Nucleic acid influenza vaccine and combined respiratory virus vaccine

Abstract

Some aspects of this disclosure relate to vaccines for seasonal influenza viruses (e.g., RNA vaccines (e.g., mRNA vaccines)) and methods of using the vaccines. Also described are combination vaccines for seasonal influenza viruses and other respiratory viruses (e.g., respiratory syncytial viruses and coronaviruses) (e.g., RNA vaccines (e.g., mRNA vaccines)) and methods of using the vaccines.

Description

[Technical Field]

[0001] Related applications This application asserts, under 35 U.S.SC § 119(e), the interests of U.S. Provisional Application No. 63 / 489,707 filed on 10 March 2023, U.S. Provisional Application No. 63 / 518,923 filed on 11 August 2023, and U.S. Provisional Application No. 63 / 582,208 filed on 12 September 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence lists The contents of the electronic sequence listing (M137870260WO00-SEQ-NTJ.xml; size: 400,770 bytes; and creation date: March 8, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Respiratory diseases, encompassing a variety of conditions affecting the gas exchange organs, pose significant health challenges worldwide. Among these, seasonal influenza, caused by influenza A and B viruses, is a common illness with substantial health and economic impacts. The sudden onset of symptoms such as fever, cough, and muscle aches can lead to prolonged absences from work and decreased productivity.

[0004] In addition to influenza, other respiratory viruses, such as human coronaviruses and human respiratory syncytial virus (hRSV), also contribute to the respiratory disease burden. Certain coronavirus strains are generally associated with mild to moderate upper respiratory tract infections. Some strains, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused global health crises, as seen in the COVID-19 pandemic.

[0005] hRSV, a negative-sense single-stranded RNA virus, is another significant concern, particularly for children and the elderly. In these populations, hRSV infection can progress to more serious conditions such as bronchitis or pneumonia. [Overview of the project]

[0006] This invention provides compositions and methods for vaccination against seasonal influenza viruses and, if necessary, other respiratory viruses such as hRSV and SARS-CoV-2. These compositions and methods are at least partially based on the discovery that specific substitutions in the hemagglutinin (HA) protein of influenza B virus (IBV) stabilize the HA protein, resulting in improved cell surface expression and immunogenicity. Stabilization has also been achieved with the HA protein of influenza A virus (IAV). Such influenza virus HA proteins enable the creation of improved influenza vaccines, for example, containing RNA encoding this influenza virus HA protein (e.g., mRNA), or other compositions for vaccination (e.g., viral vectors or protein-based vaccines).

[0007] Therefore, some embodiments relate to an influenza B / Victoria lineage virus HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of the reference influenza B / Victoria lineage virus hemagglutinin (HA) protein, in which case the influenza B / Victoria lineage virus HA protein comprises one or more of the following (a) to (s): (a) Tyrosine at position 381 and valine at position 288, (b) Cysteine ​​at position 27 and cysteine ​​at position 349, (c)Cysteine ​​at position 295 and cysteine ​​at position 328, (d) Cysteine ​​at position 399 and cysteine ​​at position 473, (e) Cysteine ​​at position 422 and cysteine ​​at position 444, (f) Cysteine ​​at position 118 and cysteine ​​at position 216, (g) Cysteine ​​at position 237 and cysteine ​​at position 261, (h)Cysteine ​​at position 363 and cysteine ​​at position 480, (i) Cysteine ​​at position 364 and cysteine ​​at position 483, (j) Cysteine ​​at position 365 and cysteine ​​at position 476, (k)Cysteine ​​at position 366 and cysteine ​​at position 479, (l) Cysteine ​​at position 367 and cysteine ​​at position 483, (m)Cysteine ​​at position 435 and cysteine ​​at position 428, (n)Cysteine ​​at position 494 and cysteine ​​at position 483, (o)Cysteine ​​at position 494 and cysteine ​​at position 480, (p) Proline at position 416, proline at position 417, proline at position 434, and proline at position 433, (q) Proline at position 434 and proline at position 433, (r) Proline at position 515, and proline at position 516, (s) Phenylalanine, ranked 473rd. The positions (a) to (s) are numbered by aligning the amino acid sequence of the reference influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0008] In some embodiments, the influenza B / Victoria lineage virus HA protein comprises one or more of the following (a) to (s): (a) Replacement of H381Y and A288V, (b) Replacement of S27C and Y349C, (c) Replacement of I295C and K328C, (d) Replacement of S399C and H473C, (e) Replacement of L422C and D444C, (f) Substitution of K118C and L216C, (g) Substitution of V237C and D261C, (h) Replacement of G363C and K480C, (i) Replacement of A364C and K483C, (j) Replacement of I365C and A476C, (k) Replacement of A366C and R479C, (l) Replacement of G367C and K483C, (m) Replacement of E435C and A428C, (n) Replacement of N494C and K483C, (o) Replacement of N494C and K480C, (p) Replacement of E416P, L417P, N434P, and H433P, (q) Replacement of N434P and H433P, (r) Replacement of T515P and F516P, and (s) Replacement for H473F.

[0009] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381 and valine at position 288.

[0010] In some embodiments, the influenza B / Victoria strain HA protein includes the substitutions H381Y and A288V.

[0011] In some embodiments, the influenza B / Victoria lineage HA protein further comprises one or more of the following (a) to (r): (a) Replacement of S27C and Y349C, (b) Replacement of I295C and K328C, (c) Replacement of S399C and H473C, (d) Replacement of L422C and D444C, (e) Replacement of K118C and L216C, (f) Replacement of V237C and D261C, (g) Replacement of G363C and K480C, (h) Replacement of A364C and K483C, (i) Replacement of I365C and A476C, (j) Replacement of A366C and R479C, (k) Replacement of G367C and K483C, (l) Replacement of E435C and A428C, (m) Replacement of N494C and K483C, (n) Replacement of N494C and K480C, (o) Replacement of E416P, L417P, N434P, and H433P, (p) Replacement of N434P and H433P, (q) Replacement of T515P and F516P, (r) Replacement of H473F.

[0012] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 422, and cysteine ​​at position 444.

[0013] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 364, and cysteine ​​at position 483.

[0014] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 367, and cysteine ​​at position 483.

[0015] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 494, and cysteine ​​at position 483.

[0016] In some embodiments, the influenza B / Victoria lineage virus HA protein contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0017] Some embodiments relate to an influenza B / Yamagata strain virus hemagglutinin (HA) protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of a reference influenza B / Yamagata strain virus HA protein, in which case the influenza B / Yamagata strain virus HA protein comprises one or more of the following (a) to (i): (a) Cysteine ​​at position 231 and cysteine ​​at position 273, (b) Cysteine ​​at position 295 and cysteine ​​at position 332, (c)Cysteine ​​at position 396 and cysteine ​​at position 510, (d) Cysteine ​​at position 239 and cysteine ​​at position 276, (e) Cysteine ​​at position 367 and cysteine ​​at position 401, (f) Cysteine ​​at position 363 and cysteine ​​at position 404, (g) Cysteine ​​at position 437 and cysteine ​​at position 429, (h)Cysteine ​​at position 451 and cysteine ​​at position 422, and (i) Tyrosine at position 381 and valine at position 290, The positions (a) to (i) are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70.

[0018] In some embodiments, the influenza B / Yamagata lineage virus HA protein comprises one or more of the following (a) to (i): (a) Replacement of A231C and G273C, (b) Replacement of K295C and I332C, (c) Replacement of A396C and L510C, (d) Replacement of V239C and V276C, (e) Replacement of I367C and S401C, (f) Replacement of F363C and E404C, (g) Substitution of E437C and G429C, (h) Replacement of D451C and K422C, and (i) Replacement of H381Y and A290V.

[0019] In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions of tyrosine at position 381 and valine at position 290.

[0020] In some embodiments, the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 239, 276, 451, and 422.

[0021] In some embodiments, the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 367, 401, 451, and 422.

[0022] In some embodiments, the influenza B / Yamagata strain virus HA protein contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0023] Some embodiments relate to an IAV H1 HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, in which case the IAV H1 HA protein comprises one or more of the following (a) to (o): (a) Cysteine ​​at position 391 and cysteine ​​at position 37, (b) Cysteine ​​at position 395 and cysteine ​​at position 36, (c)Cysteine ​​at position 461 and cysteine ​​at position 348, (d) Proline at position 404 and proline at position 416, (e) Isoleucine at position 395 and isoleucine at position 447, (f) Glycine at position 456 and isoleucine at position 402, (g) Cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, and proline at position 416, (h)Cysteine ​​at position 391, cysteine ​​at position 37, isoleucine at position 395, and isoleucine at position 447, (i) Cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, (j) Cysteine ​​at position 395, cysteine ​​at position 36, proline at position 404, and proline at position 416, (k)Cysteine ​​at position 395, cysteine ​​at position 36, glycine at position 456, and isoleucine at position 402, (l) Cysteine ​​at position 461, cysteine ​​at position 348, proline at position 404, and proline at position 416, (m)Cysteine ​​at position 461, cysteine ​​at position 348, isoleucine at position 395, and isoleucine at position 447, (n)cysteine ​​at position 461, cysteine ​​at position 348, glycine at position 456, and isoleucine at position 402, (o) cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, glycine at position 456, and isoleucine at position 402, The positions (a) to (o) are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO: 83.

[0024] In some embodiments, the IAV H1 HA protein comprises one or more of (a) to (o): (a) Replacement of K391C and L37C, (b) Replacement of K395C and V36C, (c) Replacement of N461C and G348C, (d) Replacement of N404P and H416P, (e) Replacement of K395I and E447I, (f) Replacement of D456G and K402I, (g) Replacement of K391C, L37C, N404P, H416P, (h) Replacement of K391C, L37C, K395I, E447I, (i) Replacement of K391C, L37C, D456G, and K402I. (j) Replacement of K395C, V36C, N404P, H416P, (k) Replacement of K395C, V36C, D456G, K402I, (l) Replacement of N461C, G348C, N404P, H416P, (m) Replacement of N461C, G348C, K395I, E447I, (n) Replacement of N461C, G348C, D456G, K402I, and (o) Replacement of K391C, L37C, N404P, H416P, D456G, and K402I.

[0025] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 395, and cysteine ​​at position 36.

[0026] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

[0027] In some embodiments, the IAV H1 HA protein contains glycine at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

[0028] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, and cysteine ​​at position 36.

[0029] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402.

[0030] In some embodiments, the IAV H1 HA protein contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 83.

[0031] Some embodiments relate to an IAV H1 HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, in which case the IAV H1 HA protein comprises one or more of the following (a) to (t): (a) Cysteine ​​at position 410 and cysteine ​​at position 462, (b) Cysteine ​​at position 120 and cysteine ​​at position 419, (c)Cysteine ​​at position 391 and cysteine ​​at position 37, (d) Cysteine ​​at position 395 and cysteine ​​at position 36, (e) Cysteine ​​at position 406 and cysteine ​​at position 430, (f) Cysteine ​​at position 457 and cysteine ​​at position 346, (g) Cysteine ​​at position 461 and cysteine ​​at position 348, (h) Proline at position 404 and proline at position 419, (i) Proline at position 404 and proline at position 416, (j) Proline at position 405 and proline at position 406, (k) Proline at position 415 and proline at position 416, (l) Tyrosine at position 25 and glutamic acid at position 45, (m) Tyrosine at position 370 and tryptophan at position 497, (n) Glycine at position 402, glycine at position 405, and glycine at position 407, (o) Isoleucine at position 395 and isoleucine at position 447, (p) Glycine at position 456 and glycine at position 402, (q) Cysteine ​​at position 442 and cysteine ​​at position 423, (r) Glycine at position 391, (s)Cysteine ​​at position 410, cysteine ​​at position 462, cysteine ​​at position 457, and cysteine ​​at position 346, (t)Cysteine ​​at position 391, cysteine ​​at position 37, phenylalanine at position 370, and phenylalanine at position 455, The positions (a) to (t) are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO: 95.

[0032] In some embodiments, the IAV H1 HA protein comprises one or more of (a) to (t): (a) Replacement of V410C and L462C, (b) Replacement of E120C and K419C, (c) Replacement of K391C and L37C, (d) Replacement of K395C and V36C, (e) Replacement of Q406C and D430C, (f) Replacement of S457C and L346C, (g) Replacement of N461C and G348C, (h) Replacement of N404P and K419P, (i) Replacement of N404P and H416P, (j) Replacement of T405P and Q406P, (k) Replacement of N415P and H416P, (l) Substitution of H25Y and H45E, (m) Replacement of H370Y and K497W, (n) Replacement of K402G, T405G, and F407G, (o) Substitution of K395I and E447I, (p) Replacement of D456G and K402I, (q) Replacement of L442C and N423C, (r) Replacement of K391G, (s) Replacement of V410C, L462C, S457C, L346C, and (t) Replacement of K391C, L37C, H370F, and H455F.

[0033] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 391 and cysteine ​​at position 37.

[0034] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 395 and cysteine ​​at position 36.

[0035] In some embodiments, the IAV H1 HA protein contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 95.

[0036] Some embodiments relate to an IAV H3 HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H3 hemagglutinin (HA) protein, in which case the IAV H3 HA protein comprises one or more of the following (a) to (j): (a) Cysteine ​​at position 40 and cysteine ​​at position 55, (b) Cysteine ​​at position 123 and cysteine ​​at position 421, (c)Cysteine ​​at position 260 and cysteine ​​at position 237, (d) Cysteine ​​at position 392 and cysteine ​​at position 46, (e) Cysteine ​​at position 411 and cysteine ​​at position 428, (f) Proline at position 402, proline at position 421, and proline at position 414, (g) Glycine at position 403, (h) Glycine at position 408 and glycine at position 409, (i) Isoleucine at position 396, and (j) Isoleucine at position 219 and proline at position 504, The positions (a) to (j) are numbered by aligning the reference IAV H3 HA protein amino acid sequence with SEQ ID NO: 82.

[0037] In some embodiments, the IAV H3 HA protein comprises one or more of (a) to (j): (a) Replacement of T40C and A55C, (b) Replacement of S123C and R421C, (c) Replacement of L260C and P237C, (d) Replacement of Q392C and T46C, (e) Replacement of I411C and Y428C, (f) Replacement of G402P, R421P, and E414P, (g) Replacement of K403G, (h) Replacement of F408G and H409G, (i) Substitution of K396I, and (j) Replacement of T219I and H504P.

[0038] In some embodiments, the IAV H3 HA protein contains proline at position 402, proline at position 421, and proline at position 414.

[0039] In some embodiments, the IAV H3 HA protein contains an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 82.

[0040] In some embodiments, the HA protein is a recombinant protein.

[0041] Some aspects relate to nucleic acids encoding the following: (a) Influenza B / Victoria lineage virus HA protein, (b) Influenza B / Yamagata lineage virus HA protein, (c) IAV H1 HA protein, or (d) IAV H3 HA protein.

[0042] In some embodiments, the nucleic acid is DNA, messenger ribonucleic acid (mRNA), circular ribonucleic acid (RNA), or self-amplifying ribonucleic acid (saRNA).

[0043] Some aspects relate to viral vectors, including the following: (a) Influenza B / Victoria lineage virus HA protein, and / or nucleic acid encoding influenza B / Victoria lineage virus HA protein (b) Influenza B / Yamagata lineage virus HA protein, and / or nucleic acid encoding influenza B / Yamagata lineage virus HA protein (c) IAV H1 HA protein, and / or nucleic acid encoding the IAV H1 HA protein, and / or (d) IAV H3 HA protein and / or nucleic acid encoding the IAV H3 HA protein.

[0044] Some aspects relate to vaccines, including the following: (a) HA protein, (b) nucleic acids, or (c) Virus vector.

[0045] In some embodiments, the nucleic acid is mRNA.

[0046] Some embodiments relate to messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an influenza B / Victoria lineage virus HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of the hemagglutinin (HA) protein of a reference influenza B / Victoria lineage virus, in which case the influenza B / Victoria lineage virus HA protein comprises one or more of (a) to (s): (a) Tyrosine at position 381 and valine at position 288, (b) Cysteine ​​at position 27 and cysteine ​​at position 349, (c)Cysteine ​​at position 295 and cysteine ​​at position 328, (d) Cysteine ​​at position 399 and cysteine ​​at position 473, (e) Cysteine ​​at position 422 and cysteine ​​at position 444, (f) Cysteine ​​at position 118 and cysteine ​​at position 216, (g) Cysteine ​​at position 237 and cysteine ​​at position 261, (h)Cysteine ​​at position 363 and cysteine ​​at position 480, (i) Cysteine ​​at position 364 and cysteine ​​at position 483, (j) Cysteine ​​at position 365 and cysteine ​​at position 476, (k)Cysteine ​​at position 366 and cysteine ​​at position 479, (l) Cysteine ​​at position 367 and cysteine ​​at position 483, (m)Cysteine ​​at position 435 and cysteine ​​at position 428, (n)Cysteine ​​at position 494 and cysteine ​​at position 483, (o) Cysteine ​​substitution at position 494 and cysteine ​​at position 480, (p) Proline at position 416, proline at position 417, proline at position 434, and proline at position 433, (q) Proline at position 434 and proline at position 433, (r) Proline at position 515, and proline at position 516, and (s) Phenylalanine, ranked 473rd. The positions (a) to (s) are numbered by aligning the amino acid sequence of the reference influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0047] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381 and valine at position 288.

[0048] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 422, and cysteine ​​at position 444.

[0049] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 364, and cysteine ​​at position 483.

[0050] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 367, and cysteine ​​at position 483.

[0051] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 494, and cysteine ​​at position 483.

[0052] Some embodiments relate to messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an influenza B / Yamagata strain virus HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of the hemagglutinin (HA) protein of the reference influenza B / Yamagata strain virus, in which case the influenza B / Yamagata strain virus HA protein comprises one or more of (a) to (i): (a) Cysteine ​​at position 231 and cysteine ​​at position 273, (b) Cysteine ​​at position 295 and cysteine ​​at position 332, (c)Cysteine ​​at position 396 and cysteine ​​at position 510, (d) Cysteine ​​at position 239 and cysteine ​​at position 276, (e) Cysteine ​​at position 367 and cysteine ​​at position 401, (f) Cysteine ​​at position 363 and cysteine ​​at position 404, (g) Cysteine ​​at position 437 and cysteine ​​at position 429, (h)Cysteine ​​at position 451 and cysteine ​​at position 422, and (i) Tyrosine at position 381 and valine at position 290, The positions (a) to (i) are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70.

[0053] In some embodiments, the influenza B / Yamagata strain virus HA protein contains tyrosine at position 381 and valine at position 290.

[0054] In some embodiments, the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 239, 276, 451, and 422.

[0055] In some embodiments, the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 367, 401, 451, and 422.

[0056] Some embodiments relate to messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, in which case the IAV H1 HA protein comprises one or more of the following (a) to (o): (a) Cysteine ​​at position 391 and cysteine ​​at position 37, (b) Cysteine ​​at position 395 and cysteine ​​at position 36, (c)Cysteine ​​at position 461 and cysteine ​​at position 348, (d) Proline at position 404 and proline at position 416, (e) Isoleucine at position 395 and isoleucine at position 447, (f) Glycine at position 456 and isoleucine at position 402, (g) Cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, and proline at position 416, (h)Cysteine ​​at position 391, cysteine ​​at position 37, isoleucine at position 395, and isoleucine at position 447, (i) Cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, (j) Cysteine ​​at position 395, cysteine ​​at position 36, proline at position 404, and proline at position 416, (k)Cysteine ​​at position 395, cysteine ​​at position 36, glycine at position 456, and isoleucine at position 402, (l) Cysteine ​​at position 461, cysteine ​​at position 348, proline at position 404, and proline at position 416, (m)Cysteine ​​at position 461, cysteine ​​at position 348, isoleucine at position 395, and isoleucine at position 447, (n)cysteine ​​at position 461, cysteine ​​at position 348, glycine at position 456, and isoleucine at position 402, (o) cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, glycine at position 456, and isoleucine at position 402, The positions (a) to (o) are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO: 83.

[0057] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 395, and cysteine ​​at position 36.

[0058] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

[0059] In some embodiments, the IAV H1 HA protein contains glycine at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

[0060] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, and cysteine ​​at position 36.

[0061] In some embodiments, the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402.

[0062] Some embodiments relate to messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, in which case the IAV H1 HA protein comprises one or more of the following (a) to (t): (a) Cysteine ​​at position 410 and cysteine ​​at position 462, (b) Cysteine ​​at position 120 and cysteine ​​at position 419, (c)Cysteine ​​at position 391 and cysteine ​​at position 37, (d) Cysteine ​​at position 395 and cysteine ​​at position 36, (e) Cysteine ​​at position 406 and cysteine ​​at position 430, (f) Cysteine ​​at position 457 and cysteine ​​at position 346, (g) Cysteine ​​at position 461 and cysteine ​​at position 348, (h) Proline at position 404 and proline at position 419, (i) Proline at position 404 and proline at position 416, (j) Proline at position 405 and proline at position 406, (k) Proline at position 415 and proline at position 416, (l) Tyrosine at position 25 and glutamic acid at position 45, (m) Tyrosine at position 370 and tryptophan at position 497, (n) Glycine at position 402, glycine at position 405, and glycine at position 407, (o) Isoleucine at position 395 and isoleucine at position 447, (p) Glycine at position 456 and glycine at position 402, (q) Cysteine ​​at position 442 and cysteine ​​at position 423, (r) Glycine at position 391, (s)Cysteine ​​at position 410, cysteine ​​at position 462, cysteine ​​at position 457, and cysteine ​​at position 346, (t)Cysteine ​​at position 391, cysteine ​​at position 37, phenylalanine at position 370, and phenylalanine at position 455, The positions (a) to (t) are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO: 95.

[0063] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 391 and cysteine ​​at position 37.

[0064] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 395 and cysteine ​​at position 36.

[0065] Some embodiments relate to messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H3 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H3 hemagglutinin (HA) protein, in which case the IAV H3 HA protein comprises one or more of the following (a) to (j): (a) Cysteine ​​at position 40 and cysteine ​​at position 55, (b) Cysteine ​​at position 123 and cysteine ​​at position 421, (c)Cysteine ​​at position 260 and cysteine ​​at position 237, (d) Cysteine ​​at position 392 and cysteine ​​at position 46, (e) Cysteine ​​at position 411 and cysteine ​​at position 428, (f) Proline at position 402, proline at position 421, and proline at position 414, (g) Glycine at position 403, (h) Glycine at position 408 and glycine at position 409, (i) Isoleucine at position 396, and (j) Isoleucine at position 219 and proline at position 504, The positions (a) to (j) are numbered by aligning the reference IAV H3 HA protein amino acid sequence with SEQ ID NO: 82.

[0066] In some embodiments, the IAV H3 HA protein contains proline at position 402, proline at position 421, and proline at position 414.

[0067] Some aspects relate to mRNA vaccines, including the following: (a) mRNA encoding the IAV H1 HA protein, (b) mRNA encoding the IAV H3 HA protein, and (c) mRNA encoding the HA protein of influenza B / Victoria lineage virus.

[0068] In some embodiments, the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381 and valine at position 288.

[0069] In some embodiments, the mRNA vaccine further comprises additional mRNA encoding an additional IAV H3 HA protein.

[0070] In some embodiments, the mRNA vaccine further comprises two or more additional mRNAs, each independently encoding an additional IAV H3 HA protein.

[0071] In some embodiments, the mRNA vaccine further comprises mRNA encoding the influenza B / Yamagata lineage virus HA protein described in any one of claims 45 to 48.

[0072] In some embodiments, the influenza B / Yamagata strain virus HA protein contains tyrosine at position 381 and valine at position 290.

[0073] In some embodiments, the mRNA vaccine does not contain mRNA encoding the influenza B / Yamagata lineage virus HA protein.

[0074] In some embodiments, substantially equal masses of different mRNAs encoding different influenza virus HA proteins are present in the mRNA vaccine.

[0075] In some embodiments, the mRNA vaccine further includes: (i) The first additional mRNA encoding the first IAV neuraminidase (NA) protein of the first IAV NA subtype, (ii) a second additional mRNA encoding a second IAV NA protein of a different IAV NA subtype from the first IAV NA subtype, and (iii) A third additional mRNA encoding the influenza B / Victoria lineage virus NA protein.

[0076] In some embodiments, the second IAV NA subtype is N2, and the mRNA vaccine further includes additional mRNA encoding an additional IAV N2 NA protein.

[0077] In some embodiments, the second IAV NA subtype is N2, and the mRNA vaccine further comprises two or more additional mRNAs, each encoding an additional IAV N2 NA protein.

[0078] In some embodiments, the mRNA vaccine further comprises additional mRNA encoding the influenza B / Yamagata lineage virus NA protein.

[0079] In some embodiments, the mRNA vaccine does not contain mRNA encoding the influenza B / Yamagata lineage virus NA protein.

[0080] In some embodiments, substantially equal masses of different mRNAs encoding different influenza virus NA proteins are present in the mRNA vaccine.

[0081] In some embodiments, substantially equal masses of (a) mRNA encoding the influenza virus HA protein and (b) mRNA encoding the influenza virus NA protein are present in the mRNA vaccine.

[0082] In some embodiments, the mRNA vaccine comprises (a) mRNA encoding the influenza virus HA protein and (b) mRNA encoding the influenza virus NA protein in a mass ratio of 3:1.

[0083] In some embodiments, the mRNA vaccine further comprises mRNA encoding a full-length SARS-CoV-2 spike (S) glycoprotein, which contains one or more proline substitutions relative to SEQ ID NO: 78.

[0084] In some embodiments, the embodiment further includes mRNA encoding a protein containing one or more fragments of the SARS-CoV-2 spike (S) glycoprotein.

[0085] In some embodiments, one or more fragments comprise a fusion protein including (i) the N-terminal domain (NTD) of the SARS-CoV-2 S glycoprotein, (ii) the receptor-binding domain (RBD) of the SARS-CoV-2 S glycoprotein, and (iii) a transmembrane domain.

[0086] In some embodiments, the mRNA vaccine further comprises mRNA encoding a fusion (F) glycoprotein of human respiratory syncytial virus (hRSV), or a fragment of the hRSV F glycoprotein.

[0087] In some embodiments, the mRNA vaccine further comprises a lipid delivery vehicle that contacts the mRNA.

[0088] In some embodiments, the lipid delivery vehicle is a lipid nanoparticle comprising 20-60 mol% ionic lipid, 5-25 mol% non-cationic lipid, 2-4 mol% PEG-modified lipid, and 25-55 mol% sterol.

[0089] In some embodiments, the ionic lipid is a compound of formula (IL * , 1-6 , 2 , N” , * , 4-10 , 1-6 , 1 , 1-6 , 3 , N’ , N , * , N’ , * , N , * , * , N” ) :

Chemical formula

[0090] In some embodiments, ionic lipids are [ka] That is the case.

[0091] In some embodiments, ionic lipids are [ka] That is the case.

[0092] In some embodiments, 0.25 mol% to 1.0 mol% of the PEG-modified lipid is present in the core of the lipid nanoparticles.

[0093] In some embodiments, 2.0 mol% to 2.75 mol% of the PEG-modified lipid is not present in the core of the lipid nanoparticles.

[0094] In some embodiments, the PEG-modified lipid is PEG-DMG or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratricontahexylstearate.

[0095] In some embodiments, each mRNA contains one or more chemically modified nucleotides.

[0096] In some embodiments, each mRNA contains N1-methylpseudridine.

[0097] In some embodiments, substantially all uracil nucleotides in each mRNA are modified to include N1-methylpseudridine.

[0098] In some embodiments, each mRNA contains 5-methylcytidine and 5-methyluridine.

[0099] In some embodiments, substantially all cytosine nucleotides in each mRNA are modified to contain 5-methylcytidine, and substantially all uracil nucleotides in each mRNA are modified to contain 5-methyluridine.

[0100] Some embodiments relate to methods that include administering a vaccine to a subject.

