SARS-cov-2 immunogenic compositions

Abstract

To provide RNA-containing compositions usable for inducing immune response against SARS-CoV-2 and use methods thereof.SOLUTION: Provided herein is a ribonucleic acid (RNA) comprising a nucleotide sequence encoding a polypeptide, where the polypeptide comprises: (i) a truncated S1 subdomain of SARS-CoV-2 spike (S) protein or a variant thereof; (ii) a heterologous secretory signal peptide and (iii) a homologous transmembrane domain, where optionally the polypeptide comprises, from the N-terminus to the C-terminus, the secretory signal peptide, truncated S1 subdomain and the transmembrane domain in this order. The invention further provides an immunogenic composition and therapeutic formulations, production methods and use methods thereof.SELECTED DRAWING: None

Description

Background Art

[0001] SARS-CoV-2 first emerged in 2019 and rapidly spread around the world, resulting in millions of deaths. The development and approval of the first-generation SARS-CoV-2 vaccines within less than 12 months was an epoch-making scientific achievement and helped save countless lives. Since the development of such initial vaccines, the understanding of the SARS-CoV-2 disease has further advanced, and currently, researchers have a deeper understanding of the SARS-CoV-2 virus, the progression of the disease, as well as the immune responses generated by infection and vaccines. At the same time, SARS-CoV-2 continues to evolve, and thousands of variants have emerged around the world, many of which have improved growth rates and immune evasion capabilities compared to the initial SARS-CoV-2 strains.

Summary of the Invention

[0002] In particular, the present disclosure describes 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 improved compared to first-generation approved SARS-CoV-2 vaccines (e.g., vaccines that deliver the full-length SARS-CoV-2 S protein, such as LNP-formulated RNA vaccines encoding the full-length prefusion-stabilized S protein (e.g., Comirnaty from Pfizer and SpikeVax from Moderna)). Examples of the improved immune response provided by the technologies described herein include, for example, higher neutralizing antibody titers against SARS-CoV-2 variant strains of concern and / or higher antibody titers against a broader range of SARS-CoV-2 variant strains. In some embodiments, the technologies provided herein are more potent than first-generation vaccines (e.g., can induce higher titers of neutralizing antibodies with a given concentration of RNA and / or can induce similar titers of neutralizing antibodies with 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 can be useful, for example, for extending the half-life of pharmaceutical compositions and / or eliminating the need to store and transport pharmaceutical compositions at ultra-low temperatures.

[0003] The technologies provided herein include, inter alia, immunogenic compositions (e.g., RNA compositions), methods of inducing an immune response, and methods of manufacturing immunogenic compositions. In some embodiments, the 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, the immunogenic composition delivers an immunogenic portion of the SARS-CoV-2 S protein, including, for example, the RBD, NTD, or truncated S1 subdomain of the SARS-CoV-2 virus, or variants thereof. In some embodiments, the technologies provided herein can result in an improved immune response (e.g., higher neutralizing antibody titers, improved naive B cell activation, and / or higher titers of antibodies that recognize epitopes specific to the variant strains of concern) as compared to, for example, compositions that deliver the full-length SARS-CoV-2 S protein.

[0004] In particular, the present disclosure provides certain findings regarding the design of SARS-CoV-2 antigens that can result in a significant improvement as compared to current vaccines (e.g., vaccines that deliver the full-length SARS-CoV-2 S protein). Such advantages include, for example, an improvement in antibody titers (including, in some embodiments, an improvement in neutralizing titers), an increase in efficacy, an increase in stability (e.g., an increase in thermal stability), an improvement in cross-neutralization, and / or an improvement in the persistence of the antibody response, as compared to current SARS-CoV-2 vaccines (e.g., in comparison to first-generation SARS-CoV-2 RNA vaccines that deliver the full-length spike protein (e.g., BNT162b2 (Comirnaty) and mRNA1273 (SpikeVax))).

[0005] In particular, the present disclosure provides findings regarding antigenic regions of the SARS-CoV-2 S protein, such antigenic regions being those that result in an improved immune response (e.g., increased neutralizing titers and / or a more persistent immune response) compared to the full-length S protein. In particular, the findings provided by the present disclosure are that a truncated S1 subdomain containing the endogenous sequence connecting the NTD region and the RBD region can induce an improved immune response compared to the full-length S protein or compared to a polypeptide containing the NTD and RBD connected via a heterologous flexible linker.

[0006] Also provided herein is the design of polypeptides improved for delivery by RNA, such design including the identification of preferred domains that bind to SARS-CoV-2 antigens (e.g., a secretion signal peptide and a transmembrane region), and its composition that results in a significant improvement in antigen expression and immune response. Such improved designs include, for example, an improved secretion signal peptide, a transmembrane domain, a multimerization domain, a GS linker, a nucleotide sequence, as well as combinations and configurations thereof. Each of these individually provides a significant improvement compared to existing SARS-CoV-2 vaccines, and combining them can result in an RNA with significantly improved antigen expression and / or immunogenicity.

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

[0008] In some embodiments, the improved immune response includes an improved B cell response. In some embodiments, the improved B cell immune response includes inducing an increase in the number of B cells that can recognize the SARS-CoV-2 S protein. In some embodiments, the improved B cell immune response includes an increase in the number of B cells that can recognize the RBD region of the SARS-CoV-2 S protein. In some embodiments, the improved B cell immune response includes an increase in the number of B cells that can recognize the NTD region of the SARS-CoV-2 S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% increased compared to the number of B cells induced by an RNA composition that delivers the full-length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD increases by a value in the range having a lower limit of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% and an upper limit of 150%, 200%, 250%, 300%, 350%, or 400% compared to the number of B cells induced by an RNA composition that delivers the full-length S protein. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD increases by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, the number of B cells that can recognize the S protein, NTD, or RBD increases by at least about 200%.

[0009] In some embodiments, an improved immune response includes an increase in the titer of antibodies capable of neutralizing the SARS-CoV-2 virus. In some embodiments, the titer of neutralizing antibodies increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, or 400% compared to the number of B cells induced by an RNA composition that delivers the full-length S protein. In some embodiments, the titer of neutralizing antibodies increases by a range of values having a lower limit of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% and an upper limit of 150%, 200%, 250%, 300%, 350%, or 400% compared to the number of B cells induced by an RNA composition that delivers the full-length S protein. In some embodiments, the titer of neutralizing antibodies increases by about 10% to about 1000%, about 10% to about 600%, about 10% to about 500%, about 100% to about 500%, at least about 100%, at least about 200%, at least about 400%, or at least about 500%. In some embodiments, the titer of neutralizing antibodies increases by at least about 200%. In some embodiments, the titer of neutralizing antibodies increases by at least about 400%.

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

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

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

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

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

[0015] In some embodiments, the RNA encodes a SARS-CoV-2 antigen that contains one or more mutations of a SARS-CoV-2 variant strain. In some embodiments, the SARS-CoV-2 variant strain is a strain of concern or is predicted to become a strain of concern (e.g., by a health authority including the WHO or the CDC). In some embodiments, the SARS-CoV-2 variant strain is one that is rapidly growing within a region and / or has increased immune evasion ability compared to the currently circulating SARS-CoV-2 virus. In some embodiments, the RNA encodes a SARS-CoV-2 antigen that contains one or more mutations of a variant strain for which a health authority recommends providing a seasonally updated vaccine.

[0016] In some embodiments, the SARS-CoV-2 S protein variant (or an immunogenic portion thereof) comprises one or more mutations associated with a SARS-CoV-2 variant strain having a high immune evasion ability (e.g., a variant strain of concern).

[0017] In some embodiments, the SARS-CoV-2 variant strain has been identified as having a high immune evasion ability using in vitro assays (e.g., virus neutralization assays), in silico analysis (e.g., sequence analysis and / or molecular dynamics simulations), and / or based on infection rates and / or growth rates.

[0018] In some embodiments, the SARS-CoV-2 variant strain having a high immune evasion ability is the Omicron variant strain.

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

[0020] In some embodiments, one or more mutations associated with the XBB.1.5 variant strain are T19I, Δ24-26, A27S, V83A, G142D, Δ144, 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 one or more mutations are shown relative to SEQ ID NO: 1.

[0021] In some embodiments, one or more mutations associated with the 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, where the positions of the one or more mutations are shown relative to SEQ ID NO:1.

[0022] In some embodiments, one or more mutations associated with the S1 domain of XBB.1.5 are T19I, Δ24-26, A27S, V83A, G142D, Δ144, 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, where the positions of the one or more mutations are shown relative to SEQ ID NO:1.

[0023] In some embodiments, one or more mutations associated with the XBB.1.5 variant are T19I, Δ24-26, A27S, V83A, G142D, Δ144, 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, where the positions of the one or more mutations are shown relative to SEQ ID NO:1.

[0024] In some embodiments, one or more mutations related to S1 of XBB.1.5 are T19I, Δ24-26, A27S, V83A, G142D, Δ144, 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 one or more mutations are shown relative to SEQ ID NO: 1.

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

[0026] In some embodiments, the RNA comprises a nucleotide sequence encoding 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.

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

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

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

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

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

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

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

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

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

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

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

[0038] In some embodiments, the gl2 secretion signal comprises a sequence that is at least 80% identical to SEQ ID NO: 11.

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

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

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

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

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

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

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

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

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

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

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

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

[0051] In some embodiments, the SARS-CoV-2 S protein variant (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 mutant polypeptide and the multimerization domain is adjacent to the TM domain (e.g., directly adjacent to the TM domain and / or linked to the TM domain via a GS linker).

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

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

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

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

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

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

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

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

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

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

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

[0063] In some embodiments, the G / C content is 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 35%, at least about 40%, at least about 45%, or at least about 50% higher.

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

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

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

[0067] In some embodiments, the RNA comprises a polyA sequence.

[0068] In some embodiments, the polyA sequence has a length of about 100 to 150 nucleotides.

[0069] In some embodiments, the polyA sequence is an interrupted polyA sequence.

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

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

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

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

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

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

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

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

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

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

[0080] In some embodiments, the RNA is self-amplifying RNA or trans-amplifying RNA.

[0081] In some embodiments, the composition comprises the RNA described herein, wherein the RNA is completely or partially encapsulated in a lipid nanoparticle (LNP), polyplex (PLX), lipidated polyplex (LPLX), oligosaccharide particle or polysaccharide particle, or liposome. In some embodiments, the RNA is completely or partially encapsulated in an LNP. In some embodiments, the LNP comprises a cationically ionizable lipid, a neutral lipid, a sterol, and a lipid conjugate.

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

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

[0084] In some embodiments, the immune response is induced in a subject previously administered one or more vaccines delivering a reference SARS-CoV-2 S protein one or more times.

[0085] In some embodiments, the immune response comprises a naive B cell immune response.

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

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

[0088] In some embodiments, the RNA or composition is administered to a subject previously exposed to a SARS-CoV-2 antigen (e.g., via vaccination or previous infection), where the previous infection is determined, for example, by positive PCR test results and / or antigen diagnostic test results. In some embodiments, the reference SARS-CoV-2 S protein or an immunogenic portion thereof is derived from a SARS-CoV-2 strain or variant that previously circulated or is currently circulating in the applicable jurisdiction.

[0089] In certain embodiments, the antigens described herein can be engineered to incorporate sequences and / or mutations (e.g., epitopes from the RBD, S protein, and / or S1 domain from two or more SARS-CoV-2 variants) derived from two or more SARS-CoV-2 variants. For example, in some such embodiments, mutations of one or more SARS-CoV-2 variants can be introduced into conserved epitopes of the mutant SARS-CoV-2 S protein, or an immunogenic portion thereof (e.g., the S1 domain or RBD). Such engineering can be useful, for example, to eliminate additional B cell epitopes (e.g., conserved B cell epitopes). Exemplary approaches for introducing mutations or sequences from two or more SARS-CoV-2 variants are described, for example, in a U.S. Provisional Application having U.S. Provisional Application No. 63 / 448,215, filed Feb. 24, 2023, entitled “Systems and Methods for Engineering Antigens to Promote Tailored Immune Responses”. The above application describes, inter alia, techniques related to the in silico design of custom antigens (e.g., including engineered versions of SARS-CoV2 variant proteins and portions thereof) engineered to reduce the degree of induced memory immune responses.

[0090] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide, which polypeptide comprises encoding an immunogenic fragment of the SARS-CoV-2 S protein. In some embodiments, the immunogenic fragment comprises the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. In some embodiments, the RBD of the SARS-CoV-2 S protein comprises the 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 the amino acid sequence from the corresponding region of any of those of a SARS-CoV-2 variant. In some embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises the N-terminal domain (NTD). In some embodiments, the NTD of the SARS-CoV-2 S protein comprises amino acids 14-209, 14-303, 20-318, or 20-302 of SEQ ID NO: 1, or comprises the corresponding region of any of those of the S protein of a SARS-CoV-2 variant. In some embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises the S1 domain of the SARS-CoV-2 S protein, or an immunogenic fragment thereof. In some embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises the RBD and NTD of the SARS-CoV-2 S protein. In some embodiments, the immunogenic fragment of the SARS-CoV-2 S protein comprises a truncated S1 subdomain or a variant thereof. In some embodiments, the RBD is at the C-terminus of the truncated S1 subdomain or a variant thereof (e.g., where 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 comprises the corresponding region of any of those of a SARS-CoV-2 variant).In some embodiments, the truncated S1 subdomain or a 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 comprises a corresponding region of any of those derived from the S protein of a SARS-CoV-2 variant.

[0091] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising a secretion signal peptide (e.g., a secretion signal peptide of a viral protein), and optionally, the secretion signal peptide is at the N-terminus. In some embodiments, the secretion signal peptide is the secretion signal peptide of the SARS-CoV-2 S protein. In some embodiments, the secretion signal peptide is a heterologous secretion signal peptide. In some embodiments, the heterologous secretion signal peptide is a secretion signal peptide of a viral protein that is not the SARS-CoV-2 S protein.

