Multivalent influenza mRNA vaccine

Abstract

The present invention provides an isolated mRNA molecule, comprising the following elements in sequence from a 5' end to a 3' end: (1) a 5' cap; (2) a 5'UTR region; (3) an immunogenic fragment-coding region of influenza virus hemagglutinin (HA); (4) a 3'UTR region; and (5) a PolyA tail. The PolyA and coding region sequence of the mRNA molecule are both optimized, so that an mRNA influenza vaccine has controllable quality during production and exerts optimal immune response. Also provided are a composition and vaccine comprising the mRNA, and a method for using same to induce an immune response to an influenza virus in a subject.

Description

A multivalent influenza mRNA vaccine Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to mRNA vaccines that provide protection against multiple different serotypes of influenza virus. Background Technology

[0002] Influenza viruses belong to the Orthomyxoviridae family and are RNA viruses. Influenza viruses cause seasonal epidemics and sporadic pandemics in humans. It is estimated that approximately 3 to 5 million people worldwide suffer from severe respiratory illness due to influenza viruses each year, resulting in approximately 290,000 to 650,000 deaths. The World Health Organization (WHO) and national disease control agencies recommend annual influenza vaccination, as it is one of the most effective methods of preventing influenza. However, the surface glycoproteins of the influenza virus (hemagglutinin HA and neuraminidase NA) are commonly used influenza vaccine antigens and are prone to antigenic drift (see Harrison's Infectious Diseases, 2019, Chinese 1st edition). Variations in the amino acid sequence of the glycoproteins or various glycosyltransferases of the influenza virus can lead to significant differences in the glycoproteins and their glycosylation modifications, thus easily generating new viral strains. This ability of the influenza virus to easily change its antigenic properties means that the antigenicity of newly emerging strains often does not match the immune protection of existing vaccines, resulting in generally low protective efficacy of current influenza vaccines (approximately 40%–60%) and difficulty in preventing seasonal pandemics.

[0003] Emerging during the COVID-19 pandemic, mRNA technology serves as a rapid-response vaccine development platform for addressing explosive outbreaks. Utilizing linearized plasmid DNA as a template, it is synthesized through in vitro transcription, avoiding the live-cell culture process required in traditional vaccine production. Furthermore, it features a short production cycle, simple process, and convenient structural modification, making it particularly suitable for responding to rapidly evolving outbreaks. Compared to traditional vaccines such as inactivated vaccines, live attenuated vaccines, and recombinant subunit vaccines, mRNA vaccines can induce stronger humoral and cellular immunity, and depending on the administration method, can also induce mucosal immunity, making them suitable for use as influenza vaccines. Summary of the Invention

[0004] Through in-depth research, the inventors of this application discovered that by optimizing the coding sequence and screening elements such as PolyA, the production quality of mRNA influenza vaccines can be controlled and optimal immune response can be achieved.

[0005] Specifically, this application relates to:

[0006] 1. An mRNA molecule comprising, from the 5' end to the 3' end, the following elements in sequence: (1) a 5' cap; (2) a 5' UTR region; (3) a region encoding an immunogenic fragment of influenza virus hemagglutinin HA; (4) a 3' UTR region; and (5) a PolyA tail, the sequence of which is shown in SEQ ID NO:33.

[0007] 2. According to the mRNA molecule described in item 1, the influenza virus hemagglutinin HA is recommended or selected according to the standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS).

[0008] 3. The mRNA molecule according to any one of items 1 or 2, wherein the immunogenic fragment of the influenza virus hemagglutinin HA is an immunogenic fragment of hemagglutinin HA of influenza A H1N1, an immunogenic fragment of hemagglutinin HA of influenza A H3N2, or an immunogenic fragment of hemagglutinin HA of influenza B Victoria.

[0009] 4. According to the mRNA molecule described in item 3, the influenza A H1N1 is A / Puerto Rico / 8 / 34, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022, A / Victoria / 2570 / 2019, A / Sydney / 5 / 2021, A / California / 07 / 2009, A / Michigan / 45 / 2015, A / Brisbane / 02 / 2018, A / Hawaii / 70 / 2019, A / Idaho / 07 / 2018, A / Maine / 3 8 / 2018, A / Nebraska / 15 / 2018, A / Nebraska / 14 / 2019, A / Wisconsin / 588 / 2019, A / Iowa / 33 / 2019, A / Arkansas / 28 / 2019, A / Virginia / 41 / 2019, A / Minnesota / 60 / 2019, A / Alabama / 27 / 2019 or A / Guangdong-Maonan / SWLl536 / 2019.

[0010] 5. According to the mRNA molecule described in item 3, the influenza A H3N2 is A / Aichi / 2 / 1968, A / Thailand / 8 / 2022, A / Massachusetts / 18 / 2022, A / Darwin / 9 / 2021, A / Darwin / 6 / 2021, A / Cambodia / e0826360 / 2020, A / Switzerland / 8060 / 2017, A / Switzerland / 9715293 / 2013, A / Iowa / 60 / 2018, A / South Australia / 34 / 2019, A / Hong Kong / 45 / 2019, A / Hong Kong / 2671 / 2019, A / Kansas / 14 / 2017, A / Jamaica / 60361 / 2019, A / Florida / 130 / 2019, A / Laos / 1789 / 2019, A / Vermont / 25 / 2019, A / New Jersey / 34 / 2019, A / California / 176 / 2019, A / Pennsylvania / 1026 / 2019, A / Togo / 634 / 2019,A / Kenya / 130 / 2019, A / Togo / 1307 / 2019, A / Ohio / 30 / 2019, A / Guatemala / 93 / 2019, A / Guatemala / 10 / 2019, A / Hong Kong / 4801 / 2014 or A / Singapore / INFIMH-16-0019 / 2016.

[0011] 6. The mRNA molecule according to item 3, wherein the influenza B Victoria is B / Austria / 1359417 / 2021, B / Washington / 02 / 2019, B / Colorado / 06 / 2017, B / Brisbane / 60 / 2008 or B / Colorado / 06 / 2019.

[0012] 7. According to the mRNA molecule described in item 1, the immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA of influenza A H1N1, and its amino acid sequence is shown in SEQ ID NO:1.

[0013] 8. According to the mRNA molecule described in item 1, the immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA of influenza A H3N2, and its amino acid sequence is as shown in SEQ ID NO:6 or SEQ ID NO:11.

[0014] 9. According to the mRNA molecule described in item 1, the immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA of influenza B Victoria, and its amino acid sequence is shown in SEQ ID NO:16.

[0015] 10. The immunogenic fragment coding region sequence of the influenza virus hemagglutinin HA according to item 7 is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0016] 11. The immunogenic fragment coding region sequence of the influenza virus hemagglutinin HA according to item 8, such as SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0017] 12. The mRNA according to item 9, characterized in that the immunogenic fragment coding region sequence of the influenza virus hemagglutinin HA is as shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0018] 13. The mRNA according to any one of items 1-12, wherein the 5' cap of the mRNA is m7G(5')ppp(5')(2'-OMeA)pG.

[0019] 14. The mRNA according to any one of items 1-13, wherein the sequence of the 5'UTR of the mRNA is shown in SEQ ID NO:31.

[0020] 15. The mRNA according to any one of items 1-14, wherein the sequence of the 3'UTR of the mRNA is shown in SEQ ID NO:32.

[0021] 16. The mRNA according to any one of items 1-15, wherein the mRNA further comprises a chemical modification, wherein the chemical modification is preferably a modification of all or part of the uridine nucleotide to 1-methylpseuuridine.

[0022] 17. A composition comprising:

[0023] (1) A first mRNA encoding a first immunogenic polypeptide, the first immunogenic polypeptide comprising an immunogenic fragment of H1N1 hemagglutinin HA, the first mRNA being the mRNA described in item 7 or item 10.

[0024] (2) A second mRNA encoding a second immunogenic polypeptide, the second immunogenic polypeptide comprising an immunogenic fragment of H3N2 hemagglutinin HA, the second mRNA being the mRNA described in item 8 or item 11; and

[0025] (3) A third mRNA encoding a third immunogenic polypeptide, the third immunogenic polypeptide comprising an immunogenic fragment of hemagglutinin HA from influenza B Victoria, the third mRNA being the mRNA described in item 9 or item 12.

[0026] 18. The composition described in item 17, wherein,

[0027] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0028] (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and

[0029] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0030] 19. The composition described in item 17, wherein,

[0031] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0032] (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and

[0033] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0034] 20. The composition described in item 17, wherein,

[0035] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0036] (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and

[0037] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0038] 21. The composition described in item 17, wherein,

[0039] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:5, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:5;

[0040] (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and

[0041] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:20, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:20.

[0042] 22. The composition described in item 17, wherein,

[0043] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0044] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:7, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7; and

[0045] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0046] 23. The composition described in item 17, wherein,

[0047] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0048] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:8, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8; and

[0049] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0050] 24. The composition described in item 17, wherein,

[0051] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0052] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:9, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9; and

[0053] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0054] 25. The composition described in item 17, wherein,

[0055] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0056] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10; and

[0057] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0058] 26. The composition described in item 17, wherein,

[0059] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0060] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12; and

[0061] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0062] 27. The composition described in item 17, wherein,

[0063] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0064] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13; and

[0065] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0066] 28. The composition described in item 17, wherein,

[0067] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0068] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:14, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14; and

[0069] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0070] 29. The composition described in item 17, wherein,

[0071] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0072] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:15; and

[0073] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17.

[0074] 30. The composition described in item 17, wherein,

[0075] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0076] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:7, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7; and

[0077] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0078] 31. The composition described in item 17, wherein,

[0079] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0080] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:8, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8; and

[0081] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0082] 32. The composition described in item 17, wherein,

[0083] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0084] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:9, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9; and

[0085] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0086] 33. The composition described in item 17, wherein,

[0087] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0088] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10; and

[0089] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0090] 34. The composition described in item 17, wherein,

[0091] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0092] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12; and

[0093] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0094] 35. The composition described in item 17, wherein,

[0095] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0096] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13; and

[0097] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0098] 36. The composition described in item 17, wherein,

[0099] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0100] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:14, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14; and

[0101] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0102] 37. The composition described in item 17, wherein,

[0103] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0104] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:15; and

[0105] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18.

