Multivalent antigens

Abstract

The disclosure relates to RNA molecules with multivalent capability against multiple viral strains and / or antigens and methods of making the same.

Description

MULTIVALENT ANTIGENSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application No. 63 / 638,926, filed April 25, 2024, the entire contents of which are incorporated herein by reference in their entireties.BACKGROUND

[0002] RNA is a single-stranded polynucleotide molecule that can encode a specific protein, such as a pathogen-specific antigen, upon reaching the cytoplasm. The successful synthesis of RNA by in vitro transcription (IVT) initiated research on its use as a therapeutic agent. Currently, RNA vaccines against SARS-CoV-2 (COVID-19) are widely used and have been successful in combating coronavirus.

[0003] The current RNA vaccines generally encode one antigen per RNA molecule. New variants of viruses (e.g., SARS-CoV-2 and influenza) continue to appear and vaccines are needed that can target these new variants as well as a combination of variants. To address this, multivalent vaccines are needed to provide immunity against new virus strains. Provided herein, in certain embodiments, are improved RNA vaccines with multivalent capability and efficacy against multiple viral variants.BRIEF SUMMARY

[0004] Described herein are RNA vaccines with multivalent capability against multiple variants of viruses, such as the influenza virus. Such RNA vaccines are designed to effectively integrate different sequences of genes that express the protein of different viral strains in one platform, creating a multivalent RNA vaccine. Moreover, the presence of at least two sequences encoding for proteins or protein domains with multimerization properties, such as influenza hemagglutinin (with multimerization properties, including trimerization properties, conferred by the antigen itself) and / or the T4 foldon domain (with multimerization properties, including trimerization properties), facilitates co-expression of multivalent antigens as complex multimers that may enhance immunogenicity of the vaccines. Further provided herein are methods for preparing RNA vaccines described herein, for example, by providing the RNA with an LNP composition.

[0005] In one aspect, provided herein is a purified ribonucleic acid (RNA) molecule comprising a first nucleotide sequence encoding a first multimerization domain; a second nucleotide sequence encoding a second multimerization domain, and wherein the firstnucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0006] In some embodiments, the first multimerization domain and / or the second multimerization domain are selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and an antigen, or an antigenic fragment thereof, from a virus variant.

[0007] In some embodiments, the first multimerization domain is a first antigen, or an antigenic fragment thereof, from a first virus variant.

[0008] In some embodiments, the first virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0009] In some embodiments, the first virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0010] In some embodiments, the first influenza virus variant is an influenza B virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza strains.

[0011] In some embodiments, the first virus antigen, or an antigenic fragment thereof, is a hemagglutinin (HA) protein from a Victoria lineage and Yamagata lineage influenza strains.

[0012] In some embodiments, the second multimerization domain is a second antigen, or an antigenic fragment thereof, from a second virus variant.

[0013] In some embodiments, the second virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2.

[0014] In some embodiments, the second virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0015] In some embodiments, the second influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0016] In some embodiments, the second influenza antigen is an HA protein from an influenza A virus.

[0017] In some embodiments, the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

[0018] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0019] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0020] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0021] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0022] In some embodiments, the first HA protein is encoded by any one of SEQ ID NOs: 23 or 24 and the second HA protein is encoded by any one of SEQ ID NOs: 25-28.

[0023] In some embodiments, the purified RNA further comprises a third nucleotide sequence encoding a third multimerization domain, wherein the third multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and an antigen, or an antigenic fragment thereof, from a virus variant, and wherein the third multimerization domain is after the first nucleotide sequence (3’ to the first nucleotide sequence) and before the second nucleotide sequence (5’ to the first nucleotide sequence).

[0024] In some embodiments, the third multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53.

[0025] In some embodiments, the third multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0026] In some embodiments, the third multimerization domain comprises a trimerization domain.

[0027] In some embodiments, the fibritin foldon domain is the trimerization domain from enterobacteria phage T4.

[0028] In some embodiments, the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO.: 19.

[0029] In some embodiments, the purified RNA further comprises a sequence encoding a first linker connecting the first multimerization domain to the second multimerization domain.

[0030] In some embodiments, the purified RNA further comprises a sequence encoding a second linker connecting the third multimerization domain to the second multimerization domain.

[0031] In some embodiments, the first linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

[0032] In some embodiments, the second linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

[0033] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20.

[0034] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20.

[0035] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

[0036] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

[0037] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0038] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0039] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8.

[0040] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8 .

[0041] In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0042] In some embodiments, the first linker encodes GGSG (SEQ ID NO: 38).

[0043] In some embodiments, the second linker encodes GGSG (SEQ ID NO: 38).

[0044] In some embodiments, the first linker encodes GGSLGGGGSGS (SEQ ID NO: 39).

[0045] In some embodiments, the second linker encodes GGSLGGGGSGS (SEQ ID NO: 39).

[0046] In some embodiments, the first linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22.

[0047] In some embodiments, the second linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22.

[0048] In some embodiments, the purified RNA molecule comprises about 90% identity to any one of SEQ ID NOs: 1-15.

[0049] In some embodiments, the purified RNA molecule comprises any one of SEQ ID NOs: 1-15.

[0050] In some embodiments, the purified RNA further comprises comprising a fourth sequence encoding a fourth multimerization domain after the first multimerization domain (3’ to the second multimerization domain).

[0051] In some embodiments, the fourth multimerization domain is a T4 foldon domain.

[0052] In some embodiments, the purified RNA further comprises a fifth nucleotide sequence encoding a fifth multimerization domain.

[0053] In some embodiments, the fifth multimerization domain is a third antigen, or an antigenic fragment thereof, from a third influenza virus variant.

[0054] In some embodiments, the first influenza virus variant, the second influenza virus variant, and the third influenza virus variant are different.

[0055] In some embodiments, the purified RNA further comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1) 5’ to the first nucleotide sequence.

[0056] In some embodiments, the purified RNA further comprises a 5’ UTR 3’ to the Cap 1 and 5’ to the first nucleotide sequence.

[0057] In some embodiments, the purified RNA further comprises a sequence encoding a signal peptide 3’ to the 5’ UTR and 5’ to the first nucleotide sequence.

[0058] In some embodiments, the purified RNA further comprises a sequence encoding a 3’ UTR 3’ to the second nucleotide sequence.

[0059] In one aspect, provided herein are purified ribonucleic acid (RNA) molecules comprising: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; wherein the first virus variant or the second virus variant is an influenza B variant; and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0060] In some embodiments, the first virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0061] In some embodiments, the first virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0062] In some embodiments, the first influenza virus variant is an influenza B virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza strains.

[0063] In some embodiments, the first virus antigen, or an antigenic fragment thereof, is a hemagglutinin (HA) protein from a Victoria lineage and Yamagata lineage influenza strains.

[0064] In some embodiments, the second virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0065] In some embodiments, the second virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0066] In some embodiments, the second influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8,H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7. In some embodiments, the second influenza antigen is an HA protein from an influenza A virus.

[0067] In some embodiments, the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

[0068] In some embodiments, the first HA protein comprises an HA protein from an Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus; the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus; the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus; or the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0069] In some embodiments, the first HA protein is encoded by any one of SEQ ID NOs: 23 or 24 and the second HA protein is encoded by any one of SEQ ID NOs: 25-28.

[0070] In some embodiments, the first HA protein comprises an amino acid sequence selected from SEQ ID NOs: 60-61 and the second HA protein comprises an amino acid sequence selected from SEQ ID NOs: 62-65.

[0071] In some embodiments, the purified RNA molecule further comprises a third nucleotide sequence encoding a first multimerization domain, wherein the first multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and wherein the first multimerization domain is after the first nucleotide sequence (3’ to the first nucleotide sequence) and before the second nucleotide sequence (5’ to the first nucleotide sequence).

[0072] In some embodiments, the first multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53.

[0073] In some embodiments, the first multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0074] In some embodiments, the first multimerization domain comprises a trimerization domain.

[0075] In some embodiments, the trimerization domain is the fibritin foldon domain from enterobacteria phage T4, the T4 foldon domain.

[0076] In some embodiments, the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 19.

[0077] In some embodiments, the T4 foldon domain comprises the amino acid sequence SEQ ID NO: 58.

[0078] In some embodiments, the purified RNA molecule further comprises a sequence encoding a first linker connecting the first antigen, or an antigenic fragment thereof to the first multimerization domain.

[0079] In some embodiments, the purified RNA molecule further comprises a sequence encoding a second linker connecting the first multimerization domain to the second antigen, or an antigenic fragment thereof.

[0080] In some embodiments, the first linker and the second linker each encode an amino acid sequence comprising at least 5 to about 50 amino acids.

[0081] In some embodiments, the first linker and / or the second linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20.

[0082] In some embodiments, the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

[0083] In some embodiments, the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0084] In some embodiments, the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0085] In some embodiments, the first linker and / or the second linker encodes the amino acid sequence GGSG (SEQ ID NO: 38) or GGSLGGGGSGS (SEQ ID NO: 39).

[0086] In some embodiments, the first linker and / or the second linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22.

[0087] In some embodiments, the first linker, first multimerization domain, and second linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0088] In some embodiments, the purified RNA molecule comprises about 90% identity to any one of SEQ ID NOs: 1-15.

[0089] In some embodiments, the purified RNA molecule comprises any one of SEQ ID NOs: 1-15.

[0090] In some embodiments, the purified RNA molecule further comprises a fourth sequence encoding a second multimerization domain after the second antigen, or an antigenic fragment thereof (3’ to the second antigen, or an antigenic fragment thereof).

[0091] In some embodiments, the second multimerization domain is a T4 foldon domain.

[0092] In some embodiments, the second multimerization domain comprises an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0093] In some embodiments, the purified RNA molecule further comprises a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.

[0094] In some embodiments, the third virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19.

[0095] In some embodiments, the third virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0096] In some embodiments, the third virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0097] In some embodiments, the third antigen is an HA protein from an influenza A virus.

[0098] In some embodiments, the purified RNA molecule further comprises a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, from a fourth virus variant. In some embodiments, the fourth virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0099] In some embodiments, the fourth virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0100] In some embodiments, the fourth virus variant is a coronavirus from a SARS-CoV-2 virus.

[0101] In some embodiments, the fourth antigen is a receptor-binding domain (RBD) protein.

[0102] In some embodiments, the RBD protein comprises the amino acid sequence of SEQ ID NO: 53.

[0103] In some embodiments, the purified RNA molecule further comprises a seventh sequence encoding a third multimerization domain 3’ to the third antigen, or an antigenic fragment thereof and 5’ to the fourth antigen, or antigen fragment thereof. In some embodiments, the third multimerization domain is a T4 foldon domain. In some embodiments, the T4 foldon domain comprises a sequence of SEQ ID NO: 58.

[0104] In some embodiments, the third multimerization domain comprises a sequence of any one of SEQ ID NOs: 66-68.

[0105] In some embodiments, the purified RNA further comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1) 5’ to the first nucleotide sequence.

[0106] In some embodiments, the purified RNA further comprises a 5’ UTR (SEQ ID NO: 16) 3’ to the Cap 1 and 5’ to the first nucleotide sequence.

[0107] In some embodiments, the purified RNA further comprises a sequence encoding a 3’ UTR (SEQ ID NO: 17) 3’ to the second nucleotide sequence.

[0108] In another aspect, provided herein are purified RNA molecules comprising a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 1-15.

[0109] In another aspect, provided herein are purified RNA molecules that encode a sequence comprising at least 90% identity to any one of SEQ ID NO: 45-52.

[0110] In another aspect, provided herein are purified RNA molecules comprising, from 5’ to 3’: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a first T4 foldon domain; c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; d) a fourth nucleotide sequence encoding a second T4 foldon domain; and e) a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.[oni] In another aspect, provided herein are purified RNA molecules the first antigen, or an antigenic fragment thereof, the second antigen, or an antigenic fragment thereof, and third antigen, or an antigenic fragment thereof are different antigens.

[0112] In another aspect, provided herein are purified RNA molecules the first antigen, second antigen, or third antigen are a virus variant of a virus selected from the group consisting of a Influenza virus, Paramyxoviridae (e.g., RSV, hMPV, Nipah virus, measles virus), Retroviridae (e.g., HIV), Filoviridae (e.g., Ebola virus), Orthomyxoviridae (e.g., influenza), and Coronaviridae (SARS-CoV2) families.

[0113] In another aspect, provided herein are purified RNA molecules: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); b) a second nucleotide sequence encoding a first T4 foldon domain; c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); d) a fourth nucleotide sequence encoding a second T4 foldon domain; and e) a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19), wherein at least the first, second, or third antigen is an HA protein from influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza.

[0114] In some embodiments, the purified RNA molecules further comprises a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0115] In some embodiments, the purified RNA molecules further comprises a seventh nucleotide sequence encoding a third T4 foldon domain 3’ to the third antigen, or anantigenic fragment thereof and 5 ’to the sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof.

[0116] In some embodiments, the purified RNA molecules further comprises an eighth nucleotide sequence encoding a first linker 3’ to the first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof.

[0117] In some embodiments, the purified RNA molecules further comprises: a ninth nucleotide sequence encoding a second linker 3’ to the second nucleotide sequence encoding a first T4 foldon domain; a tenth nucleotide sequence encoding a third linker 3’ to the third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof; an eleventh nucleotide sequence encoding a fourth linker 3’ to the second T4 foldon domain; a twelfth nucleotide sequence encoding a fifth linker 3’ to the third antigen, or an antigenic fragment thereof; a thirteenth nucleotide sequence encoding a sixth linker 3’ to the third T4 foldon domain.

[0118] In some embodiments, the first linker, first T4 foldon domain, and second linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0119] In some embodiments, the third linker, second T4 foldon domain, and fourth linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0120] In some embodiments, the fifth linker, second T4 foldon domain, and sixth linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0121] In some embodiments, the purified RNA, further comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1).

[0122] In some embodiments, the a first antigen, or an antigenic fragment thereof, is an HA protein from a Victoria lineage and Yamagata lineage influenza.

[0123] In some embodiments, the first antigen, or an antigenic fragment thereof, is an RBD of a SARS-CoV-2.

[0124] In some embodiments, the second antigen, or an antigenic fragment thereof, is an HA protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0125] In some embodiments, the third antigen, or an antigenic fragment thereof, is an HA protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0126] In some embodiments, the fourth antigen, or an antigenic fragment thereof, is an HA protein from an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7.

[0127] In some embodiments, the fourth antigen, or an antigenic fragment thereof, is an RBD of a SARS-CoV-2.

[0128] In another aspect, provide herein is isolated polypeptide encoded by the purified RNA molecule disclosed herein.

[0129] In another aspect, provide herein is a composition comprising a first purified RNA molecule comprising the purified RNA molecule of any disclosed herein and a delivery vehicle.

[0130] In some embodiments, the composition further comprises a second purified RNA molecule comprising the purified RNA molecule of any one disclosed herein. In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 1-15 and the second purified RNA molecule comprises any one of SEQ ID NOs: 1-15, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 1-15.

[0131] In some embodiments, the first purified RNA molecule encodes a sequence comprising any one of SEQ ID NOs: 41-52, and the second purified RNA molecule encodes a sequence comprising any one of SEQ ID NOs: 41-52, wherein the first purified RNA molecule and the second purified RNA molecule encodes a sequence comprising different sequences selected from SEQ ID NOs: 41-52.

[0132] In some embodiments, the composition, further comprises a second purified RNA molecule encoding the amino acid sequence of SEQ ID NO: 47 or 48.

[0133] In some embodiments, the first RNA molecule comprises SEQ ID NO: 4 and the second RNA molecule comprises SEQ ID NO: 3; the first RNA molecule comprises SEQ ID NO: 5 and the second RNA molecule comprises SEQ ID NO: 3; the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 7; the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 9; the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 11; the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 13; or the first RNA molecule comprises SEQ ID NO: 14 and the second RNA molecule comprises SEQ ID NO: 15.

[0134] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an aminoacid sequence at least 90% identical to SEQ ID NO: 49 or 50; or the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 51 or 52.

[0135] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0136] In some embodiments, the LNP comprises an ionizable lipid.

[0137] In some embodiments, the LNP comprises an ionizable lipid: cholesterol: DSPC: DMG-PEG2000 ratio of about 48: 40: 10: 2, and a LNP:RNA (N:P) ratio of about 8: 1.

[0138] In some embodiments, the composition comprises a particle size of no more than about 300 nanometers (nm) or no more than about 150 nm.

[0139] In some embodiments, the composition comprises a particle size of about 50 to about to 300 nm or about 50 nm to about 140 nm..

[0140] In some embodiments, the RNA is lyophilized.

[0141] In some embodiments, the RNA is adsorbed to the surface of the LNP or is encapsulated by the LNP.

[0142] In some embodiments, the RNA encapsulated by the LNP is lyophilized.

[0143] In another aspect, provided herein is a kit comprising the purified RNA of any one disclosed herein, a delivery vehicle, and instructions for use.

[0144] In another aspect, provided herein is a method of treating or preventing disease in a subject comprising administering to the subject an effective amount of the composition of any disclosed herein.

[0145] In some embodiments, the disease is caused by a virus selected from the group consisting of an influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus.

[0146] In some embodiments, the disease is caused by a coronavirus, an influenza A virus, or an influenza B virus.

[0147] In some embodiments, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0148] In some embodiments, the coronavirus is SARS-CoV-2.

[0149] In some embodiments, the influenza A virus or influenza B virus is selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0150] In some embodiments, the disease is caused by one or more viruses selected from the group consisting of an influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus.

[0151] In some embodiments, the one or more viruses includes a coronavirus, an influenza A virus, and / or an influenza B virus.

[0152] In some embodiments, the composition is administered to the subject intramuscularly.

[0153] In some embodiments, the composition is administered to the subject at least two times.

[0154] In some embodiments, the composition is administered once every one, two, three, or four weeks.

[0155] In another aspect, provided herein is a method for stimulating an immune response in a subject comprising administering to the subject an effective amount of the composition of any one disclosed herein.