[0092] In some embodiments, the polypeptide comprises a secretion signal peptide (e.g., a secretion signal peptide of a viral protein). In some embodiments, the polypeptide comprises (i) an amino acid sequence listed in Table 2, Table XXXI, or 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 the amino acid sequence listed in Table 2 or Table XXXI, and / or (ii) the RNA comprises a nucleotide sequence listed in Table 3, Table XXXI, or FIG. 15, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to the nucleotide sequence encoding the amino acid sequence listed in Table 2 or Table XXXI listed in Table 3 and / or Table XXXI and / or FIG. 15.

[0093] In some embodiments, the polypeptide comprises a SP24-Q7PUJ5_ANOGA secretion signal peptide (e.g., a secretion signal peptide comprising the amino acid sequence of MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 22), or a secretion signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MCRGLSAVLILLVSLSAQLHVVVG (SEQ ID NO: 22)).

[0094] In some embodiments, the polypeptide comprises a SP24-SP18-HEMA_CVBM secretion signal peptide (e.g., a secretion signal peptide comprising the amino acid sequence of MFLLLRFVLVSCIIGSLG (SEQ ID NO: 391), or a secretion signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MFLLLRFVLVSCIIGSLG (SEQ ID NO: 391)).

[0095] In some embodiments, the polypeptide comprises a SP25-GD_HHV1K secretion signal peptide (e.g., a secretion signal peptide comprising the amino acid sequence of MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12), or a secretion signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12)).

[0096] In some embodiments, the polypeptide comprises an SP32-GBD_HHV1K secretion signal peptide (e.g., a secretion signal peptide comprising the amino acid sequence of MHQGAPSWGRRWFVVWALLGLTLGVLVASAAP (SEQ ID NO: 38), or a secretion signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MHQGAPSWGRRWFVVWALLGLTLGVLVASAAP (SEQ ID NO: 38)).

[0097] In some embodiments, the polypeptide comprises an SP20-A7U881_HHV2 secretion signal peptide (e.g., a secretion signal peptide comprising the amino acid sequence of MARGAGLVFFVGVWVVSCLA (SEQ ID NO: 366), or a secretion signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MARGAGLVFFVGVWVVSCLA (SEQ ID NO: 366)).

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

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

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

[0101] In some embodiments, the transmembrane domain is the transmembrane domain of the SARS-CoV-2 S protein (e.g., the transmembrane domain comprises the amino acid sequence of EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to EQYIKWPWYIWLGFIAGLIAIVMVTIMLCC).

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

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

[0104] In some embodiments, the transmembrane domain can induce multimerization (e.g., trimerization).

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

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

[0107] In some embodiments, the sequence that is intrinsically 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.

[0108] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 90 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC), or comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 90.

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

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

[0111] In some embodiments, the arrangement from the N-terminus to the C-terminus of the polypeptide is (secretory signal peptide)-(fragment of S1 polypeptide)-(transmembrane domain). In some embodiments, (1) the secretory signal peptide and the fragment of the S1 polypeptide, and / or (2) the fragment of the S1 polypeptide and the transmembrane domain are linked to each other by a linker (e.g., an artificial linker, a flexible linker, a flexible linker containing a GS sequence). In some embodiments, the GS sequence includes the sequences shown in Table 5. In some embodiments, the GS sequence includes 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, the GS sequence includes the sequence of (G4S)1, (G4S)2, (G4S)3, or (G4S)4. In some embodiments, the fragment of the S1 polypeptide and the transmembrane domain are linked via a flexible linker containing 10 to 20 amino acids (e.g., about 15 amino acids).

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

[0113] In some embodiments, the RNA includes a nucleotide sequence encoding a polypeptide that includes (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) SEQ ID NO: 155, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 155.

[0114] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 160, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 160.

[0115] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 165, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 165.

[0116] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 170, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 170.

[0117] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 175, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 175.

[0118] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 180, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 180.

[0119] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 185, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 185.

[0120] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 190, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 190.

[0121] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 195, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 195.

[0122] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 200, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 200.

[0123] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 210, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 210.

[0124] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 220, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 220.

[0125] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 225, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 225.

[0126] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) the nucleotide sequence of SEQ ID NO: 230, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 230.

[0127] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) the nucleotide sequence of SEQ ID NO: 235, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 235.

[0128] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 240, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 240.

[0129] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) 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.

[0130] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 250, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 250.

[0131] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 255, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 255.

[0132] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 260, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 260.

[0133] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 265, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 265.

[0134] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 270, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 270.

[0135] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 275, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 275.

[0136] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (i) the nucleotide sequence of SEQ ID NO: 281, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% 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% identical to SEQ ID NO: 283, and / or (iii) 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% identical to SEQ ID NO: 280.

[0137] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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% 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% identical to SEQ ID NO: 288, and / or (iii) 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% identical to SEQ ID NO: 285.

[0138] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 290, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 290.

[0139] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 295, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 295.

[0140] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 300, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 300.

[0141] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 305, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 305.

[0142] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 310, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 310.

[0143] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) SEQ ID NO: 320, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 320.

[0144] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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% 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% identical to SEQ ID NO: 345, and / or (iii) the nucleotide sequence of 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% identical to SEQ ID NO: 342.

[0145] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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% 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% identical to SEQ ID NO: 350, and / or (iii) the nucleotide sequence of 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% identical to SEQ ID NO: 347.

[0146] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) the nucleotide sequence of 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.

[0147] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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) the nucleotide sequence of SEQ ID NO: 357, or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 357.

[0148] In some embodiments, the RNA comprises a 5' cap, a cap-proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence.

[0149] In some embodiments, (i) the 5' cap comprises a Cap1 structure, (ii) the 5'-UTR sequence comprises a modified human alpha-globin 5'-UTR, (iii) The 3'-UTR sequence comprises a first sequence from the amino-terminal enhancer of the split (AES) messenger RNA and a second sequence from the 12S ribosomal RNA of the mitochondrial code, (iv) The polyA sequence comprises at least 100 A nucleotides, or (v) The RNA comprises any one combination of (i)-(iv).

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

[0151] In some embodiments, the cap-proximal sequence comprises A1 and G2 of the Cap1 structure and a sequence comprising A3N4N5 at the +3, +4, and +5 positions of the RNA, respectively, where N4 and N5 are each independently selected from A, G, C, and U.

[0152] In some embodiments, the polyA sequence comprises an intermittent sequence of A nucleotides, optionally the intermittent sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, and the 30 adenine nucleotides and the 70 adenine nucleotides are separated by a linker sequence. In some embodiments, the 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, the 3'-UTR sequence comprises SEQ ID NO: 118, 647, or 648, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 118, 647, or 648.

[0153] In some embodiments, the intermittent polyA tail comprises the sequence of SEQ ID NO: 114, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 114.

[0154] In some embodiments, the sequence at the 5' end of the 3'UTR sequence (e.g., the sequence immediately adjacent to the sequence encoding the antigenic polypeptide) is CUCGAG or GGAUCCGAU.

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

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

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

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

[0159] In some embodiments, the nucleotide sequence encoding the SARS-CoV-2 S protein is encoded by a sequence with codons optimized (e.g., codons optimized for expression in human cells), and / or has an increased G / C content compared to the wild-type coding sequence.

[0160] In particular, the present specification provides a composition comprising the RNA described herein.

[0161] In some embodiments, the composition comprises RNA formulated in nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, the nanoparticles are lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise an ionizable lipid that is cationic, a sterol, a neutral lipid, and a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, the RNA is encapsulated in lipid nanoparticles (LNP), and preferably, the LNP comprises 20-60% ionizable cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid by molar ratio. In some embodiments, the nanoparticles have an average diameter of about 50-150 nm. In some embodiments, a cryoprotectant is included, and optionally, the cryoprotectant is sucrose or comprises sucrose. In some embodiments, the composition comprises a buffered aqueous solution, and optionally, the buffered aqueous solution comprises one or more of tris base, tris HCl, NaCl, KCl, Na2HPO4, and KH2PO4. In some embodiments, the composition comprises about 10 mM tris buffer and about 10% sucrose.

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

[0163] In some embodiments, the pharmaceutical composition is formulated as a multi-dose formulation in a vial, a single-dose formulation in a vial, or a pre-filled syringe.

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

[0165] In some embodiments, provided herein are methods comprising administering the RNA, composition, or pharmaceutical composition provided herein.

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

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

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

[0169] In some embodiments, the method comprises administering the RNA, composition, or pharmaceutical composition to the subject once.

[0170] In some embodiments, the method comprises administering the RNA, composition, or pharmaceutical composition described herein to the subject two or more times, optionally with a 21-day interval between the two administrations.

[0171] In some embodiments, the RNA, composition, or pharmaceutical composition described herein is administered to a subject three times, and optionally, the first and second administrations are performed at an interval of about 21 days, and the third administration is performed about 28 days after the second administration.

[0172] In some embodiments, the method described herein further comprises administering the RNA, composition, or pharmaceutical composition described herein at least about 2 months (e.g., 2 - 12 months, 2 - 10 months, 2 - 8 months, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months) after the first administration of the RNA, composition, or pharmaceutical composition described herein.

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

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

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

[0176] In some embodiments, the subject has previously been administered a first dose and a second dose of a vaccine that delivers the full-length SARS-CoV-2 S protein (e.g., a composition comprising an LNP-formulated RNA encoding the SARS-CoV-2 S protein), where the first dose and the second dose were administered at an interval of about 21 days, and optionally, the subject has previously been administered, as a booster, a monovalent or bivalent vaccine that delivers the S protein of one or more variants of SARS-CoV-2 (e.g., (i) the S protein of the Wuhan strain and the S protein of the Omicron BA.4 / 5 strain, (ii) the S protein of the XBB.1.5 variant, (iii) the S protein of the KP.2 variant, and / or (iv) the S protein of the JN.1 variant).

[0177] In some embodiments, the methods described herein include administering one or more vaccines against non-SARS-CoV-2 diseases, optionally including an RSV vaccine, an influenza vaccine, or a combination thereof.

[0178] In some embodiments, the method results in induction of an immune response against SARS-CoV-2 in a subject. In some embodiments, the immune response includes a B cell response. In some embodiments, the B cell response includes production of antibodies induced against one or more SARS-CoV-2 viruses. In some embodiments, the immune response includes a T cell response, optionally including a CD4+ T cell response and / or a CD8+ T cell response.

[0179] In some embodiments, the methods described herein are methods of preventing or reducing the likelihood of infection with a SARS-CoV-2 virus and / or treating a SARS-CoV-2 infection.

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

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

[0182] In some embodiments, the medicament is formulated to be administered to a subject according to the methods described herein.

[0183] In some embodiments, the methods provided herein are methods of inducing an immune response against a coronavirus in a subject, the method including administering an RNA or pharmaceutical composition provided herein. In some embodiments, the methods described herein induce an immune response against a SARS-CoV-2 virus.

[0184] In some embodiments, provided herein is a method for manufacturing RNA, which includes transcribing the RNA provided herein in vitro.

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

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

[0187] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide that includes (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) SEQ ID NO: 327 or a sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to SEQ ID NO: 327.

[0188] In some embodiments, the fragment of the S protein comprises one or more mutations of a SARS-CoV-2 strain or a SARS-CoV-2 variant (e.g., one or more mutations in the S protein of the variants described herein, e.g., one or more mutations associated with the variants listed in Table 1). In some embodiments, the fragment of the S protein comprises one or more mutations associated with the JN.1 variant, the KP.2 variant, or the XEC variant, or their progeny.

[0189] In some embodiments, the fragment of the S protein comprises one or more of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, 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 (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.

[0190] In some embodiments, 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, or more) of the following mutations relative to SEQ ID NO: 1: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H.

[0191] In some embodiments, 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, or more) of the following mutations relative to SEQ ID NO: 1: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L.

[0192] In some embodiments, 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, or more) of the mutations ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H with respect to SEQ ID NO: 1.

[0193] In some embodiments, the fragment of the S protein has 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) mutations among ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, or P1143L with respect to SEQ ID NO: 1.

[0194] In some embodiments, 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, or more) of the following mutations relative to SEQ ID NO: 1: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H.

[0195] In some embodiments, 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, or more) of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or E1150D when compared to SEQ ID NO: 1.

[0196] In some embodiments, 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, or more) of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H with respect to SEQ ID NO: 1.

[0197] In some embodiments, 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, or more) of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or E1150D with respect to SEQ ID NO: 1.

[0198] In some embodiments, 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, or more) of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H with respect to SEQ ID NO: 1.

[0199] In some embodiments, the fragment of the S protein has 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 relative to SEQ ID NO: 1: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, or P1143L.

[0200] In some embodiments, 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, or more) of the following mutations relative to SEQ ID NO: 1: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, or Y505H.

[0201] In some embodiments, 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, or more) of the following: T19I, Δ24-26, A27S, V83A, G142D, Δ145, 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 relative to SEQ ID NO: 1.

[0202] In some embodiments, 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, or more) of T19I, Δ24-26, A27S, V83A, G142D, Δ145, 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 with respect to SEQ ID NO: 1.

[0203] In some embodiments, 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, 65, 66, 67, or more) of ins16MPLF, T19I, R21T, T22N, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L.

[0204] In some embodiments, 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, or more) of the mutations of ins16MPLF, T19I, R21T, T22N, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, and Y505.

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

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

[0207] In some embodiments, the S1 polypeptide is (a) amino acids 1-528 of SEQ ID NO: 1 or their corresponding regions derived from the S protein of a SARS-CoV-2 variant (b) QCVMPLFNLITTTQSYTNSFTRGVYYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAISGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVFIKVCEFQFCNDPFLDVYHKNNKSWMESESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIGRDFPQGFSALEPLVDLPIGINITRFQTLLALNRSYLTPGDSSSGWTAGAADYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNVTNLCPFHEVFNATTFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSLRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPK, 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 / orQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPK, 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.

