Multivalent influenza mRNA vaccine

Abstract

Provided is an isolated mRNA molecule, which contains a nucleotide sequence encoding a chimeric immunogenic polypeptide, wherein the chimeric immunogenic polypeptide contains an immunogenic fragment of hemagglutinin (HA) of influenza A H5N1, an immunogenic fragment of hemagglutinin (HA) of influenza A H1N1, and an immunogenic fragment of hemagglutinin (HA) of influenza B Victoria, which are linked to each other. Further provided are a composition and vaccine containing the mRNA, a fusion protein encoded thereby, and a method using same for inducing an immune response to influenza virus in a subject.

Description

A polyvalent influenza mRNA vaccine Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an mRNA vaccine having a protective effect against multiple different serotypes of influenza virus. Background Art

[0002] Influenza viruses belong to the Orthomyxoviridae family and are RNA viruses. They cause seasonal epidemics and sporadic pandemics in humans. It is estimated that influenza viruses cause approximately 3 to 5 million cases of severe respiratory illness worldwide each year, resulting in approximately 290,000 to 650,000 deaths. The World Health Organization (WHO) and national disease control departments recommend annual influenza vaccination, which is one of the most effective ways to prevent influenza. However, the surface glycoproteins of influenza viruses (hemagglutinin HA and neuraminidase NA), commonly used antigens in influenza vaccines, are prone to antigenic drift (see Harrison's Journal of Infectious Diseases, 1st Chinese Edition, 2019). Variations in the amino acid sequence of glycoproteins or mutations in various influenza virus glycosyltransferases can lead to significant differences in the glycoproteins and their glycosylation modifications, facilitating the emergence of new virus strains. This ability of influenza viruses to readily alter their antigenic properties often results in a mismatch between the antigenicity of emerging strains and the immune protection provided by existing vaccines. Consequently, existing influenza vaccines generally offer low protection (approximately 40% to 60%) and are ineffective in preventing seasonal pandemics.

[0003] In recent years, influenza virus epidemics have also shown a trend of cross-infection between animals and humans. Highly pathogenic avian influenza (HPAI) viruses and other animal influenza viruses (such as H5 and H7) have crossed species and infected humans, resulting in high mortality rates. However, the current mainstream vaccines, the quadrivalent influenza vaccine targeting H1N1, H3N2, B / Victoria, and B / Yamagata, lack effective protection against highly pathogenic H5N1 and H7N9 infections.

[0004] Therefore, there is an urgent need to develop a universal vaccine that effectively protects against multiple influenza antigens. To achieve broad-spectrum efficacy in influenza vaccines, multiple influenza antigens are typically introduced into the vaccine. However, the increased number of influenza antigens poses significant challenges to both vaccine quality and production processes. Therefore, achieving broad-spectrum protection against influenza viruses with a minimal number of antigenic components has long been a key focus of influenza vaccine development.

[0005] mRNA technology emerged during the COVID-19 pandemic as a rapid-response vaccine development platform for responding to outbreaks. Using linearized plasmid DNA as a template and synthesized through in vitro transcription, it can avoid the live cell culture process used in traditional vaccine production. It also features a short production cycle, simple process, and convenient structural modification, making it particularly suitable for responding to outbreaks with rapid mutation rates. Moreover, compared with 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 form of administration, can also induce mucosal immunity, making them suitable as influenza vaccines. However, the loading capacity of a single mRNA molecule is limited, and polyvalent mRNA molecules encoding multiple influenza antigen peptides and polyvalent influenza mRNA vaccines prepared using them have not yet been reported.

[0006] Summary of the Invention

[0007] After in-depth research, the inventors of the present application found that by integrating nucleotide sequences encoding immunogenic fragments of hemagglutinin HA from influenza A H5N1, H1N1 and influenza B Victoria into the same mRNA molecule, while significantly reducing the mRNA load, it is still possible to simultaneously induce immune responses against multiple influenza virus subtypes within types A and B, thereby achieving broad-spectrum immunogenicity.

[0008] Therefore, an object of the present invention is to provide an mRNA molecule, preferably an isolated mRNA molecule, comprising a nucleotide sequence encoding a chimeric immunogenic polypeptide, wherein the chimeric immunogenic polypeptide comprises an immunogenic fragment of hemagglutinin HA of influenza A type H5N1, an immunogenic fragment of hemagglutinin HA of influenza A type H1N1, and an immunogenic fragment of hemagglutinin HA of influenza type B Victoria, which are linked together. In the isolated mRNA molecule disclosed herein, the three immunogenic fragments contained in the chimeric immunogenic polypeptide encoded thereby can be linked together in a variety of orders, and the order of linking of the immunogenic fragments includes, but is not limited to, from N-terminus to C-terminus, (1) an immunogenic fragment of hemagglutinin HA of H5N1, an immunogenic fragment of hemagglutinin HA of H1N1, and an immunogenic fragment of hemagglutinin HA of influenza type B Victoria; (2) an immunogenic fragment of hemagglutinin HA of H1N1, an immunogenic fragment of hemagglutinin HA of H5N1, ...1N1, an immunogenic fragment of hemagglutinin HA of H1N1 1, or an immunogenic fragment of the hemagglutinin HA of influenza B Victoria; (3) an immunogenic fragment of the hemagglutinin HA of influenza B Victoria, or an immunogenic fragment of the hemagglutinin HA of H5N1, or an immunogenic fragment of the hemagglutinin HA of H1N1; or (4) an immunogenic fragment of the hemagglutinin HA of influenza B Victoria, or an immunogenic fragment of the hemagglutinin HA of H1N1, or an immunogenic fragment of the hemagglutinin HA of H5N1. In a preferred embodiment, the chimeric immunogenic polypeptide comprises, from N-terminus to C-terminus, (1) the head of hemagglutinin HA of H5N1, the neck of hemagglutinin HA of H1N1, and an immunogenic fragment of hemagglutinin HA of influenza type B Victoria; (2) the head of hemagglutinin HA of H1N1, the neck of hemagglutinin HA of H5N1, and an immunogenic fragment of hemagglutinin HA of influenza type B Victoria; (3) the immunogenic fragment of hemagglutinin HA of influenza type B Victoria, the head of hemagglutinin HA of H5N1, and the neck of hemagglutinin HA of H1N1; or (4) the immunogenic fragment of hemagglutinin HA of influenza type B Victoria, the head of hemagglutinin HA of H1N1, and the neck of hemagglutinin HA of H5N1. In a preferred embodiment, the chimeric immunogenic polypeptide comprises, from N-terminus to C-terminus, (1) the head of the hemagglutinin HA of H5N1, the neck of the hemagglutinin HA of H1N1, and the full-length hemagglutinin HA of influenza type B Victoria; (2) the head of the hemagglutinin HA of H1N1, the neck of the hemagglutinin HA of H5N1, and the full-length hemagglutinin HA of influenza type B Victoria; (3) the full-length hemagglutinin HA of influenza type B Victoria, the head of the hemagglutinin HA of H5N1, and the neck of the hemagglutinin HA of H1N1; or (4) the full-length hemagglutinin HA of influenza type B Victoria, the head of the hemagglutinin HA of H1N1, and the neck of the hemagglutinin HA of H5N1.