[0156] In another aspect, provided herein is a method for preparing a RNA-LNP composition comprising: mixing an ethanol phase comprising one or more lipids and an aqueous phase comprising the purified RNA molecule of any one disclosed herein and purifying the RNA- LNP generated from step a).

[0157] In some embodiments, the one or more lipids comprises an ionizable lipid, cholesterol, a phospholipid, and / or a pegylated lipid.

[0158] In some embodiments, the N:P ratio is about 6.5 to about 9.

[0159] In some embodiments, the aqueous phase consists of tris, sodium chloride, and sucrose.

[0160] In another aspect, provided herein is a kit comprising the composition of any one of those disclosed herein, a delivery vehicle, and instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1A shows an exemplary schema of a protein encoded by an mRNA molecule described herein designed to simultaneously express two antigens from two different influenza virus variants with a sequence encoding a multimerization domain in between the two antigen sequences. SP: signal peptide; B / Aus HAecto: B / Austria / 1359417 / 2021 (B / Victoria lineage) hemagglutinin (HA) antigen, ectodomain; LI : linker 1 (GGSGGGS); T4-Fold: T4 foldon domain; L2: extended GS linker: GS[GGGGS]?; A / Dar HA[wt]: A / Darwin / 6 / 2021 (H3N2) HA antigen; wild-type, including transmembrane and cytoplasmic domains; STOP: STOP codon.

[0162] Figure IB shows an exemplary schema of how a protein encoded by the exemplary mRNA molecule from Figure 1 A multimerizes on the plasma membrane of a cell. The white ovals represent B / Austria / 1359417 / 2021, T4 foldon stabilized HA ectodomain trimers. The dashed ovals represent A / Darwin / 6 / 2021 wild-type membrane HA trimers. The black lines represent linkers.

[0163] Figure 1C shows the top view of an exemplary schema of how a protein encoded by the exemplary mRNA molecule from Figure 1 A multimerizes on the plasma membrane of a cell. The white circles with “B” represent B / Austria / 1359417 / 2021, T4 foldon stabilized HA ectodomain trimers. The dashed circles with “A” represent A / Darwin / 6 / 2021 wild-type membrane HA trimers. The black lines represent linkers.

[0164] Figure 2A shows an exemplary schema of a protein encoded by an mRNA molecule described herein designed to simultaneously express three antigens from different influenza virus variants with a sequence encoding a multimerization domain after the first and second antigen sequences. SP: signal peptide; B / Aus HAecto: B / Austria / 1359417 / 2021 (B / Victoria lineage) hemagglutinin (HA) antigen, ectodomain; LI: linker 1 (GGSGGGS); T4-Fold: T4 foldon domain; L2: extended GS linker: GS[GGGGS]?; A / Dar HA[wt]: A / Darwin / 6 / 2021 (H3N2) HA antigen; wild-type, including transmembrane and cytoplasmic domains; A / Wis HA[wt]: A / Wisconsin / 67 / 2022 (H1N1) HA antigen; wild-type, including transmembrane and cytoplasmic domains; STOP: STOP codon.

[0165] Figure 2B shows an exemplary schema of how a protein encoded by the exemplary mRNA molecule from Figure 2 A multimerizes on the plasma membrane of a cell. The white ovals represent B / Austria / 1359417 / 2021, T4 foldon stabilized HA ectodomain trimers. The closely spaced dashed ovals represent A / Massachusetts / 18 / 2022, T4 foldon stabilized HA ectodomain trimers. The widely spaced dashed ovals represent A / Wisconsin / 67 / 2022 wildtype membrane HA trimers. The black lines represent linkers.

[0166] Figure 2C shows the top view of an exemplary schema of how a protein encoded by the exemplary mRNA molecule from Figure 2A multimerizes on the plasma membrane of a cell. The white ovals represent B / Austria / 1359417 / 2021, T4 foldon stabilized HA ectodomain trimers. The closely spaced dashed ovals represent A / Massachusetts / 18 / 2022, T4 foldon stabilized HA ectodomain trimers. The widely spaced dashed ovals represent A / Wisconsin / 67 / 2022 wild-type membrane HA trimers. The black lines represent linkers.

[0167] Figure 3A shows the design of an mRNA quadrivalent vaccine, which includes four mRNA molecules, each encoding a different influenza antigen. SP: signal peptide; A / Dar HA[wt]: A / Darwin / 6 / 2021 (H3N2) HA antigen; wild-type, including transmembrane andcytoplasmic domains; A / Wis HA[wt]: A / Wisconsin / 67 / 2022 (H1N1) HA antigen; wild-type, including transmembrane and cytoplasmic domains; B / Aus HAecto: B / Austria / 1359417 / 2021 (B / Victoria lineage) hemagglutinin (HA) antigen, ectodomain; B / Phu HA[wt]: influenza B (B / Yamagata lineage) HA antigen; wild-type, including transmembrane and cytoplasmic domains; STOP: STOP codon.

[0168] Figure 3B shows the design of mRNA quadrivalent vaccines. For each design, there are two mRNA molecules. Each mRNA molecule encodes two influenza antigens from different strains, for a total of four different influenza antigens in each design. Each mRNA molecule also includes a multimerization domain, such as T4 foldon, between the two antigen sequences. SP: signal peptide; LI : linker 1 (GGSGGGS); T4-Fold: T4 foldon domain; L2: extended GS linker: GSfGGGGS]?; A / Dar HAecto - influenza A (H3N2) hemagglutinin (HA) antigen, ectodomain; A / Wis HAecto - influenza A (H1N1) HA antigen, ectodomain; B / Aus HAecto - influenza B (B / Victoria lineage) HA antigen, ectodomain; B / Phu HAecto - influenza B (B / Yamagata lineage) HA antigen, ectodomain; A / Dar HA[wt] - influenza A (H3N2) HA antigen; wild-type, including transmembrane and cytoplasmic domains; A / Wis HA[wt] - influenza A (H1N1) HA antigen; wild-type, including transmembrane and cytoplasmic domains; B / Aus HA[wt] - influenza B (B / Victoria lineage) HA antigen; wildtype, including transmembrane and cytoplasmic domains; B / Phu HA[wt] - influenza B (B / Yamagata lineage) HA antigen; wild-type, including transmembrane and cytoplasmic domains; STOP: STOP codon.

[0169] Figure 4A shows the design of an mRNA trivalent vaccine, which encodes three HA ectodomains.

[0170] Figures 4B-4D show the hemagglutination inhibition (HAI) geometric mean titer (GMT) for the tested mRNA vaccines.

[0171] Figures 5A-5C show the hemagglutination inhibition (HAI) geometric mean titer (GMT) for the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0172] Figures 5D-5E show the IgG titers against recombinant XBB.1.5 S-protein RDB for the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0173] Figure 5F shows the IgG titer ratio of IgG2a to IgGl of the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0174] Figure 6A shows two designs of an mRNA tetravalent vaccine, which encodes three HA ectodomains and one RDB protein fragment.

[0175] Figures 6B-6D show the hemagglutination inhibition (HAI) geometric mean titer (GMT) for the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0176] Figure 6E shows the total IgG titer for the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0177] Figures 7A-7F show the hemagglutination inhibition (HAI) geometric mean titer (GMT) for the tested mRNA vaccines and tested combinations of mRNA vaccines.

[0178] Figure 8 is a table showing the components of the tested mRNA vaccines.DETAILED DESCRIPTION

[0179] RNA vaccines generally employ one antigen sequence to provide immunity towards a particular virus. Variants of viruses emerge, and depending on the variant, the variant may evade the immune responses elicited by such vaccine. Therefore, it is advantageous to create a vaccine that can provide broad immunity to more than one virus variant. The RNA molecules described herein comprise multiple antigen sequences to provide immunity against multiple variants of a virus.Nucleic Acids of the Disclosure

[0180] Provided herein, in some embodiments, are purified ribonucleic acid (RNA) molecules comprising: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a multimerization domain; and c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant, wherein the first virus variant and the second virus variant are different, and wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0181] Provided herein, in some embodiments, are purified ribonucleic acid (RNA) molecules comprising: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; wherein the first virus variant or the second virus variant is an influenza B variant; and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0182] Provided herein, in some embodiments, are purified ribonucleic acid (RNA) comprising, from 5’ to 3’ : a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza or (ii) an RBD of acoronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); b) a second nucleotide sequence encoding a first T4 foldon domain; c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); d) a fourth nucleotide sequence encoding a second T4 foldon domain; e) a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19), wherein at least the first, second, or third antigen is an HA protein from influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza.

[0183] The RNA molecules described herein, in some embodiments, are messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to one type of an RNA molecule that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, RNA molecule sequences will recite “T”s in a representative DNA sequence, but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted by “U”s.

[0184] Thus, any of the RNA molecules encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA or other coding RNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U”.

[0185] It should be understood that the RNA molecules as provided herein are synthetic molecules, i.e., they are not naturally-occurring molecules. That is, the RNA molecules of thepresent disclosure are isolated (i.e., purified) RNA molecules. As is known in the art, “isolated RNA molecules or polynucleotides” refer to polynucleotides that are substantially physically separated from other cellular material (e.g., separated from cells and / or systems that produce the polynucleotides) or from other material that hinders their use in the vaccines or therapeutics of the present disclosure. Isolated RNA molecules are substantially pure in that they have been substantially separated from the substances with which they may be associated in living or viral systems. Thus, RNA molecules are not associated with living or viral systems, such as cells or viruses. The RNA molecules do not include viral components (e.g., viral capsids, viral enzymes, or other viral proteins, for example, those needed for viralbased replication), and the RNA molecules are not packaged within, encapsulated within, linked to, or otherwise associated with a virus or viral particle.

[0186] Any sequence may be codon optimized. Codon optimization methods are known in the art. Codon optimization, in some embodiments, is used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase RNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms. In some embodiments, a sequence encoding an antigen, e.g., a spike protein of SARS-CoV-2, is codon optimized. In some embodiments, a sequence encoding an antigen, e.g., an influenza hemagglutinin (HA) antigen, is codon optimized. In some embodiments, a sequence encoding an antigen, e.g., an influenza hemagglutinin (HA) antigen, that is part of an mRNA molecule is codon optimized.

[0187] In some embodiments, the RNA includes at least one RNA molecule encoding at least one antigenic polypeptide having at least one of a modification, at least one 5’ terminal cap, and formulation with a lipid nanoparticle. 5 ’-capping of polynucleotides may be completed concomitantly during the in iv' / ra-tran scription reaction using the following chemical RNA cap analogs to generate the 5 ’-guanosine cap structure according to manufacturer protocols: 3’-O-Me-m7G(5’)ppp(5’) G [the ARC A cap]; G(5’)ppp(5’)A; G(5’)ppp(5’)G; m7G(5’)ppp(5’)A; m7G(5’)ppp(5’)G. 5’-capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5’)ppp(5’)G. Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2’-0 methyl-transferase to generate: m7G(5’)ppp(5’)G-2’-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2’-O-methylation of the 5 ’-antepenultimate nucleotide using a 2’-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2’-O-methylation of the 5’- preantepenultimate nucleotide using a 2’-0 methyl-transferase. Enzymes may be derived from a recombinant source.

[0188] In some embodiments, an RNA molecule described herein comprises a Cap (e.g., m7G (Cap 0), m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or anti-reverse cap analogs (ARCA), optionally m7G or m7GpppNm— where Nm denotes any nucleotide with a 2’ O methylation). In some embodiments, an RNA molecule described herein comprises a m7G (Cap 0) cap. In some embodiments, an RNA molecule described herein comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1) cap. In some embodiments, an RNA molecule described herein comprises a N6,2'-O- dimethyladenosine (m6AM) cap. In some embodiments, an RNA molecule described herein comprises a m7G(5')ppp(5')G (mCAP) cap. In some embodiments, an RNA molecule described herein comprises anti-reverse cap analogs (ARCA).

[0189] In some embodiments, an RNA molecule described herein comprises a 5’ UTR. In some embodiments, the 5’ UTR is 5’ of the first nucleotide sequence encoding the first antigen, or the antigenic fragment thereof, from the first virus variant. In some embodiments, an RNA molecule described herein comprises a 3’ UTR. In some embodiments, the 3’ UTR is 3’ of the second nucleotide sequence encoding the second antigen, or the antigenic fragment thereof, from the second virus variant.

[0190] In some embodiments, the 5’ UTR is encoded by a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 80% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 85% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 95% identityto SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 96% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 97% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 98% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having at least about 99% identity to SEQ ID NO: 16. In some embodiments, the 5’ UTR is encoded by a sequence having 100% identity to SEQ ID NO: 16.

[0191] In some embodiments, the 3’ UTR is encoded by a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 120% sequence identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 80% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 85% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 90% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 95% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 96% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 97% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 98% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having at least about 99% identity to SEQ ID NO: 17. In some embodiments, the 3’ UTR is encoded by a sequence having 100% identity to SEQ ID NO: 17.

[0192] In some embodiments, an RNA molecule described herein comprises a nucleotide sequence encoding a signal peptide.

[0193] In some embodiments, an RNA molecule described herein comprises a polyA tail. In some embodiments, an RNA molecule described herein comprises a polyA tail encoded by SEQ ID NO: 18.

[0194] Provided herein, in certain embodiments, are purified RNA molecules comprising, from 5’ to 3’: a) a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1); b) a 5’ UTR; c) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from an influenza virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains,; d) optionally, a second nucleotidesequence encoding a first linker; e) optionally, a third nucleotide sequence encoding a T4 foldon domain; f) optionally, a fourth nucleotide sequence encoding a second linker; g) optionally, a fifth nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from an influenza virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains; h) a 3’ UTR; and i) a poly A tail.

[0195] Provided herein, in certain embodiments, are purified RNA molecules comprising, from 5’ to 3’: a) a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1); b) a 5’ UTR; c) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a H1N1 influenza virus; d) a second nucleotide sequence encoding a first linker; e) a third nucleotide sequence encoding a T4 foldon domain; f) a fourth nucleotide sequence encoding a second linker; g) a fifth nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a H3N2 influenza virus; h) a 3’ UTR; and i) a poly A tail.

[0196] Provided herein, in certain embodiments, are purified RNA molecules comprising, from 5’ to 3’: a) a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1); b) a 5’ UTR; c) a nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a H1N1 influenza virus; d) a 3’ UTR; and e) a poly A tail.

[0197] Provided herein, in certain embodiments, are purified RNA molecules comprising, from 5’ to 3’: a) a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1); b) a 5’ UTR; c) a nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a H3N2 influenza virus; d) a 3’ UTR; and e) a poly A tail.

[0198] In some embodiments, the purified RNA molecule comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 70% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 75% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 80% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 85% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 90% identity to any oneof SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 95% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 96% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 97% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 98% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having at least about 99% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises a sequence having 100% identity to any one of SEQ ID NOs: 1-15.

[0199] When transfected into mammalian cells, the modified RNAs typically have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours.

[0200] Aside from mRNA, the RNA molecules described herein can be one of several noncoding types of RNA, such as a ribosomal RNA (rRNA) or a transfer RNA (tRNA). The terms “RNA” or “RNA molecule” further encompass other coding RNA molecules, such as viral RNA, retroviral RNA, self-replicating RNA (replicon RNA), small interfering RNA (siRNA), microRNA, small nuclear RNA (snRNA), small- hairpin (sh) RNA, riboswitches, ribozymes or aptamers.

[0201] In certain embodiments, the RNA molecule is a long RNA or a long RNA molecule. The term “long RNA” as used herein typically refers to an RNA molecule, preferably as described herein, which preferably comprises at least 30 nucleotides. Alternatively, a long RNA may comprise at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or at least 500 nucleotides. A long RNA may comprise at least 1000 nucleotides or at least 2000 nucleotides. A long RNA, in the context of the present disclosure, may comprise from about 30 to about 50,000 nucleotides, from about 30 to about 20,000 nucleotides, from about 100 to about 20,000 nucleotides, from about 200 to about 20,000 nucleotides, from about 200 to about 15,000 nucleotides, from about 500 to about 20,000 nucleotides, from about 1,000 to about 15,000 nucleotides, from about 1,500 to about 10,000 nucleotides, from about 2,000 to about 5,000 nucleotides. The term “long RNA” as used herein is not limited to a certain type of RNA, but merely refers to the number of nucleotides contained in said RNA. In certain embodiments, the RNA as used herein is a long RNA.

[0202] In the present disclosure, the RNA molecule may be a coding RNA molecule encoding one or more proteins or peptides, which may be selected, without being restrictedthereto, e.g., from therapeutically active proteins or peptides, selected from adjuvant proteins, from antigens, e.g., pathogenic antigens (e.g., animal antigens, from viral antigens, from protozoan antigens, from bacterial antigens), allergenic antigens, autoimmune antigens, or further antigens, preferably as defined herein, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding RNA molecule may be transported into a cell, a tissue or an organism, and the protein may be expressed subsequently in this cell, tissue or organism.

[0203] In certain embodiments, the RNA molecule of present disclosure is an immunostimulatory RNA molecule, which is capable of inducing an immune response, preferably an innate immune response. Such an immunostimulatory RNA may be any (double-stranded or single-stranded) RNA, e.g., a coding RNA, as defined herein. In certain embodiments, the immunostimulatory RNA is a non-coding RNA. The immunostimulatory RNA may be a single-stranded, a double-stranded, or a partially double-stranded RNA, optionally a single-stranded RNA or a circular or linear RNA, preferably, a linear RNA. In various embodiments, the immunostimulatory RNA may be a linear single- stranded RNA. Even more preferably, the immunostimulatory RNA may be a long, linear single-stranded RNA.

[0204] An immunostimulatory RNA may also occur as a short RNA oligonucleotide. As used herein, an immunostimulatory RNA may be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an innate immune response and may support an adaptive immune response induced by an antigen.

[0205] In some embodiments, the RNA molecule is a self-replicating RNA.

[0206] A self-replicating RNA molecule (replicon) can, when delivered to a vertebrate cell even without any proteins, lead to the production of multiple daughter RNAs by transcription from itself (via an antisense copy which it generates from itself). A self-replicating RNA molecule can thus typically be a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA can lead to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded immunogen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the immunogen. The overall results of this sequence of transcriptions are a largeamplification in the number of the introduced replicon RNAs and so the encoded immunogen becomes a major polypeptide product of the cells.