[0106] 38. The composition described in item 17, wherein,

[0107] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0108] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:7, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7; and

[0109] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0110] 39. The composition described in item 17, wherein,

[0111] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0112] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:8, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:8; and

[0113] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0114] 40. The composition described in item 17, wherein,

[0115] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0116] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:9, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:9; and

[0117] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0118] 41. The composition described in item 17, wherein,

[0119] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0120] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:10; and

[0121] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0122] 42. The composition described in item 17, wherein,

[0123] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0124] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12; and

[0125] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0126] 43. The composition described in item 17, wherein,

[0127] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0128] (2) The second mRNA contains, for example, the immunogenic fragment coding region of influenza virus hemagglutinin HA, having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13; and

[0129] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0130] 44. The composition described in item 17, wherein,

[0131] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0132] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:14, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14; and

[0133] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0134] 45. The composition described in item 17, wherein,

[0135] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0136] (2) The second mRNA contains an immunogenic fragment coding region sequence of influenza virus hemagglutinin HA, such as SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:15; and

[0137] (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:19.

[0138] 46. ​​A composition comprising:

[0139] (1) A first mRNA encoding a first immunogenic polypeptide, the first immunogenic polypeptide comprising an immunogenic fragment of H1N1 hemagglutinin HA, the first mRNA being the mRNA described in item 7 or item 10.

[0140] (2) A second mRNA encoding a second immunogenic polypeptide, the second immunogenic polypeptide comprising an immunogenic fragment of H3N2 hemagglutinin HA, the second mRNA being the mRNA described in item 8 or item 11.

[0141] (3) A third mRNA encoding a third immunogenic polypeptide, the third immunogenic polypeptide comprising an immunogenic fragment of hemagglutinin HA from influenza B Victoria, the third mRNA being the mRNA described in item 9 or item 12.

[0142] and

[0143] (4) A fourth mRNA encoding a fourth immunogenic polypeptide, the fourth immunogenic polypeptide comprising an immunogenic fragment of influenza virus ion channel protein M2, the amino acid sequence of which is shown in SEQ ID NO:21.

[0144] 47. The composition described in item 46, wherein,

[0145] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2;

[0146] (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15;

[0147] (3) The third mRNA comprises, as shown in SEQ ID NO:17, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and

[0148] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0149] 48. The composition described in item 46, wherein,

[0150] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0151] (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15;

[0152] (3) The third mRNA comprises, as shown in SEQ ID NO:18, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, an immunogenic fragment coding region of influenza virus hemagglutinin HA; and

[0153] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0154] 49. The composition described in item 46, wherein,

[0155] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4;

[0156] (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15;

[0157] (3) The third mRNA comprises, as shown in SEQ ID NO:19, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and

[0158] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0159] 50. The composition described in item 46, wherein,

[0160] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:5, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:5;

[0161] (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15;

[0162] (3) The third mRNA comprises, as shown in SEQ ID NO:20, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and

[0163] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0164] 51. The composition described in item 46, wherein,

[0165] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0166] (2) The second mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:12, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:12;

[0167] (3) The third mRNA comprises, as shown in SEQ ID NO:18, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, an immunogenic fragment coding region of influenza virus hemagglutinin HA; and

[0168] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0169] 52. The composition described in item 46, wherein,

[0170] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0171] (2) The second mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:13, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:13;

[0172] (3) The third mRNA comprises, as shown in SEQ ID NO:18, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, an immunogenic fragment coding region of influenza virus hemagglutinin HA; and

[0173] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0174] 53. The composition described in item 46, wherein,

[0175] (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3;

[0176] (2) The second mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:14, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14;

[0177] (3) The third mRNA comprises, as shown in SEQ ID NO:18, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, an immunogenic fragment coding region of influenza virus hemagglutinin HA; and

[0178] (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:22.

[0179] 54. The composition according to any one of items 17-53, wherein the 5' cap of the mRNA is m7G(5')ppp(5')(2'-OMeA)pG.

[0180] 55. The composition according to any one of items 17-54, wherein the sequence of the 5'UTR of the mRNA is as shown in SEQ ID NO:31.

[0181] 56. The composition according to any one of items 17-55, wherein the sequence of the 3'UTR of the mRNA is as shown in SEQ ID NO:32.

[0182] 57. The composition according to any one of items 17-56, wherein the mRNA molecule further comprises chemical modification, preferably, modifying all or part of the uridine monophosphate in the nucleotide sequence with 1-methylpseuuridine monophosphate.

[0183] 58. The composition according to any one of items 17-57, wherein the content ratio of the first mRNA, the second mRNA and the third mRNA is in the range of 2 to 10:2 to 10:2 to 10 by mass, preferably in the range of 3 to 7:3 to 7:3 to 7, more preferably 5:5:5 or 2:2:2.

[0184] 59. The composition according to any one of items 46-57, wherein the content ratio of the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA is in the range of 2-10:2-10:2-10:0.5-5 by mass, preferably in the range of 2-5:2-5:2-5:0.5-2, more preferably in the range of 2:2:2:0.5, 2:2:2:1, or 2:2:2:2.

[0185] The composition according to any one of items 60-59 further includes a pharmaceutically acceptable carrier, preferably a lipid nanoparticle, wherein the mRNA is encapsulated in one or more lipid nanoparticles.

[0186] 61. The composition of claim 60, wherein each individual lipid nanoparticle is encapsulated with the first to third mRNA in substantially the same proportion, or each individual particle is encapsulated with different proportions of the first to third mRNA, or each individual particle is encapsulated with any one of the first, second, or third mRNA.

[0187] 62. The composition of claim 60, wherein each individual particle of the lipid nanoparticle is encapsulated with the first to fourth mRNA in substantially the same proportion, or each individual particle is encapsulated with different proportions of the first to fourth mRNA, or each individual particle is encapsulated with any one of the first, second, third or fourth mRNA.

[0188] 63. The composition according to any one of claims 60-62, wherein the lipid nanoparticles comprise ionizable lipids, phospholipids, structured lipids, and polyethylene glycol (PEG)-lipids.

[0189] Preferably, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (20-60):(5-25):(25-55):(0.5-5);

[0190] More preferably, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid and the PEG lipid is (40-55):(5-15):(30-50):(1-3).

[0191] 64. The composition described in item 63, wherein,

[0192] The phospholipid is selected from one or more of the following compounds: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycerol-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholestyldimethylsuccinate-sn-glycerol-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphate choline, 1,2-diaryl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diaryl-sn-glycerol-3-phosphate ethanolamine, 1,2-dithiohexaenoic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol) sodium salt (DOPG), or sphingomyelin, preferably DSPC;

[0193] The structural lipid is selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, and stigmasterol, preferably cholesterol; and / or

[0194] The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol, preferably DMG-PEG2000.

[0195] 65. The composition of any one of items 17-64 is a vaccine.

[0196] 66. A nucleic acid molecule encoding mRNA as described in any one of items 1-16, wherein the nucleic acid molecule is preferably DNA, more preferably a DNA plasmid.

[0197] 67. A method for inducing an immune response to an influenza virus in a subject, comprising administering to the subject an effective dose of an mRNA as described in any one of items 1-16, a composition as described in any one of items 17-65, or a nucleic acid molecule as described in item 66;

[0198] Preferably, the method includes administering the medication to the subject once, twice, or three times.

[0199] 68. Use of any mRNA described in any one of items 1-16, any composition described in any one of items 17-65, or the nucleic acid molecule described in item 65 in the preparation of a medicament for the prevention or treatment of influenza virus infection.

[0200] 69. Use of any mRNA described in any one of items 1-16, any composition described in any one of items 17-65, or any nucleic acid molecule described in item 66 in the prevention or treatment of influenza virus infection. Attached Figure Description

[0201] Figure 1 shows the comparison of the integrity of PolyA in plasmids containing the H1N1 antigen coding sequence when using two different PolyA.

[0202] Figure 2 shows the comparison of the integrity of PolyA in plasmids containing the H3N2 antigen coding sequence when using two different PolyA sequences.

[0203] Figure 3 shows the comparison of PolyA integrity results for plasmids containing the B / Victoria antigen coding sequence when using two different PolyA sequences.

[0204] Figure 4 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against influenza A H1N1 strain.

[0205] Figure 5 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against influenza A H3N2 strain.

[0206] Figure 6 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against the Victoria strain of influenza B.

[0207] Figure 7 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against influenza A H3N2 strain.

[0208] Figure 8 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against influenza A H1N1 strain.

[0209] Figure 9 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against the influenza A H3N2 strain.

[0210] Figure 10 shows the results of different hemagglutination inhibition assays after immunizing mice with the mRNA-LNP composition disclosed herein against the Victoria strain of influenza B. Detailed Implementation

[0211] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0212] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. For example, see Sam Brook et al., *Molecular Cloning: a Laboratory Manual*, 4th edition, Cold Spring Harbor Laboratory Press, 2012; and Ausubel et al., *Current Protocols in Molecular Biology*, Wiley Online Press, updated periodically. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are the average values ​​of three replicate experiments. Unless otherwise specified, in the following examples, the nucleotide sequences in the sequence listing are written from left to right in order from the 5' to the 3' end, and the amino acid sequences are written from left to right in order from the amino terminus to the carboxyl terminus.

[0213] definition

[0214] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure will have meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition.

[0215] As used herein, the terms “comprising” or “including” mean that a sequence, composition, or method includes the described components or steps, but does not exclude other components or steps. “consistent primarily of”, when used to define a composition or method, should exclude any other components or steps that are obviously essential to the technical effect they are intended to achieve. “composed of” should exclude other components and steps not mentioned.

[0216] Unless otherwise expressly stated, the singular forms “a / an” and “the” include plural referents. Thus, for example, a reference to “a cell” includes a combination of two or more cells, or an entire culture of cells. Unless expressly stated or obvious from the context, the term “or” is understood to be inclusive as used herein.

[0217] Unless explicitly stated or obvious from the context, as used herein, the term “about” should be understood as falling within the normal tolerance range in the field, such as within two standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the stated value. Unless otherwise obvious from the context, all numerical values ​​provided herein are modified by the term “about”.

[0218] As used herein, the term "isolated" means that a biological component (such as nucleic acid) has been substantially separated or purified from other biological components (such as other chromosomes and extrachromosomal DNA and RNA, proteins, and organelles) in the environment in which that component naturally exists (such as cells). "Isolated" nucleic acids include nucleic acids purified by standard purification methods. The term also includes nucleic acids prepared by recombinant expression in host cells and chemically synthesized nucleic acids.

