Influenza vaccines and uses thereof

Abstract

The present disclosure provides amino acid modifications to influenza hemagglutinin (HA) polypeptides for enhanced stability, increased expression yield, enhanced immunogenicity, and / or enhanced expression, or durability of expression, on the cell surface.

Description

INFLUENZA VACCINES AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 596,194 filed November 3, 2023, and U.S. Provisional Application No. 63 / 711,123 filed October 23, 2024, which is incorporated herein by reference in its entirety. This application is a continuation-in- part of International Patent Application No. PCT / US2024 / 025583, filed April 19, 2024, which claims priority to U.S. Provisional Application No. 63 / 497,404, filed April 20, 2023, and U.S. Provisional Application No. 63 / 597,625, filed November 9, 2023, each of which is incorporated by referenced in its entirety.BACKGROUND

[0002] Pathogenic agents, such as viruses, may evolve to evade detection and neutralization by a host’s immune system. Influenza hemagglutinin (HA) protein may be an important component in influenza vaccines. Influenza vaccines may require the formation of properly folded hemagglutinin (HA) trimers to retain critical conformational epitopes against which protective immune responses can be directed. Native HA protein trimers are Class 1 proteins which are inherently unstable. There exists a need for increased stability and yield of HA protein as the wild-type soluble structure of many strains may be low yield, unstable, and / or unable to reliably form trimeric complexes. Vaccines with sufficient surface expression levels and enhanced stability of HA trimers are needed to present HA as it exists in the pathogen prior to infection or as it egresses from the cell it has invaded for long enough for the immune system to recognize important protective HA epitopes.SUMMARY

[0003] The present disclosure provides an influenza A hemagglutinin (HA) polypeptide, comprising an HA1 domain and an HA2 domain, wherein the amino acid at HA2 position 26 is phenylalanine (F) or tyrosine (Y); the amino acid at HA2 position 51 is alanine (A), leucine (L), or valine (V); and / or the amino acid at HA2 position 103 is alanine (A) or leucine (L); and wherein the numbering of amino acid positions of the HA2 domain is as per SEQ ID NO: 3. In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F) or tyrosine (Y); the amino acid at HA2 position 51 is alanine (A), leucine (L), or valine (V); and the amino acid at HA2 position 103 is alanine (A) or leucine (L). In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F); the amino acid at HA2 position 51 is alanine (A), and the amino acid at HA2 position 103 is alanine (A). In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F); the amino acid at HA2 position 51 is leucine (L), and the amino acid atHA2 position 103 is leucine (L). In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81. In some embodiments, the influenza A HA polypeptide exhibits increased trimer stability as compared to an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the influenza A HA polypeptide assembles into a trimer. In some embodiments, more of the influenza A HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide exhibits an absolute increase of trimer formation of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when expressed on a cell surface than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide exhibits an absolute increase of trimer formation of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, the influenza A HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128- fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, the influenza A HA polypeptide comprises a transmembrane domain and exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20- fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression whenexpressed on a cell surface than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, the amino acid at HA2 position 26 is F. In some embodiments, the amino acid at HA2 position 26 is Y. In some embodiments, the amino acid at HA2 position 51 is A. In some embodiments, the amino acid at HA2 position 51 is L. In some embodiments, the amino acid at HA2 position 51 is V. In some embodiments, the amino acid at HA2 position 103 is A. In some embodiments, the amino acid at HA2 position 103 is L.

[0004] In another aspect, the present disclosure provides an influenza A hemagglutinin (HA) polypeptide comprising an HA1 domain and an HA2 domain, wherein the HA2 domain comprises an amino acid sequence that, relative to SEQ ID NO: 3, comprises one or more substitution mutations at one or more of HA2 position 26, HA2 position 51, and HA2 position 103, and the influenza A HA polypeptide exhibits increased trimer stability and / or cell surface expression when expressed on cell surface as compared to an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 26. In some embodiments, the amino acid at HA2 position 26 is phenylalanine or tyrosine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine. In some embodiments, the amino acid at HA2 position 26 is tyrosine. In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 51. In some embodiments, the amino acid at HA2 position 51 is alanine, leucine, or valine. In some embodiments, the amino acid at HA2 position 51 is alanine. In some embodiments, the amino acid at HA2 position 51 is leucine. In some embodiments, the amino acid at HA2 position 51 is valine. In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 103. In some embodiments, the amino acid at HA2 position 103 is alanine or leucine. In some embodiments, the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 103 is leucine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine and the amino acid HA2 position 51 is leucine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine. Insome embodiments, the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81. In some embodiments, more of the influenza A HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than the otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide exhibits an HAI titer in an hemagglutination inhibition assay that is at least 50, at least 100, or at least 200 units greater than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza A HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20- fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, the influenza A HA polypeptide further comprises a transmembrane domain. In some embodiments, the HAI domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the HAI domain comprises SEQ ID NO: 2.

[0005] In another aspect, the present disclosure provides a vaccine composition comprising the polypeptide described herein. In another aspect, the present disclosure provides a vaccine composition comprising a nucleic acid encoding for the polypeptide described herein. In some embodiments, the nucleic acid is messenger RNA (mRNA). In some embodiments, the nucleic acid comprises an RNA sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 1-2150. In some embodiments, the nucleic acid comprises an RNA sequence of any one of SEQ ID NOS: 1-2150. In some embodiments, the vaccine composition furthercomprises a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle (LNP).

[0006] In another aspect, the present disclosure provides a method of preventing an influenza infection in a subject. In some embodiments, the method comprises administering to the subject the vaccine composition described herein. In another aspect, the present disclosure provides a method of inducing an immune response in a subject. In some embodiments, the method comprises administering to the subject the vaccine composition described herein. In some embodiments, the immune response is a protective immune response. In some embodiments, the vaccine composition is administered to the subject intramuscularly, subcutaneously, intramuscularly, intranasally, or orally. In some embodiments, the vaccine composition is formulated for delivery as an LNP-encapsulated mRNA, DNA, or viral vector.

[0007] In yet another aspect, the present disclosure provides an influenza B hemagglutinin (HA) polypeptide, comprising an HA1 and a HA2 domain, wherein the amino acid at HA2 position 22 is phenylalanine, isoleucine, tryptophan, or tyrosine; and / or the amino acid at HA2 position 27 is phenylalanine, isoleucine, tryptophan, or tyrosine; and / or the amino acid at HA2 position 114 is phenylalanine, isoleucine, tryptophan, or tyrosine; wherein the numbering of amino acid positions of the HA2 is as per SEQ ID NO: 8. In some embodiments, the amino acid at HA2 position 22 is tyrosine. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 9, 26, 42, 31, 59, and 60. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 9, 26, 42, 31, 59, and 60. In some embodiments, the influenza A HA polypeptide exhibits increased trimer stability as compared to an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22. In some embodiments, more of the influenza B HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza B HA polypeptide comprises a transmembrane domain and exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression when expressed on a cell surface than the otherwise identical influenza B HA polypeptide in its soluble format that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22. In some embodiments, the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128- fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22. In some embodiments, the influenza B HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50- fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22. In some embodiments, the amino acid at HA2 position 114 is tyrosine. In some embodiments, the amino acid at HA2 position 22 is tyrosine.

[0008] In another aspect, the present disclosure provides an influenza B hemagglutinin (HA) polypeptide, comprising an HAI and an HA2 domain, wherein the HA2 domain comprises an amino acid sequence that, relative to SEQ ID NO: 8, comprises one or more substitution mutations at one or more of HA2 position 22, HA2 position 27, HA2 position 114; and the influenza B HA polypeptide exhibits increased trimer stability and / or cell surface expression when expressed on cell surface compared to an otherwise identical influenza B HA polypeptide that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114. In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 22. In some embodiments, the amino acid at HA2 position 22 is phenylalanine, tyrosine, isoleucine, or tryptophan. In some embodiments, the amino acid at HA 2 position 22 is phenylalanine. In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 27. In some embodiments, the amino acid at HA2 position 27 is phenylalanine, tyrosine, isoleucine, or tryptophan. In some embodiments, the one or more substitution mutations comprise an substitution mutation at HA2 position 114. In some embodiments, the amino acid at HA2 position 114 is phenylalanine, tyrosine, isoleucine, or tryptophan. In some embodiments, the one or more substitution mutations comprise substitution mutations at HA2 position 22, HA2 position 27, and HA2 position 114. In some embodiments, the amino acid at HA2 position 22 is phenylalanine, the amino acid at HA2 position 27 is phenylalanine, and the amino acid at HA2 position 114 is phenylalanine. In some embodiments, the one or more substitution mutations comprise substitution mutations at HA2position 22, HA2 position 238, and HA2 position 384. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 9, 12-17, 19-24, 31, 42, 53, and 58-60. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 9, 12-17, 19-24, 31, 42, 53, and 58-60. In some embodiments, more of the influenza B HA polypeptide assembles into a trimer when expressed on cell surface as compared to an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza B HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40- fold, or about 50-fold greater cell surface expression when expressed on a cell surface than the otherwise identical influenza B HA polypeptide in its soluble format that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114. In some embodiments, the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128- fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114. In some embodiments, the influenza B HA polypeptide exhibits about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza B that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114. In some embodiments, the influenza B HA polypeptide further comprises a transmembrane domain. In some embodiments, the HAI domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 7. In some embodiments, the HAI domain comprises SEQ ID NO: 7.

[0009] In another aspect, the present disclosure provide a vaccine composition comprising a polypeptide described herein. In another aspect, the present disclosure provide a vaccinecomposition comprising a nucleic acid encoding for the polypeptide described herein. In some embodiments, the nucleic acid is messenger RNA (mRNA). In some embodiments, the nucleic acid comprises an RNA sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 1-2150. In some embodiments, the nucleic acid comprises an RNA sequence of any one of SEQ ID NOS: 1-2150. In some embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the mRNA is encapsulated in a lipid nanoparticle (LNP).

[0010] In another aspect, the present disclosure provides a method of preventing an influenza infection in a subject. In some embodiments, the method comprises administering to the subject the vaccine composition described herein. In another aspect, the present disclosure provides a method of inducing an immune response in a subject. In some embodiments, the method comprises administering to the subject the vaccine composition described herein. In some embodiments, the immune response is a protective immune response. In some embodiments, the vaccine composition is administered to the subject intramuscularly, subcutaneously, intramuscularly, intranasally, orally or transdermally. In some embodiments, the vaccine composition is formulated for delivery as a LNP-encapsulated mRNA, DNA, or viral vector.

[0011] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Various features of the disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present disclosure will be obtainedby reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0014] FIG. 1 shows hemagglutination inhibition (HAI) serum titers induced by mouse vaccination with recombinant influenza B Austria / 1359417 / 2021 hemagglutinin and its mutants at 2048 ng per antigen, 512 ng per antigen, or 128 ng per antigen.

[0015] FIG. 2 shows HAI serum titers induced by mouse vaccination with recombinant influenza A A / California / 07 / 2009 hemagglutinin and its mutants at 2048 ng per antigen, 512 ng per antigen, or 128 ng per antigen.

[0016] FIG. 3 shows percentage of HA polypeptides that assemble into a trimer.

[0017] FIG. 4 shows surface expression of HA in HEK293 cells transfected with mRNA encoding wild-type or mutated HA polypeptides at various lipid nanoparticle (LNP)- encapsulated mRNA concentrations.

[0018] FIG. 5 shows HAI titer of serum from mice administered with LNP-encapsulated mRNA encoding wild-type (“WT-HAB”) or mutated (“Centi-HAB”) influenza B HA polypeptides.

[0019] FIG. 6 shows HAI serum titers from mice induced by immunization with 6 pg of LNP- encapsulated mRNA encoding wild-type soluble HA polypeptides, soluble HA polypeptides with mutations, wild-type HA polypeptides with a transmembrane domain, or HA polypeptides with mutations and a transmembrane domain.

[0020] FIG. 7 shows HAI serum titers from mice induced by immunization with 0.7 pg or 6 pg of LNP-encapsulated mRNA encoding wild-type HA polypeptides with a transmembrane domain, or HA polypeptides with mutations and a transmembrane domain.

[0021] FIG. 8 shows surface expression of HA in HEK293 cells transfected with varying concentrations of LNP-encapsulated mRNA encoding different wild-type or mutated HA polypeptides.

[0022] FIG. 9 depicts pairwise sequence identity for antigens in (or antigens expressed by nucleic acid sequences in) an exemplary influenza vaccine composition.

[0023] FIG. 10A depicts the amount of induced antibodies measured by ELISA reactive to a particular recombinant influenza hemagglutinin antigen in the serum of mice in response to vaccination with either Centi-Flu (0.031 ug of mRNA / antigen), Flu-Biv (0.25 ug / antigen), or Flu-Biv (0.031 ug / antigen). Reactivity is shown to antigens present in Centi-Flu (H3N2 Hong Kong / 1 / 1968, H3N2 Alaska / 01 / 2021), present in Centi-Flu and Flu-Biv (California / 07 / 2004), or heterologous antigens present in neither (H3N2 A / Victoria / 361 / 2011, A / Maryland / 02 / 2021). FIG. 10B depicts the amount of induced antibodies measured by ELISA reactive to a particular recombinant influenza hemagglutinin antigen in the serum of mice in response to vaccinationwith either Centi-Flu (0.25 ug of mRNA / antigen), Flu-Biv (2 ug / antigen), or Flu-Biv (0.25 ug / antigen). Reactivity is shown to antigens present in Centi-Flu (H3N2 Hong Kong / 1 / 1968, H3N2 Alaska / 01 / 2021), present in Centi-Flu and Flu-Biv (Califomia / 07 / 2004), or heterologous antigens present in neither (H3N2 A / Victoria / 361 / 2011, A / Maryland / 02 / 2021).

[0024] FIG. 11A depicts serum reactivity measured by ELISA in ferrets immunized with 1 pg of mRNA encoding influenza hemagglutinin antigens of H3N2 Alaska / 01 / 2021, H3N2 California / 07 / 2004, H3N2 Cambodia / 2020, H3N2 Indiana / 11 / 2018, H3N2 Bilthoven / 1761 / 1976, H3N2 Indiana / 08 / 2011, H3N2 Nanchang / 933 / 1995, or H3N2 Memphis / 1 / 1980, to the same antigen that the animal was immunized with; FIG. 11B depicts serum reactivity measured by ELISA in ferrets immunized with approximately 0.5 ug of mRNA per antigen encoding 8 influenza hemagglutinin antigens of H3N2 Alaska / 01 / 2021, H3N2 California / 07 / 2004, H3N2 Cambodia / 2020, H3N2 Indiana / 11 / 2018, H3N2Bilthoven / 1761 / 1976, H3N2 Nanchang / 933 / 1995, H3N2 Memphis / 1 / 1980, H3N2 Hong Kong / 1 / 1968, to the indicated recombinant protein antigens.

[0025] FIG. 12 depicts pairwise sequence identity between a set of H3N2 hemagglutinin homologous distinct antigen components.

[0026] FIG. 13 depicts the results of hemagglutinin inhibition (HAI) studies in mice immunized with a vaccine composition having twenty mRNAs encoding hemagglutinin antigens (LNP-20) at different doses.

[0027] FIG. 14 depicts the results of HAI studies in mice immunized with a vaccine composition having eighteen mRNAs encoding hemagglutinin antigens (LNP-18).

