 / 1960 H11N9) MEKTLLFAAIFLCVKA 385 Hemagglutinin of influenza A virus (strain A / Kiev / 59 / 1979) Herpesvirus Saimiriine 2 (strain 11) Glycoprotein H MTILQLFLVFLNILEA 387 Influenza A virus hemagglutinin (strain A / Silky chicken / Hong Ko ng / YU100 / 2002 H5N1 genotype X3) MEKIVLLLAIV 38KVSLV, Feline leukemia virus T cell receptor beta chain Tl 7T-22 MISWLPSVAMGSRLLCCVALCL LGAGPA 389 Influenza A virus hemagglutinin (A / Tern / Australia / G70C / l 975H11N9 strain) MEKLLLFATIILCVKA 390 Bovine coronavirus hemagglutinin esterase (Mebus strain) MFLLLRFVLVSCIIGSLG 391 Heliothis armiger entomopoxvirus a Spindolin MNKFYYICIYINILYVCVSG 392 Mumps virus FO fusion glycoprotein (RW strain) MKAFSVTCLGFAVFSSSIC 393 Homo sapiens (human) insulin MALWMRLLPLLALLALWGPDP AAA 394 Coronavirus spike glycoprotein severe acute respiratory syndrome (SARS-CoV) MFIFLLFLTLTSG 395 CD45 SP MTMYLWLKLLAFGFAFLDTEV FVTG 396 Table 3 Examples of polynucleotide sequences coding for secretory signals Signal Sequence (nucleotide) SEQ ID NO: gD of wild-type HSV-1 AUGGGGGGGGCUGCCGCCAGGUUGGGGCCG UGAUUUGUUUGUCGUCAUAGUGGGCCUCCA UGGGGUCCGCAGCAAAUAU 46 HSV-1 gD AUGggaggagccGCCGCCagacUgggaGCCGUGaUccU gUUcgUggUgaUcGUGggacUgCAUggagUgagaAGCa agUac 47 gD of HSV-1 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCG UGAUCCUGUUCGUGGUUAUCGUGGAGCAGUGGAGGAGCA398 Otidike nucleus sequence see Variant 4 gD of HSV-1 Otidike nucleus sequence gD see Variant 5 AUGGGAGGAGCCGCCGCCAGACUGGGAGCCG UGAUCCUGUGGUGAUCGUGGACUGCA UGGASVGUGAGAAGC H3-B 391 AUGCAUCAGGGCCUCCAUGGGGGGUAGAC STREET STRESS CCU 400 SARS-CoV-2-S (SP16) Variant 1 AUGGCCCUCCAUGGCH 48 SARS-CoV-2-S SP19 Variant 1 AUGUSTREQUESTIMAGE 49 SARS-CoV-2-S SP19 Variant 2 AUGUST5VIDUALIZATIONQUALIFICATION Secretory signal and chain 1 of the Ig huSec (huSec) AUGGAUGGAUUUGGAGAGAAUCCUGUUCCUC AUGGAGUUGGGGAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG outage out 52 Secretory signal e of chain 1 ourde of the Ig hu maine Secretory signal e of chain 1 orde of the Ig hu maine AUGAAAACCUGUGGUUCUUCUCCUGCUGG UGGCAGCUCCCAGAUGGGUCCUGUCC 54 Secretory signal e of chain 1 orde of the Ig huGGAGUCGUCGGACUGA UGGCAGCAGCAACAGGUGCCCACUCG 55 