[0009] In the isolated mRNA molecules disclosed herein, the three immunogenic fragments contained in the chimeric immunogenic polypeptides encoded therein can be linked together in a variety of ways, including but not limited to being directly linked by, for example, covalent bonds, or being linked by linkers. In some embodiments, the immunogenic fragment of the hemagglutinin HA of H5N1 and the immunogenic fragment of the hemagglutinin HA of H1N1 are directly linked by covalent bonds. In some embodiments, the immunogenic fragment of the hemagglutinin HA of influenza type B Victoria is linked to the immunogenic fragment of the hemagglutinin HA of H5N1 or H1N1 via a linker. In a preferred embodiment, the linker is a peptide linker. In a preferred embodiment, the linker has the structure -((G) n S) m -, wherein n is selected from 1, 2, 3, 4, 5 or 6, preferably 4; m is selected from 1, 2, 3, 4, 5 or 6, preferably 3. In a more preferred embodiment, the linker is a peptide linker having the amino acid sequence shown in SEQ ID NO: 8.

[0010] In some embodiments, the immunogenic fragment of the hemagglutinin HA of influenza A type H5N1 in the mRNA molecules disclosed herein is the head or neck portion of HA. In a preferred embodiment, the head portion of the hemagglutinin HA of H5N1 comprises the amino acid sequence set forth in SEQ ID NO:4, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:4. In some embodiments, the immunogenic fragment of the hemagglutinin HA of influenza A type H1N1 in the mRNA molecules disclosed herein is the head or neck portion of HA. In a preferred embodiment, the neck portion of the hemagglutinin HA of H1N1 comprises the amino acid sequence set forth in SEQ ID NO:6, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6. In some embodiments, the immunogenic fragment of the hemagglutinin HA of influenza type B Victoria in the mRNA molecule disclosed herein comprises the amino acid sequence shown in SEQ ID NO: 10 or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 10.

[0011] In some embodiments, the chimeric immunogenic polypeptide disclosed herein further comprises an N-terminal leader sequence, which preferably comprises an amino acid sequence as shown in SEQ ID NO: 2 or encoded by SEQ ID NO: 1, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to the sequence as shown in SEQ ID NO: 2 or encoded by SEQ ID NO: 1, and is capable of promoting the correct folding of the chimeric immunogenic polypeptide.

[0012] In some embodiments, the mRNA disclosed herein comprises the nucleotide sequence set forth in SEQ ID NO: 11, or comprises a nucleotide sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the chimeric immunogenic polypeptide encoded by the mRNA molecule disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 12, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12.

[0013] In some embodiments, the mRNA molecules disclosed herein further comprise regulatory elements such as a 5'UTR, a 3'UTR, and a poly A tail operably linked to a polynucleotide sequence encoding a chimeric immunogenic polypeptide. In a preferred embodiment, the 5'UTR comprises the sequence set forth in SEQ ID NO: 23 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto, the 3'UTR comprises the sequence set forth in SEQ ID NO: 24 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto, and the poly A tail comprises the sequence set forth in SEQ ID NO: 25 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto.

[0014] In some embodiments, the mRNA molecules disclosed herein further comprise a 5' cap. In a preferred embodiment, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.

[0015] In some embodiments, the mRNA molecules disclosed herein further comprise chemical modifications, such as modification of all or part of the uridine nucleotides in the polynucleotide sequence to 1-methylpseudouridine.

[0016] Another object of the present invention is to provide a composition comprising the isolated mRNA molecules disclosed herein.

[0017] In some embodiments, the compositions disclosed herein further comprise mRNA encoding immunogenic fragments of hemagglutinin HA of other influenza A virus subtypes, and / or mRNA encoding immunogenic fragments of hemagglutinin HA of other influenza B virus subtypes. In a preferred embodiment, the other influenza A virus subtypes are H3N2 and / or H7N9 subtypes, and the other influenza B virus subtypes are B / Yamagata subtypes.

[0018] In some embodiments, the composition of the present invention further comprises mRNA encoding an immunogenic fragment of influenza virus matrix protein M1, influenza virus ion channel protein M2, and / or influenza virus nucleoprotein NP.

[0019] In some embodiments, the compositions disclosed herein include: a first mRNA comprising a nucleotide sequence encoding a chimeric immunogenic polypeptide disclosed herein; a second mRNA encoding a second immunogenic polypeptide comprising an immunogenic fragment of H3N2 hemagglutinin HA; a third mRNA encoding a third immunogenic polypeptide comprising an immunogenic fragment of H7N9 hemagglutinin HA; and a fourth mRNA encoding a fourth immunogenic polypeptide comprising an immunogenic fragment of B / Yamagata hemagglutinin HA. In a preferred embodiment, the composition of the present invention comprises the first mRNA, the second mRNA, the third mRNA, and the fourth mRNA in a ratio by mass within the range of (2-5): (2-5): (2-5): (0.5-5). In a more preferred embodiment, the composition of the present invention comprises the first mRNA, the second mRNA, the third mRNA and the fourth mRNA in a ratio by mass of 5:5:2:0.5 or 5:5:2:2.5 or 5:2:2:0.5 or 5:5:5:5.

[0020] In some embodiments, the composition disclosed herein includes: a first mRNA comprising a nucleotide sequence encoding a chimeric immunogenic polypeptide disclosed herein; a second mRNA encoding a second immunogenic polypeptide, the second immunogenic polypeptide comprising an immunogenic fragment of H3N2 hemagglutinin HA; a third mRNA encoding a third immunogenic polypeptide, the third immunogenic polypeptide comprising an immunogenic fragment of H7N9 hemagglutinin HA; a fourth mRNA encoding a fourth immunogenic polypeptide, the fourth immunogenic polypeptide comprising an immunogenic fragment of B / Yamagata hemagglutinin HA; and a fifth mRNA encoding a fifth immunogenic polypeptide comprising an immunogenic fragment of influenza virus ion channel protein M2. In a preferred embodiment, the content ratio of the first mRNA, the second mRNA, the third mRNA, the fourth mRNA, and the fifth mRNA contained in the composition disclosed herein is in the range of (2-5): (2-5): (2-5): (0.5-5): (2-5) by mass. In a more preferred embodiment, the composition disclosed herein comprises the first mRNA, the second mRNA, the third mRNA, the fourth mRNA and the fifth mRNA in a ratio by mass of 5:5:5:5:5, 5:5:2:2:2, 5:2:2:2:2, 5:2:2:0.5:0.5, 5:5:2:0.5:2.5 or 5:2:2:0.5:3.

[0021] In some embodiments, the hemagglutinin antigen in the compositions disclosed herein is recommended or selected according to standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS).

[0022] In some embodiments, the chimeric immunogenic polypeptide described herein comprises the amino acid sequence shown in SEQ ID NO: 12 or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12.

[0023] In some embodiments, the immunogenic fragment sequence of the H3N2 hemagglutinin HA described herein comprises the amino acid sequence shown in SEQ ID NO: 14 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 14.

[0024] In some embodiments, the immunogenic fragment sequence of the hemagglutinin HA of H7N9 described herein comprises the amino acid sequence shown in SEQ ID NO: 16 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 16.

[0025] In some embodiments, the immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata described herein comprises the amino acid sequence shown in SEQ ID NO: 18 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 18.

[0026] In some embodiments, the immunogenic fragment sequence of the influenza virus ion channel protein M2 described herein comprises the amino acid sequence shown in SEQ ID NO: 20, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 20.

[0027] In some embodiments, the first mRNA described herein comprises the nucleotide sequence shown in SEQ ID NO: 11, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11.

[0028] In some embodiments, the second mRNA described herein comprises the nucleotide sequence shown in SEQ ID NO: 13, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13.

[0029] In some embodiments, the third mRNA described herein comprises the nucleotide sequence shown in SEQ ID NO: 15, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15.

[0030] In some embodiments, the fourth mRNA described herein comprises the nucleotide sequence shown in SEQ ID NO: 17, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 17.

[0031] In some embodiments, the fifth mRNA described herein comprises the nucleotide sequence shown in SEQ ID NO: 19, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 19.