[0028] FIG. 15 depicts the results of HAI studies in rats immunized with a vaccine composition having eighteen mRNAs encoding hemagglutinin antigens (LNP-18).DETAILED DESCRIPTION

[0029] The present disclosure provides amino acid modifications to influenza hemagglutinin (HA) polypeptides for enhanced stability, increased expression yield, enhanced immunogenicity, and / or enhanced expression, or durability of expression, on the cell surface. Influenza HA polypeptides may form homotrimers, but can also exist in other states, such as monomeric or in high-molecular weight aggregates. The function of prefusion influenza HA is primarily mediated by its trimeric state. After entering the cell where it exists in its post fusion state it is generally not accessible to antibody mediated protection, so antibodies directed primarily at post fusion epitopes are not functional. Furthermore, monomeric influenza HA exposes many non-native (and thus non-neutralizing) epitopes which do not exist in the actual viral HA trimer which compete for epitope recognition with functional epitopes thus crowding them out. As such, it isdesirable for influenza HA immunogens to also exist in a trimeric state. As such, in preferred embodiments of this invention, “stability” refers to the percentage of protein material by mass that is in a trimeric state (“trimer stability”). In preferred embodiments, “stability” further refers to the percentage of protein material by mass that is in a prefusion trimeric state.

[0030] In some embodiments, “enhanced immunogenicity” means higher functional antibody titers in subjects immunized with the mutated influenza HA polypeptides compared to subjects immunized with the corresponding wild-type influenza HA polypeptides. The serological response to influenza may be measured via hemagglutination inhibition (HAI), neutralization (e.g., via microneutralization of pseudovirus or live virus), serum antigen binding by ELISA, or Fc functional assays such as neutrophil, monocyte, macrophage or NK cell engagement through their Fc receptors binding to HA-specific antibodies. As such, in some embodiments, improvements to influenza HA immunogenicity from the mutations of the invention may result in increased titers in serum from immunized subjects in HAI neutralization or Fc mediated functional assays.

[0031] In some embodiments, the mutated influenza HA polypeptides may be expressed as soluble proteins. In other embodiments, the mutated influenza HA polypeptides may be encoded as nucleic acids. In preferred embodiments, the mutated influenza HA polypeptides are encoded as mRNA. In embodiments wherein the mutated influenza HA polypeptides are encoded as nucleic acids, the influenza HA may include its transmembrane domain. As such, when the nucleic acid is taken up and the influenza HA polypeptide is expressed by a cell, the influenza HA may be expressed on the cell surface. In such embodiments, the mutations of the invention may result in increased durability of cell surface expression compared to the corresponding wildtype influenza HA. For example, this may be measured as higher mean fluorescence intensity (MFI) by flow cytometry of cell surface influenza HA expression at a given time point after administration of the HA-encoding nucleic acid.

[0032] The present invention provides mutated variants of influenza HA polypeptides. The mutations may lead to enhanced trimer stability. The mutations may lead to improved expression yields. When the influenza HA polypeptides are expressed with a transmembrane domain for cell surface expression, the mutations may lead to increased durability of expression on the cell surface.

[0033] The enhanced trimer stability, increased expression yield, and / or enhanced immunogenicity may be further enhanced when the influenza HA polypeptides are expressed with a transmembrane domain on the cell surface, as opposed to as soluble influenza HA polypeptides.

[0034] In some embodiments, the present invention comprises any hemagglutinin with one or more mutations known to increase trimer stability, and a transmembrane domain. In some embodiments, the present invention describes nucleic acids encoding such a hemagglutinin such that the hemagglutinin is expressed on the cell surface. In some embodiments, said mutations may be the mutations of this application. In other embodiments, said mutations may be other mutations known in the art to increase trimer stability, e.g., in Milder et al.In some embodiments, the combination of the stabilizing mutations and transmembrane domain results in higher cell surface expression of the HA than the same construct with no stabilizing mutations. In some embodiments, this higher cell surface expression is measured 24, 48, or 96 hours after transfection. In some embodiments, this higher cell surface expression is measured after transfection of HEK293 cells.

[0035] In some embodiments, the combination of the stabilizing mutations and transmembrane domain results in higher immunogenicity than the corresponding HA immunogen with stabilizing mutations alone, or the corresponding HA immunogen with transmembrane domain alone. In some embodiments, this higher immunogenicity is measured by hemagglutination inhibition (HAI) assays, or neutralization assays (e.g. microneutralization using pseudovirus or virus), or serum antigen binding by ELISA or Fc functional antibodies using serum from immunized subjects. In some embodiments, a construct with HA stabilizing mutations and a transmembrane domain results in higher immunogenicity compared to the same construct without the transmembrane domain (i.e., soluble HA). In some embodiments, a construct with HA stabilizing mutations and a transmembrane domain results in higher immunogenicity compared to the same construct without the stabilizing mutations (i.e., the corresponding wildtype HA). In some embodiments, the combination of trimer stabilization mutations and transmembrane domain inclusion has a synergistic rather than additive effect on immunogenicity. In some embodiments, the combination of trimer stabilization mutations and transmembrane domain inclusion results in a greater fold-change increase of immunogenicity than the fold-change increase of immunogenicity from trimer stabilization mutations alone, from transmembrane domain inclusion alone, or the product of fold-change increases of trimer stabilization mutations alone and transmembrane domain inclusion alone. In some embodiments, said mutations may be the mutations of this application. In other embodiments, said mutations may be other mutations known in the art to increase trimer stability, e.g. in Milder et al. (https: / / www.pnas.org / doi / 10.1073 / pnas.2115379119).Mutations to influenza A HA Polypeptides

[0036] In one aspect, the present disclosure provides an influenza A HA polypeptide comprising an HA1 domain and an HA2 domain. The protein is post-translationally cleaved in a designated loop to yield two polypeptides, HA1 and HA2 (the full sequence is referred to as HAO). The wild-type HA2 domain may comprise SEQ ID NO: 3. The wild-type HA2 domain may comprise a histidine (H) at HA2 position 26, a lysine (K) at HA2 position 51, and a glutamic acid I at HA2 position 103. The HA2 domain may comprise an amino acid sequence that comprises one or more substitution mutations at one or more positions. The influenza A HA polypeptide may exhibit increased trimer stability when expressed on cell surface as compared to an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. The mutations may be applied to the HA2 of any influenza A hemagglutinin. The residues of any influenza A hemagglutinin corresponding to HA2 positions 26, 51, and 103 may be found by aligning the amino acid sequence of the HA2 domain of the hemagglutinin in question with SEQ ID NO: 3.

[0037] In some embodiments, the one or more positions of the one or more substitution mutations comprise HA2 position 26. In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F) or tyrosine (Y). In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F). In some embodiments, the amino acid at HA2 position 26 is tyrosine (Y). In some embodiments, the one or more positions of the one or more substitution mutations comprise HA2 position 51. In some embodiments, the amino acid at HA2 position 51 is alanine (A), leucine (L), or valine (V). In some embodiments, the amino acid at HA2 position 51 is alanine (A). In some embodiments, the amino acid at HA2 position 51 is leucine (L). In some embodiments, the amino acid at HA2 position 51 is valine (V). In some embodiments, the one or more positions of the one or more substitution mutations comprise HA2 position 103. In some embodiments, the amino acid at HA2 position 103 is alanine (A) or leucine (L). In some embodiments, the amino acid at HA2 position 103 is alanine (A). In some embodiments, the amino acid at HA2 position 103 is leucine (L). In some embodiments, the one or more positions of the one or more substitution mutations comprise HA2 positions 26 and 51. In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F) and the amino acid HA2 position 51 is leucine (L). In some embodiments, the amino acid at HA2 position 26 is phenylalanine (F) and the amino acid HA2 position 51 is alanine (A). In some embodiments, the one or more positions of the one or more substitution mutations comprise HA2 positions 26, 51 and 103. In some embodiments, the amino acid at HA2 position 26 is selected from F, Y; the amino acid at HA2 position 51 is selected from A, L, V; and the amino acid at HA2 position 103 is selected from A, L. In some embodiments, the amino acid at HA2 position 26 isphenylalanine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is alanine. In some embodiments, the amino acid at HA2 position 26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine. In some embodiments, the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine.

[0038] In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 4; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 5; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 18; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 25; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 26; and comprise one or more of the HA2substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 27; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 28; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 29; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 30; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 32; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 33; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 34; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 35; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 36; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an aminoacid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 37; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 38; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 39; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 40; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 41; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 43; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 44; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 45; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 46; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, atleast about 98%, or at least about 99% sequence identity to SEQ ID NO: 47; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 48; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 49; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 50; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 51; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 52; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 54; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 55; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 56; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 57; and comprise one or more of the HA2substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 64; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 65; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 66; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 67; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 68; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 69; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 70; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 71; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 72; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an aminoacid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 73; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 74; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 75; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 76; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 77; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 78; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 79; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 80; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 81; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 5. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 10. In someembodiments, the influenza A HA polypeptide comprises SEQ ID NO: 11. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 18. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 25. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 26. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 27. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 28. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 29. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 30. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 32. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 33. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 34. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 35. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 36. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 37. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 38. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 39. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 40. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 41. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 43. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 44. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 45. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 46. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 47. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 48. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 49. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 50. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 51. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 52. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 54. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 55. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 56. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 57. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 64. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 65. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 66. In someembodiments, the influenza A HA polypeptide comprises SEQ ID NO: 67. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 68. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 69. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 70. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 71. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 72. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 73. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 74. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 75. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 76. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 77. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 78. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 79. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 80. In some embodiments, the influenza A HA polypeptide comprises SEQ ID NO: 81.

[0039] In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2108-2127; and comprise one or more of the influenza A HA2 substitution mutations described herein.

[0040] In some embodiments, the influenza A HA polypeptide may comprise a signal peptide and the HA extracellular domain. In some embodiments, the influenza A HA polypeptide may comprise the HA extracellular domain. In some embodiments, the extracellular domain may comprise residues corresponding to amino acids 10-509 using the numbering scheme described in Figure 2 of Winter et al. (Nature 292: 72-75, 1981). In other embodiments, the extracellular domain may end at the residue corresponding to amino acid 510, 511, 512, 513, or 514, according to the same numbering scheme.

[0041] In some embodiments, the influenza A HA polypeptide further comprises a transmembrane domain. The transmembrane domain may allow the influenza A HA polypeptide to be expressed on cell surface. The influenza A HA polypeptide may comprise residues corresponding to amino acids 10-550 using the numbering scheme described in Figure 2 of Winter et al. (Nature 292: 72-75, 1981). In other embodiments, the influenza A HA polypeptide may end at a residue corresponding to an amino acid between 539 and 549, according to the same numbering scheme.

[0042] In some embodiments, the HA1 domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the HA1 domain comprises SEQ ID NO: 2.

[0043] In some embodiments, the influenza A HA polypeptide is derived from an Hl -family hemagglutinin. In some embodiments, the influenza A HA polypeptide is derived from an H3- family hemagglutinin. In some embodiments, the influenza A HA polypeptide is derived from an H5-family hemagglutinin. In some embodiments, the influenza A HA polypeptide is derived from an H7-family hemagglutinin. In some embodiments, the influenza A HA polypeptide is derived from an H9-family hemagglutinin. In some embodiments, the influenza A HA polypeptide is derived from A / California / 07 / 2009. In some embodiments, the influenza A HA polypeptide is derived from A / Alaska / 01 / 2021. In some embodiments, the influenza A HA polypeptide is derived from A / Argentina / 28302 / 2010. In some embodiments, the influenza A HA polypeptide is derived from A / Auckland / 588 / 2000. In some embodiments, the influenza A HA polypeptide is derived from A / Beijing / 262 / 1995. In some embodiments, the influenza A HA polypeptide is derived from A / Bilthoven / 1761 / 1976. In some embodiments, the influenza A HA polypeptide is derived from A / Brisbane / 59 / 2007. In some embodiments, the influenza A HA polypeptide is derived from A / California / 07 / 2004. In some embodiments, the influenza A HA polypeptide is derived from A / Cambodia / e0826360 / 2020. In some embodiments, the influenza A HA polypeptide is derived from A / Denver / 57. In some embodiments, the influenza A HA polypeptide is derived from A / Hong_Kong / l / 1968. In some embodiments, the influenza A HA polypeptide is derived from A / Indiana / 08 / 2011. In some embodiments, the influenza A HA polypeptide is derived from A / MD / 12 / 1991. In some embodiments, the influenza A HA polypeptide is derived from A / Memphis / 1 / 1980. In some embodiments, the influenza A HA polypeptide is derived from A / Nanchang / 933 / 1995. In some embodiments, the influenza A HA polypeptide is derived from A / New_Caledonia / 20 / 1999. In some embodiments, the influenza A HA polypeptide is derived from A / New_Jersey / 8 / 1976. In some embodiments, the influenza A HA polypeptide is derived from A / New_York / l / 1918. In some embodiments, the influenza A HA polypeptide is derived from A / Puerto_Rico / 8 / 1934. In some embodiments, the influenza A HA polypeptide is derived from A / Roma / 1949. In some embodiments, the influenza A HA polypeptide is derived from A / Wisconsin / 28 / 2011.2011 / 12. In some embodiments, the influenza A HA polypeptide is derived from A / Wisconsin / 588 / 2019.Mutations to Influenza B HA polypeptides

[0044] In another aspect, the present disclosure provides an influenza B HA polypeptide comprising an HA1 domain and an HA2 domain. The wild-type HA2 domain may comprise SEQ ID NO: 8. The wild-type HA2 domain may comprise histidine (H) at HA2 position 22, histidine (H) at HA2 position 27, histidine (H) at HA2 position 114. The HA2 domain may comprise an amino acid sequence that comprises one or more substitution mutations at one or more positions. The influenza B HA polypeptide may exhibit increased trimer stability when expressed on cell surface as compared to an otherwise identical influenza B HA polypeptide that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114. The mutations may be applied to the HA2 of any influenza B hemagglutinin. The residues of any influenza B hemagglutinin corresponding to HA2 positions 22, 27, and 114 may be found by aligning the amino acid sequence of the HA2 domain of the hemagglutinin in question with SEQ ID NO: 8. Equivalently, the residues of any influenza B hemagglutinin corresponding to HA2 positions 22, 27, and 114 may be found by aligning the amino acid sequence of the HA2 domain of the hemagglutinin in question with the sequence provided in Figure 2 of Berton et al. (Journal of Virology 52: 919-927, 1984), and using the numbering scheme therein.

[0045] In a preferred embodiment, the one or more substitution mutations comprise a substitution mutation at HA2 position 22. In some embodiments, the amino acid at HA2 position 22 is phenylalanine (F). In a preferred embodiment, the amino acid at HA2 position 22 is tyrosine (Y). In some embodiments, the amino acid at HA2 position 22 is isoleucine (I). In some embodiments, the amino acid at HA2 position 22 is tryptophan (W). In some embodiments, the one or more substitution mutations comprise a substitution mutation at HA2 position 27. In some embodiments, the amino acid at HA2 position 27 is phenylalanine (F). In some embodiments, the amino acid at HA2 position 27 is tyrosine (Y). In some embodiments, the amino acid at HA2 position 27 is isoleucine (I). In some embodiments, the amino acid at HA2 position 27 is tryptophan (W). In some embodiments, the one or more substitution mutations comprise an substitution mutation at HA2 position 114. In some embodiments, the amino acid at HA2 position 114 is phenylalanine (F). In some embodiments, the amino acid at HA2 position 114 is tyrosine (Y). In some embodiments, the amino acid at HA2 position 114 is isoleucine (I). In some embodiments, the amino acid at HA2 position 114 is tryptophan (W). In some embodiments, the one or more substitution mutations comprise substitution mutations at HA2 position 22, HA2 position 27, and HA2 position 114. In some embodiments, the amino acid at HA2 position 22 is phenylalanine (F), the amino acid at HA2 position 27 is phenylalanine (F), and the amino acid at HA2 position 114 is phenylalanine (F).