Secretory signal of chain 1 ourde of the Ig hu maine AUGGACUGGACCUGGAGGUUCCUUUGUGG UGGCAGCAGCUACAGGUGUCCAGUCC 56 Secretory signal of chain 1 ourde AUGGAGUUUGGGCUGAGCUGGCUUUUUCUU GUGGCGAUUCUAAAAGGUGUCCAGUGU 57 Signal sécrétoir e de la chaîne 1 ourde de l'Ig hu maine AUGGAGUUUGGGCUGAGCUGGGUUUUCCUC GUUGCUCUUUUUAGAGGUGUCCAGUGU 58 Signal secretory signal e of chain 1 ourde of the Ig hu maine AUGGACCUCCUGCACAAGAACAUGAAACACC UGUGGUUCUUCCUCCUCCUGGUGGCAGCUCC CAGAUGGGUGCUGUCC 59 Secretory signal of chain k appa of the IgGGUGCUGUCGA maine GGCUCCUGCUGCUGGCCUAGGUGCCAG AUGU 60 Secretory signal e of the k appa chain of the Ig hu maine AUGAAAUACCUAUUGCCUACGGCAGCCCGCUG GAUUGUUAUUACUCGCGGCAGCCGCCAU GGCC 61 Variant2 of the HSV-2 gl AUGCCAGGACGGAGCCUUCAGGGCUUGGCCA UACUGGGGCUUUGGGUGUGUGCAACCGGGU UGGUAGUUCGA 62 G1 of HSV-2 Variant 1 AUGCCUGCAGCACUGCAAGGACUGGCCA 63 Variant 3 of the gl of HSV-2 64 HSV-1 gD AUGGGAGGCGCAGCUGCCAGUGCUGUG UGAUCCUGGGGGGCUGC AU 65 HSV-1 gD _ajo uter AUGGGAGGCGCAGCUGCCAGUGCUGUGUG UGAUCGGAGGCGCAGCUGCCAGUGCUGUG UGAUCCUGGCGCAGCUGCCAGUGCUGUG UGAUCCUGGCGCAGCUGCCAGUGGUGCUG HSV-2 gDwt AUGGCCGCCUGACCUCCGGCGUGGGCACCG CCGCCCUGCUGGUGGCCGUGGGCCUGCG CGUGGUGUGCGCC 67 ​​gD2 du HSV-2 variant 1 68 gD of HSV-2 Variant 2 HSV-2 Variant 2 AGUGGUGUGCGCC 70 HSV-2 gD KY A wt AUGGGCCGCCUGACCUCCGGCGUGGGCACCG CCGCCCUGCUGGUGGGCCGUGGGCCUCCG CGUGGUGUGCCCAAGUACGCC 71 HSV-2 gD _K YA VariantGGUCGACUCGA 1 CUGAGCUGA CUGCUGCUGGUGGGGCCUGAGAGUCGUGUGCC 72 HSV-2 gD _K YA Variant 2 AUGGGAGACUCACAUCAGGCGUAGGAACCG CUGCCCUGUGGCCAUAUGCU 73 HSV-2 gDKYA Variant 3 AUGGGCAGACUGACCUCCGGCGUGGGCACCG CCGCCCUGCUGGUGGCCGUGGGCCUGAG AGUGGUGUGCGCC 74 HSV-2 gD _K YAL Variant 1 AUGGGCAGACUGACAUCUCAGGGCGUGGCC HSV-2 gD _K YAL Variant 2 HSV-2 gD _K YAL Variant 2 CUGCCCUGCUGGUGGGCCAUACGCCCUGG75 HSV2 _K YAL Variant 3 AUGGGCAGACUGACCUCCGGCGUGGGCACCG CCGCCCUGCUGGUGGCCGUGGGCCUGAG AGUGGUGUGCGCCAAACGCCCUG 77 gC du HSV-2 Variant 2 AUGGCCUUGGGAGAGAGUGGGGCCUUG IL2 Wild Type AUGAGACCAUGCCCUGCCCCCCCC 79 IL2 Variant 80 IL2 Variant 2 AUGACUGCAUGCCUGCUGCACAGC 81 IL2 Variant 3 AUGAGAAUGCCUGCCCUGCCCCU 82 AUGACUGCUGCUGCCA1 variant UCCUCGGACUGGGUUUGGCCAACAGGCCU G 83 12 Variant 2UACUGGGGGCUUUGGGUGUGCAACCGGUGUC