[0207] One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon. These replicons can be +-stranded RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell. The replicase can be translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic --strand copies of the +-strand delivered RNA. These --strand transcripts can themselves be transcribed to give further copies of the +-stranded parent RNA and also to give a subgenomic transcript which encodes the immunogen. Translation of the subgenomic transcript can thus lead to in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a SemLiki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild-type virus sequences can be used e.g., the attenuated TC83 mutant of VEEV has been used in replicons.

[0208] A self-replicating RNA molecule can encode (i) an RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) an immunogen. The polymerase can be an alphavirus replicase e.g., comprising one or more of alphavirus proteins nspl, nsp2, nsp3 and nsp4.

[0209] Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polyprotein, a self-replicating RNA molecule described herein can lack one or more or all alphavirus structural proteins. Thus, a self-replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule generally does not perpetuate itself in infectious form. The alphavirus structural proteins which are used for perpetuation in wildtype viruses are typically absent from self-replicating RNAs described herein and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.

[0210] Thus a self-replicating RNA molecule may have two open reading frames. The first (5’) open reading frame encodes a replicase; the second (3’) open reading frame encodes an immunogen. In some embodiments the RNA has additional (e.g., downstream) open reading frames, e.g., to encode further immunogens (see below) or to encode accessory polypeptides.

[0211] Self-replicating RNA molecules can have various lengths, but they are typically 5,000-25,000 nucleotides long, e.g., 8,000-15,000 nucleotides, or 9,000-12,000 nucleotides.

[0212] A self-replicating RNA molecule described herein may have a 5’ cap e.g., a 7- methylguanosine). This cap can enhance in vivo translation of the RNA. The 5’ nucleotide of a RNA molecule useful with the present disclosure may have a 5’ triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5’ -to-5’ bridge. In some embodiments, the RNA cap includes m7G (Cap 0), m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or anti-reverse cap analogs (ARCA), optionally m7G or m7GpppNm— where Nm denotes any nucleotide with a 2’ O methylation.

[0213] A self-replicating RNA molecule may have a 3’ poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3’ end.

[0214] A self-replicating RNA molecule will typically be single-stranded. Single-stranded RNAs can generally initiate an adjuvant effect by binding to TLR7, TLR8, RNA helicases and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, and this receptor can also be triggered by dsRNA which is formed either during replication of a single-stranded RNA or within the secondary structure of a single-stranded RNA.

[0215] A self-replicating RNA molecule described herein can be prepared by in vitro transcription (IVT). IVT can use a cDNA template created and propagated in plasmid form in bacteria, or created synthetically (for example, by gene synthesis and / or polymerase chainreaction (PCR) engineering methods). For instance, a DNA-dependent RNA polymerase (such as the bacteriophage T7, T3 or SP6 RNA polymerases) can be used to transcribe the RNA from a DNA template. Appropriate capping and poly-A addition reactions can be used as required (although the replicon’s poly-A is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5’ nucleotide(s) and, in some embodiments, these requirements must be matched with the requirements of the encoded replicase, to ensure that the IVT -transcribed RNA can function efficiently as a substrate for its self-encoded replicase.

[0216] In some embodiments, the RNA molecule is not a self-replicating RNA.

[0217] In some embodiments, the RNA of the present disclosure comprises a purified RNA molecule, such as a messenger RNA (mRNA). RNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template.”

[0218] In vitro transcription of RNA is known in the art and is described, e.g., in International Publication WO2014 / 152027, which is incorporated by reference herein in its entirety. For example, in some embodiments, the RNA transcript is generated using a nonamplified, linearized DNA template in an in vitro transcription reaction to generate the RNAtranscript. In some embodiments the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA transcript is purified via chromatographic methods, e.g., use of an oligo dT substrate. Some embodiments exclude the use of DNase. In some embodiments the RNA transcript is synthesized from a non-amplified, linear DNA template coding for the gene of interest via an enzymatic in vitro transcription reaction utilizing a T7 phage RNA polymerase and nucleotide triphosphates of the desired chemistry. Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides.

[0219] In some embodiments, an in vitro transcription template encodes a 5’ untranslated (UTR) region, contains an open reading frame, and encodes a 3’ UTR and a poly A tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the RNA encoded by the template.Antigens

[0220] The RNA molecules described herein can be designed to contain or encode a substance that produces an immune response in a subject. After administration of the RNA molecule, the RNA is translated in vivo. The RNA may elicit an immune response against an antigen, virus, and / or viral antigen. The immune response may comprise an antibody response. The RNA will typically elicit an immune response which recognizes the corresponding antigen such as a viral polypeptide. The RNA will typically comprise one or more sequences of a surface polypeptide, e.g., an adhesin, a hemagglutinin, an envelope glycoprotein, a spike glycoprotein, a receptor-binding domain (RBD). In some embodiments, the RNA elicits an immune response against receptor binding domains (RBDs) of SARS- CoV-2. In some embodiments, the RNA elicits an immune response against an antigen of influenza A or influenza B virus. In some embodiments, the RNA elicits an immune response against hemagglutinin (HA) of influenza A or influenza B virus. In some embodiments, the RNA elicits an immune response against receptor binding domains (RBDs) of SARS-CoV-2 and elicits an immune response against an antigen of influenza A or influenza B virus.

[0221] In some embodiments, the RNA molecules provided herein comprise a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant, a second nucleotide sequence encoding a multimerization domain, and a thirdnucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant, wherein the first virus variant and the second virus variant are different, and wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction. In some embodiments, the RNA molecules provided herein comprise a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first influenza virus variant, a second nucleotide sequence encoding a multimerization domain, and a third nucleotide sequence encoding a second influenza antigen, or an antigenic fragment thereof, from a second virus variant, wherein the first influenza virus variant and the second influenza virus variant are different, and wherein the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction. In some embodiments, the virus is selected from Orthomyxoviruses, such as influenza virus; Rhabdoviruses, such as rabies virus; Picomaviruses, such as poliovirus; Poxviruses, such as vaccinia virus; Rotavirus; respiratory syncytial virus (RSV), and coronaviruses, such as COVID-19.

[0222] In some embodiments, the virus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2.

[0223] In some embodiments, the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu-1, Alpha, Beta, Gamma, Delta, Epsilon Eta, Iota, Kappa, 1.617.3, Mu, Zeta, and Omicron. In some embodiments, the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu- 1 and Delta.

[0224] In some embodiments, the first antigen, or the antigenic fragment thereof, or the second antigen, or the antigenic fragment thereof, comprises a viral envelope protein, viral spike protein, viral membrane protein, or viral capsid protein. In some embodiments, the first antigen, or the antigenic fragment thereof, or the second antigen, or the antigenic fragment thereof, comprises a viral spike protein.

[0225] In some embodiments, the first antigen, or the antigenic fragment thereof, or the second antigen, or the antigenic fragment thereof, comprises a receptor-binding domain (RBD). In some embodiments, the RBD domain is a RBD domain from Wuhan-Hu- 1 variant or Delta variant. In some embodiments, the first antigen, or the antigenic fragment thereof, comprises the RBD domain from the Delta variant and the second antigen, or the antigenic fragment thereof, comprises the RBD domain of the Wuhan-Hu-1 variant.

[0226] In some embodiments, the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu-1, Alpha, Beta, Gamma, Delta, Epsilon Eta, Iota, Kappa, 1.617.3, Mu, Zeta, Omicron, H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains. In some embodiments, the first virus variant or the second virus variant is selected from the group consisting of Wuhan-Hu- 1, Alpha, Beta, Gamma, Delta, Epsilon Eta, Iota, Kappa, 1.617.3, Mu, Zeta, Omicron, H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0227] In some embodiments, the virus is an influenza A virus or an influenza B virus. In some embodiments, the influenza A virus or influenza B virus is selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0228] In some embodiments, the first antigen, or the antigenic fragment thereof, or the second antigen, or the antigenic fragment thereof, comprises an RNA polymerase subunit, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein 1 (Ml), matrix protein 2 (M2), non-structural protein NS1, or non-structural protein NEP.

[0229] In some embodiments, the first antigen, or the antigenic fragment thereof, or the second antigen, or the antigenic fragment thereof, comprises a hemagglutinin (HA) protein.

[0230] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 97% sequenceidentity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having the nucleic acid sequence of any one of SEQ ID NOs: 23-28.

[0231] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence of any one of SEQ ID NOs: 23-24.

[0232] In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequencehaving at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the second antigen, or the antigenic fragment thereof comprises a sequence having the nucleic acid sequence of any one of SEQ ID NOs: 25-28.

[0233] In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 70% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 90% sequenceidentity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-28. In some embodiments, the first antigen, or the antigenic fragment thereof comprises a sequence set forth in of any one of SEQ ID NOs: 23-24 and the second antigen, or the antigenic fragment thereof comprises a sequence set forth in any one of SEQ ID NOs: 25-28.

[0234] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from H3N2 and the second antigen, or the antigenic fragment thereof, comprises an HA protein from H1N1.

[0235] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from B (Yamagata lineage) and the second antigen, or the antigenic fragment thereof, comprises an HA protein from A (H3N2) influenza strain.

[0236] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from B (Victoria) lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from A (H3N2) influenza strain.

[0237] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from B (Victoria) lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from A (H1N1) influenza strain.

[0238] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from B (Yamagata) lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from A (H1N1) influenza strain.

[0239] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from Yamagata lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from an influenza A (H3N2) strain.

[0240] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from Victoria lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from an influenza A (H3N2) strain.

[0241] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from Victoria lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from an influenza A (H1N1) strain.

[0242] In some embodiments, the first antigen, or the antigenic fragment thereof, comprises an HA protein from Yamagata lineage and the second antigen, or the antigenic fragment thereof, comprises an HA protein from an influenza A (H1N1) strain.

[0243] Provided herein are purified ribonucleic acid (RNA) molecules comprising a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; a second nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; wherein the first virus variant or the second virus variant is an influenza B variant; and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0244] In some embodiments, the first virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0245] In some embodiments, the first virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0246] In some embodiments, the first influenza virus variant is an influenza B virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza strains.

[0247] In some embodiments, the first virus antigen, or an antigenic fragment thereof, is a hemagglutinin (HA) protein from a Victoria lineage and Yamagata lineage influenza strains.

[0248] In some embodiments, the second virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2.

[0249] In some embodiments, the second virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0250] In some embodiments, the second influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0251] In some embodiments, the second influenza antigen is an HA protein from an influenza A virus.

[0252] In some embodiments, the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

[0253] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0254] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0255] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0256] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0257] In some embodiments, the first HA protein is encoded by any one of SEQ ID NOs: 23 or 24 and the second HA protein is encoded by any one of SEQ ID NOs: 25-28.

[0258] In some embodiments, the first HA protein comprises an amino acid sequence selected from SEQ ID NOs: 60-61 and the second HA protein comprises an amino acid sequence selected from SEQ ID NOs: 62-65.

[0259] In some embodiments, the purified RNA further comprises a third nucleotide sequence encoding a first multimerization domain, wherein the first multimerization domainis selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and wherein the third multimerization domain is after the first nucleotide sequence (3 ’ to the first nucleotide sequence) and before the second nucleotide sequence (5’ to the first nucleotide sequence).

[0260] In some embodiments, the first multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53.

[0261] In some embodiments, the first multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0262] In some embodiments, the first multimerization domain comprises a trimerization domain. In some embodiments, the fibritin foldon domain is the trimerization domain from enterobacteria phage T4. In some embodiments, the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments, the T4 foldon domain comprises the amino acid sequence SEQ ID NO: 58. In some embodiments, the purified RNA further comprises a fourth sequence encoding a second multimerization domain after the second antigen, or an antigenic fragment thereof (3’ to the second antigen, or an antigenic fragment thereof).

[0263] In some embodiments, the second multimerization domain is a T4 foldon domain.

[0264] In some embodiments, the purified RNA further comprises a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.

[0265] In some embodiments, the first influenza virus variant, the second influenza virus variant, and the third virus variant are different.

[0266] In some embodiments, the third virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19

[0267] In some embodiments, the third virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0268] In some embodiments, the third virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0269] In some embodiments, the third antigen is an HA protein from an influenza A virus.

[0270] In some embodiments, the purified RNA further comprises a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, from a fourth virus variant. In some embodiments, the fourth virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2. In some embodiments, the fourth virus variant is a coronavirus from a SARS-CoV-2 virus. In some embodiments, the fourth antigen is a receptor-binding domain (RBD) protein.

[0271] In some embodiments, the RBD protein comprises the amino acid sequence of SEQ ID NO: 53.

[0272] In some embodiments, the fourth virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0273] In some embodiments, the purified RNA further comprises a seventh sequence encoding a third multimerization domain after the third antigen, or an antigenic fragment thereof. In some embodiments, the third multimerization domain is a T4 foldon domain.

[0274] Provided herein is an isolated polypeptide encoded by the purified RNA molecule of any of one disclosed herein.

[0275] In some embodiments, the purified RNA molecule encodes a sequence comprising at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 45- 52. In some embodiments, the purified RNA molecule encodes a sequence comprising at least 90% identity to any one of SEQ ID NOs: 45-52. In some embodiments, the purified RNA molecule encodes a sequence comprising at least 95% identity to any one of SEQ ID NOs: 45-52. In some embodiments, the purified RNA molecule encodes a sequence comprising at least 97% identity to any one of SEQ ID NOs: 45-52. In some embodiments, the purified RNA molecule encodes a sequence comprising at least 98% identity to any one of SEQ ID NOs: 45-52. In some embodiments, the purified RNA molecule encodes a sequence comprising at least 99% identity to any one of SEQ ID NOs: 45-52. In some embodiments, the purified RNA molecule encodes a sequence comprising 100% identity to any one of SEQ ID NOs: 45-52.

[0276] Provided herein are compositions of a first purified RNA molecule and a second purified RNA molecule. In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 1-15 and the second purified RNA molecule comprises any one ofSEQ ID NOs: 1-15, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 1-15.

[0277] In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 41-52, and the second purified RNA molecule comprises any one of SEQ ID NOs: 41- 52, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 41-52.

[0278] In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 41-46 and 49-52, and the second purified RNA molecule comprises a SEQ ID NOs: 47 or 48.

[0279] In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 8. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 9. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 10. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 11. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 12. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 15.

[0280] In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 8. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 9. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 10. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 11. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 12. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 7 and the second RNA molecule comprises SEQ ID NO: 15.

[0281] In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 9. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO:10. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 11. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 12. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 15.

[0282] In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO: 10. In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO:11. In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO: 12. In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO: 13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 9 and the second RNA molecule comprises SEQ ID NO: 15.

[0283] In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 12. In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO:13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 15. In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 12.

[0284] In some embodiments, the first RNA molecule comprises SEQ ID NO: 11 and the second RNA molecule comprises SEQ ID NO: 13. In some embodiments, the first RNA molecule comprises SEQ ID NO: 11 and the second RNA molecule comprises SEQ ID NO:14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 11 and the second RNA molecule comprises SEQ ID NO: 15. In some embodiments, the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 13. Insome embodiments, the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 15. In some embodiments, the first RNA molecule comprises SEQ ID NO: 13 and the second RNA molecule comprises SEQ ID NO: 14. In some embodiments, the first RNA molecule comprises SEQ ID NO: 13 and the second RNA molecule comprises SEQ ID NO: 15. In some embodiments, the first RNA molecule comprises SEQ ID NO: 14 and the second RNA molecule comprises SEQ ID NO: 15.

[0285] In some embodiments, the first RNA molecule comprises SEQ ID NO: 4 and the second RNA molecule comprises SEQ ID NO: 3.

[0286] In some embodiments, the first RNA molecule comprises SEQ ID NO: 5 and the second RNA molecule comprises SEQ ID NO: 3.

[0287] In some embodiments, the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 7.

[0288] In some embodiments, the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 9.

[0289] In some embodiments, the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 11.

[0290] In some embodiments, the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 13

[0291] In some embodiments, the first RNA molecule comprises SEQ ID NO: 14 and the second RNA molecule comprises SEQ ID NO: 15.

[0292] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 41 or 42 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 43 or 44. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 41 or 42 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 41 or 42 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 41 or 42 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 49 or 50. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 41 or 42 and thesecond RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 51 or 52.

[0293] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 43 or 44 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 43 or 44 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 43 or 44 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 49 or 50. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 43 or 44 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 51 or 52.

[0294] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 49 or 50. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 51 or 52.

[0295] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46..

[0296] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 45 or 46.Table A. RNA molecule sequences encoding a first influenza HA antigen or a second influenza HA antigen.Antigen sequences (regular font) are separated by linker sequences (bold) flanking T4 foldon sequence (bold and underlined).Table B. Influenza HA antigens encoded by mRNA constructsLinker Sequences

[0297] Linker sequences can be used in an RNA molecule to separate different components of the RNA molecule described herein. The linker sequence can join two antigens, or antigenic fragments thereof or an antigen and an oligomerization domain. It is understood that the linker sequence is not a sequence that naturally separates a first and second RNA molecule, if the first and second RNA molecule happen to naturally exist in combination together.

[0298] In some embodiments, the RNA molecule described herein comprises a sequence encoding a first linker connecting the first antigen, or the antigenic fragment thereof, to a multimerization domain. In some embodiments, the RNA molecule described herein comprises a sequence encoding a second linker connecting the second antigen, or the antigenic fragment thereof, to a multimerization domain.

[0299] In some embodiments, the first linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids. In some embodiments, one or both of the first linker and the second linker encodes an amino acid sequence comprising about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 aminoacids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0300] In some embodiments, the second linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids. In some embodiments, one or both of the first linker and the second linker encodes an amino acid sequence comprising about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, about 5 to about 10 amino acids, about 10 to about 50 amino acids, about 15 to about 50 amino acids, about 20 to about 50 amino acids, about 25 to about 50 amino acids, about 30 to about 50 amino acids, about 35 to about 50 amino acids, about 40 to about 50 amino acids, or about 45 to about 50 amino acids.