[0046] In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 13; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 14; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 15; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 16; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 17; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 20; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at leastabout 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 21; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 22; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 23; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 24; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 31; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 42; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 53; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 58; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 59; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99% sequence identity to SEQ ID NO: 60; and comprise one or more of the HA2 substitution mutations described herein. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 9. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 12. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 13. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 14. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 15. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 16. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 17. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 19. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 20. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 21. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 22. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 23. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 24. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 31. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 42. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 53. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 58. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 59. In some embodiments, the influenza B HA polypeptide comprises SEQ ID NO: 60.

[0047] In some embodiments, the influenza A HA polypeptide comprises an amino acid sequence with at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2128; and comprises one or more of the influenza B HA2 substitution mutations described herein.

[0048] In some embodiments, the influenza B HA polypeptide may comprise a signal peptide and the HA extracellular domain. In some embodiments, the influenza A HA polypeptide may comprise the HA extracellular domain. In some embodiments, the extracellular domain may comprise residues corresponding to amino acids 1-346 (HA1) and 1-186 (HA2) using the numbering scheme described in Figure 2 of Berton et al. (Journal of Virology 52: 919-927, 1984). In other embodiments, the extracellular domain may end at a residue corresponding to HA2 residue 187, 188, 189, or 190, according to the same numbering scheme.

[0049] In some embodiments, the influenza B HA polypeptide further comprises a transmembrane domain. The transmembrane domain may allow the influenza B HA polypeptide to be expressed on cell surface. In some embodiments, the transmembrane domain mayadditionally or alternatively increase trimer stability. The influenza B HA polypeptide may comprise residues corresponding to amino acids 1-346 of HA1 and 1-223 of HA2 using the numbering scheme described in Figure 2 of Berton et al. (Journal of Virology 52: 919-927, 1984).

[0050] In some embodiments, the HA1 domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 7. In some embodiments, the HA1 domain comprises SEQ ID NO: 7.

[0051] In some embodiments, a polypeptide of the invention comprises a signal peptide and the stabilized extracellular domain. In Table 1, signal peptide sequences are underlined. In each such case, an alternative polypeptide of the invention does not comprise the underlined signal sequence. In some embodiments, a polypeptide of the invention may further comprise a different signal peptide. In some embodiments, a polypeptide of the invention may comprise a sequence of Table 1 followed by an HA transmembrane domain. In some embodiments, said HA transmembrane domain is a wild-type transmembrane domain. In some embodiments, said HA transmembrane domain is a wild-type transmembrane domain from the same strain as the extracellular domain. In some embodiments, said HA transmembrane domain is a wild-type transmembrane domain from the same strain as the extracellular domain as specified in the “clone name” column of Table 1. In Table 1, unless otherwise specified, the “clone name” column identifies the influenza strain from which the HA is derived, and further specifies the mutations numbered relative to the HA2 domain.Table 1. Sequences of HA extracellular domain polypeptidesProperties of the stabilized influenza HAs

[0052] In some embodiments, an influenza HA of the invention assembles as a homotrimer. In some embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the influenza HA polypeptide assembles into a trimer when expressed as a soluble recombinant protein. The percent trimer may be evaluted using SEC-MALS or SEC-HPLC. In some embodiments, a mutated influenza HA polypeptide of the invention has an absolute increase in trimer percentage of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% when compared to the corresponding wild-type influenza HA polypeptide.

[0053] In some embodiments, an influenza HA of the invention may comprise a transmembrane domain, be encoded as a nucleic acid, and express on the cell surface. In some embodiments, a mutated influenza HA polypeptide of the invention exhibits at least about 2- fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression than an otherwise identical influenza HA polypeptide. In some embodiments, the cell surface expression is measured by flow cytometry using an HA-binding antibody. In some embodiments, the HA-binding antibody is a pan-HA binding antibody. In some embodiments, the pan-HA binding antibody is CR9114. In some embodiments, cell surface expression is measured as mean fluorescence intensity. In some embodiments, cell surface expression is measured in a cell line 24, 48, or 96 hours after transfection with the nucleic acid. In some embodiments, the cell line is HEK293 or a derivative ofHEK293.

[0054] In some embodiments, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the influenza HA polypeptide assembles into a trimer when expressed on a cell surface. In some embodiments, more of the influenza HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, the influenza HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20- fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza HA polypeptide in its soluble format that lacks a transmembrane domain. In some embodiments, expression on the cell surface is determined by flow cytometry using a trimer-specific pan HA antibody.

[0055] In some embodiments, an influenza HA polypeptide of the invention induces an HAI titer in serum from immunized subjects measured in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza HA polypeptide in its soluble format that lacks a transmembrane domain.

[0056] In some embodiments, an influenza A HA polypeptide of the invention induces an HAI titer that is at least about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 128-fold, or about 256-fold greater when expressed on a cell surface than an otherwise identical influenza HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103. In some embodiments, an influenza A HA polypeptide of the invention induces an HAI titer that is at least about 2-fold, about 4- fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 128-fold, or about 256-fold greater when expressed on a cell surface than an otherwise identical influenza HA polypeptide in its soluble form that lacks a transmembrane domain, and that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

[0057] In some embodiments, an influenza B HA polypeptide of the invention induces an HAI titer that is at least about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 128-fold, or about 256-fold greater when expressed on a cell surface than an otherwise identical influenza HA polypeptide that has a histidine at HA2 position 22, a histidine at HA2 position 27, and a histidine at HA2 position 114. In some embodiments, an influenza B HA polypeptide of the invention induces an HAI titer that is at least about 2-fold, about 4-fold, about 8-fold, about 16-fold, about 32-fold, about 64-fold, about 128-fold, or about 256-fold greater when expressed on a cell surface than an otherwise identical influenza HA polypeptide in its soluble form that lacks a transmembrane domain, and that has a histidine at HA2 position 22, a histidine at HA2 position 27, and a histidine at HA2 position 114.

[0058] In some embodiments, an influenza B HA polypeptide of the invention exhibits an absolute increase in trimer percentage of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% when expressed on a cell surface than an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 22 and a histidine at HA2 position 27. In some embodiments, an influenza A HA polypeptide of the invention exhibits an absolute increase in trimer percentage of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% when expressed on acell surface than an otherwise identical influenza A HA polypeptide that has histidine at HA2 position 26, lysine at HA2 position 51, glutamic acid at HA2 position 103.Vaccine Composition

[0059] The present disclosure provides a vaccine composition comprising the HA polypeptide described herein. The present disclosure also provides a vaccine composition comprising a nucleic acid encoding for the HA polypeptide described herein. In some embodiments, the nucleic acid is messenger RNA (mRNA). In some embodiments, uridine in the mRNA is substituted with N1 -methylpseudouridine. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid comprises an RNA sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 1000-1020. In some embodiments, the nucleic acid comprises an RNA sequence of any one of SEQ ID NOS: 1-1020. In some embodiments, the nucleic acid encodes for a polypeptide corresponding to any one of SEQ ID NOS: 4-5, 9-81. In some embodiments, the nucleic acid encodes for a polypeptide with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NO: 4-5, 9-81, wherein the polypeptide comprises one or more of the HA2 substitution mutations described herein.

[0060] In some embodiments, the nucleic acid is an mRNA. In some embodiments, the vaccine composition further comprises a pharmaceutically acceptable carrier, excipient, diluent and / or buffer. In some embodiments, the nucleic acid is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the LNP comprises ALC-0315. In some embodiments, the compositions described herein can be administered to a subject in need of treatment or prevention of a condition. In certain circumstances, it will be desirable to deliver the vaccine composition as described herein in suitably formulated pharmaceutical compositions disclosed herein intramuscularly, intravenously, intranasally or intradermally.

[0061] In some embodiments, the plurality of antigens comprises the amino acid sequences of any one of SEQ ID NOS: 2108-2128, as disclosed in Table 4. In some embodiments, an antigen of the plurality of antigens comprises the extracellular domain of the antigen. In some embodiments, an antigen of the plurality of antigens further comprises a transmembrane domain. In some embodiments, the transmembrane domain comprises the sequence of any one of SEQ ID NOS: 2043-2063 or 2130-2150. In some embodiments, the transmembrane domain comprises the sequence of any one of SEQ ID NOS: 2043-2063. In some embodiments, the transmembrane domain may be coupled to an extracellular domain of the antigen. In some embodiments, the vaccine composition comprises nucleic acid sequences encoding any one of SEQ ID NOS: 2085- 2105. In some embodiments, the vaccine composition comprises one or more nucleic acids. Insome embodiments, each nucleic acid of the nucleic acid vaccine may encode a signal peptide of an influenza hemagglutinin. In some embodiments, each nucleic acid may encode an extracellular domain of an influenza hemagglutinin. In some embodiments, each nucleic acid may encode a transmembrane domain of an influenza hemagglutinin. In some embodiments, each nucleic acid may encode a signal peptide, an extracellular domain, and a transmembrane domain, wherein the signal peptide, the extracellular domain, and the transmembrane domain are each operably linked with one another. In some embodiments, each nucleic acid may encode an open reading frame coding for a signal peptide, extracellular domain, and transmembrane domain. In some embodiments, each nucleic acid encodes only a signal peptide and an extracellular domain of an antigen. In some embodiments, the nucleic acid encodes an antigen extracellular domain that is not connected to a signal peptide. In some embodiments, the nucleic acid encodes an antigen extracellular domain that is not connected to a transmembrane domain. In some embodiments, the nucleic acid encodes an antigen extracellular domain that is not connected to a signal peptide or a transmembrane domain. In some embodiments, a nucleic acid of the vaccine composition may comprise any of SEQ ID NOs 1000-1020. In some embodiments, the antigen extracellular domain may have 1-5 amino acid mutations relative to the wild type sequence to increase the stability of the antigen. In some embodiments, the antigen extracellular domain may have 3 amino acid mutations relative to the wild type sequence to increase the stability of the antigen.

[0062] In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2050, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2052, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2053, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2054, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2055, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2056, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2057, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2058, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2059, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2060, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domaincomprises the sequence of SEQ ID NO: 2061, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2062, or a sequence at least 70% identical thereto. In some embodiments, the transmembrane domain comprises the sequence of SEQ ID NO: 2063, or a sequence at least 70% identical thereto.Nucleic acid vaccine compositions

[0063] The present disclosure provides a nucleic acid vaccine composition comprising one or more nucleic acids encoding for a plurality of antigens as disclosed herein. In some embodiments, the one or more nucleic acids are a messenger RNA (mRNA). In some embodiments, the nucleic acid vaccine composition comprises a nucleic acid having any one of the sequences described in Table 2. In some embodiments, the nucleic acid vaccine composition comprises a nucleic acid having any one of SEQ ID NOS: 1000-1020. In some embodiments, the nucleic acid encodes a signal peptide sequence. In some embodiments, the nucleic acid encodes a transmembrane domain. In some embodiments, the nucleic acid encodes an extracellular domain.

[0064] Any nucleic acid sequence in Table 2 may be followed by a stop codon (e.g., TGA). Each nucleic acid sequence encodes for a signaling peptide sequence (denoted with single underlining), a stabilized extracellular sequence, and a transmembrane domain sequence (denoted with double underlining). In some embodiments, nucleic acid sequences of the invention may encode for the denoted signal peptide sequence and denoted stabilized extracellular sequence. In other embodiments, nucleic acid sequences of the invention may encode for the denoted stabilized extracellular sequence. In other embodiments, nucleic acid sequences of the invention may encode for the denoted stabilized extracellular sequence and denoted transmembrane domain sequence.Table 2. mRNA sequences encoding signal peptide, HA ectodomain, and HA transmembrane domain with stabilizing mutations.

[0065] Table 3 depicts antigen amino acid sequences of the invention. Furthermore, these antigen amino acid sequences may be encoded by nucleic acids of a vaccine composition of the invention. In some embodiments, a vaccine composition comprises nucleic acids encoding any one of (or all of) the sequences in Table 3. Each amino acid sequence comprises a signaling peptide sequence (denoted with single underlining), a stabilized extracellular sequence, and a transmembrane domain sequence (denoted with double underlining). In some embodiments, polypeptides of the invention may comprise the denoted signal peptide sequence and denoted stabilized extracellular sequence. In other embodiments, polypeptides of the invention may comprise the denoted stabilized extracellular sequence. In other embodiments, amino acid sequences of the invention may comprise the denoted stabilized extracellular sequence and denoted transmembrane domain sequence.

[0066] In some embodiments, the one or more nucleic acids encoding for an antigen encode a signal peptide, extracellular domain, and transmembrane domain. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2085 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2086 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2087 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2088 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2089 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2090 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2091 or a sequence at least 90% identicalthereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2092 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2093 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2094 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2095 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2096 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2097 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2098 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2099 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2100 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2101 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2102 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2103 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2104 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2105 or a sequence at least 90% identical thereto.

[0067] Table 4 depicts antigen amino acid sequences of the invention. Furthermore, these antigen amino acid sequences may be encoded by nucleic acids of a vaccine composition of the invention. Each amino acid sequence comprises a stabilized extracellular sequence.Table 4 - Stabilized amino acid sequences of antigen extracellular domains

[0068] In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2119or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 90% identical thereto.

[0069] In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for anantigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 95% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 95% identical thereto.

[0070] In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence atleast 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2124or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 98% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 98% identical thereto.

[0071] In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for anantigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 99% identical thereto. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 99% identical thereto.

[0072] In some embodiments, the one or more nucleic acids encode four or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2108-2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode six or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 21 OS- 2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode eight or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2108-2115, 2124- 2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode antigens comprising SEQ ID NOS: 2108-2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode four or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-3271, or sequences atleast 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode six or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode eight or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode antigens comprising SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the one or more nucleic acids encode an antigen comprising SEQ ID NO: 2128, or a sequence at least 90% identical thereto.

[0073] In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2085. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2086. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2087. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2088. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2089. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2090. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2091. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2092. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2093. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2094. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2095. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2096. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2097. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQID NO: 2098. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2099. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2100. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2101. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2102. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2103. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2104. In some embodiments, the one or more nucleic acids encode for an antigen comprising or consisting of an amino acid sequence corresponding to SEQ ID NO: 2105.

[0074] In some embodiments, the one or more nucleic acids are RNAs and comprise an RNA sequence selected from SEQ ID NOS: 1000-1020. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1000, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1001, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1002, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1003, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1004, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1005, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1006, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1007, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1008, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1009, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1010, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1011, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1012,or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1013, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1014, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1015, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1016, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1017, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1018, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1019, or a sequence at least 90% identical thereto. In some embodiments, the one or more nucleic acids comprise RNA corresponding to SEQ ID NO: 1020, or a sequence at least 90% identical thereto.