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

[0209] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising an NTD, an RBD, and / or a truncated S1 subdomain, or a variant thereof, the polypeptide further comprising one or more T cell epitopes derived from a SARS-CoV-2 protein other than the S protein. In some embodiments, the polypeptide comprises one or more T cell epitopes derived from the nucleocapsid (N) protein, NS9b protein, membrane (M) protein, ORF1ab protein, ORF3a protein, ORF9b protein, or NSP1-4 of SARS-CoV-2, or any combination thereof. In some embodiments, the polypeptide comprises the following (i), (ii), (iii), (iv), (v), (vi). (i) One or more T cell epitopes derived from the N protein and one or more T cell epitopes derived from the NS9b protein, (ii) One or more T cell epitopes derived from the N protein and one or more T cell epitopes derived from the M protein, (iii) One or more T cell epitopes derived from the N protein, one or more T cell epitopes derived from the M protein, and one or more T cell epitopes derived from the NS9b protein, (iv) One or more T cell epitopes derived from the M protein, one or more T cell epitopes derived from NSP2, one or more T cell epitopes derived from NSP3, one or more T cell epitopes derived from NSP1, and one or more T cell epitopes derived from the N protein, (v) One or more T cell epitopes derived from NSP2, one or more T cell epitopes derived from NSP1, one or more T cell epitopes derived from NSP3, one or more T cell epitopes derived from the N protein, one or more T cell epitopes derived from NSP4, and one or more T cell epitopes derived from the M protein, (vi) One or more T-cell epitopes derived from the N protein, one or more T-cell epitopes derived from NSP1, one or more T-cell epitopes derived from NSP2, one or more T-cell epitopes derived from NSP3, and one or more T-cell epitopes derived from NSP4. In some embodiments, the RNA encodes a polypeptide, and the polypeptide (a) A truncated S1 subdomain, or a variant thereof, and the SARS-CoV-2 transmembrane domain, (b) A truncated S1 subdomain, or a variant thereof, and the HSV-1 gD secretion signal peptide, (c) A truncated S1 subdomain or a variant thereof, a transmembrane domain, and the HSV-1 gD secretion signal peptide, (d) A truncated S1 subdomain or a variant thereof, the transmembrane domain of the SARS-CoV-2 S protein, and a secretion signal peptide, or (e) A truncated S1 subdomain or a variant thereof, the transmembrane domain of the SARS-CoV-2 S protein, and the HSV-1 gD secretion signal peptide.

[0210] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide, and the polypeptide (a) (i) The amino acids 20-528 of SEQ ID NO: 1 (e.g., amino acids 1-528, 14-528, 17-528, and 50-528, etc.) or the sequence of the corresponding region of the S protein of a SARS-CoV-2 variant, or (ii) the amino acids 20-541 of SEQ ID NO: 1 (e.g., amino acids 1-541, 14-541, 17-541, and 20-541, etc.) or the sequence of the corresponding region of the S protein of a SARS-CoV-2 variant, or a variant of any of them, comprising a truncated S1 subdomain, (b) A secretory signal peptide comprising the amino acid sequence of MGGAAARLGAVILFVVIVGLHGVRG, or a secretory signal peptide comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more identical to MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO: 12), and / or (c) It comprises a transmembrane domain comprising the 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.

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

[0212] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising (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% 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% identical to SEQ ID NO: 652, and / or (iii) 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% identical to SEQ ID NO: 649.

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

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

[0215] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising a truncated S1 subdomain or a variant thereof, a transmembrane domain, and a secretion signal peptide, and the arrangement of the polypeptide from the N-terminus to the C-terminus is (secretion signal peptide)-(truncated S1 subdomain)-(transmembrane domain), and the truncated S1 subdomain or a variant thereof and the transmembrane domain are linked via a sequence containing a GS linker, optionally, the GS linker contains about 10 - 20 residues (e.g., about 15 residues or about 20 residues). BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0258] Specific Definitions Generally, the terms used herein follow the meanings understood in the art, unless explicitly indicated otherwise. Explicit definitions of specific terms are provided below. The meanings of these and other terms in specific examples throughout this specification will be apparent to those skilled in the art from the context.

[0259] To better understand the present invention, certain terms are first defined. Further definitions of the following terms and other terms are set forth throughout this specification.

[0260] About: As used herein with respect to a value, the term "about" refers to a value that approximates the recited value in relation thereto. Generally, the appropriate degree of variation encompassed by "about" in that context will be apparent to those skilled in the art with familiarity in the context. For example, in some embodiments, the term "about" may encompass a range of values 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 recited value.

[0261] Agent (Factor): As used herein, the term "agent" (or "factor") may refer to a physical entity or phenomenon. In some embodiments, an agent can be characterized by certain features and / or effects. In some embodiments, an agent can be, for example, a small molecule, polypeptide, nucleic acid, sugar, lipid, metal, or a compound, molecule, or entity of any chemical class including combinations or complexes thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that includes a polymer. In some embodiments, this term may refer to a compound or entity that includes one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that substantially excludes a particular polymer or polymer moiety. In some embodiments, this term may refer to a compound, molecule, or entity that substantially excludes any polymer or polymer moiety.

[0262] Amino acid: As used herein, the term "amino acid" refers, in the broadest sense, to a compound and / or substance that can be incorporated into (e.g., by the formation of one or more peptide bonds), is incorporated into (e.g., by the formation of one or more peptide bonds), or has been incorporated into (e.g., by the formation of one or more peptide bonds) a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is a non-natural amino acid. In some embodiments, the amino acid is a D-amino acid. In some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid (including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide) may include a structural modification relative to the normal structure described above. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., substitution of an amino group, a carboxylic acid group, one or more protons, and a hydroxyl group) relative to the normal structure. In some embodiments, such modifications cause, for example, the circulating half-life of a polypeptide containing the modified amino acid to change compared to that of a polypeptide containing the unmodified amino acid but otherwise the same. In some embodiments, such modifications do not significantly change the relevant activity of a polypeptide containing the modified amino acid compared to that of a polypeptide containing the unmodified amino acid but otherwise the same. As will be apparent 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.

[0263] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is a polypeptide that includes one or more structural elements whose amino acid sequences are recognized by those skilled in the art as complementarity-determining regions (CDRs), or includes such a polypeptide. In some embodiments, an antibody agent is a polypeptide that includes at least one CDR (e.g., at least one heavy-chain CDR and / or at least one light-chain CDR) whose amino acid sequence is substantially identical to that present in a reference antibody, or includes such a polypeptide. In some embodiments, the included CDR is substantially identical to the reference CDR in that the sequence is either identical to the reference CDR or includes 1 to 5 amino acid substitutions. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that while at least one amino acid within the included CDR is deleted, added, or substituted compared to the reference CDR, otherwise the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR are deleted, added, or substituted compared to the reference CDR, otherwise the included CDR has the same amino acid sequence as the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs is substituted as compared to the reference CDRs, while otherwise the included CDRs have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs are deleted, added, or substituted as compared to the reference CDRs, while otherwise the included CDRs have the same amino acid sequence as the reference CDRs. In some embodiments, the antibody agent is a polypeptide that includes structural elements recognized by those skilled in the art as immunoglobulin variable domains in its amino acid sequence, or includes such a polypeptide. In some embodiments, the antibody agent is a polypeptide that includes structural elements recognized by those skilled in the art as corresponding to CDR1, CDR2, and CDR3 of the antibody variable domain in its amino acid sequence, or includes such a polypeptide. In some such embodiments, the antibody agent is a polypeptide or a set of polypeptides whose combined amino acid sequence(s) includes structural elements recognized by those skilled in the art as corresponding to both the variable region CDRs of the heavy and light chains (e.g., heavy chain CDR1, CDR2, and / or CDR3 and light chain CDR1, CDR2, and / or CDR3), or includes such a polypeptide or a set of polypeptides. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or almost homologous to the immunoglobulin binding domain. In some embodiments, the antibody agent can be a polyclonal antibody preparation or can include a polyclonal antibody preparation. In some embodiments, the antibody agent can be a monoclonal antibody preparation or can include it. In some embodiments, the antibody agent can include one or more constant region sequences specific to a particular organism such as camel, human, mouse, primate, rabbit, rat, etc. In many embodiments, the antibody agent can include one or more constant region sequences specific to humans. In some embodiments, the antibody agent can include one or more sequence elements that can be recognized by those skilled in the art as humanized sequences, primatized sequences, chimeric sequences, etc.In some embodiments, the antibody agent can be a standard antibody (e.g., can include two heavy chains and two light chains). In some embodiments, the antibody agent is an intact IgA antibody, IgG antibody, IgE antibody, or IgM antibody, a bispecific or multispecific antibody (e.g., Zybodies® etc.), a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, an Fd’ fragment, an Fd fragment, and antibody fragments such as isolated CDRs or sets thereof, a single-chain Fv, a polypeptide-Fc fusion, a single-domain antibody (e.g., a shark single-domain antibody such as IgNAR or a fragment thereof), a camelid antibody, a masked antibody (e.g., Probody®), Small Modular ImmunoPharmaceuticals (“SMIPs™”), a single-chain diabody or tandem diabody (TandAb®), a VHH, an anti-klotho®, a Nanobody®, a minibody, a BiTE®, ankyrin repeat protein or DARPIN®, an Avmer®, a DART, a TCR-like antibody, an Adnectin®, an Affilin®, a Trans-body®, an Affibody®, a TrimerX®, a MicroProtein, a Fynomer®, a Centyrin®, and a KALBITOR®, but is not limited thereto. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it has when produced naturally. In some embodiments, the antibody can include covalent modifications (e.g., glycan attachment), a payload (e.g., a detectable moiety, a therapeutic agent moiety, a catalytic moiety, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0264] Antigen: As will be apparent to those skilled in the art upon reading this specification, the term "antigen" refers to a molecule that is recognized by the immune system (e.g., in certain embodiments, the adaptive immune system) and elicits an antigen-specific immune response. In some embodiments, the antigen-specific immune response can be or can include the production of antibodies and / or antigen-specific T cells. In some embodiments, the antigen is a peptide or polypeptide that contains at least one epitope to which an immune response can occur. In one embodiment, the antigen is presented by cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. In one embodiment, the antigen or its processed product, e.g., a T cell antigen, is bound by a T cell receptor or a B cell receptor, or by an immunoglobulin molecule, e.g., an antibody. Thus, the antigen or its processed product can specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a parasitic antigen. According to the present disclosure, in some embodiments, the antigen can be delivered by an RNA molecule as described herein. In some embodiments, the peptide antigen or polypeptide antigen can be, for example, 2 to 100 amino acids in length, such as 5 amino acids in length, 10 amino acids in length, 15 amino acids in length, 20 amino acids in length, 25 amino acids in length, 30 amino acids in length, 35 amino acids in length, 40 amino acids in length, 45 amino acids in length, or 50 amino acids in length. In some embodiments, the peptide antigen or polypeptide antigen can be more than 50 amino acids. In some embodiments, the peptide antigen or polypeptide antigen can be more than 100 amino acids. In some embodiments, the antigen is recognized by immune effector cells. In some embodiments, when the antigen is recognized by immune effector cells, it can induce the stimulation, priming, and / or proliferation of immune effector cells having an antigen receptor that recognizes the antigen in the presence of an appropriate co-stimulatory signal. With respect to embodiments of the present disclosure, in some embodiments, the antigen can be presented on or can be present on the surface of a cell, such as an antigen-presenting cell. In one embodiment, the "antigen" is presented by diseased cells, such as virus-infected cells. In one embodiment, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC.In one embodiment, a TCR, when expressed by a T cell and / or present on the T cell, binds to an antigen presented by a cell such as an antigen-presenting cell, resulting in the stimulation, priming, and / or proliferation of the T cell. In one embodiment, a TCR, when expressed by a T cell and / or present on the T cell, binds to an antigen presented on a diseased cell, resulting in the cytolysis and / or apoptosis of the diseased cell. Here, it is preferable that the T cell releases cytotoxic factors such as perforin and granzyme.

[0265] Related: Two events or entities are "related" to each other if, as the term is used herein, the presence, level, degree, type, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be related to a particular disease, disorder, or illness if its presence, level, and / or form correlates with the incidence, prevalence, severity, stage, etc. of the disease, disorder, or illness (e.g., across a relevant population). In some embodiments, two or more entities are physically "related" to each other if they interact directly or indirectly so as to be physically proximate to and / or maintain a proximate state with respect to each other. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other. In some embodiments, two or more entities that are physically related to each other are not covalently bonded to each other but are non-covalently related by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0266] Binding: As will be apparent to those skilled in the art upon reading this specification, the term "binding" typically refers to non-covalent bonds between or within entities or moieties. In some embodiments, binding data is represented by "IC 50 " As understood in the art, IC 50is the concentration of an agent to be evaluated in a binding assay, which is the concentration of the agent at which binding of a reference agent to a known related binding partner is found to be inhibited by 50%. In some embodiments, the assay is performed under conditions such that the IC 50 value approximates the K D value. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO94 / 20127 and WO94 / 03205, as well as 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 can be expressed as compared to binding by a reference standard peptide. For example, binding can be expressed as its IC 50 compared to the IC 50It can be based on. Binding can also be determined using other assay systems. Such assays include those using live cells (e.g., Cepellini 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)), those using cell-free systems with detergent lysates (e.g., Cerundlo et al., J. Immunol 21:2069 (1991)), those using immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994), Marshall et al., J. Immunol. 152:4946 (1994)), those using ELISA systems (e.g., Reay et al., EMBO J. 11:2829 (1992)), those using surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)), those using high-flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and those using measurements of the stabilization or assembly of class I MHC (e.g., Ljungren 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)).

[0267] Cap: As used herein, the term "cap" typically refers to a structure that contains or consists essentially of a nucleoside-5'-triphosphate attached to the 5' end of a capless RNA (e.g., a capless RNA having a 5'-diphosphate). In some embodiments, the cap is or includes a guanine nucleotide. In some embodiments, the cap is or includes a native RNA 5' cap (including, for example but without limitation, a 7-methylguanosine cap having a structure referred to as "m7G"). In some embodiments, the cap is or includes a synthetic cap analog that is similar to an RNA cap structure and has the ability to stabilize RNA when bound to the RNA, and such synthetic cap analogs include, for example but without limitation, anti-reverse cap analogs (ARCA) known in the art. As will be apparent to those skilled in the art, methods for ligating a cap to the 5' end of an RNA are known in the art. For example, in some embodiments, capped RNA can be obtained by in vitro capping an RNA having a 5'-triphosphate group or an RNA having a 5'-diphosphate group using a capping enzyme system (including, for example but without limitation, a vaccinia capping enzyme system or a Saccharomyces cerevisiae capping enzyme system). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template in the presence of a dinucleotide cap analog or a trinucleotide cap analog.