[0032] In some embodiments, the compositions disclosed herein comprise (1) a nucleotide sequence encoding a chimeric immunogenic polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12 or comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12, (2) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of H3N2, wherein the immunogenic fragment sequence of the hemagglutinin HA of H3N2 comprises the amino acid sequence set forth in SEQ ID NO: 14 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14, (3) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of H7N9, wherein the immunogenic fragment sequence of the hemagglutinin HA of H7N9 comprises the amino acid sequence set forth in SEQ ID NO: 16 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: NO:16 has a sequence with at least 80%, 90%, 95%, 99% sequence identity and (4) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata, wherein the immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata comprises the amino acid sequence shown in SEQ ID NO:18 or comprises a sequence with at least 80%, 90%, 95%, 99% sequence identity to SEQ ID NO:18.

[0033] In some embodiments, the compositions disclosed herein comprise (1) a nucleotide sequence encoding a chimeric immunogenic polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12 or comprising an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 12, (2) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of H3N2, wherein the immunogenic fragment sequence of the hemagglutinin HA of H3N2 comprises the amino acid sequence set forth in SEQ ID NO: 14 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 14, (3) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of H7N9, wherein the immunogenic fragment sequence of the hemagglutinin HA of H7N9 comprises the amino acid sequence set forth in SEQ ID NO: 16 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: NO:16 has a sequence with at least 80%, 90%, 95%, or 99% sequence identity, (4) a nucleotide sequence encoding an immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata, the immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata comprising the amino acid sequence shown in SEQ ID NO:18 or comprising a sequence with at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:18, and (5) a nucleotide sequence encoding an immunogenic fragment sequence of the influenza virus ion channel protein M2, the immunogenic fragment sequence of the influenza virus ion channel protein M2 comprising the amino acid sequence shown in SEQ ID NO:20 or comprising a nucleotide sequence with at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:20.

[0034] In some embodiments, the compositions disclosed herein comprise: (1) a first mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 11, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11; (2) a second mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 13, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13; (3) a third mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 15, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15; and (4) a fourth mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 17, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 17.

[0035] In some embodiments, the compositions disclosed herein comprise: (1) a first mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 11, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11; (2) a second mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 13, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 13; (3) a third mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 15, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 15; (4) a fourth mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 17, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 17; and (5) a fifth mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 19, or comprising a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: NO: 19 has a sequence with at least 80%, 90%, 95%, or 99% sequence identity.

[0036] In some embodiments, the mRNA molecules in the compositions disclosed herein further comprise regulatory elements such as a 5'UTR, a 3'UTR, and a poly A tail operably linked to the coding nucleotide sequence. In a preferred embodiment, the 5'UTR comprises a sequence as set forth in SEQ ID NO: 23 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto, the 3'UTR comprises a sequence as set forth in SEQ ID NO: 24 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto, and the poly A tail comprises a sequence as set forth in SEQ ID NO: 25 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity thereto.

[0037] In some embodiments, the mRNA molecule in the composition disclosed herein further comprises a 5' cap, preferably the 5' cap comprises m7G(5')ppp(5')(2'-OMeA)pG.

[0038] In some embodiments, the mRNA molecules in the compositions disclosed herein further comprise chemical modifications, such as modification of all or part of the uridine nucleotides in the nucleotide sequence to 1-methylpseudouridine.

[0039] In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In a preferred embodiment, the carrier is a lipid nanoparticle, and the mRNA disclosed herein is encapsulated in one or more lipid nanoparticles.

[0040] In some embodiments, each individual particle in the lipid nanoparticles encapsulates one or more of the first to fifth mRNAs in substantially the same proportions. In other embodiments, individual particles in the lipid nanoparticles may encapsulate one or more of the first to fifth mRNAs in different proportions. In other embodiments, individual particles in the lipid nanoparticles encapsulate any one of the first, second, third, fourth, or fifth mRNAs, respectively.

[0041] In some embodiments, each individual particle in the lipid nanoparticles is encapsulated with the first to fourth mRNAs in substantially the same proportions. In other embodiments, individual particles in the lipid nanoparticles may be encapsulated with the first to fourth mRNAs in different proportions. In still other embodiments, individual particles in the lipid nanoparticles are respectively encapsulated with any one of the first, second, third, or fourth mRNAs.

[0042] In some embodiments, each individual particle in the lipid nanoparticles is encapsulated with the first to fifth mRNAs in substantially the same proportions. In other embodiments, individual particles in the lipid nanoparticles may be encapsulated with the first to fifth mRNAs in different proportions. In still other embodiments, individual particles in the lipid nanoparticles are respectively encapsulated with any one of the first, second, third, fourth, or fifth mRNAs.

[0043] In some embodiments, the lipid nanoparticles comprise ionizable lipids, phospholipids, structural lipids, and polyethylene glycol (PEG)-lipids. In a preferred embodiment, the molar ratio of the sum of the ionizable lipids, phospholipids, and the structural lipids in the lipid nanoparticles to the PEG lipids is (40-65): (35-65): (1-3). In a more preferred embodiment, the lipid nanoparticles comprise ionizable lipids, phospholipids, the structural lipids, and the PEG lipids in a molar ratio of (40-55): (5-15): (30-50): (1-3).

[0044] In some embodiments, the lipid nanoparticles comprise an ionizable lipid, a phospholipid, a structural lipid, and a polyethylene glycol (PEG)-lipid. In a preferred embodiment, the lipid nanoparticles comprise the ionizable lipid, the phospholipid, the structural lipid, and the PEG-lipid in a molar ratio of (20-60):(5-25):(25-55):(0.5-5).

[0045] In some embodiments, the lipid nanoparticles comprise one or more of the following phospholipid compounds: dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DUPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholestyldimethylsuccinate-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso In a preferred embodiment, the lipid nanoparticles comprise DSPC.

[0046] In some embodiments, the lipid nanoparticles comprise one or more of the following structural lipids: cholesterol, coprostanol, sitosterol, ergosterol, and stigmasterol. In a preferred embodiment, the structural lipid is cholesterol.

[0047] In some embodiments, the lipid nanoparticles comprise one or more of the following PEG lipids: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol. In a preferred embodiment, the lipid nanoparticles comprise DMG-PEG2000.

[0048] In some embodiments, the compositions disclosed herein are vaccines.

[0049] Another object of the present invention is to provide a nucleic acid molecule encoding the mRNA disclosed herein, wherein the nucleic acid molecule is preferably DNA, more preferably a DNA plasmid.

[0050] Another object of the present invention is to provide a fusion protein comprising the amino acid sequence encoded by the first mRNA disclosed herein, which can be used to prevent or treat influenza virus infection. Also provided is a composition comprising the fusion protein disclosed herein.

[0051] Another object of the present invention is to provide a method for inducing an immune response to influenza virus in a subject, comprising administering to the subject an effective dose of the isolated mRNA, composition, vector, or fusion protein disclosed herein. In a preferred embodiment, the method disclosed herein comprises administering to the subject two or three effective doses of the isolated mRNA, composition, nucleic acid molecule, or fusion protein disclosed herein.

[0052] Another object of the present invention is to provide use of the isolated mRNA, composition, nucleic acid molecule or fusion protein disclosed herein in the preparation of a medicament for preventing or treating influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 shows the results of a hemagglutination inhibition test after immunizing mice with the mRNA-LNP compositions of groups G1, G3, and G4 prepared in Example 2 (ns: not significant, *: p<0.05, using Multiple t test);

[0054] FIG2 shows the results of a hemagglutination inhibition test after mice were immunized with the mRNA-LNP compositions of groups G1, G2, and G5 prepared in Example 2;

[0055] Figure 3 shows the IgG titers of anti-H1N1, anti-H7N9, anti-H5N1, anti-H3N2, anti-B / Victoria, and anti-B / Yamagata that can be simultaneously detected in the mouse serum when mice were immunized with the mRNA-LNP compositions of groups G1 and G6 prepared in Example 2. DETAILED DESCRIPTION

[0056] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. For details, please refer to, for example, Sambrook et al., Molecular Cloning: a Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Publishing, updated from time to time. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The quantitative tests in the following examples, unless otherwise specified, are the average values ​​of three repeated experiments. In the following examples, unless otherwise specified, the nucleotide sequences in the sequence table are written from left to right in the order of 5' to 3' end, and the amino acid sequences are written from left to right in the order of amino terminus to carboxyl terminus.