[0075] In some embodiments, the nucleic acid vaccine composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the one or more nucleic acids that encode a plurality of antigens are present in the nucleic acid vaccine composition in an amount of less than about 5 micrograms (pg) such as, for example, from about 1 ng to about 5 pg. In some embodiments, the one or more nucleic acids that encode a plurality of antigens are present in the nucleic acid vaccine composition in an amount of less than about 3 micrograms (pg) such as, for example, from about 1 ng to about 3 pg. In some embodiments, the one or more nucleic acids that encode a plurality of antigens are present in the nucleic acid vaccine composition in an amount of less than about 5 pg, less than about 4 pg, less than about 3 pg, less than about 2 pg, or less than about 1 pg. In some embodiments, each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 5 micrograms (pg), such as from about 1 ng to about 5 pg. In some embodiments, each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 3 pg, such as from about 1 ng to about 3 pg. In some embodiments, the plurality of antigens comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 distinct antigens. In some embodiments, the one or more nucleic acids encode for no more than 30, no more than 25, or no more than 20 distinct antigens. In some embodiments, the one or more nucleic acids that encode a plurality of antigens are present in the nucleic acid vaccine composition in an amount of more than 1 ng. In some embodiments, the plurality of antigens comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17distinct antigens. In some embodiments, the plurality of antigens comprises no more than 30 antigens. In some embodiments, the plurality of antigens comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 antigens. In some embodiments, the plurality of antigens comprises no more than 25 antigens. In some embodiments, the plurality of antigens comprises no more than 20 antigens.

[0076] In some embodiments, the one or more nucleic acids encode for SEQ ID NOS: 21 OS- 2128. In some embodiments, the one or more nucleic acids further encode a transmembrane domain. In some embodiments, the transmembrane domain comprises any one of SEQ ID NOS: 2043-2063. In some embodiments, the one or more nucleic acids encode each of SEQ ID NOS: 2043-2063. In some embodiments, the one or more nucleic acids further encode a signaling peptide. In some embodiments, the one or more nucleic acids comprise any one of SEQ ID NOS: 1000-1020. In some embodiments, the one or more nucleic acids comprise each of SEQ ID NOS: 1000-1020. In some embodiments, the one or more nucleic acids encode any one of SEQ ID NOS: 2085-2105. In some embodiments, the one or more nucleic acids encode each of SEQ ID NOS: 2085-2105. In some embodiments, the one or more nucleic acids encode any one of SEQ ID NOS: 2085-2104. In some embodiments, the one or more nucleic acids encode each of SEQ ID NOS: 2085-2104. In some embodiments, the one or more nucleic acids encode any one of SEQ ID NOS: 2085-2100. In some embodiments, the one or more nucleic acids encode each of SEQ ID NOS: 2085-2100. In some embodiments, the vaccine composition comprises twenty- one nucleic acid sequences encoding SEQ ID NOS: 2108-2128, or sequences at least 90% identical thereto. In some embodiments, the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 2108-2128, or sequences at least 95% identical thereto. In some embodiments, the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 2108-2128, or sequences at least 98% identical thereto. In some embodiments, the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 2108-2128, or sequences at least 99% identical thereto.Table 5, Amino acid sequences of transmembrane domainsMultivalent Vaccine Compositions

[0077] The present disclosure provides vaccine compositions and methods for treating influenza. In some embodiments, the vaccine composition comprises one or more antigens. In some other embodiments, the vaccine composition comprises nucleic acids that encode for the plurality of antigens. In some embodiments, the plurality of antigens includes one or more influenza proteins, or derivatives thereof. In some embodiments, the one or more antigens comprise hemagglutinin proteins, or derivatives thereof. In some embodiments, the hemagglutinin proteins or derivatives thereof comprises one or more hemagglutinin subtypes selected from Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, H17, or Hl 8. In some embodiments, the one or more antigens include hemagglutinin proteins from multiple subtypes, such as from Hl and H3. In some embodiments, the antigens include hemagglutinin proteins from the influenza B subtype (HAB).

[0078] In some embodiments, upon administration of the vaccine to a subject, the vaccine elicits an immune response in the subject yet none of the plurality of distinct antigens is individually expressed at a level that is sufficient to elicit an immune response in a subject by itself.

[0079] In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 90% identical thereto. In someembodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 90% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 90% identical thereto.

[0080] In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequencecorresponding to SEQ ID NO: 2112 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 95% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 95% identical thereto.

[0081] In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 98% identical thereto.In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequencecorresponding to SEQ ID NO: 2126 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 98% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 98% identical thereto.

[0082] In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2108 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2109 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2110 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2111 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2112 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2113 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2114 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2115 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2116 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2117 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2118 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2119 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2120 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2121 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2122 or a sequence at least 99% identical thereto. In some-n-embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2123 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2124 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2125 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2126 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2127 or a sequence at least 99% identical thereto. In some embodiments, the plurality of antigens comprises a polypeptide with amino acid sequence corresponding to SEQ ID NO: 2128 or a sequence at least 99% identical thereto.

[0083] In some embodiments, the vaccine composition comprises four or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2108-2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises six or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2108-2115, 2124- 2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises eight or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2108-2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises antigens comprising SEQ ID NOS: 2108-2115, 2124-2125, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises four or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises six or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises eight or more distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises antigens comprising SEQ ID NOS: 2116-2123, 2126-2127, or sequences at least 90% identical to any one of such sequences. In some embodiments, the vaccine composition comprises an antigen comprising SEQ ID NO: 2128, or a sequence at least 90% identical thereto.

[0084] In some embodiments, each antigen of a plurality of antigens is present in the vaccine composition in an amount of less than about 5 micrograms (pg). In some embodiments, each antigen of a plurality of antigens is present in the vaccine composition in an amount of less than about 5 pg, less than about 4 pg, less than about 3 pg, less than about 2 pg, or less than about 1 pg. In some embodiments, the plurality of antigens comprises at least 6, at least 8, at least 10, at least 12, at least 14, at least 16, or at least 17 distinct antigens. In some embodiments, the plurality of antigens comprises no more than 30 antigens. In some embodiments, the plurality of antigens comprises no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 antigens. In some embodiments, the plurality of antigens comprises no more than 25 antigens. In some embodiments, the plurality of antigens comprises no more than 20 antigens.

[0085] In some embodiments, the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 2108-2128, or sequences at least 90% identical thereto. In some embodiments, the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 2108-2128, or sequences at least 95% identical thereto. In some embodiments, the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 2108-2128, or sequences at least 98% identical thereto. In some embodiments, the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 2108-2128, or sequences at least 99% identical thereto. In some embodiments, the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 2108-2128.

[0086] A vaccine composition comprising twenty-one distinct antigens, wherein each of the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 2108-2128, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto. In some embodiments, each of the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 2108-2128. In some embodiments, each of the twenty-one distinct antigens comprises or consists of a sequence selected from SEQ ID NOS: 2085-2105 A nucleic acid vaccine composition comprising one or more nucleic acids encoding for at least twenty-one distinct antigens, wherein each the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 2108-2128, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, each of the at least twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 2108-2128. In some embodiments, each of the twenty-one distinct antigens comprises or consists of a sequenceselected from SEQ ID NOS: 2085-2105. In some embodiments, the vaccine composition comprises each of SEQ ID NOS: 1000-1020.Method of Use

[0087] The present disclosure provides a method for preventing an influenza infection in a subject. In some embodiments, the method comprises administering to the subject a vaccine composition as described herein. In another aspect, the present disclosure provides a method of inducing an immune response (e.g., a protective immune response) in a subject by administering to the subject a vaccine composition as disclosed herein. Generation of an immune response via administration of a vaccine composition as described herein can do one or more of: (1) protect a human against influenza virus infection, (2) decrease the likelihood of infection (or decrease the severity of one or more symptoms associated with infection) by an influenza virus, or (3) enhance the immune system of the subject to resist subsequent infection by an influenza virus (4) decrease the risk of serious infection, hospitalization and death from influenza itself or its complications, and 5) decrease the spread of influenza from immunized individuals to other individuals (transmission blocking). The vaccine composition may be administered intramuscularly, subcutaneously, intramuscularly, intranasally, orally, or trans-dermally. The nucleic acid vaccine composition can be formulated for delivery as a mRNA / LNP, or DNA. An infection rate in subjects receiving the vaccine may be reduced by 2% (or more) relative or more relative to a placebo control or relative to a subject who did not receive the vaccine composition.Certain Definitions

[0088] All terms are intended to be understood as they would be understood by a person skilled in the art. 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 the disclosure pertains.

[0089] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0090] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details arecapable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0091] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0092] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0093] The term “about” means a range within 10% of a given value.

[0094] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0095] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0096] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range arespecifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0097] The term “pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia (U.S.P.) or other generally recognized pharmacopeia for use in animals, including humans.

[0098] The term “subject” refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.

[0099] The term “optional” or “optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0100] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0101] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0102] The “percent sequence identity” between a reference amino acid sequence and a query amino sequence (i.e., the amino sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference amino acid sequence) is determined by optimally aligning the sequences using the Needleman-Wunsch alignment algorithm with a gap existence penalty of 11 and a gap extension penalty of 1 and comparing the sequences. The number of exact matches, divided by the total number of positions in the alignment (which corresponds with the number of amino acids in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query amino acid sequence and the reference amino acid sequence (i.e., percent sequence identity = (# of exact matches / (total # of positions in alignment)* 100). An alignment using the Needleman-Wunsch alignment algorithm(with a gap existence penalty of 11 and a gap extension penalty of 1) can be generated using the “Global Align” BLAST program availa-ble at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0103] The “percent sequence identity” between a reference nucleic acid sequence and a query nucleic acid sequence (i.e., the nucleic acid sequence being analyzed to determine whether it is within a particular percent sequence identity with the reference nucleic acid sequence) is determined by optimally aligning the sequences using the Needleman-Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap extension penalty of 2) and comparing the aligned nucleic acids. The number of exact matches divided by the total number of nucleotides in the alignment (which corresponds with the number of nucleotides in the reference sequence plus any gaps in the reference sequence when aligned with the query sequence) is determined and expressed as a percentage. This is the percent sequence identity between the query nucleic acid sequence and the reference nucleic acid sequence (i.e., percent sequence identity = (# of exact matches) / (total # of nucleotides in the alignment)* 100). An alignment using the Needleman-Wunsch alignment algorithm (with match / mismatch scores of 2,-3, a gap existence penalty of 5, and a gap ex -tension penalty of 2) can be generated using the “Global Align” BLAST program available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.EXAMPLES

[0104] Example 1: Immunization with recombinant HA polypeptides increased hemagglutination inhibition (HAI) activity.

[0105] HAI (hemagglutination inhibition) was measured using serum from mice immunized with the indicated dose of recombinant influenza B Austria / 1359417 / 2021 hemagglutinin with the indicated HA2 mutations. WT denotes the wild-type sequence (shaded column), as shown in FIG. 1. The mutations improved HAI activity up to 16-fold. Hl 14Y and H22Y were more effective than Hl 14W. The Hl 14Y and H22Y mutations improved HAI activity over multiple dose levels. The results are depicted in FIG. 1.

[0106] HAI (hemagglutination inhibition) was measured using serum from mice immunized with indicated dose of recombinant influenza A A / California / 07 / 2009 hemagglutinin with the indicated HA2 mutations. WT denotes wild type sequence (grey column), as shown in FIG. 2. The mutations improved HAI activity up to 2-fold. The H26F / K51A / E103A and H26F / K51L / E103L mutations improved HAI activity over multiple dose levels. Both these sets of mutations of this invention were superior to a previously disclosed stabilizing mutation, H26W / K5H / E103I, in US17 / 594,576 and Milder et al. (PNAS 2022, DOI:10.1073 / pnas.2115379119) (black column), as shown in FIG. 2. These previously disclosedmutations in fact reduced HAI levels by 2-fold at both the 512 ng / Ag and 128 ng / Ag dose levels.The results are depicted in FIG. 2.

[0107] FIG. 3 and Table 6 depict percentages of HA polypeptides that assemble into a trimer.Table 6. Percentages of trimers and yield for HA polypeptides

[0108] Example 2: HA polypeptides improve cell surface expression of transmembrane HAs when delivered as mRNA.

[0109] Each mRNA encoding for a specific influenza HA polypeptide (based on A / California / 2009) was encapsulated as LNP and each LNP was transfected into HEK293 cells. Cells were incubated for 24h, 48h and 96h. Cells were harvested at each time point and stained with pan anti-HA antibody (clone CR9114) labeled with fluorescent fluorophore BV421. Cells were detected by flow cytometry and data were analyzed using FlowJo to identify and mean fluorescent intensity (MFI). FIG. 4 depicts that cells incubated with mRNAs encoding the H26F / K51L / E103L mutant expressed about 8 to about 200 times more cell-surface HA compared to cells derived from mice administered with mRNAs encoding the previously- described H26W7K5H / E103I mutant. FIG. 4 also depicts that cells incubated with mRNAs encoding the H26F / K51L / E103L mutant expressed about 8 to about 350 times more cell-surface HA compared to cells derived from mice administered with mRNAs encoding wild-type HA polypeptide or the previously-described H26W7K5H / E103I mutant.

[0110] Example 3: Mutations in the HA polypeptides synergize with transmembrane antigen expression in induction of anti-HA immunogenicity.

[0111] HA inhibition (HAI) assay was used to assess the presence and concentration of antibodies that can inhibit virus hemagglutination in serum samples of mice administered with mRNAs encoding HA polypeptides. FIG. 5 depicts that H22Y (“Centi-HAB” in FIG. 5) exhibited a titer that was at least 16-fold greater than the wild-type HA (“WT-HAB” in FIG. 5).

[0112] FIG. 6 depicts that the wild-type HA polypeptides of H1N1 (A / Califomia / 04 / 2009), H3N2 (A / Alaska / 01 / 2021), and Influenza B (B / Michigan / 01 / 2021) expressed on the cell surface induce about 2- to 30-fold higher titers than the soluble wild-type HA polypeptides lacking a transmembrane domain. As such, the transmembrane domain modification alone improves immunogenicity over the soluble HA polypeptide. The soluble HA polypeptides with mutations lacking a transmembrane domain exhibited a titer that was no more than 2-fold greater than the soluble wild-type HA polypeptides lacking a transmembrane domain. By contrast, the HA polypeptides with mutations that were expressed on the cell surface exhibited at least 500-foldhigher titers than the soluble wild-type HA polypeptides lacking a transmembrane domain; and at least 8-fold higher titers than the wild-type HA polypeptides with a transmembrane domain. The mutations in H1N1 and H3N2 were H22W, K51I, and E103I. The mutations in influenza B were H22Y. FIG. 6 demonstrates that mutations in the HA polypeptides demonstrated to increase trimer stability synergize with transmembrane antigen expression in inducing potent immunogenicity.

[0113] FIG. 7 depicts that when 0.7 pg mRNA (left) or 6 pg mRNA (right) encoding HA polypeptides expressed on the cell surface were used for immunization, HAI titer of the HA polypeptides with mutations of this invention was at least 4-fold greater than the ones of the wild-type HA polypeptides. The mutations in H1N1 and H3N2 were H22F, K51L, and E103L. The mutations in influenza B were H22Y. FIG. 7 demonstrates that stabilizing mutations of the invention improve HAI titers in the transmembrane format, even at low doses of mRNA.

[0114] FIG. 8 depicts the comparative cell-surface expression of A / Califomia / 2009 HA with different sets of stabilizing mutations on HEK293 cells, in the same experimental setup as described for FIG. 4. “WT” denotes the wild type; “WII” denotes previously-described H26W / K5H / E103I; “FLA” denotes H26F / K51L / E103A; “FAA” denotes H26F / K51A / E103A; “FA” denotes H26F / K51A; “FLL” denotes H26F / K51L / E103L. All proposed mutation combinations of this invention result in significantly higher, and more durable, cell-surface expression compared to the corresponding wild-type HA, or the “WII” variant previously described in the art.