[0101] In some embodiments, subjects have not been vaccinated against the influenza virus for at least six months prior to vaccine administration. [Brief explanation of the drawing]

[0102] [Figure 1A]This graph shows the in vitro binding of polyclonal serum antibodies to the protein expressed from mRNA encoding mutant influenza B virus hemagglutinin 72 hours after transfection of cells using transIT with 250 ng mRNA / 1 × 10⁶ cells. Substitutions in Figure 1A are numbered according to the post-cleavage morphology of the HA protein. [Figure 1B] This graph shows the in vitro binding of an anti-hemagglutinin (CR8059) antibody to the protein expressed from mRNA encoding mutant influenza B virus hemagglutinin 72 hours after transfection of cells using transIT with 250 ng mRNA / 1 × 10⁶ cells. Substitutions in Figure 1B are numbered according to the post-cleavage morphology of the HA protein. [Figure 1C] This graph shows the in vitro binding of an anti-hemagglutinin (CR8059) antibody to the protein expressed from mRNA encoding mutant influenza B virus hemagglutinin 72 hours after transfection of cells using transIT with 100 ng mRNA / 1 × 10⁶ cells. Substitutions in Figure 1C are numbered according to the post-cleavage morphology of the HA protein. [Figure 1D] This graph shows the in vitro binding of polyclonal serum antibodies to the protein expressed from mRNA encoding mutant influenza B virus hemagglutinin 72 hours after transfection of cells using transIT with 100 ng mRNA / 1 × 10⁶ cells. Substitutions in Figure 1D are numbered according to the post-cleavage morphology of the HA protein. [Figure 2]A is a graph showing the antibody binding titer (increase relative to wild type) 21 days after injection from mice injected with a 0.25 μg dose of mRNA encoding mutant influenza B virus hemagglutinin. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. B is a graph showing the antibody binding titer (increase relative to wild type) 36 days after injection from mice injected with a 0.25 μg dose of mRNA encoding mutant influenza B virus hemagglutinin. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 3] A is a graph showing the HAI titer of serum collected 21 days after injection from mice injected with 0.25 μg of mRNA encoding influenza B virus hemagglutinin protein. Substitutions in the figure are numbered according to the morphology after cleavage of the HA protein. B is a graph showing the HAI titer of serum collected 36 days after injection from mice injected with 0.25 μg of mRNA encoding influenza B virus hemagglutinin protein. Substitutions in the figure are numbered according to the morphology after cleavage of the HA protein. [Figure 4] A is a graph showing the antibody binding titer 21 days after injection in mice administered a dose of 0.0625 μg of mRNA encoding mutant IBV hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. B is a graph showing the antibody binding titer 36 days after injection in mice administered a dose of 0.0625 μg of mRNA encoding mutant IBV hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 5]A is a graph showing the HAI titer of serum collected 21 days after injection from mice injected with 0.0625 μg of mRNA encoding influenza B virus hemagglutinin protein. Substitutions in the figure are numbered according to the morphology after cleavage of the HA protein. B is a graph showing the HAI titer of serum collected 36 days after injection from mice injected with 0.0625 μg of mRNA encoding influenza B virus hemagglutinin protein. Substitutions in the figure are numbered according to the morphology after cleavage of the HA protein. [Figure 6] A is a graph showing the antibody binding titer 21 days after injection in mice administered a dose of 0.25 μg of mRNA encoding the mutant IBV hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. B is a graph showing the antibody binding titer 36 days after injection in mice administered a dose of 0.25 μg of mRNA encoding the encoded mutant IBV hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 7] A is a graph showing the antibody binding titer 21 days after injection in mice administered with a dose of 0.0625 μg of mRNA encoding the mutant IBV hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. B is a graph showing the antibody binding titer 36 days after injection in mice administered with a dose of 0.0625 μg of mRNA encoding the mutant B hemagglutinin protein. Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 8] This figure shows in vitro expression data 72 hours after transfecting cells with mRNA encoding mutant influenza B virus hemagglutinin protein (mutations to the protein are shown on the X axis). Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 9]This figure shows the binding titer of IgG antibodies in mice 36 days after administration of mRNA encoding mutant influenza B virus hemagglutinin protein at a dose of 0.25 μg (mutations to the protein are shown on the X axis). Substitutions in the figure are numbered according to the cleavage morphology of the HA protein. [Figure 10A] This graph shows the effect of stabilizing mutations in IBV HA on the in vitro expression of 4×HA. The graph shows the expression levels after staining with CR8059 (an IBV HA-specific antibody). [Figure 10B] This graph shows the effect of stabilizing mutations in IBV HA on the in vitro expression of 4×HA. The graph shows the expression levels after staining with 5E04 (H3 HA-specific antibody). [Figure 10C] This graph shows the effect of stabilizing mutations in IBV HA on the in vitro expression of 4×HA. The graph shows the expression levels after staining with 2B06 (H1 HA-specific antibody). [Figure 11A] This shows the HAI titer present in samples derived from mice administered with either an influenza vaccine (4×HA), a SARS-CoV-2 vaccine (NTD-RBD-HAtm), or a mixed vaccine (NTD-RBD-hAtm) containing mRNA encoding both influenza virus and coronavirus antigens. [Figure 11B] This shows the neutralizing antibody titer present in samples derived from mice administered with either an influenza vaccine (4×HA), a SARS-CoV-2 vaccine (NTD-RBD-HAtm), or a mixed vaccine (NTD-RBD-hAtm) containing mRNA encoding both influenza virus and coronavirus antigens. [Figure 12A]The following mutant influenza B / Victoria lineage virus HA proteins containing the following amino acid mutations are shown to stabilize the amino acid sequence (SEQ ID NO: 71) of the wild-type influenza B / Austria / 1359417 / 2021 (B / Victoria lineage) virus HA protein: (i) H381Y-A288V, (ii) H381Y-A288V-L422C-D444C, (iii) H381Y-A288V-A364C-K483C, (iv) H381Y-A288V-G367C-K483C, and (v) H381Y-288V-N494C-K483C. The figure shows the in vitro surface expression levels of proteins expressed by mRNA encoding either the wild-type or mutant protein after staining with polyclonal serum antibody or CR8059 (influenza B virus HA-specific antibody). [Figure 12B] The following amino acid mutations in the HA protein of mutant influenza B / Victoria lineage viruses, including (i) H381Y-A288V, (ii) H381Y-A288V-L422C-D444C, (iii) H381Y-A288V-A364C-K483C, (iv) H381Y-A288V-G367C-K483C, and (v) H381Y-288V-N494C-K483C are shown to stabilize the HA protein of mutant influenza B / Victoria lineage viruses containing the following amino acid mutations relative to the amino acid sequence (SEQ ID NO: 71) of the wild-type influenza B / Austria / 1359417 / 2021 (B / Victoria lineage) virus: (i) H381Y-A288V-L422C-D444C, (iii) H381Y-A288V-A364C-K483C, (iv) H381Y-A288V-G367C-K483C, and (v) H381Y-288V-N494C-K483C. The figure shows the melting temperatures of soluble proteins in PBS for wild-type and mutant proteins. [Figure 12C]The following mutant influenza B / Victoria lineage virus HA proteins containing the following amino acid mutations are shown to stabilize the HA protein of the wild-type influenza B / Austria / 1359417 / 2021 (B / Victoria lineage) virus HA protein (SEQ ID NO: 71): (i) H381Y-A288V, (ii) H381Y-A288V-L422C-D444C, (iii) H381Y-A288V-A364C-K483C, (iv) H381Y-A288V-G367C-K483C, and (v) H381Y-288V-N494C-K483C. The figure summarizes the selection criteria for each candidate, with asterisks indicating the mutant with the best performance based on polyclonal serum antibody binding, CR8059 antibody binding, melting temperature, and trimer formation. [Figure 13] The mutant influenza B / Yamagata lineage virus HA protein containing the following amino acid mutations stabilizes the amino acid sequence (SEQ ID NO: 70) of the wild-type influenza B / Phuket / 3073 / 2013 (B / Yamagata lineage) virus HA protein: (i) V239C-V276C-D451C-K422C, and (ii) I367C-S401C-D451C-K422C. A indicates the in vitro surface expression level of the protein expressed by a high dose of mRNA (0.5 μg mRNA) encoding either the wild-type or candidate protein after staining with polyclonal serum antibody, CR8059 monoclonal antibody (influenza B virus HA-specific antibody), or monoclonal antibody 042 (influenza B virus HA head-specific antibody). B shows the in vitro surface expression level of proteins expressed by low-dose mRNA (0.1 μg mRNA) encoding either the wild-type or mutant protein after staining with polyclonal serum antibody, CR8059 monoclonal antibody, or monoclonal antibody 042. C shows the melting temperature of soluble proteins in PBS for the wild-type and mutant proteins. [Figure 14A]The mutant IAV H3 HA protein exhibits a stabilizing effect on the amino acid sequence (SEQ ID NO: 82) of the wild-type influenza A / Darwin / 6 / 2021(H3N2) virus HA protein, with the following amino acid mutations: (i) S123C-R421C, Q392C-T46C, and G402-R421P-E414P. The figure shows the in vitro surface expression levels of proteins expressed by mRNA encoding either the wild-type or candidate protein after staining with polyclonal serum antibody or 5E04 monoclonal antibody (influenza A virus H3 HA-specific antibody). [Figure 14B] The mutant IAV H3 HA protein exhibits a stabilizing effect on the amino acid sequence (SEQ ID NO: 82) of the wild-type influenza A / Darwin / 6 / 2021(H3N2) virus HA protein, with the following amino acid mutations: (i) S123C-R421C, Q392C-T46C, and G402-R421P-E414P. The figure shows the melting temperatures of the soluble proteins in PBS for the wild-type and mutant proteins. [Figure 14C] The mutant stabilization effect of mutant IAV H3 HA proteins containing the following amino acid mutations relative to the amino acid sequence (SEQ ID NO: 82) of the wild-type influenza A / Darwin / 6 / 2021(H3N2) virus HA protein is demonstrated: (i) S123C-R421C, Q392C-T46C, and G402-R421P-E414P. The figure summarizes the selection criteria for each, with asterisks indicating the mutant with the best performance based on polyclonal serum antibody binding, 5E04 binding, melting temperature, and trimer formation. [Figure 15]This shows the HA-specific IgG titer of serum collected 21 days post-injection from mice injected with 5 μg / mL mRNA encoding wild-type or mutant IAV H1 HA protein. Candidate amino acid mutations included (i) N404P-H416P, (ii) K395C-V36C, (iii) D456C-D402I-K395C-V36C, (iv) D456C-D402I-K391C-L37C, and (vi) N404P-H416-K391C-L37C-D456G-K402I relative to the amino acid sequence (SEQ ID NO: 83) of the wild-type influenza A / wisconsin / 67 / 2022(H1N1)pdm09 virus HA protein. [Figure 16A] The mutant IAV H1 HA protein containing the following amino acid mutations stabilizes the amino acid sequence (SEQ ID NO: 95) of the wild-type influenza A / Sydney / 5 / 2021(H1N1)pdm09 virus HA protein: (i) K391C-L37C and (ii) K395C-V36C. The figure shows the in vitro surface expression levels of proteins expressed by mRNA encoding either the wild-type or mutant protein after staining with polyclonal serum antibody and 2B06 monoclonal antibody (influenza A virus H1 HA-specific antibody). [Figure 16B] The mutant IAV H1 HA protein exhibits stabilization effects on the amino acid sequence (SEQ ID NO: 95) of the wild-type influenza A / Sydney / 5 / 2021(H1N1)pdm09 virus HA protein, with the following amino acid mutations: (i) K391C-L37C and (ii) K395C-V36C. The figure shows the melting temperatures of the soluble proteins in PBS for the wild-type and candidate proteins. [Figure 16C]The following mutant IAV H1 HA proteins exhibit stabilization effects on the amino acid sequence (SEQ ID NO: 95) of the wild-type influenza A / Sydney / 5 / 2021(H1N1)pdm09 virus HA protein: (i) K391C-L37C and (ii) K395C-V36C. The figure summarizes the selection criteria for each mutant, with asterisks indicating the mutant with the best performance based on polyclonal serum antibody binding, 2B06 binding, melting temperature, and trimer formation. [Figure 17A] The figure shows reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6. The figure shows local reactiongenicity events in all participants (left), young adults (18-49 years old, center), or elderly (50-75 years old, right) who received one of the eight interventions (FLUBLOK®, 4×HA [50 μg], 4×HA / 4×NA (1:1 HA:NA) [50, 100, or 150 μg], or 4×HA / 4×NA (3:1 HA:NA) [25, 50, or 100 μg]). [Figure 17B] The figure shows the reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6. The figure shows systemic reactiongenicity events in all participants (left), young adults (18-49 years old, center), or elderly (50-75 years old, right) who received the eight trial interventions (FLUBLOK®, 4×HA [50 μg], 4×HA / 4×NA (1:1 HA:NA) [50, 100, or 150 μg], or 4×HA / 4×NA (3:1 HA:NA) [25, 50, or 100 μg]. [Figure 17C] The reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6 are shown. The figure shows the HAI titers for H1N1, H3N2, B / Victoria, and B / Yamagata HA proteins for each participant who received the trial intervention, as a multiple increase from day 1 to day 29, depending on the dose of the administered HA component. [Figure 17D]The reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6 are shown. The figure shows the NAI titers for H1N1, H3N2, B / Victoria, and B / Yamagata NA proteins for each participant who received the trial intervention, as a multiple increase from day 1 to day 29, depending on the dose of the administered NA component. [Figure 17E] The reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6 are shown. The figure shows strain-specific hemagglutination and neuraminidase inhibitory titers induced in all participants who received FLUBLOK®, 4×HA (50 μg), or 4×HA / 4×NA (1:1HA:NA) (50 μg). [Figure 17F] The reactiongenicity and immunogenicity data from the Phase 1 clinical trial described in Example 6 are shown. The figure shows strain-specific hemagglutination and neuraminidase inhibitory titers induced in participants aged 50-75 years who were administered FLUBLOK®, 4×HA (50 μg), or 4×HA / 4×NA (3:1HA:NA) (50 μg). [Figure 18] A shows the geometric mean titer (GMT) ratios for each of the four vaccine-compatible strains in adults aged 18 years or older who were administered a 4×HA[50μg]mRNA vaccine containing stabilizing substitutions (such as those present in SEQ ID NOs. 85, 87, 91, and 94) in the encoded HA protein. B shows the GMT ratios for each of the four vaccine-compatible strains in adults aged 18 years or older who were administered a 4×HA[50μg]mRNA vaccine in which the encoded HA protein did not contain substitutions in a previous trial, relative to the wild-type amino acid sequence. [Figure 19A] This figure shows representative alignments of exemplary influenza virus HA protein sequences within IAV HA subtypes and IBV lineages. The figure also shows multiple alignments of the influenza A / (H1N1)pdm09 HA protein. [Figure 19B]This figure shows representative alignments of exemplary influenza virus HA protein sequences within IAV HA subtypes and IBV lineages. The figure also shows multiple alignments of the influenza A / (H3N2)HA protein. [Figure 19C] This figure shows representative alignments of exemplary influenza virus HA protein sequences within IAV HA subtypes and IBV lineages. The figure also shows multiple alignments of influenza B / Victoria lineage virus HA proteins. [Figure 19D] This figure shows representative alignments of exemplary influenza virus HA protein sequences within IAV HA subtypes and IBV lineages. The figure also shows multiple alignments of influenza B / Yamagata lineage virus HA proteins. [Figure 19E] The figure shows pairwise alignments to identify substitution residues in the amino acid sequences of other reference HA proteins. The figure shows pairwise alignments of the HA protein amino acid sequences of influenza B / Brisbane / 60 / 2008 (B / Victoria lineage) virus and influenza B / Austria / 1359417 / 2021 (B / Victoria lineage) virus, SEQ ID NOs. 157 and 71, respectively. Highlights indicate residues H381 and A288 in SEQ ID NOs. 71 and the corresponding residues (after alignment) H384 and A291 in SEQ ID NOs. 157. [Figure 19F] The figure shows pairwise alignment to identify substitution residues in the amino acid sequences of other reference HA proteins. The figure shows pairwise alignment of the amino acid sequences of the HA proteins of influenza A / Beijing / 262 / 1995(H1N1) virus and influenza A / Wisconsin / 67 / 2022(H1N1)pdm09 virus, with highlighting indicating residues L37 and K391 of SEQ ID NO: 83 and the corresponding residues (after alignment) L37 and G390 of SEQ ID NO: 123. [Figure 19G]The figure shows pairwise alignments to identify substitution residues in the amino acid sequences of other reference HA proteins. The figure shows pairwise alignments of influenza B / Austria / 1359417 / 2021 virus HA proteins with and without the signal peptide (SEQ ID NO: 71). [Figure 19H] The figure shows pairwise alignments indicating substitutions present in the amino acid sequences of the modified HA protein. The figure shows the alignment of the IAV H1 HA protein, including the K391C and L37C substitutions present in SEQ ID NO: 94, to the amino acid sequence of the influenza A / Wisconsin / 67 / 2022(H1N1)pdm09 virus HA protein of SEQ ID NO: 83. [Figure 19I] The figure shows pairwise alignments indicating substitutions present in the amino acid sequences of the modified HA protein. The figure shows the alignment of the IAV H3 HA protein, including the G402P, R421P, and E414P substitutions present in SEQ ID NO: 91, to the amino acid sequence of the influenza A / Darwin / 6 / 2021(H3N2) virus HA protein of SEQ ID NO: 82. [Figure 19J] The figure shows pairwise alignments indicating substitutions present in the amino acid sequences of the modified HA protein. The figure shows the amino acid sequence alignment of the influenza B / Victoria lineage HA protein, including the H381Y, A288V, N494C, and K483C substitutions present in SEQ ID NO: 87, to the amino acid sequence of the influenza B / Austria / 1359417 / 2021 virus HA protein of SEQ ID NO: 71. [Figure 19K] The figure shows pairwise alignments indicating substitutions present in the amino acid sequences of the modified HA protein. The figure shows the alignment of the influenza B / Yamagata strain HA protein, including the V239C, V276C, D451C, and K422C substitutions present in SEQ ID NO: 85, to the amino acid sequence of the influenza B / Phuket / 1359417 / 2021 virus HA protein of SEQ ID NO: 70. [Modes for carrying out the invention]

[0103] Respiratory viruses are common pathogens of disease and have a significant impact on global morbidity and mortality. Vaccines against respiratory viruses are designed to stimulate an immune response that protects against the virus. Various types of vaccines exist, including nucleic acid vaccines (e.g., DNA, and RNA, e.g., auto-amplified RNA or mRNA vaccines) that stimulate an immune response using genetic instructions for antigenic polypeptide production. Protein-based vaccines use antigenic polypeptides or fragments thereof derived from either inactivated viruses or purified subunits. Attenuated live vaccines use attenuated live viruses that contain or encode antigenic polypeptides, while viral vector vaccines use viruses to deliver antigenic polypeptides and / or nucleic acids encoding antigenic polypeptides to cells. Mutations that stabilize antigenic polypeptides can enhance the efficacy of these diverse types of vaccines, resulting in improved immune responses and protection against viruses. Some embodiments relate, for example, to compositions and methods in which vaccine efficacy is improved by increased stability of the viral antigen. Preferred compositions include mRNA vaccines.

[0104] Seasonal influenza virus Seasonal influenza is a contagious respiratory illness caused by the influenza virus, resulting in annual outbreaks with symptoms ranging from mild to severe, and potentially leading to complications, especially in high-risk groups. While not theoretically bound, influenza viruses are thought to spread via respiratory droplets and aerosols, but may also spread through contact with surfaces (vectors). Once in the respiratory tract, virions bind to cells via hemagglutinin (HA) protein, which binds to sialic acid receptors on the cell surface. The virions then enter the cell, where they replicate and produce new virions that bud off the cell membrane. NA protein on the virion surface cleaves sialic acid from the cell surface, releasing free virions that can then infect other cells. This process continues until the immune system can clear the infection.

[0105] Influenza viruses belong to the Orthomyxoviridae family and are classified into types A, B, C, and D. Of these, influenza A and B viruses pose a significant health concern for humans.

[0106] Influenza A virus (IAV) can be further classified based on two surface proteins: hemagglutinin (sometimes referred to as H or HA) and neuraminidase (sometimes referred to as N or NA). There are 18 known HA subtypes and 11 known N subtypes. However, only the H1, H2, and H3 subtypes, as well as the N1 and N2 subtypes (e.g., A / (H1N1), A(H1N2), A(H2N2), and A(H3N2)), cause widespread human illness. Frequent mutation and reassortment give rise to the genetic diversity of influenza A viruses, which can lead to novel strains with pandemic potential.

[0107] Influenza B virus (IBV) is not classified into subtypes, but it can be broken down into lineages and strains within those lineages. Currently, two lineages are circulating in humans: B Yamagata (e.g., B / Yamagata / 16 / 1988) and B / Victoria (e.g., B / Victoria / 2 / 1987).

[0108] Table IV-1 shows a non-exclusive list of isolates from the A / (H1N1) subtype, A / (H3N2) subtype, B / Victoria lineage, and B / Yamagata lineage.

[0109] A key challenge in controlling influenza through vaccination is the rapid evolution of the virus, which can alter its antigenic properties and result in evasion of existing immunity (e.g., through infection or vaccination with previously circulating strains). For this reason, influenza vaccines need to be regularly updated to match currently circulating strains. However, a lack of stability related to vaccine antigens can limit the effectiveness of such strain-matched vaccines. Therefore, several embodiments relate to compositions and methods for improving the stability of influenza virus antigenic polypeptides.

[0110] Influenza virus HA proteins may contain one or more mutations compared to a reference influenza virus HA protein. With respect to proteins containing one or more mutations (e.g., substitutions), the "reference protein" (e.g., reference HA protein) refers to the protein into which the mutation is introduced in order to produce the mutated protein.

[0111] The reference HA protein may be the HA protein of an influenza virus isolate. “Influenza virus isolate” or “influenza virus isolate” refers to an influenza virus collected from an infected host and grown in cell culture. The amino acid sequence of the protein of a viral isolate (e.g., HA) may be determined by sequencing the viral RNA from a genomic segment of the isolate (e.g., segment 4) and / or from cells infected with the isolate. As used herein, the reference HA protein amino acid sequence of an influenza virus isolate is the HA protein amino acid sequence encoded by the consensus nucleotide sequence of segment 4 of the isolate. Those skilled in the art will understand that a replicating virus (e.g., in a cell culture) forms a population of virions, each virion containing a genome that may have one or more mutations relative to the consensus nucleotide sequence (or, in the case of a virus with a segmented genome such as influenza virus, a set of consensus nucleotide sequences), and that a viral genome can be defined based on its consensus nucleotide sequence (or, in the case of a segmented viral genome, a set of consensus nucleotide sequences of a genomic segment). See, for example, Domingo et al., Gene. 1985. 40(1):1-8; and Kuroda et al., PLoS ONE. 2010. 5(4):e10256. Even if a small number of genomes within the isolate's virion population may encode amino acid sequences of HA proteins not encoded by the consensus nucleotide sequence of genome segment 4 of the isolate, the amino acid sequence of the isolate's reference HA protein is understood not to encompass the amino acid sequences of such other HA proteins. The reference HA protein may be a modified HA protein not encoded by the consensus nucleotide sequence of genome segment 4 of a natural influenza virus isolate.

[0112] Those skilled in the art will understand that mutations may be applied to the amino acid sequence of any B / Victoria lineage HA protein, B / Yamagata lineage HA protein, H1 HA protein, or H3 HA protein that exist at the time of filing of this specification. Those skilled in the art will also understand that mutations may be applied to the amino acid sequence of any B / Victoria lineage HA protein, B / Yamagata lineage HA protein, H1 HA protein, or H3 HA protein that do not yet exist at the time of filing of this specification. Indeed, due to the ongoing evolution of influenza viruses, public health authorities update the influenza virus isolates recommended for inclusion in seasonal vaccines each year. Therefore, for example, if a particular influenza virus isolate is recommended for inclusion in a seasonal influenza vaccine, those skilled in the art can apply the mutations described below to the HA protein of that influenza virus isolate to generate variants of the HA protein of that recommended isolate.

[0113] Recommendations for candidate vaccine viruses are typically made annually by the World Health Organization (WHO) after reviewing monitoring data and discussing candidate vaccine viruses (usually early in the calendar year, e.g., February). (World Health Organization. (2023, February 24). Recommended composition of influenza virus vaccines for use in the 2023-2024 northern hemisphere influenza season: Questions and answers.)

[0114] For example, the WHO recommended including the following viruses in vaccines for use during the 2023-2024 influenza season in the Northern Hemisphere: Egg-based vaccines: A / Victoria / 4897 / 2022(H1N1)pdm09 virus, A / Darwin / 9 / 2021(H3N2) virus, and Virus of type B / Austria / 1359417 / 2021 (B / Victoria lineage).

[0115] Cell culture-based or recombinant-based vaccines: A / Wisconsin / 67 / 2022(H1N1)pdm09 virus, A / Darwin / 6 / 2021(H3N2) virus, and Virus of type B / Austria / 1359417 / 2021 (B / Victoria lineage). (Same as above).

[0116] For any type of quadrivalent vaccine (egg-based, cell culture-based, or recombinant-based), the WHO recommends including a B / Phuket / 3073 / 2013 (B / Yamagata lineage)-like virus as the B / Yamagata lineage component. (Ibid.) Regarding the term "~-like virus," the recommended vaccine virus is representative of the antigenic group of viruses expected to be widely circulated during the upcoming influenza season, and multiple candidate vaccine viruses possessing antigenically similar HA proteins to the recommended vaccine virus may be available. (Ibid.) The term "~-like virus" is included in recommendations to allow the use of other candidate vaccine viruses during manufacturing. (Ibid.)

[0117] Sequence information of influenza viruses recommended for inclusion in seasonal influenza vaccines (e.g., amino acid sequences of HA and NA proteins and nucleotide sequences of corresponding genomic segments) is usually available in public databases such as GenBank and GISAID.

[0118] Since HA proteins within a given subtype or lineage are similar to HA proteins of other subtypes or lineages, a person skilled in the art will understand that mutations described for H1 HA proteins may be applied to existing or later H1 HA proteins, mutations described for H3 HA proteins may be applied to existing or later H3 HA proteins, mutations described for B / Victoria lineage HA proteins may be applied to existing or later B / Victoria lineage HA proteins, and mutations described for B / Yamagata lineage HA proteins may be applied to existing or later B / Yamagata lineage HA proteins.

[0119] As of the filing date of this specification, seasonal influenza vaccines are quadrivalent and are intended to induce immunity against influenza A / (H1N1)pdm09 virus, influenza A / (H3N2) virus, influenza B / Victoria lineage virus, and influenza B / Yamagata lineage virus. However, recombination of influenza A viruses may lead to the formation of novel IAVs, which may become dominant in the population and replace the predominantly circulating influenza A / (H1N1)pdm09 and A / (H3N2) subtypes. Those skilled in the art will understand that if mutations are disclosed with respect to the H1 HA of the influenza A / (H1N1)pdm09 virus (e.g., influenza A / Wisconsin / 67 / 2022(H1N1)pdm09 and A / Sydney / 5 / 2021(H1N1)pdm09 viruses), such mutations may also be applicable to the H1 HA protein of other IAV subtypes (e.g., A / (H1N2)) that contain H1 HA. Similarly, the mutations disclosed with respect to the H3 HA of the influenza A / (H3N2) virus (e.g., influenza A / Darwin / 6 / 2021(H3N2) virus) may also be applicable to the H3 HA protein of other IAV subtypes that contain H3 HA (e.g., A / (H3N8)).

[0120] The influenza virus HA protein is discussed in the following section titled "Influenza Virus HA Protein". Those skilled in the art will understand that the influenza virus proteins discussed herein are useful in several types of vaccine compositions. In some embodiments, the composition comprises one or more influenza virus proteins. In some embodiments, the composition comprises one or more nucleic acids (e.g., mRNA) encoding one or more influenza virus proteins. These and other vaccine compositions are discussed in the following section titled "Vaccine Compositions".