[0058] definition

[0059] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0060] As used herein, the terms "comprising" or "including" mean that sequences, compositions, and methods include the recited components or steps, but do not exclude other components or steps. "Consisting essentially of," when used to define compositions and methods, should be construed to exclude any other components or other steps that are clearly important for the technical effect to be achieved. "Consisting of" should be construed to exclude other components and steps not mentioned.

[0061] Unless the content clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells, or an entire culture of cells. Unless explicitly specified or obvious from the context, as used herein, the term "or" is understood to be inclusive.

[0062] Unless expressly provided or obvious from the context, otherwise as used herein, the term "about" should be understood to be within the normal tolerance range of the field, for example, within 2 standard deviations of the mean. "About" can be understood to be 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 value. Unless otherwise obvious from the context, all numerical values ​​provided herein are modified by the term "about".

[0063] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid) has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which the component naturally occurs. "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.

[0064] As used herein, the term "immunogenic polypeptide" refers to proteins (including glycoproteins) and peptides that are capable of eliciting an immune response in a mammal, including peptides that, upon administration to a host, are capable of eliciting humoral and / or cellular immune responses against the polypeptide and / or homologous polypeptides comprising an amino acid sequence that is 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 specific polypeptide. Therefore, the "immunogenic polypeptide" herein includes the full-length sequence of the polypeptide, its analogs or immunogenic fragments thereof. The term "immunogenic fragment" herein may be at least one polypeptide fragment selected from the following amino acid sequences: a protein / polypeptide fragment that comprises at least one epitope or antigenic determinant, or consists essentially of at least one epitope or antigenic determinant, or consists of at least one epitope or antigenic determinant, thereby being capable of eliciting an immune response. Herein, an "immunogenic fragment" may include deletions, substitutions and / or additions relative to the polypeptide sequence from which it is derived, as long as the fragment can function to elicit an immune response against the polypeptide.

[0065] In some embodiments, the immunogenic polypeptide is the full length, fragment, derivative or variant of the influenza virus hemagglutinin HA antigen. For example, in some embodiments, HA is wild-type HA. In other embodiments, HA is a modified HA, for example, comprising at least one amino acid substitution, deletion and / or insertion, so that its primary structure is different from that of wild-type HA. In some specific embodiments, the mutation of HA is T2191, H371Y, I494M, H504P, M362L, HA0, APB, TB or VASP, or any combination thereof. In some embodiments, the mutation of HA is the generation of a disulfide bond in the stem of HA to connect adjacent protomers, the deletion of a cleavage site, and / or the replacement of a polybasic cleavage site (HPAI) with an LPAI sequence.

[0066] 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 antigens and NA antigens recommended or selected by GISRS are included in the influenza virus vaccine for the Southern Hemisphere or Northern Hemisphere influenza vaccine of the year in which the vaccine is manufactured and distributed. In some embodiments, HA antigens and NA antigens are selected using a hemagglutinin inhibition (HAI) assay to identify circulating influenza viruses that are antigenically similar to the influenza viruses of the previous season's vaccine, optionally wherein the influenza viruses are considered antigenically similar if the HAI titers of the influenza viruses differ by two-fold or less.

[0067] The term "chimeric" has the general meaning understood in the art, and refers to the formation of a new entity (such as a new polypeptide or nucleic acid molecule) by linking at least two components (such as amino acid sequences or nucleotide sequences) from different sources. When describing the immunogenic polypeptides herein, "chimeric" means that the immunogenic polypeptide to which it refers contains at least two epitopes or antigenic determinants from different antigens, or contains at least two epitopes or antigenic determinants from the same antigen but linked together in an altered order of linkage.

[0068] As used herein, "linker" refers to a peptide sequence or non-peptide structure that connects two polypeptide fragments to each other. In some embodiments, the linker is a flexible peptide linker, which allows the two amino acid fragments to have a certain degree of mobility. The addition of Ser and Thr allows the linker to form hydrogen bonds with water molecules, giving the linker stability in aqueous solution, thereby reducing the interaction between the linker and the two proteins before and after. Common flexible peptide linkers are composed of Gly and Ser residues ("GS" linker). In addition to the GS linker, there are some other flexible linkers, such as (Gly)8, etc., which are all known in the art. In some embodiments, the linker is a rigid linker, which can be used to completely isolate two connected proteins and maintain their independent functions. Commonly used rigid linkers include α-helical structure peptides, (XP) netc., where P represents proline, X can be any amino acid, preferably Ala, Lys, or Glu, and n represents the number of times XP is repeated. Those skilled in the art can independently adjust and select different linkers based on specific application scenarios and fusion protein 3D structural requirements.

[0069] As used herein, the "head" and "neck" of hemagglutinin (HA) refer to the globular head domain and neck domain (stem domain or stalk domain) of the hemagglutinin protein, respectively. The corresponding hemagglutinin (HA) fragments are known to those skilled in the art (for example, they can be obtained from public databases such as GenBank), or can be determined according to the method described in, for example, the document "Influenza Viruses Expressing Chimeric Hemagglutinins: Globular Head and Stalk Domains Derived from Different Subtypes doi:10.1128 / JVI.00137-12".

[0070] As used herein, the term "identity" refers to the percentage of identical residues that two or more nucleic acids or polypeptide sequences have in a specified region. Methods for determining the percentage identity between two amino acid sequences or nucleotide sequences are well known in the art. For example, the identity between two sequences can be calculated as follows: the two sequences are optimally aligned in a specified region, the number of positions where identical residues occur in the two sequences is determined as the number of matching positions, the number of matching positions is divided by the total number of positions in the specified region, and the result is multiplied by 100. When the two sequences are different in length, or the alignment produces one or more staggered ends and a specific comparison region only includes a single sequence, the residues of the single sequence are included in the denominator of the calculation but are not included in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be performed manually or using a computer sequence algorithm (such as BLAST or BLAST 2.0).

[0071] As used herein, the term "5' cap" is located at the 5' end of the mRNA and comprises a methylated guanylate, which is linked to the 5' end of the mRNA via pyrophosphate to form a 5', 5'-triphosphate connection with its adjacent nucleotides. There are usually three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, m7G5'ppp5'NmpNmpNp), which are referred to as type O, type I, and type II, respectively. Type O refers to that the ribose of the terminal nucleotide is not methylated, type I refers to that the ribose of one terminal nucleotide is methylated, and type II refers to that the ribose of both terminal nucleotides is methylated. Herein, a preferred 5' cap is m7G(5')ppp(5')(2'-OMeA)pG (available from, for example, TriLink Biotechnologies).

[0072] As used herein, the term "Poly A tail" or "Poly A sequence" refers to an uninterrupted or interrupted sequence of adenylate 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 according to actual needs. In mRNA, in the presence of a 3'-UTR, the Poly-A sequence is connected to the 3' end of the 3'-UTR. An uninterrupted poly-A tail is characterized by continuous adenylate residues. The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80 or at least 100 and at most 500, at most 400, at most 300, at most 200 or at most 150 adenylate residues (A), in particular about 120 A's. Typically, the vast majority of nucleotides in the poly A tail are adenosine, where the vast majority refers to 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, but the remaining nucleotides are allowed to be nucleotides other than A, such as U (uridylic acid), G (guanylic acid), or C (cytidylic acid).