[0115] Example 4: Trimer percentage and Tm of HA polypeptides.Table 7. Purity measured by SEC-HPLC, and SEC-MALS, and Tm of HA polypeptidesTable 8. Trimer percentage measured by SEC-HPLC, and SEC-MALS, and Tm of HA polypeptidesIn this table, “N.D.” means not determined.

[0116] Example 5: Serum activity to mRNA antigens in mice. Mice were injected with either Centi-Flu (mRNA encoding a mixture of 16 hemagglutinin antigens, including eight Hl, corresponding to SEQ ID NOs. 2-9; and eight H3, corresponding to SEQ ID NOs. 16-22 and 24) or Flu-Biv (mRNA encoding a mixture of 2 hemagglutinin antigens, including one Hl and one H3. All mRNA vaccine compositions were complexed in lipid nanoparticles (LNPs).Specifically, the same LNP composition as BNT162b2 was used. The serum was collected 16 days after immunization and measured by ELISA for antibodies that bind to recombinant hemagglutinin proteins of H3N2 Hong Kong / 1 / 1968 (present in Centi-Flu), H3N2 California / 07 / 2004 (present in Centi-Flu and Flu-Biv), H3N2 Alaska / 01 / 2021 (present in Centi- Flu), H3N2 A / Victoria / 361 / 2011 (heterologous to both Centi-Flu and Flu-Biv), or H3N2 A / Maryland / 02 / 2021 (heterologous to both Centi-Flu and Flu-Biv). FIG. 10A suggests that at a lower matched per-antigen dose, Flu-Biv induced no response while Centi-Flu induced a robust response. For example, when the mice were injected with Centi-Flu LNP at 0.031 pg per antigen (=0.5 ug total mRNA) or Flu-Biv at 0.031 pg per antigen (=0.063 ug total mRNA), Flu-Biv induced no response, while Centi-Flu induced a robust response. Furthermore, this difference cannot be explained by the total antigen dose, as Flu-Biv at 0.25 ug per antigen (=0.5 ug total mRNA) still exhibits a weaker response in all unmatched antigens than Centi-Flu at 0.031 ug / antigen (=0.5 ug total mRNA). FIG. 10B suggests that at a higher per-antigen dose, Flu-Biv induced a weak response, while Centi-Flu induced a more potent response. For example, when the mice were injected with Centi-Flu LNP at 0.25 pg per antigen (=4 ug total mRNA) or Flu- Biv at 0.25 pg per antigen (=0.5 ug total mRNA), Flu-Biv induced a weak response, while Centi- Flu induced a robust response. Furthermore, this difference cannot be explained by the total antigen dose, as Flu-Biv at 2 ug per antigen (=4 ug total mRNA) still exhibits a weaker response in all unmatched antigens than Centi-Flu at 0.25 ug / antigen (=4 ug total mRNA). Therefore, it isthe unique combination of (1) using six or more homologous distinct antigen components and (2) mRNA encoding each component at a low dose, that achieves the desired serological effect.

[0117] One example set of eight homologous distinct antigen components encoded by mRNA in the Centi-Flu vaccine composition are A / Hong Kong / 1 / 1968, A / Nanchang / 933 / 1995, A / California / 07 / 2004, A / Memphis / 1 / 1980, A / Alaska / 01 / 2021, A / Indiana / 11 / 2018, A / Cambodia / e0826360 / 2020, and A / Bilthoven / 1971 / 1976. FIG. 12 shows that any two of these eight H3N2 homologous distinct antigen components have pairwise sequence identity less than 96%, and greater than 80%. Any subset of six of these eight constitute sets of six homologous distinct antigen components with pairwise sequence identity less than 96% and greater than 80%.

[0118] mRNA generation from DNA plasmids and subsequent encapsulation in lipid nanoparticles (LNPs) was performed using methods well-known in the art, summarized here. DNA plasmids were constructed that comprise, under the SP6 promoter, the same 5’ UTR as used in the clinically approved BNT162b2 vaccine (SEQ ID NO: 2106), DNA encoding an antigen component, the same 3’ UTR as used in BNT162b2 (SEQ ID NO: 2107), and a poly- adenosine(120) tail. An alternative 3’ UTR from human HBA1 is shown as SEQ ID NO: 2129. The plasmids were linearized using a Notl restriction site and utilized as templates for in vitro transcription (IVT) using SP6 RNA polymerase and a mix of nucleoside triphosphates. Nl- methyl-pseudouridine-5’ -triphosphate was used instead of uridine-5’ -triphosphate for IVT. The Vaccinia enzymatic capping system (New England BioLabs) was used to generate capped mRNA. Purified mRNA was further encapsulated in the same LNP formulation as BNT162b2 (46.3% ionizable lipid ALC-0315, 9.4% phospholipid DSPC, 1.6% ALC-0159 PEG-lipid, and 42.7% cholesterol) to obtain the LNP-encapsulated RNA.

[0119] Codon optimization of nucleic acid sequences may be performed using various methods well-known in the art, including LinearDesign.Table 9. Table of UTR Sequences

[0120] Serum ELISA assays in all Examples were performed as follows: The indicated proteins (5ug / mL) were added to microtiter plates (CoStar), in coating buffer (0.1 M sodium bicarbonate, pH 8.6). After incubation at 4°C overnight and blocking with 3% bovine serum albumin (BSA) in PBS, for 1 hour at 37°C, serially diluted serum / plasma in blocking buffer was added to individual wells and incubated for 1 hour at 37°C. Then, plates were washed three times with 0.05% PBST. Horseradish peroxidase (HRP)-conjugated anti-IgG secondary antibody was added to wells and incubated for 1 hour at 37°C. After washing three times with PBST, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic (Thermo Fisher Scientific Inc., Waltham, MA, USA) or 3,3',5,5'-tetramethylbenzidineliquid substrate system (Thermo Fisher Scientific Inc.) was added to the wells. Absorbance was measured at 405 or 650 nm, respectively, using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific Inc.).

[0121] Example 6: Serum activity to mRNA antigens in ferret.

[0122] Ferrets were immunized once with mRNA encoding any one of eight distinct antigens complexed with a lipid nanoparticle (LNP). Specifically, ferrets were immunized with 1 pg of LNP-encapsulated mRNA encoding the hemagglutinin corresponding to H3N2 Alaska / 01 / 202, H3N2 California / 07 / 2004, H3N2 Cambodia / 2020, H3N2 Indiana / 11 / 2018, H3N2 Bilthoven / 1761 / 1976, H3N2 Nanchang / 933 / 1995, or H3N2 Memphis / 1 / 1980. Serum was obtained 28 days after immunization. Serum reactivity to the recombinant protein of each antigen with which the ferrets were immunized was measured, as shown in FIG. 11 A. The antigen-matched serum reactivity (i.e., serum from mice immunized with a given antigen tested against that same antigen) was very weak in all cases (EC50 not attained in any group even at the highest tested serum dilution of 1 : 100).

[0123] Ferrets were immunized with LNP-encapsulated mRNAs encoding the hemagglutinin corresponding to 8 H3N2 antigens, each at about 0.5 pg per antigen (total of 4 pg of H3N2 antigens). Serum reactivity to the recombinant protein of each antigen with which the ferrets were immunized was measured, as shown in FIG. 1 IB. The Centi-Flu immunized ferrets exhibited potent responses against all immunized antigens, as shown in FIG. 1 IB. It suggeststhat the single antigen at a low per-antigen dose (1 ug per antigen) is insufficient, but Centi-Flu at an even lower per-antigen dose (0.5 ug per antigen) induces potent and broad reactivity. Therefore, it is the unique combination of (1) using six or more homologous distinct antigen components and (2) mRNA encoding each component at a low dose, that achieves the desired serological effect.

[0124] All LNP-encapsulated RNA constructs were generated as described in Example 5. Serum ELISA was performed as described in Example 5.

[0125] One exemplary set of eight homologous distinct antigen components encoded by mRNA in the Centi-Flu vaccine composition are A / Hong Kong / 1 / 1968, A / Nanchang / 933 / 1995, A / California / 07 / 2004, A / Memphis / 1 / 1980, A / Alaska / 01 / 2021, A / Indiana / 11 / 2018, A / Cambodia / e0826360 / 2020, and A / Bilthoven / 1971 / 1976. FIG. 12 shows that any two of these eight H3N2 homologous distinct antigen components have pairwise sequence identity less than 96%, and greater than 80%. Any subset of six of these eight constitute sets of six homologous distinct antigen components with pairwise sequence identity less than 96% and greater than 80%.

[0126] Example 7: Hemagglutination Inhibition Tests on Immunized Mice

[0127] Mice were immunized twice (dO, d21) with an LNP-encapsulated mixture of 20 mRNAs (“LNP20”). Specifically, the 20 mRNAs encode antigens corresponding to the antigens of SEQ ID NOS: 2085-2104, provided in Table 3; for example, using mRNA sequences provided in Table 2 (SEQ ID NOS: 1000-1020). Each mRNA of the mixture encoded for one influenza hemagglutinin (10 Hl antigens, 10 H3 antigens). Mice were immunized with a total mRNA dose of 18 pg, 6 pg, 2 pg, or 0.7 pg. Hemagglutination inhibition (HAI) was tested using serum drawn at 35 days post-immunization (dpi). HAI was tested against strains included in the LNP20 mixture (H1N1 A / Puerto Rico / 8 / 1934, H1N1 A / Califomia / 04 / 2009, and H3N2 A / Alaska 01 / 2021); as well as against heterologous strains not included in the LNP20 mixture (H1N1 A / Connecticut / 01 / 2021, H1N1 A / New Caledonia / 20 / 99, H3N2 A / Fujian / 411 / 2002, H3N2 A / Darwin / 9 / 2021, and B / Michigan / 01 / 2021). HAI titers >40 were observed for all strains except B / Michigan / 01 / 2021, which is expected since LNP20 did not include mRNA encoding any influenza B hemagglutinin antigens.

[0128] The results are depicted in FIG. 13. This demonstrates the potent and broad humoral response induced by LNP20.

[0129] Example 8: Hemagglutination Inhibition Tests on Immunized Mice

[0130] Mice were immunized twice (dO, d21) with an LNP-encapsulated mixture of 18 mRNAs (“LNP18”). Each mRNA encoded for one influenza hemagglutinin (8x Hl, 8x H3, 2x HAB). Specifically, the mRNAs of LNP18 encode antigens corresponding to the antigens ofSEQ ID NOS: 2085-2100 provided in Table 3. LNP18 further comprises mRNAs encoding two HAB strains: B / Guangdong-Maonan / 316 / 2021 and B / Utah / 02 / 2012. Mice were immunized with a total mRNA dose of 12 pg. Hemagglutination inhibition (HAI) was tested using serum drawn at 63 days post-immunization (dpi). HAI was tested against strains included in the LNP20 mixture (H1N1 A / Brisbane / 59 / 2007, H1N1 A / California / 04 / 2009, and H3N2 A / Alaska / 01 / 2021); as well as against heterologous strains not included in the LNP20 mixture (all others). HAI titers >40 were observed for 13 / 16 strains, and >32 for 15 / 16 strains.

[0131] The results are depicted in FIG. 14. This demonstrates the potent and broad humoral response induced by LNP18.

[0132] Example 9: Hemagglutination Inhibition Tests on Immunized Rats

[0133] Rats were immunized twice (dO, d21) with an LNP-encapsulated mixture of 18 mRNAs (“LNP18”). Each mRNA encoded for one influenza hemagglutinin (8x Hl, 8x H3, 2x HAB). Specifically, the mRNAs of LNP18 encode antigens corresponding to the antigens of SEQ ID NOS: 2085-2100 provided in Table 3. LNP18 further comprises mRNAs encoding two HAB strains: B / Guangdong-Maonan / 316 / 2021 and B / Utah / 02 / 2012. Rats were immunized with a total mRNA dose of 12 pg. Hemagglutination inhibition (HAI) was tested using serum drawn at 63 days post-immunization (dpi). HAI was tested against strains included in the LNP20 mixture (H1N1 A / Brisbane / 59 / 2007, H1N1 A / California / 04 / 2009, and H3N2 A / Alaska / 01 / 2021); as well as against heterologous strains not included in the LNP20 mixture (all others). HAI titers >40 were observed for 12 / 16 strains, and >32 for 14 / 16 strains.

[0134] The results are depicted in FIG. 15. This demonstrates the potent and broad humoral response induced by LNP18.

[0135] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.Lists of Embodiments

[0136] The following list of embodiments of the invention are to be considered as disclosing various features of the invention, which features can be considered to be specific to the particular embodiment under which they are discussed, or which are combinable with the various otherfeatures as listed in other embodiments. Thus, simply because a feature is discussed under one particular embodiment does not necessarily limit the use of that feature to that embodiment.

[0137] Embodiment 1. A vaccine composition comprising a plurality of distinct antigens, the vaccine composition comprising at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3 or a sequence at least 70% identical to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16 or a sequence at least 70% identical to SEQ ID NO: 16; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18 or a sequence at least 70% identical to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19 or a sequence at least 70% identical to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21 or a sequence at least 70% identical to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24 or a sequence at least 70% identical to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38 or a sequence at least 70% identical to SEQ ID NO: 38; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39 or a sequence at least 70% identical to SEQ ID NO: 39; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40 or a sequence at least 70% identical to SEQ ID NO: 40; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41 or a sequence at least 70% identical to SEQ ID NO: 41; or an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42 or a sequence at least 70% identical to SEQ ID NO: 42.

[0138] Embodiment 2. The vaccine composition of Embodiment 1, wherein the vaccine composition comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41; or an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42.

[0139] Embodiment 3. The vaccine composition of Embodiment 1 or 2, wherein the plurality of distinct antigens comprises eight distinct antigens, each of which comprises amino acidsequences selected from the group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42 or sequences at least 90% identical thereto.

[0140] Embodiment 4. The vaccine composition of any one of Embodiments 1-3, wherein the plurality of distinct antigens comprises twelve distinct antigens, each of which comprises amino acid sequences selected from the group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42 or sequences at least 90% identical thereto.

[0141] Embodiment 5. The vaccine composition of any preceding Embodiment, wherein the plurality of distinct antigens comprises eighteen distinct antigens, each of which comprises amino acid sequences selected from the group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42 or sequences at least 90% identical thereto.

[0142] Embodiment 6. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3.

[0143] Embodiment 7. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18.

[0144] Embodiment 8. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19.

[0145] Embodiment 9. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21.

[0146] Embodiment 10. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24.

[0147] Embodiment 11. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38.

[0148] Embodiment 12. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39.

[0149] Embodiment 13. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40.

[0150] Embodiment 14. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41.

[0151] Embodiment 15. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42.

[0152] Embodiment 16. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 2.

[0153] Embodiment 17. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 4.

[0154] Embodiment 18. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 5.

[0155] Embodiment 19. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 6.

[0156] Embodiment 20. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 7.

[0157] Embodiment 21. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 8.

[0158] Embodiment 22. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16.

[0159] Embodiment 23. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 17.

[0160] Embodiment 24. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 20.

[0161] Embodiment 25. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 22.

[0162] Embodiment 26. The vaccine composition of any preceding Embodiment, wherein the plurality of distinct antigens comprises eighteen distinct antigens selected from the group consisting of SEQ ID NOS: 2-9, 16-22, 24, 38-42, or sequences at least 70% identical thereto.