[0301] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20. In some embodiments, n is an integer from 2 to 18, from 2 to 16, from 2 to 14, from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 20, from 6 to 20, from 8 to 20, from 10 to 20, from 12 to 20, from 14 to 20, from 16 to 20, or from 18 to 20.

[0302] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20. In some embodiments, n is an integer from 2 to 18, from 2 to 16, from 2 to 14, from 2 to 12, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 20, from 6 to 20, from 8 to 20, from 10 to 20, from 12 to 20, from 14 to 20, from 16 to 20, or from 18 to 20.

[0303] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

[0304] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

[0305] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0306] In some embodiments, one or both of the first linker and the second linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0307] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8. In some embodiments, z is between 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0308] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8. In some embodiments, z is between 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0309] In some embodiments, the first linker encodes GGSG (SEQ ID NO: 38). In some embodiments, the second linker encodes GGSG (SEQ ID NO: 38).

[0310] In some embodiments, first linker encodes GGSLGGGGSGS (SEQ ID NO: 39). In some embodiments, the second linker encodes GGSLGGGGSGS (SEQ ID NO: 39).

[0311] In some embodiments, the first linker is encoded by the nucleotide sequence of any one of SEQ ID NOs: 20-22.

[0312] In some embodiments, the second linker is encoded by the nucleotide sequence of any one of SEQ ID NOs: 20-22.

[0313] In some embodiments, the first or second linker encode an amino acid sequence of any one of SEQ ID NOS: 54-57.

[0314] In some embodiments, the first linker, first multimerization domain, and second linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.Table C. Amino acid sequences of linkersFusion Proteins

[0315] The present disclosure relates generally to a strategy for multimerizing protein antigens in vaccine-related or other immunotherapeutic constructs. The strategy, in some embodiments, involves creating nucleic acid constructs with oligomerization motifs and a linker sequence separating two or more antigens such that the encoded fusion protein can form a dimeric, trimeric, tetrameric, or higher order complex from a monomer polypeptide encoded by a single nucleic acid construct.

[0316] This strategy for multimerizing proteins can be exploited with proteins including viral, bacterial, parasitic, autoimmune, and tumor antigens. This platform can be used to create multimeric fusion proteins comprising multiple copies of a single antigen of interest. For example, a homodimer, homotrimer or tetramer can be created using two, three, or four copies of the same antigen with a dimerization, trimerization or tetramerization domain.

[0317] Alternatively, this platform can be used to create multimeric fusion proteins comprising two or more different antigens of interest. For example, a heterodimer can be created with a first antigen linked to a second different antigen (or a heterotrimer comprising two or three different antigens).

[0318] One aspect described herein is directed to an RNA molecule comprising a first antigen, an oligomerization, or multimerization, domain, and a second antigen. One aspect described herein is directed to a RNA comprising a first antigen, a first linker sequence, an oligomerization domain, a second linker sequence, and a second antigen, wherein the first linker sequence joins the first antigen to the oligomerization domain, and the second linker joins the oligomerization domain to the second antigen. In one embodiment, the first and second antigens are the same. In another embodiment, the first and second antigens are different. The first and second antigens can be viral antigens, bacterial antigens, parasite antigens, autoimmune antigens, or tumor antigens. In one embodiment, the first and second antigens comprise a polypeptide and / or a polysaccharide. In one embodiment, the RNA forms a multimeric protein when expressed in a host cell. In another embodiment, the first and second antigens do not occur naturally as a multimeric protein.

[0319] In one embodiment, the RNA forms a multimeric protein when expressed in a host cell. In another embodiment, the first and second antigens do not occur naturally as a multimeric protein.

[0320] In some embodiments, the oligomerization or multimerization domain is selected from the group consisting of a dimerization domain, a trimerization domain, and a tetramerization domain. In some embodiments, the oligomerization domain is a dimerization domain. In other embodiments, the oligomerization domain is a trimerization domain. In some embodiments, the oligomerization domain is a tetramerization domain.

[0321] In some embodiments, the multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53. In some embodiments, the multimerization domain comprises a leucine zipper or a fibritin foldon domain. In some embodiments, the dimerization domain is a leucine zipper domain, including but not limited to a yeast GCN4 leucine zipper domain or a derivative thereof. In some embodiments, the trimerization domain is a T4 bacteriophage fibritin motif. In some embodiments, the trimerization domain is a eukaryotic GNC4 transcription factor motif or a derivative thereof. In some embodiments, the fibritin foldon domain is the trimerization domain from enterobacteria phage T4 or a derivative thereof. In some embodiments, the trimerization domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments, the T4 foldon domain comprises the amino acid sequence SEQ ID NO: 58(GYIPEAPRDGQAYVRKDGEWVLLSTFL).

[0322] In some embodiments, the multimerization domain comprises an amino acid sequence of any one of SEQ ID NOs: 66-68. In some embodiments, the multimerization domain comprises an amino acid sequence 90% identical to any one of SEQ ID NOs: 66-68. In some embodiments, the multimerization domain comprises an amino acid sequence 95% identical to any one of SEQ ID NOs: 66-68. In some embodiments, the multimerization domain comprises an amino acid sequence 98% identical to any one of SEQ ID NOs: 66-68. In some embodiments, the multimerization domain comprises an amino acid sequence 100% identical to any one of SEQ ID NOs: 66-68.

[0323] In some embodiments, the oligomerization domain is located between any antigens.

[0324] In one aspect, provided herein is a polypeptide encoded by any embodiment of an RNA described herein.

[0325] Given the disclosure of this application, one of skill in the art could substitute any antigen of interest into the RNA molecules described herein.Lipid Compositions and Methods of Preparing

[0326] Provided herein, in some embodiments, are compositions of an RNA molecule described herein and a delivery vehicle. Further provided herein are methods relating to the RNA molecule described herein and a delivery vehicle. In some embodiments, an RNA delivery vehicle is a nanoparticle (e.g., LNP).

[0327] A delivery vehicle can be non-virion particles, i.e., they are not a virion. Thus, in some embodiments, the delivery vehicle does not comprise a protein capsid. By avoiding the need to create a capsid, a delivery vehicle does not utilize a packaging cell line, thus permitting easier up-scaling for commercial production and minimizing the risk that dangerous infectious viruses will inadvertently be produced. Various materials are suitable delivery particles which can deliver RNA to a vertebrate cell in vivo. Exemplary delivery materials are (i) amphiphilic lipids which can form liposomes and (ii) non-toxic and biodegradable polymers which can form microparticles. Other delivery methods may include, but are not limited to, exosomes and cationic nano-emulsions.

[0328] Where delivery is by liposome, the RNA can be encapsulated or adsorbed; where delivery is by polymeric microparticle, the RNA can be encapsulated or adsorbed. A third delivery material is the particulate reaction product of a polymer, a crosslinker, a RNA, and a charged monomer. In certain embodiments, the delivery particle described herein comprises a liposome adsorbing RNA molecules described herein.

[0329] In some embodiments, the RNA is encapsulated within LNPs. This means that RNA inside the particles is separated from any external medium by the delivery material, and encapsulation has been found to protect RNA from RNase digestion. Encapsulation can take various forms. For example, in some embodiments, the delivery material forms an outer layer around an aqueous RNA-containing core. In some embodiments, the composition of the RNA encapsulated by the LNP is lyophilized.

[0330] In some embodiments, RNA is adsorbed to the surface of the LNPs. This means, in some embodiments, that RNA is not separated from any external medium by the delivery material, unlike the RNA genome of a natural virus.

[0331] In some embodiments, the LNP has a pH of about 5 to about 7. In some embodiments, the LNP has a pH of about 5.2 to about 6.8. In some embodiments, the LNP has a pH of about 5.4 to about 6.6. In some embodiments, the LNP has a pH of about 5.5 to about 6.5. In some embodiments, the LNP has a pH of about 5.2 to about 6.4. In some embodiments, the LNP has a pH of about 5 to about 6.2.

[0332] In some embodiments, the RNA of the present disclosure is formulated in lipid nanoparticles (LNPs) having a diameter of about 40 nanometer (nm) to about 600 nm. In some embodiments, the LNP has a diameter of about 50 to about 600 nm, about 100 to about 600 nm, about 150 to about 600 nm, about 200 to about 600 nm, about 250 to about 600 nm, or about 300 to about 600 nm. In some embodiments, the LNP has a diameter of about 50 to about 550 nm, about 50 to about 500 nm, about 50 to about 450 nm, about 50 to about 400 nm, about 50 to about 350 nm, or about 50 to about 300 nm.

[0333] In some embodiments, the LNP has a diameter no more than about 300 nm. In some embodiments, the LNP has a diameter no more than about 250 nm. In some embodiments, the LNP has a diameter no more than about 200 nm. In some embodiments, the LNP has a diameter no more than about 190 nm. In some embodiments, the LNP has a diameter no more than about 180 nm. In some embodiments, the LNP has a diameter no more than about 170 nm. In some embodiments, the LNP has a diameter no more than about 160 nm. In some embodiments, the LNP has a diameter no more than about 150 nm. In some embodiments, the LNP has a diameter no more than about 140 nm. In some embodiments, the LNP has a diameter no more than about 130 nm. In some embodiments, the LNP has a diameter no more than about 120 nm.

[0334] Further provided herein are methods of preparing an RNA-LNP composition comprising: a) mixing an ethanol phase comprising one or more lipids and an aqueous phase comprising the RNA molecules described herein; and b) purifying the RNA-LNP generated from step a).

[0335] In some embodiments, the one or more lipids comprises an ionizable lipid. In some embodiments, the one or more lipids comprises cholesterol. In some embodiments, the one or more lipids comprises a phospholipid. In some embodiments, the one or more lipids is pegylated.

[0336] In some embodiments, the LNP:RNA (N:P) ratio is about 6 to about 10. In some embodiments, the N:P ratio is about 6.5 to about 9, about 7 to about 9, about 7.5 to about 9, about 8 to about 9, about 6 to about 9.5, about 6 to about 9, about 6 to about 8.5, or about 6 to about 8.

[0337] In some embodiments, the aqueous phase comprises tris, sodium chloride, sucrose, or combinations thereof. In some embodiments, the aqueous phase comprises tris. In some embodiments, the aqueous phase comprises sodium chloride. In some embodiments, the aqueous phase comprises sucrose. In some embodiments, the aqueous phase comprises or consists of tris, sodium chloride, and sucrose.

[0338] In some embodiments, a lipid nanoparticle (LNP) formulation described herein comprises, consists essentially of, or consists of (i) a neutral phospholipid (ii) a sterol, e.g., cholesterol; (iii) a pegylated lipid optionally and (iv) a ionizable lipid with the molar ratio within ranges of neutral phospholipid: 5%-20%, sterol: 18.5%-58.5%, pegylated lipid: 1% - 4%, ionizable lipid: 30% - 70%, the total summation of the mole ratio of the lipids is 100%, optionally 10:40.5:1.5:48.

[0339] In some embodiments, the LNP comprises a DSPC: cholesterol: DMG-PEG 2000: and an ionizable lipid with the mole ratio within the ranges of DSPC: 5%-20%, cholesterol: 18.5% - 58.5%, DMG-PEG 2000: 1% - 4%, ionizable lipid: 30% - 70%, and the total summation of the mole ratio of the lipids is 100%.

[0340] In some embodiments, the lipid is a cationic lipid, also called ionizable lipid. In some embodiments, useful cationic lipids generally contain a nitrogen atom that is positively charged under physiological conditions e.g., as a tertiary or quaternary amine. This nitrogen can be in the hydrophilic head group of an amphiphilic surfactant. The lipid may be selected from, but is not limited to, l,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3’-[N- (N’,N’-Dimethylaminoethane)-carbamoyl] cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA e.g., the bromide), l,2-Dimyristoyl-3-Trimethyl- AmmoniumPropane (DMTAP), dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP). Other useful cationic lipids are: benzalkonium chloride (BAK), benzethonium chloride, cetramide (which contains tetradecyltrimethylammonium bromide and possibly small amounts of dedecyltrimethylammonium bromide and hexadecyltrimethyl ammonium bromide), cetylpyridinium chloride (CPC), cetyl trimethylammonium chloride (CTAC), N, N’, N’- polyoxyethylene (10)-N-tallow-l,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecyl-ammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecyl ammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctylammonium chloride), N,N-dimethyl-N-[2 (2-methyl-4-(l,l,3,3tetramethylbutyl)- phenoxy]-ethoxy)ethyl]-benzenemetha-naminium chloride (DEBDA), dialkyldimetylammonium salts, [1 -(2, 3-dioleyloxy)-propyl]-N,N,N, trimethylammonium chloride, l,2-diacyl-3-(trimethylammonio)propane (acyl group=dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), l,2-diacyl-3-(dimethylammonio)propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), l,2-dioleoyl-3-(4’-trimethyl-ammonio)butanoyl-sn- glycerol, 1,2-dioleoyl 3 -succinyl-sn -glycerol choline ester, cholesteryl (4’- trimethylammonio)butanoate), N-alkyl pyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic bolaform electrolytes (C12Me6; C12BU6), dialkylglycetylphosphorylcholine, lysolecithin, L-a dioleoylphosphatidylethanolamine, cholesterol hemisuccinate choline ester, lipopolyamines, including but not limited to dioctadecylamidoglycylspermine (DOGS), dipalmitoyl phosphatidylethanol-amidospermine (DPPES), lipopoly-L (or D)-lysine (LPLL, LPDL), poly (L (or D)-lysine conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate ester with pendant amino group (CnGluPhCnN ), ditetradecyl glutamate ester with pendant amino group (Cl 2GluPhCnNl), cationic derivatives of cholesterol, including but not limited to cholesteryl-3 P-oxysuccinamidoethylenetrimethylammonium salt, cholesteryl-3 P- oxysuccinamidoethylenedimethylamine, cholesteryl-3 P- carboxyamidoethylenetrimethylammonium salt, and cholesteryl-3 P- carboxy amidoethylenedimethylamine.

[0341] In some embodiments, the lipid has the following structure:lipid 1.

[0342] In some embodiments, the lipid has the following structure:lipid 2.

[0343] In some embodiments, an RNA delivery vehicle is a nanoparticle that comprises at least one lipid. In some embodiments, the lipid may be a neutral lipid. In some embodiments, the lipid is a phospholipid. The phospholipid may be selected from, but is not limited to, DDPC, l,2-Didecanoyl-sn-Glycero-3 -phosphatidylcholine, DEP A, 1,2-Dierucoyl-sn- Glycero-3 -Phosphate, DEPC, l,2-Erucoyl-sn-Glycero-3 -phosphatidylcholine, DEPE, 1,2- Dierucoyl-sn-Glycero-3 -phosphatidylethanolamine, DEPG, 1,2-Dipalmitoylphosphatidylglycerol, DLOPC, l,2-Linoleoyl-sn-Glycero-3 -phosphatidylcholine, DLPA, l,2-Dilauroyl-sn-Glycero-3 -Phosphate, DLPC, l,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine, DLPE, l,2-Dilauroyl-sn-Glycero-3 -phosphatidylethanolamine, DLPG1.2-Dilauroyl-sn-Glycero-3(phosphorylglycerol), DLPS l,2-Dilauroyl-sn-Glycero-3- phosphatidylserine, DMG, l,2-Dimyristoyl-sn-glycero-3 -phosphoethanolamine, DMPA, 1,2- Dimyristoyl-sn-Glycero-3-Phosphate, DMPC, l,2-Dimyristoyl-sn-Glycero-3- phosphatidylcholine, DMPE, l,2-Dimyristoyl-sn-Glycero-3 -phosphatidylethanolamine, DMPG, l,2-Dimyristoyl-sn-glycero-3 -phosphoglycerol, DMPS, 1,2-Dimyristoyl-sn-Glycero- 3-phosphatidylserine, DOPA, l,2-Dioleoyl-sn-Glycero-3-Phosphate, DOPC, 1,2-Dioleoyl-sn- Glycero-3 -phosphatidylcholine, DOPE, l,2-Dioleoyl-sn-Glycero-3- phosphatidylethanolamine, DOPG, 1,2-Dipalmitoylphosphatidylglycerol, DOPS, 1,2- Dioleoyl-sn-Glycero-3-phosphatidylserine, DPP A, l,2-Dipalmitoyl-sn-Glycero-3 -Phosphate, DPPC, l,2-Dipalmitoyl-sn-Glycero-3 -phosphatidylcholine, DPPE, 1,2-Dipalmitoyl-sn- Glycero-3 -phosphatidylethanolamine, DPPG, l,2-Dipalmitoyl-sn-glycero-3 -phosphoglycerol, DPPS, l,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine, DPyPE, 1,2-diphytanoyl-sn- glycero-3 -phosphoethanolamine, DSP A, l,2-Distearoyl-sn-Glycero-3 -Phosphate, DSPC, 1,2- Distearoyl-sn-Glycero-3-phosphatidylcholine, DSPE, l,2-Diostearpyl-sn-Glycero-3- phosphatidylethanolamine, DSPG, l,2-Distearoyl-sn-Glycero-3- phosphorylglycerol, DSPS,1.2-diundecanoyl-sn-glycero-phosphocholine, DUPC, l,2-Distearoyl-sn-Glycero-3- phosphatidylserine, EPC, Egg-PC, HEPC, Hydrogenated Egg PC, HSPC, High purity Hydrogenated Soy PC, HSPC, Hydrogenated Soy PC, Lysopc Myristic, 1-Myristoyl-sn- Glycero-3 -phosphatidylcholine, LYSOPC PALMITIC, l-Palmitoyl-sn-Glycero-3- phosphatidylcholine, LYSOPC STEARIC, l-Stearoyl-sn-Glycero-3-phosphatidylcholine, Milk Sphingomyelin, MPPC, l-Myristoyl,2-palmitoyl-sn-Glycero 3 -phosphatidyl choline, MSPC, 1 -Myristoyl, 2-stearoyl-sn-Glycero-3 -phosphatidylcholine, PMPC, 1 -Palmitoyl, 2- myristoyl-sn-Glycero-3-phosphatidylcholine, POPC, 1 -Palmitoyl, 2-oleoyl-sn-Gly cero-3- phosphatidylcholine, POPE, l-Palmitoyl-2-oleoyl-sn-Glycero-3 -phosphatidylethanolamine, POPG, l,2-Dioleoyl-sn-Glycero-3-Phospho-rac-(l-glycerol)], PSPC, 1 -Palmitoyl, 2-stearoyl- sn-Glycero-3-phosphatidylcholine, SMPC, l-Stearoyl,2-myristoyl-sn-Glycero-3- phosphatidylcholine, SOPC, l-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine, SPPC, 1- Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine.