U 84 12 Variant 3 AUGCCUGGAAGAUCUCUGCAGGGACUGGGACUGCAA UUCUGGGACUGGGUGUGUGCAACAGGAC UG 85 gE of HSV-2 Variant 2 AUGGCACGGGGAGCCGGAUUGGUGUUCUUU GUGGGCGUGUGGGUGGUGCUGCUUGGCA GCCGCACCACGCACCUCU 86 gE of HSV-2 Variant 3 AUGGCUAGAGGUGCCGGCCUGGUGUUCUUG UUGGCGUGUGGGUCGUGUCCCUGUCUGGCU 401 gD of HSV-1 ( strain F) AUGGGGGGGCUGCCGCCAGGUUGGGGGCCG UGAUUUUGUUUGUCGUCAUAGUCGGCCUCCA UGGGGUCCGCGGC 87 gD of HSV-1 (strain 1) AUGGGGCGUUUGACCUCCGGCGUCGGGACGG CGGCCCUGCUAGUUGUCGCGGUGGGACUCCG CGUCGUCUGCGCC 88 HA protein of influenza A virus (strain A / Canard / Ireland / 113 / 1983 H5N 8) UGUCCCUGGCCAGAUCU 402 HA protein of influenza virus A (strain A / Turkey / Ontario 0 / 7732 / 1966 H 5N9)_1 AUGGAAGAGAUCGUGCUGCUGUUCGCCAUCG UGUCCCUGGCCAGAUCU 403 HA protein of influenza A virus (strain A / Turkey / Ontari 0 / 7732 / 1966 H 5N9)_2 AUGGAACGGAUCGUGAUCGCCCUGGCCAUCA UCUCUGUGGUCAAGGGA 404 Herpesvirus bo vin 1.1 (strain P8-2) AUGCAGGGACCUACACUGGCUGCUGGAG 405 Anopheles gam biae (mosquito AUGUGUAGCACUGCUGGCCGCCUCCUCC0606006 African malaria vector) U stilago maydis virus P4 AUGAACACCCAGAUCCUGAUCCUGGCCACCA GCGCCUUCUGUGUGUUAGAGCU 408 Influenza A heme agglutinin virus (strain A / Shearwater / Australia / 1975 H5N3) GI glycoprotein of herpesv irus 1 pig type 1 (strain Rice) AUGAGGCCCUUCCUGCUGAGAGCUGCUCAAC UGCUGGCUCUGCUUGCCCUGGCUCUUUCU 410 Hemagglutinin of influenza A virus (strain A / Wilson-HlN / Smith HlN1933) AUGAAGGCCAAGCUGCUGGUGCUGCUGUACG CCUUUGUGGCCACUGAUGCU 411 Hemagglutinin of influenza A virus (strain A / Turkey / Ireland / 1378 / 198 3 H5N8) Envelope glycoprotein of v AUGGGCAUCUGGAAGUGGCUGGUUAUGGCC AGCCUUGUGUGUGGCCUGUGCUGACA 413 irus Hantaan (s ouche 76-118) Influenza A virus (strain A / Equine / New M arket / 1 / 1977 H 7N7) (Ogden sub-type and Concan strain) 415 Vaccine virus complement protein (only Western R reserve) The UGCUNCUGUGUGUGUGUCU 416 Sported Sported Sported Adoninvious Glycoprot and E3 of E3 of 18.5 knate 18.5 kn grass A (shoe. A / Gull / Maryla nd / 704 / 1977 H 13N6) AUGGCCCUGAACGUGAUCGCCACACUGACCC 418 Hemagglutinin from the G rippe A virus (strain A / Japan / 305 / 19) 57 CUGCUACAUGCAUHC2 CAGCCGUGCGGGGA 419...