[0268] Cell-mediated immunity: The terms "cell-mediated immunity", "cellular immunity", "cell immune response", or similar terms are intended to encompass a cellular response against cells that feature the expression of an antigen, particularly cells that feature the presentation of an antigen having class I MHC or class II MHC. The cellular response is associated with immune effector cells, particularly T cells or T lymphocytes that act as either "helper" or "killer". Helper T cells (CD4 +T cells, also known as CD4 T cells, play a central role by regulating killer cells (cytotoxic T cells, cytolytic T cells, also called CD8 + T cells, CD8 T cells, or CTLs), killing diseased cells such as virus-infected cells, and preventing the production of further diseased cells.

[0269] Combined administration: As used herein, the term "combined administration" refers to the use of a pharmaceutical composition described herein (e.g., an immunogenic composition, e.g., a vaccine) and an additional therapeutic agent. The combination of a pharmaceutical composition described herein (e.g., an immunogenic composition, e.g., a vaccine) and an additional therapeutic agent may be administered simultaneously or separately (e.g., sequentially in any order). In some embodiments, the pharmaceutical composition described herein (e.g., an immunogenic composition, e.g., a vaccine) and the additional therapeutic agent may be combined in one pharmaceutically acceptable carrier, or they may be present in separate carriers and delivered to the target cells, or administered to the subject at different times. Each of these situations is considered to be within the meaning of "combined administration" or "combination", provided that the pharmaceutical composition described herein (e.g., an immunogenic composition, e.g., a vaccine) and the additional therapeutic agent are delivered or administered at times close enough to each other to each have at least some biologically overlapping effect(s) on the target cells or the subject being treated.

[0270] Codon optimized: As used herein, the term "codon optimized" preferably refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism without changing the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, in some embodiments, the coding region is codon optimized to provide optimal expression in a subject being treated using the RNA molecules described herein. In some embodiments, codon optimization can be performed such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons". In some embodiments, codon optimization can include increasing the guanosine / cytosine (G / C) content of the coding region of the RNA described herein relative to the G / C content of the corresponding coding sequence of the wild-type RNA. Here, it is preferred that the amino acid sequence encoded by the RNA is not modified relative to the corresponding amino acid sequence.

[0271] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously. In some embodiments, those regimens may be administered sequentially (e.g., all of the "doses" of the first regimen are administered before any dose of the second regimen is administered). In some embodiments, those agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy can include administering one or more agents or modalities in combination to a subject that is receiving another agent(s) or modality(ies). For clarity, combination therapy does not require that the individual agents be administered together (or even necessarily simultaneously) in a single composition, although in some embodiments, two or more agents or their active moieties may be administered together in a composite composition.

[0272] Comparable: As used herein, the term "comparable" means that two or more agents, entities, situations, sets of conditions, etc., need not be identical to each other, but are sufficiently similar to be comparable, such that it is apparent to one of ordinary skill in the art that reasonable conclusions can be drawn based on the differences or similarities recognized between them. In some embodiments, comparable sets of conditions, situations, individuals, or groups are characterized by a plurality of substantially identical features and one or a few different features. One of ordinary skill in the art will understand, in any given context, what degree of identity is required for two or more such agents, substances, situations, sets of conditions, etc., to be considered comparable. For example, as will be apparent to one of ordinary skill in the art, if a set of situations, individuals, or groups has a sufficient number and variety of substantially identical features such that it is reasonable to conclude that differences in results or observed phenomena obtained under or by different sets of situations, solids, or groups are due to or indicative of differences in those features, then such sets of situations, individuals, or groups are comparable to each other.

[0273] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" can be used to specify the position / identity of a structural element of a compound or composition relative to another compound or composition (e.g., a suitable reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) can be specified as "corresponding to" a certain residue in a suitable reference polymer. For example, as will be apparent to those skilled in the art, for simplicity, residues in a polypeptide are often specified using a standard numbering system based on a reference related polypeptide, whereby, for example, the amino acid "corresponding to" the residue at position 190 need not actually be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue at position 190 of the reference polypeptide. Those skilled in the art can readily understand how to identify the "corresponding" amino acid. For example, those skilled in the art are familiar with various sequence alignment methods. Such sequence alignment methods include, for example, software programs such as 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 BLAST, and these can be used to identify, for example, the "corresponding" residues in polypeptides and / or nucleic acids according to the present disclosure. Or as will be apparent to those skilled in the art, in some examples, the term "corresponding to" can be used to indicate that an event or entity shares a suitable similarity with another event or entity (e.g., a suitable reference event or entity). As an example, a gene or protein in one organism can be described as "corresponding to" a gene or protein from another organism.The purpose is, in some embodiments, to show that it performs a similar role or function, and / or that it exhibits a certain degree of sequence identity or homology, and / or that it shares certain characteristic sequence elements.

[0274] As is well known to those skilled in the art, sequence alignment methods allow for consideration of, for example, "gaps" within a sequence and / or "repetitive" residues. Further, as will be apparent to those skilled in the art, in some cases it may not be possible to uniquely determine the exact position of a sequence change relative to a reference sequence. For example, if the reference sequence contains two or more consecutive identical residues and the altered sequence has one less of those residues, it is not possible to assign a deletion of a specific single residue within the reference sequence. This is because deleting any one of the consecutive identical residues results in the same altered sequence. Thus, as will be apparent to those skilled in the art, it is conventional to arbitrarily assign a deletion as a residue missing one of the positions of the reference residues. By way of 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 missing, one skilled in the art cannot determine whether amino acid 144 or 145 is missing in the altered sequence. However, one skilled in the art understands that either deletion results in the same polypeptide sequence, and thus can uniquely determine the sequence of a polypeptide described as having a deletion at position 144 or 145 of SEQ ID NO: 1 (i.e., as will be apparent to those skilled in the art, a polypeptide described as having a deletion at the position corresponding to position 144 of SEQ ID NO: 1 and a polypeptide described as having a deletion at the position corresponding to position 145 of SEQ ID NO: 1 have the same amino acid sequence).

[0275] Origin: In the context of an amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide), "derived from" refers to a structural analog of the specified 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. An amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, as will be apparent to those skilled in the art, an antigen suitable for use in the present disclosure can be modified to have a sequence that is different from the native or original sequence from which the antigen is derived while retaining the desired activity of the original sequence.

[0276] Design: As used herein, the term "design" refers to (i) the structure of the agent being the result of human hand or being selected by human hand, (ii) the agent being produced by a process that requires human hand, and / or (iii) the agent being different from natural substances and other known agents.

[0277] Dosage regimen: As will be apparent to those skilled in the art, the term "dosage regimen" can be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject over a period of time. In some embodiments, a given therapeutic agent may have a recommended dosage regimen that may include one or more administrations. In some embodiments, the dosage regimen includes multiple administrations, each administration being temporally spaced from the others. In some embodiments, the individual administrations are separated from each other by equal intervals of time. In some embodiments, the dosage regimen includes multiple administrations and at least two different periods separating the individual administrations. In some embodiments, all of the administrations within a dosage regimen are of the same unit dose. In some embodiments, the different administrations within a dosage regimen are of different amounts. In some embodiments, the dosage regimen includes a first administration at a first dose and one or more additional administrations at a second dose different from the first dose that follows it. In some embodiments, the dosage regimen includes a first administration at a first dose and one or more additional administrations at a second dose that is the same as the first dose that follows it. In some embodiments, the dosage regimen is associated with a desired or beneficial outcome when administered within an appropriate population (i.e., is a therapeutic dosage regimen).

[0278] Code: As used herein, the term "code" or "encode" refers to the sequence information of a first molecule inducing the production of a second molecule having a predetermined nucleotide sequence (e.g., mRNA) or a predetermined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process involving a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, if a gene, cDNA, or RNA molecule (e.g., mRNA) encodes a polypeptide, the polypeptide is produced in a cell or other biological system by transcription and translation of the RNA (e.g., mRNA) corresponding to that gene. In some embodiments, the coding region of an RNA molecule encoding a target antigen refers to the coding strand, and its nucleotide sequence is identical to the RNA (e.g., mRNA) sequence of such target antigen. In some embodiments, the coding region of an RNA molecule encoding a target antigen refers to the non-coding strand of such target antigen, which can be used as a template for transcription of a gene or cDNA.

[0279] Manipulation: Generally, the term "manipulation" refers to a manner that is manipulated by a human hand. For example, when a polynucleotide is considered to be "manipulated", two or more sequences that are not linked together in that order in nature are manipulated by a human hand to be directly linked to each other in the manipulated polynucleotide, and / or certain residues within the polynucleotide are not present naturally, and / or by the action of a human hand, are linked to something or a portion that is not linked in nature.

[0280] Epitope: As used herein, the term "epitope" refers to the portion specifically recognized by an immunoglobulin (e.g., an antibody or a receptor) binding component. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are exposed on the surface when the antigen assumes an appropriate three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen assumes such a structure. In some embodiments, at least some such chemical atoms are groups that are physically separated from each other when the antigen assumes a different three-dimensional structure (e.g., becomes linear). Thus, in some embodiments, an epitope of an antigen can include a continuous or discontinuous fragment of the antigen. In some embodiments, the epitope is a T cell epitope or includes a T cell epitope. In some embodiments, the epitope is about 5 to about 30 amino acids in length, or about 10 to about 25 amino acids in length, or about 5 to about 15 amino acids in length, or about 5 to about 12 amino acids in length, or about 6 to about 9 amino acids in length.

[0281] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, the expression of a nucleic acid sequence includes one or more of the following. (1) Generation of an RNA template from a DNA sequence (e.g., by transcription), (2) Processing of the RNA transcript (e.g., by splicing, editing, etc.), (3) Translation of the RNA into a polypeptide or protein, and / or (4) Post-translational modification of the polypeptide or protein.

[0282] 5 prime untranslated region: As used herein, the term "5 prime untranslated region" or "5'UTR" refers to a sequence of an RNA (e.g., mRNA) molecule that is between the transcription start site and the start codon of the coding region of the RNA. In some embodiments, the "5'UTR" is a sequence of an RNA (e.g., mRNA) molecule that starts at the transcription start site and ends 1 nucleotide (nt) before the start codon (usually AUG) of the coding region of the RNA molecule (e.g., in its native context).

[0283] Fragment: As used herein, the term "fragment" with respect to a nucleic acid sequence (e.g., an RNA sequence) or an amino acid sequence can typically be a fragment of a reference sequence. In some embodiments, the reference sequence is, for example, the full-length sequence of a nucleic acid sequence or an amino acid sequence. Thus, a fragment typically refers to a sequence that is identical to the corresponding strand within the reference sequence. In some embodiments, a fragment comprises a continuous strand of nucleotides or amino acid residues corresponding to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the full length of the reference sequence from which it is derived. In some embodiments, the term "fragment" with respect to an amino acid sequence (a peptide or polypeptide) refers to a sequence that represents a portion of the amino acid sequence, e.g., an amino acid sequence that is truncated at the N-terminus and / or C-terminus. In some embodiments, a fragment of an amino acid sequence comprises at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0284] Homologous: As used herein, the terms "homologous" or "homolog" refer 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 to be "homologous" to each other when 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 when 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., including residues having related chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are typically classified as being similar to each other as "hydrophobic" amino acids or "hydrophilic" amino acids and / or as having "polar" or "nonpolar" side chains. Substituting one amino acid for another of the same type is often considered to be an "isologous" substitution.

[0285] Humoral immunity: As used herein, the terms "humoral immunity" or "humoral immune response" refer to antibody production and associated processes, including, for example, Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell generation. This also refers to the effector functions of antibodies, including, for example, pathogen neutralization, classical complement activation, and opsonin-facilitated pathogen elimination and phagocytosis.

[0286] 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 polypeptide molecules are considered to be "substantially identical" to each other when 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. For example, the percent identity of two nucleic acid sequences or polypeptide sequences can be calculated by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Then, the nucleotides at corresponding positions are compared. If the position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity of two nucleotide sequences can be determined using the algorithm of Meyers and Miller (1989) incorporated into the ALIGN program (version 2.0).In some exemplary embodiments, the nucleic acid sequence comparison performed by the ALIGN program uses the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can alternatively be determined by the GAP program of the GCG software package using the NWSgapdna.CMP matrix.

[0287] Immunologically equivalent: The term "immunologically equivalent" means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit the same or substantially the same immunological properties and / or exert the same or substantially the same immunological effects, for example, with respect to the type of immunological effect. For the purposes of the present disclosure, in some embodiments, the term "immunologically equivalent" is used with respect to the immunological effects or properties of an antigen or antigen variant used for immunization. For example, if an amino acid sequence is immunologically equivalent to a reference amino acid sequence, the amino acid sequence induces an immune response having the reaction specificity of the reference amino acid sequence when exposed to the immune system of a subject.

[0288] In one embodiment, the antigen receptor is an antibody or a B cell receptor that binds to an epitope of an antigen. In one embodiment, the antibody or B cell receptor binds to the native epitope of the antigen.

[0289] Increase, induce, or reduce: As used herein, these terms or grammatically equivalent comparative terms indicate that the value is relative to a comparable reference measurement. For example, in some embodiments, an evaluation value achieved by a provided pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) may be "increased" compared to an evaluation value obtained by a comparable reference pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). Alternatively or additionally, in some embodiments, an evaluation value achieved in a subject is "increased" compared to an evaluation value obtained in the same subject under different conditions (e.g., before or after something, or in the presence or absence of something (e.g., administration of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein), or in a different comparable subject (e.g., a subject different from the subject of interest pre-exposed to a certain condition (e.g., in the absence of administration of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein)). In some embodiments, the comparative term refers to a statistically appropriate difference (e.g., one having a spread and / or magnitude sufficient to obtain statistical relevance). Determining the degree and / or spread of difference necessary or sufficient to obtain such statistical significance in a given situation is known to those of skill in the art or can be readily done by those of skill in the art. In some embodiments, the term "reduce" or an equivalent term refers to a reduction in the level of the evaluation value of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or more compared to a comparable reference. In some embodiments, the term "reduce" or an equivalent term refers to complete or substantially complete inhibition, i.e., a reduction to zero or substantially zero. In some embodiments, the terms "increase" or "induce" refer to an increase in the level of the evaluation 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 compared to a comparable reference.