[0073] As used herein, the terms "individual," "subject," "patient," "host," "one in need thereof," or similar expressions refer to any mammal or non-mammal. Mammals include, but are not limited to, cats, other vertebrates such as rodents, humans, non-human primates such as cows, horses, dogs, pigs, sheep, goats, giraffes, deer, camels, sheep, rats, mice, hares, and rabbits.

[0074] In the vaccination method of the present application, the vaccinated subject may have been exposed to influenza virus. As used herein, the terms exposed, exposure, etc. indicate that the subject has been in contact with a person or animal known to be infected with influenza virus. The vaccine of the present application can be administered using techniques well known to those skilled in the art. Preparation and administration techniques can be found in, for example, the 18th edition of "Leishman's Pharmaceutical Encyclopedia". The vaccine can be administered by means including but not limited to traditional syringes, needle-free injection devices, or micro-jet 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 the present application can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer.

[0075] The term "lipid nanoparticles" (LNP) as used herein refers to particles having at least one nanometer size comprising at least one lipid. In a preferred embodiment, the lipids include, but are not limited to, neutral phospholipids and polyethylene glycol-lipids. The term "neutral phospholipids" as used herein refers to uncharged, non-phosphoglyceride phospholipid molecules. The term "polyethylene glycol-lipid" as used herein refers to molecules comprising a lipid portion and a polyethylene glycol portion.

[0076] The lipid nanoparticles generally comprise ionizable lipids, non-cationic lipids, sterols and PEG lipid components and a target nucleic acid, such as mRNA. The lipid nanoparticles of the present disclosure can be produced using components, compositions and methods as generally 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 / US2017 / 037551, PCT / US2017 / 037552, PCT / US2017 / 052352, PCT / US2016 / 068300, PCT / US2017 / 037551, PCT / US2015 / 027400, PCT / US2016 / 047406, PCT / US2016000129, PCT / US2016 / 014280, PCT / US2017 / 037551, PCT / US2017 / 037551, PCT / US2017 / 0 S2016 / 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, all of which are incorporated herein by reference in their entirety.

[0077] As used herein, "neutralizing antibodies" refer to antibodies that prevent influenza viruses from completing a round of replication. A round of replication refers to the life cycle of the virus, starting with the virus attaching to the host cell and ending with the newly formed virus budding from the host cell. This life cycle includes, but is not limited to, the steps of attachment to the cell, internalization, lysis and rearrangement, fusion of the viral membrane with the endosomal membrane, release of viral ribonucleoprotein into the cytoplasm, formation of new virus particles, and budding of virus particles from the host cell membrane. Neutralizing antibodies are antibodies that inhibit one or more of the above steps. "Broadly neutralizing antibodies" are antibodies that neutralize more than one type, group, subtype and / or strain of influenza virus. For example, broadly neutralizing antibodies induced by the HA protein of influenza A virus can neutralize influenza B or C viruses.

[0078] As used herein, "influenza virus" refers to any strain of influenza virus that can cause disease in animals or human subjects. See, for example, Fields, B. et al., Fields' Virology, 4th edition, 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) that can cause disease in animals or human subjects. Influenza A viruses include different subtypes, which are generally defined by the type of hemagglutinin (HA) and / or neuraminidase (NA) of the virus. It is known in the art that there are 18 different HA subtypes (H1 to H18) and 11 different NA subtypes (N1 to N11), see, for example, CDC, Types of Influennza Viruses, 2019. As non-limiting examples of influenza A virus strains, there can be mentioned, 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.

[0079] Influenza B viruses include any influenza B virus strain known in the art, examples of which include, but are not limited to, influenza B virus strains from Aichi Prefecture, Akita Prefecture, Alaska, Ann Arbor, Argentina, Bangkok, Beijing, Belgium, Bonn, Brazil, Buenos Aires, Canada, Chaco, Chiba, Chongqing, CNIC, Cordoba, Czechoslovakia, Oku, Durban, Finland, Fujian, Fukuoka, Genoa, Guangdong, Guangzhou, Hanover, Harbin, Hawaii, Hebei, Henan, Hiroshima, Hong Kong, Houston, Hunan, Ibaraki, India, Israel, Johannesburg, Kagoshima, Kanagawa, Kansas, Kharkiv, Kobe, Takone, Lazio, Lee, Leningrad, Lisbon, Los Angeles, Lusaka, Lyon, Malaysia, Maputo, Madupi 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, circulating recombinant 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, 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), Buenos Aires / 9 / 95, Buenos Aires / SW16 / 97, Buenos Aires / VL518 / 99, Canada / 464 / 2001, Canada / 464 / 2002, Chaco / 366 / 00, Chaco / R113 / 00, Cheju / 303 / 03, Chiba / 447 / 98, Chongqing / 3 / 2000, clinical isolate SA1Thailand / 2002, clinical isolate SA10Thailand / 2002, clinical isolate SA101Philippines / 2002, clinical isolate SA100 110Philippines / 2002), clinical isolate SA112Philippines / 2002, clinical isolate SA113Philippines / 2002, clinical isolate SA114Philippines / 2002, clinical isolate SA2Thailand / 2002, clinical isolate SA20Thailand / 2002, clinical isolate SA38Philippines / 2002, clinical isolate SA39Thailand / 2002, clinical isolate SA99Philippines / 2002, CNIC / 27 / 20 01 strain, Colorado / 2597 / 2004 strain, Cordoba / VA418 / 99 strain, Czechoslovakia / 16 / 89 strain, Czechoslovakia / 69 / 90 strain, Daeku / 10 / 97 strain, Daeku / 45 / 97 strain, Daek u / 47 / 97 strain, Daeku / 9 / 97 strain, B / Du / 4 / 78 strain, B / Durban / 39 / 98 strain, Durban / 43 / 98 strain, Durban / 44 / 98 strain, B / Durban / 52 / 98 strain, Durban / 55 / 98 strain, Durban / 56 / 98 strains, England / 1716 / 2005 strains, England / 2054 / 2005) strains, England / 23 / 04 strains, Finland / 154 / 2002 strains, Finland / 159 / 2002 strains, Finland / 160 / 2002 strains, Finl and / 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, Gua 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 Kong / 110 / 99 shares, Hong Kong / 11 15 / 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 strain,Kadoma / 1076 / 99, Kadoma / 122 / 99, Kagoshima / 15 / 94, Kansas / 22992 / 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, 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 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 Rochester / 02 / 2001. The sequences of the above virus strains are known in the art and can be obtained from GenBank.

[0080] The hemagglutination inhibition (HAI) test is a classic laboratory procedure used to classify or type hemagglutinating viruses and further characterize the antigenic characteristics of influenza virus isolates. To determine HAI, serial dilutions of virus are prepared in a 96-well microtiter plate with a U- or V-bottom. For example, the most concentrated sample in the first well can be diluted two-fold (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 containing no virus. Different rows of the plate typically contain different viruses and the same dilution pattern. After the 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, the assay can be analyzed to distinguish between agglutinating and non-agglutinating wells. The relative concentration, or titer, of the virus sample is determined based on the well in which the last agglutination was observed before precipitation.

[0081] Serological methods such as HAI detection can be used as epidemiological and immunological studies of viruses and vaccines, evaluate antibody responses after vaccination, measure the effectiveness of candidate vaccines, and identify antigenically similar influenza viruses. Viruses with HAI titers that differ by two times or less dilution can be considered as antigenically similar viruses. In some instances, mRNA vaccines disclosed herein have HAI titers that increase by 2, 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control (for example, compared to subjects administering traditional quadrivalent influenza vaccines). In some instances, HAELISA is measured to check the HA antibody titers (for example, IgG antibody titers) produced by administering candidate vaccines.