[0163] Embodiment 27. The vaccine composition of any preceding Embodiment, wherein the plurality of distinct antigens comprises at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NO: 2-9, 16-22, 24, 38-42, or sequences at least 70% identical thereto.

[0164] Embodiment 28. The vaccine composition of any preceding Embodiment, further comprising a pharmaceutically acceptable carrier.

[0165] Embodiment 29. The vaccine composition of any preceding Embodiment, wherein each antigen of the plurality of distinct antigens is present in the vaccine composition in an amount of less than about 5 micrograms (pg), such as between 1 ng and 5 pg.

[0166] Embodiment 30. The vaccine composition of Embodiment 29, wherein each antigen of the plurality of distinct antigens is present in the vaccine composition in an amount of less than about 3 pg, such as between 1 ng and 3 pg.

[0167] Embodiment 31. The vaccine composition of any preceding Embodiment, wherein the plurality of antigens comprises at 6, at least 8, at least 10, at least 12, at least 14, at least 16 or at least 17 distinct antigens.

[0168] Embodiment 32. The vaccine composition of any preceding Embodiment, wherein the composition has no more than 30, no more than 25, or no more than 20 distinct antigens.

[0169] Embodiment 33. The vaccine composition of any preceding Embodiment, wherein the plurality of antigens are collectively sufficient to elicit an immune response in a subject yet each individual antigen of the plurality of antigens in present in the composition in an amount that is insufficient to elicit in an immune response in a subject.

[0170] Embodiment 34. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises SEQ ID NOS: 2-9, 16-22, 24, and 38-42.

[0171] Embodiment 35. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 108-128, or sequences at least 90% identical thereto.

[0172] Embodiment 36. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 108-128, or sequences at least 95% identical thereto.

[0173] Embodiment 37. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 108-128, or sequences at least 98% identical thereto.

[0174] Embodiment 38. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 108-128, or sequences at least 99% identical thereto.

[0175] Embodiment 39. The vaccine composition of any preceding Embodiment, wherein the vaccine composition comprises twenty-one different sequences selected from the group consisting of SEQ ID NOS: 108-128.

[0176] Embodiment 40. A nucleic acid vaccine composition comprising one or more nucleic acids encoding for a plurality of distinct antigens, wherein the plurality of distinct antigens comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3 or a sequence at least 70% identical to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16 or a sequence at least 70% identical to SEQ ID NO: 16; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18 or a sequence at least 70% identical to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19 or a sequence at least 70% identical to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21 or a sequence at least 70% identical to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24 or a sequence at least 70% identical to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38 or a sequence at least 70% identical to SEQ ID NO: 38. an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39 or a sequence at least 70% identical to SEQ ID NO: 39. an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40 or a sequence at least 70% identical to SEQ ID NO: 40. an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41 or a sequence at least 70% identical to SEQ ID NO: 41; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42 or a sequence at least 70% identical to SEQ ID NO: 42.

[0177] Embodiment 41. The nucleic acid vaccine composition of Embodiment 40, wherein: the plurality of distinct antigens comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24; an antigencomprising an amino acid sequence corresponding to SEQ ID NO: 38; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41; or an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42.

[0178] Embodiment 42. The nucleic acid vaccine composition of Embodiment 40 or 41, wherein the one or more nucleic acids comprise messenger RNA (mRNA).

[0179] Embodiment 43. The nucleic acid vaccine composition of Embodiment 42, wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

[0180] Embodiment 44. The nucleic acid vaccine composition of any one of Embodiments 40-43, wherein each nucleic acid encoding for each distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 5 pg.

[0181] Embodiment 45. The nucleic acid vaccine composition of any one of Embodiments 40-44, wherein each nucleic acid encoding for each distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 3 pg.

[0182] Embodiment 46. The nucleic acid vaccine composition of any one of Embodiments 40-44, wherein the one or more nucleic acids encode eight distinct antigens, each of which is selected from a group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42.

[0183] Embodiment 47. The nucleic acid vaccine composition of any one of Embodiments 40-44, wherein the one or more nucleic acids encode twelve distinct antigens, each of which is selected from a group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42.

[0184] Embodiment 48. The nucleic acid vaccine composition of any one of Embodiments 40-44, wherein the one or more nucleic acids encode eighteen distinct antigens, each of which is selected from a group consisting of SEQ ID NOS: 2-9, 16-22, 24 and 38-42.

[0185] Embodiment 49. The nucleic acid vaccine composition of any one of Embodiments 40-48, wherein the one or more nucleic acids encode for at least five distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 38-42.

[0186] Embodiment 50. The nucleic acid vaccine composition of any one of Embodiments 40-49, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3.

[0187] Embodiment 51. The nucleic acid vaccine composition of any one of Embodiments 40-50, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18.

[0188] Embodiment 52. The nucleic acid vaccine composition of any one of Embodiments 40-51, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19.

[0189] Embodiment 53. The nucleic acid vaccine composition of any one of Embodiments 40-52, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21.

[0190] Embodiment 54. The nucleic acid vaccine composition of any one of Embodiments 40-53, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24.

[0191] Embodiment 55. The nucleic acid vaccine composition of any one of Embodiments 40-54, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38.

[0192] Embodiment 56. The nucleic acid vaccine composition of any one of Embodiments 40-55, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39.

[0193] Embodiment 57. The nucleic acid vaccine composition of any one of Embodiments 40-56, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40.

[0194] Embodiment 58. The nucleic acid vaccine composition of any one of Embodiments 40-57, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41.

[0195] Embodiment 59. The nucleic acid vaccine composition of any one of Embodiments 40-58, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42.

[0196] Embodiment 60. The nucleic acid vaccine composition of any one of Embodiments 40-59, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 2.

[0197] Embodiment 61. The nucleic acid vaccine composition of any one of Embodiments 40-60, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 4.

[0198] Embodiment 62. The nucleic acid vaccine composition of any one of Embodiments 40-61, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 5.

[0199] Embodiment 63. The nucleic acid vaccine composition of any one of Embodiments 40-62, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 6.

[0200] Embodiment 64. The nucleic acid vaccine composition of any one of Embodiments 40-63, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 7.

[0201] Embodiment 65. The nucleic acid vaccine composition of any one of Embodiments 40-64, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 8.

[0202] Embodiment 66. The nucleic acid vaccine composition of any one of Embodiments 40-65, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16.

[0203] Embodiment 67. The nucleic acid vaccine composition of any one of Embodiments 40-66, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 17.

[0204] Embodiment 68. The nucleic acid vaccine composition of any one of Embodiments 40-67, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 20.

[0205] Embodiment 69. The nucleic acid vaccine composition of any one of Embodiments 40-68, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 22.

[0206] Embodiment 70. The nucleic acid vaccine composition of any one of Embodiments 40-69, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 38.

[0207] Embodiment 71. The nucleic acid vaccine composition of any one of Embodiments 40-70, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 39.

[0208] Embodiment 72. The nucleic acid vaccine composition of any one of Embodiments 40-71, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 40.

[0209] Embodiment 73. The nucleic acid vaccine composition of any one of Embodiments 40-72, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 41.

[0210] Embodiment 74. The nucleic acid vaccine composition of any one of Embodiments 40-73, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 42.

[0211] Embodiment 75. The nucleic acid vaccine composition of any one of Embodiments 40-74, wherein the one or more nucleic acids encode for at least eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2-9, 16- 22, 24, 38-42.

[0212] Embodiment 76. The nucleic acid vaccine composition of any one of Embodiments 40-75, wherein the plurality of distinct antigens comprises at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 2-9, 16-22, 24, 38-42.

[0213] Embodiment 77. The nucleic acid vaccine composition of any one of Embodiments 40-76, further comprising a pharmaceutically acceptable carrier.

[0214] Embodiment 78. The nucleic acid vaccine composition of any one of Embodiments 40-77, wherein each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 5 micrograms (pg), such as from about 1 ng to about 5 pg.

[0215] Embodiment 79. The nucleic acid vaccine composition of Embodiment 78, wherein each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 3 pg, such as from about 1 ng to about 3 pg.

[0216] Embodiment 80. The nucleic acid vaccine composition of any one of Embodiments 40-79, wherein the plurality of antigens comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 distinct antigens.

[0217] Embodiment 81. The nucleic acid vaccine composition of any one of Embodiments 40-80, wherein the one or more nucleic acids encode for no more than 30, no more than 25, or no more than 20 distinct antigens.

[0218] Embodiment 82. The nucleic acid vaccine composition of any one of Embodiments 40-81, wherein, upon administration of the vaccine to a subject, the vaccine elicits an immune response in the subject yet none of the plurality of distinct antigens is individually expressed at a level that is sufficient to elicit an immune response in a subject by itself.

[0219] Embodiment 83. The nucleic acid vaccine composition of any one of Embodiments 40-82, wherein the nucleic acid vaccine composition is formulated as a LNP-encapsulated mRNA, DNA, or viral vector.

[0220] Embodiment 84. The nucleic acid vaccine composition of any one of Embodiments 42-83, wherein the one or more nucleic acids encode for SEQ ID NOS: 2-9, 16-22, 24, and 38- 42.

[0221] Embodiment 85. The nucleic acid vaccine composition of any one of Embodiments 40-84, wherein the one or more nucleic acids further encode a transmembrane domain.

[0222] Embodiment 86. The nucleic acid vaccine composition of Embodiment 85, wherein the transmembrane domain comprises any one of SEQ ID NOS: 43-63.

[0223] Embodiment 87. The nucleic acid vaccine composition of any one of Embodiments 40-86, wherein the one or more nucleic acids encode each of SEQ ID NOS: 43-63.

[0224] Embodiment 88. The nucleic acid vaccine composition of any one of Embodiments 40-87, wherein the one or more nucleic acids further encode a signaling peptide.

[0225] Embodiment 89. The nucleic acid vaccine composition of any one of Embodiments 40-88, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64-84.

[0226] Embodiment 90. The nucleic acid vaccine composition of any one of Embodiments 40-89, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64-84.

[0227] Embodiment 91. The nucleic acid vaccine composition of any one of Embodiments 40-90, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-105.

[0228] Embodiment 92. The nucleic acid vaccine composition of any one of Embodiments 40-91, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-105.

[0229] Embodiment 93. The nucleic acid vaccine composition of any one of Embodiments 40-92, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64-83.

[0230] Embodiment 94. The nucleic acid vaccine composition of any one of Embodiments 40-93, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64-83.

[0231] Embodiment 95. The nucleic acid vaccine composition of any one of Embodiments 40-94, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64, 67-79, and 82-83 .

[0232] Embodiment 96. The nucleic acid vaccine composition of any one of Embodiments 40-95, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64, 67-79, and 82- 83.

[0233] Embodiment 97. The nucleic acid vaccine composition of any one of Embodiments 40-94, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-104.

[0234] Embodiment 98. The nucleic acid vaccine composition of any one of Embodiments 40-95, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-104.

[0235] Embodiment 99. The nucleic acid vaccine composition of any one of Embodiments 40-94, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-100.

[0236] Embodiment 100. The nucleic acid vaccine composition of any one of Embodiments 40-95, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-100.

[0237] Embodiment 101. The nucleic acid vaccine composition of any one of Embodiments 40-100, wherein the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 108-128, or sequences at least 90% identical thereto.

[0238] Embodiment 102. The nucleic acid vaccine composition of any one of Embodiments 40-101, wherein the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 108-128, or sequences at least 95% identical thereto.

[0239] Embodiment 103. The nucleic acid vaccine composition of any one of Embodiments 40-102, wherein the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 108-128, or sequences at least 98% identical thereto.

[0240] Embodiment 104. The nucleic acid vaccine composition of any one of Embodiments 40-103, wherein the vaccine composition comprises twenty-one nucleic acid sequences encoding SEQ ID NOS: 108-128, or sequences at least 99% identical thereto.

[0241] Embodiment 105. A vaccine composition comprising a plurality of distinct antigens, the vaccine composition comprising at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3 or a sequence at least 70% identical to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16 or a sequence at least 70% identical to SEQ ID NO: 16; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18 or a sequence at least 70% identical to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19 or a sequence at least 70% identical to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21 or a sequence at least 70% identical to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23 or a sequence at least 70% identical to SEQ ID NO: 23; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24 or a sequence at least 70% identical to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25 or a sequence at least 70% identical to SEQ ID NO: 25; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27 or a sequence at least 70% identical to SEQ ID NO: 27; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29 or a sequence at least 70% identical to SEQ ID NO: 29; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33 or a sequence at least 70% identical to SEQ ID NO: 33; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34 or a sequence at least 70% identical to SEQ ID NO: 34; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36 or a sequence at least 70% identical to SEQ ID NO: 36; and anantigen comprising an amino acid sequence corresponding to SEQ ID NO: 37 or a sequence at least 70% identical to SEQ ID NO: 37.

[0242] Embodiment 106. The vaccine composition of Embodiment 105, wherein: the vaccine composition comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 13; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36; and an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 37.

[0243] Embodiment 107. The vaccine composition of Embodiment 106, wherein the plurality of distinct antigens comprises eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 1-8 or sequences at least 70% identical thereto.

[0244] Embodiment 108. The vaccine composition of any one of Embodiments 105-107, wherein the plurality of distinct antigens comprises eight distinct antigens, wherein the eight distinct antigens correspond to amino acid sequences selected from the group consisting of SEQ ID NOS: 16-23 or sequences at least 70% identical thereto.

[0245] Embodiment 109. The vaccine composition of any one of Embodiments 105-108, wherein the plurality of distinct antigens comprises eight distinct antigens, wherein the eight distinct antigens correspond to amino acid sequences selected from the group consisting of SEQ ID NOS: 1-15 or sequences at least 70% identical thereto.

[0246] Embodiment 110. The vaccine composition of any one of Embodiments 105-109, wherein the plurality of distinct antigens comprises six distinct antigens, wherein the six distinct antigens correspond to amino acid sequences selected from the group consisting of SEQ ID NOS: 16-28 or sequences at least 70% identical thereto.

[0247] Embodiment 111. The vaccine composition of any one of Embodiments 105-110, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3.

[0248] Embodiment 112. The vaccine composition of any one of Embodiments 105-111, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 13.

[0249] Embodiment 113. The vaccine composition of any one of Embodiments 105-112, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18.

[0250] Embodiment 114. The vaccine composition of any one of Embodiments 105-113, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19.

[0251] Embodiment 115. The vaccine composition of any one of Embodiments 105-114, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21.

[0252] Embodiment 116. The vaccine composition of any one of Embodiments 105-115, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23.

[0253] Embodiment 117. The vaccine composition of any one of Embodiments 105-116, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24.

[0254] Embodiment 118. The vaccine composition of any one of Embodiments 105-117, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25.

[0255] Embodiment 119. The vaccine composition of any one of Embodiments 105-118, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27.

[0256] Embodiment 120. The vaccine composition of any one of Embodiments 105-119, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29.

[0257] Embodiment 121. The vaccine composition of any one of Embodiments 105-120, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33.

[0258] Embodiment 122. The vaccine composition of any one of Embodiments 105-121, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34.

[0259] Embodiment 123. The vaccine composition of any one of Embodiments 105-122, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36.

[0260] Embodiment 124. The vaccine composition of any one of Embodiments 105-123, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 37.

[0261] Embodiment 125. The vaccine composition of any one of Embodiments 105-124, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 1.

[0262] Embodiment 126. The vaccine composition of any one of Embodiments 105-125, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 2.