[0290] Ionizable: The term "ionizable" refers to a compound, group, or atom that becomes charged at a specific pH. In relation to ionizable amino lipids, such a lipid, or its functional group or atom, has a positive charge at a specific pH. In some embodiments, the ionizable amino lipid is positively charged at an acidic pH. In some embodiments, the ionizable amino lipid is predominantly neutral at physiological pH values, e.g., in some embodiments, around 7.0 - 7.4, but is positively charged at lower pH values. In some embodiments, the ionizable amino lipid can have a pKa in the range of about 5 to about 7.

[0291] Isolated: The term "isolated" means alteration or removal from a natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting substances in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-native environment such as a host cell.

[0292] Lipid: As used herein, the terms "lipid" and "lipid-like substance" are broadly defined as molecules that contain one or more hydrophobic moieties or groups, and optionally one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphilic molecules.

[0293] RNA lipid nanoparticles: As used herein, the term "RNA lipid nanoparticles" refers to nanoparticles comprising at least one lipid and RNA molecule(s). In some embodiments, the RNA lipid nanoparticles comprise at least one ionizable amino lipid. In some embodiments, the RNA lipid nanoparticles comprise at least one ionizable amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, the average size (e.g., Z-average) of the RNA lipid nanoparticles described herein can be from about 100 nm to 1000 nm, or from about 200 nm to 900 nm, or from about 200 nm to 800 nm, or from about 250 nm to about 700 nm. In some embodiments of the present disclosure, the average size (e.g., Z-average) of the RNA lipid nanoparticles can be from about 30 nm to about 200 nm, or from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, or from about 70 nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticles is determined by measuring the particle diameter. In some embodiments, the RNA lipid nanoparticles can be prepared by mixing the lipids with the RNA molecules described herein.

[0294] Lipidoid: As used herein, "lipidoid" refers to lipid-like molecules. In some embodiments, the lipidoid is an amphiphilic molecule having one or more lipid-like physical properties. In the context of the present disclosure, the term lipid is considered to encompass lipidoids.

[0295] Nanoparticles: As used herein, the term "nanoparticles" refers to particles having an average size suitable for parenteral administration. In some embodiments, the nanoparticles have a longest dimension (e.g., diameter) of less than 1,000 nanometers (nm). In some embodiments, the nanoparticles can be characterized by a longest dimension (e.g., diameter) of less than 300 nm. In some embodiments, the nanoparticles can be characterized by a longest dimension (e.g., diameter) of less than 100 nm. In many embodiments, the nanoparticles can be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 1 μm and about 500 nm, or between about 1 nm and 1,000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is less than about 1,000 nm, about 500 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm, and in many cases greater than about 1 nm. In many embodiments, the nanoparticles are substantially spherical, and thus their longest dimension can be their diameter. In some embodiments, the nanoparticles have a diameter of less than 100 nm, as defined by the National Institutes of Health.

[0296] Naturally occurring: As used herein, the term "naturally occurring" refers to the ability of an entity to exist in nature. For example, a polypeptide or nucleic acid that exists in a living organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by a person in a laboratory is a naturally occurring entity.

[0297] Neutralization: As used herein, the term "neutralization" refers to the event in which a binding agent, such as an antibody, binds to the biological active site of a virus, such as a receptor-binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term "neutralization" refers to the event in which a binding agent eliminates or significantly reduces the ability to infect cells.

[0298] Nucleic acid particles: "Nucleic acid particles" can be used to deliver nucleic acids to a target site of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can include at least one cationic lipid or a lipid ionizable to a cation or at least one cationic lipid-like material or a lipid-like material ionizable to a cation, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acids. In some embodiments, the nucleic acid particles are lipid nanoparticles. In some embodiments, the nucleic acid particles are lipoplex particles.

[0299] Nucleic acid / Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is DNA or contains DNA. In some embodiments, the nucleic acid is RNA or contains RNA. In some embodiments, the nucleic acid is peptide nucleic acid (PNA) or contains peptide nucleic acid (PNA). In some embodiments, the nucleic acid is a single-stranded nucleic acid or contains a single-stranded nucleic acid. In some embodiments, the nucleic acid is a double-stranded nucleic acid or contains a double-stranded nucleic acid. In some embodiments, the nucleic acid contains both single-stranded and double-stranded fragments. In some embodiments, the nucleic acid contains a backbone having one or more phosphodiester bonds. In some embodiments, the nucleic acid contains a backbone having both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may contain a backbone having one or more phosphorothioate bonds or 5'-N-phosphoramidite bonds and / or one or more peptide bonds, such as "peptide nucleic acid". In some embodiments, the nucleic acid contains one or more, or all, of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid contains one or more, or all, of the unnatural residues. In some embodiments, the unnatural residues include nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof).In some embodiments, the unnatural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) relative to that of the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., polymerization based on a complementary template), replication in a recombinant cell or system in vivo or in vitro, or chemical synthesis. In some embodiments, the 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, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000, or more residues or nucleotides.

[0300] Nucleotide: As used herein, the term "nucleotide" refers to the meaning recognized in the art. When a particular number of nucleotides is used, for example, as an indication of the size of a polynucleotide, the specific number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0301] Patient: As used herein, the term "patient" refers to any organism that has or is at risk of having a disease, disorder, or illness. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is a human. In some embodiments, the patient has or is predisposed to one or more diseases, disorders, or illnesses. In some embodiments, the patient exhibits one or more symptoms of a disease, disorder, or illness. In some embodiments, the patient has been diagnosed with one or more diseases, disorders, or illnesses. In some embodiments, the disease, disorder, or illness suitable for the provided technology is or includes HSV infection. In some embodiments, the patient has received or has been administered a particular therapy for diagnosing and / or treating a disease, disorder, or illness. In some embodiments, the patient is a patient who has or is susceptible to HSV infection.

[0302] PEG-conjugated lipid: The term "PEG-conjugated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

[0303] 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 dose appropriate for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a suitable population. In some embodiments, the pharmaceutical composition can be formulated specifically for parenteral administration, such as by subcutaneous, intramuscular, or intravenous injection, for example, as a sterile solution or suspension formulation.

[0304] Pharmaceutically effective amount: The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves a desired response or desired effect, either alone or in conjunction with additional dosages. When treating a particular disease, in some embodiments, the desired response relates to suppression of the course of the disease. In some embodiments, such inhibition may include slowing the progression of the disease and / or interrupting or reversing the progression of the disease. In some embodiments, the desired response in treating a disease may be or may include delaying or preventing the onset of the disease or illness. The effective amount of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein depends, for example, on the disease or illness being treated, the severity of such disease or illness, the individual parameters of the patient (e.g., age, physiological state, size and weight, treatment period, type of concomitant therapy (if any), particular route of administration, and like factors, etc.). Accordingly, the dosage of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) described herein may depend on such various parameters. If the patient's response is insufficient with the initial dosage, a higher dosage (or an effectively higher dosage achieved by a different, more localized route of administration) can be used.

[0305] PolyA sequence: As used herein, the term "polyA sequence" or "polyA tail" typically refers to a continuous or intermittent sequence of adenylate residues located at the 3' end of an RNA molecule. PolyA sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. A continuous polyA sequence is characterized by consecutive adenylate residues. In nature, continuous polyA sequences are typical. The RNAs disclosed herein may have a polyA sequence, which may be attached to the free 3' end of the RNA by a template-independent RNA polymerase post-transcriptionally or may be encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0306] Polypeptide: As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has a naturally occurring amino acid sequence. In some embodiments, the polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, the polypeptide has an amino acid sequence that has been engineered in that it has been designed and / or produced by human action. In some embodiments, the polypeptide can comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide can comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, the polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide can comprise only D-amino acids. In some embodiments, the polypeptide can comprise only L-amino acids. In some embodiments, the polypeptide can include one or more pendant groups or other modifications, for example, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof, can modify one or more amino acid side chains or include one or more pendant groups or other modifications attached to one or more amino acid side chains. In some embodiments, such pendant groups or modifications include acetylation, amidation, lipidation, methylation, pegylation, etc. (including combinations thereof). In some embodiments, the polypeptide can be cyclic and / or can include a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not include a cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide can be or can include a stapled polypeptide. In some embodiments, the term "polypeptide" may be assigned to the name of a reference polypeptide, activity, or structure, in which case it is used to refer to polypeptides that share the relevant activity or structure and can thus be considered members of the same class or family of polypeptides.For each such class, the specification shows exemplary polypeptides within the class for which the amino acid sequence and / or function are known and / or which should be known to those of ordinary skill in the art. In some embodiments, such exemplary polypeptides are reference polypeptides for a class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity with the reference polypeptide of that class (in some embodiments, with all polypeptides within that class), share a common sequence motif (e.g., a characteristic sequence element), and / or share a common activity (in some embodiments, an equivalent level or activity within a specified range). For example, in some embodiments, the polypeptides of the members have an overall degree of sequence homology or identity with the reference polypeptide of at least about 30-40%, often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or the polypeptides of the members include at least one region (e.g., a conserved region that may be or include a characteristic sequence element in some embodiments) that exhibits very high sequence identity (often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%). Such conserved regions typically include at least 3-4, often up to 20 or more amino acids, and in some embodiments, the conserved region includes at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids. In some embodiments, related polypeptides may comprise or consist of fragments of the parent polypeptide.

[0307] Prevention: As used herein, the term "prevent" or "preventing," when used in connection with the occurrence of a disease, disorder, and / or illness, means reducing the risk that the disease, disorder, and / or illness will occur and / or delaying the occurrence of one or more features or symptoms of the disease, disorder, or illness. Prevention may be considered achieved if the onset of a disease, disorder, or illness is delayed for a predefined period of time.

[0308] Recombinant: As used in connection with the present disclosure, the term "recombinant" means "produced by genetic engineering." In some embodiments, "recombinant" entities such as recombinant nucleic acids, in connection with the present disclosure, do not occur in nature.

[0309] Reference: As used herein, the term "reference" indicates 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, the reference or control is tested and / or measured substantially simultaneously with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible expression medium. Typically, as will be apparent to those of ordinary skill in the art, the reference or control is measured or characterized under conditions or circumstances comparable to those being evaluated. It will be apparent to those of ordinary skill in the art what circumstances exist such that there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0310] Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA includes both single-stranded and double-stranded fragments. In some embodiments, the RNA may include a backbone structure as described in the above definition of "nucleic acid / polynucleotide". The RNA can be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments where the RNA is mRNA, the RNA typically includes a polyA region at its 3' end. In some embodiments where the RNA is mRNA, the RNA typically includes, at its 5' end, a cap structure known in the art for recognizing the mRNA and binding it to ribosomes to initiate translation. In some embodiments, the RNA is synthetic RNA. Examples of synthetic RNA include RNA synthesized in vitro (e.g., by enzymatic synthesis and / or chemical synthesis methods).

[0311] Ribonucleotide: As used herein, the term "ribonucleotide" encompasses both unmodified and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides include, for example, (a) terminal modifications such as 5'-terminal modifications (e.g., phosphorylation, dephosphorylation, ligation, inverse ligation, etc.), 3'-terminal modifications (e.g., ligation, inverse ligation, etc.), (b) base modifications (e.g., modified bases, stabilized bases, destabilized bases, or substitutions with bases that base pair with partners having an expanded repertoire, or substitutions with conjugated bases), (c) sugar modifications (e.g., at the 2'- or 4'-position) or sugar replacements, and (d) modifications of the phosphodiester bond or substitutions, such as internucleoside bond modifications including but not limited to these. The term "ribonucleotide" also encompasses ribonucleotide triphosphates including modified and unmodified ribonucleotide triphosphates.

[0312] Risk: As is apparent from the context, the risk of a disease, disorder, and / or illness refers to the likelihood that a particular individual will develop that disease, disorder, and / or illness. 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 to 100%. In some embodiments, the risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk for a disease, disorder, illness, and / or onset. In some embodiments, the reference sample or group of reference samples is derived from an individual comparable to a particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or greater. In some embodiments, the risk can reflect one or more genetic traits, which can, for example, make an individual more (or less) likely to develop a particular disease, disorder, and / or illness. In some embodiments, the risk can reflect one or more epigenetic events or traits and / or one or more lifestyle or environmental events or environmental characteristics.

[0313] RNA lipoplex particle: As used herein, the term "RNA lipoplex particle" refers to a complex comprising a liposome (particularly a cationic liposome) and an RNA molecule. Without wishing to be bound by a particular theory, electrostatic interactions between the positively charged liposome and the negatively charged RNA result in complex formation and the spontaneous formation of RNA lipoplex particles. In some embodiments, the positively charged liposome can comprise a cationic lipid (e.g., in some embodiments, DOTMA) and other lipids (e.g., in some embodiments, DOPE). In some embodiments, the RNA lipoplex particle is a nanoparticle.

[0314] Selective or specific: As used herein with respect to an agent having activity, the terms "selective" or "specific" mean that the agent shows a difference among entities, states, or cells that are capable of being targeted. For example, in some embodiments, when an agent is said to "specifically" bind to its target, the agent preferentially binds to the target in the presence of one or more competing other targets. In many embodiments, the specific interaction varies depending on the presence of specific structural features of the entity being targeted (e.g., epitope, cleft, binding site). Of course, specificity need not be absolute. In some embodiments, specificity can be evaluated in comparison to the specificity of the target binding site for one or more other entities that are capable of being targeted (e.g., competitors). In some embodiments, the property can be evaluated in comparison to the specificity of a reference specific binding site. In some embodiments, the property can be evaluated in comparison to the specificity of a reference specific binding site.

[0315] Stable: As used herein, the term "stable" with respect to the present disclosure refers to the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as a whole and / or its components meeting or exceeding certain acceptable criteria. For example, in some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) does not exhibit unacceptable levels of microbial growth and shows no or substantially no degradation or deterioration of the bioactive molecule component(s). In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the integrity of the RNA molecule being maintained at least about 90% or more. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to at least 90% or more (e.g., including at least 95%, at least 96%, at least 97%, or more) of the RNA molecules being encapsulated and maintained within lipid nanoparticles. In some embodiments, a stable pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) refers to the formulation being in a state where it can induce the desired immune response when administered to the subject. In some embodiments, the pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) is stable for a predetermined period under specific conditions.