[0121] Therefore, discussions of influenza virus proteins can also apply to nucleic acids encoding the above influenza virus proteins, unless otherwise evident from the context. Thus, disclosures relating to specific polypeptide mutations also relate to nucleic acids encoding those polypeptides having those mutations, unless otherwise evident from the context. Similarly, disclosures relating to mRNA encoding mutant influenza virus proteins may also relate to mutant influenza virus proteins. Therefore, if a composition containing mRNA encoding an influenza virus protein having a specific mutation is disclosed, a person skilled in the art can infer that the influenza virus protein itself and compositions containing the influenza virus protein are also disclosed. In some embodiments, the influenza virus protein is a recombinant protein. A "recombinant protein" refers to a protein produced in a different organism that does not naturally produce the protein or its variants. Non-limiting examples of organisms that can produce recombinant proteins include bacteria (e.g., Escherichia coli), yeast (e.g., Saccharomyces cerevisiae), and mammalian cells. [Table 1-1] [Table 1-2]

[0122] Influenza virus HA protein Surprisingly, influenza virus HA proteins with one or more mutations (such as substitutions) from the wild-type HA protein sequence were found to exhibit improved stability, surface expression, and / or immunogenicity compared to wild-type influenza virus HA proteins. While not theoretically bound, such mutations in influenza virus HA proteins may stabilize the three-dimensional structure of the HA protein.

[0123] The mutations that may be applicable to the HA protein of influenza viruses are listed in the following Tables HA-1 to HA-5. For clarity, the mutations are described using amino acid numbering corresponding to the specific HA amino acid sequences described (e.g., recent vaccine strains A / Wisconsin / 67 / 2022(H1N1)pdm09 (SEQ ID NO: 83), A / Darwin / 6 / 2021(H3N2) (SEQ ID NO: 82), B / Austria / 1359417 / 2021(B / Victoria lineage) (SEQ ID NO: 71), and B / Phuket / 3073 / 2013(B / Yamagata lineage) (SEQ ID NO: 70), as well as additional strain A / Sydney / 5 / 2021(H1N1)pdm09 (SEQ ID NO: 95)). Those skilled in the art will understand that the mutations disclosed in relation to the described HA amino acid sequence may be applied to other HA amino acid sequences of its IAV HA subtype (e.g., H1, H3) or to other HA amino acid sequences of its IBV lineage (i.e., B / Victoria or B / Yamagata lineage). To apply the numbered mutations disclosed in relation to the described amino acid sequence (e.g., SEQ ID NO: 71) to a different reference HA protein, those skilled in the art may align the amino acid sequence of that different reference HA protein to the described amino acid sequence to which the mutation is numbered. For example, to apply the H381Y substitution (described amino acid sequence) numbered according to SEQ ID NO: 71 to a reference HA protein, those skilled in the art would align the amino acid sequence of the reference HA protein to SEQ ID NO: 71 and introduce tyrosine (Y) to the residue of the reference HA protein corresponding to H381 in SEQ ID NO: 71. An example of such alignment is shown in Figure 19E (amino acid sequence of influenza B / Brisbane / 60 / 2008 virus HA protein of SEQ ID NO: 157), which shows that residue H384 of SEQ ID NO: 157 aligns with residue H381 of SEQ ID NO: 71. Therefore, since residue 384 of SEQ ID NO: 157 aligns with H381 of SEQ ID NO: 71, a person skilled in the art would replace residue 384 of SEQ ID NO: 157 with tyrosine in order to apply the H381Y substitution (or generally a tyrosine substitution at position 381) to SEQ ID NO: 157.Another example is shown in Figure 19F, which illustrates the alignment of the amino acid sequence of the influenza A / Beijing / 262 / 1995(H1N1) virus HA protein of SEQ ID NO: 123 with the amino acid sequence of the influenza A / Wisconsin / 62 / 2022(H1N1)pdm09 virus HA protein. While both SEQ ID NO: 123 and 83 contain the L37 residue, the residue G390 in SEQ ID NO: 123 is aligned with K391 in SEQ ID NO: 83. Therefore, since G390 is aligned with K391 in SEQ ID NO: 83, a person skilled in the art would substitute G390 in SEQ ID NO: 123 with cysteine ​​(C) in order to apply the K391C substitution (numbering according to SEQ ID NO: 83) to the amino acid sequence of SEQ ID NO: 123.

[0124] Influenza virus HA proteins may contain one or more substitutions compared to a reference HA protein. For example, the influenza B / Victoria lineage virus HA protein having the amino acid sequence of SEQ ID NO: 77 contains tyrosine and valine substitutions compared to the sequence of the reference influenza B / Victoria lineage virus HA protein of SEQ ID NO: 71. Those skilled in the art will understand that the substitutions disclosed in relation to the described amino acid sequence (e.g., SEQ ID NO: 71) may be referred to in the form X1[#]X2, where X1 is the amino acid at the [#] position in the described amino acid sequence and X2 is the amino acid introduced to replace X1 at the [#] position. For example, the tyrosine substitution at position 381 may also be called the H381Y substitution, since, when SEQ ID NO: 71 is used as the described amino acid sequence, the histidine (H) at position 381 of SEQ ID NO: 71 is substituted with tyrosine (Y). Such substitutions are sometimes referred to as "X2 substitutions at position [#]", meaning that the protein contains an X2 residue at position [#] (numbered by alignment to the described sequence), regardless of whether the residue at position [#] in the reference amino acid sequence was X1 or a different residue other than X2. Those skilled in the art will understand that substitutions disclosed in the form of X1[#]X2 can also be applied to the reference amino acid sequence in the form of "X2 substitutions at position [#]", regardless of the residue present in the reference amino acid sequence (numbered by alignment to the described sequence). In the preceding paragraph, Sequence ID 71 (HA protein of influenza B / Austria / 1359417 / 2021 (B / Victoria lineage) virus) is used as the reference HA protein amino acid sequence, but the reference HA protein may be the HA protein of another influenza virus isolate, or a modified HA protein not encoded by the genome of the isolate.

[0125] The influenza virus HA protein may contain specific residues at specific positions (these positions are numbered according to the described amino acid sequence (e.g., SEQ ID NO: 71)). A person skilled in the art may align the sequence of the HA protein with the described amino acid sequence to determine whether the HA protein contains specific residues at positions corresponding to specific positions in the described amino acid sequence, similar to determining the positions of substitutions numbered according to the described amino acid sequence. For example, to determine whether the HA protein contains tyrosine at position 381 and valine at position 288 (the positions are numbered by aligning with SEQ ID NO: 71), a person skilled in the art would align the amino acid sequence of the HA protein with SEQ ID NO: 71, determine whether the amino acid of the HA protein aligned to residue 381 of SEQ ID NO: 71 is tyrosine, and determine whether the amino acid of the HA protein aligned to residue 288 of SEQ ID NO: 71 is valine.

[0126] Unless otherwise evident from the context, mutations herein are numbered according to the full-length amino acid sequence of the influenza virus HA protein (e.g., SEQ ID NOs. 70, 71, 82, 83, and 95), with each sequence containing the signal peptide of the HA protein (i.e., residue 1 in each sequence is methionine encoded by the start codon of the ORF encoding the HA protein). Those skilled in the art will understand that other numbering schemes exist to refer to other forms or subunits of the HA protein, e.g., the form after signal peptide cleavage (residue 1 being the N-terminal amino acid after signal peptide cleavage). For example, those skilled in the art will understand that a reference to the A270V+H363Y substitution in the influenza B / Austria / 1359417 / 2021 virus HA protein corresponds to A288V+H381Y in SEQ ID NO: 71, because SEQ ID NO: 71 contains the signal peptide protein at its N-terminus, and after containing the signal peptide, the A and H residues separated by 93 amino acids are located at positions 288 and 381 of SEQ ID NO: 71, respectively.

[0127] Some embodiments relate to HA proteins comprising one or more mutations selected from (i) the introduction of one or more cysteines (at least one introduced cysteine ​​forming a disulfide bond), (ii) one or more proline or glycine substitutions in the B-loop of the HA protein, (iii) one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues, (iv) one or more substitutions of pH-sensitive histidine in the HA protein, and / or (v) mutations or removals of polynucleotide cleavage sites in the HA protein.

[0128] In some embodiments, the HA protein comprises one or more substitutions of pH-sensitive histidine in the HA protein, and one or more substitutions of charged or polar cavity-lining residues to hydrophobic residues.

[0129] In some embodiments, the HA protein includes an interprotomer disulfide bond formed by at least one introduced cysteine ​​and one or more proline substitutions in the B loop.

[0130] In some embodiments, the HA protein includes an interprotomer disulfide bond formed by at least one introduced cysteine, and one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues.

[0131] In some embodiments, the HA protein includes an interprotomer disulfide bond formed by at least one introduced cysteine, one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues, and one or more proline substitutions in the B-loop.

[0132] In some embodiments, the HA protein includes an interprotomeral disulfide bond formed by at least one introduced cysteine ​​and an intraprotomeral disulfide bond formed by at least one introduced cysteine, wherein the introduced cysteine(s) of the interprotomeral disulfide bond are located at different positions from the introduced cysteine(s) of the intraprotomeral disulfide bond.

[0133] In some embodiments, the HA protein comprises a protomer disulfide bond formed by at least one introduced cysteine, one or more substitutions of pH-sensitive histidine in the HA protein, and one or more substitutions of charged or polar cavity-lining residues to hydrophobic residues.

[0134] In some embodiments, the HA protein contains one or more cysteine ​​substitutions such that a disulfide bond is formed between the introduced cysteines. In some embodiments, the disulfide bond is an interprotomeral disulfide bond covalently linking two protomers of an HA protein containing multiple protomers (e.g., three protomers). In some embodiments, the disulfide bond is an intraprotomeral disulfide bond covalently linking two residues of a single protomer of the HA protein. In some embodiments, the disulfide bond is located in the head region of the HA protein (also referred to as the "globular head," "head," or HA1 subunit). In some embodiments, the disulfide bond is located in the stalk region of the HA protein (also referred to as the "stem," "stalk," or HA2 subunit). Translation of an open reading frame encoding a full-length HA protein sequence generates a precursor HA0 polypeptide, which is then cleaved (e.g., by a serine protease) to produce a disulfide-bonded HA1 subunit (N-terminus of the cleavage site) and an HA2 subunit (C-terminus of the cleavage site). See, for example, Wang et al., J Virol. 89(20):10602-10611. The locations of the cleavage sites in IAV H1 HA, IAV H3 HA, influenza B / Victoria lineage virus HA, and influenza B / Yamagata lineage HA proteins are known in the art, and those skilled in the art can identify the residues in a given reference full-length HA protein sequence that correspond to the HA1 and HA2 subunits of that reference HA protein.

[0135] In some embodiments, the HA protein contains one or more proline substitutions in the B-loop of the HA protein. In some embodiments, the HA protein contains one or more glycine substitutions in the B-loop of the HA protein. The HA protein contains multiple subdomains, including a B-loop that enables fusion of the viral envelope with the endosomal membrane. Proline and glycine are among the residues considered helix disruptors, and the introduction of proline or glycine residues can stabilize or destabilize the protein by altering its conformation (LYu et al. 1990. Science. 250(4981), 669-673). In some embodiments, the HA protein contains two, three, or four proline substitutions in the B-loop. The locations of the B-loop in IAV H1 HA, IAV H3 HA, influenza B / Victoria lineage virus HA, and influenza B / Yamagata lineage HA proteins are known in the art, and those skilled in the art can identify the residues corresponding to the B-loop of a given reference full-length HA protein sequence. For example, see Maire et al., Biochim Biophys Acta. 2014. 1838(4): 1153-1168.

[0136] In some embodiments, the HA protein contains one or more substitutions of pH-sensitive histidine residues. The protonation state and charge of certain histidine residues in the influenza virus HA protein are pH-sensitive, and as a result, histidine residues that do not deprotonate at neutral pH may become protonated as the pH of the endosome decreases, and thus become positively charged. Such protonation may result in a conformational change that allows the viral envelope to fuse with the endosomal membrane. For example, the H5 HA1 residue His184 is located near a positively charged residue in the pre-fusion HA protein structure, and its protonation state acts as a molecular switch that triggers a conformational change in HA. See, for example, Kampmann et al., Structure. 2006. 14(10): 1481-1487; Mair et al., J Virol. 2014. 88(22): 13189-13200. Therefore, substitution of one or more pH-sensitive histidines can inhibit or prevent conformational changes that occur at low pH (e.g., transition to the post-fusion conformation), thereby stabilizing the HA protein.

[0137] In some embodiments, the HA protein involves substitution or removal of a polynucleotide cleavage site. Cell infection by influenza virus involves cleavage of immature HA monomers at the cleavage site, but different cleavage motifs of influenza viruses confer distinct tissue and cell specificity to which the virus can replicate, resulting in different pathogenicities (Klenk et al. 1975. Virology. 68:426-439). Highly pathogenic avian influenza (HPAI) viruses typically possess polynucleotide HA cleavage sites (e.g., RKTR (SEQ ID NO: 120) or RKKR (SEQ ID NO: 121)) which are features lacking in low pathogenic avian influenza (LPAI) viruses. In some embodiments, the mutation is a substitution from a polynucleotide cleavage site of the HPAI virus to a protease cleavage site of the LPAI virus. In some embodiments, the mutation is a deletion of a polynucleotide cleavage site. In some embodiments, the mutation is a deletion of an HPAI polynucleotide cleavage site. In some embodiments, the mutation is a substitution from a polynucleotide cleavage site to an amino acid sequence that does not contain a polynucleotide cleavage site. In some embodiments, the mutation is a substitution of one or more arginine residues in the HPAI polybasic cleavage site for one or more nonbasic residues (e.g., glycine and / or serine).

[0138] In some embodiments, the HA protein includes one or more substitutions from polar or charged cavity-lining residues to hydrophobic residues. In some embodiments, the HA protein includes one or more substitutions from polar or charged cavity-lining residues to glycine. The interior of a protein may contain tightly packed side chains that affect the protein's stability, with larger cavities being less stable than narrower cavities (Bueno et al. J Mol Bio. 2006. 358(3): 701-712). Protein stability can be increased by such cavity-filling mutations by substituting small cavity-lining residues for larger hydrophobic residues. Any suitable hydrophobic residue may be used in such substitutions, such as serine, alanine, valine, phenylalanine, histidine, leucine, methionine, valine, or tryptophan.

[0139] Influenza B virus HA protein Some embodiments relate to the HA protein of influenza B virus. The HA protein of influenza B virus may contain one or more mutations (e.g., substitutions) to the amino acid sequence of a reference influenza B virus HA protein. The reference IBV HA protein may be the HA protein of an IBV isolate. The reference IBV HA protein may be a modified IBV HA protein. Non-limiting examples of mutations include (i) the introduction of one or more cysteines (at least one introduced cysteine ​​forming a disulfide bond), (ii) one or more proline or glycine substitutions in the B-loop of the HA protein, (iii) one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues, (iv) one or more substitutions of pH-sensitive histidine in the HA protein, and / or (v) mutations or removal of polynucleotide cleavage sites in the HA protein.

[0140] B / Victoria lineage HA Protein Some embodiments relate to influenza B / Victoria virus HA proteins containing amino acid substitutions compared to a reference influenza B / Victoria virus HA protein. The reference influenza B / Victoria virus HA protein may be the HA protein of an influenza B / Victoria virus isolate. The reference influenza B / Victoria virus HA protein may be a modified influenza B / Victoria virus HA protein. Non-limiting examples of influenza B / Victoria virus isolates include B / Guangdong / 7 / 1997, B / Hong Kong / 330 / 2001, B / Malaysia / 2506 / 2004, B / Brisbane / 60 / 2008, B / Colorado / 06 / 2017, B / Washington / 02 / 2019, and B / Austria / 1359417 / 2021. Representative amino acid sequences of influenza B / Victoria virus HA proteins from previous seasonal influenza vaccines are shown as SEQ ID NOs. 154-160.

[0141] Those skilled in the art will understand that, for example, the mutation disclosed with respect to the described sequence of influenza B / Austria / 1359417 / 2021 virus HA may be applicable to other influenza B / Victoria lineage virus HA proteins or other reference influenza B / Victoria lineage virus HA proteins. For example, when applying an H381Y substitution to an influenza B / Victoria lineage virus HA protein, those skilled in the art would align the amino acid sequence of the reference HA protein to the sequence of the B / Austria / 1359417 / 2021 virus HA protein of SEQ ID NO: 71, and introduce a Y (tyrosine) residue in the amino acid sequence of the reference HA protein that aligns to H381 (histidine at position 381) of SEQ ID NO: 71.

[0142] Exemplary substitutions that may be present in the HA protein of the influenza B / Victoria lineage are shown in Table HA-1 below. If the reference HA protein sequence (e.g., of the isolate) already contains the substitutions mentioned in the table, it will be understood that some embodiments may include the residues listed below without making substitutions at those specific positions. For example, if the isolate already contains tyrosine at position 381, embodiments containing 381Y and 288V can be obtained by substituting only 288V. [Table 2]

[0143] In preferred embodiments, the influenza B / Victoria lineage HA protein includes a tyrosine substitution at position 381 and a valine substitution at position 288 (e.g., H381Y, A288V). These preferred substitutions can be combined with one or more additional substitutions listed in the table above.

[0144] In some embodiments, the influenza B / Victoria virus HA protein includes a tyrosine substitution at position 381 and a valine substitution at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes a tyrosine substitution at position 381 and a valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes the substitutions H381Y and A288V compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The H381Y substitution is a substitution of a pH-sensitive histidine residue, and A290V is a cavity-filling substitution.

[0145] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 27 and 349, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 27 and 349 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions of S27C and Y349C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions of S27C and Y349C result in the formation of an intraprotomer disulfide bond in the head region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0146] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 295 and 328, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 295 and 328 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions I295C and K328C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions I295C and K328C result in the formation of an intraprotomer disulfide bond in the head region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0147] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 399 and cysteine ​​at position 473, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 399 and cysteine ​​substitutions at position 473 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions S399C and H473C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions S399C and H473C result in the formation of an intraprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0148] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 422 and cysteine ​​at position 444, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 422 and cysteine ​​substitutions at position 444 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions L422C and D444C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions L422C and D444C result in the formation of an intraprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein further includes the substitutions H381Y and A288V compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes the substitution of tyrosine at position 381 and valine at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 422, and cysteine ​​at position 444, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0149] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 118 and 216, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 118 and 216 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes K118C and L216C substitutions compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The K118C and L216C substitutions result in the formation of an interprotomer disulfide bond in the head region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0150] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 237 and cysteine ​​at position 261, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 237 and cysteine ​​substitutions at position 261 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions V237C and D261C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions V237C and D261C result in the formation of an interprotomer disulfide bond in the head region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0151] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 363 and cysteine ​​at position 480, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 363 and cysteine ​​substitutions at position 480 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions G363C and K480C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions G363C and K480C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0152] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 364 and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 364 and cysteine ​​substitutions at position 483 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions A364C and K483C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions A364C and K483C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 364, and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0153] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 365 and cysteine ​​at position 476, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 365 and cysteine ​​substitutions at position 476 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions I365C and A476C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions I365C and A476C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0154] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 366 and cysteine ​​at position 479, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 366 and cysteine ​​substitutions at position 479 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions A366C and R479C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions A366C and R479C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0155] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 367 and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 367 and cysteine ​​substitutions at position 483 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions G367C and K483C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions G367C and K483C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 367, and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0156] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 435 and cysteine ​​at position 428, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 435 and cysteine ​​substitutions at position 428 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions E435C and A428C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions E435C and A428C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria lineage virus HA protein further includes the substitutions H381Y and A288V compared to the reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0157] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 494 and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 494 and cysteine ​​substitutions at position 483 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions N494C and K483C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions N494C and K483C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 494, and cysteine ​​at position 483, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria lineage virus HA protein with SEQ ID NO: 71.

[0158] In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​at position 494 and cysteine ​​at position 480, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes cysteine ​​substitutions at position 494 and cysteine ​​substitutions at position 480 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions N494C and K480C compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. The substitutions N494C and K480C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0159] In some embodiments, the influenza B / Victoria virus HA protein includes proline at position 416, proline at position 417, proline at positions 434 and 433, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes proline substitutions at position 416, proline substitutions at position 417, proline substitutions at position 434 and 433 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes substitutions E416P, L417P, N434P and H433P compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. Proline is a residue considered a helix disruptor, and the three-dimensional structure of the protein can be altered by substituting a residue in the B-loop of the HA protein with proline (e.g., E416P, L417P, N434P, and H433P). In some embodiments, the influenza B / Victoria virus HA protein also includes a tyrosine substitution at position 381 and a valine substitution at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes a tyrosine substitution at position 381 and a valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein further includes the substitutions H381Y and A288V compared to the reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0160] In some embodiments, the influenza B / Victoria virus HA protein includes proline at position 434 and proline at position 433, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes proline substitutions at position 434 and proline substitutions at position 433 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes N434P and H433P substitutions compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0161] In some embodiments, the influenza B / Victoria virus HA protein includes proline at position 515 and proline at position 516, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes proline substitutions at position 515 and proline substitutions at position 516 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein includes T515P and F516P substitutions compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. Proline is a residue considered a helix disruptor, and the conformation of the protein can be altered by substituting one or more residues in the B-loop of the HA protein with proline (e.g., T515P and F516P). In some embodiments, the influenza B / Victoria virus HA protein also includes a tyrosine substitution at position 381 and a valine substitution at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes a tyrosine substitution at position 381 and a valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71.In some embodiments, the influenza B / Victoria lineage virus HA protein further includes the substitutions H381Y and A288V compared to the reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0162] In some embodiments, the influenza B / Victoria virus HA protein contains phenylalanine at position 473, the position numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein contains a phenylalanine substitution at position 473 compared to a reference influenza B / Victoria virus HA protein, the position numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein contains an H473F substitution compared to a reference influenza B / Victoria virus HA protein, in which case the numbering of the substituted amino acid in the reference HA protein corresponds to the numbering in SEQ ID NO: 71. Substitution of a pH-sensitive histidine (e.g., H473) can inhibit or prevent conformational changes that occur at low pH (e.g., during viral fusion), thereby stabilizing the HA protein. In some embodiments, the influenza B / Victoria virus HA protein also includes tyrosine at position 381 and valine at position 288, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes tyrosine substitution at position 381 and valine substitution at position 288 compared to a reference influenza B / Victoria virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Victoria virus HA protein with SEQ ID NO: 71. In some embodiments, the influenza B / Victoria virus HA protein further includes H381Y and A288V substitutions compared to a reference HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 71.

[0163] The substitutions described in this subsection for B / Victoria strain HA proteins may be combined. For example, any pair of cysteine ​​substitutions may be combined with one or more proline substitutions and / or pH-sensitive histidine substitutions in the B loop. Pairs of cysteine ​​substitutions that form different disulfide bonds (e.g., interprotomer and intraprotomer, head region and slump region) may also be combined. In some embodiments, influenza B / Victoria strain HA proteins include one or more pH-sensitive histidine substitutions in the HA protein, and one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues.

[0164] In some embodiments, the influenza B / Victoria strain HA protein contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 71.

[0165] B / Yamagata strain HA protein Some embodiments relate to influenza B / Yamagata lineage virus HA proteins containing amino acid substitutions compared to a reference influenza B / Yamagata lineage virus HA protein. The reference influenza B / Yamagata lineage virus HA protein may be the HA protein of an influenza B / Yamagata lineage virus isolate. The reference influenza B / Yamagata lineage virus HA protein may be a modified influenza B / Yamagata lineage virus HA protein. Non-limiting examples of influenza B / Yamagata lineage virus isolates include B / Beijing / 184 / 1993, B / Sichuan / 379 / 1999, B / Shanghai / 361 / 2002, B / Florida / 4 / 2006, B / Wisconsin / 1 / 2010, B / Massachusetts / 2 / 2012, and B / Phuket / 3073 / 2013. Representative amino acid sequences of influenza B / Yamagata lineage virus HA proteins from previous seasonal influenza vaccines are shown as SEQ ID NOs: 164-167.

[0166] Those skilled in the art will understand that, for example, the mutation disclosed with respect to the described sequence of influenza B / Phuket / 3073 / 2013 virus HA may be applied to other influenza B / Yamagata lineage virus HA proteins or other reference influenza B / Victoria lineage virus HA proteins. For example, when applying the A231C substitution to another influenza B / Yamagata lineage virus HA protein, those skilled in the art would align the amino acid sequence of that reference HA protein to the sequence of influenza B / Phuket / 3073 / 2013 virus HA protein of SEQ ID NO: 70 and introduce C into the amino acid residue of the reference HA protein that is aligned to A231 of SEQ ID NO: 70.

[0167] Table HA-2 below shows exemplary substitutions that may be present in the HA protein of the influenza B / Yamagata strain. If the reference HA protein sequence (e.g., of the isolate) already contains the substitutions mentioned in the table, it will be understood that some embodiments may include the residues listed below without making substitutions at those specific positions. For example, if the isolate already contains tyrosine at position 381, embodiments containing 381Y and 290V can be obtained by substituting only 290V. [Table 3]

[0168] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 231 and 273, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 231 and 273 compared to a reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions A231C and G273C compared to a reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions A231C and G273C result in the formation of an intraprotomer disulfide bond in the head region of the HA protein.

[0169] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 295 and 332, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 295 and 332 compared to a reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes K295C and I332C substitutions compared to a reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The K295C and I332C substitutions result in the formation of an intraprotomer disulfide bond in the head region of the HA protein.

[0170] In some embodiments, the influenza B / Yamagata strain HA protein includes cysteine ​​at position 396 and cysteine ​​at position 510, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain HA protein includes cysteine ​​substitutions at position 396 and cysteine ​​substitutions at position 510 compared to a reference influenza B / Yamagata strain HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain HA protein includes substitutions A396C and L510C compared to a reference influenza B / Yamagata strain HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions A396C and L510C result in the formation of an intraprotomer disulfide bond in the stalk region of the HA protein.

[0171] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 239 and cysteine ​​at position 276, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 239 and cysteine ​​substitutions at position 276 compared to a reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions V239C and V276C compared to a reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions V239C and V276C result in the formation of an interprotomer disulfide bond in the head region of the HA protein. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 239, 276, 451, and 422, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70.

[0172] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 367 and cysteine ​​at position 401, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 367 and cysteine ​​substitutions at position 401 compared to the reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions I367C and S401C compared to the reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions I367C and S401C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 367, 401, 451, and 422, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70.

[0173] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 363 and cysteine ​​at position 404, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 363 and cysteine ​​substitutions at position 404 compared to the reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions F363C and E404C compared to the reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions F363C and E404C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein.

[0174] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 437 and 429, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 437 and 429 compared to the reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes substitutions E437C and G429C compared to the reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The substitutions E437C and G429C result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein.

[0175] In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​at position 451 and 422, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes cysteine ​​substitutions at position 451 and 422 compared to the reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes D451C and K422C substitutions compared to the reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. The D451C and K422C substitutions result in the formation of an interprotomer disulfide bond in the stalk region of the HA protein.

[0176] In some embodiments, the influenza B / Yamagata strain virus HA protein includes a tyrosine substitution at position 381 and a valine substitution at position 290, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes a tyrosine substitution at position 381 and a valine substitution at position 290 compared to a reference influenza B / Yamagata strain virus HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70. In some embodiments, the influenza B / Yamagata strain virus HA protein includes the substitutions H381Y and A290V compared to a reference influenza B / Yamagata strain virus HA protein, in which case the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70. H381Y is a substitution that replaces a pH-sensitive histidine residue, and A290V is a cavity-filling substitution.

[0177] The substitutions described in this subsection relating to the B / Yamagata strain HA protein may be combined. For example, any pair of cysteine ​​substitutions may be combined with one or more pH-sensitive histidine substitutions and / or cavity-filling substitutions. Pairs of cysteine ​​substitutions forming different disulfide bonds (e.g., interprotomer and intraprotomer, head region and slump region) may also be combined. In some embodiments, the influenza B / Yamagata strain HA protein includes one or more pH-sensitive histidine substitutions in the HA protein, and one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues.

[0178] In some embodiments, the influenza B / Yamagata strain virus HA protein includes the substitutions H381Y and A290V compared to the reference influenza B / Yamagata strain virus HA protein, in which case, in addition to one or more other substitutions described in this section, the numbering of the substituted amino acids in the reference HA protein corresponds to the numbering in SEQ ID NO: 70.