[0219] As used herein, the term "immunogenic polypeptide" refers to proteins (including glycoproteins) and peptides capable of inducing an immune response in mammals, including, upon administration to a host, an immune-active peptide capable of inducing a humoral and / or cellular type immune response against the polypeptide and / or against homologous polypeptides containing an amino acid sequence highly identical (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the particular polypeptide. Therefore, "immunogenic polypeptide" as used herein includes the full-length sequence of a polypeptide, its analogues, or immunogenic fragments thereof. The term "immunogenic fragment" herein may be a polypeptide fragment selected from at least one of the following amino acid sequences: containing at least one epitope or antigenic determinant, or substantially composed of at least one epitope or antigenic determinant, or composed of at least one epitope or antigenic determinant, thereby capable of inducing an immune response. In this document, "immunogenic fragment" may include deletions, substitutions, and / or additions relative to the polypeptide sequence from which it originates, provided that the fragment can induce an immune response against the polypeptide.

[0220] In some embodiments, the immunogenic peptide is the full-length, fragment, derivative, or variant of the influenza virus hemagglutinin (HA) antigen. For example, in some embodiments, the HA is wild-type HA. In other embodiments, the HA is modified HA, for example, comprising a substitution, deletion, and / or insertion of at least one amino acid such that its primary structure differs from that of wild-type HA. In some specific embodiments, the mutation in HA is T2191, H371Y, I494M, H504P, M362L, HA0, APB, TB, or VASP, or any combination thereof. In some embodiments, the mutation in HA is the formation of a disulfide bond at the stem of the HA to connect adjacent protomers, the deletion of a cleavage site, and / or the replacement of a multi-base cleavage site (HPAI) with an LPAI sequence.

[0221] Influenza antigens can be recommended or selected based on standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS). In some embodiments, the HA and NA antigens recommended or selected by GISRS are included in the influenza virus vaccine manufactured and distributed for the current year's Southern or Northern Hemisphere influenza vaccine. In some embodiments, hemagglutinin inhibition (HAI) assays are used to select HA and NA antigens to identify circulating influenza viruses that are antigenically similar to the influenza virus in the previous season's vaccine; optionally, influenza viruses are considered antigenically similar if their HAI titers differ by a factor of two or less.

[0222] In some embodiments, the immunogenic peptide is the full-length, fragment, derivative, or variant of the influenza virus ion channel protein M2 antigen. In some embodiments, the influenza virus ion channel protein M2 may be derived from any influenza A subtype, including but not limited to H1N1, H3N2, H5N1, H7N9, etc.

[0223] As used herein, the term "identity" refers to the percentage of identical residues in a specified region of two or more nucleic acid or polypeptide sequences. Methods for determining the percentage of identity between two amino acid or nucleotide sequences are well known in the art. For example, the identity between two sequences can be calculated as follows: perform a best-fit alignment of the two sequences in a specified region, determine the number of positions in both sequences where identical residues appear as the number of matching positions, divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100. In cases where the two sequences differ in length, or where the alignment produces one or more staggered ends, or where a particular comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or using computer sequence algorithms such as BLAST or BLAST 2.0.

[0224] As used herein, the term "5' cap" is located at the 5' end of mRNA and contains a methylated guanosine monophosphate (GMP) linked to the 5' end of the mRNA via pyrophosphate, forming a 5',5'-triphosphate link with the adjacent nucleotide. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O indicates that the ribose of the terminal nucleotide is unmethylated, type I indicates that the ribose of the terminal nucleotide is methylated, and type II indicates that the ribose of both terminal nucleotides is methylated. In this document, the preferred 5' cap is m7G(5')ppp(5')(2'-OMeA)pG (available from, for example, TriLink Biotechnologies).

[0225] As used herein, the term "PolyA tail" or "PolyA sequence" refers to a continuous or interrupted sequence of adenosine residues typically located at the 3' end of an RNA molecule. Poly-A tails or Poly-A sequences are known to those skilled in the art and can be selected as needed. In mRNA, in the presence of a 3'-UTR, the Poly-A sequence is attached to the 3' end of the 3'-UTR. A continuous poly-A tail is characterized by a continuous sequence of adenosine residues. A poly-A tail can be of any length. In some embodiments, a poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 adenosine residues (A), particularly about 120 A residues. Typically, the vast majority of nucleotides in the PolyA tail are adenosine, meaning 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% of the nucleotides. However, the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridine monophosphate), G (guanylic acid), or C (cytidine monophosphate).

[0226] As used herein, the terms “individual,” “subject,” “host,” or similar expressions refer to any mammal or non-mammal. Mammals include, but are not limited to, cats, other vertebrates such as rodents, humans, and non-human primates such as cattle, horses, dogs, pigs, sheep, goats, giraffes, deer, camels, sheep, rats, mice, hares, and rabbits.

[0227] In the vaccination method of this application, the subject being vaccinated may have already been exposed to the influenza virus. As used herein, the terms exposed, exposure, etc., mean that the subject has been in contact with a person or animal known to be infected with the influenza virus. The vaccine of this application can be administered using techniques well known to those skilled in the art. Formulation and administration techniques can be found, for example, in Reich's Complete Book of Pharmacy, 18th edition. The vaccine can be administered by means including, but not limited to, conventional syringes, needle-free injection devices, or microjet bombardment gene guns. Suitable routes of administration include, but are not limited to, parenteral administration, such as intramuscular, intradermal, subcutaneous, or intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. For injection, the nanoparticles of this application can be formulated in an aqueous solution, preferably in a physiologically compatible buffer solution, such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0228] As used herein, the term "lipid nanoparticles" (LNPs) refers to particles having a size on the nanometer scale that comprise at least one lipid. In preferred embodiments, the lipids include, but are not limited to, neutral phospholipids and polyethylene glycol-lipids. As used herein, the term "neutral phospholipid" refers to an uncharged, non-glycerophosphate phospholipid molecule. As used herein, the term "polyethylene glycol-lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety.

[0229] The lipid nanoparticles typically comprise ionizable lipids, noncationic lipids, sterols, and PEG lipid components, as well as target nucleic acids, such as mRNA. The lipid nanoparticles of this disclosure can be produced using components, compositions, and methods commonly known in the art, see, for example, PCT / US2016 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / U The entirety of PCT / US2016 / 014280, PCT / US2017 / 038426, PCT / US2014 / 027077, PCT / US2014 / 055394, PCT / US2016 / 52117, PCT / US2012 / 069610, PCT / US2017 / 027492, PCT / US2016 / 059575 and PCT / US2016 / 069491 are incorporated herein by reference.

[0230] As used herein, a "neutralizing antibody" refers to an antibody that prevents an influenza virus from completing one round of replication. One round of replication refers to the viral life cycle, from the moment the virus attaches to a host cell to the moment newly formed viruses bud from the host cell. This life cycle includes, but is not limited to, the steps of cell attachment, internalization, lysis and rearrangement, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoproteins into the cytoplasm, formation of new viral particles, and budding of viral particles from the host cell membrane. A neutralizing antibody is an antibody that inhibits one or more of these steps. A "broadly neutralizing antibody" is an antibody that neutralizes one or more types, groups, subtypes, and / or strains of influenza virus. For example, a broadly neutralizing antibody induced by the HA protein of influenza A virus can neutralize influenza B or C virus.

[0231] As used herein, “influenza virus” means any strain of influenza virus capable of causing disease in animal or human subjects. See, for example, Fields, B. et al., Fields’ Virology, 4th ed., Philadelphia: Lippincott Williams and Wilkins; ISBN: 0781718325, 2001. Specifically, the term primarily includes any strain of influenza A virus (or influenza A virus) and influenza B virus (or influenza B virus) capable of causing disease in animal or human subjects. Influenza A virus includes different subtypes, usually defined by the type of hemagglutinin (HA) and / or neuraminidase (NA) of the virus. Eighteen different HA subtypes (H1 to H18) and eleven different NA subtypes (N1 to N11) are known in the art; see, for example, CDC, Types of Influenza Viruses, 2019. Non-limiting examples of influenza A virus strains include, for example, H1N1, H5N1, H3N2, H6N2, H7N3, H7N7, H9N2, H10N4, and H10N5.Including but not limited to: A / Puerto Rico / 8 / 34, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022, A / Victoria / 2570 / 2019, A / Sydney / 5 / 2021, A / California / 07 / 2009, A / Michigan / 45 / 2015, A / Brisbane / 02 / 2018, A / Hawaii / 70 / 2019, A / Idaho / 07 / 2018, A / Maine / 38 / 2018, A / Nebraska / 15 / 2018, A / Nebraska / 14 / 2019, A / Wisconsin / 588 / 2019, A / Iowa / 33 / 2019, A / Arkansas / 28 / 2019, A / Virginia / 41 / 2019, A / Minnesota / 60 / 2019, A / Alabama / 27 / 2019 or A / Guangdong-Maonan / SWLl536 / 2019; A / Aichi / 2 / 1968, A / Thailand / 8 / 2022, A / Massachusetts / 18 / 2022, A / Darwin / 9 / 2021, A / Darwin / 6 / 2021, A / Cambodia / e0826360 / 2020, A / Switzerland / 8060 / 2017, A / Switzerland / 9715293 / 2013, A / Iowa / 60 / 2018, A / South Australia / 34 / 2019, A / Hong Kong / 45 / 2019, A / Hong Kong / 2671 / 2019, A / Kansas / 14 / 2017, A / Jamaica / 60361 / 2019, A / Florida / 130 / 2019, A / Laos / 1789 / 2019, A / Vermont / 25 / 2019, A / New Jersey / 34 / 2019, A / California / 176 / 2019, A / Pennsylvania / 1026 / 2019, A / Togo / 634 / 2019, A / Kenya / 130 / 2019, A / Togo / 1307 / 2019, A / Ohio / 30 / 2019, A / Guatemala / 93 / 2019, A / Guatemala / 10 / 2019, A / Hong Kong / 4801 / 2014 or A / Singapore / INFIMH-16-0019 / 2016.