[0082] As used herein, "N:P ratio" or "N / P ratio" is the molar ratio of ionizable nitrogen atoms in the ionizable lipid to phosphate groups in the RNA.

[0083] Example

[0084] Below in conjunction with specific embodiment, the present invention is further described in detail, and the embodiment provided is only for illustrating the present invention, rather than for limiting the scope of the present invention. Those skilled in the art can make various modifications or adjustments based on the teachings of the present invention, which do not deviate from the spirit and scope of the present invention.

[0085] Biosafety

[0086] The present invention uses four influenza viruses with pathogenicity (A / Puerto Rico / 8 / 34(H1N1), A / Aichi / 2 / 1968(X31)(H3N2), B / Brisbane / 60 / 2008(B / Victoria), and B / Phuket / 3073 / 2013(B / Yamagata)). The experimental process was carried out in a biological level 2 laboratory located in the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences. The strains A / Puerto Rico / 8 / 34(H1N1), B / Brisbane / 60 / 2008(B / Victoria), and B / Phuket / 3073 / 2013(B / Yamagata) were obtained from the Institute of Medical Biology, Chinese Academy of Medical Sciences, and the strain A / Aichi / 2 / 1968(X31)(H3N2) was obtained from the National Influenza Center, Institute of Virology, Chinese Center for Disease Control and Prevention. All experimental materials, operations, and waste disposal complied with the safety regulations of the laboratory.

[0087] All experimental operations and waste disposal of the present invention are in compliance with the relevant provisions of the WHO "Laboratory Biosafety Manual (Fourth Edition)" (LABORATORY BIOSAFETY MANUAL, FOURTH EDITION), China's "General Requirements for Laboratory Biosafety" standard (GB19489-2208) and the "Regulations on Biosafety Management of Pathogenic Microorganism Laboratories".

[0088] Experimental materials, reagents, instruments and experimental methods

[0089] Unless otherwise specified, the reagents, instruments, genes, and enzymes used in the present invention are all commercially available. The relevant nucleic acid chains, genes, and enzymes can also be obtained using conventional molecular biology experimental techniques based on information from public databases. Unless otherwise specified, nucleotide sequences described in the specification are written from 5' to 3', and amino acid sequences are written from amino to carboxyl termini. In the event of any discrepancy between the specification and the sequence listing, the sequence described in the specification shall prevail.

[0090] Example 1:

[0091] Suzhou Genewise Biotechnology Co., Ltd. was commissioned to synthesize double-stranded DNA plasmids 1 to 7 for preparing mRNA.

[0092] Plasmids 1 to 7 contain the nucleotide sequences shown in SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21, respectively, encoding the immunogenic polypeptide BW having the amino acid sequence shown in SEQ ID NO: 10, the chimeric immunogenic polypeptide 51-BW having the amino acid sequence shown in SEQ ID NO: 12, the H3N2 hemagglutinin HA having the amino acid sequence shown in SEQ ID NO: 14, the H7N9 hemagglutinin HA having the amino acid sequence shown in SEQ ID NO: 16, the B / Yamagata hemagglutinin HA having the amino acid sequence shown in SEQ ID NO: 18, the M2 protein having the amino acid sequence shown in SEQ ID NO: 20, and the 51 chimeric polypeptide having the amino acid sequence shown in SEQ ID NO: 22.

[0093] According to the manufacturer's instructions, using the reaction system shown in Table 1, plasmids 1 to 7 were digested with restriction enzyme SapI (purchased from NEB) and reacted at 37° C. for 3 hours to obtain the corresponding linearized plasmid templates.

[0094] Table 1: SapI restriction enzyme digestion reaction system:

[0095] Purify and recover the linearized plasmid template using the Linearized Plasmid Recovery and Purification Kit DP205-02 (purchased from TIANGEN) according to the manufacturer's instructions. Specifically, add 5 volumes of PB Buffer to the linearization reaction system and mix thoroughly. Activate the filter column with 200 μL of PS buffer, centrifuge at 12,000 × g for 1 minute, and discard the filtrate. Apply the mixed reaction mixture to the column and centrifuge at 12,000 × g for 1 minute. Discard the filtrate and wash away any impurities that did not adhere to the column with 700 μL of wash buffer and centrifuge at 12,000 × g for 1 minute. Complete the purification by eluting the linearized plasmid template with 50 μL of RNase-free ddH2O into a clean 1.5 mL EP tube (12,000 × g for 2 minutes). Quantify the purified linearized plasmid using a Nanodrop.

[0096] Purified linearized plasmid templates 1 to 7 were capped and in vitro transcribed using T7 polymerase and the CleanCap kit, respectively, according to the manufacturer's instructions. Specifically, the capping and in vitro transcription reaction systems shown in Table 2 below were added to a 1.5 mL EP tube and placed in a 37°C incubator with rotation (10 rpm) for 3 hours.

[0097] Table 2: mRNA capping and in vitro transcription reaction system:

[0098] After the in vitro transcription reaction is complete, add 20 μL of DNase I to the tube, mix thoroughly by pipetting, and incubate at 37°C for 15 minutes. Then, add 50% of the volume of LiCl to the reaction system and let it stand overnight at -20°C. Transfer to a centrifuge precooled to 4°C, centrifuge at 12,000 × g for 15 minutes, and discard the supernatant. After washing with 700 μL of 70% ethanol and centrifuging again (12,000 × g, 5 minutes), the white precipitate is redissolved in 1000 μL of RNase-free water, mixed thoroughly, and quantified using a Nanodrop to determine the amount of recovered mRNA.

[0099] The prepared mRNA contains the following elements from the 5' end to the 3' end: (1) 5' cap; (2) 5' UTR region (as shown in SEQ ID NO: 23); (3) antigen coding region; (4) 3' UTR region (as shown in SEQ ID NO: 24); (5) Poly A tail (as shown in SEQ ID NO: 25).

[0100] Example 2:

[0101] To the mRNA stock solution purified in Example 1, add acetic acid solution to a final acetic acid concentration of 20 mmol / L and a final mRNA concentration of 200 μg / ml, stir and mix, and use as an mRNA working solution. The mRNA working solution and the mixed lipid solution (prepared according to Table 3) are mixed at a flow ratio of 2: 1 to 4: 1 through a T mixing device to prepare LNP. Then, the LNP is diluted 2 to 5 times with 2 mmol / L acetic acid solution, and then replaced with 2 mmol / L acetic acid solution for not less than 3 times, and the feed solution is concentrated to the target concentration. Sucrose solution is added to adjust the osmotic pressure, and Tris solution is used to adjust the pH to 7.0 to 8.0 to obtain mRNA-lipid nanoparticles (LNP) containing the mRNA prepared and purified in Example 1.

[0102] In the prepared mRNA-LNPs, the N / P ratio of mRNA to ionizable lipid was 5.2.

[0103] Table 3: Fat-mixed solution formula

[0104] The mRNA-LNPs prepared as described above were mixed according to the mass ratios shown in Table 4 below to prepare the mRNA-LNP compositions shown in each group.

[0105] Table 4:

[0106] Note:

[0107] 1. 51-BW represents the mRNA encoding the 51-BW chimeric polypeptide;

[0108] 2. 51 represents mRNA encoding the 51 chimeric polypeptide; BW represents mRNA encoding the hemagglutinin HA of the B / victoria strain; the molecular weight of the mRNA encoding the 51-BW chimeric polypeptide is approximately 1.08*10^6Da, the molecular weight of the mRNA encoding the 51 chimeric polypeptide is approximately 5.23*10^5Da, and the molecular weight of the mRNA encoding the BW polypeptide is approximately 5.40*10^5Da; the molecular weights were calculated using SNAPGENE software.