[0179] In some embodiments, the influenza B / Yamagata strain HA protein contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 70.

[0180] Influenza A virus HA protein Some embodiments relate to the HA protein of influenza A virus. The HA protein of influenza A virus may contain one or more mutations (e.g., substitutions) to the amino acid sequence of the HA protein of the influenza A virus isolate.

[0181] H1 HA protein Some embodiments relate to IAV H1 HA proteins containing amino acid substitutions relative to a reference IAV H1 HA protein. The reference IAV H1 HA protein may be the HA protein of an isolate of an influenza A virus of an IAV subtype containing the H1 HA protein, such as the influenza A / (H1N1)pdm09 virus. The H1 HA protein is present in, for example, the A / (H1N1)pdm09 subtype influenza A virus, but other IAV subtypes that express the H1 HA protein have been identified (such as the H1N2 subtype, which is unique to pigs and can also be observed in humans). Non-exclusive examples of influenza A / (H1N1) pdm09 virus isolates (and related influenza A / (H1N1) virus isolates preceding the 2009 influenza A / (H1N1) virus pandemic) include A / Beijing / 262 / 1995(H1N1), A / New Caledonia / 20 / 1999(H1N1), A / Solomon Islands / 3 / 2006(H1N1), A / Brisbane / 59 / 2007(H1N1), A / California / 7 / 2009(H1N1)pdm09, and A / Michigan. Examples include / 45 / 2015(H1N1)pdm09, A / Brisbane / 02 / 2018(H1N1)pdm09, A / Guangdong-Maonan / SWL1536 / 2019(H1N1)pdm09, A / Hawaii / 70 / 2019(H1N1)pdm09, A / Victoria / 2570 / 2019(H1N1)pdm09, A / Wisconsin / 588 / 2019(H1N1)pdm09, A / Victoria / 4897 / 2022(H1N1)pdm09, and A / Wisconsin / 67 / 2022(H1N1)pdm09. The representative amino acid sequences of the influenza A / (H1N1)pdm09 virus HA protein used in previous seasonal influenza vaccines are shown as SEQ ID NOs. 122-135.

[0182] Those skilled in the art will understand that, for example, the mutation disclosed with respect to the described sequence of influenza A / wisconsin / 67 / 2022(H1N1)pdm09 virus HA may be applicable to other IAV H1 HA proteins. For example, when applying a K391C substitution to another IAV H1 HA protein, those skilled in the art would align the amino acid sequence of that reference HA protein to the sequence of influenza A / wisconsin / 67 / 2022(H1N1)pdm09 virus HA protein of SEQ ID NO: 83, and introduce C at the residue of the amino acid sequence of the reference HA protein that aligns to K391 in SEQ ID NO: 83. Similarly, the mutation disclosed with respect to the reference sequence of influenza A / sydney / 5 / 2021(H1N1)pdm09 virus HA may be applied to a different reference HA protein by aligning the amino acid sequence of the reference HA protein to the amino acid sequence of SEQ ID NO: 95. Such alignment allows a person skilled in the art to identify residues in the amino acid sequence of a reference HA protein that correspond to mutant residues in the influenza A / sydney / 5 / 2021(H1N1)pdm09 virus HA protein (for example, identifying the residue in the amino acid sequence of the reference HA protein that corresponds to V410 in the amino acid sequence of the A / sydney / 5 / 2021(H1N1)pdm09 virus HA protein, and determining where to introduce C when applying the V410C substitution).

[0183] Exemplary substitutions that may be present in the IAV H1 HA protein are shown in Tables HA-3 and HA-4 below. If the reference HA protein sequence (e.g., of the isolate) already contains the substitutions mentioned in the table, it will be understood that some embodiments may include the residues listed below without making substitutions at those specific positions. For example, if the isolate already contains isoleucine at position 395, embodiments containing 395I and 447I can be obtained by substituting only 447I. [Table 4] [Table 5]

[0184] Some embodiments relate to an IAV H1 HA protein having one or more substitutions relative to a reference H1 HA protein, where the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering of Sequence ID No. 83, which represents the HA protein of the influenza A / wisconsin / 67 / 2022(H1N1)pdm09 virus isolate.

[0185] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391 and position 37, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391 and position 37 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes K391C and L37C substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. The K391C and L37C substitutions result in the formation of protomer-interprotomer disulfide bonds in the stalk region of the HA protein.

[0186] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 395 and cysteine ​​at position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 395 and cysteine ​​substitutions at position 36 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes K395C and V36C substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. The K395C and V36C substitutions result in the formation of protomer disulfide bonds in the stalk region of the HA protein.

[0187] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 461 and 348, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 461 and 348 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains substitutions of N461C and G348C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. The substitutions of N461C and G348C result in the formation of protomer disulfide bonds in the stalk region of the HA protein.

[0188] In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains substitutions N404P and H416P compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. N404P and H416P are proline substitutions within the B-loop of the HA protein, stabilizing the HA protein by altering its conformation. In some embodiments, the IAV H1 HA protein includes proline at position 404, proline at position 416, cysteine ​​at position 395, and cysteine ​​at position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes proline at position 404, proline at position 416, cysteine ​​at position 391, and cysteine ​​at position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes proline at position 404, proline at position 416, cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83.

[0189] In some embodiments, the IAV H1 HA protein contains isoleucine at position 395 and isoleucine at position 447, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains isoleucine substitutions at position 395 and isoleucine substitutions at position 447 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains substitutions of K395I and E447I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. The substitutions of K395I and E447I are replacements of charged lysine and glutamate residues with hydrophobic isoleucine residues.

[0190] In some embodiments, the IAV H1 HA protein contains glycine at position 456 and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains glycine substitution at position 456 and isoleucine substitution at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein contains D456G and K402I substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. The D456G and K402I substitutions are replacements of charged aspartic acid and lysine residues with uncharged glycine and uncharged hydrophobic isoleucine residues. In some embodiments, the IAV H1 HA protein includes glycine at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83.

[0191] In some embodiments, the amino acid substitutions include glycine at position 456 and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the influenza B / Victoria lineage virus HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes a cysteine ​​residue at position 456 and isoleucine substitutions at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes the substitutions D456C and K402I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83.

[0192] In some embodiments, the IAV H1 HA protein contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 83.

[0193] Some embodiments relate to an IAV H1 HA protein having one or more substitutions relative to a reference H1 HA protein, where the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in Sequence ID No. 95, which represents the HA protein of the influenza A / Sydney / 5 / 2021(H1N1)pdm09 virus isolate.

[0194] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 410 and 462, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 410 and 462 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains substitutions of V410C and L462C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions of V410C and L462C result in the formation of intraprotomer disulfide bonds in the HA protein.

[0195] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 120 and 419, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 120 and 419 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes substitutions of E120C and K419C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions of E120C and K419C result in the formation of protomer-interprotomer disulfide bonds in the HA protein.

[0196] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 391 and position 37, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 391 and position 37 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains K391C and L37C substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The K391C and L37C substitutions result in the formation of protomer disulfide bonds in the HA protein.

[0197] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 395 and position 36, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 395 and position 36 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains K395C and V36C substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The K395C and V36C substitutions result in the formation of protomer disulfide bonds in the HA protein.

[0198] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 406 and 430, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 406 and 430 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains substitutions of Q406C and D430C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions of Q406C and D430C result in the formation of protomer disulfide bonds in the HA protein.

[0199] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 457 and 346, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 457 and 346 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains substitutions at S457C and L346C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions at S457C and L346C result in the formation of protomer disulfide bonds in the HA protein.

[0200] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 461 and 348, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 461 and 348 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains substitutions of N461C and G348C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions of N461C and G348C result in the formation of protomer disulfide bonds in the HA protein.

[0201] In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 419, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 419 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains substitutions of N404P and K419P compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The substitutions of N404P and K419P are proline substitutions in the B loop of the HA protein.

[0202] In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains proline substitutions at position 404 and position 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains N404P and H416P substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The N404P and H416P substitutions are proline substitutions in the B loop of the HA protein.

[0203] In some embodiments, the IAV H1 HA protein contains proline substitutions at positions 405 and 406, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains proline substitutions at positions 405 and 406 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains T405P and Q406P substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The T405P and Q406P substitutions are proline substitutions in the B-loop of the HA protein.

[0204] In some embodiments, the IAV H1 HA protein contains proline substitutions at positions 415 and 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains proline substitutions at positions 415 and 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains N415P and H416P substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The N415P and H416P substitutions are proline substitutions in the B-loop of the HA protein.

[0205] In some embodiments, the IAV H1 HA protein contains tyrosine at position 25 and glutamic acid at position 45, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains tyrosine substitution at position 25 and glutamic acid substitution at position 45 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains H25Y and H45E substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The H25Y and H45E substitutions are pH-sensitive histidine substitutions.

[0206] In some embodiments, the IAV H1 HA protein contains tyrosine at position 370 and tryptophan at position 497, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains tyrosine substitution at position 370 and tryptophan substitution at position 497 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains H370Y and K497W substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The H370Y substitution is a pH-sensitive histidine substitution, and the K497W substitution is a cavity-filling substitution.

[0207] In some embodiments, the IAV H1 HA protein includes glycine at position 402, 405, and 407, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes glycine substitutions at position 402, 405, and 407 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes K402G, T405G, and F407G substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The K402G, T405G, and F407G substitutions are glycine substitutions in the B-loop of the HA protein.

[0208] In some embodiments, the IAV H1 HA protein contains isoleucine at position 395 and position 447, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains isoleucine substitutions at position 395 and position 447 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains K395I and E447I substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The K395I and E447I substitutions are cavity-filling substitutions. In some embodiments, the IAV H1 HA protein contains glycine at position 456 and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains glycine substitution at position 456 and isoleucine substitution at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains D456G and K402I substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The D456G substitution is a glycine substitution in the B loop, and the K402I substitution is a cavity-filling substitution.

[0209] In some embodiments, the IAV H1 HA protein contains cysteine ​​at position 442 and position 423, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains cysteine ​​substitutions at position 442 and position 423 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains L442C and N423C substitutions compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The L442C and N423C substitutions result in the formation of protomer disulfide bonds in the HA protein.

[0210] In some embodiments, the IAV H1 HA protein contains a glycine substitution at position 391, the position of which is numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains a glycine substitution at position 391 compared to the reference IAV H1 HA protein, the position of which is numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein contains a K391G substitution compared to the reference IAV H1 HA protein, the numbering of the substituted amino acid in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95. The K391G substitution is a glycine substitution in the B loop of the HA protein.

[0211] In some embodiments, the IAV H1 HA protein contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 95.

[0212] The substitutions described in this subsection for the IAV H1 HA protein may be combined. For example, any pair of cysteine ​​substitutions may be combined with one or more proline substitutions in the B-loop, glycine substitutions in the B-loop, pH-sensitive histidine substitutions, and / or cavity-filling substitutions. Pairs of cysteine ​​substitutions that form different disulfide bonds (e.g., interprotomer and intraprotomer, head region and slump region) may also be combined. Preferred combinations of substitutions are discussed below.

[0213] In some embodiments, the IAV H1 HA protein includes an interprotomeral disulfide bond formed by at least one introduced cysteine ​​and one or more proline substitutions in the B-loop. In some embodiments, the IAV H1 HA protein includes an interprotomeral disulfide bond formed by at least one introduced cysteine ​​and one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues. In some embodiments, the IAV H1 HA protein includes an interprotomeral disulfide bond formed by at least one introduced cysteine, one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues and one or more proline substitutions in the B-loop.

[0214] In some embodiments, the IAV H1 HA protein includes an interprotomeral disulfide bond formed by at least one introduced cysteine ​​and an intraprotomeral disulfide bond formed by at least one introduced cysteine, wherein the introduced cysteine(s) of the interprotomeral disulfide bond are located at different positions from the introduced cysteine(s) of the intraprotomeral disulfide bond.

[0215] In some embodiments, the IAV H1 HA protein comprises an interprotomer disulfide bond formed by at least one introduced cysteine, one or more substitutions of pH-sensitive histidine in the HA protein, and one or more substitutions of charged or polar cavity-lining residues to hydrophobic residues.

[0216] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, and proline at position 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391, cysteine ​​substitutions at position 37, proline substitutions at position 404, and proline substitutions at position 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions K391C, L37C, N404P, and H416P compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0217] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391, cysteine ​​at position 37, isoleucine at position 395, and isoleucine at position 447, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391, cysteine ​​substitutions at position 37, isoleucine substitutions at position 395, and isoleucine substitutions at position 447 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions K391C, L37C, K395I, and E447I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0218] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391, cysteine ​​substitutions at position 37, glycine substitutions at position 456, and isoleucine substitutions at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions K391C, L37C, D456G, and K402I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0219] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 395, cysteine ​​at position 36, proline at position 404, and proline at position 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 395, cysteine ​​substitutions at position 36, proline substitutions at position 404, and proline substitutions at position 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions of K395C, V36C, N404P, and H416P compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0220] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 395, cysteine ​​at position 36, glycine at position 456, and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 395, cysteine ​​substitutions at position 36, glycine substitutions at position 456, and isoleucine substitutions at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions K395C, V36C, D456G, and K402I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0221] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 461, cysteine ​​at position 348, proline at position 404, and proline at position 416, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 461, cysteine ​​substitutions at position 348, proline substitutions at position 404, and proline substitutions at position 416 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions N461C, G348C, N404P, and H416P compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0222] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 461, cysteine ​​at position 348, isoleucine at position 395, and isoleucine at position 447, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 461, cysteine ​​substitutions at position 348, isoleucine substitutions at position 395, and isoleucine substitutions at position 447 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions N461C, G348C, K395I, and E447I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0223] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 461, cysteine ​​at position 348, glycine at position 456, and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 461, cysteine ​​substitutions at position 348, glycine substitutions at position 456, and isoleucine substitutions at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes substitutions N461C, G348C, D456G, and K402I compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0224] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, proline at position 416, glycine at position 456, and isoleucine at position 402, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391, cysteine ​​substitutions at position 37, proline substitutions at position 404, proline substitutions at position 416, glycine substitutions at position 456, and isoleucine substitutions at position 402 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes the substitutions K391C, L37C, N404P, H416P, D456G, and K402I compared to the reference IAV H1 HA protein, where the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 83.

[0225] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 410, 462, 457, and 346, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 410, 462, 457, and 346 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes substitutions V410C, L462C, S457C, and L346C compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95.

[0226] In some embodiments, the IAV H1 HA protein includes cysteine ​​at position 391, cysteine ​​at position 37, phenylalanine at position 370, and phenylalanine at position 455, the positions of which are numbered by aligning the amino acid sequence of the IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes cysteine ​​substitutions at position 391, cysteine ​​substitutions at position 37, phenylalanine substitutions at position 370, and phenylalanine substitutions at position 455 compared to the reference IAV H1 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO: 95. In some embodiments, the IAV H1 HA protein includes substitutions K391C, L37C, H370F, and H455F compared to the reference IAV H1 HA protein, the numbering of the substituted amino acids in the reference IAV H1 HA protein corresponds to the numbering in SEQ ID NO: 95.

[0227] H3 HA protein Some embodiments relate to IAV H3 HA proteins comprising amino acid substitutions relative to a reference IAV H3 HA protein. The reference IAV H3 HA protein may be the HA protein of an influenza A virus of an IAV subtype containing an H3 HA protein, such as the influenza A / (H3N2) virus. While the H3 HA protein is present in, for example, the H3N2 subtype of influenza A virus, other IAV subtypes expressing the H3 HA protein have been identified (such as the H3N8 subtype, which is specific to birds, horses, and dogs). Non-exclusive examples of influenza A / (H3N2) virus isolates include A / Sydney / 5 / 97(H3N2), A / Moscow / 10 / 1999(H3N2), A / Fujian / 411 / 2002(H3N2), A / California / 7 / 2004(H3N2), A / Wisconsin / 67 / 2005(H3N2), A / Brisbane / 10 / 2007(H3N2), A / Perth / 16 / 2009(H3N2), A / Victoria / 361 / 2011(H3N2), and A / Texas / 50 / 2012(H3N2). 2) Examples include A / Switzerland / 9715293 / 2013(H3N2), A / Hong Kong / 4801 / 2014(H3N2), A / Singapore / INFIMH-16-0019 / 2016(H3N2), A / Kansas / 14 / 2017(H3N2), A / Hong Kong / 2671 / 2019(H3N2), A / Hong Kong / 45 / 2019(H3N2), A / Cambodia / e0826360 / 2020(H3N2), A / Darwin / 9 / 2021(H3N2), and A / Darwin / 6 / 2021(H3N2). Representative amino acid sequences of the influenza A / (H3N2) virus HA protein used in seasonal influenza vaccines to date are shown as sequence numbers 136-153.

[0228] Those skilled in the art will understand that, for example, the mutation disclosed with respect to the described sequence of influenza A / Darwin / 6 / 2021(H3N2) virus HA may be applied to other influenza A / (H3N2) virus HA proteins or other reference IAV H3 HA proteins. For example, when applying a T40C substitution to another IAV H3 HA protein, those skilled in the art would align the amino acid sequence of that reference HA protein to the sequence of influenza A / Darwin / 6 / 2021(H3N2) virus HA protein of SEQ ID NO: 82 and introduce C into the residue of the amino acid sequence of the reference HA protein that is aligned to T40 of SEQ ID NO: 82.

[0229] Exemplary substitutions that may be present in the IAV H1 HA protein are shown in Table HA-5 below. If the reference HA protein sequence (e.g., of the isolate) already contains the substitutions mentioned in the table, it will be understood that some embodiments may contain the residues listed below without making substitutions at those specific positions. For example, if the isolate already contains isoleucine at position 219, embodiments containing 219I and H504P can be obtained by substituting only 504P. [Table 6]

[0230] In some embodiments, the IAV H3 HA protein includes cysteine ​​at position 40 and position 55, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes cysteine ​​substitutions at position 40 and position 55 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes T40C and A55C substitutions compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The T40C and A55C substitutions result in the formation of an intraprotomer disulfide bond in the head region of the HA protein.

[0231] In some embodiments, the IAV H3 HA protein includes cysteine ​​at position 123 and cysteine ​​at position 421, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes cysteine ​​substitutions at position 123 and cysteine ​​substitutions at position 421 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes substitutions of S123C and R421C compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The substitutions of S123C and R421C result in the formation of protomer disulfide bonds in the head region of the HA protein.

[0232] In some embodiments, the IAV H3 HA protein includes cysteine ​​at position 260 and 237, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes cysteine ​​substitutions at position 260 and 237 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes L260C and P237C substitutions compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The L260C and P237C substitutions result in the formation of protomer-interprotomer disulfide bonds in the head region of the HA protein.

[0233] In some embodiments, the IAV H3 HA protein includes cysteine ​​at position 392 and cysteine ​​at position 46, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes cysteine ​​substitutions at position 392 and cysteine ​​substitutions at position 46 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes substitutions of Q392C and T46C compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The substitutions of Q392C and T46C result in the formation of protomer disulfide bonds in the stalk region of the HA protein.

[0234] In some embodiments, the IAV H3 HA protein includes cysteine ​​at position 411 and 428, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes cysteine ​​substitutions at position 411 and 428 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes substitutions I411C and Y428C compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The substitutions I411C and Y428C result in the formation of protomeral disulfide bonds in the stalk region of the HA protein.

[0235] In some embodiments, the IAV H3 HA protein includes proline at position 402, position 421, and position 414, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes proline substitutions at position 402, position 421, and position 414 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein includes substitutions G402P, R421P, and E414P compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The substitutions G402P, R421P, and E414P are proline substitutions in the B loop of the HA protein.

[0236] In some embodiments, the IAV H3 HA protein contains a glycine substitution at position 403, the position of which is numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains a glycine substitution at position 403 compared to the reference IAV H3 HA protein, the position of which is numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains a K403G substitution compared to the reference IAV H3 HA protein, the numbering of the substituted amino acid in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The K403G substitution is a glycine substitution in the B loop of the HA protein.

[0237] In some embodiments, the IAV H3 HA protein contains glycine at positions 408 and 409, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains glycine substitutions at positions 408 and 409 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains K408G and H409G substitutions compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The F408G and H409G substitutions are glycine substitutions in the B loop of the HA protein.

[0238] In some embodiments, the IAV H3 HA protein contains isoleucine at position 396, the position of which is numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains an isoleucine substitution at position 396 compared to the reference IAV H3 HA protein, the position of which is numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains a K396I substitution compared to the reference IAV H3 HA protein, the numbering of the substituted amino acid in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The K396I substitution is a cavity-filling substitution.

[0239] In some embodiments, the IAV H3 HA protein contains isoleucine at position 219 and proline at position 504, the positions of which are numbered by aligning the amino acid sequence of the IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains isoleucine substitution at position 219 and proline substitution at position 504 compared to the reference IAV H3 HA protein, the positions of which are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO: 82. In some embodiments, the IAV H3 HA protein contains T219I and H504P substitutions compared to the reference IAV H3 HA protein, the numbering of the substituted amino acids in the reference IAV H3 HA protein corresponds to the numbering in SEQ ID NO: 82. The T219I substitution is a cavity-filling substitution, and the H504P substitution is a proline substitution in the B-loop of the HA protein.

[0240] The substitutions described in this subsection relating to the IAV H3 HA protein may be combined. For example, any pair of cysteine ​​substitutions may be combined with one or more proline substitutions in the B-loop, glycine substitutions in the B-loop, pH-sensitive histidine substitutions, and / or cavity-filling substitutions. Pairs of cysteine ​​substitutions that form different disulfide bonds (e.g., interprotomer and intraprotomer, head region and slump region) may also be combined. In some embodiments, the IAV H3 HA protein includes one or more pH-sensitive histidine substitutions in the HA protein, and one or more substitutions from charged or polar cavity-lining residues to hydrophobic residues.

[0241] In some embodiments, the IAV H3 HA protein contains an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 82.

[0242] Characterization of viruses and antigens The sequences of influenza viruses and their proteins may be classified as belonging to a given IAV subtype or IBV lineage using any suitable classification method. Non-limiting examples of classification methods include sequence-based analysis (e.g., sequence comparison and phylogenetic tree construction) and antigen characterization (e.g., HAI assay, microneutralization test, and immunofluorescence).

[0243] Sequence analysis The amino acid sequence of the influenza virus antigen may be analyzed and classified as belonging to a given IAV subtype or IBV lineage. For example, the HA amino acid sequence may be classified as an H1 subtype, H3 subtype, B / Victoria lineage, or B / Yamagata lineage based on its similarity to existing H1 HA, H3 HA, B / Victoria lineage HA, or B / Yamagata lineage HA amino acid sequences. Alternatively, the HA amino acid sequence may be fitted into a phylogenetic tree of existing HA amino acid sequences and classified as belonging to a given IAV subtype or IBV lineage according to its most likely location within the phylogenetic tree.

[0244] Similarly, the nucleotide sequence of an influenza virus genome segment may be compared to an existing genome segment and classified as belonging to a given IAV subtype or IBV lineage based on the degree of identity with the existing sequence of a given subtype or lineage. Alternatively, the nucleotide sequence of an influenza virus genome segment may be fitted to a phylogenetic tree of existing genome segment nucleotide sequences and classified as belonging to a given IAV subtype or IBV lineage according to the most likely location within the phylogenetic tree.

[0245] Determination of antigen characteristics Serological methods such as HAI testing are useful in many epidemiological and immunological studies, as well as in evaluating antibody responses after vaccination. Serological methods are also useful in situations where viral identification cannot be performed (e.g., after viral shedding has stopped). HAI testing is used to identify circulating influenza viruses that are antigenically similar to the influenza virus of the previous season.

[0246] The hemagglutination inhibition (HAI) test is a classic experimental procedure for classifying or subtyping hemagglutinating viruses and determining the antigenic characteristics of influenza virus isolates, provided that the reference antiserum used contains antibodies against the currently circulating virus (see, for example, Pedersen JC Methods Mol Biol. 2014;1161:11-25). The antiserum used is based on an antigen preparation derived from either a wild-type strain or a highly proliferative reasortant prepared using a wild-type strain or an antigenically equivalent strain. For example, if effective hemagglutinin inhibition by H1 HA-specific serum or antibody is not effective with H3 HA-specific serum or antibody, it indicates that the influenza virus expresses HA belonging to the H1 subtype.

[0247] Microneutralization assays are highly sensitive and specific assays for detecting virus-specific neutralizing antibodies against influenza viruses in human and animal serum, and in some embodiments, include the detection of human antibodies against avian subtypes. Once a novel virus is identified, testing can be performed immediately before the purified viral protein can be used in other assays. For example, if influenza virus cells are neutralized by serum or antibodies specific to H1 HA but not by serum or antibodies specific to H3 HA, it indicates that the influenza virus expresses HA belonging to the H1 subtype.

[0248] Immunofluorescence (IFA) staining of virus-infected cells in original clinical specimens and field isolates is a rapid and highly sensitive method for diagnosing respiratory infections and other viral infections. In some embodiments, IFA staining is performed on isolates rather than original clinical specimens, so that the present virus can be amplified first and used for other tests as needed. For example, if influenza virus-infected cells are stained with an antibody specific to H1 HA but not neutralized with an antibody specific to H3 HA, it indicates that the influenza virus is expressing HA belonging to the H1 subtype.

[0249] Vaccine composition Some embodiments relate to compositions for use as vaccines against seasonal influenza viruses and, optionally, other respiratory viruses (e.g., coronaviruses and / or respiratory syncytial viruses).

[0250] Some embodiments relate to compositions comprising nucleic acids (e.g., RNA (e.g., mRNA)) encoding respiratory virus (e.g., influenza virus, RSV, SARS-CoV-2) antigens, wherein nucleic acids encoding different antigens are present in specific ratios. The “ratio” of two nucleic acids (e.g., encoding proteins A and B) may refer to a molar ratio (the number of nucleic acid molecules encoding protein A relative to the number of nucleic acid molecules encoding protein B) or a mass ratio (the mass of nucleic acid encoding protein A relative to the mass of nucleic acid encoding protein B). Unless otherwise stated or made clear from the context, any reference to nucleic acids (e.g., RNA (e.g., mRNA)) being present in a certain “ratio” refers to the mass ratio of the nucleic acids.

[0251] Multivalent vaccine Some embodiments relate to multivalent vaccines containing components that protect a subject from multiple influenza viruses. A multivalent influenza vaccine may contain 3 (trivalent), 4 (quadrivalent), 5 (pentavalent), or more (e.g., octavalent) components, each independently designed to protect against one of several influenza virus strains. For example, a trivalent vaccine may contain RNA(s) encoding influenza A / (H1N1) virus protein, influenza A / (H3N2) virus protein, and influenza B / Victoria lineage virus protein. Some trivalent compositions contain RNA(s) encoding two influenza A virus HA proteins and one influenza B virus HA protein. A quadrivalent vaccine may contain RNA(s) encoding three influenza A virus proteins (e.g., HA proteins) and one influenza B virus protein (e.g., HA protein). Some quadrivalent vaccines contain mRNA encoding two influenza A virus proteins (e.g., HA proteins) and two influenza B virus proteins (e.g., HA proteins).

[0252] In some embodiments, the polyvalent vaccine contains three mRNAs: a first mRNA encoding the influenza A / (H1N1) virus HA protein, a second mRNA encoding the influenza A / (H3N2) virus HA protein, and a third mRNA encoding the influenza B / Victoria lineage virus HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1.

[0253] In some embodiments, the polyvalent vaccine contains four mRNAs: a first mRNA encoding the influenza A / (H1N1) virus HA protein, a second mRNA encoding the influenza A / (H3N2) virus HA protein, a third mRNA encoding the influenza B / Victoria lineage virus HA protein, and a fourth mRNA encoding the influenza B / Yamagata lineage virus HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1.