[0316] Subject: As used herein, the term "subject" refers to an organism to which the compositions described herein are administered (e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes). Typical subjects include animals (e.g., mice, rats, rabbits, non-human primates, household pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject is susceptible to a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject exhibits no symptoms or features of a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject has one or more characteristics specific to being susceptible to a disease, disorder, or illness (e.g., HSV infection) or one or more characteristics specific to the risk of a disease, disorder, or illness (e.g., HSV infection). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who is undergoing and / or has undergone diagnosis and / or treatment.

[0317] Affected: An individual who is "affected with" a disease, disorder, and / or illness has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or illness.

[0318] Prone to contracting: An individual who is "prone to contracting" a disease, disorder, and / or illness is an individual who has a higher risk of developing the disease, disorder, and / or illness than the general population. In some embodiments, an individual who is prone to contracting a particular disease, disorder, and / or illness may not have been diagnosed with the disease, disorder, and / or illness. In some embodiments, an individual who is prone to contracting a particular disease, disorder, and / or illness may exhibit symptoms of the disease, disorder, and / or illness. In some embodiments, an individual who is prone to contracting a particular disease, disorder, and / or illness may not exhibit symptoms of the disease, disorder, and / or illness. In some embodiments, an individual who is prone to contracting a particular disease, disorder, and / or illness will develop the disease, disorder, and / or illness. In some embodiments, an individual who is prone to contracting a particular disease, disorder, and / or illness will not develop the disease, disorder, and / or illness.

[0319] Synthetic: As used herein, the term "synthetic" refers to an entity being artificial, or being made by human involvement, or being derived from synthesis rather than occurring naturally. For example, in some embodiments, a synthetic nucleic acid or synthetic polynucleotide refers to a nucleic acid molecule that has been chemically synthesized (e.g., in some embodiments, by solid-phase synthesis). In some embodiments, the term "synthetic" refers to an entity being made outside of a living cell. For example, in some embodiments, a synthetic nucleic acid or synthetic polynucleotide refers to a nucleic acid molecule (e.g., RNA) that has been generated by in vitro transcription using a template.

[0320] Treatment: The term "treatment" refers to the administration or provision of an agent or intervention that has a therapeutic effect and / or exhibits a desired biological and / or pharmacological effect (e.g., such an effect has been statistically demonstrated to be possible when administered to an appropriate population). In some embodiments, a therapeutic agent can be any substance used to alleviate, improve, reduce, 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 illness. In some embodiments, a therapeutic agent or treatment is a medical intervention (e.g., surgery, radiation therapy, phototherapy) that can be performed to alleviate, reduce, 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 illness.

[0321] 3 prime untranslated region: As used herein, the term "3 prime untranslated region" or "3'UTR" refers to the sequence of an RNA (e.g., mRNA) molecule that begins following the stop codon of the coding region of an open reading frame sequence. In some embodiments, the 3'UTR begins, for example, immediately following the stop codon of the coding region of the open reading frame sequence in its natural context. In some embodiments, the 3'UTR does not begin, for example, immediately following the stop codon of the coding region of the open reading frame sequence in its natural context.

[0322] Threshold level (e.g., pass / fail criteria): As used herein, the term "threshold level" refers to a level used as a reference for classifying the results of a measurement (e.g., measurement results obtained in an assay) and / or obtaining information about such results. For example, in some embodiments, the threshold level is a value that defines a boundary between two subsets of a population (e.g., batches that meet quality control criteria vs. batches that do not meet quality control criteria) measured in an assay. Thus, values above the threshold level define one subset of the population, and values below the threshold level define another subset of the population. The threshold level can be determined based on one or more control samples or relative to a population of control samples. The threshold level can be determined before, simultaneously with, or after performing the measurement of interest. In some embodiments, the threshold level can be a range of values.

[0323] Treatment: As used herein, the terms "treating," "treatment," or "treat" refer to any method being used to partially or completely alleviate, ameliorate, reduce, inhibit, prevent, partially or completely delay the onset of, partially or completely reduce the severity of, and / or partially or completely reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or illness. Treatment may be administered to a subject who does not exhibit symptoms of the disease, disorder, and / or illness. In some embodiments, treatment may be administered to a subject who exhibits only early signs of a disease, disorder, and / or illness, for the purpose of, e.g., reducing the risk of developing a medical condition associated with the disease, disorder, and / or illness. In some embodiments, treatment may be administered to a subject in the later stages of a disease, disorder, and / or illness.

[0324] Vaccination: As used herein, the term "vaccination" refers to the administration of a composition intended to elicit an immune response (e.g., against a disease-related agent (e.g., a disease-causing agent)). In some embodiments, vaccination can be performed before, during, and / or after exposure to the disease-related agent, and in certain embodiments, can be performed before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination includes administering the vaccine composition multiple times at appropriate intervals. In some embodiments, vaccination elicits an immune response against an infectious agent.

[0325] Vaccine: As used herein, the term "vaccine" refers to a composition that, when administered to a subject, induces an immune response. In some embodiments, the induced immune response results in protective immunity.

[0326] Variant: As used herein with respect to a molecule, e.g., a nucleic acid, protein, or small molecule, the term “variant” refers to a molecule that exhibits significant structural identity with a reference molecule while being structurally different from the reference molecule (e.g., different in the presence or absence or level of one or more chemical moieties as compared to the reference entity). In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a “variant” of a reference molecule is based on the degree of its structural identity with the reference molecule. As will be apparent to those of skill in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a molecule that shares one or more such characteristic structural elements with the reference molecule but is different from the reference molecule in at least one aspect. In some embodiments, a variant polypeptide or variant nucleic acid differs from a reference polypeptide or reference nucleic acid as a result of one or more differences in the amino acid or nucleotide sequence and / or differences in chemical moieties (e.g., carbohydrate, lipid, phosphate group) that are covalent constituents of the polypeptide or nucleic acid (e.g., attached to the backbone of the polypeptide or nucleic acid). In some embodiments, a variant polypeptide or variant nucleic acid exhibits an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with the reference polypeptide or reference nucleic acid. In some embodiments, a variant polypeptide or variant nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or reference nucleic acid. In some embodiments, the reference polypeptide or reference nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or variant nucleic acid shares one or more of the biological activities of the reference polypeptide or reference nucleic acid. In some embodiments, a variant polypeptide or variant nucleic acid lacks one or more of the biological activities of the reference polypeptide or reference nucleic acid. In some embodiments, a variant polypeptide or variant nucleic acid has a reduced level of one or more biological activities as compared to the reference polypeptide or reference nucleic acid.In some embodiments, when the polypeptide or nucleic acid of interest is considered a “variant” of a reference polypeptide or reference nucleic acid, it has an amino acid sequence or nucleotide sequence that is identical to that of the reference but has a small number of sequence changes at specific positions. Typically, in a variant, 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 are substituted, inserted, or deleted as compared to the reference. In some embodiments, the variant polypeptide or variant nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to the reference. In many cases, the variant polypeptide or variant nucleic acid has a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of functional residues (i.e., residues involved in a particular biological activity) that are substituted, inserted, or deleted as compared to the reference. In some embodiments, the variant polypeptide or variant nucleic acid has about 5, about 4, about 3, about 2, or about 1 or fewer additions or deletions as compared to the reference, and in some embodiments, there are no additions or deletions. In some embodiments, the variant polypeptide or variant nucleic acid has 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, less than about 6, typically less than about 5, about 4, about 3, or about 2 additions or deletions. In some embodiments, the reference polypeptide or reference nucleic acid is a naturally occurring one.

[0327] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of binding to and transporting another nucleic acid. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop that can ligate additional DNA segments. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. Further, certain vectors can induce the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". In some embodiments, for example, for the production or manipulation of recombinant DNA, for oligonucleotide synthesis, for tissue culture and transformation (e.g., electroporation, lipofection), known techniques can be used. Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications, or as generally accomplished in the art, or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art, and as described in various general and more specific references cited and described throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2012)) (which is incorporated herein by reference for all purposes).

[0328] All documents and similar materials cited in this application, including but not limited to patents, patent applications, papers, books, treatises, and web pages, are hereby expressly incorporated by reference in their entirety regardless of the format of such documents and similar materials. If one or more of the incorporated documents and similar materials are different from or conflict with this application (including, without limitation, defined terms, usage of terms, described techniques, etc.), this application shall prevail. Section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described in any way.

[0329] In some embodiments, the present disclosure provides techniques (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 techniques provided by the present disclosure can be used to reduce immune imprinting effects and / or induce a stronger, novel immune response (e.g., as compared to other vaccination methods).

[0330] Overview of SARS-CoV-2 The SARS-CoV-2 spike (S) protein can be proteolytically cleaved into an S1 (685 amino acids) subunit and an S2 (588 amino acids) subunit. The S1 of SARS-CoV-2 contains a receptor-binding domain (RBD), which mediates the entry of the virus into host cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.

[0331] COVID-19 generally presents with cough and fever, and ground-glass opacities or patchy opacities can be seen on its chest X-ray. However, there are many patients without fever or X-ray changes, and the infection may be asymptomatic, which is related to the suppression of transmission. For symptomatic patients, the progression of the disease may lead to acute respiratory distress syndrome requiring mechanical ventilation, subsequent multiple organ failure, and death. Common symptoms in hospitalized patients (listed from high to low frequency) include fever, dry cough, shortness of breath, fatigue, muscle pain, nausea / vomiting or diarrhea, headache, malaise, and runny nose. Olfactory impairment (anosmia) or gustatory impairment (ageusia) can be symptoms that occur alone in about 3% of individuals with COVID-19.

[0332] This disease can occur in all age groups. In particular, the case fatality rate (CFR) increases when over 60 years old. Comorbidities are also associated with an increase in CFR, and such diseases include cardiovascular diseases, diabetes, and chronic respiratory diseases. Healthcare workers are more numerous among COVID-19 patients because they are occupationally exposed to infected patients.

[0333] In most cases, molecular testing is used to detect SARS-CoV-2 and confirm the infection. Reverse transcription polymerase chain reaction (RT-PCR) testing targeting SARS-CoV-2 viral RNA is one method for diagnosing suspected cases of COVID-19. The samples to be tested are collected using swabs from the nose and / or throat.

[0334] SARS-CoV-2 variants Since the initial discovery of SARS-CoV-2, numerous variants have emerged worldwide. The emergence of these newly circulating variants of SARS-CoV-2 has raised significant concerns regarding the geographical and temporal effectiveness of vaccine interventions. The emergence of the Omicron (B.1.1.529) variant, which contains several mutations in the S protein, is of particular concern. As used herein, "SARS-CoV-2 variant" refers to a SARS-CoV-2 virus that has acquired one or more mutations that differ from the Wuhan strain of SARS-CoV-2, which first emerged in 2019. Variants can be identified by virologists and / or healthcare institutions using an appropriate classification system, such as, for example, the Pango or NextClade classification systems (examples of which are described herein).

[0335] In some embodiments, the disclosure refers to SARS-CoV-2 variants that are circulating and / or rapidly spreading in the applicable jurisdiction. In some embodiments, such variants can be identified based on publicly available data (e.g., data provided by the GISAID Initiative database: https: / / www.gisaid.org, and / or data provided by the World Health Organization WHO (e.g., provided at https: / / www.who.int / activities / tracking-SARS-CoV-2-variants)). In some embodiments, such variants refer to the variants disclosed herein.

[0336] The Omicron BA.1 variant was first reported to the WHO on November 24, 2021 and detected in South Africa. Omicron and its sub-lineages have had a major impact on the epidemiology of the COVID-19 pandemic since their first emergence (WHO Technical Advisory Group on SARS-CoV-2 Virus Evolution (TAG-VE), Classification of Omicron (B.1.1.259), SARS-CoV-2 Variants of Concern (2021), WHO Headquarters (HQ), WHO Health Emergencies Programme, Strengthening Response to the Omicron SARS-CoV-2 Variant, Technical Briefing and Priority Actions for Member States (2022)). Significant changes occurred in the spike (S) glycoprotein of the first Omicron variant BA.1, resulting in the loss of many neutralizing antibody epitopes (M. Hoffmann et al., “The Omicron variant is highly resistant against antibody mediated neutralization: Implications for control of the COVID-19 pandemic”, Cell 185, 447-456.e11 (2022)), and BA.1 has become capable of partially evading immunity based on previously established SARS-CoV-2 wild-type strains (Wuhan-Hu-1) (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)).

[0337] As a result, breakthrough infections with Omicron in vaccinated individuals are more common than previously feared variants of concern (VOCs). While Omicron BA.1 has been replaced by the BA.2 variant strain in many countries around the world, other variant strains 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. Target. Ther. 7, 241 (2022), H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns, Cell Host Microbe 7, 241 (2022)). Omicron BA.2.12.1 subsequently replaced BA.2 and became dominant in the United States. Meanwhile, BA.4 and BA.5 replaced BA.2 in Europe, parts of Africa, and Asia / Pacific (H. Gruell et al., “SARS-CoV-2 Omicron sublineages exhibit distinct antibody escape patterns,” Cell Host Microbe 7, 241 (2022), European Centre for Disease Prevention and Control, Weekly COVID-19 country overview-Country overview report: Week 31 2022 (2022), J. Hadfield et al., “Nextstrain: Real-time tracking of pathogen evolution,” Bioinformatics 34, 4121-4123 (2018)).Currently, Omicron BA.5 is dominant worldwide, including in the United States (Centers for Disease Control and Prevention. COVID Data Tracker. Atlanta, GA: US Department of Health and Human Services, CDC; 2022, August 12. https: / / covid.cdc.gov / coviddata-tracker (2022)).