[0232] Influenza B virus includes any influenza B virus strain known in the art, examples of which include, but are not limited to, influenza B virus strains originating from the following locations: Aichi Prefecture, Akita Prefecture, Alaska, Ann Arbor, Argentina, Bangkok, Beijing, Belgium, Bonn, Brazil, Buenos Aires, Canada, Chaco, Chiba, Chongqing, CNIC, Córdoba, Czechoslovakia, Durban, Finland, Fujian, Fukuoka, Genoa, Guangdong, Guangzhou, Hanover, Harbin, Hawaii, Hebei, Henan, Hiroshima, Hong Kong, Houston, Hunan, Ibaraki, India, Israel, Johannesburg, Kagoshima, Kanagawa, Kansas, Khazkov, Kobe, Kona, Lazio, Li, Leningrad, Lisbon, Los Angeles, Lusaka, Lyon, Malaysia, Maputo, Madbouly. Lata, Maryland, Memphis, Michigan, Mie, Milan, Minsk, Nagasaki, Nagoya, Nanchang, Nashville, Nebraska, Netherlands, New York, NIB, Ningxia, Norway, Oman, Oregon, Osaka, Oslo, Panama, Paris, Parma, Perugia, Philippines, Busan, Quebec, Rochester, Rome, Saga, Seoul, Shandong, Shanghai, Shenzhen, Shiga, Shizuoka, Sichuan, Siena, Singapore, South Carolina, South Dakota, Spain, Stockholm, Switzerland, Taiwan, Texas, Tokushima, Tokyo, Trento, Trieste, United Kingdom, Ushuaia, USSR, Utah, Victoria, Vienna, Wuhan, Xuanwu, Yamagata, Yamanashi, Yunnan, and their hybrid subtypes, cyclic recombination forms, clinical and field isolates. Exemplary influenza B virus strains include, but are not limited to: Akita / 27 / 2001, Akita / 5 / 2001, Alaska / 16 / 2000, Alaska / 1777 / 2005, Argentina / 69 / 2001, Arizona / 146 / 2005, Arizona / 148 / 2005, Bangkok / 163 / 90, Bangkok / 34 / 99, Bangkok / 460 / 03, Bangkok / 54 / 99, Barcelona / 215 / 03, Beijing / 15 / 84, Beijing / 184 / 93, Beijing / 243 / 97, Beijing / 43 / 75, Beijing / 5 / 76, Beijing / 76 / 98, and Belgium / WV. 106 / 2002 strain, Belgium / WV 107 / 2002 strain, Belgium / WV 109 / 2002 strain, Belgium / WV114 / 2002 strain, Belgium / WV122 / 2002 strain, Bonn / 43 strain, Brazil / 952 / 2001 strain, Brisbane / 60 / 2008 (B / Victoria) strain, Bucharest / 795 / 03 strain,Buenos Aires / 161 / 00 strain, Buenos Aires / 9 / 95 strain, Buenos Aires / SW16 / 97 strain, Buenos Aires / VL518 / 99 strain, Canada / 464 / 2001 strain, Canada / 464 / 2002 strain, Chaco / 366 / 00 strain, Chaco / R113 / 00 strain, Cheju / 303 / 03 strain, Chiba / 447 / 98 strain, Chongqing / 3 / 2000 strain, clinical isolate SA1 Thailand / 2002, clinical isolate SA10 Thailand / 2002, clinical isolate SA100 Philippines / 2002, clinical isolate SA101 Philippines / 2002, clinical isolate SA110 Philippines / 2002), clinical isolate SA112 Philippines / 2002, clinical isolate SA113 Philippines / 2002, clinical isolate SA114 Philippines / 2002, Clinical isolate SA2; Thailand / 2002, Clinical isolate SA20; Thailand / 2002, Clinical isolate SA38; Philippines / 2002, Clinical isolate SA39; Thailand / 2002, Clinical isolate SA99; Philippines / 2002, CNIC / 27 / 2001; Colorado / 2597 / 2004; Cordoba / VA418 / 99; Czechoslovakia / 16 / 89; Czechoslovakia / 69 / 90; Daeku / 10 / 97; Daeku / 45 / 97; Daeku / 47 / 97; Daeku / 9 / 97; B / Du / 4 / 78; B / Durban / 39 / 98; Durban / 43 / 98; Durban / 44 / 98; B / Durban / 52 / 98; D urban / 55 / 98 strain, Durban / 56 / 98 strain, England / 1716 / 2005 strain, England / 2054 / 2005) strain, England / 23 / 04 strain, Finland / 154 / 2002 strain, Finland / 159 / 2002 strain, F inland / 160 / 2002 strain, Finland / 161 / 2002 strain, Finland / 162 / 03 strain, Finland / 162 / 2002 strain, Finland / 162 / 91 strain, Finland / 164 / 2003 strain, Finland / 172 / 91 strain,Finland / 173 / 2003 strain, Finland / 176 / 2003 strain, Finland / 184 / 91 strain, Finland / 188 / 2003 strain, Finland / 190 / 2003 strain, Finland / 220 / 2003 strain, Finland / WV5 / 2002 strain, Fujian / 36 / 82 strain, Geneva / 5079 / 03 strain, Genoa / 11 / 02 strain, Genoa / 2 / 02 strain, Genoa / 21 / 02 strain, Genova / 54 / 02 strain, Genova / 55 / 02 strain, Guangdong / 05 / 94 strain, Guangdong / 08 / 93 strain, Guangdong / 5 / 94 strain, Guangdong / 55 / 89 strain, Guangdong / 8 / 93 strain, Guangzhou / 7 / 97 strain, Guangzhou / 86 / 92 strain, Guangzhou / 87 / 92 strain, Gyeonggi / 592 / 2005 strain, Hannover / 2 / 90 strain, Harbin / 07 / 94 strain, Hawaii / 10 / 2001 strain, Hawaii / 1990 / 2004 strain, Hawaii / 38 / 2001 strain, Hawaii / 9 / 2001 strain, Hebei / 19 / 94 strain, Hebei / 3 / 94 strain, Henan / 22 / 97 strain, Hiroshima / 23 / 2001 strain, Hong Kong Hong Kong / 110 / 99 shares, Hong Kong / 1115 / 2002 shares, Hong Kong / 112 / 2001 shares, Hong Kong / 123 / 2001 shares, Hong Kong / 1351 / 2002 shares, Hong Kong / 1434 / 2002 shares, Hong Kong / 147 / 99 shares, Hong Kong / 156 / 99 shares, Hong Kong / 157 / 99 shares, Hong Kong / 22 / 2001 shares, Hong Kong / 22 / 89 shares, Hong Kong / 336 / 2001 shares, Hong Kong / 666 / 2001 shares, Hong Kong / 9 / 89 strain, Houston / 1 / 91 strain, Houston / 1 / 96 strain, Houston / 2 / 96 strain, Hunan / 4 / 72 strain, Ibaraki / 2 / 85 strain, ncheon / 297 / 2005 strain, India / 3 / 89 strain, India / 77276 / 2001 strain, Israel / 95 / 03 strain, Israel / WV 187 / 2002 strain, Japan / 1224 / 2005 strain, Jiangsu / 10 / 03 strain, Johannesburg / 1 / 99 strain,Johannesburg / 96 / 01, Kadoma / 1076 / 99, Kadoma / 122 / 99, Kagoshima / 15 / 94, Kansas / 229 / 92 / 99, Khazkov / 224 / 91, Kobe / 1 / 2002, Kouchi / 193 / 99, Lazio / 1 / 02, Lee / 40, Leningrad / 129 / 91, Lissabon / 2 / 90, Los Angeles / 1 / 02, Lusaka / 270 / 99, Lyon / 1271 / 96, Malaysia / 83077 / 2001, Maputo / 1 / 99, Mar del Plata Plata / 595 / 99, Maryland / 1 / 01, Memphis / 1 / 01, Memphis / 12 / 97-MA, Michigan / 22572 / 99, Mie / 1 / 93, Milano / 1 / 01, Minsk / 318 / 90, Moscow / 3 / 03, Nagoya / 20 / 99, Nanchang / 1 / 00, Nashville / 107 / 93, Nashville / 45 / 91, Nebraska / 2 / 01, Netherland / 801 / 90, Netherlands / 429 / 98, New York / 1 / 2002, NIB / 48 / 90, Ningxia / 45 / 83, Norway / 1 / 84, Oman / 16299 / 2001, Os aka / 1059 / 97 strain, Osaka / 983 / 97-V2 strain, Oslo / 1329 / 2002 strain, Oslo / 1846 / 2002 strain, Panama / 45 / 90 strain, Paris / 329 / 90 strain, Parma / 23 / 02 strain, Perth / 211 / 2001 strain, Peru / 1364 / 2004 strain, Philippines / 5072 / 2001 strain, Phuket / 3073 / 2013 strain, Pusan / 270 / 99 strain, Qu Quebec / 173 / 98 strain, Quebec / 465 / 98 strain, Quebec / 7 / 01 strain, Roma / 1 / 03 strain, Saga / S172 / 99 strain, Seoul / 13 / 95 strain, Seoul / 37 / 91 strain, Shangdong / 7 / 97 strain, Shanghai / 361 / 2002 strain, Shiga / T30 / 98 strain, Sichuan / 379 / 99 strain, Singapore / 222 / 79 strain, Spain / WV27 / 2002 strain,Stockholm / 10 / 90 strain, Switzerland / 5441 / 90 strain, Taiwan / 0409 / 00 strain, Taiwan / 0722 / 02 strain, Taiwan / 97271 / 2001 strain, Tehran / 80 / 02 strain, Tokyo / 6 / 98 strain, Trieste / 28 / 02 strain, Ulan Ude / 4 / 02 strain, United The following strains were identified: Kingdom / 34304 / 99, USSR / 100 / 83, Victoria / 103 / 89, Vienna / 1 / 99, Wuhan / 356 / 2000, WV194 / 2002, Xuanwu / 23 / 82, Yamagata / 1311 / 2003, Yamagata / K500 / 2001, Alaska / 12 / 96, GA / 86, NAGASAKI / 1 / 87, Tokyo / 942 / 96, and strain Rochester / 02 / 2001. The sequences of all these viral strains are known in the art and are available from GenBank.