[0254] In some embodiments, the polyvalent vaccine contains eight mRNAs: a first mRNA encoding the IAV H1 HA protein, a second mRNA encoding the IAV H3 HA protein, a third mRNA encoding the influenza B / Victoria lineage virus HA protein, a fourth mRNA encoding the influenza B / Yamagata lineage virus HA protein, a fifth mRNA encoding the IAV N1 NA protein, a sixth mRNA encoding the IAV N2 NA protein, a seventh mRNA encoding the influenza B / Victoria lineage virus NA protein, and an eighth mRNA encoding the influenza B / Yamagata lineage virus NA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1:1:1:1:1. In some embodiments, the mRNAs are present in a mass ratio of 3:3:3:3:1:1:1:1 (i.e., the mRNA encoding the HA protein is present in three times the amount (mass) of the mRNA encoding the NA protein).

[0255] In some embodiments, the polyvalent vaccine contains five mRNAs: a first mRNA encoding the IAV H1 HA protein, a second mRNA encoding the first IAV H3 HA protein, a third mRNA encoding the second IAV H3 HA protein, a fourth mRNA encoding the influenza B / Victoria lineage virus HA protein, and a fifth mRNA encoding the influenza B / Yamagata lineage HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1:1.

[0256] In some embodiments, the polyvalent vaccine comprises four mRNAs: a first mRNA encoding the IAV H1 HA protein, a second mRNA encoding the first IAV H3 HA protein, a third mRNA encoding the second IAV H3 HA protein, and a fourth mRNA encoding the influenza B / Victoria lineage virus HA protein, the vaccine not containing the mRNA encoding the influenza B / Yamagata lineage virus HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1.

[0257] In some embodiments, the polyvalent vaccine contains six mRNAs, namely, a first mRNA encoding the IAV H1 HA protein, a second mRNA encoding the first IAV H3 HA protein, a third mRNA encoding the second IAV H3 HA protein, a fourth mRNA encoding the third IAV H3 HA protein, a fifth mRNA encoding the influenza B / Victoria lineage virus HA protein, and a sixth mRNA encoding the influenza B / Yamagata lineage HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1:1:1.

[0258] In some embodiments, the polyvalent vaccine comprises five mRNAs: a first mRNA encoding the IAV H1 HA protein, a second mRNA encoding the first IAV H3 HA protein, a third mRNA encoding the second IAV H3 HA protein, a fourth mRNA encoding the third IAV H3 HA protein, and a fifth mRNA encoding the influenza B / Victoria lineage virus HA protein, the vaccine not containing mRNA encoding the influenza B / Yamagata lineage HA protein. In some embodiments, these mRNAs are present in a mass ratio of 1:1:1:1:1.

[0259] Some embodiments relate to a polyvalent vaccine comprising mRNA encoding multiple H3 HA proteins derived from distinct influenza A / (H3N2) viruses. The distinct H3 HA proteins may belong to different clades of the A / (H3N2) subtype. In some embodiments, each H3 HA protein encoded by the vaccine mRNA is derived from an influenza virus of a distinct clade within the A / (H3N2) subtype. In some embodiments, each H3 HA protein encoded by the vaccine mRNA differs from each other H3 HA proteins encoded by other mRNAs of the vaccine by at least 1, at least 2, at least 3, at least 4, at least 5, 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, at least 17, at least 18, at least 19, and at least 20 substitutions. If two H3 HA proteins have different lengths, the number of substitutions between their amino acid sequences is calculated after aligning the amino acid sequences, as discussed in the section titled "Protein Variants and Alignment."

[0260] In some embodiments of the polyvalent vaccine, the influenza B / Victoria lineage virus HA protein is the influenza B / Victoria lineage virus HA protein described in the section titled "B / Victoria lineage HA protein". In some embodiments, the influenza B / Victoria lineage HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0261] In some embodiments of the polyvalent vaccine, the influenza B / Yamagata strain virus HA protein is the influenza B / Yamagata strain virus HA protein described in the section titled "B / Yamagata strain HA protein". In some embodiments, the influenza B / Yamagata strain HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0262] In some embodiments of the polyvalent vaccine, the IAV H1 HA protein is the IAV H1 HA protein described in the section titled "H1 HA Protein". In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 95.

[0263] In some embodiments of the polyvalent vaccine, the IAV H3 HA protein is the IAV H3 HA protein described in the section titled "H3 HA Protein". If the polyvalent vaccine contains multiple H3 HA proteins (for example, multiple mRNAs encoding different H3 HA proteins), each different H3 HA protein may contain the same or different substitutions as those described in the section titled "H3 HA Protein". In some embodiments, one or more H3 HA proteins do not contain the substitutions described in the section titled "H3 HA Protein". In some embodiments, the IAV H3 HA protein contains an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82. In some embodiments, each IAV H3 HA protein contains an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82.

[0264] Combination vaccine Some embodiments of vaccines include combination vaccines. As used herein, “combination vaccine” means a vaccine comprising one or more components for inducing an immune response against (i) one or more influenza viruses and (ii) one or more viruses other than influenza viruses. As stated above, discussions relating to combination vaccines comprising one or more RNAs (e.g., mRNA) encoding proteins of different viruses may, in particular, also apply to combination vaccines comprising the same protein (e.g., an isolated protein or a protein present in a viral vector). Those skilled in the art will understand that in the case of a combination vaccine comprising one or more RNAs (e.g., mRNA) encoding two or more proteins, the two or more proteins may be encoded by a single RNA in the combination vaccine or by different RNAs.

[0265] For example, a combination vaccine may contain one or more RNAs encoding antigens of viruses from different families (e.g., a first antigen of influenza virus (Orthomyxoviridae) and a second antigen of coronavirus (Coronaviriade) or respiratory syncytial virus (Pneumoviridae), respectively). In some embodiments, the composition contains one or more RNAs (e.g., mRNA) encoding at least one influenza virus antigen and at least one antigen of a different virus (e.g., coronavirus or a virus derived from Pneumoviridae (e.g., respiratory syncytial virus (RSV))). In some embodiments, the different virus is SARS-CoV-2, i.e., in some embodiments, the composition contains at least one influenza virus antigen and at least one SARS-CoV-2 antigen. It comprises one or more RNAs that collectively encode. In some embodiments, the different virus is human respiratory syncytial virus (hRSV), i.e., in some embodiments, the composition comprises one or more RNAs that collectively encode at least one influenza virus antigen and at least one hRSV antigen. In some embodiments, the composition comprises at least one influenza virus antigen and one or more RNAs that collectively encode at least one antigen each of two different viruses (e.g., coronavirus and hRSV). In some embodiments, the different viruses are SARS-CoV-2 and hRSV, i.e., in some embodiments, the composition comprises one or more RNAs that collectively encode at least one influenza virus antigen, at least one SARS-CoV-2 antigen, and at least one hRSV antigen.

[0266] With respect to the SARS-CoV-2 antigens of the mixed vaccine, in some embodiments, the mixed vaccine comprises 1, 2, 3, 4, 5, or 6 RNAs (e.g., mRNA) encoding different coronavirus antigens, each antigen containing at least one mutation and / or at least one deletion of the amino acid sequence of SEQ ID NO: 78. In some embodiments, the mixed vaccine comprises RNA encoding the wild-type SARS-CoV-2 S protein antigen or an antigenic fragment thereof. In some embodiments, the mixed vaccine comprises RNA encoding a fusion protein comprising at least two domains of the SARS-CoV-2 spike(S) protein and less than the full-length spike protein. In some embodiments, the mixed vaccine comprises a first RNA encoding a first fusion protein comprising at least two domains of the SARS-CoV-2 spike(S) protein and less than the full-length S protein, and a second RNA encoding a second fusion protein comprising at least two domains of the SARS-CoV-2 spike(S) protein and less than the full-length S protein. In some embodiments, the two RNAs are present in the mixed vaccine in a 1:1 ratio. In some embodiments, the two RNAs are present in the mixed vaccine in a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0267] With respect to antigens of viruses derived from Pneumoviridae, in some embodiments, the mixed vaccine comprises 1, 2, 3, 4, 5, or 6 RNAs (e.g., mRNA) encoding different antigens of a virus derived from Pneumoviridae (e.g., RSV), each antigen containing at least one mutation and / or at least one deletion compared to a reference Pneumoviridae virus antigen. In some embodiments, the mixed vaccine comprises RNA encoding the wild-type hRSV F glycoprotein antigen or an antigenic fragment thereof. In some embodiments, the mixed vaccine comprises RNA encoding an hRSV F glycoprotein variant lacking a cytoplasmic tail. In some embodiments, the mixed vaccine comprises RNA encoding an hRSV F glycoprotein variant lacking a cytoplasmic tail and further containing one or more modifications compared to the wild-type hRSV F glycoprotein. In some embodiments, the mixed vaccine comprises a first RNA encoding a first hRSV F glycoprotein variant lacking a cytoplasmic tail, and a second RNA encoding a second hRSV F glycoprotein variant lacking a cytoplasmic tail and further containing one or more modifications compared to the wild-type hRSV F glycoprotein. In some embodiments, the two RNAs are present in the mixed vaccine in a 1:1 ratio. In some embodiments, the two RNAs are present in the mixed vaccine in a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0268] In some embodiments, the mixed vaccine comprises one or more RNAs (e.g., mRNA) encoding the influenza virus antigen (e.g., mRNA encoding the HA antigen) and one or more RNAs (e.g., mRNA) encoding the antigen of at least one different respiratory virus (e.g., mRNA encoding the SARS-CoV-2 fusion protein or hRSV F glycoprotein). In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 10:1. In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 15:1. In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 20:1. In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 25:1. In some embodiments, the ratio of RNA encoding the influenza virus antigen to RNA encoding at least one different respiratory virus antigen in the mixed vaccine is 30:1.

[0269] In some embodiments, the mixed vaccine comprises (i) one or more RNAs (e.g., mRNAs) encoding one or more influenza virus proteins, (ii) one or more RNAs (e.g., mRNAs) encoding one or more SARS-CoV-2 proteins, and (iii) one or more RNAs (e.g., mRNAs) encoding one or more hRSV proteins. In some embodiments, each of (i) RNAs (or more) encoding influenza virus proteins, (ii) RNAs (or more) encoding SARS-CoV-2 proteins, and (iii) RNAs (or more) encoding hRSV proteins are present in substantially equal masses in the mixed vaccine. In some embodiments, RNAs (or more) encoding influenza virus proteins are present in three times the mass of RNAs (or more) encoding SARS-CoV-2 proteins and three times the mass of RNAs (or more) encoding hRSV proteins in the mixed vaccine. In some embodiments, RNA(s) encoding influenza virus proteins(s) are present in the mixed vaccine at a mass of four times that of RNA(s) encoding SARS-CoV-2 proteins(s) and four times that of RNA(s) encoding hRSV proteins(s). In some embodiments, RNA(s) encoding influenza virus proteins(s) are present in the mixed vaccine at a mass of five times that of RNA(s) encoding SARS-CoV-2 proteins(s) and five times that of RNA(s) encoding hRSV proteins(s). In some embodiments, RNA(s) encoding influenza virus proteins(s) are present in the mixed vaccine at a mass of twice that of RNA(s) encoding SARS-CoV-2 proteins(s) and four times that of RNA(s) encoding hRSV proteins(s).In some embodiments, RNA(s) encoding influenza virus proteins(s) are present in the mixed vaccine at a mass of four times that of RNA(s) encoding SARS-CoV-2 proteins(s) and twice that of RNA(s) encoding hRSV proteins(s). In some embodiments, RNA(s) encoding influenza virus proteins(s) are present in the mixed vaccine at a mass of 0.5 times that of RNA(s) encoding SARS-CoV-2 proteins(s) and 0.5 times that of RNA(s) encoding hRSV proteins(s). In some embodiments, the mass ratio of (i) RNA(s) encoding influenza virus proteins(s), (ii) RNA(s) encoding SARS-CoV-2 proteins(s), and (iii) RNA(s) encoding hRSV proteins(s) in the mixed vaccine is 4:1:1, 4:2:1, 4:3:2, 4:3:3, or 2:1:1.

[0270] In some embodiments, the mixed vaccine contains mRNA polynucleotides, each polynucleotide encoding a different respiratory viral antigenic polypeptide. In some embodiments, the first, second, and third mRNA polynucleotides are present in the mixed vaccine in a 1:1:1 ratio. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides from a first virus (e.g., influenza virus), a second virus, and a third virus in a 4:1:1 ratio. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides from a first virus (e.g., influenza virus), a second virus, and a third virus in a 3:1:1 ratio. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides from a first virus (e.g., influenza virus), a second virus, and a third virus in a 5:1:1 ratio. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides in a ratio of 4:2:1 for a first virus (e.g., influenza virus), a second virus, and a third virus. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides in a ratio of 1:2:1 for a first virus (e.g., influenza virus), a second virus, and a third virus. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides in a ratio of 1:2:2 for a first virus (e.g., influenza virus), a second virus, and a third virus. In some embodiments, the mixed vaccine contains mRNA polynucleotides encoding respiratory viral antigenic polypeptides in the ratios of the first virus, the second virus, and the third virus of 8:2:2, 4:1:1, 4:2:2, 4:2:1, 4:3:2, 4:3:3, 4:3:2, or 4:2:2.

[0271] In some embodiments, each mRNA polynucleotide in the mixed vaccine is complementary to and does not interfere with the others in the mixed vaccine. That is, the antigens produced by the administration of the mixed vaccine do not significantly interfere with the immune response to other antigens produced in response to the vaccine, and as a result, the ability of the antigens to elicit a protective immune response in the subject is not diminished. In some embodiments, the mixed vaccine is additive with respect to neutralizing antibodies compared to the individual antigens in the vaccine.

[0272] Therefore, a composition (e.g., an RNA vaccine (e.g., an mRNA vaccine)) may target one or more antigens of the same strain / species, or one or more antigens of different strains / species (e.g., antigens that induce immunity against organisms found in the same geographical area with a high risk of respiratory virus (e.g., influenza virus and / or coronavirus and / or respiratory syncytial virus) infection).

[0273] A mixed vaccine comprising RNA (e.g., mRNA) polynucleotides encoding at least two respiratory viral antigenic polypeptides derived from at least two different respiratory viridae families (e.g., influenza virus of Orthomyxoviridae, respiratory syncytial virus of Pneumoviridae, and coronavirus of Coronaviridae) may be used for the treatment and / or prevention of respiratory viral infections. In some embodiments, the mixed vaccine comprises mRNA polynucleotides encoding antigens derived from Orthomyxoviridae (e.g., influenza virus antigen) and Coronaviridae (e.g., SARS-CoV-2). In some embodiments, the mixed vaccine comprises mRNA polynucleotides encoding antigens derived from Orthomyxoviridae (e.g., influenza virus) and Pnemoviridae (e.g., human respiratory syncytial virus). In some embodiments, the composition comprises RNA (e.g., mRNA) polynucleotides encoding at least three respiratory antigenic polypeptides derived from at least three different respiratory viruses. In some embodiments, the three different viruses are derived from the families Orthomyxoviridae (e.g., influenza virus), Coronaviridae (e.g., SARS-CoV-2), and Pneumoviridae (e.g., human respiratory syncytial virus).

[0274] In some embodiments, one or more RNAs (e.g., mRNAs) encoding polypeptides derived from at least two different respiratory virus families are encapsulated in a single lipid nanoparticle. Some embodiments include one or more RNAs (e.g., mRNAs) encoding polypeptides derived from at least two different respiratory virus families, and the composition includes lipid nanoparticles encapsulating one or more RNAs (e.g., mRNAs) encoding polypeptides derived from a single respiratory virus family.

[0275] In some embodiments, the mixed vaccine comprises a combination of proteins or nucleic acids (e.g., RNA (e.g., mRNA)) that collectively encode a combination of (i) IAV H1 HA protein, (ii) IAV H3 HA protein, (iii) influenza B / Victoria lineage virus HA protein, and (iv) SARS-CoV-2 S protein or fragments thereof.

[0276] In some embodiments, the mixed vaccine comprises a combination of proteins or nucleic acids (e.g., RNA (e.g., mRNA)) that collectively encode a combination of (i) IAV H1 HA protein, (ii) IAV H3 HA protein, (iii) influenza B / Victoria lineage virus HA protein, and (iv) hRSV F protein or fragments thereof.

[0277] In some embodiments, the mixed vaccine comprises a combination of proteins or nucleic acids (e.g., RNA (e.g., mRNA)) that collectively encode a combination of (i) IAV H1 HA protein, (ii) IAV H3 HA protein, (iii) influenza B / Victoria lineage virus HA protein, (iv) SARS-CoV-2 S protein or a fragment thereof, and (v) hRSV protein or a fragment thereof.

[0278] In some embodiments, the combination includes two IAV H3 HAs. In some embodiments, the combination includes three IAV H3 HAs. In some embodiments, the combination includes four, five, six, seven, eight, nine, or ten IAV H3 HAs.

[0279] In some embodiments, the combination further includes influenza B / Yamagata lineage HA protein. In some embodiments, the combination does not include influenza B / Yamagata lineage HA protein.

[0280] In some embodiments, the combination further comprises (i) IAV N1 NA protein, (ii) IAV N2 NA protein, and (iii) influenza B / Victoria lineage NA protein. In some embodiments, the combination further comprises influenza B / Yamagata lineage NA protein. In some embodiments, the combination does not contain influenza B / Yamagata lineage NA protein.

[0281] In some embodiments of the mixed vaccine, the influenza B / Victoria lineage virus HA protein is the influenza B / Victoria lineage virus HA protein described in the section titled "B / Victoria lineage HA protein". In some embodiments, the influenza B / Victoria lineage HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 71.

[0282] In some embodiments of the mixed vaccine, the influenza B / Yamagata lineage virus HA protein is the influenza B / Yamagata lineage virus HA protein described in the section titled "B / Yamagata lineage HA protein". In some embodiments, the influenza B / Yamagata lineage HA protein includes an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0283] In some embodiments of the mixed vaccine, the IAV H1 HA protein is the IAV H1 HA protein described in the section titled "H1 HA Protein". In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0284] In some embodiments of the mixed vaccine, the IAV H3 HA protein is the IAV H3 HA protein described in the section titled "H3 HA Protein". If the mixed vaccine contains multiple H3 HA proteins (for example, multiple mRNAs encoding different H3 HA proteins), each different H3 HA protein may contain the same or different substitutions as those described in the section titled "H3 HA Protein". In some embodiments, one or more H3 HA proteins do not contain the substitutions described in the section titled "H3 HA Protein". In some embodiments, the IAV H3 HA protein contains an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82. In some embodiments, each IAV H3 HA protein contains an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 82.

[0285] In some embodiments, the SARS-CoV-2 S protein or a fragment thereof is a full-length S glycoprotein as described in the "Coronavirus" section below. In some embodiments, the SARS-CoV-2 S protein or a fragment thereof is a protein comprising one or more fragments of the SARS-CoV-2 S glycoprotein as described in the "Coronavirus" section below.

[0286] In some embodiments, the hRSV F protein or a fragment thereof is the hRSV protein or fragment thereof described in the following section, “Respiratory Syncytial Virus (hRSV)”.

[0287] coronavirus Some embodiments of combination vaccines contain coronavirus antigens or nucleic acids encoding coronavirus antigens. Coronaviruses are a family of enveloped, positive-sense single-stranded RNA viruses that infect mammals and birds. Notable human coronaviruses that cause respiratory illness include 229E, NL63, OC43, HKU1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). The name coronavirus comes from the crown-like protrusions on the surface formed by the viral spike glycoprotein (S protein). The S protein binds to host receptors and mediates the entry of the virus into cells. Other viral structural proteins include the envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. SARS-CoV-2 belongs to the genus Betacoronavirus and is the causative agent of COVID-19. It has a size of 29.8–30 kb (see, e.g., Chan et al. 2000 (above); Kim et al. 2020 Cell, May 14;181(4):914–921.e10.). The SARS-CoV-2 genome is organized into specific genes encoding structural and non-structural proteins (Nsps). The order of structural proteins in the genome is 5'-replicase(open reading frame (ORF)1 / ab)-structural protein[spike(S)-envelope I-membrane(M)-nucleocapsid(N)]-3'. The coronavirus genome includes a variety of open reading frames encoding accessory proteins, non-structural proteins, and structural proteins (Song et al. 2019 Viruses;11(1):p.59). Most antigen epitopes are located in structural proteins (Cui et al. 2019 Nat. Rev. Microbiol.; 17(3): 181-192). The four main structural proteins are the spike surface glycoprotein (S), the small envelope protein (E), the matrix protein (M), and the nucleocapsid protein (N).Because the S protein contributes to cell targeting and viral entry, and can induce neutralizing antibodies (NAbs) and protective immunity, it can be considered one of the most important targets in coronavirus vaccine development among all structural proteins.

[0288] Variant virus strains of SARS-CoV-2 can emerge from time to time. These strains may emerge, for example, from season to season. Therefore, in exemplary embodiments, a vaccine may be designed to combat seasonal coronavirus strains and is itself a vaccine for use in the upcoming Northern Hemisphere or Southern Hemisphere season. A vaccine may be designed to combat viruses that are predicted to circulate or spread in the upcoming virus season, based on an understanding of the coronavirus circulating at a particular point in time.

[0289] A preferred protein is the spike (S) protein present on the surface of coronaviruses, including SARS-CoV-2. An example of the wild-type SARS-CoV-2 spike protein is provided by the amino acid sequence of SEQ ID NO: 78. In some embodiments, the SARS-CoV-2 antigen is a full-length S protein. In some embodiments, the SARS-CoV-2 antigen comprises a full-length S protein having two or more proline substitutions. In some embodiments, the S protein contains two proline substitutions at residues corresponding to K986V and V987P in SEQ ID NO: 78. In some embodiments, the S protein does not contain any proline substitutions.

[0290] In some embodiments, the SARS-CoV-2 antigen does not contain a full-length S protein. For example, in some embodiments, the SARS-CoV-2 antigen includes the receptor-binding domain (RBD) of the S protein. In some embodiments, the SARS-CoV-2 antigen includes a fusion protein containing the RBD and N-terminal domain (NTD) of the S protein. In some embodiments, the fusion protein includes the RBD and NTD of the S protein, as well as a transmembrane domain (TD). In some embodiments, the fusion protein contains NTD-RBD-TD in the order of N-terminus to C-terminus. In some embodiments, the RBD and NTD are linked via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a glycine-serine (GS) linker.

[0291] In some embodiments, TD is a betacoronavirus TD. In some embodiments, TD is a heterologous TD. In some embodiments, TD is a non-betacoronavirus TD. In some embodiments, TD is an influenza virus hemagglutinin (HA) TD. In some embodiments, TD is an influenza A virus H1 HA TD.

[0292] In some embodiments, the SARS-CoV-2 antigen includes at least one mutation present in the S protein of an epidemic SARS-CoV-2 isolate, compared to the amino acid sequence of the S protein of SEQ ID NO: 78. In some embodiments, the SARS-CoV-2 antigen includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations present in the S protein of an epidemic SARS-CoV-2, compared to SEQ ID NO: 78. In some embodiments, the SARS-CoV-2 antigen comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 78.

[0293] In some embodiments, the SARS-CoV-2 antigen includes at least one mutation present in the RBD of the S protein of an epidemic SARS-CoV-2 isolate, compared to the S protein of SEQ ID NO: 78. In some embodiments, the SARS-CoV-2 spike protein antigen includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBD mutations, compared to SEQ ID NO: 78. Since RBD corresponds to amino acids 330–528 in SEQ ID NO: 78, a person skilled in the art will understand that "mutations present in the RBD of the S protein" compared to the S protein of SEQ ID NO: 78 means (i) a substitution at the position corresponding to any one of amino acids 330–528 in SEQ ID NO: 78, (ii) a deletion of any one or more amino acids 330–528 in SEQ ID NO: 78, and / or (iii) an insertion of one or more amino acids at the positions of amino acids 330–528 in SEQ ID NO: 78. In some embodiments, the SARS-CoV-2 antigen comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 60.

[0294] Mutations present in the SARS-CoV-2 antigen of the combination vaccine may be present in circulating SARS-CoV-2 isolates. For example, at the time of filing this specification, isolates of the JN.1 variant are circulating. SARS-CoV-2 subvariant JN.1 (also known as BA.2.86.1.1) is a subvariant (sublineage) of the SARS-CoV-2 "Omicron" variant, first observed in August 2023, and is closely related to BA.2.86. The mutation observed in this variant, in particular the L455F "FLip" mutation, which is also observed in XBB lineage variants (e.g., HK.3 and EG.5.1), is considered to have a high probability of immune evasion. The mutations observed in JN.1 include A31D, V238L, K1155R, N1708S, A1892T, V24F, R252K, T35I, ins16MPLF, R21T, S50L, Δ69-70, V127F, F157S, R158G, N211del, L212I, V213G, L216F, H245N, A264D, I332V, K356T, R403K, V445H, N450D, L452W, L455S, N481K, V483del, E484K, E554K, A570V, P621S, P681H, S939F, and P1143L.

[0295] Several sub-variants of the XBB variant have also recently been identified: HV.1, JD.1.1, HK.3, and EG.5. These sub-variants include T19I, L24S, del25 / 27, V83A, G142D, del144 / 144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, F456L, N460K, They share several mutations, including S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, and some variants include additional mutations such as Q52H, F157L, L452R, L455F, and A475V.

[0296] Those skilled in the art will understand that the SARS-CoV-2 antigen of a combination vaccine may include mutations present in any SARS-CoV-2 S protein existing at the time of this specification. Those skilled in the art will understand that the amino acid sequences of SARS-CoV-2 S proteins that do not exist at the time of filing this specification may be analyzed with respect to mutations relative to SEQ ID NO: 78, and those mutations may be applied to the SARS-CoV-2 antigen of a combination vaccine.

[0297] In some embodiments, the mixed vaccine comprises two or more coronavirus antigens, or nucleic acids encoding two or more coronavirus antigens, for example, SARS-CoV-2 antigens derived from different SARS-CoV-2 variant strains. In some embodiments, the composition comprises one or more RNAs that collectively encode two or more SARS-CoV-2 variant antigens.

[0298] Respiratory syncytial virus (RSV) Some embodiments of combination vaccines contain human respiratory syncytial virus antigen or nucleic acids encoding human respiratory syncytial virus antigen. Human respiratory syncytial virus (hRSV; also known as human orthopneumovirus) is a negative-sense single-stranded ribonucleic acid (RNA) virus belonging to the family Pneumoviridae. The virus exists in at least two antigenic subgroups known as group A and group B.

[0299] The hRSV envelope contains three surface glycoproteins: F, G, and SH. The G and F proteins are targets of protective antigens and neutralizing antibodies. However, the F protein is more conserved across hRSV strains and types (A and B). The hRSV F protein is a type I fusion glycoprotein that is well conserved across clinical isolates, including between the hRSV-A and hRSV-B antigenic subgroups. The F protein transitions between a pre-fusion state and a more stable post-fusion state, thereby facilitating entry into target cells. The hRSV F glycoprotein is initially synthesized as the F0 precursor protein. hRSV F0 folds into a trimer, which is activated by furin cleavage to become a mature pre-fusion protein containing F1 and F2 subunits (Bolt, et al., Virus Res., 68:25, 2000). While the target for neutralizing monoclonal antibodies lies on the post-fusion structure of the F protein, the neutralizing antibody response primarily targets the pre-fusion structure of the F protein in humans naturally infected with hRSV (Magro M et al., Proc Natl Acad Sci USA 2012;109(8):3089-94; Ngwuta JO et al., Sci Transl Med 2015;7(309):309ra162). Consistent with this, hRSV F protein stabilized at the pre-fusion structure elicits a greater neutralizing immune response in animal models than hRSV F protein stabilized at the post-fusion structure (McLellan et al., Science, 342:592-598, 2013). Therefore, stabilized pre-fusion hRSV F protein is a good candidate for inclusion in hRSV vaccines. Other RSV proteins include low molecular weight hydrophobic (SH), matrix (M), nucleocapsid (N), phosphoprotein (P), polymerase (L), and non-structural NS1 / NS2 proteins. Preferred proteins are F glycoproteins, such as the hRSV F protein having the amino acid sequence of SEQ ID NO: 98, which is present on the surface of hRSV, including wild-type and mutant strains.