[0344] In some embodiments, an RNA delivery vehicle is a nanoparticle that comprises at least one lipid. In some embodiments, the lipid may be a PEGylated lipid (PEG). In some embodiments, the PEGylated lipid comprises a polyethylene glycol moiety. In some embodiments, a PEG lipid includes but is not limited to PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides,PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, or combinations thereof. In some embodiments, a PEGylated lipid is PEG-c- DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEGylated lipid is DMG-PEG, i.e. PEG-conjugated 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol). In some embodiments, the lipid is DMG-PEG 2000, i.e. l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000. In some embodiments, the pegylated lipid comprises l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol -2000 (DMG-PEG 2000), DMG-PEG 3500, DMG-PEG 5000, DTDAM-PEG 2000(ALC-0159), DTDAM-PEG 5000, DMG-C-PEG 2000, DMG-C-PEG 5000, DSG-PEG 2000, DSG-PEG 5000, DPG-PEG 2000, DPG-PEG 5000, or any combination thereof.

[0345] In some embodiments, the lipid is a structural lipid. In some embodiments, the structural lipid includes, but is not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, or combinations thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is cholesterol, a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0346] In certain embodiments, provided herein is a pharmaceutically acceptable composition comprising the RNA-LNP described herein, and a pharmaceutically acceptable carrier.

[0347] In some embodiments, pharmaceutically acceptable salts of the lipids described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, trifluoroacetate, and undecanoate.Methods of Treatment

[0348] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and / or treatment of diseases or conditions in humans and other mammals.

[0349] The disclosure herein provides a method of mixing the lyophilized RNA with a liquid LNP solution to make a pharmaceutical composition. In certain embodiments, the liquid LNP solution is added to the lyophilized RNA. In certain embodiments, the lyophilized RNA and liquid LNP solution are mixed at room temperature. In certain embodiments, the lyophilized RNA and liquid LNP solution are mixed prior to clinical use. In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state is administered to the subject at least two times. In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state is administered to the subject two times, three times, or four times. In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state is administered to the subject once every two, three, or four weeks.

[0350] In some embodiments, the lyophilized RNA encapsulated in the LNP is mixed with sterile water. In some embodiments, the lyophilized RNA encapsulated in the LNP and sterile water are administered to the subject intramuscularly. In some embodiments, the lyophilized RNA encapsulated in the LNP is administered to the subject at least two times. In some embodiments, the lyophilized RNA encapsulated in the LNP is administered to the subject two times, three times, or four times. In some embodiments, the lyophilized RNA encapsulated in the LNP is administered to the subject once every two, three, or four weeks. Prophylactic protection from an antigen can be achieved following administration of an RNA vaccine or a therapeutic of the present disclosure. In certain embodiments, it is sufficient to administer the vaccine or therapeutic twice. It is possible, although less desirable, to administer the vaccine or therapeutic to an infected individual to achieve a therapeutic response.

[0351] In some embodiments, the RNA is encapsulated in an LNP in a sterile buffer. In some embodiments, the sterile buffer is water. In some embodiments, the sterile buffer comprises salts. In some embodiments, the RNA encapsulated in an LNP is administered to the subject at least two times. In some embodiments, the RNA encapsulated in an LNP is equilibrated at room temperature for about 5-20 minutes or 10-15 minutes before administration. In some embodiments, the RNA encapsulated in an LNP is administered to the subject two times, three times, or four times. In some embodiments, the RNA encapsulated in an LNP is administered to the subject once every two, three, or four weeks.

[0352] A method of eliciting an immune response in a subject against an antigen is provided in aspects of the present disclosure. The method involves administering to the subject a RNA vaccine or therapeutic comprising a RNA molecule having an open reading frame encoding at least one antigenic polypeptide and a delivery vehicle, such as an LNP, thereby inducing in the subject an immune response. An “anti-antigenic polypeptide antibody” is a serum antibody the binds specifically to the antigenic polypeptide.

[0353] A “prophylactically effective dose” as used herein is a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the therapeutically effective dose is a dose listed in a package insert for the vaccine or therapeutic. A traditional vaccine, as used herein, refers to a vaccine other than the RNA vaccine or therapeutic of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and / or is registered by a national drug regulatory body, for example, the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

[0354] A method of treating or preventing disease in a subject comprising administering to the subject an effective amount the RNA-lipid composition described herein is provided in this disclosure.

[0355] In some embodiments, the disease is caused by a virus is selected from the group consisting of a influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus.

[0356] In some embodiments, the disease is caused by coronavirus. In some embodiments, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the coronavirus is SARS-CoV-2.

[0357] In some embodiments, the disease is caused by one or more viruses selected from the group consisting of an influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus. In some embodiments, the one or more viruses includes a coronavirus, an influenza A virus, and / or an influenza B virus. In some embodiments, the one or more viruses includes at least a coronavirus and an influenza B virus. In some embodiments, the one or more viruses includes at least a coronavirus and an influenza A virus

[0358] In some embodiments, the RNA adsorbed to the surface of the LNP is in a liquid state, and optionally at a temperature of 2-8 °C.

[0359] In some embodiments, the RNA and LNP is administered to the subject intramuscularly.

[0360] In some embodiments, the lyophilized RNA encapsulated in the LNP is mixed with sterile water.

[0361] In some embodiments, the lyophilized RNA encapsulated in the LNP and sterile water are administered to the subject intramuscularly.

[0362] In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state or lyophilized RNA encapsulated in the LNP is administered to the subject at least two times.

[0363] In some embodiments, the RNA adsorbed to the surface of the LNP in a liquid state or lyophilized RNA encapsulated in the LNP is administered once every one, two, three, or four weeks.

[0364] In some embodiments, the RNA vaccine or therapeutic is administered to a subject (e.g., parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal)). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. In some embodiments, the RNA vaccine or therapeutic is administered intramuscularly. In some embodiments, the RNA vaccine or therapeutic is administered intravenously. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0365] In some embodiments, the vaccine or therapeutic is administered to the subject at a dose of 1 pg -100 pg, optionally 5, 30, or 50 pg. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0366] In some embodiments, the vaccine or therapeutic is administered to the subject more than once. In some embodiments, the vaccine or therapeutic is administered to the subject at least two times. In some embodiments, the second dose is administered to the subject about 8 weeks following the initial or prime dose. In some embodiments, the second dose is administered to the subject about 7 weeks following the prime dose. In some embodiments, the second dose is administered to the subject about 6 weeks following the prime dose. Insome embodiments, the second dose is administered to the subject about 5 weeks following the prime dose. In some embodiments, the second dose is administered to the subject about 4 weeks following the prime dose. In some embodiments, the second dose is administered to the subject about 3 weeks following the prime dose. In some embodiments, the second dose is administered to the subject about 2 weeks following the prime dose. In some embodiments, the second dose is administered to the subject about 1 week following the prime dose.Kits

[0367] In some embodiments, the present disclosure also provides kits comprising: i) an RNA; ii) a delivery vehicle, such as a liquid LNP solution; iii) instructions for mixing the RNA with the delivery vehicle to prepare an immunogenic composition; and iv) instructions for administration of the immunogenic composition to stimulate an immune response against the antigen in a mammalian subject, such as a human subject in need thereof.

[0368] In some embodiments, the RNA is stored at or below about -50 °C In some embodiments, the RNA is stored at or below about -60 °C. In some embodiments, the RNA is stored at or below about -70 °C. In some embodiments, the RNA is stored at about -80 °C. In some embodiments, the RNA-LNP vaccine is stored at or below about 10 °C. In some embodiments, the RNA-LNP vaccine is stored at or below about 0 °C. In some embodiments, the RNA-LNP vaccine is stored at or below about -10 °C. In some embodiments, the RNA- LNP vaccine is stored at or below about -15 °C. In some embodiments, the RNA-LNP vaccine is stored at or below about -20 °C.Embodiments

[0369] In one aspect, provided herein is purified ribonucleic acid (RNA) molecule comprising a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, of a hemagglutinin (HA) protein from a first influenza virus variant; a second nucleotide sequence encoding a first multimerization domain, and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0370] In some embodiments, the virus is an influenza A virus or an influenza B virus.

[0371] In some embodiments, the influenza A virus or influenza B virus is selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0372] In some embodiments, the first multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and a second antigen, or an antigenic fragment thereof, from a second influenza virus variant.

[0373] In some embodiments, the first multimerization domain is a second antigen, or an antigenic fragment thereof, from a second influenza virus variant, wherein the first influenza virus variant and the second influenza virus variant are different.

[0374] In some embodiments, the second influenza antigen is an HA protein.

[0375] In some embodiments, the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

[0376] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0377] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0378] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0379] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0380] In some embodiments, the first HA protein comprises any one of SEQ ID NOs: 23 or 24 and the second HA protein comprises any one of SEQ ID NOs: 25-28.

[0381] In some embodiments, the first HA protein comprises an amino acid sequence selected from SEQ ID NOs: 60-61 and the second HA protein comprises an amino acid sequence selected from SEQ ID NOs: 62-65.

[0382] In some embodiments, the purified RNA further comprises a third nucleotide sequence encoding a second multimerization domain, wherein the second multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, and a tetramerization domain, and wherein the second multimerization domain is after the first nucleotide sequence and before the second nucleotide sequence.

[0383] In some embodiments, the second multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53.

[0384] In some embodiments, the second multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0385] In some embodiments, the second multimerization domain comprises a trimerization domain. In some embodiments, the fibritin foldon domain is the trimerization domain from enterobacteria phage T4. In some embodiments, the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO.: 19.

[0386] In some embodiments, the purified RNA molecule further comprises a sequence encoding a first linker connecting the first antigen, or the antigenic fragment thereof, to the second multimerization domain.

[0387] In some embodiments, the purified RNA molecule further comprises a sequence encoding a second linker connecting the second multimerization domain to the first multimerization domain.

[0388] In some embodiments, the first linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids. In some embodiments, the second linker encodes an amino acid sequence comprising at least 5 to about 50 amino acids.

[0389] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20. In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20.

[0390] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6. In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6. In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3. In some embodiments, wherein the second linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

[0391] In some embodiments, the first linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8.

[0392] In some embodiments, the second linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8.

[0393] In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0394] In some embodiments, the first linker encodes GGSG (SEQ ID NO: 38). In some embodiments, the second linker encodes GGSG (SEQ ID NO: 38). In some embodiments, the first linker encodes GGSLGGGGSGS (SEQ ID NO: 39). In some embodiments, the second linker encodes GGSLGGGGSGS (SEQ ID NO: 39).

[0395] In some embodiments, the first linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22. In some embodiments, the second linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22.

[0396] In some embodiments, the purified RNA molecule comprises about 90% identity to any one of SEQ ID NOs: 1-15. In some embodiments, the purified RNA molecule comprises any one of SEQ ID NOs: 1-15.

[0397] In some embodiments, the purified RNA molecule further comprises a fourth sequence encoding a third multimerization domain after the first multimerization domain.

[0398] In some embodiments, the third multimerization domain is a T4 foldon domain.

[0399] In some embodiments, the purified RNA molecule further comprises a fifth nucleotide sequence encoding a fourth multimerization domain.

[0400] In some embodiments, the fourth multimerization domain is a third antigen, or an antigenic fragment thereof, from a third influenza virus variant.

[0401] In some embodiments, the first influenza virus variant, the second influenza virus variant, and the third influenza virus variant are different.

[0402] In some embodiments, the purified RNA further comprises a sequence encoding a first linker connecting the first antigen, or an antigenic fragment thereof to the first multimerization domain.

[0403] In some embodiments, the purified RNA further comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1) 5’ to the first nucleotide sequence.

[0404] In some embodiments, the purified RNA further comprises a 5’ UTR 3’ to the Cap 1 and 5’ to the first nucleotide sequence.

[0405] In some embodiments, the purified RNA further comprises a sequence encoding a signal peptide 3’ to the 5’ UTR and 5’ to the first nucleotide sequence.

[0406] In some embodiments, the purified RNA further comprises a sequence encoding a 3’ UTR 3’ to the second nucleotide sequence.

[0407] Provided herein is a purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 1-15. Provided herein is a purified RNA molecule comprising a sequence having at least 95% sequence identity to the sequence of any one of SEQ ID NOs: 1-15. Provided herein is a purified RNA molecule comprising a sequence comprising any one of SEQ ID NOs: 1-15.

[0408] Provided herein is a purified RNA comprising, from 5’ to 3’: a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap l);a 5’ UTR;a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, of a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza; optionally, a second nucleotide sequence encoding a first linker; optionally, a third nucleotide sequence encoding a T4 foldon domain; optionally, a fourth nucleotide sequence encoding a second linker; optionally, a fifth nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, of a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7 influenza strains; a 3’ UTR; and a poly A tail.

[0409] In some embodiments, the purified RNA further comprises a sixth nucleotide sequence encoding a third linker and a seventh nucleotide sequence encoding a second T4 foldon domain.

[0410] In some embodiments, the purified RNA further comprises an eighth nucleotide sequence encoding a antigen, or an antigenic fragment thereof, of a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0411] Provided herein are purified ribonucleic acid (RNA) molecules comprising a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; a second nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; wherein the first virus variant or the second virus variant is an influenza B variant; and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

[0412] In some embodiments, the first virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0413] In some embodiments, the first virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0414] In some embodiments, the first influenza virus variant is an influenza B virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza strains.

[0415] The purified RNA of any one of claims 3 or 4, wherein the first virus antigen, or an antigenic fragment thereof, is a hemagglutinin (HA) protein from a Victoria lineage and Yamagata lineage influenza strains.

[0416] In some embodiments, the second virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2.

[0417] The purified RNA of claim 1, wherein the second virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0418] In some embodiments, the second influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0419] In some embodiments, the second influenza antigen is an HA protein from an influenza A virus.

[0420] In some embodiments, the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

[0421] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0422] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus.

[0423] In some embodiments, the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0424] In some embodiments, the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

[0425] In some embodiments, the first HA protein is encoded by any one of SEQ ID NOs: 23 or 24 and the second HA protein is encoded by any one of SEQ ID NOs: 25-28.

[0426] In some embodiments, the first HA protein comprises an amino acid sequence selected from SEQ ID NOs: 60-61 and the second HA protein comprises an amino acid sequence selected from SEQ ID NOs: 62-65.

[0427] In some embodiments, the purified RNA further comprises a third nucleotide sequence encoding a first multimerization domain, wherein the first multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and wherein the third multimerization domain is after the first nucleotide sequence (3 ’ to the first nucleotide sequence) and before the second nucleotide sequence (5’ to the first nucleotide sequence).

[0428] In some embodiments, the first multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53.

[0429] In some embodiments, the first multimerization domain comprises a leucine zipper or a fibritin foldon domain.

[0430] In some embodiments, the first multimerization domain comprises a trimerization domain. In some embodiments, the fibritin foldon domain is the trimerization domain from enterobacteria phage T4. In some embodiments, the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 19. In some embodiments, the T4 foldon domain comprises the amino acid sequence SEQ ID NO: 58.

[0431] In some embodiments, the purified RNA further comprises a fourth sequence encoding a second multimerization domain after the second antigen, or an antigenic fragment thereof (3’ to the second antigen, or an antigenic fragment thereof).

[0432] In some embodiments, the second multimerization domain is a T4 foldon domain.

[0433] In some embodiments, the second multimerization domain comprises an amino acid sequence of any one of SEQ ID NOs: 66-68.

[0434] In some embodiments, the purified RNA further comprises a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.

[0435] In some embodiments, the first influenza virus variant, the second influenza virus variant, and the third influenza virus variant are different.

[0436] In some embodiments, the third influenza virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0437] In some embodiments, the third influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

[0438] In some embodiments, the third influenza antigen is an HA protein from an influenza A virus.

[0439] In some embodiments, the purified RNA further comprises a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, from a fourth virus variant. In some embodiments, the fourth virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2. In some embodiments, the fourth virus variant is a coronavirus from a SARS-CoV-2 virus. In some embodiments, the fourth antigen is a receptor-binding domain (RBD) protein.

[0440] In some embodiments, the RBD protein comprises the amino acid sequence of SEQ ID NO: 53.

[0441] In some embodiments, the purified RNA further comprises a seventh sequence encoding a third multimerization domain after the third antigen, or an antigenic fragment thereof. In some embodiments, the third multimerization domain is a T4 foldon domain.

[0442] In some embodiments, the third multimerization domain comprises a sequence of any one of SEQ ID NOs: 66-68.

[0443] Provided herein is an isolated polypeptide encoded by the purified RNA molecule of any of one disclosed herein.

[0444] Provided herein is a composition comprising a first purified RNA molecule comprising the purified RNA molecule of any of one disclosed herein and a delivery vehicle.

[0445] In some embodiments, the purified RNA further comprises a second purified RNA molecule comprising the purified RNA molecule of any of one disclosed herein.

[0446] In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 1-15 and the second purified RNA molecule comprises any one of SEQ ID NOs: 1-15, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 1-15.

[0447] In some embodiments, the first RNA molecule comprises SEQ ID NO: 4 and the second RNA molecule comprises SEQ UD NO: 2; the first RNA molecule comprises SEQ ID NO: 5 and the second RNA molecule comprises SEQ UD NO: 2; the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ UD NO: 7; the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ UD NO: 9; the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ UD NO: 11; the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ UD NO: 13; or the first RNA molecule comprises SEQ ID NO: 14 and the second RNA molecule comprises SEQ UD NO: 15.

[0448] In some embodiments, the first purified RNA molecule comprises any one of SEQ ID NOs: 41-52, and the second purified RNA molecule comprises any one of SEQ ID NOs: 41- 52, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 41-52.

[0449] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 45 or 46. In some embodiments, the first RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 45 or 46.