[0338] Omicron has acquired many mutations in the S glycoprotein (amino acid exchanges, insertions, or deletions), some of which are shared by all Omicron VOCs, while others are specific to one or more Omicron sub-lineages. Antigenically, BA.2.12.1 shows high similarity to BA.2 but not to BA.1, while BA.4 and BA.5 are quite different from their ancestor BA.2 and even more different from BA.1, consistent with their phylogeny ((A.Z. Mykytyn et al., “Antigenic cartography of SARS-CoV-2 reveals that Omicron BA.1 and BA.2 are antigenically distinct,” Sci. Immunol. 7, Eabq4450 (2022)). What makes BA.1 significantly different from other Omicron VOCs are Δ143-145, L212I, or ins214EPE in the S glycoprotein N-terminal domain and G446S or G496S in the receptor-binding domain (RBD). And the amino acid mutations T376A, D405N, and R408S in the RBD are common to BA.2 and its descendants but not present in BA.1. Furthermore, some mutations are specific to individual VOCs that are descendants of BA.2, such as L452Q in BA.2.12.1, and L452R and F486V in BA.4 and BA.5 (BA.4 and BA.5 encode the same S sequence). Most of these common mutations and VOC-specific mutations have been shown to play important roles in immune evasion from monoclonal antibodies and polyclonal sera produced against the wild-type S glycoprotein.In particular, BA.4 / BA.5-specific mutations are strongly involved in the immune evasion of these VOCs (P. Wang et al., “Antibodyess 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 subvarits BA.2.12.1, BA.4, & BA.5. Nature 608, 603-608 (2022)).

[0339] SARS-CoV-2 variant compatibility In some embodiments, the antigen utilized as described herein is a fragment or domain of a viral polypeptide, or an antigenic fragment thereof, or comprises a fragment or domain of a viral polypeptide, or an antigenic fragment thereof. In some embodiments, the antigen utilized as described herein is a membrane-bound antigen (e.g., an antigenic fragment thereof fused to a membrane-binding portion such as a transmembrane portion). In some embodiments, the pharmaceutical composition provided (e.g., an immunogenic composition, e.g., a vaccine) contains or delivers an antigen sequence, and the antigen sequence is one or more antibody epitopes and / or one or more CD4 T cell epitopes and / or one or more CD8 T cell epitopes or comprises one or more antibody epitopes and / or one or more CD4 T cell epitopes and / or one or more CD8 T cell epitopes.

[0340] In some embodiments, the antigen utilized as described herein contains one or more variant sequences compared to a relevant reference antigen. For example, in some embodiments, a protease cleavage site is removed or blocked. Alternatively or additionally, in some embodiments, truncated antigens are utilized, 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.

[0341] In some embodiments, the antigens utilized as described herein include multimerization elements (e.g., heterologous multimerization elements).

[0342] In some embodiments, the antigens utilized as described herein include membrane-binding elements such as transmembrane domains (e.g., homologous membrane-binding elements).

[0343] In some embodiments, the antigens utilized as described herein include secretion signals (e.g., homologous secretion signals).

[0344] In some embodiments, the sequences utilized may include one or more mutations associated with viral variants (e.g., variants predicted to be prevalent and / or highly immune-evasive). In some embodiments, the sequences utilized include one or more mutations associated with concerning variants (e.g., concerning variants identified by the WHO). In some embodiments, the sequences utilized include one or more mutations associated with viral variants determined or predicted to have high immune evasion (e.g., high immune evasion against the immune response generated in subjects administered a previously approved vaccine and / or high immune evasion against previously circulating viral variants).

[0345] In particular, this specification describes specific SARS-CoV-2 antigens for use in inducing an immunogenic response. In some embodiments, the SARS-CoV-2 antigen comprises an immunogenic portion of a full-length SARS-CoV-2 polypeptide (e.g., the S1 domain of the SARS-CoV-2 S protein, a truncated S1 subdomain of the SARS-CoV-2 S protein, and / or the RBD of the SARS-CoV-2 S protein). In some embodiments, such an antigen is delivered as a protein antigen to induce an immunogenic response. In some embodiments, such an antigen is delivered using RNA (e.g., modRNA encoding the S1 domain of the SARS-CoV-2 S protein, a truncated S1 subdomain, and / or the RBD, formulated into LNP particles) to induce an immunogenic response.

[0346] The full-length SARS-CoV-2 S protein, including the "wild-type" or "Wuhan" sequence used in this disclosure, has a sequence corresponding to that of the first detected SARS-CoV-2 strain, which consists of 1273 amino acids and has the amino acid sequence according to SEQ ID NO: 1 below.

[0347] Unless otherwise indicated, the numbering of positions in the SARS-CoV-2 S protein shown in this specification is associated with the amino acid sequence of SEQ ID NO: 1. Those skilled in the art reading this disclosure can understand and identify the corresponding positions in the SARS-CoV-2 S protein variant from the positions shown for the amino acid sequence of SEQ ID NO: 1 (i.e., those skilled in the art shown the positions relative to SEQ ID NO: 1 or other variants can identify the corresponding positions in the S protein sequence or a fragment thereof of another SARS-CoV-2 variant). Also, as will be apparent to those skilled in the art, a fragment of the SARS-CoV-2 S protein containing one or more mutations of a variant contains only such mutations within the fragment region. For example, if a truncated S1 subdomain contains an amino acid sequence corresponding to amino acids 20 to 528 of SEQ ID NO: 1 and contains one or more mutations of a SARS-CoV-2 variant, it will be apparent to those skilled in the art that this truncated S1 subdomain contains only such mutations located within the corresponding region of the SARS-CoV-2 variant.

[0348] In certain embodiments, the spike (S) protein or fragment thereof described herein can be modified to stabilize the prototypical prefusion conformation. Specific mutations that stabilize the prefusion conformation are known in the art and are disclosed, for example, in WO2021243122A2 and Hsieh, Ching-Lin, et al. (“Structure-based design of prefusion-stabilized SARS-CoV-2 spikes,” Science 369.6510 (2020):1501-1505) (the entire contents of each are incorporated herein by reference). In some embodiments, the SARS-CoV-2 S protein can be stabilized by introducing one or more proline mutations. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at a position corresponding to residue 986 and / or 987 of SEQ ID NO:1. In some embodiments, the 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, the SARS-CoV-2 S protein comprises a proline substitution at a position corresponding to each of residues 817, 892, 899, and 942 of SEQ ID NO:1. In some embodiments, the SARS-CoV-2 S protein comprises a proline substitution at a position corresponding to each of residues 817, 892, 899, 942, 986, and 987 of SEQ ID NO:1.

[0349] In some embodiments, stabilization of the prototypical prefusion conformation of the SARS-CoV-2 S protein can be obtained by introducing two consecutive proline substitutions at residues 986 and 987. Specifically, the spike (S) protein-stabilized protein variant is obtained by replacing the amino acid residue at position 986 with proline and also replacing the amino acid residue at position 987 with proline. In one embodiment, the SARS-CoV-2 S protein variant with a stabilized prototypical prefusion conformation comprises the amino acid sequence shown in SEQ ID NO:2 below. (SEQ ID NO: 2).

[0350] One of ordinary skill in the art is aware of various SARS-CoV-2 spike variants and / or resources that document them. For example, the following strains, the amino acid sequences of their SARS-CoV-2 S proteins, and in particular, modifications thereof relative to the amino acid sequence of the wild-type SARS-CoV-2 S protein (e.g., SEQ ID NO: 1) are useful in the present disclosure.

[0351] B.1.1.7 (“Variant of Concern 202012 / 01” (VOC-202012 / 01)

[0352] B.1.1.7 (“Alpha variant”) is a SARS-CoV-2 variant first detected in the United Kingdom in October 2020 from samples taken the previous month, and began to spread rapidly by mid-December. This is associated with a significant increase in the COVID-19 infection rate. This increase is thought to be due, at least in part, to the N501Y mutation within the receptor-binding domain of the spike glycoprotein required for binding to human cell ACE2. B.1.1.7 is characterized by 23 mutations. Thirteen of these are non-synonymous mutations, four are deletions, and six are synonymous mutations (e.g., there are 17 mutations that change the protein and six that do not). Changes in the spike protein in B.1.1.7 include deletions 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H.

[0353] B.1.351 (501.V2)

[0354] The B.1.351 lineage (the "beta variant"), commonly known as the South African COVID-19 variant, has increased infectivity compared to the original Wuhan strain. The B.1.351 variant is characterized by multiple spike protein changes, including L18F, D80A, D215G, deletion 242-244, R246I, K417N, E484K, N501Y, D614G, and A701V. In the spike region of the B.1.351 genome, there are three particularly interesting mutations: K417N, E484K, and N501Y.

[0355] B.1.1.298 (cluster 5)

[0356] B.1.1.298 was discovered in the northern Jutland region of Denmark and is thought to have spread from mink to humans via mink farms. Several different mutations have been identified in the spike protein of this virus. Specific mutations include deletion 69-70, Y453F, D614G, I692V, M1229I, and optionally S1147L.

[0357] P.1 (B.1.1.248)

[0358] Lineage B.1.1.248 (the "gamma variant"), known as the Brazilian (Brazilian) variant, is one of the SARS-CoV-2 variants named the P.1 lineage. P.1 has several S protein mutations (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F) and is similar to the South African variant B.1.351 at certain key RBD positions (K417, E484, N501).

[0359] B.1.427 / B.1.429 (CAL.20C)

[0360] Lineage B.1.427 / B.1.429 (also known as CAL.20C, "Epsilon variant") is characterized by the mutations S13I, W152C, L452R, and D614G in the S protein. Of these, the L452R mutation is of particular interest. The CDC has listed B.1.427 / B.1.429 as a "variant of concern."

[0361] B.1.525

[0362] B.1.525 ("Eta variant") has the same E484K mutation seen in the P.1 and B.1.351 variants, as well as the same ΔH69 / ΔV70 deletion seen in B.1.1.7 and B.1.1.298. It also has the mutations D614G, Q677H, and F888L.

[0363] B.1.526

[0364] B.1.526 ("Iota variant") was detected as a new lineage of virus isolates in New York and has the same mutations as previously reported variants. The commonly shared set of spike mutations for this lineage is L5F, T95I, D253G, E484K, D614G, and A701V.

[0365] B.1.1.529

[0366] B.1.529 (the "Omicron variant") was first detected in South Africa in November 2021. Omicron grows at a rate approximately 70 times that of the Delta variant and has rapidly become the dominant strain of SARS-CoV-2 worldwide. Since its first detection, several Omicron sub-lineages have emerged. The current Omicron variants of concern are listed below, along with the specific characteristic mutations associated with their respective S proteins. The S proteins of BA.4 and BA.5 have the same set of characteristic mutations. Therefore, in the table below, there is one horizontal row designated as "BA.4 or BA.5", and in some embodiments, the present disclosure refers to the "BA.4 / 5" S protein. Similarly, the S proteins of the BA.4.6 and BF.7 Omicron variants have the same set of characteristic mutations. Therefore, in the table below, there is one horizontal row designated as "BA.4.6 or BF.7".

[0367] The JN.1 variant emerged in Luxembourg in August 2023. It is a descendant of the BA.2.86 variant. BA.2.86 initially attracted the attention of health authorities because it had a large number of mutations in the S protein (approximately 30 more than other co-circulating variants at the time). However, BA.2.86 did not become the dominant circulating SARS-CoV-2 variant. In contrast to BA.2.86, the JN.1 variant (and its descendants) has the ability to efficiently transmit among humans, which is thought to be due to the acquisition of the L455S mutation (at the position shown relative to SEQ ID NO: 1) in the S protein. JN.1 has rapidly become the dominant SARS-CoV-2 variant, increasing from less than 5% in November 2023 to 60% of cases by January 2024. Since the first appearance of the JN.1 variant, descendants including the JN.1.2, JN.1.6, JN.1.7, KP.2, KP.3, and XEC variants have continued to be identified. These have acquired additional mutations relative to JN.1 and are thought to further increase the infectivity and / or transmissibility of the SARS-CoV-2 variant.

[0368] Since the emergence of JN.1, descendants of JN.1 have arisen and rapidly replaced the JN.1 variant. These descendants of JN.1 include the "SLip" variants (e.g., including JN.1.16) containing the L455S and F456L mutations, as well as the "FLiRT" variants (e.g., including KS.1.1, KP.2) containing mutations related to the SLip variants and the additional R346T mutation. And the FLuQE variant (e.g., KP.3.3) is a descendant of the FLiRT variant and contains an additional Q493E mutation in addition to the same mutations. Position 455 also continues to be a mutation hot spot, and "FLip" includes the L455F and F456L mutations. In some embodiments, the S protein or a fragment thereof includes one or more mutations related to the Slip variants, FLiRT variants, and / or Flip variants.

[0369] The XEC variant is a hybrid of the KS.1.1 variant and the KP.3.3 variant. Explanations regarding the emergence of the JN.1 variant and its descendants are shown, for example, in E. Topol, “Are We FLiRTing With A New Covid Wave?,” April 18, 2024 (accessible at erictopol.substack.com / p / are-we-flirting-with-a-new-covid), and Sankaran, V. “New Covid XEC variant starting to spread in Europe - what we know,” September 4, Independent (accessible at www.independent.co.uk / news / science / covid-variant-xec-europe-symptoms-b2613485.html).

[0370]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0371] In some embodiments, the SARS-CoV-2 S protein described herein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations (e.g., one or more mutations of the Omicron variant strains described in Table 1, e.g., each of the mutations associated with the predetermined XBB, JN.1, KP.2, or XEC variants in Table 1 above) specific to a particular Omicron variant strain.

[0372] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., an RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1)) or a variant thereof, and 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 (e.g., one or more mutations associated with the SARS-CoV-2 variant strains described in Table 1).

[0373] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., the RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1)) or a variant thereof, wherein the fragment of the S protein comprises at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the mutations associated with a given SARS-CoV-2 variant in the S protein fragment region. For example, in some embodiments, the construct comprises at least 5% of the mutations associated with a SARS-CoV-2 variant within the region corresponding to amino acids 1-528 of the S protein.

[0374] In some embodiments, the RNA comprises a nucleotide sequence encoding a polypeptide comprising a fragment of the S protein (e.g., the RBD or a truncated S1 polypeptide (e.g., the region corresponding to amino acids 1-528 of SEQ ID NO: 1)), wherein the fragment of the S protein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or more) mutations associated with a SARS-CoV-2 variant.