[0300] hRSV commonly causes bronchitis. Most infected adults develop mild, cold-like symptoms such as congestion, low fever, and wheezing. Infants and children may experience more severe symptoms, including bronchitis and pneumonia. The disease can be transmitted between people through contact with respiratory secretions.

[0301] Some embodiments relate to stabilized pre-fusion RSV F proteins that include mutations that prevent the transfer of the protein to its post-fusion conformation. For example, in some embodiments, the stabilized pre-fusion RSV F protein includes proline residue (e.g., S215P substitution) and / or isoleucine (e.g., N67I substitution) substitutions. As an example, the DS-Cav1 variant, which is a stabilized pre-fusion RSV F protein, contains an additional disulfide bond (S155C / S290C) as well as two cavity-filling mutations (S190F / V207L). Another stabilized pre-fusion RSV F protein is PR-DM, which contains one proline substitution (S215P) and one mutation (N67I) in the F2 subunit.

[0302] In some embodiments, the stabilized pre-fusion hRSV F glycoprotein variant lacks a cytoplasmic tail. In some embodiments, the cytoplasmic tail contains 20-30, 20-25, 15-30, 15-25, 15-20, 10-30, 10-25, 10-20, 10-15, 5-30, 5-25, 5-20, or 5-15 amino acids from the C-terminus of the hRSV F glycoprotein variant. In some embodiments, the cytoplasmic tail contains 25 amino acids from the C-terminus of the hRSV F glycoprotein (e.g., CKARSTPVTLSKDQLSGINNIAFSN (SEQ ID NO: 101)). In some embodiments, the cytoplasmic tail contains 20 amino acids from the C-terminus of the hRSV F glycoprotein (e.g., TPVTLSKDQLSGINNIAFSN (SEQ ID NO: 102)). In some embodiments, the cytoplasmic tail contains 15 amino acids from the C-terminus of the hRSV F glycoprotein (e.g., SKDQLSGINNIAFSN (SEQ ID NO: 103)). In some embodiments, the cytoplasmic tail contains 10 amino acids from the C-terminus of the hRSV F glycoprotein (e.g., SGINNIAFSN (SEQ ID NO: 104)).

[0303] In some embodiments, the stabilized pre-fusion hRSV F glycoprotein variant lacks a cytoplasmic tail, in which case this RSV F glycoprotein variant has at least 80%, at least 85%, at least 90%, and at least 95% identity with wild-type hRSV F glycoprotein (e.g., wild-type hRSV F glycoprotein containing the sequence of SEQ ID NO: 98), and lacks a cytoplasmic tail. In some embodiments, the stabilized pre-fusion hRSV F glycoprotein variant lacks a cytoplasmic tail, in which case this RSV F glycoprotein variant has at least 80%, at least 85%, at least 90%, and at least 95% identity with the sequence of SEQ ID NO: 99. In some embodiments, the stabilized pre-fusion hRSV F glycoprotein variant contains the sequence of SEQ ID NO: 99.

[0304] In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes modifications selected from the group consisting of P102X substitution, amino acid substitutions 104-144 by a linker molecule, A149X substitution, S155X substitution, S190X substitution, V207X substitution, S290X substitution, L373X substitution, I379X substitution, M447X substitution, and Y458X substitution, where X is any amino acid other than the amino acid sequence to be substituted in the amino acid sequence of the wild-type hRSV F glycoprotein (e.g., A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V). In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes modifications selected from the group consisting of P102A substitution, amino acid substitutions 104-144 by a linker molecule, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, I379V substitution, M447V substitution, and Y458C substitution, compared to the wild-type hRSV F glycoprotein (SEQ ID NO: 98). In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes a P102A substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes amino acid substitutions 104-144 by a linker molecule. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes an A149C substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the S155C substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the S190F substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the V207L ​​substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the S290C substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the L373R substitution.In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the I379V substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the M447V substitution. In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further comprises the Y458C substitution.

[0305] In some embodiments, the hRSV F glycoprotein variant lacking a cytoplasmic tail further includes the following modifications compared to the wild-type hRSV F glycoprotein: P102A substitution, substitution of amino acids 104-144 by a linker molecule, A149C substitution, S155C substitution, S190F substitution, V207L ​​substitution, S290C substitution, L373R substitution, I379V substitution, M447V substitution, and Y458C substitution.

[0306] In some embodiments, the hRSV F glycoprotein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 99. In some embodiments, the hRSV F glycoprotein comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the hRSV F glycoprotein consists of the amino acid sequence of SEQ ID NO: 99.

[0307] In some embodiments, the composition comprises RNA having an ORF encoding the hRSV F glycoprotein, wherein the ORF comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence of SEQ ID NO: 113. In some embodiments, the ORF comprises the nucleotide sequence of SEQ ID NO: 113.

[0308] In some embodiments, the hRSV F glycoprotein comprises an F1 subunit and an F2 subunit, wherein the F1 subunit has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 106, and the F2 subunit has at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 107. In some embodiments, the F1 subunit comprises the amino acid sequence of SEQ ID NO: 106, and the F2 subunit comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the F1 subunit consists of the amino acid sequence of SEQ ID NO: 106, and the F2 subunit consists of the amino acid sequence of SEQ ID NO: 107. In some embodiments, the hRSV F glycoprotein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 108. In some embodiments, the hRSV F glycoprotein comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the hRSV F glycoprotein consists of the amino acid sequence of SEQ ID NO: 108.

[0309] In some embodiments, the hRSV F glycoprotein comprises an F1 subunit and an F2 subunit, wherein the F1 subunit has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 109, and the F2 subunit has at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 110. In some embodiments, the F1 subunit comprises the amino acid sequence of SEQ ID NO: 109, and the F2 subunit comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the F1 subunit comprises the amino acid sequence of SEQ ID NO: 109, and the F2 subunit comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the F1 subunit consists of the amino acid sequence of SEQ ID NO: 109, and the F2 subunit consists of the amino acid sequence of SEQ ID NO: 110. In some embodiments, the hRSV F glycoprotein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 111. In some embodiments, the hRSV F glycoprotein comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the hRSV F glycoprotein consists of the amino acid sequence of SEQ ID NO: 111.

[0310] In some embodiments, the hRSV F glycoprotein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 112.

[0311] Protein variants and alignments Some embodiments relate to proteins having one or more mutations (e.g., substitutions) relative to a reference amino acid sequence and / or numbered according to the described amino acid sequence.

[0312] Several embodiments relate to amino acid or nucleotide sequences having a specified percentage of sequence identity with respect to a comparison amino acid or nucleotide sequence. The term "identity" refers to the relationship between two or more polypeptide (e.g., antigen) or polynucleotide (nucleic acid) sequences, determined by comparing the sequences. Identity also refers to the degree of sequence relevance between or between sequences, determined by the number of matching amino acid or nucleic acid residues in chains of two or more amino acid residues or nucleic acid residues. The "percentage of identity (%)" or "percentage of sequence identity (%)" applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the second sequence's amino acid or nucleic acid sequence after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of identity.

[0313] The degree of sequence identity between a candidate sequence (e.g., a sequence present in a claimed protein or nucleic acid) and a comparison sequence (e.g., a sequence having an array sequence as specified herein) is calculated by (i) aligning the candidate sequence and the comparison sequence, (ii) determining the number of matching residues (amino acids or nucleotides) between the aligned candidate sequence and the comparison sequence, and (iii) dividing the number of matching residues by the length of the comparison sequence (including any gaps introduced into the comparison sequence when the two sequences are aligned).

[0314] For example, alignment of the candidate amino acid sequence of the influenza B / Brisbane / 60 / 2008 virus HA protein (SEQ ID NO: 157) with the comparative amino acid sequence of the influenza B / Austria / 1359417 / 2021 virus HA protein (SEQ ID NO: 71) reveals 572 matching residues (Figure 19E). Although the amino acid length of SEQ ID NO: 71 is only 582, an internal gap of 3 amino acids is introduced into the alignment of SEQ ID NO: 71 (Figure 19E), making the denominator 585. Therefore, the candidate sequence SEQ ID NO: 157 has 572 / 585 = 97.8% sequence identity with the comparative sequence SEQ ID NO: 71.

[0315] Those skilled in the art will understand that, in order to determine whether a candidate protein or nucleic acid contains an amino acid or nucleotide sequence that has a given degree of sequence identity with respect to a comparison sequence, the denominator (the length of the comparison sequence plus internal gaps) used to calculate sequence identity does not need to include the gap shown at the end of the comparison sequence in the alignment. This is because such a gap is added if the candidate sequence contains additional amino acids or nucleotides that extend beyond the portion aligned to the N-terminus and / or C-terminus (amino acid sequence), or the 5' or 3' end (nucleotide sequence) of the comparison sequence. For example, when the amino acid sequence of the influenza B / Austria / 1359417 / 2021 virus HA protein of SEQ ID NO: 71, which contains a signal peptide, is aligned to its signal peptide-cleaved form of SEQ ID NO: 174, a gap is created at the N-terminus of the comparison sequence where the signal peptide is absent (Figure 19F). Candidate sequence SEQ ID NO: 71, aligned to comparison sequence SEQ ID NO: 174, contains 567 matches. Since comparison sequence SEQ ID NO: 174 has 567 amino acids, a protein with the full-length amino acid sequence of SEQ ID NO: 71 still contains an amino acid sequence that is 100% identical to SEQ ID NO: 174 (567 / 567=100%).

[0316] When alignment between two sequences is intended, the Needleman-Wunsch algorithm for global alignment of two sequences is used to align the first sequence (e.g., candidate sequence) to the second sequence (e.g., comparison sequence). Needleman & Wunsch, J Mol Biol. 1970. 48:443-453. When aligning two protein sequences, the Needleman-Wunsch algorithm uses the BLOSUM62 substitution score matrix, a Gap Open penalty of 10, a Gap Extend penalty of 0.5, and no End Gap penalty. When aligning two nucleotide sequences, the alignment uses the DNAFULL substitution score matrix, a Gap Open penalty of 10, a Gap Extend penalty of 0.5, and no End Gap penalty. Those skilled in the art will understand that, at the time of filing this application, these parameters are the default parameters for the EMBOSS Needle pairwise comparison tool provided by the European Bioinformatics Institute (see ebi.ac.uk). These parameters may be used to obtain global alignment using other suitable alignment programs such as BLAST, or the Needleman-Wunsch algorithm may be implemented in a scripting language (e.g., Python).

[0317] Linkers and cleavable peptides Some embodiments of the protein include a linker between at least one pair of protein segments. The linker may be, for example, a cleavage linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linkers, P2A linkers, T2A linkers, E2A linkers, and combinations thereof (see, e.g., WO2017 / 127750). This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the Art (see, e.g., Kim, J. Het al., PLoS ONE 2011;6:e18556).

[0318] In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GS linker. A GS linker is a polypeptide linker comprising glycine and serine amino acid repeats. They contain flexible and hydrophilic residues and can be used to carry out the fusion of protein subunits without interfering with the folding and function of the protein domain or forming secondary structures. In some embodiments, the protein comprises a GS linker having a length of 3 to 20 amino acids. For example, a GS linker may have a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (or at least that length). In some embodiments, the GS linker is 15 amino acids long (or at least 15 amino acids long) (e.g., GGSGGSGGSGGSGGG (SEQ ID NO: 114)). In some embodiments, the GS linker is 8 amino acid long (or at least 8 amino acid long) (e.g., GGGSGGGS (SEQ ID NO: 115)). In some embodiments, the GS linker is 7 amino acid long (or at least 7 amino acid long) (e.g., GGGGGG (SEQ ID NO: 116)). In some embodiments, the GS linker contains the amino acid sequence GGGSGG (SEQ ID NO: 117). In some embodiments, the GS linker is 4 amino acid long (or at least 4 amino acid long) (e.g., GGGS (SEQ ID NO: 118)). In some embodiments, the GS linker contains (GGGS)n (SEQ ID NO: 118), where n is any integer from 1 to 5. In some embodiments, the GS linker is 4 amino acid long (or at least 4 amino acid long) (e.g., GSGG (SEQ ID NO: 119)). In some embodiments, the GS linker contains (GSGG)n (SEQ ID NO: 119), where n is any integer from 1 to 5. In some embodiments, the linker is, for example, a glycine linker (e.g., GGG) having a length of 3 amino acids (or at least 3 amino acids). In some embodiments, the protein encoded by RNA (e.g., mRNA) comprises two or more linkers, which may be the same as or different from one another.Those skilled in the art will understand that other linkers may be suitable for use with proteins.

[0319] Signal peptide In some embodiments, the protein contains a signal peptide. The signal peptide comprises the N-terminal 15-60 amino acids of the protein. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides ribosomes to the rough endoplasmic reticulum (ER) membrane and initiates transmembrane transport of the elongated peptide chain for processing. ER processing produces a mature protein, and the signal peptide is either cleaved from the precursor protein or retained uncleaved by the host cell's ER-resident signal peptidase, and functions as a membrane anchor. The signal peptide may also facilitate the targeting of the protein to the cell membrane.

[0320] The length of a signal peptide can range from 15 to 60 amino acids. For example, the length of a signal peptide can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In the globe embodiment, the lengths of the signal peptides are 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50. The amino acid lengths are 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acid lengths.

[0321] Signal peptides derived from heterologous genes (other than, for example, influenza virus HA, influenza virus NA, hRSV F, and SARS-CoV-2 S glycoprotein) can also be used in proteins.

[0322] The intrinsic signal peptide of a protein can be determined using any suitable method, such as a signal peptide prediction tool. These tools employ bioinformatics algorithms, such as neural networks, machine learning, and / or language model-based approaches, in combination with an annotated protein database (e.g., UniProt), to predict the signal peptide sequence within a given amino acid sequence. See, for example, Teufel et al., Nat Biotechnol. 2022. 40(7):1023-1025 (SignalP6.0).

[0323] Table SP-1 shows non-exclusive examples of signal peptides for HA and NA proteins from isolates of influenza A / (H1N1) subtype, A / (H3N2) subtype, B / Victoria lineage, and B / Yamagata lineage. [Table 7]

[0324] nucleic acid A composition comprising nucleic acid is provided. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA such as self-amplified RNA, circular RNA, or mRNA. Preferably, the nucleic acid comprises mRNA.

[0325] Unless otherwise stated, in the nucleic acid sequences described in this application, "T" may be listed in representative DNA sequences, but when the sequence represents RNA, "T" is replaced with "U". Accordingly, for any DNA disclosed herein and identified by a particular sequence, the corresponding RNA sequence in which each "T" in the DNA sequence is replaced with "U" is also disclosed.

[0326] Messenger RNA (mRNA) Messenger RNA (mRNA) is RNA that encodes (at least one) protein or a fragment thereof, and can be translated to produce an encoded protein or fragment in vitro, in vivo, in situ, or ex vivo. mRNA includes an open reading frame (ORF) that encodes the protein or a fragment thereof. In some embodiments, mRNA further includes a 5' untranslated region (UTR), a 3' UTR, a poly-A tail, and / or a 5' cap analogue.

[0327] The disclosed mRNAs may encode a single protein or fragment, or they may be polycistronic constructs that separately encode multiple proteins or fragments within the same mRNA molecule. Furthermore or alternatively, the mRNAs of this disclosure may encode a fusion protein or a fragment thereof.

[0328] i. Open Reading Frame (ORF) An open reading frame (ORF) is a continuous segment of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (plural possible) (e.g., TAA, TAG, TGA, UAA, UAG, UGA, UGAUGA, or UGAUAAUAG). For clarity, the stop codon itself is not considered part of the ORF. ORFs typically encode proteins or fragments thereof.

[0329] ii. Untranslated Region (UTR) In some embodiments, mRNA includes one or more regions or portions that act or function as untranslated regions. The 5' untranslated region (5'UTR) is a region of mRNA located upstream of the start codon (i.e., 5') and does not encode a polypeptide. The 3' untranslated region (3'UTR) is a region of mRNA located downstream of the stop codon (i.e., 3') and also does not encode a polypeptide.

[0330] The 5'UTR begins at the transcription start site and may continue to the start codon, but may not include the start codon. The 3'UTR begins immediately after the stop codon and may continue to the transcription termination signal. Various 5'UTR and 3'UTR sequences are known. Exemplary UTR sequences include SEQ ID NOs. 1-35 (5'UTR) and 36-44 (3'UTR), which are shown in Tables S-1 (5'UTR) and S-2 (3'UTR) in the "Exemplary Sequences" section. In some embodiments, the 5'UTR includes the sequences shown in Table S-1, or sequences, variants, or fragments thereof that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the 5'UTR sequences shown in Table S-1. In some embodiments, the 3'UTR includes the sequence shown in Table S-2, or a sequence, variant, or fragment thereof that has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the 3'UTR sequence shown in Table S-2.

[0331] Each RNA species in a multivalent RNA composition may contain an IDR sequence that is not a sequence isomer of the IDR sequence of another RNA species in the multivalent RNA composition (for example, an IDR sequence may not have the same number of adenosine nucleotides, cytosine nucleotides, guanine nucleotides, and (and therefore the same mass) of uracil nucleotides as another IDR sequence in the composition (even if these sequences are different)).

[0332] Each RNA species in a multivalent RNA composition may contain IDR sequences having a different mass than the IDR sequences of other RNA species in the multivalent RNA composition. For example, the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da. Using IDR sequences with different masses makes it possible to distinguish RNA fragments containing different IDR sequences using mass-based analytical methods (e.g., mass spectrometry) that do not require reverse transcription, amplification, or sequencing of RNA.

[0333] Each RNA species in an RNA composition may contain IDR sequences of different lengths. For example, each IDR sequence may have a length independently selected from 0 to 25 nucleotides. Since nucleic acid length affects the rate at which nucleic acids pass through a chromatography column, using IDR sequences of different lengths for different RNA species makes it possible to distinguish RNA fragments with different IDR sequences using chromatography-based methods (e.g., LC-UV).

[0334] The combination of features may be included in the flanking region or incorporated within other features. For example, the ORF may be flanked by a 5'UTR that may contain a potent Kozak translation initiation signal and / or a 3'UTR that may contain an oligo(dT) sequence for template addition of a poly(A) tail. The 5'UTR may contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes (e.g., 5'UTRs described in US2010 / 0293625 and WO2015 / 085318).

[0335] In some embodiments, double, triple, or quadruple UTRs, such as 5'UTR or 3'UTR, may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are coded consecutively or substantially consecutively. For example, a double 3'UTR of β-globin may be used as described in US2010 / 0129877.

[0336] For the purposes of this disclosure, the UTR may also include one or more translation enhancer elements (TEEs). In non-limiting examples, TEEs may include those described in US2009 / 0226470, incorporated herein by reference, and those known in the art.

[0337] iii. Poly A Tail In some embodiments, the mRNA contains a 3'-poly-A tail. The poly-A tail may contain 10 to 300 adenosine monophosphates. In some cases, the poly-A tail may contain up to approximately 400 adenine nucleotides. For example, the poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine nucleotides. In some embodiments, the poly-A tail contains 50 to 250 adenosine nucleotides. In some embodiments, the poly-A tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides. In some embodiments, the poly-A tail has a length of 100 nucleotides.

[0338] In some embodiments, the mRNA may contain two polyA sequences separated by an intervening nucleotide sequence. In some embodiments, the intervening nucleotide sequence contains 3 or fewer, 2 or fewer, 1 or fewer adenosine nucleotides, or none at all. In some embodiments, the intervening sequence contains 3 adenosine nucleotides. In some embodiments, the nucleotide length of the intervening sequence is 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, or 10 or fewer. In some embodiments, the intervening sequence consists of 10 nucleotides. In some embodiments, the intervening sequence contains the sequence GCAUAUGACU. In some embodiments, the intervening sequence does not begin with an adenosine nucleotide and does not end with an adenosine nucleotide. In some embodiments, the first polyA sequence contains at least 15, at least 20, at least 25, or at least 30 consecutive adenosine nucleotides. In some embodiments, the second polyA sequence contains at least 55, at least 60, at least 65, or at least 70 consecutive adenosine nucleotides. In some embodiments, the first polyA sequence contains 30 consecutive adenosine nucleotides. In some embodiments, the second polyA sequence contains 70 adenosine nucleotides.

[0339] iv. 5'Cap In some embodiments, the mRNA includes a 5' end cap or "5' end cap". The cap analog may be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, the cap analog is a dinucleotide cap. In some embodiments, the cap analog is a trinucleotide cap. In some embodiments, the cap analog is a tetranucleotide cap.

[0340] According to the manufacturer's protocol, for example, the following chemical RNA cap analogs may be used to simultaneously complete the 5'-capping of polynucleotides during an in vitro transcription reaction to generate a 5'-guanosine cap structure (New England BioLabs, Ipswich, MA). Alternatively, for example, a vaccinia virus capping enzyme may be used to complete the 5'-capping of modified mRNA after transcription to generate a "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The Cap1 structure may be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')G-2'-O-methyl. After generating the Cap2 structure from the Cap1 structure, the third nucleotide from the 5' end may be 2'-O-methylated using 2'-O methyltransferase. After generating the Cap3 structure from the Cap2 structure, the fourth nucleotide from the 5' end may be 2'-O-methylated using 2'-O methyltransferase. The enzymes may be derived from recombinant sources. Other cap analogs such as 7mG(5')ppp(5')NlmpNp cap may be used.

[0341] chemical modification mRNA can contain chemically unmodified nucleotides (i.e., unmodified nucleotides), chemically modified nucleotides, or both. Chemically unmodified nucleotides are standard ribonucleotides consisting of adenosine, guanosine, cytidine, and uridine.

[0342] Some embodiments of mRNA include modified nucleosides and / or nucleotides. “Nucleoside” refers to a compound comprising a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleic acid base”). “Nucleotide” refers to a nucleoside bonded to a phosphate group. Modifications to nucleotides or nucleosides may be present in the sugar or nucleic acid base. Modified nucleotides may be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly.

[0343] In some embodiments, the modified nucleic acid bases in mRNA include N1-methyl-pseudridine (m1ψ), N1-ethyl-pseudridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-uridine (m5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, the modified nucleic acid bases in mRNA include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, mRNA contains a combination of at least two (e.g., two, three, four, or more) of any of the above modified nucleic acid bases, including but not limited to chemical modifications.

[0344] In some embodiments, the mRNA contains a 1-methylpseudridine (m1ψ) substitution at one or more or all of the uridine positions of the mRNA.

[0345] In some embodiments, the mRNA includes 1-methyl-pseudruridine (m1ψ) substitutions at one or more uridine positions and 5-methylcytidine substitutions at one or more cytidine positions.

[0346] In some embodiments, the mRNA contains pseudouridine (ψ) substitutions at one or more or all uridine positions.

[0347] In some embodiments, the mRNA includes pseudouridine (ψ) substitutions at one or all uridine positions and 5-methylcytidine substitutions at one or all cytidine positions.

[0348] In some embodiments, the mRNA contains uridine at one or more or all of the uridine positions.

[0349] In some embodiments, the mRNA contains 5-methyluridine and 5-methylcytidine at one or more or all of the uridine and cytidine positions, respectively.

[0350] In some embodiments, mRNA is uniformly modified with a specific modification (e.g., completely modified, modified throughout the entire sequence). For example, mRNA can be uniformly modified with 1-methylpseudridine, which means replacing all uridine residues in the mRNA sequence with 1-methylpseudridine. Similarly, nucleic acids can be uniformly modified by replacing any type of nucleoside residue present in the sequence with a modifying residue such as the above residue. In some embodiments, ORFs are uniformly modified with a specific modification such as 1-methylpseudridine. In some embodiments, uniform modification does not involve an mRNA cap. For example, a cap with a different modification from the rest of the mRNA can be added to the mRNA simultaneously with or after transcription.

[0351] Codon optimization In some embodiments, ORFs encoding a protein or a fragment thereof are codon-optimized. Methods for codon optimization are known in the art. For example, one or more ORFs of any of the sequences described below may be codon-optimized. In some embodiments, codon optimization may be used to ensure proper folding by matching codon frequencies in the target organism and the host organism, to improve RNA stability or reduce secondary structures by biasing the GC content, to minimize tandem repeat codon or base sequences that may impair gene construction or expression, to customize transcription and translation regulatory regions, to insert or remove protein transport sequences, to remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, to add, remove or shuffle protein domains, to insert or delete restriction sites, to modify ribosome binding sites and RNA degradation sites, to adjust the translation rate so that various domains of the protein fold correctly, or to reduce or eliminate problematic secondary structures within a polynucleotide. Codon optimization tools, algorithms, and services are publicly known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In some embodiments, an open reading frame (ORF) sequence is optimized using an optimization algorithm.

[0352] In some embodiments, the codon-optimized sequence shares less than 95%, less than 90%, less than 85%, less than 80%, or less than 75% sequence identity with respect to the open reading frame of a natural or wild-type sequence (e.g., a natural or wild-type mRNA sequence encoding a protein or a fragment thereof). In some embodiments, the codon-optimized sequence shares 65% to 85% sequence identity with respect to a naturally occurring or wild-type sequence (e.g., a natural or wild-type RNA or DNA sequence encoding a protein or a fragment thereof).

[0353] In some embodiments, the codon-optimized sequence encodes an antigen that is immunogenic to the same extent as, or more immunogenic than, the protein or fragment encoded by the non-codon-optimized sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% higher).

[0354] Self-amplifying RNA In some embodiments, the RNA is self-amplifying RNA. Self-amplifying RNA is RNA that codes for one or more proteins, either alone or in combination, that can replicate the self-amplifying RNA. In some embodiments, the proteins coded by the self-amplifying RNA are non-structural proteins nsP1, nsP2, nsP3, and nsP4, which form RNA-dependent RNA polymerases (RdRp), or replicases, that can replicate the self-amplifying RNA. By coding for proteins that can replicate the RNA, self-amplifying RNA can self-amplify within a cell under conditions that the cell can translate the RNA to produce the coded protein(s). Self-amplifying RNA is sometimes referred to as an RNA replicon.

[0355] When self-amplified RNA is translated, one or more encoded viral non-structural proteins are translated. “Viral non-structural proteins” are proteins encoded by the virus but are not part of the viral particle. With respect to self-amplified RNA, viral non-structural proteins replicate the nucleotide sequence encoding the desired protein from the self-amplified RNA via a subgenomic viral promoter. Such replication, driven by a viral subgenomic promoter using viral non-structural proteins, enhances the expression level of the encoded protein. In some embodiments, viral non-structural proteins are derived from single-stranded positive sense RNA viruses. In some embodiments, viral non-structural proteins are derived from alphaviruses belonging to the Togaviridae family. In some embodiments, the alphaviruses are Sindbis or Venezuelan encephalitis virus. In some embodiments, the viral non-structural protein is RNA-dependent RNA polymerase (RdRp) polyprotein P1234 (also referred to as NSP1-4).

[0356] Upon translation, P1234 is rapidly cleaved by autoproteolytic activity derived from the nsP2 (proteinase) portion of the polyprotein to form P123 and nsP4. Alphaviral RNA synthesis occurs in the cell's plasma membrane, where nsPs, along with the alphaviral RNA, form membrane invaginations (i.e., "microspheres"). These microspheres contain dsRNA, which is produced by replicating the viral genomic RNA of the "+" strand into the antigenomic RNA of the "-" strand. The "-" strand acts as a template from which additional "+" strand genomic RNA (synthesized from the 5'UTR) or shorter sub-sequences of the genomic RNA (referred to as subgenomic RNA) are synthesized from the subgenomic viral promoter region located near the end of the non-structural protein ORF. The "+" strand genomic RNA and subgenomic RNA are transported from the microspheres into the cytoplasm, where they are translated by endogenous ribosomes. The transported "+" strand genomic RNA associates with nsPs, forming additional spheres, thus potentially leading to an exponential increase in replicon RNA.