[0450] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 49 or 50. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 49 or 50. In some embodiments, the first RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 49 or 50.

[0451] In some embodiments, the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 51 or 52. In some embodiments, the first RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 95% identical to SEQ ID NO: 51 or 52. In some embodiments, the first RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence 100% identical to SEQ ID NO: 51 or 52.

[0452] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0453] In some embodiments, the LNP comprises an ionizable lipid.

[0454] In some embodiments, the LNP comprises an ionizable lipid: cholesterol: DSPC: DMG-PEG2000 ratio of about 48: 40: 10: 2, and an LNP:RNA (N:P) ratio of about 8: 1.

[0455] In some embodiments, the composition comprises a particle size of no more than about 300 nanometers (nm). In some embodiments, the composition comprises a particle size of about 50 to about to 300 nm. In some embodiments, the composition comprises a particle size of no more than about 150 nm. In some embodiments, the composition comprises a particle size of about 50 nm to about 140 nm.

[0456] In some embodiments, the RNA is lyophilized. In some embodiments, the RNA is adsorbed to the surface of the LNP. In some embodiments, the RNA is encapsulated by the LNP. In some embodiments, the RNA encapsulated by the LNP is lyophilized.

[0457] Provided herein is a kit comprising the purified RNA of any one disclosed herein, a delivery vehicle, and instructions for use.

[0458] Provided herein is a method of treating or preventing disease in a subject comprising administering to the subject an effective amount of the composition of any one disclosed herein.

[0459] In some embodiments, the disease is caused by a virus is selected from the group consisting of a influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus. In some embodiments, the disease is caused by a coronavirus, an influenza A virus, or an influenza B virus.

[0460] In some embodiments, the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

[0461] In some embodiments, the coronavirus is SARS-CoV-2.

[0462] In some embodiments, the influenza A virus or influenza B virus is selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

[0463] In some embodiments, the composition is administered to the subject intramuscularly.

[0464] In some embodiments, the composition is administered to the subject at least two times. In some embodiments, the composition is administered once every one, two, three, or four weeks.

[0465] Provided herein is a method for stimulating an immune response in a subject comprising administering to the subject an effective amount of the composition of any one disclosed herein.

[0466] Provided herein is a method for preparing a RNA-LNP composition comprising: a) mixing an ethanol phase comprising one or more lipids and an aqueous phase comprising the purified RNA molecule of any one disclosed herein; and b) purifying the RNA-LNP generated from step a).

[0467] In some embodiments, the one or more lipids comprises an ionizable lipid. In some embodiments, the one or more lipids comprises cholesterol. In some embodiments, the one or more lipids comprises a phospholipid. In some embodiments, the one or more lipids is pegylated. In some embodiments, the N:P ratio is about 6.5 to about 9. In some embodiments, the aqueous phase consists of tris, sodium chloride, and sucrose.

[0468] Provided herein is kit comprising the composition of any one disclosed herein, a delivery vehicle, and instructions for use.Definitions

[0469] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0470] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations used herein have their conventional meaning within the chemical arts.

[0471] Throughout the description, where systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are systems of the present invention that consist essentially of, or consist of, the recited components, andthat there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0472] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0473] Further, it should be understood that elements and / or features of an apparatus or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular component of a system, that component can be used in various embodiments of systems of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0474] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.

[0475] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0476] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0477] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additionalunrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0478] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10%, ±5%, ±3% or ±2% variation from the nominal value unless otherwise indicated or inferred from the context.

[0479] At various places in the present specification, variable or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual sub-combination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 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, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0480] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0481] As a general matter, formulations specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.

[0482] The term “dried RNA” or as used herein has to be understood as RNA that has been lyophilized, or spray-dried, or spray-freeze dried as defined herein to obtain a temperature stable dried RNA (powder).

[0483] “Cryoprotectants” are known in the art and include without limitation, sucrose, trehalose, and glycerol. A cryoprotectant exhibiting low toxicity in biological systems is generally used.

[0484] The terms “lyophilization” include the related terms “cryodesiccation,” “lyophilizing,” or “freeze drying,” and typically relates to a process which allows reduction of a solvent (e.g., water) content of a frozen sample (preferably a solution containing an RNA molecule and a cryoprotectant as described herein) in one or more steps via sublimation. In the context of the present disclosure, lyophilization is typically carried out by freezing a sample and subsequently drying the sample via sublimation, optionally by reducing thesurrounding pressure and / or by heating the sample so that the solvent sublimes directly from the solid phase to the gas phase.

[0485] “Polynucleotide,” “nucleic acid,” or “nucleotide” are used interchangeably herein and refer to chains of nucleotides of any length, and comprise DNA and RNA. In some embodiments, the nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that is incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure is imparted before or after assembly of the chain. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, a polynucleotide is further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications comprise, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates) and with charged linkages (e.g., phosphorothioates, phosphorodithioates), those containing pendant moi eties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine), those with intercalators (e.g., acridine, psoralen), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). In some embodiments, any of the hydroxyl groups ordinarily present in the sugars are replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or are conjugated to solid supports. In some embodiments, the 5’ and 3’ terminal OH is phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. In some embodiments, polynucleotides also contain analogous forms of ribose or deoxyribose sugars, comprising, for example, 2’-O-methyl-, 2’-O-allyl, 2’ -fluoro- or 2’ -azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. In some embodiments, one or more phosphodiester linkages are replaced by alternative linking groups. These alternative linking groups comprise, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (0)NRi (“amidate”), P(O)R, P(O)OR’, CO orCH2 (“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, comprising RNA and DNA. In a polynucleotide, when referring to a T, a T means U (Uracil) in RNA and T (Thymine) in DNA.

[0486] The term “messenger RNA” (mRNA) refers to one type of a polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

[0487] As used herein, the terms “termini” or “terminus,” when referring to polypeptides, RNA molecules, or polynucleotides, refers to an extremity of a polypeptide, RNA molecule, or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide, RNA molecule, or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These proteins comprise multiple N-termini and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0488] A “5’ untranslated region” (5’ UTR) refers to a region of an RNA (e.g., mRNA or other coding RNA) that is directly upstream (i.e., 5’) from the start codon (i.e., the first codon of an RNA, such as mRNA or other coding RNA, transcript translated by a ribosome) that does not encode a polypeptide.

[0489] A “3’ untranslated region” (3’ UTR) refers to a region of an RNA (e.g., mRNA or other coding RNA) that is directly downstream i.e., 3’) from the stop codon (i.e., the codon of an RNA, such as mRNA or other coding RNA, transcript that signals a termination of translation) that does not encode a polypeptide.

[0490] An “open reading frame” is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)), and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA) and typically encodes a polypeptide (e.g., protein). It will be understood that the sequences may further comprise additional elements, e.g., 5’ and3’ UTRs, but that those elements, unlike the ORF, need not necessarily be present in a vaccine or a therapeutic of the present disclosure.

[0491] A “polyA tail” is a region of RNA (e.g., mRNA or other coding RNA) that is downstream, e.g., directly downstream (i.e., 3’), from the 3’ UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates (SEQ ID NO: 40). For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect RNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and / or export of the RNA from the nucleus and translation.

[0492] The term “dose” as used herein in reference to an immunogenic composition refers to a measured portion of the immunogenic composition taken by (administered to or received by) a subject at any one time.

[0493] The term “immunization” refers to a process that increases a mammalian subject’s reaction to an antigen and therefore improves its ability to resist or overcome infection and / or resist disease.

[0494] The term “vaccination” as used herein refers to the introduction of a vaccine into a body of a subject, preferably, a mammalian subject such as a human.

[0495] The term “prophylactically effective dose” includes the related terms “effective dose” and “therapeutically effective dose,” and as used herein refers to a dose that prevents infection with the virus at a clinically acceptable level.

[0496] The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 32 carbon atoms (“C1-C32 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C1-C10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C1-C9 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C1-C7 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-C5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-C4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C1-C3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C1-C2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“Ci alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6 alkyl”). In some embodiments,an alkyl group has 1 to 30 carbon atoms (“C1-C30 alkyl”). In some embodiments, an alkyl group has 1 to 22 carbon atoms (“C1-C22 alkyl”). In some embodiments, an alkyl group has 5 to 10 carbon atoms (“C5-C10 alkyl”). In some embodiments, an alkyl group has 7 to 17 carbon atoms (“C7-C17 alkyl”). In some embodiments, an alkyl group has 10 to 32 carbon atoms (“C10-C32 alkyl”).

[0497] The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C20 alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-C10 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C2-C8 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C2-C6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C2-C5 alkenyl”).

[0498] The term “suitable protective agent” includes the related terms “cryoprotectant” and “protective agent”, and does not cause or enhance degradation of the RNA.EXAMPLESExample 1. Hemagglutination Inhibition (HAI) by multivalent vaccines

[0499] This Example describes the hemagglutination inhibition by a purified RNA molecule (e.g., mRNA) described herein of various influenza strains.

[0500] Multivalent vaccines, such as seasonal influenza vaccines, tend to include antigens with different immunogenicity profiles and different levels of immunodominance of the antigen epitopes (see, e.g., Liu et al., 2018, Sanchez-de Prada et al., 2022). Influenza A strains have been studied more and have historically better vaccine efficacy than the influenza B counterparts. Influenza B is less studied as it does not carry the risk of a pandemic that influenza A strains have. Despite this, influenza B is relevant to human health and carries an increased risk to the very young and the very old (see, e.g., Cardenas -Garcia et al., 2020). Influenza type B viruses have two lineages the Victoria and Yamagata. Trivalent vaccines have a long history of mismatching the influenza B component with the actively circulating strain. While known quadrivalent vaccine designs allow for incorporation of both B lineages and have a lower mismatch rate, immune responses generated to influenza B vaccine components are often lower than these to influenza A encoded antigens, thus reducing the vaccines efficacy for protection against the B strains (see, e.g., Rowe et al., 2024).

[0501] While commercially approved vaccines such as Fluzone and Fluad meet protection thresholds for both A and B influenza strains, the recorded antibody titers and seroconversion rates are consistently lower for both B lineage than for the A subtype strains. This problem appears to be amplified in mRNA vaccines with multiple vaccine candidates failing noninferiority criteria for the B influenza strains (see, .e.g., Modema, 2023; Sanofi, 2023). The quadrivalent influenza mRNA vaccine candidates disclosed herein provide proof of concept for an antigen design approach that overcomes low antigenicity of B influenza strain-encoded hemagglutinin (HA) antigens and permits optimization of the immune responses elicited by multivalent vaccines through the novel, quaternary structure-based multivalent antigen design. The HA antigen multimers designed by this approach proved to represent potent antigens with balanced immunogenicity of influenza A and B HA antigens. This approach may be broadly applied to antigen design for different vaccines, especially the multivalent vaccines against viral pathogens.

[0502] Test Article Preparation for Administration: Before administration, mRNA components and LNP dispersion of a vaccine were equilibrated to room temperature for about 15 minutes, but not exceeding 1 hour. Immediately before use, the LNP dispersion wasgently shaken for 5 to 10 seconds, and 1.0 mL of LNP dispersion was extracted by a needled- syringe combination and added to the lyophilized mRNA. After addition, the vial was inverted repeatedly for approximately 30 seconds to mix. The suspension was incubated at room temperature for 10 minutes and then administered.

[0503] Female mice of specific pathogen free (SPF) grade were randomly assigned into groups (6 mice per group) and immunized with the test articles by intramuscular (i.m.) administration on Day 0 and Day 21. Blood samples were collected on Day -1 for pre- immune serum collection, and on Day 14 (post-prime immunization serum collection), Day- 35 (post-boost immunization serum collection) and Day-42 (terminal bleed, back-up serum sample). The mice were checked daily for appearance, clinical signs, behavioral activities, animal posture, diet, fur, irritative reaction, respiratory status, and deaths. The mice were also weighed regularly.

[0504] The vehicle control group received blank LNP dispersion via i.m. administration. Mice in positive control groups were immunized with of 50 pL doses of quadrivalent influenza vaccines: Fluad or Fluzone. Fluad quadrivalent vaccine is an inactivated virus eggbased influenza vaccine prepared by combining four virus antigens with the MF59 adjuvant. Each 0.5 mL dose is formulated to contain 60 pg of total HA or 15 pg of HA from each of the following four influenza strains recommended for the 2023-24 season: an A / Victoria / 4897 / 2022 (HlNl)pdmO9-like virus, an A / Darwin / 9 / 2021 (H3N2)-like virus, a B / Austria / 1359417 / 2021-like virus, and B / Phuket / 3073 / 2013 -like virus. Fluzone quadrivalent vaccine is an inactivated virus egg-based influenza vaccine prepared by combining four virus antigens. Each 0.5 mL dose is formulated to contain 60 pg of total HA or 15 pg of HA from each of the four influenza strains recommended for the 2023-24 season listed above. The 50 pL doses of the commercial vaccines administered to mice were equivalent to 1 / 1 Oth of the human dose. Similarly, the doses of the mRNA molecules disclosed herein were equivalent to approximately 1 / 1 Oth of the expected human dose of the mRNA vaccine (50 -100 pg mRNA), which permitted direct comparison of in vivo efficacy of the mRNA vaccines against Fluad and Fluzone.

[0505] Mice in the mRNA QIV group received a quadrivalent mRNA influenza vaccine with an mRNA molecule encoding influenza A (H3N2) hemagglutinin (HA) antigen, an mRNA molecule encoding influenza A (H1N1) HA antigen, an mRNA molecule encoding influenza B (B / Victoria lineage) HA antigen, and an mRNA encoding influenza B (B / Yamagata lineage) HA antigen, as detailed in Fig. 3A.

[0506] Mice in the other test groups received a combination of mRNA molecules, each encoding two antigens from different influenza virus variants, as detailed in Fig. 3B. An exemplary single mRNA molecule encoding two antigens from different influenza virus variants is shown in Fig. 1A.Serum Preparation

[0507] Blood samples (-100 pL per mice) were collected into Eppendorf tubes, allowed to clot prior to centrifugation, and maintained on ice. After centrifugation at 2,500 rpm for 10 minutes at 4 °C, the supernatants were immediately transferred to new tubes and stored at temperature below -20 °C.Measurement of Hemagglutination Inhibition (HAI) Titers

[0508] The HAI procedure was derived from the WHO Manual for the laboratory diagnosis and virological surveillance of influenza. One volume of the aliquoted sera was incubated with 3 volumes of the Receptor Destroying Enzyme II (RDE) reconstituted in 0.85% NaCl overnight (18 hrs) at 37 °C, and heat inactivated the next day by incubating for 60 minutes at 56 °C. RDE treated sera were stored at 4 °C. Red blood cells (RBCs) were washed and brought to a final working concentration of 0.5%.

[0509] Prior to the HAI assay, HAU testing (titration and back titration) was performed for each influenza virus used, with the serially (two-fold) diluted virus and 50 pL of RBCs in 96 well plate format. After mixing and incubating for 60 minutes at room temperature, plates were tilted about 90 degrees. The agglutination and titer results were recorded and confirmed in a back titration assay.

[0510] For the HAI tests, 1 :20 diluted RDE treated sera were serially (two-fold) diluted using 96 well plates and mixed with the 8 HAU / 50 pl of virus and 50 pL of RBCs. After 60 minutes of incubation, plates were tilted, and results were recorded.Strain Specific Mouse Serum HAI Titers

[0511] To evaluate the levels of HAI titers induced by the quadrivalent vaccines tested, mouse sera collected on Day 14 and Day 35 (the peak of the HA specific IgG responses) were RDE treated and incubated with the respective viral antigens and RBCs to determine the level of hemagglutination inhibition (HAI). HAI titers were reported as Log2 values. The results are summarized in Table 1A, Table IB, Table 1C, Table ID, and Table 2 below.

[0512]

[0513] Table 1A. Hemagglutination inhibition (HAI) titer against influenza A (H1N1): comparison of in vivo immunogenicity of commercial vaccines, Fluzone and Fluad, versus RNA molecules disclosed herein of flu mRNA vaccines.mRNA QIV - quadrivalent mRNA influenza vaccine;Mean values among more than two groups were determined using ANOVA, P < 0.05 indicate statistically significant differences (highlighted in bold).(1)A / Victoria / 2570 / 2019 (H1N1) is the HA matched strain used for HAI assay for immunization experiments with HA antigen derived from A / Wisconsin / 588 / 2019 (H1N1) pdm09-like 2022-23 seasonal vaccine strain.(2)A / Georgia / 12 / 2022 (H1N1) is the HA matched strain used for HAI assay for immunization experiments with HA antigen derived from A / Wisconsin / 67 / 2022 (H1N1) pdm09-like 20232- 24 seasonal vaccine strain.

[0514] Table IB Hemagglutination inhibition (HAI) titer against influenza A (H3N2): comparison of in vivo immunogenicity of commercial vaccines, Fluzone and Fluad, versus RNA molecules disclosed herein of flu mRNA vaccines.QIV - quadrivalent influenza vaccine;Mean values among more than two groups were determined using ANOVA, P < 0.05 indicate statistically significant differences (highlighted in bold).

[0515] Table 1C Hemagglutination inhibition (HAI) titer against influenza B (Victoria lineage): comparison of in vivo immunogenicity of commercial vaccines, Fluzone and Fluad, versus RNA molecules disclosed herein of flu mRNA vaccines.mRNA QIV - quadrivalent influenza vaccine;Mean values among more than two groups were determined using ANOVA, P < 0.05 indicate statistically significant differences (highlighted in bold).

[0516] Table ID Hemagglutination inhibition (HAI) titer against influenza B (Yamagata lineage): comparison of in vivo immunogenicity of commercial vaccines, Fluzone and Fluad, versus RNA molecules disclosed herein of flu mRNA vaccines.mRNA QIV - quadrivalent influenza vaccine;Mean values among more than two groups were determined using ANOVA, P < 0.05 indicate statistically significant differences (highlighted in bold).