[0233] Hemagglutination inhibition (HAI) testing is a classic laboratory procedure used to classify or type hemagglutinating viruses and further determine the antigenic characteristics of influenza virus isolates. To determine HAI, serially diluted viruses are prepared in a 96-well microtiter plate with a U-shaped or V-shaped bottom. For example, the most concentrated sample in the first well can be diluted using 1 / 5 of the stock solution, and subsequent wells can be diluted two-fold (1 / 10, 1 / 20, 1 / 40, etc.). The last well serves as a negative control without virus. Different rows of the titration plate typically contain different viruses and follow the same dilution pattern. After serial dilution, a standardized concentration of red blood cells (RBS) is added to each well and gently mixed. The plate is incubated at room temperature. After the incubation period, an analytical assay can be performed to distinguish between agglutinated and non-agglutinated wells. The relative concentration or titer of the virus sample is determined based on the wells where the last agglutination occurred before precipitation was observed.

[0234] Serological methods such as HAI assays can be used for epidemiological and immunological studies of viruses and vaccines, assessing antibody responses after vaccination, measuring the effectiveness of candidate vaccines, and identifying antigenically similar influenza viruses. Viruses with HAI titers differing by two times or less in dilution can be considered antigenically similar. In some instances, HA ELISA assays are used to examine HA antibody titers (e.g., IgG antibody titers) generated by administration of candidate vaccines.

[0235] As used in this article, the “N:P ratio” or “N / P ratio” is the molar ratio of ionizable nitrogen atoms in lipids to phosphate groups in RNA.

[0236] Example

[0237] The present invention will now be described in further detail with reference to specific embodiments. The given examples are merely illustrative and not intended to limit the scope of the invention. Those skilled in the art can make various modifications or adjustments based on the teachings of the present invention without departing from the spirit and scope of the invention.

[0238] Biosafety

[0239] This invention utilizes three pathogenic influenza viruses (A / Puerto Rico / 8 / 34 (H1N1), A / Aichi / 2 / 1968 (X31) (H3N2), B / Brisbane / 60 / 2008 (B / Victoria)), A / Hong Kong / 4801 / 2014 (H3N2), and A / Michigan / 45 / 205). The experimental procedures were conducted in a biosafety level 2 laboratory at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, or at Renjing (Suzhou) Biotechnology Co., Ltd. (A / Hong Kong / 4801 / 2014 (H3N2), A / Michigan / 45 / 205, A / Puerto Rico / 8 / 34 (H1N1), A / Aichi / 2 / 1968 (X31) ... The Rico / 8 / 34 (H1N1) and B / Brisbane / 60 / 2008 (B / Victoria) strains were obtained from the Institute of Medical Biology, Chinese Academy of Medical Sciences, and the A / Aichi / 2 / 1968 (X31) (H3N2) strain was obtained from the National Influenza Center, Institute of Virology, Chinese Center for Disease Control and Prevention. All experimental materials, procedures, and waste disposal complied with the laboratory's safety regulations.

[0240] All experimental operations and waste disposal procedures in this invention comply with the relevant provisions of the WHO Laboratory Biosafety Manual (Fourth Edition), the Chinese General Requirements for Laboratory Biosafety (GB19489-2208), and the Regulations on Biosafety Management of Pathogenic Microorganism Laboratories.

[0241] Experimental materials, reagents, instruments and experimental methods

[0242] Unless otherwise specified, all reagents, instruments, genes, and enzymes used in this invention are commercially available. Related nucleic acid chains, genes, and enzymes can also be obtained using conventional molecular biology experimental techniques based on information from publicly available databases. Unless otherwise specified, the nucleotide sequences described in the specification are written from the 5' to the 3' end, and the amino acid sequences are written from the amino terminus to the carboxyl terminus. In case of discrepancies between the sequences in the specification and the sequence listing, the sequences described in the specification shall prevail.

[0243] Example 1: Screening of PolyA

[0244] The effects of SEQ ID NO:33 (self-designed) and SEQ ID NO:34 (existing technology) on the stability of each mRNA sequence (containing SEQ ID NO:4 encoding H1N1, SEQ ID NO:9 encoding H3N2, and SEQ ID NO:19 encoding B / Victoria, respectively) were investigated.

[0245] Monoclonal screening

[0246] 1. Construct a vector containing the coding region of influenza HA antigen protein.

[0247] The vector uses the E. coli cloning vector pUC57 as its backbone. Between the Xba I restriction site and the EcoR I restriction site at its multiple cloning site, the T7 promoter sequence, 5'UTR (SEQ ID NO:31), Kozak sequence (GCCACC), HA protein coding sequence (SEQ ID NO:4, SEQ ID NO:9 or SEQ ID NO:19), 3'UTR (SEQ ID NO:32), and PolyA shown in SEQ ID NO:33 (self-designed) or SEQ ID NO:34 (prior art) are arranged in sequence to obtain 6 plasmids (each HA protein coding sequence corresponds to two PolyA).

[0248] 2. Small-scale bacterial culture to detect the stability of two poly(A) variants of the influenza HA antigen protein in Escherichia coli.

[0249] The vector plasmids containing the H1N1, H3N2, and B / Victoria HA protein coding sequences constructed in step 1 were confirmed to be correct by sequencing and then transformed into E. coli DH5α. The transformed plates were grown at 37°C to complete plasmid extraction and sequencing. After sequencing, the stability and base deletion of different poly(A) variants were analyzed and calculated based on the sequencing results; stability was expressed as the percentage of clones that did not undergo any base changes, with a higher percentage indicating greater stability.

[0250] As shown in Figures 1 and 2, for mRNA sequences encoding the H1N1 antigen, the percentages of intact PolyA in the self-designed and existing technology groups were 80% and 72% (i.e., 40 / 50 and 36 / 50), respectively. For mRNA sequences encoding the H3N2 antigen, the percentages of intact PolyA in the self-designed and existing technology groups were 84% and 80% (i.e., 42 / 50 and 40 / 50), respectively. It is evident that while the integrity of PolyA in the self-designed group is slightly higher than that in the existing technology group for mRNA sequences encoding H1N1 or H3N2 antigens, there is no significant difference between the two.

[0251] As shown in Figure 3, for the mRNA sequence encoding the B / Victoria antigen, the proportion of complete PolyA in the self-designed and existing technology groups was 38% and 8% respectively (i.e., 19 / 50 and 4 / 50). It can be seen that for the mRNA sequence encoding the B / Victoria antigen, the integrity of the self-designed PolyA is higher than that of the existing technology.

[0252] Passage stability test

[0253] To further investigate the impact of the self-designed PolyA and the existing PolyA technology on passage stability, and to assess the impact on subsequent process scale-up and industrial production, we conducted passage studies on monoclonal clones containing complete PolyA (following the aforementioned "monoclonal screening study") to observe the differences in stability during the passage process.

[0254] The strain was passaged as follows:

[0255] 1. Subculture: Each time the bacterial culture is inoculated into fresh culture medium and cultured once, it is counted as one subculture.

[0256] 2. Subculturing conditions: 1 ml of bacterial culture is inoculated into 50 ml of LB liquid medium, the final concentration of kanamycin is 50 ug / ml, and the culture time on a shaker is about 4-6 hours;

[0257] 3. Shaking incubation conditions: 37℃, 220rpm;

[0258] 4. Preservation of culture: After about 4-6 hours of subculturing, take 500 μL of bacterial culture and mix it with 500 μL of 40% glycerol. Store it in a refrigerator at -80°C. Continue to culture the remaining bacterial culture overnight.

[0259] 5. Plasmid extraction: Extract plasmids from the bacterial culture that has been cultured overnight (Qingke Plasmid Extraction Kit, catalog number: TSP502-200), and sequence them.

[0260] The passage stability results of PolyA for the mRNA sequences encoding H1N1, H3N2, and B / Victoria antigens are as follows:

[0261] (1) For the mRNA sequence encoding the H1N1 antigen

[0262] Prior art PolyA group (SEQ ID NO:34): After 6 passages, only 1 of the 5 single clones maintained the integrity of PolyA;

[0263] Self-designed PolyA group (SEQ ID NO:33): After 6 passages, 4 out of 5 single clones maintained the integrity of PolyA.

[0264] (2) For the mRNA sequence encoding the H3N2 antigen

[0265] Prior art PolyA group (SEQ ID NO:34): After 6 passages, all 5 single clones had PolyA deletions to varying degrees.

[0266] Self-designed PolyA group (SEQ ID NO:33): After 6 passages, all 5 single clones maintained the integrity of PolyA.

[0267] (3) For the mRNA sequence encoding the B / Victoria antigen

[0268] Existing technology PolyA group (SEQ ID NO:34): After one passage, the tails of all three monoclonal clones are completely lost;

[0269] The self-designed PolyA group (SEQ ID NO:33): after one passage, the tails of all 5 monoclonal clones were complete.

[0270] in conclusion

[0271] In summary, the PolyA independently designed in this invention can significantly improve the stability of plasmid replication and passage, and is applicable to various types of mRNA encoding influenza antigens.

[0272] Example 2: Optimization of the coding sequence

[0273] We commissioned Suzhou Genewiz Biotechnology Co., Ltd. to synthesize a double-stranded DNA plasmid for the preparation of mRNA.

[0274] The plasmids contain the nucleotide sequences shown in SEQ ID NO:2, 3, 4, 5, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 19, 20, 22, 24, 26, 28, and 30, respectively encoding hemagglutinin HA of H1N1 (encoded by any one of SEQ ID NO:2-5) having the amino acid sequence shown in SEQ ID NO:1, hemagglutinin HA of H3N2 (encoded by any one of SEQ ID NO:7-10) having the amino acid sequence shown in SEQ ID NO:6, hemagglutinin HA of H3N2 (encoded by any one of SEQ ID NO:12-15) having the amino acid sequence shown in SEQ ID NO:11, hemagglutinin HA of B / Victoria (encoded by any one of SEQ ID NO:17-20) having the amino acid sequence shown in SEQ ID NO:16, M2 (encoded by SEQ ID NO:22) having the amino acid sequence shown in SEQ ID NO:21, and hemagglutinin HA of B / Victoria (encoded by any one of SEQ ID NO:17-20), respectively. Hemagglutinin HA of H1N1 with the amino acid sequence shown in SEQ ID NO:23 (encoded by SEQ ID NO:24), hemagglutinin HA of H3N2 with the amino acid sequence shown in SEQ ID NO:25 (encoded by SEQ ID NO:26), hemagglutinin HA of B / Victoria with the amino acid sequence shown in SEQ ID NO:27 (encoded by SEQ ID NO:28), and hemagglutinin HA of B / Yamagata with the amino acid sequence shown in SEQ ID NO:29 (encoded by SEQ ID NO:30).