[0357] Viral non-structural proteins facilitate the replication of nucleotide sequences encoding desired proteins via subgenomic viral promoters (also referred to herein as “subgenomic promoters”). “Subgenomic viral promoter” refers to a promoter that drives the transcription of subgenomic mRNA. Typically, mRNA is transcribed from genomic DNA and episomal DNA (e.g., plasmids). Some viruses can transcribe subgenomic mRNA from RNA replicons produced from their genomic RNA. Many positive-sense RNA viruses produce subgenomic mRNA as one of the common infection techniques used by these viruses, generally transcribing delayed viral genes. Subgenomic viral promoters range from 20 nucleotides (Sindobis virus) to over 100 nucleotides (beet necrotic leaf vein yellowing virus) and are usually located upstream of transcription initiation. In some embodiments, the nucleotide lengths of the subgenome virus promoter are 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5 The subgenome virus promoters are 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more. Subgenome virus promoters are described in the art, for example, in PCT Publication WO2016 / 040359 and in Wagner et al., Chemical Biology, DOI:10.1038 / s41589-018-0146-9 (2018).

[0358] Circular RNA In some embodiments, the RNA is circular RNA. Circular RNA is RNA that does not have a 5' or 3' terminal nucleotide. Every nucleotide in circular RNA is covalently bonded to both (1) a 5' neighboring nucleotide and (2) a 3' neighboring nucleotide. In circular RNA having a nucleotide sequence containing all nucleotides in 5' to 3' order, the last nucleotide in the nucleotide sequence is covalently bonded to the first nucleotide in the nucleotide sequence.

[0359] A circular RNA may be a circular mRNA containing one or more 5'UTRs, an open reading frame, and one or more 3'UTRs. A circular RNA may contain a polyA region, as described in the section titled "PolyA Tail." Since circular RNA does not have ends like linear mRNA, those skilled in the art will understand that a polyA tail, when incorporated into a circular RNA, is referred to as a polyA region.

[0360] Circular RNA may contain an intra-sequence ribosome entry site (IRES). Because circular RNA does not contain a 5' from which ribosomes can initiate translation, as capped linear mRNA does, the inclusion of an IRES enables the translation of one or more open reading frames from the circular RNA. The IRES element attracts the eukaryotic ribosome translation initiation complex, thereby promoting translation initiation. See, for example, Kaufman et al., Nucleic Acid Res. 1991 19:4485-4490; Gurtu et al., Biochem Biophys Res Commun. 1996.229:295-298; Rees et al., BioTechniques. 1996.20:102-110; Kobayashi et al., BioTechniques. 1996.21:399-402; and Mosser et al., BioTechniques. 1997.22:150-161. A large number of IRES sequences are available, including a wide variety of virus-derived sequences such as picornavirus leader sequences (Jang et al., J Virol. 1989. 63: 1651-1660), polio leader sequences, hepatitis A virus leader sequences, hepatitis C virus IRESs, human rhinovirus type 2 IRESs (Dobrikova et al., Proc Natl Acad Sci US A. 2003. 100(25): 15125-15130), foot-and-mouth disease virus-derived IRES elements (Ramesh et al., Nucleic Acid Res. 1996. 24: 2697-2700), and giardiavirus IRESs (Garlapati et al., J Biol Chem. 2004. 279(5): 3389-3397).Furthermore, or alternatively, circular RNAs include yeast-derived IRES sequences, as well as human angiotensin II1 receptor IRESs (Martin et al., Mol Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al., Mol Cell Biol. 2004. 24(17):7622-7635), and vascular endothelial growth factor (VEGF) IRESs (Baranick et al., Proc Natl Acad Sci USA. 2008. 105(12):4733-4738, Stein et al., Mol Cell Biol. 1998. 18(6):3112-3119, Bert et al. It may contain any of the various nonviral IRES sequences, such as al., RNA. 2006. 12(6): 1074-1083) and insulin-like growth factor II (IGF-II) IRES (Pedersen et al., Biochem J. 2002. 363(Pt I): 37-44). These elements are commercially available as plasmids, for example, from Clontech (Mountain Review, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA), and GeneCopoeia (Rockville, MD). See also IRESite: a database of experimentally validated IRES structures. In some embodiments, the circular RNA contains coxsackievirus B3 (CVB3) IRES. See Gharbi et al., PLoS One. 2022. 17(10): e0274162. In some embodiments, the circular RNA contains EMCV IRES. In some embodiments, the circular RNA includes Salivirus IRES. See Sweeney et al., J Virol. 2012. 86(3):1468-1486. ​​In some embodiments, the Salivirus IRES is present in or derived from Salivirus FHB (SaliFHB). See GenBank accession number KM023140.1.

[0361] Viral vector Some embodiments relate to viral vectors containing or encoding influenza virus proteins. In some embodiments, the protein is contained within the viral vector. In some embodiments, the viral vector contains nucleic acids encoding the protein.

[0362] Any suitable virus may be used as a viral vector. Non-limiting examples of viruses that can be used as viral vectors include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses (AAVs), varicella-stomatitis virus (VSV), herpesviruses, Rous sarcoma virus, measles virus, poxvirus, gammavirus, alphavirus, mouse stem cell virus, Moloney's mouse leukemia virus, and bovine leukemia virus. In some embodiments, the viral vector is a VSV vector. In some embodiments, the viral vector is a measles virus vector. In some embodiments, the viral vector is an adenovirus vector. These and other viral vectors suitable for the expression of heterologous proteins (i.e., proteins not naturally expressed by the virus from which the viral vector originates) are known in the art.

[0363] In some embodiments, the viral vector comprises an influenza B / Victoria strain HA protein, or a nucleic acid encoding an influenza B / Victoria strain HA protein. In some embodiments, the influenza B / Victoria strain virus HA protein is the influenza B / Victoria strain virus HA protein described in the section titled "B / Victoria strain HA protein". In some embodiments, the influenza B / Victoria strain HA protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0364] In some embodiments, the viral vector comprises an influenza B / Yamagata strain HA protein, or a nucleic acid encoding an influenza B / Yamagata strain HA protein. In some embodiments, the influenza B / Yamagata strain virus HA protein is the influenza B / Yamagata strain virus HA protein described in the section titled "B / Yamagata strain HA protein". In some embodiments, the influenza B / Yamagata strain HA protein comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 70.

[0365] In some embodiments of the viral vector, the viral vector includes the IAV H1 HA protein, or a nucleic acid encoding the IAV H1 HA protein. In some embodiments, the IAV H1 HA protein is the IAV H1 HA protein described in the section titled "H1 HA Protein". In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the IAV H1 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0366] In some embodiments, the viral vector includes the IAV H3 HA protein, or a nucleic acid encoding the IAV H3 HA protein. In some embodiments, the IAV H3 HA protein is the IAV H3 HA protein described in the section titled "H3 HA Protein". If a mixed vaccine or polyvalent vaccine contains multiple H3 HA proteins (for example, containing multiple viral vectors containing or encoding different H3 HA proteins), each different H3 HA protein may contain the same or different substitutions as those described in the section titled "H3 HA Protein". In some embodiments, one or more H3 HA proteins do not contain the substitutions described in the section titled "H3 HA Protein". In some embodiments, the IAV H3 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 82. In some embodiments, each IAV H3 HA protein includes an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 82.

[0367] nucleic acid production RNA in vitro transcription (IVT) cDNA encoding RNA polynucleotides can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO / 2014 / 152027 (to the extent that it discloses an IVT method). In some embodiments, RNA is prepared according to one or more of the methods described in WO2018 / 053209 and WO2019 / 036682 (each of which is introduced herein by reference to the extent that it discloses an RNA production method).

[0368] In some embodiments, the RNA transcript is produced using an unamplified linearized DNA template in an in vitro transcription reaction for generating the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of RNA polynucleotides, e.g., influenza virus mRNA, but not limited to these. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells, are transfected using the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, e.g., a T7 promoter located 5' to the gene of interest and operably ligated to the gene of interest.

[0369] In some embodiments, the in vitro transcription template encodes the 5' untranslated (UTR) region, includes an open reading frame, and encodes the 3' UTR and poly-A tail. The specific nucleic acid sequence composition and length of the in vitro transcription template depend on the mRNA encoded by the template.

[0370] In vitro transcription systems typically include a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase.

[0371] NTPs may be manufactured in-house, selected from suppliers, or synthesized. NTPs may be selected from natural and non-natural NTPs, and from unmodified NTPs (e.g., ATP, GTP, UTP, CTP) or modified NTPs.

[0372] Any number of RNA polymerases or variants may be used to transcribe the RNA. The polymerases may be selected from, but are not limited to, phage RNA polymerases, e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or mutant polymerases, e.g., but not limited to chemically modified nucleic acids and / or nucleotides, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides. Some embodiments exclude the use of DNases.

[0373] In some embodiments, RNA transcripts are capped via enzymatic capping. In some embodiments, the RNA contains a 5' end cap, e.g., 7mG(5')ppp(5')NlmpNp.

[0374] In some embodiments, the RNA polymerase is wild-type RNA polymerase. In some embodiments, the RNA polymerase is an RNA polymerase variant as described in WO2020 / 172239, which is incorporated herein by reference to the extent described for RNA polymerase variants. The RNA polymerase variant may contain at least one amino acid substitution with respect to wild-type (WT) RNA polymerase. WT T7 RNA polymerase is represented by SEQ ID NO: 81. In some embodiments, the RNA polymerase is a variant RNA polymerase containing any one amino acid sequence from SEQ ID NOs: 176-179.

[0375] purification Nucleic acid purification may include, but is not limited to, nucleic acid cleanup, quality assurance, and quality control. Cleanup may be carried out by, but is not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), Poly-T beads, LNA® oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), HPLC-based purification methods, such as, but is not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), and / or tangential flow filtration, as known in the art. The term “purified,” when used in relation to nucleic acids, for example, “purified nucleic acid,” means that it has been separated from at least one impurity. An “impurity” is any substance that makes another one unsuitable, impure, or substandard. Therefore, purified nucleic acids (e.g., DNA and RNA) exist in a form or configuration different from that found in nature, or in a form or configuration different from that which existed before being subjected to processing or purification.

[0376] lipid composition In some embodiments, nucleic acids are formulated as lipid compositions, such as compositions comprising lipid nanoparticles, liposomes, and / or lipoplexes. In some embodiments, the lipid composition (e.g., lipid nanoparticles, liposomes, and / or lipoplexes) does not contain protamine. In some embodiments, the lipid composition contains protamine. In some embodiments, nucleic acids are formulated as lipid nanoparticle (LNP) compositions. Lipid nanoparticles typically contain ionic lipids (e.g., ionic aminolipids), noncationic lipids (e.g., phospholipids), structural lipids (e.g., sterols), and PEG-modified lipid components together with the nucleic acid cargo of interest. Lipid nanoparticles can be produced using components, compositions, and methods generally known in the art, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 038426, P See CT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575, PCT / US2016 / 069491, PCT / US2016 / 069493, and PCT / US2014 / 66242 (all of which are incorporated herein by reference in their entirety).

[0377] In some embodiments, the lipid nanoparticles consist of ionic lipids in a molar ratio of 20-60%, noncationic lipids in a molar ratio of 5-25%, structural lipids in a molar ratio of 25-55%, and PEG-modified lipids in a molar ratio of 0.5-15%.

[0378] In some embodiments, the lipid nanoparticles consist of ionic lipids in a molar ratio of 20-60%, noncationic lipids in a molar ratio of 5-30%, structural lipids in a molar ratio of 10-55%, and PEG-modified lipids in a molar ratio of 0.5-15%.

[0379] In some embodiments, the lipid nanoparticles contain 40-50 mol%, optionally 45-50 mol%, of ionic lipids, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol%, for example, about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0380] In some embodiments, the lipid nanoparticles contain 20-60 mol% of ionic lipids. For example, the lipid nanoparticles may contain 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% of ionic lipids. In some embodiments, the lipid nanoparticles contain 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% of ionic lipids. In some embodiments, the lipid nanoparticles contain 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% of ionic lipids.

[0381] In some embodiments, the lipid nanoparticles contain 45 to 55 mole percent (mol%) of ionic lipids. For example, the lipid nanoparticles may contain 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% of ionic lipids.

[0382] Ionic lipids In some embodiments, the ionic lipid is expressed by formula (IL * ) compounds [ka] or a salt thereof, in the formula: R 1-OH, optionally substituted with one or more oxos, or -NR N -C 4-10 Cycloalkenyl, or -N(R N’ R N” ) and R N is H or C 1-6 It is alkyl, R N’ is H or C 1-6 It is alkyl, R N” is H or C 1-6 It is alkyl, o is 1, 2, 3, or 4. n is 4, 5, 6, 7, or 8. m is 4, 5, 6, 7, or 8. M is -C(=O)-O- * Or -OC(=O)- * And in the formula, * R 2 This shows a connection to, M' is -C(=O)-O- * Or -OC(=O)- * And in the formula, * R 3 This shows a connection to, R 2 teeth, [ka] or -(C 1-6 Alkylene)-(C 3-8 Cycloalkyl)-C 1-6 It is alkyl, R 2a is -H or C 1-10 It is alkyl, R 2b is -H or C 1-10 It is alkyl, R 2c C 1-8 Alkyl or C 2-8 It is alkenyl, R 3 teeth, [ka] and R 3a is H or C 1-10 alkyl, R 3b is H or C 1-8 alkyl, R 3c is C 1-10 alkyl or C 2-8 alkenyl.

[0383] In some embodiments, the ionic lipid is of formula (IL ** -I): [Chemical formula] or a salt thereof, wherein: R 1 is -OH, o is 2, 3, or 4, n is 4, 5, 6, 7, or 8, M is -C(=O)-O- * wherein, * is a bond to R 2 and m is​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​1 is -NR optionally substituted with one or more oxo N -C 4-10 cycloalkenyl, or -N(R N’ R N” ), and R N is H, R N’ is C 1-2 alkyl, R N’’ is H, o is 2, 3, or 4, n is 6, 7, or 8, M is -C(=O)-O- * and in the formula, * is the bond to R 2 , m is 6, 7, or 8, M’ is -C(=O)-O-<00001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​2 This shows a connection to, m is 6, 7, or 8. M' is -C(=O)-O- * And in the formula, * R 3 This shows a connection to, R 2b C 3-5 It is alkyl, R 2c C 2-4 It is alkyl, R 3a C 7-10 It is alkyl, R 3c C 4-6 It is alkyl.

[0386] In some embodiments, the ionic lipid is expressed by formula (IL * -I): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is the variable IL * As defined, R 3a C 1-8 It is alkyl.

[0387] In some embodiments, the ionic lipid is expressed by formula (IL * -Ia): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is, formula IL * As defined, R 3a C 1-8 It is alkyl.

[0388] In some embodiments, the ionic lipid is expressed by formula (IL * -Ia'): [ka] or a salt thereof, in the formula: o, M, M', R 2c , and R 3c is the variable IL * As defined, R 3a C 1-8 It is alkyl.

[0389] In some embodiments, the ionic lipid is expressed by formula (IL * -Iia): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is, formula IL * As defined, R 3a C 1-8 It is alkyl.

[0390] In some embodiments, the ionic lipid is expressed by formula (IL * -II'): [ka] or a salt thereof, in the formula: o, M, M', R 2c , and R 3c is the variable IL * As defined, R 3a C 1-8 It is alkyl.

[0391] In some embodiments, the ionic lipid is expressed by formula (IL * -III): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0392] In some embodiments, the ionic lipid is expressed by formula (IL * -IIIa): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is the variable IL * As defined, R 2b C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0393] In some embodiments, the ionic lipid is expressed by formula (IL * -IIIa): [ka] or a salt thereof, in the formula: R 1 ,o,M,M',R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0394] In some embodiments, the ionic lipid is expressed by formula (IL * -IIIa'): [ka] or a salt thereof, in the formula: R 1 ,o,M,M',R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0395] In some embodiments, the ionic lipid is expressed by formula (IL * -IIIb): [ka] or a salt thereof, in the formula: R 1 ,o,M,M',R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0396] In some embodiments, the ionic lipid is expressed by formula (IL * -IIIb'): [ka] or a salt thereof, in the formula: R 1 ,o,M,M',R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0397] In some embodiments, the ionic lipid is expressed by formula (IL * -IV): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is the variable IL * As defined, R 2b C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0398] In some embodiments, the ionic lipid is expressed by formula (IL * -Iva): [ka] or a salt thereof, in the formula: R 1 o, m, n, M, M', R 2c , and R 3c is the variable IL * As defined, R 2b C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0399] In some embodiments, the ionic lipid is expressed by formula (IL * -Iva'): [ka] or a salt thereof, in the formula: o, M, M', R 2c , and R 3c is the variable IL * As defined, R 2a C 1-8 It is alkyl, R 3a C 1-8 It is alkyl.

[0400] Variables o and R of ionic lipids 1 , R N , R N’ , R N’’ In some embodiments of ionic lipids, o is 1.

[0401] In some embodiments of ionic lipids, o is 2.

[0402] In some embodiments of ionic lipids, o is 3.

[0403] In some embodiments of ionic lipids, o is 4.

[0404] Several embodiments of ionic lipids, R 1 It is -OH.

[0405] Several embodiments of ionic lipids, R N H is H.

[0406] Several embodiments of ionic lipids, R N It is methyl.

[0407] Several embodiments of ionic lipids, R N It is ethyl.

[0408] Several embodiments of ionic lipids, R 1 -NR N-Cyclobutenyl, and cyclobutenyl is optionally one or more oxo or -N(R) N’ R N’’ It will be replaced with ).

[0409] Several embodiments of ionic lipids, R N’ H is H.

[0410] Several embodiments of ionic lipids, R N’ It is methyl.

[0411] Several embodiments of ionic lipids, R N’ It is ethyl.

[0412] Several embodiments of ionic lipids, R N’’ H is H.

[0413] Several embodiments of ionic lipids, R N’’ It is methyl.

[0414] Several embodiments of ionic lipids, R N’’ It is ethyl.

[0415] Several embodiments of ionic lipids, R N’ H is R N’’ It is methyl.

[0416] Several embodiments of ionic lipids, R 1 teeth, [ka] That is the case.

[0417] Several embodiments of ionic lipids, R 1 teeth, [ka] That is the case.

[0418] Variables m and n of ionic lipids In some embodiments of ionic lipids, m is 4.

[0419] In some embodiments of ionic lipids, m is 5.

[0420] In some embodiments of ionic lipids, m is 6.

[0421] In some embodiments of ionic lipids, m is 7.

[0422] In some embodiments of ionic lipids, m is 8.

[0423] In some embodiments of ionic lipids, m is 4.

[0424] In some embodiments of the ionic lipid, n is 5.

[0425] In some embodiments of the ionic lipid, n is 6.

[0426] In some embodiments of ionic lipids, n is 7.

[0427] In some embodiments of ionic lipids, n is 8.

[0428] In some embodiments of the ionic lipid, n is 5 and m is 7.

[0429] In some embodiments of the ionic lipid, n is 7 and m is 7.

[0430] In some embodiments of the ionic lipid, m is 6 and n is 6.

[0431] Variables M and M' of ionic lipids In some embodiments of ionic lipids, M is -OC(=O)- * And in the formula, *R 2 This indicates a connection to [the specified location].

[0432] In some embodiments of ionic lipids, M is -C(=O)-O- * And in the formula, * R 2 This indicates a connection to [the specified location].

[0433] In some embodiments of ionic lipids, M' is -OC(=O)- * And in the formula, * R 3 This indicates a connection to [the specified location].

[0434] In some embodiments of ionic lipids, M' is -C(=O)-O- * And in the formula, * R 3 This indicates a connection to [the specified location].

[0435] In some embodiments of ionic lipids, M is -OC(=O)- * And in the formula, * R 2 This indicates a bond to, and M' is -C(=O)-O- * And, * R 3 This indicates a connection to [the specified location].

[0436] Variable R of ionic lipids 2 , R 2a , R 2b , R 2c Several embodiments of ionic lipids, R 2 teeth, [ka] That is the case.

[0437] Several embodiments of ionic lipids, R 2a It is hydrogen.

[0438] Several embodiments of ionic lipids, R 2a It is methyl.

[0439] Several embodiments of ionic lipids, R 2a It is ethyl.

[0440] Several embodiments of ionic lipids, R 2a It is propyl.

[0441] Several embodiments of ionic lipids, R 2a It is butyl.

[0442] Several embodiments of ionic lipids, R 2a It is pentyl.

[0443] Several embodiments of ionic lipids, R 2a It is hexyl.

[0444] Several embodiments of ionic lipids, R 2a It is heptyl.

[0445] Several embodiments of ionic lipids, R 2a It is Octill.

[0446] Several embodiments of ionic lipids, R 2b It is hydrogen.

[0447] Several embodiments of ionic lipids, R 2b It is methyl.

[0448] Several embodiments of ionic lipids, R 2b It is ethyl.

[0449] Several embodiments of ionic lipids, R 2b It is propyl.

[0450] Several embodiments of ionic lipids, R 2b It is butyl.

[0451] Several embodiments of ionic lipids, R 2b It is pentyl.

[0452] Several embodiments of ionic lipids, R 2b It is hexyl.

[0453] Several embodiments of ionic lipids, R 2b It is heptyl.

[0454] Several embodiments of ionic lipids, R 2b It is Octill.

[0455] Several embodiments of ionic lipids, R 2a is hydrogen, and R 2b It is hydrogen.

[0456] Several embodiments of ionic lipids, R 2a It is hexyl, and R 2b It is hydrogen.

[0457] Several embodiments of ionic lipids, R 2a is octyl, R 2b It is hydrogen.

[0458] Several embodiments of ionic lipids, R 2a is hydrogen, and R 2b It is butyl.

[0459] Several embodiments of ionic lipids, R 2c It is methyl.

[0460] Several embodiments of ionic lipids, R 2c It is ethyl.

[0461] Several embodiments of ionic lipids, R 2c It is propyl.

[0462] Several embodiments of ionic lipids, R 2c It is butyl.

[0463] Several embodiments of ionic lipids, R 2c It is pentyl.

[0464] Several embodiments of ionic lipids, R 2c It is hexyl.

[0465] Several embodiments of ionic lipids, R 2c It is heptyl.

[0466] Several embodiments of ionic lipids, R 2c It is Octill.

[0467] Several embodiments of ionic lipids, R 2 is, -(C 1-6 Alkylene)-(C 3-8 Cycloalkyl)-C 1-6 It is alkyl.

[0468] Several embodiments of ionic lipids, R 2 is, -(C 1-6 Alkylene)-(cyclohexyl)-C 1-6 It is alkyl.

[0469] Several embodiments of ionic lipids, R 2 is, -(C 1-6 Alkylene)-(cyclopentyl)-C 1-6 It is alkyl.

[0470] Variable R of ionic lipids 3 , R 3a , R 3b , and R 3c Several embodiments of ionic lipids, R 3 teeth, [ka] That is the case.

[0471] Several embodiments of ionic lipids, R 3a It is hydrogen.

[0472] Several embodiments of ionic lipids, R 3a It is methyl.

[0473] Several embodiments of ionic lipids, R 3a It is ethyl.

[0474] Several embodiments of ionic lipids, R 3a It is propyl.

[0475] Several embodiments of ionic lipids, R 3a It is butyl.

[0476] Several embodiments of ionic lipids, R 3a It is pentyl.

[0477] Several embodiments of ionic lipids, R 3a It is hexyl.

[0478] Several embodiments of ionic lipids, R 3a It is heptyl.

[0479] Several embodiments of ionic lipids, R 3a It is Octill.

[0480] Several embodiments of ionic lipids, R 3b It is hydrogen.

[0481] Several embodiments of ionic lipids, R 3b It is methyl.

[0482] Several embodiments of ionic lipids, R 3b It is ethyl. 【048...

Claims

1. An influenza B / Victoria lineage virus HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of the reference influenza B / Victoria lineage virus hemagglutinin (HA) protein, wherein the influenza B / Victoria lineage virus HA protein is as follows (a) to (s), i.e., (a) Tyrosine at position 381 and valine at position 288, (b) Cysteine ​​at position 27 and cysteine ​​at position 349, (c) Cysteine ​​at position 295 and cysteine ​​at position 328, (d) Cysteine ​​at position 399 and cysteine ​​at position 473, (e) Cysteine ​​at position 422 and cysteine ​​at position 444, (f) Cysteine ​​at position 118 and cysteine ​​at position 216, (g) Cysteine ​​at position 237 and cysteine ​​at position 261, (h) Cysteine ​​at position 363 and cysteine ​​at position 480, (i) Cysteine ​​at position 364 and cysteine ​​at position 483, (j) Cysteine ​​at position 365 and cysteine ​​at position 476, (k) Cysteine ​​at position 366 and cysteine ​​at position 479, (l) Cysteine ​​at position 367 and cysteine ​​at position 483, (m) Cysteine ​​at position 435 and cysteine ​​at position 428, (n) Cysteine ​​at position 494 and cysteine ​​at position 483, (o) Cysteine ​​at position 494 and cysteine ​​at position 480, (p) Proline at position 416, proline at position 417, proline at position 434, and proline at position 433, (q) Proline at position 434 and proline at position 433, (r) Proline at position 515 and proline at position 516, (s) Contains one or more phenylalanine at position 473, The influenza B / Victoria virus HA protein, wherein the positions (a) to (s) are numbered by aligning them with SEQ ID NO: 71 of the amino acid sequence of the reference influenza B / Victoria virus HA protein.

2. The aforementioned influenza B / Victoria lineage virus HA protein is as follows (a) to (s), (a) Replacement of H381Y and A288V, (b) Substitution of S27C and Y349C, (c) Substitution of I295C and K328C, (d) Substitution of S399C and H473C, (e) Replacement of L422C and D444C, (f) Substitution of K118C and L216C, (g) Substitution of V237C and D261C, (h) Replacement of G363C and K480C, (i) Replacement of A364C and K483C, (j) Replacement of I365C and A476C, (k) Replacement of A366C and R479C, (l) Substitution of G367C and K483C, (m) Replacement of E435C and A428C, (n) Substitution of N494C and K483C, (o) Replacement of N494C and K480C, (p) Substitution of E416P, L417P, N434P, and H433P, (q) Replacement of N434P and H433P, (r) Replacement of T515P and F516P, and The influenza B / Victoria lineage virus HA protein according to claim 1, comprising one or more substitutions of (s)H473F.

3. The influenza B / Victoria lineage virus HA protein according to claim 1 or 2, wherein the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381 and valine at position 288.

4. The influenza B / Victoria lineage HA protein according to claim 1 or 2, wherein the influenza B / Victoria lineage HA protein comprises the substitutions of H381Y and A288V.

5. The following (a) to (r), that is, (a) Replacement of S27C and Y349C, (b) Substitution of I295C and K328C, (c) Substitution of S399C and H473C, (d) Replacement of L422C and D444C, (e) Substitution of K118C and L216C, (f) Substitution of V237C and D261C, (g) Substitution of G363C and K480C, (h) Replacement of A364C and K483C, (i) Replacement of I365C and A476C, (j) Replacement of A366C and R479C, (k) Replacement of G367C and K483C, (l) Replacement of E435C and A428C, (m) Replacement of N494C and K483C, (n) Replacement of N494C and K480C, (o) Replacement of E416P, L417P, N434P, and H433P, (p) Replacement of N434P and H433P, (q) Replacement of T515P and F516P, (r) Replacement of H473F The influenza B / Victoria lineage virus HA protein according to claim 3 or 4, further comprising one or more of the above.

6. The influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 5, wherein the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 422, and cysteine ​​at position 444.

7. The influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 5, wherein the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 364, and cysteine ​​at position 483.

8. The influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 5, wherein the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 367, and cysteine ​​at position 483.

9. The influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 5, wherein the influenza B / Victoria lineage virus HA protein comprises tyrosine at position 381, valine at position 288, cysteine ​​at position 494, and cysteine ​​at position 483.

10. The influenza B / Victoria virus HA protein according to any one of claims 1 to 9, wherein the influenza B / Victoria virus HA protein comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:

71.

11. An influenza B / Yamagata strain virus HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of the reference influenza B / Yamagata strain virus hemagglutinin (HA) protein, wherein the influenza B / Yamagata strain virus HA protein is (a) to (i) below, i.e., (a) Cysteine ​​at position 231 and cysteine ​​at position 273, (b) Cysteine ​​at position 295 and cysteine ​​at position 332, (c) Cysteine ​​at position 396 and cysteine ​​at position 510, (d) Cysteine ​​at position 239 and cysteine ​​at position 276, (e) Cysteine ​​at position 367 and cysteine ​​at position 401, (f) Cysteine ​​at position 363 and cysteine ​​at position 404, (g) Cysteine ​​at position 437 and cysteine ​​at position 429, (h) cysteine ​​at position 451 and cysteine ​​at position 422, and (i) Tyrosine at position 381 and valine at position 290 Includes one or more of the following: The positions (a) to (i) above are numbered by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO: 70, and this is the influenza B / Yamagata strain virus HA protein.