[0517] Table 2 Summary of hemagglutination inhibition (HAI) titers against seasonal influenza strains: comparison of in vivo immunogenicity of Fluad or Fluzone to mRNA QIV and combination of mRNA molecules disclosed herein, such as C21+C23.A / Wis - A / Wisconsin / 67 / 2022 (H1N1) pdm09-like virus,A / Dar - A / Darwin / 6 / 2021 (H3N2)-like virus,B / Aus - B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus, B / Phu - B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus.Conclusions

[0518] The HAI analysis indicated that the vaccine candidate designs C26+C27 (secreted) and C21+C23 (membrane) consistently showed high immunogenic responses. Notably, the upstream position within the dumbbell structures included HA ectodomains derived from B influenza strains, while the downstream position of dumbbell included HA ectodomain derived from A influenza strains. These vaccine candidates are likely forming primary upstream HAI dumbbell trimers stabilized by T4 foldon domains as well as forming higher order multimers assembled via cross-trimerization of the membrane bound downstream influenza HA antigen domains with the same domains of the neighboring membrane bound molecules (Figure 1C). In this design, the RNA molecules with a B influenza antigen as the first antigen and an A influenza antigen as the second antigen outperformed the C8 + C3 design, indicating that the antigen design can be applied to improve immunogenicity of selected antigens, particularly in the context of multivalent vaccine development. This was a surprising result as it is common to design quadrivalent vaccines similar to the design of C8 + C3, where the antigens of influenza A and influenza B are on separate mRNA molecules, which allows for the ability to control the ratio of the two mRNA molecules. Specifically, such as design would permit to increase the relative content of B-B dumbbell encoding mRNA versus A-A dumbbell encoding mRNA in the quadrivalent vaccine and to overcome the underperformance of B influenza HA antigens by gene dosage adjustment.

[0519] Immunogenicity of A / Wis and A / Dar HA antigens was comparable across the mRNA QIV and the combination of mRNA molecules disclosed herein (no statistically significant differences of HAI titers are noted, see Table 1A and Table IB).

[0520] Immunogenicity of B / Aus and B / Phu HA antigens in the candidates C26+C27 (secreted) and C21+C23 (membrane) was statistically higher than in the mRNA QIV vaccine, see Table 1C and Table ID. Of note, the same B influenza strain derived antigens expressed from a candidate B-B dumbbell construct C3 are less immunogenic than the mRNA QIV vaccine: the effect is statistically significant for the downstream dumbbell ectodomain, B / Aus (Table 1C, groups C8+C3 and Cl 1 and C3), while the ~ 2-fold lower geometric mean HAI titers for the C3 upstream dumbbell, B / Phu, vs. the mRNA QIV vaccine did not reach the statistical significance. Additional statistically significant differences (C18+C20 group vs. the 1stGen mRNA vaccine in Table 1C, and C12+C13 group vs. the 1stGen mRNA vaccine in Table ID) indicate that in the context of certain antigen combinations, different structural features such as the linker lengths or the upstream vs. downstream position of the immune dominant or subdominant antigen may differentially contribute to the overall immunogenicity of the complex multivalent antigens.

[0521] The lead vaccine candidate C21+C23 that incorporates all features of the complex multimeric antigen design described in this disclosure, namely the presence of the multimerization domains within a single monomeric antigen dumbbell that permit the primary trimerization of the dumbbell monomers and the secondary trimerization of these primary dumbbell trimers with the adjacent primary dumbbell trimers that can lead to the quaternary structured complex antigen formation, has a balanced immunogenicity profile for all four HA antigens, with B influenza mean HAI titers comparable to Fluad (an adjuvanted commercial vaccine), exceeding over 8-fold HAI titers elicited by Fluzone and exceeding over 4-fold HAI titers elicited by the mRNA QIV vaccine (Table 2), while keeping A influenza mean HAI titers at comparable levels with Fluad and 1stGen mRNA vaccine. Therefore, these key features contributed to the overall most improved immunogenicity of the multivalent influenza mRNA vaccine.Example 2 - Trivalent Influenza mRNA vaccines

[0522] While a bivalent HA dumbbell design is compatible with a quadrivalent format as described in Example 1, using it for a vaccine that includes three influenza strains was less straightforward. To this end, trivalent mRNA vaccines of two formats were tested: (1) BVAHS + AH3 AHI mRNA vaccine with the AH3 antigen encoded by both mRNA species and (2) BVAHSAHI mRNA vaccine in which a single mRNA species encodes all three HA antigens (trivalent HA dumbbell design, Figure 4A). The sequences of the tested constructs are provided in Table 3B.

[0523] In the first format, the AH3 antigen was selected to be expressed from each mRNA species to increase its expression level and immunogenicity. The immunogenicity profiles of the quadrivalent vaccines shown in Tables 1A-1D indicate that, among the extended Gly-rich linker variants of BVAHS + BYAHI mRNA vaccines, the lowest HAI GMTs were induced by AH3, while the same, strain matched AH3 antigen included in the Fluad vaccine elicited the highest HAI GMT observed in this study. Additionally, the data in Tables 1A-1D showed that AH3 antigen was immunogenic in both BVAH3 and AH3 AHI configurations. Both BVAH3 and AH3 AHI were expressed as membrane proteins based on marginally higher HAI titers induced by the membrane, extended linker BYAHI + BVAH3 mRNA vaccine versus its secreted counterpart (Tables 1A-1D). The trivalent BVAHSAHI antigen expressed as a secreted protein was tested.

[0524] The trivalent influenza mRNA vaccines were evaluated against the 2024 / 25 trivalent Fluzone HD vaccine since the 2024 / 25 Fluad vaccine used previously was not commercially distributed at the time the in vivo experiments were performed (Figure 4B-4D). HAI GMTs for all tested mRNA vaccines were higher than for Fluzone HD across all three seasonal strains (B / Austria / 1359417 / 2021, A / Califomia / 123 / 2022 (H3N2), and A / Georgia / 12 / 2002 (H1N1)). For vaccines expressing the trivalent HA dumbbell BVAHSAHI, the differences were statistically significant across all seasonal strains. For the bivalent BVAHS+AHSAHI vaccine groups, the HAI GMTs against B / Austria / 1359417 / 2021 were comparable with Fluzone HD (Figures 4B-4D). Due to the robust performance and reduced manufacturing complexity, the vaccine composed of the mRNA encoding the trivalent BVAH3 AHI HA antigen was selected for further studies.

[0525] Table 3 A. Descriptions of the tested constructs

[0526] Table 3B. Sequences of tested constructsIllAntigen sequences (italicized font) are separated by linker sequences (underlined) flanking the T4 foldon sequence (bold and underlined), signal peptides sequences are bold.Example 3 -Multivalent influenza / covid mRNA vaccines

[0527] A COVID-19 mRNA vaccine was tested in combination with a vaccine including dumbbell antigens from seasonal influenza. In the SARS-CoV-2 dumbbell two identical receptor binding domains (RBDs) of the S-protein, derived from the Omicron XBB.1.5 variant, were connected by a T4 foldon domain flanked with Gly-rich linkers (COVXBBCOVXBB; SEQ ID NO: 47 or 48). The details of the protein and mRNA design of these constructs can be seen in Table 4A and Figure 8.

[0528] SARS-CoV-2 pseudovirus neutralization assay: COVID-19 neutralizing antibody titers were measured in mouse sera using 293T-ACE2 cells and SARS-CoV-2 Omicron XBB.1.5 pseudoviral particles. 293T-ACE2 cells express hACE2 receptors and are permissive to infection by the S-protein pseudotyped viral particles. XBB.1.5 pseudotyped viral particles contain the luciferase reporter gene, permitting pseudovirus viral entry measurements via the luciferase activity assay. Pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 hr before adding it to 3e4 reporter cells plated in 96-well plate format. Equal, 50 pL volumes of the cell suspension and pseudovirus / sera mixes were added into each well. The plates were centrifuged at 700 rpm for 15 min at 4°C. The cells were cultured with pseudovirus and diluted sera for additional 48 hrs. The 96-well plates were equilibrated to room temperature for 30 min, 100 pL supernatant was removed fromeach well and 100 pL / well bio-Lite luciferase detection reagent was added. The plates were shaken for 3 min. and the luminescence was immediately measured by a microplate reader.

[0529] CO VID-19 IgG titer assay. Mouse sera were analyzed for RBD-specific antibody responses by ELISA. Briefly, 96-well ELISA plates were precoated overnight with 1 pg / mL of SARS-CoV-2 XBB.1.5 (Omicron) spike protein RBD in carbonate buffer pH 9.6 at 4 °C and blocked with an assay buffer for 1 hr at 37 °C. Plates were washed three times with PBST. Serially diluted mouse sera were added to the plates and incubated at 37°C for 2 hrs, followed by three washes. Bound antibodies were incubated with HRP-conjugated goat antimouse IgG (1 : 15000), anti -mouse IgGl (1 : 12000) or anti-mouse IgG2a (1 :5000) for 1 hr at 37°C. The enzymatic reaction was performed with ELISA TMB stabilized chromogen and stopped by ELISA stop solution. The absorbance at 450 nm was measured by using a plate reader.

[0530] To perform a direct comparison of the influenza / COVID-19 combination vaccine with Spikevax, a commercial LNP mRNA vaccine, BVAHSAHI (SEQ ID NO: 45 or 46) and COVXBBCOVXBB mRNAs were premixed at 1 : 1 weight ratio and formulated as a traditional encapsulated LNP mRNA vaccine. The combination vaccine was tested at low and high doses (3 pg and 12 pg total mRNA per injection, respectively) corresponding to 1 / 10thof the estimated human vaccine dose in the 30 and 120 pg range.

[0531] The encapsulated LNP mRNA vaccines, expressing BVAHSAHI HA antigen alone or in combination with the COVXBBCOVXBB dumbbell, elicited strong and balanced antibody responses against all three influenza HA antigens (Figure 5A-5C). In line with the previous experiment (RTU LNP formulated, Figure 4), all encapsulated LNP mRNA vaccines that included the 6 pg dose of BVAHSAHI mRNA performed better than Fluzone HD ( 1 / 10thof the human dose). The HAI GMT differences between the Fluzone HD vaccine group and both the influenza only and the combination vaccine groups containing a 6 pg dose of BVAHSAHI mRNA were statistically significant across all three seasonal influenza strains(B / Austria / 1359417 / 2021, A / Califomia / 123 / 2022 (H3N2), and A / Georgi a / 12 / 2002 (H1N1). Further, the low dose combination mRNA vaccine group (containing 1.5 pg of BVAHSAHI mRNA) also performed better than FluzoneHD across the three seasonal influenza strains. In this case the HAI titer differences reached statistical significance for B / Austria / 1359417 / 2021 but not for A / Georgia / 12 / 2002 (H1N1) or A / Califomia / 123 / 2022 (H3N2).

[0532] To assess COVID-19 immunogenicity responses, neutralizing antibody titers were measured for individual mouse sera using a SARS-CoV-2 pseudovirus neutralization assay performed on HEK-293 ACE2 cells (Figure 5D). Total IgG titers against recombinantXBB.1.5 S-protein RDB were determined for the same serum samples by a capture ELISA assay (Figure 5E). Additionally, the capture ELISA assay, utilizing IgG subclass specific detection antibodies, was used to test IgGl and IgG2a titers in pooled mouse serum samples (Figure 5F).

[0533] Significantly higher neutralizing antibody titers were induced in mice immunized with the high dose combination mRNA vaccine (containing 6 pg of COVXBBCOVXBB mRNA per dose) than with Spikevax (5 pg mRNA per dose, Figure 5D). The titers elicited by the low dose vaccine (1.5 pg of COVXBBCOVXBB mRNA per dose) remained higher than the titers induced by Spikevax, however these differences were not statistically significant. An analogous pattern was observed for total IgG titers (Figure 5E), where both the high and low dose groups containing the COVXBBCOVXBB mRNA performed better than Spikevax, but the differences were not statistically significant.

[0534] For all tested mRNA vaccines, including Spikevax, the titer ratio of IgG2a to IgGl was consistent, indicating a balanced Thl and Th2 response profile following the vaccination with Spikevax, COVXBBCOVXBB COVID-19 only vaccine, or BVAHSAHI+COVXBBCOVXBB combination vaccine (Figure 5F).

[0535] In mouse in vivo preclinical studies, this vaccine (combination vaccine comprising two mRNAs, one encoding a SARS-CoV-2 S-protein RBD dumbbell and another encoding a trivalent influenza HA dumbbell) performed better than both a commercial COVID-19 mRNA vaccine and a commercial inactivated influenza virus comparator vaccine.

[0536] Table 4 A. Descriptions of the tested constructs

[0537] Table 4B. Sequences of COV constructAntigen sequences (italicized font) are separated by linker sequences (underlines) flanking the T4 foldon sequence (bold and underlined), signal peptides sequences are bold.Example 4 - Single mRNA Species Encoding Quadrivalent Influenza / COVID-19Combination Vaccine

[0538] Both bivalent and trivalent influenza HA dumbbell antigen formats showed strong immunogenicity. At the same time there was little to no effect of COVXBBCOVXBB antigenco-expression on HAI GMTs elicited by the dumbbell HAs. Therefore, we sought to test two quadrivalent vaccine designs in which a single mRNA species coded for three influenza antigens and a SARS-CoV-2 antigen. Antigen details can be seen in Table 5A and in Figure 8. Briefly the COVXBB antigen representing a single RBD fragment was placed in the 1st(upstream) or 4th(downstream) position in addition to the three HA antigens organized as a BVAHSAHI fusion protein (quadrivalent chimeric dumbbell design, Figure 6A). Since the T4 foldon / extended Gly-rich linker fragment was added to the designs to maintain the same type of fusion protein scaffold, the quadrivalent mRNA vaccines included three T4 foldon domains within each chimeric antigen.

[0539] The experimental quadrivalent mRNA vaccines were evaluated against the trivalent BVAHSAHI influenza vaccine shown in Figures 5D-5F and 6B-6D. The position of COVXBB antigen within the quadrivalent dumbbell affected HAI GMTs of the included HA antigens. When COVXBB antigen was in the 1stposition, upstream of the three HA antigens, the HAI titers induced by the downstream HA antigens Bv and AH3 were significantly lower these seen in response to the trivalent BVAHSAHI dumbbell. However, when COVXBB antigen was in the 4thposition, downstream of the three HA antigens, the B / Austria / 1359417 / 2021 and A / Califomia / 123 / 2022 (H3N2) titers were comparable to titers in the trivalent BVAHSAHI dumbbell group. Interestingly, the A / Georgia / 12 / 20222 (H1N1) titers were comparable across all three vaccine groups (Figures 6B-6D).

[0540] To assess COVID-19 responses, total IgG titers were determined for pooled mouse sera (Figure 6E).

[0541] Table 5A. Descriptions of the tested constructs

[0542] Table 5B. Sequences of tetravalent constructsAntigen sequences (italicized font) are separated by linker sequences (underlines) flanking the T4 foldon sequence (bold and underlined), signal peptides sequences are bold.Example 5 - Trivalent mRNA vaccine

[0543] A trivalent mRNA vaccine is made similar to Example 2. Briefly, the trivalent vaccine comprises a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant, a second nucleotide sequence encoding a first T4foldon domain, a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant, a fourth nucleotide sequence encoding a second T4 foldon domain, and a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant. The first virus variant, the second virus variant, and the third virus variant are variants of viruses from the Paramyxoviridae (e.g., RSV, hMPV, Nipah virus, measles virus), Retroviridae (e.g., HIV), Filoviridae (e.g., Ebola virus), Orthomyxoviridae (e.g., influenza), and Coronaviridae (SARS-CoV2) families.

[0544] The trivalent mRNA that includes the first antigen or antigenic fragment thereof, from a first virus variant, the second antigen, or an antigenic fragment thereof, from a second virus variant, and the third antigen, or an antigenic fragment thereof, from a third virus variant include one or more trimerizing Type I viral fusion proteins.Example 6 - Quadrivalent mRNA vaccine

[0545] A quadrivalent mRNA vaccine is made similar to Example 4. Briefly, the quadrivalent vaccine comprises a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant, a second nucleotide sequence encoding a first T4 foldon domain, a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant, a fourth nucleotide sequence encoding a second T4 foldon domain, a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant, a sixth nucleotide encoding a third T4 foldon domain. The first virus variant, the second virus variant, the third virus variant, and the fourth virus variant are variants of viruses from the Paramyxoviridae (e.g., RSV, hMPV, Nipah virus, measles virus), Retroviridae (e.g., HIV), Filoviridae (e.g., Ebola virus), Orthomyxoviridae (e.g., influenza), and Coronaviridae (SARS-CoV2) families.

[0546] The quadrivalent mRNA that includes the first antigen or antigenic fragment thereof, from a first virus variant, the second antigen, or an antigenic fragment thereof, from a second virus variant, and the third antigen, or an antigenic fragment thereof, from a third virus variant include one or more trimerizing Type I viral fusion proteinsReferences:Cardenas-Garcia S, Caceres CJ, Rajao D, Perez DR. Reverse genetics for influenza B viruses and recent advances in vaccine development. Curr Opin Virol. 2020 Oct;44: 191-202. doi: 10.1016 / j.coviro.2020.10.005. Epub 2020 Nov 27. PMID: 33254031; PMCID: PMC8693393.Liu STH, Behzadi MA, Sun W, Freyn AW, Liu WC, Broecker F, Albrecht RA, Bouvier NM, Simon V, Nachbagauer R, Krammer F, Palese P. Antigenic sites in influenza Hl hemagglutinin display species-specific immunodominance. J Clin Invest. 2018 Nov1 ; 128(11):4992-4996. doi: 10.1172 / JCI122895. Epub 2018 Oct 8. PMID: 30188868; PMCID: PMC6205383.Modema, February 16, 2023, Modema Announces Interim Phase 3 Safety and Immunogenicity Results for mRNA-1010, a Seasonal Influenza Vaccine Candidate, https: / / investors.modernatx.com / news / news-details / 2023 / Moderna-Announces-Interim- Phase-3 -Safety-and-Immunogenicity-Results-for-mRNA- 1010-a-Seasonal-Influenza- V accine-Candidate / default, aspx.Rowe T, Davis W, Wentworth DE, Ross T. Differential interferon responses to influenza A and B viruses in primary ferret respiratory epithelial cells. J Virol. 2024 Feb 20;98(2):e0149423. doi: 10.1128 / jvi.01494-23. Epub 2024 Jan 31. PMID: 38294251;PMCID: PMC10878268.Sanchez-de Prada L, Sanz-Munoz I, de Lejarazu RO, Eiros JM, Garcia-Sastre A, Aydillo T. Immunodominance hierarchy after seasonal influenza vaccination. Emerg Microbes Infect. 2022 Dec; l l(l):2670-2679. doi: 10.1080 / 22221751.2022.2135460. PMID: 36219456;PMCID: PMC9639523.Sanofi, June 29, 2023, https: / / www.reuters.com / business / healthcare-pharmaceuticals / sanofi- says-its-back-drawing-board-mma-flu-vaccines-2023-06-29INCORPORATION BY REFERENCE

[0547] The entire disclosure of each of the patent documents and scientific articles cited herein are incorporated by reference for all purposes.EQUIVALENTS

[0548] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWe claim1. A purified ribonucleic acid (RNA) molecule comprising: a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; wherein the first virus variant or the second virus variant is an influenza B variant; and wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to each other in a 5’ -to -3’ direction.