[0109] 3.H3N2 indicates the mRNA encoding the hemagglutinin HA of the H3N2 strain;

[0110] 4.H7N9 represents the mRNA encoding the hemagglutinin HA of the H7N9 strain;

[0111] 5. BP represents the mRNA encoding the hemagglutinin HA of the B / Yamagata strain;

[0112] 6. M2 represents the mRNA encoding the M2 polypeptide.

[0113] Example 3:

[0114] The mRNA-LNP compositions prepared in Example 2 were used to immunize 6- to 8-week-old female BALB / c mice (SPF grade), and the immune protective response against influenza virus induced by each composition was evaluated.

[0115] Specifically, mice were randomly divided into groups of five and received intramuscular injections of the mRNA-LNP composition (doses shown in Table 4). A booster injection was given on day 14. On day 28, approximately 200 μL of blood was collected from the mouse orbital cavity. The blood was incubated at 37°C for 60 minutes and then refrigerated at 4°C for 120 minutes. Serum was separated by centrifugation at 3000 rpm for 10 minutes and used for subsequent neutralizing antibody assays and hemagglutination inhibition tests.

[0116] Example 4:

[0117] The mouse sera from each group, prepared according to Example 3, were mixed with receptor-destroying enzyme (RDE) at a volume ratio of 1:3. The reaction was inactivated in a 37°C water bath for 12 h and then in a 56°C water bath for 30 min. The H5N1 and H7N9 hemagglutination tests were performed using commercially available avian influenza virus H5 or H7 subtype hemagglutination inhibition test antigens (Harbin Veken Biotechnology Co., Ltd.).

[0118] In a V-shaped 96-well plate, add PBS at a rate of 25 μL / well. Then, add 25 μL of the virus stock solution to the first well and mix thoroughly with the PBS by pipetting. Then, draw 25 μL of the mixture from this well and add it to the second well and mix thoroughly. Then, dilute the virus by 2-fold in 12 steps. Subsequently, add 25 μL of 1% guinea pig blood to each well containing the virus gradient dilution solution, mix thoroughly, and let it stand at room temperature for 30-45 minutes. The number of virus particles corresponding to the lowest concentration well where hemagglutination is observed is considered a hemagglutination unit. The dose of virus used in the hemagglutination inhibition test is four hemagglutination units.

[0119] In a V-shaped 96-well plate, add PBS at a rate of 25 μL / well. Add 25 μL of RDE-treated serum to the first well and mix thoroughly with PBS by pipetting. Perform eight dilution gradients using the same procedure as in 1.2. Add 25 μL / well of the diluted virus (4 hemagglutination units) to each well and mix with the serially diluted serum at a 1:1 ratio. Incubate at room temperature for 1 hour. Subsequently, add 50 μL / well of 1% guinea pig blood to the serum-virus mixture, mix thoroughly, and incubate at room temperature for 45 minutes. Observe for hemagglutination.

[0120] The final serum dilution factor corresponding to the well immediately preceding the well in which hemagglutination was first observed was used as the hemagglutination titer. If hemagglutination was observed in the first well, the result was considered negative. The results are shown in Figures 1 and 2.

[0121] Figure 1 shows that, compared to the blank LNP control, the mRNA-LNP compositions of the G3 and G4 groups can significantly enhance the hemagglutination inhibition effect of the serum of the immunized mice against H1, H5 and family B hemagglutinin antigens. Further comparison of the results of the G3 and G4 groups shows that compared with the mice immunized with a mixture of 51 and BW (G4 group), the mice immunized with the exemplary chimeric immunogenic polypeptide 51-BW of the present application alone (G3 group) showed the same level of hemagglutination inhibition effect against H1 and H5 hemagglutinin antigens, and the hemagglutination inhibition effect of the mice in the G3 group against influenza virus Victoria type B was significantly better, and the difference was statistically significant. This shows that further chimerization of the 51 chimeric protein with the BW protein can unexpectedly enhance the immune effect in vivo.

[0122] Figure 2 shows that the sera from groups G1 and G2, which served as negative controls, were unable to inhibit hemagglutination caused by any virus strain. In contrast, a significant hemagglutination inhibition effect was observed in the sera obtained from mice immunized with the mRNA-LNP composition from group G5, and this inhibition effect also covered influenza A viruses H1N1, H3N2, H5N1, H7N9, and influenza B viruses Yamagata and Victoria. The above results further validate the potent and broad-spectrum immune protective activity of the chimeric antigen polypeptides and mRNA-LNP compositions disclosed herein. Furthermore, it demonstrates that those skilled in the art can adjust the protective effect against individual virus strains by increasing or decreasing the types and ratios of influenza antigens in the composition according to actual conditions, while substantially maintaining the broad-spectrum immune protective activity.

[0123] Example 5:

[0124] According to the manufacturer's instructions, an ELISA kit (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was used to measure the antibody titer in the mouse serum after immunization of mice with the mRNA-LNP combination. Each antigen shown in Table 5 (all purchased from Yiqiao Shenzhou) was diluted to 2 μg / mL with 1x coating buffer (pH 9.0) and added to a 96-well ELISA plate at 100 μL / well. Cover the plate with a sealing film and let it stand at 4°C overnight. Then, discard the supernatant, wash twice with 200 μL / well of washing buffer, add blocking buffer, let it stand for 2 hours, and then let it stand at room temperature to dry to obtain a coated 96-well plate. The coated plate can be sealed with a sealing film and stored in a cold storage with a desiccant until use.

[0125] Table 5

[0126] Before use, wash the coated ELISA plate once with 200 μL / well of washing buffer. After diluting the serum of the mice in group G6 prepared according to Example 3 with dilution buffer, add 100 μL / well to the ELISA plate, cover with a film, and incubate at 37°C for 2 hours. Then, discard the serum, wash 4 times with 200 μL / well of washing buffer, and then add 100 μL / well of mouse secondary antibody at a ratio of 1:2500. Cover with a sealing film and incubate at 37°C for 1 hour. Then discard the secondary antibody and wash 4 times with 200 μL / well of washing buffer. Add 100 μL / well of color developing solution, let it stand at room temperature in the dark for 20 minutes, and then add 50 μL / well of stop solution (diluted once) to stop color development. Read the measurement value at a wavelength of 450 nm. The results are shown in Figure 3.

[0127] Figure 3 shows that after immunizing mice with the mRNA-LNP composition of the G6 group, not only can the mice be induced to produce significantly increased total IgG against influenza virus, but also the proportion of IgG against antigens of six different virus strains (covering influenza A virus H1N1, H3N2, H5N1, H7N9 and common influenza B virus Yamagata, Victoria) is roughly the same. This shows that the mRNA composition disclosed herein can effectively express all antigens including trivalent chimeric antigen polypeptides in vivo, and thus can successfully induce an immune response to all parent antigens constituting the chimeric polypeptide. This can greatly reduce the dosage of mRNA. It also shows that after the chimeric antigen polypeptide herein is made into a mixture or composition with other influenza virus antigens, the expression of the chimeric antigen polypeptide and the immunogenicity of each constituent antigen unit it contains are not adversely affected by other influenza virus antigens, nor do they interfere with the expression and immunogenicity of other influenza virus antigens, and have the potential to be suitable for making a multivalent influenza vaccine (especially a multivalent vaccine for both type A and type B influenza viruses), thereby achieving a broad spectrum of influenza virus immunity.

[0128] The sequences used in the above examples of the present application are as follows. It should be understood that the following sequences are merely exemplary sequences of the embodiments of the present application and are not intended to limit the present application. The nucleic acid sequences in the following sequence listing may represent DNA sequences or RNA sequences. When representing RNA sequences, "T" represents uridine.