12. The aforementioned influenza B / Yamagata strain virus HA protein is as follows (a) to (i), (a) Replacement of A231C and G273C, (b) Substitution of K295C and I332C, (c) Replacement of A396C and L510C, (d) Replacement of V239C and V276C, (e) Replacement of I367C and S401C, (f) Replacement of F363C and E404C, (g) Substitution of E437C and G429C, (h) Substitution of D451C and K422C, and (i) Replacement of H381Y and A290V The influenza B / Yamagata strain virus HA protein according to claim 11, comprising one or more of the above.

13. The influenza B / Yamagata strain virus HA protein according to claim 11 or 12, wherein the influenza B / Yamagata strain virus HA protein comprises a tyrosine substitution at position 381 and a valine substitution at position 290.

14. The influenza B / Yamagata strain virus HA protein according to claim 11 or 12, wherein the influenza B / Yamagata strain virus HA protein comprises cysteine ​​at position 239, cysteine ​​at position 276, cysteine ​​at position 451, and cysteine ​​at position 422.

15. The influenza B / Yamagata strain virus HA protein according to claim 11 or 12, wherein the influenza B / Yamagata strain virus HA protein comprises cysteine ​​at position 367, cysteine ​​at position 401, cysteine ​​at position 451, and cysteine ​​at position 422.

16. The influenza B / Yamagata strain virus HA protein according to any one of claims 11 to 15, wherein the influenza B / Yamagata strain virus HA protein comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:

70.

17. An IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of the reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, wherein the IAV H1 HA protein is (a) to (o) below, i.e., (a) Cysteine ​​at position 391 and cysteine ​​at position 37, (b) Cysteine ​​at position 395 and cysteine ​​at position 36, (c) Cysteine ​​at position 461 and cysteine ​​at position 348, (d) Proline at position 404 and proline at position 416, (e) Isoleucine at position 395 and isoleucine at position 447, (f) Glycine at position 456 and isoleucine at position 402, (g) cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, and proline at position 416, (h) Cysteine ​​at position 391, cysteine ​​at position 37, isoleucine at position 395, and isoleucine at position 447, (i) cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, (j) Cysteine ​​at position 395, cysteine ​​at position 36, proline at position 404, and proline at position 416, (k) cysteine ​​at position 395, cysteine ​​at position 36, glycine at position 456, and isoleucine at position 402, (l) cysteine ​​at position 461, cysteine ​​at position 348, proline at position 404, and proline at position 416, (m) Cysteine ​​at position 461, cysteine ​​at position 348, isoleucine at position 395, and isoleucine at position 447, (n) cysteine ​​at position 461, cysteine ​​at position 348, glycine at position 456, and isoleucine at position 402, (o) Cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, glycine at position 456, and isoleucine at position 402 Includes one or more of the following: The IAV H1 HA protein, wherein the positions (a) to (o) above are numbered by aligning the amino acid sequence of the reference IAV H1 HA protein with SEQ ID NO:

83.

18. The IAV H1 HA protein is as follows (a) to (o), (a) Replacement of K391C and L37C, (b) Substitution of K395C and V36C, (c) Replacement of N461C and G348C, (d) Replacement of N404P and H416P, (e) Replacement of K395I and E447I, (f) Replacement of D456G and K402I, (g) Substitution of K391C, L37C, N404P, H416P, (h) Substitution of K391C, L37C, K395I, E447I, (i) Replacement of K391C, L37C, D456G, and K402I. (j) Replacement of K395C, V36C, N404P, H416P, (k) Replacement of K395C, V36C, D456G, K402I, (l) Replacement of N461C, G348C, N404P, H416P, (m) Replacement of N461C, G348C, K395I, E447I, (n) Substitution of N461C, G348C, D456G, K402I, and (o) K391C, L37C, N404P, H416P, D456G, K402I The IAV H1 HA protein according to claim 17, comprising one or more of the substitutions.

19. The IAV H1 HA protein according to claim 17 or 18, wherein the IAV H1 HA protein comprises proline at position 404, proline at position 416, cysteine ​​at position 395, and cysteine ​​at position 36.

20. The IAV H1 HA protein according to claim 17 or 18, wherein the IAV H1 HA protein comprises cysteine ​​at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

21. The IAV H1 HA protein according to claim 17 or 18, wherein the IAV H1 HA protein contains glycine at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

22. The IAV H1 HA protein according to claim 17 or 18, wherein the IAV H1 HA protein comprises proline at position 404, proline at position 416, cysteine ​​at position 391, and cysteine ​​at position 36.

23. The IAV H1 HA protein according to claim 17 or 18, wherein the IAV H1 HA protein comprises proline at position 404, proline at position 416, cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402.

24. The IAV H1 HA protein according to any one of claims 17 to 23, wherein the IAV H1 HA protein comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:

83.

25. An IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of the reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, wherein the IAV H1 HA protein is (a) to (t) below, i.e., (a) Cysteine ​​at position 410 and cysteine ​​at position 462, (b) Cysteine ​​at position 120 and cysteine ​​at position 419, (c) Cysteine ​​at position 391 and cysteine ​​at position 37, (d) Cysteine ​​at position 395 and cysteine ​​at position 36, (e) Cysteine ​​at position 406 and cysteine ​​at position 430, (f) Cysteine ​​at position 457 and cysteine ​​at position 346, (g) Cysteine ​​at position 461 and cysteine ​​at position 348, (h) Proline at position 404 and proline at position 419, (i) Proline at position 404 and proline at position 416, (j) Proline at position 405 and proline at position 406, (k) Proline at position 415 and proline at position 416, (l) Tyrosine at position 25 and glutamic acid at position 45, (m) Tyrosine at position 370 and tryptophan at position 497, (n) Glycine at position 402, glycine at position 405, and glycine at position 407, (o) Isoleucine at position 395 and isoleucine at position 447, (p) Glycine at position 456 and glycine at position 402, (q) Cysteine ​​at position 442 and cysteine ​​at position 423, (r) Glycine, ranked 391st. (s) cysteine ​​at position 410, cysteine ​​at position 462, cysteine ​​at position 457, and cysteine ​​at position 346, and (t) Cysteine ​​at position 391, cysteine ​​at position 37, phenylalanine at position 370, and phenylalanine at position 455 Includes one or more of the following: The IAV H1 HA protein, wherein the positions (a) to (t) above are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO:

95.

26. The aforementioned IAV H1 HA protein is (a) to (t) below, that is, (a) Replacement of V410C and L462C, (b) Substitution of E120C and K419C, (c) Replacement of K391C and L37C, (d) Substitution of K395C and V36C, (e) Replacement of Q406C and D430C, (f) Replacement of S457C and L346C, (g) Substitution of N461C and G348C, (h) Substitution of N404P and K419P, (i) Replacement of N404P and H416P, (j) Replacement of T405P and Q406P, (k) Substitution of N415P and H416P, (l) Substitution of H25Y and H45E, (m) Replacement of H370Y and K497W, (n) Replacement of K402G, T405G, and F407G, (o) Replacement of K395I and E447I, (p) Replacement of D456G and K402I, (q) Replacement of L442C and N423C, (r) Substitution of K391G, (s) Replacement of V410C, L462C, S457C, L346C, and (t) Replacement of K391C, L37C, H370F, and H455F The IAV H1 HA protein according to claim 25, comprising one or more of the above.

27. The IAV H1 HA protein according to claim 25 or 26, wherein the IAV H1 HA protein contains cysteine ​​at position 391 and cysteine ​​at position 37.

28. The IAV H1 HA protein according to claim 25 or 26, wherein the IAV H1 HA protein contains cysteine ​​at position 395 and cysteine ​​at position 36.

29. The IAV H1 HA protein according to any one of claims 25 to 28, wherein the IAV H1 HA protein comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:

95.

30. An IAV H3 HA protein having an amino acid sequence that includes amino acid substitutions to the amino acid sequence of the reference influenza A virus (IAV) H3 hemagglutinin (HA) protein, wherein the IAV H3 HA protein is (a) to (j) below, i.e., (a) Cysteine ​​at position 40 and cysteine ​​at position 55, (b) Cysteine ​​at position 123 and cysteine ​​at position 421, (c) Cysteine ​​at position 260 and cysteine ​​at position 237, (d) Cysteine ​​at position 392 and cysteine ​​at position 46, (e) Cysteine ​​at position 411 and cysteine ​​at position 428, (f) Proline at position 402, proline at position 421, and proline at position 414, (g) Glycine at position 403, (h) Glycine at position 408 and glycine at position 409, (i) Isoleucine at position 396, and (j) Isoleucine at position 219 and proline at position 504 Includes one or more of the following: The IAV H3 HA protein, wherein the positions (a) to (j) are numbered by aligning the amino acid sequence of the reference IAV H3 HA protein with SEQ ID NO:

82.

31. The aforementioned IAV H3 HA protein is (a) to (j) below, that is, (a) Replacement of T40C and A55C, (b) Substitution of S123C and R421C, (c) Replacement of L260C and P237C, (d) Substitution of Q392C and T46C, (e) Replacement of I411C and Y428C, (f) Replacement of G402P, R421P, and E414P, (g) Substitution of K403G, (h) Replacement of F408G and H409G, (i) Substitution of K396I, and (j) Replacement of T219I and H504P The IAV H3 HA protein according to claim 30, comprising one or more of the above.

32. The IAV H3 HA protein according to claim 30 or 31, wherein the IAV H3 HA protein contains proline at position 402, proline at position 421, and proline at position 414.

33. The IAV H3 HA protein according to any one of claims 30 to 32, wherein the IAV H3 HA protein comprises an amino acid sequence having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:

82.

34. The HA protein according to any one of claims 1 to 33, wherein the HA protein is a recombinant protein.

35. (a) Influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 10, (b) Influenza B / Yamagata strain virus HA protein according to any one of claims 11 to 16 (c) The IAV H1 HA protein according to any one of claims 17 to 29, or (d) IAV H3 HA protein according to any one of claims 30 to 33 Nucleic acids that code for something.

36. The nucleic acid according to claim 35, wherein the nucleic acid is DNA, messenger ribonucleic acid (mRNA), cyclic ribonucleic acid (RNA), or self-amplifying ribonucleic acid (saRNA).

37. (a) the influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 10, and / or the nucleic acid encoding the influenza B / Victoria lineage virus HA protein according to any one of claims 1 to 10, (b) the influenza B / Yamagata strain virus HA protein according to any one of claims 11 to 16, and / or the nucleic acid encoding the influenza B / Yamagata strain virus HA protein according to any one of claims 11 to 16. (c) The IAV H1 HA protein according to any one of claims 17 to 29, and / or a nucleic acid encoding the IAV H1 HA protein according to any one of claims 17 to 29, and / or (d) The IAV H3 HA protein according to any one of claims 30 to 33, and / or the nucleic acid encoding the IAV H3 HA protein according to any one of claims 30 to 33. A viral vector containing [unspecified].

38. (a) The HA protein according to any one of claims 1 to 34, (b) The nucleic acid according to any one of claims 35 to 36, or (c) The viral vector according to claim 37 A vaccine containing [this ingredient].

39. The vaccine according to claim 38, wherein the nucleic acid is mRNA.

40. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an influenza B / Victoria lineage virus HA protein having an amino acid sequence including amino acid substitutions to the amino acid sequence of a reference influenza B / Victoria lineage virus hemagglutinin (HA) protein, wherein the influenza B / Victoria lineage virus HA protein is as follows (a) to (s), i.e., (a) Tyrosine at position 381 and valine at position 288, (b) Cysteine ​​at position 27 and cysteine ​​at position 349, (c) Cysteine ​​at position 295 and cysteine ​​at position 328, (d) Cysteine ​​at position 399 and cysteine ​​at position 473, (e) Cysteine ​​at position 422 and cysteine ​​at position 444, (f) Cysteine ​​at position 118 and cysteine ​​at position 216, (g) Cysteine ​​at position 237 and cysteine ​​at position 261, (h) Cysteine ​​at position 363 and cysteine ​​at position 480, (i) Cysteine ​​at position 364 and cysteine ​​at position 483, (j) Cysteine ​​at position 365 and cysteine ​​at position 476, (k) Cysteine ​​at position 366 and cysteine ​​at position 479, (l) Cysteine ​​at position 367 and cysteine ​​at position 483, (m) Cysteine ​​at position 435 and cysteine ​​at position 428, (n) Cysteine ​​at position 494 and cysteine ​​at position 483, (o) Cysteine ​​substitution at position 494 and cysteine ​​at position 480, (p) Proline at position 416, proline at position 417, proline at position 434, and proline at position 433, (q) Proline at position 434 and proline at position 433, (r) Proline at position 515, and proline at position 516, (s) Phenylalanine, ranked 473rd Includes one or more of the following: The mRNA wherein the positions (a) to (s) above are numbered by aligning the amino acid sequence of the reference influenza B / Victoria lineage virus HA protein with SEQ ID NO:

71.

41. The mRNA according to claim 40, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381 and valine at position 288.

42. The mRNA according to claim 40, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 422, and cysteine ​​at position 444.

43. The mRNA according to claim 40, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 364, and cysteine ​​at position 483.

44. The mRNA according to claim 40, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 367, and cysteine ​​at position 483.

45. The mRNA according to claim 40, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381, valine at position 288, cysteine ​​at position 494, and cysteine ​​at position 483.

46. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an influenza B / Yamagata strain virus HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of the hemagglutinin (HA) protein of the reference influenza B / Yamagata strain virus, wherein the influenza B / Yamagata strain virus HA protein is as follows (a) to (i), i.e., (a) Cysteine ​​at position 231 and cysteine ​​at position 273, (b) Cysteine ​​at position 295 and cysteine ​​at position 332, (c) Cysteine ​​at position 396 and cysteine ​​at position 510, (d) Cysteine ​​at position 239 and cysteine ​​at position 276, (e) Cysteine ​​at position 367 and cysteine ​​at position 401, (f) Cysteine ​​at position 363 and cysteine ​​at position 404, (g) Cysteine ​​at position 437 and cysteine ​​at position 429, (h) cysteine ​​at position 451 and cysteine ​​at position 422, and (i) Tyrosine at position 381 and valine at position 290 Includes one or more of the following: The mRNA is numbered at positions (a) to (i) above by aligning the amino acid sequence of the reference influenza B / Yamagata strain virus HA protein with SEQ ID NO:

70.

47. The mRNA according to claim 46, wherein the influenza B / Yamagata strain virus HA protein contains tyrosine at position 381 and valine at position 290.

48. The mRNA according to claim 46, wherein the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 239, cysteine ​​at position 276, cysteine ​​at position 451, and cysteine ​​at position 422.

49. The mRNA according to claim 46, wherein the influenza B / Yamagata strain virus HA protein contains cysteine ​​at position 367, cysteine ​​at position 401, cysteine ​​at position 451, and cysteine ​​at position 422.

50. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, wherein the IAV H1 HA protein is as follows (a) to (o), i.e., (a) Cysteine ​​at position 391 and cysteine ​​at position 37, (b) Cysteine ​​at position 395 and cysteine ​​at position 36, (c) Cysteine ​​at position 461 and cysteine ​​at position 348, (d) Proline at position 404 and proline at position 416, (e) Isoleucine at position 395 and isoleucine at position 447, (f) Glycine at position 456 and isoleucine at position 402, (g) cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, and proline at position 416, (h) Cysteine ​​at position 391, cysteine ​​at position 37, isoleucine at position 395, and isoleucine at position 447, (i) cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402, (j) Cysteine ​​at position 395, cysteine ​​at position 36, proline at position 404, and proline at position 416, (k) cysteine ​​at position 395, cysteine ​​at position 36, glycine at position 456, and isoleucine at position 402, (l) cysteine ​​at position 461, cysteine ​​at position 348, proline at position 404, and proline at position 416, (m) Cysteine ​​at position 461, cysteine ​​at position 348, isoleucine at position 395, and isoleucine at position 447, (n) cysteine ​​at position 461, cysteine ​​at position 348, glycine at position 456, and isoleucine at position 402, (o) Cysteine ​​at position 391, cysteine ​​at position 37, proline at position 404, glycine at position 456, and isoleucine at position 402 Includes one or more of the following: The mRNA wherein the positions (a) to (o) above are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO:

83.

51. The mRNA according to claim 50, wherein the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 395, and cysteine ​​at position 36.

52. The mRNA according to claim 50, wherein the IAV H1 HA protein contains cysteine ​​at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

53. The mRNA according to claim 50, wherein the IAV H1 HA protein contains glycine at position 456, isoleucine at position 402, cysteine ​​at position 395, and cysteine ​​at position 36.

54. The mRNA according to claim 50, wherein the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, and cysteine ​​at position 36.

55. The mRNA according to claim 50, wherein the IAV H1 HA protein contains proline at position 404, proline at position 416, cysteine ​​at position 391, cysteine ​​at position 37, glycine at position 456, and isoleucine at position 402.

56. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H1 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H1 hemagglutinin (HA) protein, wherein the IAV H1 HA protein is (a) to (t) below, i.e., (a) Cysteine ​​at position 410 and cysteine ​​at position 462, (b) Cysteine ​​at position 120 and cysteine ​​at position 419, (c) Cysteine ​​at position 391 and cysteine ​​at position 37, (d) Cysteine ​​at position 395 and cysteine ​​at position 36, (e) Cysteine ​​at position 406 and cysteine ​​at position 430, (f) Cysteine ​​at position 457 and cysteine ​​at position 346, (g) Cysteine ​​at position 461 and cysteine ​​at position 348, (h) Proline at position 404 and proline at position 419, (i) Proline at position 404 and proline at position 416, (j) Proline at position 405 and proline at position 406, (k) Proline at position 415 and proline at position 416, (l) Tyrosine at position 25 and glutamic acid at position 45, (m) Tyrosine at position 370 and tryptophan at position 497, (n) Glycine at position 402, glycine at position 405, and glycine at position 407, (o) Isoleucine at position 395 and isoleucine at position 447, (p) Glycine at position 456 and glycine at position 402, (q) Cysteine ​​at position 442 and cysteine ​​at position 423, (r) Glycine, ranked 391st. (s) cysteine ​​at position 410, cysteine ​​at position 462, cysteine ​​at position 457, and cysteine ​​at position 346, and (t) Cysteine ​​at position 391, cysteine ​​at position 37, phenylalanine at position 370, and phenylalanine at position 455 Includes one or more of the following: The mRNA wherein the positions (a) to (t) above are numbered by aligning the reference IAV H1 HA protein amino acid sequence with SEQ ID NO:

95.

57. The mRNA according to claim 56, wherein the IAV H1 HA protein contains cysteine ​​at position 391 and cysteine ​​at position 37.

58. The mRNA according to claim 56, wherein the IAV H1 HA protein contains cysteine ​​at position 395 and cysteine ​​at position 36.

59. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding an IAV H3 HA protein having an amino acid sequence containing amino acid substitutions to the amino acid sequence of a reference influenza A virus (IAV) H3 hemagglutinin (HA) protein, wherein the IAV H3 HA protein is (a) to (j) below, i.e., (a) Cysteine ​​at position 40 and cysteine ​​at position 55, (b) Cysteine ​​at position 123 and cysteine ​​at position 421, (c) Cysteine ​​at position 260 and cysteine ​​at position 237, (d) Cysteine ​​at position 392 and cysteine ​​at position 46, (e) Cysteine ​​at position 411 and cysteine ​​at position 428, (f) Proline at position 402, proline at position 421, and proline at position 414, (g) Glycine at position 403, (h) Glycine at position 408 and glycine at position 409, (i) Isoleucine at position 396, and (j) Isoleucine at position 219 and proline at position 504 Includes one or more of the following: The mRNA wherein the positions (a) to (j) are numbered by aligning the reference IAV H3 HA protein amino acid sequence with SEQ ID NO:

82.

60. The mRNA according to claim 59, wherein the IAV H3 HA protein contains proline at position 402, proline at position 421, and proline at position 414.

61. (a) mRNA encoding the IAV H1 HA protein according to any one of claims 49 to 57, (b) mRNA encoding the IAV H3 HA protein according to claim 58 or 59, and (c) mRNA encoding the influenza B / Victoria virus HA protein according to any one of claims 39 to 44 mRNA vaccine containing this component.

62. The mRNA vaccine according to claim 61, wherein the influenza B / Victoria lineage virus HA protein contains tyrosine at position 381 and valine at position 288.

63. The mRNA vaccine according to claim 61 or 62, further comprising additional mRNA encoding an additional IAV H3 HA protein.

64. The mRNA vaccine according to claim 61 or 62, further comprising two or more additional mRNAs, each independently encoding an additional IAV H3 HA protein.

65. The mRNA vaccine according to any one of claims 61 to 64, further comprising mRNA encoding the influenza B / Yamagata lineage virus HA protein according to any one of claims 45 to 48.

66. The mRNA vaccine according to claim 65, wherein the influenza B / Yamagata lineage virus HA protein contains tyrosine at position 381 and valine at position 290.

67. The mRNA vaccine according to any one of claims 61 to 64, wherein the mRNA vaccine does not contain mRNA encoding the influenza B / Yamagata lineage virus HA protein.

68. The mRNA vaccine according to any one of claims 61 to 67, wherein substantially equal masses of different mRNAs encoding different influenza virus HA proteins are present in the mRNA vaccine.

69. (i) A first additional mRNA encoding the first IAV neuraminidase (NA) protein of the first IAV NA subtype, (ii) A second additional mRNA encoding a second IAV NA protein of a second IAV NA subtype different from the first IAV NA subtype, and (iii) A third additional mRNA encoding the influenza B / Victoria lineage virus NA protein. The mRNA vaccine according to any one of claims 61 to 68, further comprising:

70. The mRNA vaccine according to claim 69, wherein the second IAV NA subtype is N2, and the mRNA vaccine further comprises additional mRNA encoding an additional IAV N2 NA protein.

71. The mRNA vaccine according to claim 69, wherein the second IAV NA subtype is N2, and the mRNA vaccine further comprises two or more additional mRNAs, each encoding an additional IAV N2 NA protein.

72. The mRNA vaccine according to any one of claims 69 to 71, further comprising additional mRNA encoding the influenza B / Yamagata lineage virus NA protein.

73. The mRNA vaccine according to any one of claims 69 to 71, wherein the mRNA vaccine does not contain mRNA encoding the influenza B / Yamagata lineage virus NA protein.

74. The mRNA vaccine according to any one of claims 69 to 73, wherein substantially equal masses of different mRNAs encoding different influenza virus NA proteins are present in the mRNA vaccine.

75. The mRNA vaccine according to claim 74, wherein substantially equal masses of (a) mRNA encoding influenza virus HA protein and (b) mRNA encoding influenza virus NA protein are present in the mRNA vaccine.

76. The mRNA vaccine according to claim 74, wherein the mRNA vaccine comprises (a) mRNA encoding influenza virus HA protein and (b) mRNA encoding influenza virus NA protein in a mass ratio of 3:

1.

77. The mRNA vaccine according to any one of claims 61 to 76, further comprising mRNA encoding a full-length SARS-CoV-2 spike (S) glycoprotein, comprising one or more proline substitutions relative to sequence number 78.

78. The mRNA vaccine according to any one of claims 61 to 77, further comprising mRNA encoding a protein containing one or more fragments of the SARS-CoV-2 spike (S) glycoprotein.

79. The mRNA vaccine according to claim 78, wherein one or more fragments comprise a fusion protein comprising (i) the N-terminal domain (NTD) of the SARS-CoV-2 S glycoprotein, (ii) the receptor-binding domain (RBD) of the SARS-CoV-2 S glycoprotein, and (iii) the transmembrane domain.

80. The mRNA vaccine according to any one of claims 61 to 79, further comprising a fusion (F) glycoprotein of human respiratory syncytial virus (hRSV), or mRNA encoding a fragment of the hRSV F glycoprotein.

81. The mRNA vaccine according to any one of claims 61 to 80, further comprising a lipid delivery vehicle that contacts the mRNA.

82. The mRNA vaccine according to claim 81, wherein the lipid delivery vehicle is lipid nanoparticles containing 20-60 mol% ionic lipids, 5-25 mol% noncationic lipids, 2-4 mol% PEG-modified lipids, and 25-55 mol% sterols.

83. The aforementioned ionic lipid is of formula (IL ＊ ) compounds 【Chemistry 1】 or its salt, in the formula: R １ However, -OH, and -NR substituted with one or more oxos of any choice. Ｎ -C ４－１０ Cycloalkenyl, or -N(R) Ｎ’ R Ｎ” ) and R Ｎ However, H or C １－６ It is alkyl, R Ｎ’ is H or C １－６ is alkyl, R Ｎ” However, H or C １－６ It is alkyl, o is 1, 2, 3, or 4, n is 4, 5, 6, 7, or 8. m is 4, 5, 6, 7, or 8. M is -C(=O)-O-* or -OC(=O)-*, where * is R ２ This shows a connection to, M' is -C(=O)-O-* or -OC(=O)-*, where * is R ３ This shows a connection to, R ２ but, 【Chemistry 2】 or -(C １－６ Alkylene) - (C ３－８ Cycloalkyl)-C １－６ It is alkyl, R ２ａ However, -H or C １－１０ It is alkyl, R ２ｂ However, -H or C １－１０ It is alkyl, R ２ｃ However, C １－８ Alkyl or C ２－８ It is alkenyl, R ３ but, 【Transformation 3】 And, R ３ａ However, H or C １－１０ It is alkyl, R ３ｂ However, H or C １－８ It is alkyl, R ３ｃ However, C １－１０ Alkyl or C ２－８ The mRNA vaccine according to claim 82, wherein the mRNA vaccine is an alkenil.

84. The aforementioned ionic lipids 【Chemistry 4】 The mRNA vaccine according to claim 82 or 83.

85. The aforementioned ionic lipids 【Transformation 5】 The mRNA vaccine according to claim 82 or 83.

86. The mRNA vaccine according to any one of claims 82 to 85, wherein 0.25 mol% to 1.0 mol% of the PEG-modified lipid is present in the core of the lipid nanoparticles.

87. The mRNA vaccine according to claim 86, wherein 2.0 mol% to 2.75 mol% of the PEG-modified lipid is not present in the core of the lipid nanoparticles.

88. The mRNA vaccine according to any one of claims 82 to 87, wherein the PEG-modified lipid is PEG-DMG or 134-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78,81,84,87,90,93,96,99,102,105,108,111,114,117,120,123,126,129,132-tetratetracontaoxatetratriacontahectylstearate.

89. Each mRNA comprises one or more chemically modified nucleotides, wherein each mRNA is an mRNA according to any one of claims 40 to 60 or an mRNA vaccine according to any one of claims 61 to 88.

90. Each mRNA comprises N1-methylpseudridine, wherein each mRNA is an mRNA according to any one of claims 40 to 60 or an mRNA vaccine according to any one of claims 61 to 89.

91. The mRNA according to any one of claims 40 to 60 or the mRNA vaccine according to any one of claims 61 to 90, wherein substantially all of the uracil nucleotides of each mRNA are modified to include N1-methylpseudridine.

92. Each mRNA comprises 5-methylcytidine and 5-methyluridine, wherein each mRNA is an mRNA according to any one of claims 40 to 60 or an mRNA vaccine according to any one of claims 61 to 89.

93. The mRNA according to any one of claims 40 to 60 or the mRNA vaccine according to any one of claims 61 to 89 and 92, wherein substantially all of the cytosine nucleotides of each mRNA are modified to contain 5-methylcytidine, and substantially all of the uracil nucleotides of each mRNA are modified to contain 5-methyluridine.

94. A method comprising administering the mRNA vaccine described in any one of claims 61 to 93 to a target.

95. The method according to claim 94, wherein the subject has not been vaccinated against the influenza virus for at least six months prior to the administration of the vaccine.

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