2. The purified RNA of claim 1, wherein the first virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

3. The purified RNA of claim 1, wherein the first virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

4. The purified RNA of claim 3, wherein the first influenza virus variant is an influenza B virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza strains.

5. The purified RNA of any one of claims 3 or 4, wherein the first virus antigen, or an antigenic fragment thereof, is a hemagglutinin (HA) protein from a Victoria lineage and Yamagata lineage influenza strains.

6. The purified RNA of claim 1, wherein the second virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

7. The purified RNA of claim 1, wherein the second virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

8. The purified RNA of claim 7, wherein the second influenza virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

9. The purified RNA of claim 8, wherein the second influenza antigen is an HA protein from an influenza A virus.

10. The purified RNA of claim 7 or 8, wherein the first HA protein is an HA protein from an influenza B virus and the second HA protein is an HA protein from an influenza A virus.

11. The purified RNA of claim 10, wherein: a. the first HA protein comprises an HA protein from an Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus; b. the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H3N2) strain virus; c. the first HA protein comprises an HA protein from a Yamagata lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus; or d. the first HA protein comprises an HA protein from a Victoria lineage virus and the second HA protein comprises an HA protein from an influenza A (H1N1) strain virus.

12. The purified RNA of claim 10, wherein the first HA protein is encoded by any one of SEQ ID NOs: 23 or 24 and the second HA protein is encoded by any one of SEQ ID NOs: 25-28.

13. The purified RNA of claim 10, wherein the first HA protein comprises an amino acid sequence selected from SEQ ID NOs: 60-61 and the second HA protein comprises an amino acid sequence selected from SEQ ID NOs: 62-65.

14. The purified RNA of any one of claims 1-10, further comprising a third nucleotide sequence encoding a first multimerization domain, wherein the first multimerization domain is selected from the group consisting of a dimerization domain, trimerization domain, a tetramerization domain, and wherein the first multimerization domain is after the first nucleotide sequence (3’ to the firstnucleotide sequence) and before the second nucleotide sequence (5’ to the first nucleotide sequence).

15. The purified RNA of claim 14, wherein the first multimerization domain is selected from the group consisting of enterobacteria phage T4, GCN4pII, GCN4- pLI, and p53.

16. The purified RNA of claim 14 or 15, wherein the first multimerization domain comprises a leucine zipper or a fibritin foldon domain.

17. The purified RNA of any one of claims 14-16, wherein the first multimerization domain comprises a trimerization domain.

18. The purified RNA of claim 17, wherein the trimerization domain is the fibritin foldon domain from enterobacteria phage T4, the T4 foldon domain.

19. The purified RNA of claim 18, wherein the T4 foldon domain is encoded by a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 19.

20. The purified RNA of claim 18, wherein the T4 foldon domain comprises the amino acid sequence SEQ ID NO: 58.

21. The purified RNA molecule of any one claims 1-20, further comprising a sequence encoding a first linker connecting the first antigen, or an antigenic fragment thereof to the first multimerization domain.

22. The purified RNA molecule of any one of claims 1-21, further comprising a sequence encoding a second linker connecting the first multimerization domain to the second antigen, or an antigenic fragment thereof.

23. The purified RNA molecule of claim 21 or 22, wherein the first linker and the second linker each encode an amino acid sequence comprising at least 5 to about 50 amino acids.

24. The purified RNA molecule of claim 21 or 22, wherein the first linker and / or the second linker encodes an amino acid sequence selected from the group consisting of (GS)n (SEQ ID NO: 29), (G2S)n (SEQ ID NO: 30), (G3S)n (SEQ ID NO: 31), (G4S)n (SEQ ID NO: 32), and (G)n (SEQ ID NO: 33), and wherein n is an integer from 2 to 20.

25. The purified RNA molecule of claim 21 or 22, wherein the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 34) or (GGSGGE)n (SEQ ID NO: 35), and wherein n is an integer from 2 to 6.

26. The purified RNA molecule of claim 21 or 22, wherein the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 36) and (GGGGGPGGGGP)n (SEQ ID NO: 37), and wherein n is an integer from 1 to 3.

27. The purified RNA molecule of claim 21 or 22, wherein the first linker and / or the second linker encodes the amino acid sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n, and (GzX)n, wherein z is between 1 and 20, and wherein n is at least 8, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline.

28. The purified RNA of claim 21 or 22, wherein the first linker and / or the second linker encodes the amino acid sequence GGSG (SEQ ID NO: 38) or GGSLGGGGSGS (SEQ ID NO: 39).

29. The purified RNA of claim 21 or 22, wherein the first linker and / or the second linker is encoded by the nucleotide sequence of any one of SEQ ID NO: 20-22.

30. The purified RNA of claim 22, wherein the first linker, first multimerization domain, and second linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

31. The purified RNA of any one of claims 1-30, wherein the purified RNA molecule comprises about 90% identity to any one of SEQ ID NOs: 1-15.

32. The purified RNA of any one of claims 1-31, wherein the purified RNA molecule comprises any one of SEQ ID NOs: 1-15.

33. The purified RNA molecule of any one of claims 14- 32, further comprising a fourth sequence encoding a second multimerization domain after the second antigen, or an antigenic fragment thereof (3’ to the second antigen, or an antigenic fragment thereof).

34. The purified RNA molecule of claim 33, wherein the second multimerization domain is a T4 foldon domain.

35. The purified RNA molecule of claim 34, wherein the second multimerization domain comprises an amino acid sequence of any one of SEQ ID NOs: 66-68.

36. The purified RNA molecule of any one of claims 33-35, further comprising a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.

37. The purified RNA molecule of claim 36, wherein the third virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63(alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS- CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19.

38. The purified RNA molecule of claim 36, wherein the third virus variant is an influenza A or influenza B virus selected from the group consisting of HINT, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

39. The purified RNA molecule of claim 38, wherein the third virus variant is an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, and H10N7.

40. The purified RNA molecule of claim 39, wherein the third antigen is an HA protein from an influenza A virus.

41. The purified RNA molecule of claim 35, further comprising a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, from a fourth virus variant.

42. The purified RNA molecule of claim 41, wherein the fourth virus variant is a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS- CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

43. The purified RNA molecule of claim 41, wherein the fourth virus variant is an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

44. The purified RNA molecule of claim 41, wherein the fourth virus variant is a coronavirus from a SARS-CoV-2 virus.

45. The purified RNA molecule of claim 44, wherein the fourth antigen is a receptorbinding domain (RBD) protein.

46. The purified RNA molecule of claim 45, wherein the RBD protein comprises the amino acid sequence of SEQ ID NO: 53.

47. The purified RNA molecule of claim 41, further comprising a seventh sequence encoding a third multimerization domain 3’ to the third antigen, or an antigenic fragment thereof and 5’ to the fourth antigen, or antigen fragment thereof.

48. The purified RNA molecule of claim 47, wherein the third multimerization domain is a T4 foldon domain.

49. The purified RNA of claim 48, wherein the T4 foldon domain comprises a sequence of SEQ ID NO: 58.

50. The purified RNA of claim 48 or 49, wherein the third multimerization domain comprises a sequence of any one of SEQ ID NOs: 66-68.

51. The purified RNA of any one of claims 1-48, wherein the purified RNA further comprises a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1) 5’ to the first nucleotide sequence.

52. The purified RNA of claim 51, wherein the purified RNA further comprises a 5’ UTR (SEQ ID NO: 16) 3’ to the Cap 1 and 5’ to the first nucleotide sequence.

53. The purified RNA of claim 52, wherein the purified RNA further comprises a sequence encoding a 3’ UTR (SEQ ID NO: 17) 3’ to the sixth nucleotide sequence.

54. A purified RNA molecule comprising a sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 1-15.

55. A purified RNA molecule that encodes a sequence comprising at least 90% identity to any one of SEQ ID NO: 45-52.

56. A purified RNA comprising, from 5’ to 3’ : a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, from a first virus variant; b) a second nucleotide sequence encoding a first T4 foldon domain; c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, from a second virus variant; d) a fourth nucleotide sequence encoding a second T4 foldon domain; and e) a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, from a third virus variant.

57. The purified RNA of claim 56, wherein the first antigen, or an antigenic fragment thereof, the second antigen, or an antigenic fragment thereof, and third antigen, or an antigenic fragment thereof are different antigens.

58. The purified RNA of claim 56 or 57, wherein the first antigen, second antigen, or third antigen are a virus variant of a virus selected from the group consisting of a Influenza virus, Paramyxoviridae (e.g., RSV, hMPV, Nipah virus, measles virus), Retroviridae (e.g., HIV), Filoviridae (e.g., Ebola virus), Orthomyxoviridae (e.g., influenza), and Coronaviridae (SARS-CoV2) families.

59. The purified RNA of claim 56 or 58, wherein:a) a first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); b) a second nucleotide sequence encoding a first T4 foldon domain; c) a third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19); d) a fourth nucleotide sequence encoding a second T4 foldon domain; and e) a fifth nucleotide sequence encoding a third antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19), wherein at least the first, second, or third antigen is an HA protein from influenza virus selected from the group consisting of Victoria lineage and Yamagata lineage influenza.

60. The purified RNA of claim 59, further comprising a sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof, of (i) a hemagglutinin (HA) protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains or (ii) an RBD of a coronavirus selected from the groupconsisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

61. The purified RNA of claim 60, further comprising a seventh nucleotide sequence encoding a third T4 foldon domain 3’ to the third antigen, or an antigenic fragment thereof and 5 ’to the sixth nucleotide sequence encoding a fourth antigen, or an antigenic fragment thereof.

62. The purified RNA of claim 61, further comprising an eighth nucleotide sequence encoding a first linker 3 ’ to the first nucleotide sequence encoding a first antigen, or an antigenic fragment thereof.

63. The purified RNA of claim 62, further comprising: a. a ninth nucleotide sequence encoding a second linker 3’ to the second nucleotide sequence encoding a first T4 foldon domain; b. a tenth nucleotide sequence encoding a third linker 3’ to the third nucleotide sequence encoding a second antigen, or an antigenic fragment thereof; c. an eleventh nucleotide sequence encoding a fourth linker 3’ to the second T4 foldon domain; d. a twelfth nucleotide sequence encoding a fifth linker 3’ to the third antigen, or an antigenic fragment thereof; e. a thirteenth nucleotide sequence encoding a sixth linker 3’ to the third T4 foldon domain.

64. The purified RNA of claim 63, wherein the first linker, first T4 foldon domain, and second linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

65. The purified RNA of claim 63, wherein the third linker, second T4 foldon domain, and fourth linker comprise an amino acid sequence of any one of SEQ ID NOs: 66-68.

66. The purified RNA of claim 63, wherein the fifth linker, second T4 foldon domain, and sixth linker comprise an amino acid sequence of any one of SEQ ID NOs: 66- 68.

67. The purified RNA of any one of claims 56 -66, further comprising a m7GpppNm-, where Nm denotes any nucleotide with a 2’ O methylation (Cap 1).

68. The purified RNA of any one of claims 56 -67, wherein the a first antigen, or an antigenic fragment thereof, is an HA protein from a Victoria lineage and Yamagata lineage influenza.

69. The purified RNA of any one of claims 56 -67, wherein the first antigen, or an antigenic fragment thereof, is an RBD of a SARS-CoV-2.

70. The purified RNA of claim 68 or 69, wherein the second antigen, or an antigenic fragment thereof, is an HA protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

71. The purified RNA of any one of claims 68-70, wherein the third antigen, or an antigenic fragment thereof, is an HA protein from an influenza A or influenza B virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

72. The purified RNA of any one of claims 68-71, wherein the fourth antigen, or an antigenic fragment thereof, is an HA protein from an influenza A virus selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7.

73. The purified RNA of any one of claims 68-71, wherein the fourth antigen, or an antigenic fragment thereof, is an RBD of a SARS-CoV-2.

74. An isolated polypeptide encoded by the purified RNA molecule of any of claims 1-73.

75. A composition comprising a first purified RNA molecule comprising the purified RNA molecule of any of claims 1-73 and a delivery vehicle.

76. The composition of claim 75, further comprising a second purified RNA molecule comprising the purified RNA molecule of any of claims 1-73.

77. The composition of claim 76, wherein the first purified RNA molecule comprises any one of SEQ ID NOs: 1-15 and the second purified RNA molecule comprises any one of SEQ ID NOs: 1-15, wherein the first purified RNA molecule and the second purified RNA molecule comprise different sequences selected from SEQ ID NOs: 1-15.

78. The composition of claim 76, wherein the first purified RNA molecule encodes a sequence comprising any one of SEQ ID NOs: 41-52, and the second purifiedRNA molecule encodes a sequence comprising any one of SEQ ID NOs: 41-52, wherein the first purified RNA molecule and the second purified RNA molecule encodes a sequence comprising different sequences selected from SEQ ID NOs: 41-52.

79. The composition of claim 75, further comprising a second purified RNA molecule encoding the amino acid sequence of SEQ ID NO: 47 or 48.

80. The composition of claim 76, wherein: a. the first RNA molecule comprises SEQ ID NO: 4 and the second RNA molecule comprises SEQ ID NO: 3; b. the first RNA molecule comprises SEQ ID NO: 5 and the second RNA molecule comprises SEQ ID NO: 3; c. the first RNA molecule comprises SEQ ID NO: 6 and the second RNA molecule comprises SEQ ID NO: 7; d. the first RNA molecule comprises SEQ ID NO: 8 and the second RNA molecule comprises SEQ ID NO: 9; e. the first RNA molecule comprises SEQ ID NO: 10 and the second RNA molecule comprises SEQ ID NO: 11; f. the first RNA molecule comprises SEQ ID NO: 12 and the second RNA molecule comprises SEQ ID NO: 13; or g. the first RNA molecule comprises SEQ ID NO: 14 and the second RNA molecule comprises SEQ ID NO: 15.

81. The composition of claim 76, wherein the first RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 47 or 48 and the second RNA molecule encodes an amino acid sequence at least 90% identical to SEQ ID NO: 45 or 46.

82. The composition of any one of claims 75-81, wherein the delivery vehicle comprises a lipid nanoparticle (LNP).

83. The composition of claim 82, wherein the LNP comprises an ionizable lipid.

84. The composition of claim 83, wherein the LNP comprises an ionizable lipid: cholesterol: DSPC: DMG-PEG2000 ratio of about 48: 40: 10: 2, and a LNP:RNA (N:P) ratio of about 8: 1.

85. The composition of any one of claims 75-84, wherein the composition comprises a particle size of no more than about 300 nanometers (nm) or no more than about 150 nm.

86. The composition of any one of claims 75-84, wherein the composition comprises a particle size of about 50 to about to 300 nm or about 50 nm to about 140 nm..

87. The composition of any one of claims 75-86, wherein the RNA is lyophilized.

88. The composition of claim 87, wherein the RNA is adsorbed to the surface of the LNP or is encapsulated by the LNP.

89. The composition of claim 87, wherein the RNA encapsulated by the LNP is lyophilized.

90. A kit comprising the purified RNA of any one of claims 1-73, a delivery vehicle, and instructions for use.

91. A method of treating or preventing disease in a subject comprising administering to the subject an effective amount of the composition of any of claims 75-89.

92. The method of claim 91, wherein the disease is caused by a virus selected from the group consisting of an influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus.

93. The method of claim 91, wherein the disease is caused by a coronavirus, an influenza A virus, or an influenza B virus.

94. The method of claim 93, wherein the coronavirus is selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19).

95. The method of claim 94, wherein the coronavirus is SARS-CoV-2.

96. The method of claim 93, wherein the influenza A virus or influenza B virus is selected from the group consisting of H1N1, H2N2, H1N2, H3N2, H5N1, H5N2, H5N8, H5N9, H7N2, H7N3, H7N7, H9N2, H10N3, H10N7, Victoria lineage, and Yamagata lineage influenza strains.

97. The method of claim 91, wherein the disease is caused by one or more viruses selected from the group consisting of an influenza virus, rabies virus, respiratory syncytial virus (RSV) and coronavirus.

98. The method of claim 97, wherein the one or more viruses includes a coronavirus, an influenza A virus, and / or an influenza B virus.

99. The method of any one of claims 91-98, wherein the composition is administered to the subject intramuscularly.

100. The method of claim 91-99, wherein the composition is administered to the subject at least two times.

101. The method of claim 100, wherein the composition is administered once every one, two, three, or four weeks.

102. A method for stimulating an immune response in a subject comprising administering to the subject an effective amount of the composition of any of claims 75-89.

103. A method for preparing a RNA-LNP composition comprising: a) mixing an ethanol phase comprising one or more lipids and an aqueous phase comprising the purified RNA molecule of any one of claims 1-73 and b) purifying the RNA-LNP generated from step a).

104. The method of claim 103, wherein the one or more lipids comprises an ionizable lipid, cholesterol, a phospholipid, and / or a pegylated lipid.

105. The method of any one of claims 103-104, wherein the N:P ratio is about 6.5 to about 9.

106. The method of any one of claims 103-105, wherein the aqueous phase consists of tris, sodium chloride, and sucrose.

107. A kit comprising the composition of any one of claims 75-89, a delivery vehicle, and instructions for use.