[0263] Embodiment 127. The vaccine composition of any one of Embodiments 105-126, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 4.

[0264] Embodiment 128. The vaccine composition of any one of Embodiments 105-127, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 5.

[0265] Embodiment 129. The vaccine composition of any one of Embodiments 105-128, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 6.

[0266] Embodiment 130. The vaccine composition of any one of Embodiments 105-129, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 7.

[0267] Embodiment 131. The vaccine composition of any one of Embodiments 105-130, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 8.

[0268] Embodiment 132. The vaccine composition of any one of Embodiments 105-131, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16.

[0269] Embodiment 133. The vaccine composition of any one of Embodiments 105-132, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 17.

[0270] Embodiment 134. The vaccine composition of any one of Embodiments 105-133, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 20.

[0271] Embodiment 135. The vaccine composition of any one of Embodiments 105-134, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 22.

[0272] Embodiment 136. The vaccine composition of any one of Embodiments 105-135, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 26.

[0273] Embodiment 137. The vaccine composition of any one of Embodiments 105-136, wherein the vaccine composition comprises an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 28.

[0274] Embodiment 138. The vaccine composition of any one of Embodiments 105-137, wherein the plurality of distinct antigens comprises eighteen distinct antigens selected from the group consisting of SEQ ID NOS: 1-8, 16-23, and 29, or sequences at least 70% identical thereto.

[0275] Embodiment 139. The vaccine composition of any one of Embodiments 105-138, wherein the plurality of distinct antigens comprises at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NO: 1-8, 16-23, and 29, or sequences at least 70% identical thereto.

[0276] Embodiment 140. The vaccine composition of any one of Embodiments 105-139, further comprising a pharmaceutically acceptable carrier.

[0277] Embodiment 141. The vaccine composition of any one of Embodiments 105-140, wherein each antigen of the plurality of distinct antigens is present in the vaccine composition in an amount of less than about 5 micrograms (pg), such as between 1 ng and 5 pg.

[0278] Embodiment 142. The vaccine composition of Embodiment 141, wherein each antigen of the plurality of distinct antigens is present in the vaccine composition in an amount of less than about 3 pg, such as between 1 ng and 3 pg.

[0279] Embodiment 143. The vaccine composition of any one of Embodiments 105-142, wherein the plurality of antigens comprises at 6, at least 8, at least 10, at least 12, at least 14, at least 16 or at least 17 distinct antigens.

[0280] Embodiment 144. The vaccine composition of any one of Embodiments 105-143, wherein the composition has no more than 30, no more than 25, or no more than 20 distinct antigens.

[0281] Embodiment 145. The vaccine composition of any one of Embodiments 105-144, wherein the plurality of antigens are collectively sufficient to elicit an immune response in a subject yet each individual antigen of the plurality of antigens in present in the composition in an amount that is insufficient to elicit in an immune response in a subject.

[0282] Embodiment 146. A nucleic acid vaccine composition comprising one or more nucleic acids encoding for a plurality of distinct antigens, wherein the plurality of distinct antigens comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3 or a sequence at least 70% identical to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 13 or a sequence at least 70% identical to SEQ ID NO: 13; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18 or a sequence at least 70% identical to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19 or a sequence at least 70% identical to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21 or a sequence at least 70% identical to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23 or a sequence at least 70% identical to SEQ ID NO: 23; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24 or a sequence at least 70% identical to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25 or a sequence at least 70% identical to SEQ ID NO: 25; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27 or a sequence at least 70% identical to SEQ ID NO: 27; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29 or a sequence at least 70% identical to SEQ ID NO: 29; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33 or a sequence at least 70% identical to SEQ ID NO: 33; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34 or a sequence at least 70% identical to SEQ ID NO: 34; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36 or a sequence at least 70% identical to SEQ ID NO: 36; and an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 37 or a sequence at least 70% identical to SEQ ID NO: 37.

[0283] Embodiment 147. The nucleic acid vaccine composition of Embodiment 146, wherein: the plurality of distinct antigens comprises at least one of: an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 13; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23; anantigen comprising an amino acid sequence corresponding to SEQ ID NO: 24; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34; an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36; and an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 37.

[0284] Embodiment 148. The nucleic acid vaccine composition of Embodiment 146 or 147, wherein the one or more nucleic acids comprise messenger RNA (mRNA).

[0285] Embodiment 149. The nucleic acid vaccine composition of Embodiment 148, wherein the mRNA is encapsulated in lipid nanoparticles (LNPs).

[0286] Embodiment 150. The nucleic acid vaccine composition of any one of Embodiments 146-149, wherein each nucleic acid encoding for each distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 5 pg.

[0287] Embodiment 151. The nucleic acid vaccine composition of any one of Embodiments 146-150, wherein each nucleic acid encoding for each distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 3 pg.

[0288] Embodiment 152. The nucleic acid vaccine composition of any one of Embodiments 146-151, wherein the one or more nucleic acids encode for at least eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 1-8.

[0289] Embodiment 153. The nucleic acid vaccine composition of any one of Embodiments 146-152, wherein the one or more nucleic acids encode for at least eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 16-23.

[0290] Embodiment 154. The nucleic acid vaccine composition of any one of Embodiments 146-153, wherein the one or more nucleic acids encode for at least eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 1-15.

[0291] Embodiment 155. The nucleic acid vaccine composition of any one of Embodiments 146-154, wherein the one or more nucleic acids encode for at least six distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 16-28.

[0292] Embodiment 156. The nucleic acid vaccine composition of any one of Embodiments 146-155, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 3.

[0293] Embodiment 157. The nucleic acid vaccine composition of any one of Embodiments 146-156, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 13.-I l l-

[0294] Embodiment 158. The nucleic acid vaccine composition of any one of Embodiments 146-157, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 18.

[0295] Embodiment 159. The nucleic acid vaccine composition of any one of Embodiments 146-158, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 19.

[0296] Embodiment 160. The nucleic acid vaccine composition of any one of Embodiments 146-159, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 21.

[0297] Embodiment 161. The nucleic acid vaccine composition of any one of Embodiments 146-160, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 23.

[0298] Embodiment 162. The nucleic acid vaccine composition of any one of Embodiments 146-161, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 24.

[0299] Embodiment 163. The nucleic acid vaccine composition of any one of Embodiments 146-162, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 25.

[0300] Embodiment 164. The nucleic acid vaccine composition of any one of Embodiments 146-163, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 27.

[0301] Embodiment 165. The nucleic acid vaccine composition of any one of Embodiments 146-164, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 29.

[0302] Embodiment 166. The nucleic acid vaccine composition of any one of Embodiments 146-165, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 33.

[0303] Embodiment 167. The nucleic acid vaccine composition of any one of Embodiments 146-166, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 34.

[0304] Embodiment 168. The nucleic acid vaccine composition of any one of Embodiments 146-167, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 36.

[0305] Embodiment 169. The nucleic acid vaccine composition of any one of Embodiments 146-168, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 37.

[0306] Embodiment 170. The nucleic acid vaccine composition of any one of Embodiments 146-169, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 1.

[0307] Embodiment 171. The nucleic acid vaccine composition of any one of Embodiments 146-170, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 2.

[0308] Embodiment 172. The nucleic acid vacScine composition of any one of Embodiments 146-171, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 4.

[0309] Embodiment 173. The nucleic acid vaccine composition of any one of Embodiments 146-172, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 5.

[0310] Embodiment 174. The nucleic acid vaccine composition of any one of Embodiments 146-173, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 6.

[0311] Embodiment 175. The nucleic acid vaccine composition of any one of Embodiments 146-174, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 7.

[0312] Embodiment 176. The nucleic acid vaccine composition of any one of Embodiments 146-175, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 8.

[0313] Embodiment 177. The nucleic acid vaccine composition of any one of Embodiments 146-176, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 16.

[0314] Embodiment 178. The nucleic acid vaccine composition of any one of Embodiments 146-177, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 17.

[0315] Embodiment 179. The nucleic acid vaccine composition of any one of Embodiments 146-178, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 20.

[0316] Embodiment 180. The nucleic acid vaccine composition of any one of Embodiments 146-179, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 22.

[0317] Embodiment 181. The nucleic acid vaccine composition of any one of Embodiments 146-180, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 26.

[0318] Embodiment 182. The nucleic acid vaccine composition of any one of Embodiments 146-181, wherein the one or more nucleic acids encode for an antigen comprising an amino acid sequence corresponding to SEQ ID NO: 28.

[0319] Embodiment 183. The nucleic acid vaccine composition of any one of Embodiments 146-182, wherein the one or more nucleic acids encode for at least eight distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 1-8, 16- 23, or 29.

[0320] Embodiment 184. The nucleic acid vaccine composition of any one of Embodiments 146-183, wherein the plurality of distinct antigens comprises at least 6, at least 8, at least 10, at least 12, at least 14, or at least 16 distinct antigens comprising amino acid sequences selected from the group consisting of SEQ ID NOS: 1-8, 16-23, or 29.

[0321] Embodiment 185. The nucleic acid vaccine composition of any one of Embodiments 146-184, further comprising a pharmaceutically acceptable carrier.

[0322] Embodiment 186. The nucleic acid vaccine composition of any one of Embodiments 146-185, wherein each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 5 micrograms (pg), such as from about 1 ng to about 5 pg.

[0323] Embodiment 187. The nucleic acid vaccine composition of Embodiment 186, wherein each nucleic acid encoding a distinct antigen is present in the nucleic acid vaccine composition in an amount of less than about 3 pg, such as from about 1 ng to about 3 pg.

[0324] Embodiment 188. The nucleic acid vaccine composition of any one of Embodiments 146-187, wherein the plurality of antigens comprises at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 distinct antigens.

[0325] Embodiment 189. The nucleic acid vaccine composition of any one of Embodiments 146-188, wherein the one or more nucleic acids encode for no more than 30, no more than 25, or no more than 20 distinct antigens.

[0326] Embodiment 190. The nucleic acid vaccine composition of any one of Embodiments 146-189, wherein, upon administration of the vaccine to a subject, the vaccine elicits an immuneresponse in the subject yet none of the plurality of distinct antigens is individually expressed at a level that is sufficient to elicit an immune response in a subject by itself.

[0327] Embodiment 191. The nucleic acid vaccine composition of any one of Embodiments 146-190, wherein the nucleic acid vaccine composition is formulated as an LNP-encapsulated mRNA or DNA or viral vector.

[0328] Embodiment 192. The nucleic acid vaccine composition of any one of Embodiments 146-191, wherein the one or more nucleic acids further encode a transmembrane domain.

[0329] Embodiment 193. The nucleic acid vaccine composition of Embodiment 192, wherein the transmembrane domain comprises any one of SEQ ID NOS: 43-63.

[0330] Embodiment 194. The nucleic acid vaccine composition of any one of Embodiments 146-193, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 43-63.

[0331] Embodiment 195. The nucleic acid vaccine composition of any one of Embodiments 146-194, wherein the one or more nucleic acids further encode a signaling peptide.

[0332] Embodiment 196. The nucleic acid vaccine composition of any one of Embodiments 146-195, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64-84.

[0333] Embodiment 197. The nucleic acid vaccine composition of any one of Embodiments 146-196, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64-84.

[0334] Embodiment 198. The nucleic acid vaccine composition of any one of Embodiments 146-197, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-105.

[0335] Embodiment 199. The nucleic acid vaccine composition of any one of Embodiments 146-198, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-105.

[0336] Embodiment 200. The nucleic acid vaccine composition of any one of Embodiments 146-199, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64-83.

[0337] Embodiment 201. The nucleic acid vaccine composition of any one of Embodiments 146-200, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64-83.

[0338] Embodiment 202. The nucleic acid vaccine composition of any one of Embodiments 146-201, wherein the one or more nucleic acids comprise any one of SEQ ID NOS: 64, 67-79, and 82-83 .

[0339] Embodiment 203. The nucleic acid vaccine composition of any one of Embodiments 146-202, wherein the one or more nucleic acids comprise each of SEQ ID NOS: 64, 67-79, and 82-83.

[0340] Embodiment 204. The nucleic acid vaccine composition of any one of Embodiments 146-203, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-104.

[0341] Embodiment 205. The nucleic acid vaccine composition of any one of Embodiments 146-204, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-104.

[0342] Embodiment 206. The nucleic acid vaccine composition of any one of Embodiments 146-205, wherein the one or more nucleic acids encode any one of SEQ ID NOS: 85-100.

[0343] Embodiment 207. The nucleic acid vaccine composition of any one of Embodiments 146-206, wherein the one or more nucleic acids encode each of SEQ ID NOS: 85-100.

[0344] Embodiment 208. A vaccine composition comprising twenty-one distinct antigens, wherein each of the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 108-128, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity thereto.

[0345] Embodiment 209. The vaccine composition of Embodiment 208, wherein each of the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 108-128.

[0346] Embodiment 210. The vaccine composition of Embodiment 208 or 209, wherein each of the twenty-one distinct antigens comprises or consists of a sequence selected from SEQ ID NOS: 85-105

[0347] Embodiment 211. A nucleic acid vaccine composition comprising one or more nucleic acids encoding for at least twenty-one distinct antigens, wherein each the twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 108-128, or a sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0348] Embodiment 212. The nucleic acid vaccine composition of Embodiment 211, wherein each of the at least twenty-one distinct antigens comprises a sequence selected from SEQ ID NOS: 108-128.

[0349] Embodiment 213. The nucleic acid vaccine composition of Embodiment 211 or 212, wherein each of the twenty-one distinct antigens comprises or consists of a sequence selected from SEQ ID NOS: 85-105.

[0350] Embodiment 214. The nucleic acid vaccine composition of any one of Embodiments 211-213, wherein the vaccine composition comprises each of SEQ ID NOS: 64-84.

[0351] Embodiment 215. A method for preventing an influenza infection in a subject, comprising administering to the subject the vaccine composition of any preceding Embodiment.

[0352] Embodiment 216. A method of inducing an immune response in a subject, the method comprising administering to the subject the vaccine composition of any preceding Embodiment.

[0353] Embodiment 217. The method of Embodiment 216, wherein the immune response is a protective immune response.

[0354] Embodiment 218. The method of Embodiment 216 or 217, wherein the vaccine composition is administered to the subject intramuscularly, subcutaneously, intramuscularly, intranasally, or orally.

[0355] Embodiment 219. The method of any one of Embodiments 216-218, wherein the nucleic acid vaccine composition is formulated for delivery as a LNP-encapsulated mRNA or DNA or viral vector.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An influenza A hemagglutinin (HA) polypeptide, comprising an HA1 domain and an HA2 domain, wherein:(a) the amino acid at HA2 position 26 is phenylalanine (F) or tyrosine (Y);(b) the amino acid at HA2 position 51 is alanine (A), leucine (L), or valine (V); and / or(c) the amino acid at HA2 position 103 is alanine (A) or leucine (L); and wherein the numbering of amino acid positions of the HA2 domain is as per SEQ ID NO: 3.

2. The influenza A HA polypeptide of claim 1, wherein:(a) the amino acid at HA2 position 26 is phenylalanine (F) or tyrosine (Y);(b) the amino acid at HA2 position 51 is alanine (A), leucine (L), or valine (V); and(c) the amino acid at HA2 position 103 is alanine (A) or leucine (L).

3. The influenza A HA polypeptide of claim 1, wherein:(a) the amino acid at HA2 position 26 is phenylalanine (F);(b) the amino acid at HA2 position 51 is alanine (A), and(c) the amino acid at HA2 position 103 is alanine (A).