Claims

Claims

1. A composition comprising an RNA molecule comprising a nucleotide sequence that encodes a polypeptide comprising: (i) a truncated S1 subdomain of a S protein of SARS-CoV-2; (ii) a heterologous secretory signal peptide; and (iii) a homologous transmembrane domain, wherein the RNA comprises a modified uridine in place of each uridine; and optionally, wherein the truncated S1 domain comprises amino acids 20 to 528 of SEQ ID NO: 1 or a corresponding region of an S protein of a variant of SARS-CoV-2.

2. The composition of claim 1, wherein the N-terminal to C-terminal orientation of the polypeptide is (secretory signal peptide)-(truncated SI subdomain)-(transmembrane domain).

3. A composition according to claim 1 or 2, wherein: (i) the secretory signal peptide comprises: (a) an amino acid sequence of MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 22) or an amino acid sequence which 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); (b) an 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); (c) 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);(d) an 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 MHQGAPSWGRRWFVVWAL LGLTLGVLVASAAP (SEQ ID NO: 38); or (e) an 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); (ii) the transmembrane domain comprises: (f) an amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC MTSCCSCLKGCCSCGSCC), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 90; or (g) an amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC MTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence that is at least 70%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 89;and (iii) the polypeptide does not comprise a soluble multimerization domain (e.g., a trimerization domain, including, for example, a T4 fibritin domain).;

4. A composition according to any one of claims 1 to 3, wherein the truncated S1 subdomain and the transmembrane domain are connected by a flexible linker, wherein the flexible linker comprises about 10 to about 20 amino acids (e.g., about 15 amino acids), optionally wherein the flexible linker comprises a (GRS)2, (G4S)3 or (G4S)4 sequence.

5. A composition according to any one of claims 1 to 4, wherein the RNA comprises: (i) the nucleotide sequence of SEQ ID NO: 236 or a sequence which 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; the nucleotide sequence of SEQ ID NO: 238 or a sequence which 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 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; or (ii) 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; 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 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.

6. A composition according to any one of claims 1 to 5, wherein the fragment of an S protein comprises one or more mutations of a SARS-CoV-2 variant (e.g., a JN.1, KP.2 or XEC variant, or a descendant thereof).

7. A composition according to any one of claims 1 to 6, wherein the polypeptide also comprises one or more T cell epitopes from a SARS-CoV-2 protein that is not an S protein, optionally wherein the one or more T cell epitopes from a SARS-CoV-2 nucleocapsid (N) protein, an NS9b protein, a membrane (M) protein, an ORFlab protein, an ORF3a protein, an ORF9b protein or NSP1-4, or any combination thereof.

8. A composition according to any preceding claim, wherein: (i) the RNA comprises a 5' cap, a cap-proximal sequence, a 5' UTR sequence, a 3' UTR sequence and a polyA sequence; and (ii) the modified uridine is Nl-methyl-pseudouridine; and optionally wherein the 5' cap comprises m27,3'-OGppp(ml2'-O)ApG.

9. A composition according to any one of claims 1 to 8, wherein the RNA is formulated in a nanoparticle, optionally wherein the nanoparticle is a lipid nanoparticle, a polyplex (PLX), a lipidated polyplex (LPLX), a liposome or a polysaccharide nanoparticle.

10. A composition according to any one of claims 1 to 9, comprising a cryoprotectant, optionally wherein the cryoprotectant is or comprises sucrose.

11. A composition according to any one of claims 1 to 10, wherein the composition comprises at least one unit dose of RNA molecules encapsulated in the LNP, wherein the unit dose comprises about 100 pg or less of total RNA or 90 pg, about 60 pg, about 30 pg, about 25 pg, about 20 pg, about 10 pg, about 6 pg, about 5 pg or about 3 pg of total RNA.

12. A composition according to any one of claims 1 to 11, for use in a method of inducing an immune response against SARS-CoV-2 in a subject, the method comprising administering the composition to the subject.

13. A composition for use according to claim 12, wherein: (i) the subject is 12 years of age or older and the composition is formulated to provide a dose of 30 μg of RNA, (ii) the subject is 5 years of age to less than 12 years of age, and the composition is formulated to provide a dose of 10 μg of RNA, or (iii) the subject is 6 months of age to less than 5 years of age, and the composition is formulated to provide a dose of 3 μg of RNA; and optionally, wherein the composition is administered in a volume of about 200 μl to about 300 μl.

14. Use according to claim 12 or 13, wherein a single dose or two or more doses of the composition are administered to the subject.

15. Use according to any one of claims 12 to 14, further comprising administering one or more vaccines against a disease unrelated to SARS-CoV-2, wherein the one or more vaccines optionally comprise an RSV vaccine, an influenza vaccine or a combination thereof.

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