[0275] According to the manufacturer's instructions, using the reaction system shown in Table 1, the plasmids were digested with the restriction enzyme SapI (purchased from NEB) and reacted at 37°C for 3 hours to obtain the corresponding linearized plasmid templates.

[0276] Table 1: SapI restriction enzyme digestion reaction system:

[0277] According to the manufacturer's instructions, the linearized plasmid template was purified and recovered using the Linearized Plasmid Recovery and Purification Kit DP205-02 (purchased from TIANGEN). Specifically, 5 volumes of PB Buffer were added to the linearization reaction system and mixed thoroughly. The filter column was activated with 200 μL of PS buffer, centrifuged at 12000×g for 1 min, and the filtrate was discarded. The mixed reaction solution was loaded onto the column, centrifuged at 12000×g for 1 min, and the filtrate was discarded. 700 μL of washing buffer was used to wash away any unattached impurities by centrifugation at 12000×g for 1 min. The linearized plasmid template was eluted with 50 μL of RNase-free ddH2O into a clean 1.5 mL EP tube (12000×g, 2 min) to complete the purification. The purified linearized plasmid was quantified using Nanodrop.

[0278] According to the manufacturer's instructions, T7 polymerase and the CleanCap kit were used to cap and transcribe the purified linearized plasmid templates 1 to 16, respectively. Specifically, the capping and in vitro transcription reaction systems shown in Table 2 were added to 1.5 mL EP tubes and incubated at 37°C with rotation for 3 hours (10 rpm).

[0279] Table 2: mRNA capping and in vitro transcription reaction system:

[0280] After the in vitro transcription reaction was completed, 20 μL of DNase I was added to the tube, mixed by pipetting, and reacted at 37°C for 15 min. Then, 50% volume of LiCl was added to the reaction system, and the mixture was incubated overnight at -20°C. The mixture was then transferred to a centrifuge pre-cooled to 4°C and centrifuged at 12000×g for 15 min, and the supernatant was discarded. The mixture was washed with 700 μL of 70% ethanol and centrifuged again (12000×g, 5 min). The white precipitate was redissolved in 1000 μL of RNase-free water, mixed, and quantified using Nanodrop to determine the amount of mRNA recovered.

[0281] The prepared mRNA contains the following elements from the 5' end to the 3' end: (1) 5' cap; (2) 5' UTR region (SEQ ID NO:35 is the 5' UTR corresponding to the coding sequences SEQ ID NO:24, 26, 28, 30, and SEQ ID NO:31 is the 5' UTR corresponding to the remaining coding sequences); (3) antigen coding region; (4) 3' UTR region (SEQ ID NO:36 is the 3' UTR corresponding to the coding sequences SEQ ID NO:24, 26, 28, 30, and SEQ ID NO:32 is the 3' UTR corresponding to the remaining coding sequences); (5) PolyA tail (SEQ ID NO:37 is the PolyA tail corresponding to the coding sequences SEQ ID NO:24, 26, 28, 30, and SEQ ID NO:33 is the PolyA tail corresponding to the remaining coding sequences).

[0282] For example, the sequence of the full-length mRNA used for translation preparation in the embodiments of this application is shown in SEQ ID NO:38 (starting from the transcription start site sequence AGG and ending at the poly A tail). For the mRNA sequence used for translation to produce other influenza antigens, its structure and composition are the same as SEQ ID NO:38, except that the ORF sequence is replaced by any one of SEQ ID NO:3-5, 7-10, 12-15, 17-20, 22.

[0283] Replace SEQ ID NO:2 with SEQ ID NO:3 to obtain sequence SEQ ID NO:39

[0284] Replace SEQ ID NO:2 with SEQ ID NO:4 to obtain sequence SEQ ID NO:40

[0285] Replace SEQ ID NO:2 with SEQ ID NO:5 to obtain sequence SEQ ID NO:41

[0286] Replace SEQ ID NO:2 with SEQ ID NO:7 to obtain sequence SEQ ID NO:42

[0287] Replace SEQ ID NO:2 with SEQ ID NO:8 to obtain sequence SEQ ID NO:43

[0288] Replace SEQ ID NO:2 with SEQ ID NO:9 to obtain sequence SEQ ID NO:44

[0289] Replace SEQ ID NO:2 with SEQ ID NO:10 to obtain sequence SEQ ID NO:45

[0290] Replace SEQ ID NO:2 with SEQ ID NO:12 to obtain sequence SEQ ID NO:46

[0291] Replace SEQ ID NO:2 with SEQ ID NO:13 to obtain sequence SEQ ID NO:47

[0292] Replace SEQ ID NO:2 with SEQ ID NO:14 to obtain sequence SEQ ID NO:48

[0293] Replace SEQ ID NO:2 with SEQ ID NO:15 to obtain sequence SEQ ID NO:49

[0294] Replace SEQ ID NO:2 with SEQ ID NO:17 to obtain sequence SEQ ID NO:50

[0295] Replace SEQ ID NO:2 with SEQ ID NO:18 to obtain sequence SEQ ID NO:51

[0296] Replace SEQ ID NO:2 with SEQ ID NO:19 to obtain sequence SEQ ID NO:52

[0297] Replace SEQ ID NO:2 with SEQ ID NO:20 to obtain sequence SEQ ID NO:53

[0298] Replace SEQ ID NO:2 with SEQ ID NO:22 to obtain sequence SEQ ID NO:54

[0299] Example 3:

[0300] Acetic acid solution was added to the purified mRNA stock solution obtained in Example 2 until the final acetic acid concentration was 20 mmol / L and the final mRNA concentration was 200 μg / ml. The mixture was stirred until homogeneous to obtain the mRNA working solution. The mRNA working solution and the lipid mixture (prepared according to Table 3) were mixed in a T-flow mixing apparatus at a flow ratio of 2:1 to 4:1 to prepare LNPs. Then, the LNPs were diluted 2 to 5 times with 2 mmol / L acetic acid solution, and then replaced at least 3 times with 2 mmol / L acetic acid solution to concentrate the solution to the target concentration. Sucrose solution was added to adjust the osmotic pressure, and the pH was adjusted to 7.0 to 8.0 with Tris solution to obtain mRNA-lipid nanoparticles (LNPs) containing one of the mRNAs prepared and purified in Example 2.

[0301] In the prepared mRNA-LNP, the N / P ratio of mRNA to ionizable lipids was 5.2.

[0302] Table 3: Formula for Mixed Fat Solution

[0303] The mRNA-LNPs prepared as described above were mixed according to the mass ratios shown in Table 4 to prepare the mRNA-LNP compositions shown in each group (in Table 4, "mRNA-X" represents the full-length mRNA corresponding to the coding sequence SEQ ID NO:X).

[0304] Table 4:

[0305] Example 4:

[0306] The mRNA-LNP compositions shown in Example 3 were prepared and used to immunize 6- to 8-week-old female BALB / c mice (SPF grade) to evaluate the immune protective response against influenza virus induced by each composition.

[0307] Specifically, mice were randomly divided into groups of five, and each group received an intramuscular injection of the mRNA-LNP composition (dosage shown in Table 4). A booster injection was given on day 14. On day 28, approximately 200 μl of blood was collected from the orbital region of the mice. The blood was incubated at 37°C for 60 min, followed by freezing at 4°C for 120 min. The blood was centrifuged at 3000 rpm for 10 min, and the serum was separated for subsequent neutralizing antibody assays and hemagglutination inhibition assays.

[0308] Example 5:

[0309] The mouse serum from each group prepared according to Example 4 was mixed with receptor-destroying enzyme (RDE) at a volume ratio of 1:3, and the reaction was terminated by inactivation in a water bath at 37°C for 12 h and then in a water bath at 56°C for 30 min.

[0310] Add PBS at a rate of 25 μL / well to a 96-well V-shaped plate. Then, add 25 μL of the viral stock solution to the first well and mix thoroughly with PBS via pipetting. Next, pipette 25 μL of the mixture from the first well and add it to the second well, mixing well after each of the 12 2-fold dilutions. Subsequently, add 25 μL of 1% guinea pig blood to each well containing the viral gradient dilutions, mix well, and incubate at room temperature for 30–45 min. The number of viral particles corresponding to the lowest concentration from which hemagglutination is observed is defined as one hemagglutination unit. The viral load used in the hemagglutination inhibition assay is four hemagglutination units.

[0311] Add PBS at 25 μL / well to a 96-well V-shaped plate. Then add 25 μL of RDE-treated serum to the first well and mix with PBS by pipetting. Add another 25 μL of the mixture to the second well and mix. Repeat this 2-fold dilution for eight wells. Add 25 μL / well of the diluted virus (four hemagglutination units) to each well and mix with the serially diluted serum at a 1:1 ratio. Incubate at room temperature for 1 hour. Then, add 50 μL / well of 1% guinea pig blood to the serum-virus mixture, mix, and incubate at room temperature for 45 minutes. Observe for hemagglutination.

[0312] The final serum dilution factor corresponding to the well immediately preceding the well where observable hemagglutination occurs is taken as the hemagglutination titer. If hemagglutination is observed in the first well, the result is considered negative. The results are shown in Figures 4 to 6.

[0313] Figure 4 shows that, against influenza A H1N1 virus, groups G2-G8 had significantly higher hemagglutination inhibition titers compared to group G1 (LNP blank).

[0314] Compared to group G2 (the known quadrivalent mRNA influenza vaccine), the hemagglutination inhibition titers of groups G4, G5, and G8 were slightly lower, group G6 was comparable, while group G7 was significantly higher.

[0315] Compared to the G3 group (H1N1-HA+H3N2-HA+BW-HA, all wild-type mRNA), the G4-G8 groups (H1N1-HA+H3N2-HA+BW-HA, all optimized mRNA) all showed significantly better hemagglutination inhibition effects.

[0316] Figure 5 shows that, against influenza A H3N2 virus, groups G2-G8 had significantly higher hemagglutination inhibition titers compared to group G1 (LNP blank).

[0317] Compared to the G2 group (the known quadrivalent mRNA influenza vaccine), the hemagglutination inhibition titer of the G8 group was slightly lower, the G6 group was comparable, while the G4, G5, and G7 groups were significantly higher.