4. The influenza A HA polypeptide of claim 1, wherein:(a) the amino acid at HA2 position 26 is phenylalanine (F);(b) the amino acid at HA2 position 51 is leucine (L), and(c) the amino acid at HA2 position 103 is leucine (L).

5. The influenza A HA polypeptide of claim 1 or claim 2, wherein the influenza A HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81.

6. The influenza A HA polypeptide of any one of claims 1-3, wherein the influenza A HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25- 30, 32-41, 43-52, 54-57, and 64-81.

7. The influenza A HA polypeptide of any one of claims 1-6, wherein the influenza A HA polypeptide exhibits increased trimer stability as compared to an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

8. The influenza A HA polypeptide of any one of claims 1-7, wherein at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the influenza A HA polypeptide assembles into a trimer.

9. The influenza A HA polypeptide of any one of claims 1-8, wherein more of the influenza A HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

10. The influenza A HA polypeptide of any one of claims 1-9, wherein the influenza A HA polypeptide exhibits an absolute increase of trimer formation of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% when expressed on a cell surface than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

11. The influenza A HA polypeptide of any one of claims 1-9, wherein the influenza A HA polypeptide exhibits an absolute increase of trimer formation of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

12. The influenza A HA polypeptide of any one of claims 1-10, wherein the influenza A HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

13. The influenza A HA polypeptide of any one of claims 1-10, wherein the influenza A HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identicalinfluenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

14. The influenza A HA polypeptide of any one of claims 1-12, wherein the influenza A HA polypeptide comprises a transmembrane domain and exhibits at least about 2-fold, about 5- fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression when expressed on a cell surface than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

15. The influenza A hemagglutinin polypeptide of any one of claims 1-14, wherein the amino acid at HA2 position 26 is F.

16. The influenza A hemagglutinin polypeptide of any one of claims 1-14, wherein the amino acid at HA2 position 26 is Y.

17. The influenza A hemagglutinin polypeptide of any one of claims 1-16, wherein the amino acid at HA2 position 51 is A.

18. The influenza A hemagglutinin polypeptide of any one of claims 1-16, wherein the amino acid at HA2 position 51 is L.

19. The influenza A hemagglutinin polypeptide of any one of claims 1-16, wherein the amino acid at HA2 position 51 is V.

20. The influenza A hemagglutinin polypeptide of any one of claims 1-19, wherein the amino acid at HA2 position 103 is A.

21. The influenza A hemagglutinin polypeptide of any one of claims 1-19, wherein the amino acid at HA2 position 103 is L.

22. An influenza A hemagglutinin (HA) polypeptide comprising an HA1 domain and an HA2 domain, wherein: the HA2 domain comprises an amino acid sequence that, relative to SEQ ID NO: 3, comprises one or more substitution mutations at one or more of HA2 position 26, HA2 position 51, and HA2 position 103, and the influenza A HA polypeptide exhibits increased trimer stability and / or cell surface expression when expressed on cell surface as compared to an otherwise identicalinfluenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

23. The influenza A HA polypeptide of claim 22, wherein the one or more substitution mutations comprise a substitution mutation at HA2 position 26.

24. The influenza A HA polypeptide of claim 23, wherein the amino acid at HA2 position 26 is phenylalanine or tyrosine.

25. The influenza HA polypeptide of claim 23 or claim 24, wherein the amino acid at HA2 position 26 is phenylalanine.

26. The influenza HA polypeptide of claim 23 or claim 24, wherein the amino acid at HA2 position 26 is tyrosine.

27. The influenza A HA polypeptide of any one of claims 22-26, wherein the one or more substitution mutations comprise a substitution mutation at HA2 position 51.

28. The influenza A HA polypeptide of any one of claims 22-27, wherein the amino acid at HA2 position 51 is alanine, leucine, or valine.

29. The influenza A HA polypeptide of claim 28, wherein the amino acid at HA2 position 51 is alanine.

30. The influenza A HA polypeptide of claim 28, wherein the amino acid at HA2 position 51 is leucine.

31. The influenza A HA polypeptide of claim 28, wherein the amino acid at HA2 position 51 is valine.

32. The influenza A HA polypeptide of any one of claims 22-31, wherein the one or more substitution mutations comprise a substitution mutation at HA2 position 103.

33. The influenza A HA polypeptide of any one of claims 22-32, wherein the amino acid at HA2 position 103 is alanine or leucine.

34. The influenza A HA polypeptide of claim 33, wherein the amino acid at HA2 position 103 is alanine.

35. The influenza A HA polypeptide of claim 33, wherein the amino acid at HA2 position 103 is leucine.

36. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position26 is phenylalanine and the amino acid HA2 position 51 is leucine.

37. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position26 is phenylalanine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine.

38. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is alanine, and the amino acid at HA2 position 103 is alanine.

39. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is alanine.

40. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position26 is phenylalanine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine.

41. The influenza A HA polypeptide of claim 22, wherein the amino acid at HA2 position 26 is tyrosine, the amino acid HA2 position 51 is leucine, and the amino acid at HA2 position 103 is leucine.

42. The influenza A HA polypeptide of any one of claims 22-41, wherein the influenza A HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81.

43. The influenza A HA polypeptide of any one of claims 22-42, wherein the influenza A HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 4, 5, 10, 11, 18, 25-30, 32-41, 43-52, 54-57, and 64-81.

44. The influenza A HA polypeptide of any one of claims 22-43, wherein more of the influenza A HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

45. The influenza A HA polypeptide of any one of claims 22-44, wherein the influenza A HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than the otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

46. The influenza A HA polypeptide of any one of claims 22-45, wherein the influenza A HA polypeptide exhibits an HAI titer in an hemagglutination inhibition assay that is at least 50, at least 100, or at least 200 units greater than an otherwise identical influenza A HA polypeptide in its soluble format that lacks a transmembrane domain.

47. The influenza A HA polypeptide of any one of claims 22-44, wherein the influenza A HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza A HA polypeptide that has a histidine at HA2 position 26, a lysine at HA2 position 51, and a glutamic acid at HA2 position 103.

48. The influenza A HA polypeptide of any one of the preceding claims, further comprising a transmembrane domain.

49. The influenza A HA polypeptide of any one of the preceding claims, wherein the HAI domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 2.

50. The influenza A HA polypeptide of any one of the preceding claims, wherein the HAI domain comprises SEQ ID NO: 2.

51. A vaccine composition comprising the polypeptide of any one of the preceding claims.

52. A vaccine composition comprising a nucleic acid encoding for the polypeptide of any one of the preceding claims.

53. The vaccine composition of claim 51 or 52, wherein the nucleic acid is messenger RNA (mRNA).

54. The vaccine composition of any one of claims 52-53, wherein the nucleic acid comprises an RNA sequence with at least about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 1- 2150.

55. The vaccine composition of any one of claims 52-54, wherein the nucleic acid comprises an RNA sequence of any one of SEQ ID NOS: 1-2150.

56. The vaccine composition of any one of claims 51-55, further comprising a pharmaceutically acceptable carrier.

57. The vaccine composition of any one of claims 53-56, wherein the nucleic acid is encapsulated in a lipid nanoparticle (LNP).

58. A method of preventing an influenza infection in a subject, the method comprising administering to the subject the vaccine composition of any one of claims 51-57.

59. A method of inducing an immune response in a subject, the method comprising administering to the subject the vaccine composition of any one of claims 51-57.

60. The method of claim 59, wherein the immune response is a protective immune response.

61. The method of any one of claims 58-60, wherein the vaccine composition is administered to the subject intramuscularly, subcutaneously, intramuscularly, intranasally, or orally.

62. The method of any one of claims 58-61, wherein the vaccine composition is formulated for delivery as an LNP-encapsulated mRNA, DNA, or viral vector.

63. A influenza B hemagglutinin (HA) polypeptide, comprising an HA1 and aHA2 domain, wherein:(a) the amino acid at HA2 position 22 is phenylalanine, isoleucine, tryptophan, or tyrosine; and / or(b) the amino acid at HA2 position 27 is phenylalanine, isoleucine, tryptophan, or tyrosine; and / or(c) the amino acid at HA2 position 114 is phenylalanine, isoleucine, tryptophan, or tyrosine; wherein the numbering of amino acid positions of the HA2 is as per SEQ ID NO: 8.

64. The influenza B HA polypeptide of claim 63, wherein:(a) the amino acid at HA2 position 22 is tyrosine.

65. The influenza B HA polypeptide of claim 63 or claim 64, wherein the influenza B HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 9, 26, 42, 31, 59, and 60.

66. The influenza B HA polypeptide of any one of claims 63-65, wherein the influenza B HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 9, 26, 42, 31, 59, and 60.

67. The influenza B HA polypeptide of any one of claims 63-66, wherein the influenza A HA polypeptide exhibits increased trimer stability as compared to an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22.

68. The influenza B HA polypeptide of any one of claims 63-67, wherein more of the influenza B HA polypeptide assembles into a trimer when expressed on a cell surface as compared to an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain.

69. The influenza B HA polypeptide of any one of claims 58-68, wherein the influenza B HA polypeptide comprises a transmembrane domain and exhibits at least about 2-fold, about 5- fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression when expressed on a cell surface than the otherwise identical influenza B HA polypeptide in its soluble format that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22.

70. The influenza B HA polypeptide of any one of claims 58-69, wherein the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain.

71. The influenza B HA polypeptide of any one of claims 58-69, wherein the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identicalinfluenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22.

72. The influenza B HA polypeptide of any one of claims 63-68, wherein the influenza B HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza B HA polypeptide that has a histidine at HA2 position 114 and a histidine at HA2 position 27 and a histidine at HA2 position 22.

73. The influenza B hemagglutinin polypeptide of any one of claims 63-72, wherein the amino acid at HA2 position 114 is tyrosine.

74. The influenza B hemagglutinin polypeptide of any one of claims 63-72, wherein the amino acid at HA2 position 22 is tyrosine.

75. A influenza B hemagglutinin (HA) polypeptide, comprising an HA1 and an HA2 domain, wherein: the HA2 domain comprises an amino acid sequence that, relative to SEQ ID NO:8, comprises one or more substitution mutations at one or more of HA2 position 22, HA2 position 27, HA2 position 114,; and the influenza B HA polypeptide exhibits increased trimer stability and / or cell surface expression when expressed on cell surface compared to an otherwise identical influenza B HA polypeptide that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114.

76. The influenza B HA polypeptide of claim 75, wherein the one or more substitution mutations comprise a substitution mutation at HA2 position 22.

77. The influenza B HA polypeptide of claim 75 or 76, wherein the amino acid at HA2 position 22 is phenylalanine, tyrosine, isoleucine, or tryptophan.

78. The influenza B HA polypeptide of any one of claims 75-77, wherein the amino acid at HA 2 position 22 is phenylalanine.

79. The influenza B HA polypeptide of any one of claims 75-78, wherein the one or more substitution mutations comprise a substitution mutation at HA2 position 27.

80. The influenza B HA polypeptide of claim 79, wherein the amino acid at HA2 position 27 is phenylalanine, tyrosine, isoleucine, or tryptophan.

81. The influenza B HA polypeptide of any one of claims 75-80, wherein the one or more substitution mutations comprise an substitution mutation at HA2 position 114.

82. The influenza B HA polypeptide of claim 81, wherein the amino acid at HA2 position 114 is phenylalanine, tyrosine, isoleucine, or tryptophan.

83. The influenza B HA polypeptide of any one of claims 72-79, wherein the one or more substitution mutations comprise substitution mutations at HA2 position 22, HA2 position 27, and HA2 position 114.

84. The influenza B HA polypeptide of any one of claims 75-83, wherein the amino acid at HA2 position 22 is phenylalanine, the amino acid at HA2 position 27 is phenylalanine, and the amino acid at HA2 position 114 is phenylalanine.

85. The influenza B HA polypeptide of any one of claims 75-84, wherein the one or more substitution mutations comprise substitution mutations at HA2 position 22, HA2 position 238, and HA2 position 384.

86. The influenza B HA polypeptide of any one of claims 75-85, wherein the influenza B HA polypeptide comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 9, 12-17, 19-24, 31, 42, 53, and 58-60.

87. The influenza B HA polypeptide of any one of claims 75-86, wherein the influenza B HA polypeptide comprises an amino acid sequence of any one of SEQ ID NOS: 9, 12-17, 19-24, 31, 42, 53, and 58-60.

88. The influenza B HA polypeptide of any one of claims 63-87, wherein more of the influenza B HA polypeptide assembles into a trimer when expressed on cell surface as compared to an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain.

89. The influenza B HA polypeptide of any one of claims 63-88, wherein the influenza B HA polypeptide exhibits at least about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater cell surface expression when expressed on a cell surface than the otherwise identical influenza B HA polypeptide in its soluble format that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114.

90. The influenza B HA polypeptide of any one of claims 63-89, wherein the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide in its soluble format that lacks a transmembrane domain.

91. The influenza B HA polypeptide of any one of claims 63-89, wherein the influenza B HA polypeptide induces an HAI titer in serum from immunized subjects in an hemagglutination inhibition assay that is at least about 2-fold, at least about 4-fold, at least about 8-fold, at least about 16-fold, at least about 32-fold, at least about 64-fold, at least about 128-fold, at least about 256-fold, at least about 512-fold, or at least about 1024-fold greater than an otherwise identical influenza B HA polypeptide that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114.

92. The influenza B HA polypeptide of any one of claims 63-88, wherein the influenza B HA polypeptide exhibits about 2-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold greater trimer formation when expressed on a cell surface than an otherwise identical influenza B that has histidine at HA2 position 22, histidine at HA2 position 27, histidine at HA2 position 114,.

93. The influenza B HA polypeptide of any one of claims 63-92, further comprising a transmembrane domain.

94. The influenza B HA polypeptide of any one of claims 63-93, wherein the HAI domain comprises an amino acid sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 7.

95. The influenza B HA polypeptide of any one of claims 63-94, wherein the HAI domain comprises SEQ ID NO: 7.

96. A vaccine composition comprising a polypeptide of any one of claims 63-95.

97. A vaccine composition comprising a nucleic acid encoding for the polypeptide of any one of claims 63-95.

98. The vaccine composition of claim 96 or 97, wherein the nucleic acid is messenger RNA (mRNA).

99. The vaccine composition of any one of claims 96-98, wherein the nucleic acid comprises an RNA sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to any one of SEQ ID NOS: 1- 2150.

100. The vaccine composition of any one of claims 96-99, wherein the nucleic acid comprises an RNA sequence of any one of SEQ ID NOS: 1-2150.

101. The vaccine composition of any one of claims 96-100, further comprising a pharmaceutically acceptable carrier.

102. The vaccine composition of any one of claims 96-101, wherein the mRNA is encapsulated in a lipid nanoparticle (LNP).

103. A method of preventing an influenza infection in a subject, the method comprising administering to the subject the vaccine composition of any one of claims 94-102.

104. A method of inducing an immune response in a subject, the method comprising administering to the subject the vaccine composition of any one of claims 94-102.

105. The method of claim 104, wherein the immune response is a protective immune response.

106. The method of any one of claims 103-105, wherein the vaccine composition is administered to the subject intramuscularly, subcutaneously, intramuscularly, intranasally, orally or transdermally.

107. The method of any one of claims 103-106, wherein the vaccine composition is formulated for delivery as a LNP-encapsulated mRNA, DNA, or viral vector.

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