[0375] In some embodiments, the RNA described herein encodes an immunogenic fragment of the SARS-CoV-2 S protein or a variant of an immunogenic fragment of the SARS-CoV-2 S protein that contains one or more mutations specific to the JN.1, JN.1.2, JN.1.6, KP.2, KP.3, XEC, and / or JN.1.7 variants (e.g., one or more of the mutations described herein). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 455 of SEQ ID NO: 1 (e.g., L455S). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 455 of SEQ ID NO: 1 (e.g., L455F). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 456 of SEQ ID NO: 1 (e.g., F456L). In some embodiments, the one or more mutations include mutations at positions corresponding to positions 455 and 456 of SEQ ID NO: 1 (e.g., F456L and L455F). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 346 of SEQ ID NO: 1 (e.g., R346T). In some embodiments, the one or more mutations include a mutation at a position corresponding to position 1104 of SEQ ID NO: 1 (e.g., V1104L). In some embodiments, the one or more mutations include mutations at positions corresponding to positions 346 and 1104 of SEQ ID NO: 1 (e.g., R346T and V1104L).

[0376] In some embodiments, one or more mutations specific to the KP.2 variant include 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 (e.g., all of them)) of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, and P1143L. In some embodiments, one or more mutations specific to the KP.2 variant include R346T and V1104L, where the positions are shown relative to SEQ ID NO: 1. In some embodiments, one or more mutations specific to the KP.2 variant include R346T, F456L, and / or V1104L, where the positions are shown relative to SEQ ID NO: 1.

[0377] In some embodiments, one or more mutations specific to the KP.3 variant include one or more of ins16MPLF, T19, Δ24-26, A27S, S50L, Δ69 / 70, V127F, G142D, Δ144, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q493E, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L, or combinations thereof (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., all of them)). In some embodiments, one or more mutations specific to the KP.3 variant include F456L, Q493E, and / or V1104L, where the positions are shown relative to SEQ ID NO: 1.

[0378] In some embodiments, one or more mutations specific to the XEC variant include one or more of ins16MPLF, T19I, R21T, T22N, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L (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, 67, or more (e.g., all of them)), where the mutations are shown relative to SEQ ID NO: 1. In some embodiments, one or more mutations specific to the XEC variant include T22N, F59S, F456L, Q493E, and / or V1104L, where the positions are shown relative to SEQ ID NO: 1.

[0379] In some embodiments, 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, 65, or 66 or more) of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or M1229I.In some embodiments, 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, or more) of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, L212I, V213G, L216F, H245N, A264D, I332V, G339H, R446T, K356T, S371F, S373P, S375F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, L455S, F456L, N460K, S477N, T478K, N481K, Δ483, E484K, F486P, Q498R, N501Y, Y505H.

[0380] In some embodiments, one or more mutations specific to the JN.1 variant include ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, or 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 (e.g., all of them)) of any combination thereof, where the mutations are shown relative to SEQ ID NO: 1.In some embodiments, one or more mutations specific to the JN.1 variant include ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L with respect to SEQ ID NO: 1. In some embodiments, one or more mutations specific to JN.1 include L455S.

[0381] In some embodiments, a fragment of the S protein (e.g., the RBD or truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with the JN.2 variant. In some embodiments, the fragment of the S protein comprises one or more of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L (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).In some embodiments, 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, or more) of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, and Y505H.

[0382] In some embodiments, one or more mutations specific to the JN.1.2 variant include one or more of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and M1229I (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 them)), where the mutations are shown relative to SEQ ID NO: 1. In some embodiments, one or more mutations specific to the JN.1.2 variant include M1229I, where the position is shown relative to SEQ ID NO: 1.

[0383] In some embodiments, one or more mutations of the JN.1.6 variant include one or more of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, and P1143L (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 them)), where the mutations are shown relative to SEQ ID NO: 1. In some embodiments, one or more mutations of the JN.1.6 variant include R346T.

[0384] In some embodiments, one or more mutations of the JN.1.7 variant include one or more of ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ211, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and E1150D (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 them)), where the mutations are shown relative to SEQ ID NO: 1.

[0385] In some embodiments, a fragment of the S protein (e.g., the RBD or truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with the JN.6 variant. In some embodiments, the fragment of the S protein comprises one or more of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, T572I, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L, and E1150D (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).In some embodiments, 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, or more) of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H.

[0386] In some embodiments, a fragment of the S protein (e.g., the RBD or truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with the JN.6 variant. In some embodiments, the fragment of the S protein comprises one or more of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, V1104L, P1143L (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).In some embodiments, 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, or more) of the following mutations: ins16MPLF, T19I, R21T, Δ24-26, A27S, S50L, Δ69-70, V127F, G142D, Δ145, F157S, R158G, Δ201, 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, Δ483, E484K, F486P, Q498R, N501Y, and Y505H.

[0387] In some embodiments, a fragment of the S protein (e.g., the RBD or truncated S1 polypeptide described herein) or a variant thereof comprises one or more mutations associated with the XBB.1.5 variant strain. In some embodiments, 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, or more) of the following mutations: T19I, Δ24-26, A27S, V83A, G142D, Δ145, 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, 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, or more) of the following mutations: T19I, Δ24-26, A27S, V83A, G142D, Δ145, 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.

[0388] In some embodiments, the S protein encoded by the RNA molecule comprises the majority of the mutations associated with the KP.2, KP.3, or XEC variant strains and one or more additional mutations. In some embodiments, the 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 (e.g., all) of the mutations associated with the KP.2, KP.3, or XEC variant strains (e.g., the mutations shown herein). In some embodiments, the S protein comprises the majority of the mutations associated with the JN.1 variant strain (e.g., those described herein) and one or more additional mutations. In some embodiments, the S protein comprises the majority of the mutations associated with the JN.1 variant strain (e.g., those described herein) and one or more additional mutations associated with descendants of the JN.1 variant strain (e.g., the descendants described herein). In some embodiments, the S protein comprises the majority of the mutations associated with the JN.1 variant strain (e.g., those described herein) and one or more additional mutations associated with descendants of the JN.1 variant strain with increasing prevalence (e.g., the descendants described herein).

[0389] In some embodiments, the S protein comprises the majority of the mutations associated with the JN.1 variant strain (e.g., those described herein) and (a) ...

Claims

1. A ribonucleic acid (RNA) comprising a nucleotide sequence encoding a polypeptide, said polypeptide comprising: (i) a truncated S1 subdomain of the SARS-CoV-2 spike (S) protein or a mutant thereof; (ii) a heterologous secretory signal peptide, and (iii) a cognate transmembrane domain; Optionally, the RNA, wherein the arrangement from N-terminus to C-terminus of the polypeptide is (secretory signal peptide)-(truncated S1 subdomain)-(transmembrane domain).

2. 2. The RNA of claim 1, wherein the truncated S1 subdomain comprises amino acids 20-528 of SEQ ID NO:1 or the corresponding region of an S protein of a SARS-CoV-2 mutant strain, and optionally the truncated S1 domain comprises amino acids 14-528 of SEQ ID NO:1, amino acids 17-528 of SEQ ID NO:1, amino acids 14-541 of SEQ ID NO:1, amino acids 17-541 of SEQ ID NO:1, or amino acids 20-541 of SEQ ID NO:1, or the corresponding region of an S protein of a SARS-CoV-2 mutant strain.

3. The secretory signal peptide is (i) the 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 SEQ ID NO:22; (ii) the amino acid sequence of MFLLLRFVLVSCIIGSLG (SEQ ID NO: 391), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 391; (iii) an amino acid sequence of MGGAAARLGAVILFVVIVGLHGVRG (SEQ ID NO:12), 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 SEQ ID NO:12; (iv) the amino acid sequence of MHQGAPSWGRRRWFVVWALLGLTLGVLVASAAP (SEQ ID NO: 38), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 38; (v) the amino acid sequence of MARGAGLVFFVGVWVVSCLA (SEQ ID NO: 366), or an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 366; The RNA of claim 1 or 2.

4. The polypeptide is (i) the 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; or (ii) the amino acid sequence of SEQ ID NO: 89 (EQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT), or an amino acid sequence which is at least 70%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 89; The RNA according to any one of claims 1 to 3, comprising:

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

6. 6. The RNA of any one of claims 1 to 5, wherein the truncated S1 subdomain or variant thereof and the transmembrane domain are linked to each other by a flexible linker, optionally comprising from about 10 to about 20 amino acids, further optionally comprising about 15 amino acids.

7. The flexible linker is ４ S) ２ , (G ４ S) ３ , or (G ４ S) ４ The RNA of claim 6, comprising the sequence:

8. 8. The RNA of any one of claims 1 to 7, wherein the RNA comprises a nucleotide sequence encoding a polypeptide comprising: (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% 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% identical to SEQ ID NO:238; and / or (iii) 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% identical to SEQ ID NO:

235.

9. 8. The RNA of any one of claims 1 to 7, wherein the RNA comprises a nucleotide sequence encoding a polypeptide comprising: (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% 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% identical to SEQ ID NO:330; and / or (iii) 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% identical to SEQ ID NO:

327.

10. The RNA of any one of claims 1 to 9, wherein the truncated S1 subdomain or variant thereof comprises one or more mutations of a SARS-CoV-2 variant, and optionally the truncated S1 domain or variant thereof comprises one or more mutations associated with a variant listed in Table 1 (e.g., a JN.1 variant, a KP.2 variant, or a XEC variant, or a progeny thereof).

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

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

13. The RNA comprises a 5' cap, a cap proximal sequence, a 5' UTR sequence, a 3' UTR sequence, and a polyA sequence, and optionally (i) the 5' cap comprises a Cap1 structure; (ii) the 5'-UTR sequence comprises a modified human alpha-globin 5'-UTR; (iii) the 3'-UTR sequence comprises a first sequence from an amino-terminal enhancer of split (AES) messenger RNA and a second sequence from a mitochondrially encoded 12S ribosomal RNA; (iv) the polyA sequence comprises at least 100 A nucleotides; or (v) The RNA of any one of the preceding claims, wherein the RNA comprises any one combination of (i) to (iv).

14. the 5' cap comprising a Cap1 structure, and the Cap1 structure comprises m7(3'OMeG)(5')ppp(5')(2'OMeA1)pG2, where A1 is position +1 of the RNA and G2 is position +2 of the RNA, and optionally the cap proximal sequence is １ and G ２ and A at positions +3, +4, and +5 of the RNA, respectively. ３ N ４ N ５ and a sequence comprising: ４ and N ５ is independently selected from A, G, C, and U.

15. 15. The RNA of any one of claims 13 to 14, wherein the polyA sequence comprises an interrupted sequence of A nucleotides, optionally comprising 30 adenine nucleotides followed by 70 adenine nucleotides, the 30 adenine nucleotides and the 70 adenine nucleotides being separated by a linker sequence.

16. and / or the 5'-UTR sequence comprises SEQ ID NO: 112 or 113 or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 112 or 113; and / or the 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% identical to SEQ ID NO: 118, 647, or 648; and / or 16. The RNA of any one of claims 13 to 15, wherein the interrupted poly-A 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.

17. The RNA of any one of claims 13 to 16, wherein the sequence at the 5' end of the 3'UTR sequence (e.g., the sequence immediately adjacent to the sequence encoding an antigenic polypeptide) is CUCGAG or GGAUCCGAU.

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

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

20. 10. The RNA of any one of the preceding claims, wherein the nucleotide sequence encoding the SARS-CoV-2 S protein is encoded by a codon-optimized sequence (e.g., codon-optimized for expression in human cells) and / or has an increased G / C content compared to a wild-type coding sequence.

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

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

23. The cryoprotectant may further comprise a cryoprotectant, optionally comprising sucrose and / or an aqueous buffer solution, optionally comprising an aqueous buffer solution such as Tris base, Tris HCl, NaCl, KCl, Na ２ H.P.O. ４ , and K.H. ２ P.O. ４ and optionally, the aqueous buffer solution comprises about 10 mM Tris buffer and about 10% sucrose.

24. A pharmaceutical composition comprising: (i) the RNA according to any one of claims 1 to 20 or the composition according to any one of claims 21 to 23; and (ii) a pharma- ceutically acceptable excipient.

25. 25. The pharmaceutical composition of claim 24, wherein the pharmaceutical composition is formulated as a multi-dose formulation in a vial, a single-dose formulation in a vial, or a pre-filled syringe.

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

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

28. (i) the subject is 12 years of age or older, and the method comprises administering 30 μg of the RNA; or (ii) the subject is between 5 and 12 years of age and the method comprises administering 10 μg of the RNA; or (iii) the subject is between 6 months and less than 5 years old, and the method comprises administering 3 μg of the RNA.

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

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

31. 30. The method of any one of claims 27 to 29, wherein the method comprises administering to the subject the RNA, composition, or pharmaceutical composition two or more times, optionally wherein the two administrations are administered at intervals of about 21 days.

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

33. 33. The method of any one of claims 27-32, further comprising administering one or more vaccines against a non-SARS-CoV-2 disease, optionally wherein the one or more vaccines comprise an RSV vaccine, an influenza vaccine, or a combination thereof.

34. 34. The method of any one of claims 27-33, wherein the method results in the induction of an immune response in the subject against SARS-CoV-2, optionally the immune response comprising the generation of antibodies and / or a T cell response (e.g., a CD4+ T cell response and / or a CD8+ T cell response) directed against one or more SARS-CoV-2 viruses.

35. 35. The method of any one of claims 27 to 34, wherein the method is a method of preventing SARS-CoV-2 infection, a method of reducing the likelihood of SARS-CoV-2 infection, a method of preventing or reducing adverse symptom changes associated with SARS-CoV-2 infection (which may, for example, result in a reduced length of hospitalization), a method of increasing the transition to experiencing asymptomatic SARS-CoV-2 infection, and / or a method of treating SARS-CoV-2 infection.

36. 36. The RNA of any one of claims 1 to 20, the composition of any one of claims 21 to 23, or the pharmaceutical composition of any one of claims 24 to 26, for use in inducing an immune response in a subject, optionally said use comprising carrying out according to the method of any one of claims 27 to 35.

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

38. A method for producing RNA, comprising in vitro transcribing the RNA according to any one of claims 1 to 27.

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