[0318] Compared to group G3 (H1N1-HA+H3N2-HA+BW-HA, all wild-type mRNA), groups G4-G7 (H1N1-HA+H3N2-HA+BW-HA, all optimized mRNA) showed significantly better hemagglutination inhibition, while group G8 was comparable.

[0319] Figure 6 shows that, against influenza B Victoria virus, groups G2-G8 had significantly higher hemagglutination inhibition titers compared to group G1 (LNP blank).

[0320] Compared to the G2 group (the known quadrivalent mRNA influenza vaccine), the hemagglutination inhibition titer of the G5 group was comparable, while that of the G4, G6, G7, and G8 groups was significantly higher.

[0321] Compared to group G3 (H1N1-HA+H3N2-HA+BW-HA, all wild-type mRNAs), groups G4-G8 (H1N1-HA+H3N2-HA+BW-HA, all codon-optimized mRNAs) were also included.

[0322] in conclusion

[0323] The above results demonstrate that the mRNA trivalent vaccine of this invention has excellent protective effects against all types of influenza viruses, and the protective effect can be significantly improved after codon optimization.

[0324] Example 6:

[0325] Based on the results of Example 5, a comprehensive evaluation was conducted. The protective effect of mRNA-3:mRNA-8:mRNA-18 (corresponding to groups G6 and G7 in Example 5) was relatively good. However, considering the high prevalence of H3N2, we replaced the H3N2 antigen sequence for further validation. Mouse immunization and sample collection procedures were the same as in Example 4, and the neutralizing antibody assay and hemagglutination inhibition assay were the same as in Example 5. Specific grouping information is shown in Table 5.

[0326] The mRNA-LNP prepared according to Example 3 was mixed with the mass ratios shown in Table 5 to prepare the mRNA-LNP compositions shown in each group (in Table 5, "mRNA-X" represents the full-length mRNA corresponding to the coding sequence SEQ ID NO:X).

[0327] Table 5:

[0328] As shown in Figure 7, compared with the blank group G1 (LNP), groups G2-G5 all showed significantly higher hemagglutination inhibition titers against influenza A H3N2 virus.

[0329] Compared to group G5 (using wild-type mRNA sequence), groups G2 and G3 had significantly higher hemagglutination inhibition titers, while group G4 had relatively lower hemagglutination inhibition titers.

[0330] It is evident that even with the replacement of the H3N2 antigen, it can still provide effective protection against the H3N2 influenza A virus, with groups G2 and G3 being the best.

[0331] Example 7

[0332] Based on the results of Example 6, it was found that the protective effect of mRNA-3:mRNA-12:mRNA-18 (corresponding to group G2 in Example 6) was better. Since the M2 antigen is also an antigen in influenza vaccines, to further expand the protective effect of the vaccine, we attempted to add the M2 antigen to the mRNA-3:mRNA-12:mRNA-18 combination to verify the specific protective effect. The mouse immunization and sample collection procedures were the same as in Example 4, and the neutralizing antibody assay and hemagglutination inhibition assay were the same as in Example 5. Specific grouping information is shown in Table 6.

[0333] The mRNA-LNP prepared according to Example 3 was mixed with the mass ratios shown in Table 6 to prepare the mRNA-LNP compositions shown in each group (in Table 6, "mRNA-X" represents the full-length mRNA corresponding to the coding sequence SEQ ID NO:X).

[0334] Table 6:

[0335] As shown in Figure 8, for influenza A H1N1 virus, compared with the G1 group (LNP blank), both the G2 group and the G3 group have significantly higher hemagglutination inhibition titers, but the G2 group with added M2 antigen has significantly higher hemagglutination inhibition titers.

[0336] As shown in Figure 9, for influenza A H3N2 virus, compared with the G1 group (LNP blank), both the G2 and G3 groups have significantly higher hemagglutination inhibition titers, with the hemagglutination inhibition titers of the G2 and G3 groups being comparable.

[0337] As shown in Figure 10, for influenza B Victoria virus, compared with the G1 group (LNP blank), both the G2 and G3 groups had significantly higher hemagglutination inhibition titers, with the hemagglutination inhibition titer of the G3 group being lower than that of the G2 group.

[0338] It is evident that the addition of the M2 antigen increases the hemagglutination inhibition titer against influenza A, and since influenza A has a stronger pathogenicity, the addition of the M2 antigen significantly increases the value of the vaccine.

[0339] The sequences used in the above embodiments of this application are shown in the following sequence listing. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences, and when they represent RNA sequences, "t" represents uridine.

[0340] Sequence List:

Claims

1. An mRNA, characterized in that, The following elements are included sequentially from the 5' end to the 3' end: (1) a 5' cap; (2) a 5' UTR region; (3) an influenza virus immunogenic fragment coding region; (4) a 3' UTR region; and (5) a poly A tail, the sequence of which is shown in SEQ ID NO:

33.

2. The mRNA according to claim 1, characterized in that, The immunogenic fragment of the influenza virus is the immunogenic fragment of influenza virus hemagglutinin HA.

3. The mRNA according to claim 2, characterized in that, The immunogenic fragments of the influenza virus are immunogenic fragments of hemagglutinin HA from influenza A H1N1, influenza A H3N2, influenza B Victoria, and / or influenza B Yamagata.

4. An mRNA composition comprising: First mRNA, second mRNA and third mRNA; the first mRNA, second mRNA and third mRNA all have the following structure: from the 5' end to the 3' end, they contain the following elements in sequence: (1) 5' cap; (2) 5' UTR region; (3) the immunogenic fragment coding region of influenza virus hemagglutinin HA; (4) 3' UTR region; (5) poly A tail, the sequence of which is shown in SEQ ID NO:33; The immunogenic fragment of influenza virus hemagglutinin HA in the first mRNA is the immunogenic fragment of hemagglutinin HA of influenza A H1N1. The immunogenic fragment of influenza virus hemagglutinin HA in the second mRNA is the immunogenic fragment of hemagglutinin HA of influenza A H3N2. The immunogenic fragment of the influenza virus hemagglutinin HA in the third mRNA is the immunogenic fragment of the hemagglutinin HA of influenza B (Victoria).

5. An mRNA composition comprising: The first mRNA, second mRNA, third mRNA, and fourth mRNA; the first mRNA, second mRNA, third mRNA, and fourth mRNA all have the following structure: from the 5' end to the 3' end, they sequentially contain the following elements: (1) a 5' cap; (2) a 5' UTR region; (3) a region encoding the immunogenic fragment of influenza virus hemagglutinin HA; (4) a 3' UTR region; (5) a poly A tail, the sequence of which is shown in SEQ ID NO:33; The immunogenic fragment of influenza virus hemagglutinin HA in the first mRNA is the immunogenic fragment of hemagglutinin HA of influenza A H1N1. The immunogenic fragment of influenza virus hemagglutinin HA in the second mRNA is the immunogenic fragment of hemagglutinin HA of influenza A H3N2. The immunogenic fragment of influenza virus hemagglutinin HA in the third mRNA is the immunogenic fragment of hemagglutinin HA from influenza B Victoria. The immunogenic fragment of the influenza virus hemagglutinin HA in the fourth mRNA is the immunogenic fragment of hemagglutinin HA from type B Yamagata.

6. The influenza virus is based on the mRNA or mRNA composition described in any of the preceding claims, and is recommended or selected according to the standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS).

7. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The type A influenza H1N1 is A / Puerto Rico / 8 / 34, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022, A / Victoria / 2570 / 2019, A / Sydney / 5 / 2021, A / Cali fornia / 07 / 2009, A / Michigan / 45 / 2015, A / Brisbane / 02 / 2018, A / Hawaii / 70 / 2019, A / Idaho / 07 / 2018, A / Maine / 3 8 / 2018, A / Nebraska / 15 / 2018, A / Nebraska / 14 / 2019, A / Wisconsin / 588 / 2019, A / Iowa / 33 / 2019, A / Arkansas / 28 / 2019, A / Virginia / 41 / 2019, A / Minnesota / 60 / 2019, A / Alabama / 27 / 2019 or A / Guangdong-Maonan / SWLl536 / 2019.

8. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The type A influenza H3N2 is A / Aichi / 2 / 1968, A / Thailand / 8 / 2022, A / Massachusetts / 18 / 2022, A / Darwin / 9 / 2021, A / Darwin / 6 / 2021 , A / Cambodia / e0826360 / 2020, A / Switzerland / 8060 / 2017, A / Switzerland / 9715293 / 2013, A / Iowa / 60 / 2018, A / South Australia / 34 / 2019、A / Hong Kong / 45 / 2019、A / Hong Kong / 2671 / 2019, A / Kansas / 14 / 2017, A / Jamaica / 60361 / 2019, A / Florida / 130 / 2019, A / Laos / 1789 / 2019, A / Vermont / 25 / 2019, A / New Jersey / 34 / 2019, A / California / 176 / 2019, A / Pennsylvania / 1026 / 2019, A / Togo / 634 / 2019,A / Kenya / 130 / 2019, A / Togo / 1307 / 2019, A / Ohio / 30 / 2019, A / Guatemala / 93 / 2019, A / Guatemala / 10 / 2019, A / Hong Kong / 4801 / 2014 or A / Singapore / INFIMH-16-0019 / 2016.

9. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The influenza B virus, Victoria, is referred to as B / Austria / 1359417 / 2021, B / Washington / 02 / 2019, B / Colorado / 06 / 2017, B / Brisbane / 60 / 2008, or B / Colorado / 06 / 2019.

10. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA of influenza A H1N1, and its amino acid sequence is shown in SEQ ID NO:

1.

11. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA of influenza A H3N2, and its amino acid sequence is shown in SEQ ID NO:6 or SEQ ID NO:

11.

12. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is the immunogenic fragment of hemagglutinin HA from influenza B (Victoria), and its amino acid sequence is shown in SEQ ID NO:

16.

13. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is an immunogenic fragment of hemagglutinin HA of influenza A H1N1, and the coding region sequence of the immunogenic fragment of the influenza virus hemagglutinin HA is as shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5, or a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; or the full-length mRNA sequence of the immunogenic fragment of the hemagglutinin HA of influenza A H1N1 is as shown in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41, or a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:

41.

14. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is an immunogenic fragment of hemagglutinin HA from influenza A H3N2, and the coding region sequence of the immunogenic fragment of the influenza virus hemagglutinin HA is as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; ... the full-length mRNA sequence of the immunogenic fragment encoding hemagglutinin HA of influenza A H3N2 is as shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID The sequence shown in NO:48 or SEQ ID NO:49, or a sequence having at least 80%, 90%, 95%, or 99% sequence identity with the sequences shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:

49.

15. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The immunogenic fragment of the influenza virus hemagglutinin HA is an immunogenic fragment of hemagglutinin HA from influenza B (Victoria), and the coding region sequence of the immunogenic fragment of the influenza virus hemagglutinin HA is as shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; or the full-length mRNA sequence encoding the immunogenic fragment of hemagglutinin HA from influenza B (Victoria) is as shown in SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:53, or has at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 or SEQ ID NO:

53.

16. The mRNA composition according to any one of the preceding claims, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2; (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:17, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

17.

17. The mRNA composition according to any one of the preceding claims, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3; (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:18, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

18.

18. The mRNA composition according to any one of the preceding claims, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4; (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:19, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

19.

19. The mRNA composition according to any one of the preceding claims, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:5, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:5; (2) The second mRNA comprises, as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or an immunogenic fragment coding region of influenza virus hemagglutinin HA having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; and (3) The third mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:20, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

20.

20. An mRNA composition comprising: (1) A first mRNA encoding a first immunogenic polypeptide, the first immunogenic polypeptide comprising an immunogenic fragment of H1N1 hemagglutinin HA, the first mRNA being the mRNA of claim 10 or 13. (2) A second mRNA encoding a second immunogenic polypeptide, the second immunogenic polypeptide comprising an immunogenic fragment of H3N2 hemagglutinin HA, the second mRNA being the mRNA of claim 11 or 14. (3) A third mRNA encoding a third immunogenic polypeptide, said third immunogenic polypeptide comprising an immunogenic fragment of hemagglutinin HA from influenza B Victoria, said third mRNA being the mRNA of claim 12 or 15; and (4) A fourth mRNA encoding a fourth immunogenic polypeptide, the fourth immunogenic polypeptide comprising an immunogenic fragment of influenza virus ion channel protein M2, the amino acid sequence of which is shown in SEQ ID NO:

21.

21. The mRNA composition of claim 20, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:2, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:2; (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (3) The third mRNA comprises, as shown in SEQ ID NO:17, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

22.

22. The mRNA composition of claim 20, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:3, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:3; (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (3) The third mRNA comprises, as shown in SEQ ID NO:18, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

22.

23. The mRNA composition of claim 20, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:4, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:4; (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (3) The third mRNA comprises, as shown in SEQ ID NO:19, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

22.

24. The mRNA composition of claim 20, characterized in that, (1) The first mRNA contains the immunogenic fragment coding region sequence of influenza virus hemagglutinin HA as shown in SEQ ID NO:5, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:5; (2) The second mRNA contains the immunogenic fragment coding region of influenza virus hemagglutinin HA as shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (3) The third mRNA comprises, as shown in SEQ ID NO:20, or has at least 80%, 90%, 95%, or 99% sequence identity with influenza virus hemagglutinin HA; and (4) The fourth mRNA contains the immunogenic fragment coding region sequence of influenza virus ion channel protein M2 as shown in SEQ ID NO:22, or having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

22.

25. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The 5' cap of the mRNA is selected from any one of m7G(5')ppp(5')(2'-OMeA)pG, m7(3'OMeG)(5')ppp(5')m6(2'OMeA)pG, m7(3'AcmG)(5')ppp(5')(2'OMeA)pG and m7G(5')vppp(5')(2'OMeA)pG, with m7G(5')ppp(5')(2'-OMeA)pG being preferred.

26. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The 5'UTR sequence of the mRNA is shown in SEQ ID NO:

31.

27. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The 3'UTR sequence of the mRNA is shown in SEQ ID NO:

32.

28. The mRNA or mRNA composition according to any one of the preceding claims, characterized in that, The mRNA further comprises a chemically modified nucleoside, optionally selected from chemically modified uridine; optionally, some or all of the uridine in the mRNA is chemically modified uridine; preferably, all of the uridine in the mRNA is chemically modified uridine; optionally, the chemically modified uridine is selected from pseudouridine, N1-methyl-pseudouridine, 2-thiouridine, 4-thiouridine, 2-thio-1-methyl-1-diaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5- The chemically modified uridine is selected from any one or more of the following: azauridine, 2-thio-dihydropseuuridine, 2-thio-pseuuridine, 4-methoxy-2-thio-pseuuridine, 4-methoxy-pseuuridine, 4-thio-pseuuridine, 5-azauridine, dihydropseuuridine, 5-methoxyuridine, 5-methyluridine, and 2-methoxyuridine. Preferably, the chemically modified uridine is a pseudouridine or N1-methylpseuuridine. More preferably, all uridine acid in the chemical modification is modified to N1-methylpseuuridine.

29. The mRNA composition according to any one of the preceding claims, characterized in that, The content ratio of the first mRNA, the second mRNA, and the third mRNA, by mass, is in the range of 2 to 10:2 to 10:2 to 10, preferably in the range of 3 to 7:3 to 7:3 to 7, and more preferably in the range of 5:5:5 or 2:2:

2.

30. The mRNA composition according to any one of the preceding claims, characterized in that, The content ratio of the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA, by mass, is in the range of 2-10:2-10:2-10:0.5-5, preferably in the range of 2-5:2-5:2-5:0.5-2, and more preferably in the range of 2:2:2:0.

5.

31. The mRNA or mRNA composition according to any one of the preceding claims, wherein the mRNA is encapsulated in one or more lipid nanoparticles.

32. The mRNA composition according to any one of the preceding claims, wherein, Each individual lipid nanoparticle encapsulates a first to a third mRNA in substantially the same proportion, or each individual particle encapsulates a first to a third mRNA in different proportions, or each individual particle encapsulates any one of the first, second, or third mRNA.

33. The mRNA composition according to any one of the preceding claims, wherein, Each individual lipid nanoparticle encapsulates the first to fourth mRNAs in substantially the same proportion, or each individual particle encapsulates the first to fourth mRNAs in different proportions, or each individual particle encapsulates any one of the first, second, third, or fourth mRNAs.

34. The composition according to any one of claims 31-33, wherein, The lipid nanoparticles comprise ionizable lipids, phospholipids, structural lipids, and polyethylene glycol (PEG)-lipids. Preferably, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid, and the PEG lipid is (20-60):(5-25):(25-55):(0.5-5); More preferably, the molar ratio of the ionizable lipid, the phospholipid, the structural lipid and the PEG lipid is (40-55):(5-15):(30-50):(1-3).

35. The composition according to claim 34, wherein, The phospholipid is selected from one or more of the following compounds: dilauroyl lecithin (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl lecithin (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearate phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycerol-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholestyldimethylsuccinate-sn-glycerol-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-divinyl-sn-glycerol-3-phosphate choline, 1,2-diaryl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-divinyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diaryl-sn-glycerol-3-phosphate ethanolamine, 1,2-dithiohexaenoic acid-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-(1-glycerol) sodium salt (DOPG), or sphingomyelin, preferably DSPC; The structural lipid is selected from one or more of cholesterol, coccosterol, sitosterol, ergosterol, and stigmasterol, preferably cholesterol; and / or The PEG lipid is selected from one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol, preferably DMG-PEG2000.

36. The composition according to any one of the preceding claims is a vaccine.

37. A nucleic acid molecule encoding mRNA as described in any of the preceding claims, wherein the nucleic acid molecule is preferably DNA, more preferably a DNA plasmid.

38. A method for inducing an immune response to an influenza virus in a subject, comprising administering to the subject an effective dose of any of the preceding mRNAs, any of the preceding mRNA compositions, or the nucleic acid molecule as described in claim 37; Preferably, the method includes administering the medication to the subject once, twice, or three times.

39. Use of the mRNA described in any of the preceding claims, the mRNA composition described in any of the preceding claims, or the nucleic acid molecule described in claim 37 in the preparation of a medicament for the prevention or treatment of influenza virus infection.

40. Use of any of the preceding mRNAs, any of the preceding mRNA compositions, or any of the preceding nucleic acid molecules as described in claim 37 in the prevention or treatment of influenza virus infection.

41. The method according to claim 38, the use according to claims 39-40, wherein the influenza virus is selected from one or more of the following influenza strains: A / Puerto Rico / 8 / 34, A / Victoria / 4897 / 2022, A / Wisconsin / 67 / 2022, A / Victoria / 2570 / 2019, A / Sydney / 5 / 2021, A / Cali fornia / 07 / 2009, A / Michigan / 45 / 2015, A / Brisbane / 02 / 2018, A / Hawaii / 70 / 2019, A / Idaho / 07 / 2018, A / Maine / 3 8 / 2018, A / Nebraska / 15 / 2018, A / Nebraska / 14 / 2019, A / Wisconsin / 588 / 2019, A / Iowa / 33 / 2019, A / Arkansas / 28 / 2019, A / Virginia / 41 / 2019, A / Minnesota / 60 / 2019, A / Alabama / 27 / 2019 or A / Guangdong-Maonan / SWLl536 / 2019; A / Aichi / 2 / 1968, A / Thailand / 8 / 2022, A / Massachusetts / 18 / 2022, A / Darwin / 9 / 2021, A / Darwin / 6 / 2021, A / Cambodia / e0826360 / 2020, A / Switzerland / 8060 / 2017, A / Switzerland / 9715293 / 2013, A / Iowa / 60 / 2018, A / South Australia / 34 / 2019, A / Hong Kong / 45 / 2019, A / Hong Kong / 2671 / 2019, A / Kansas / 14 / 2017, A / Jamaica / 60361 / 2019, A / Florida / 130 / 2019, A / Laos / 1789 / 2019, A / Vermont / 25 / 2019, A / New Jersey / 34 / 2019, A / California / 176 / 2019, A / Pennsylvania / 1026 / 2019, A / Togo / 634 / 2019, A / Kenya / 130 / 2019, A / Togo / 1307 / 2019, A / Ohio / 30 / 2019, A / Guatemala / 93 / 2019, A / Guatemala / 10 / 2019, A / Hong Kong / 4801 / 2014 or A / Singapore / INFIMH-16-0019 / 2016; B / Austria / 1359417 / 2021, B / Washington / 02 / 2019, B / Colorado / 06 / 2017, B / Brisbane / 60 / 2008 or B / Colorado / 06 / 2019.

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