[0129] SEQ ID NO.1 (nucleotide sequence of the folding region)

[0130] SEQ ID NO.2 (amino acid sequence of the folding region)

[0131] SEQ ID NO.3 (nucleotide sequence of the HA head of H5N1)

[0132] SEQ ID NO.4 (amino acid sequence of the HA head of H5N1)

[0133] SEQ ID NO.5 (nucleotide sequence of the HA neck of H1N1)

[0134] SEQ ID NO.6 (amino acid sequence of the HA neck of H1N1)

[0135] SEQ ID NO.7 (Nucleotide sequence of the flexible linker region)

[0136] SEQ ID NO.8 (amino acid sequence of the flexible linker region)

[0137] SEQ ID NO.9 (nucleotide sequence of HA coding region of BW)

[0138] SEQ ID NO.10 (amino acid sequence of the HA coding region of BW)

[0139] SEQ ID NO.11 (nucleotide sequence of 51-BW)

[0140] SEQ ID NO.12 (amino acid sequence of 51-BW)

[0141] SEQ ID NO.13 (H3N2 HA coding region nucleotide sequence)

[0142] SEQ ID NO.14 (H3N2 HA coding region amino acid sequence)

[0143] SEQ ID NO.15 (H7N9 HA coding region nucleotide sequence)

[0144] SEQ ID NO.16 (H7N9 HA coding region amino acid sequence)

[0145] SEQ ID NO.17 (BP HA coding region nucleotide sequence)

[0146] SEQ ID NO.18 (BP HA coding region amino acid sequence)

[0147] SEQ ID NO.19 (M2 protein nucleotide sequence)

[0148] SEQ ID NO.20 (M2 protein amino acid sequence)

[0149] SEQ ID NO.21 (protein nucleotide sequence of 51 chimeric antigen)

[0150] SEQ ID NO.22 (amino acid sequence of protein of 51 chimeric antigen)

[0151] SEQ ID NO.23 (5'UTR)

[0152] SEQ ID NO.24 (3'UTR)

[0153] SEQ ID NO.25 (Poly A)

Claims

1. An mRNA comprising a nucleotide sequence encoding a chimeric immunogenic polypeptide, wherein: The chimeric immunogenic polypeptide comprises an immunogenic fragment of hemagglutinin HA of influenza A type H5N1, an immunogenic fragment of hemagglutinin HA of influenza A type H1N1 and an immunogenic fragment of hemagglutinin HA of influenza B type Victoria linked together.

2. The mRNA of claim 1, wherein The immunogenic fragments contained in the chimeric immunogenic polypeptide are linked together from N-terminus to C-terminus in an order selected from any one of the following: (1) immunogenic fragments of hemagglutinin HA of H5N1, immunogenic fragments of hemagglutinin HA of H1N1, and immunogenic fragments of hemagglutinin HA of influenza B Victoria; (2) immunogenic fragments of hemagglutinin HA of H1N1, immunogenic fragments of hemagglutinin HA of H5N1, and immunogenic fragments of hemagglutinin HA of influenza B Victoria; (3) an immunogenic fragment of the hemagglutinin HA of influenza B Victoria, an immunogenic fragment of the hemagglutinin HA of H5N1, or an immunogenic fragment of the hemagglutinin HA of H1N1; or (4) An immunogenic fragment of the hemagglutinin HA of influenza B Victoria, an immunogenic fragment of the hemagglutinin HA of H1N1, and an immunogenic fragment of the hemagglutinin HA of H5N1.

3. The mRNA according to claim 1 or 2, wherein The immunogenic fragments are directly connected or connected through a linker. Preferably, the immunogenic fragment of the hemagglutinin HA of H5N1 and the immunogenic fragment of the hemagglutinin HA of H1N1 are directly linked by a covalent bond; and The immunogenic fragment of the hemagglutinin HA of influenza type B Victoria is connected to the immunogenic fragment of the hemagglutinin HA of influenza type A via a linker; Preferably, the linker is a peptide linker; More preferably, the linker is -((G) n S) m -A linker of the structure shown, wherein n is selected from 1, 2, 3, 4, 5 or 6, and m is selected from 1, 2, 3, 4, 5 or 6; Further preferably, the linker is a peptide linker having an amino acid sequence shown in SEQ ID NO:

8.

4. The mRNA according to any one of claims 1 to 3, wherein The immunogenic fragment of the hemagglutinin HA of influenza A H5N1 is the head of HA, and the head of HA preferably comprises the amino acid sequence as shown in SEQ ID NO:4 or comprises an amino acid sequence having at least 80%, 90%, 95%, 99% sequence identity with SEQ ID NO:4; Optionally, the immunogenic fragment of the hemagglutinin HA of influenza A H1N1 is the neck of HA, and the neck of HA preferably comprises the amino acid sequence as shown in SEQ ID NO: 6 or comprises an amino acid sequence having at least 80%, 90%, 95%, 99% sequence identity with SEQ ID NO: 6; Optionally, the immunogenic fragment of the hemagglutinin HA of influenza type B Victoria comprises the amino acid sequence as shown in SEQ ID NO: 10 or comprises an amino acid sequence having at least 80%, 90%, 95%, 99% sequence identity with SEQ ID NO:

10.

5. The mRNA according to any one of claims 1 to 4, wherein The chimeric immunogenic polypeptide comprises the head of the hemagglutinin HA of influenza A type H5N1, the neck of the hemagglutinin HA of influenza A type H1N1 and the full-length hemagglutinin HA of influenza B type Victoria linked together; Preferably, the immunogenic polypeptide comprises the amino acid sequence as shown in SEQ ID NO:12 or comprises an amino acid sequence having at least 80%, 90%, 95%, 99% sequence identity with SEQ ID NO:

12.

6. The mRNA according to any one of claims 1 to 5, wherein The chimeric immunogenic polypeptide further comprises an N-terminal leader sequence, which preferably comprises an amino acid sequence as shown in SEQ ID NO:2 or encoded by SEQ ID NO:1, or comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity with the sequence as shown in SEQ ID NO:2 or encoded by SEQ ID NO:1, and is capable of promoting the correct folding of the chimeric immunogenic polypeptide.

7. The mRNA according to any one of claims 1 to 6, comprising the nucleotide sequence as shown in SEQ ID NO: 11 or comprising a nucleotide sequence having at least 80%, 90%, 95%, 99% sequence identity with SEQ ID NO:

11.

8. The mRNA according to any one of claims 1 to 7, further comprising regulatory elements such as 5'UTR, 3'UTR, poly A tail operably linked to the coding nucleotide sequence, Preferably, the 5'UTR comprises a sequence as shown in SEQ ID NO: 23 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 23, the 3'UTR comprises a sequence as shown in SEQ ID NO: 24 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 24, and the poly A tail comprises a sequence as shown in SEQ ID NO: 25 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

25. 9 . The mRNA according to claim 1 , further comprising a 5′ cap, wherein the 5′ cap is preferably m7G(5′)ppp(5′)(2′-OMeA)pG.

10. The mRNA according to any one of claims 1 to 9, further comprising a chemical modification, wherein the chemical modification is preferably modification of all or part of the uridine nucleotides in the nucleotide sequence to 1-methylpseudouridine.

11. A composition comprising the mRNA of any one of claims 1 to 10.

12. The composition of claim 11, further comprising: mRNA encoding immunogenic fragments of hemagglutinin HA of other influenza A virus subtypes, preferably encoding immunogenic fragments of hemagglutinin HA of H3N2 and / or H7N9 subtypes; and / or mRNA encoding an immunogenic fragment of hemagglutinin HA of other influenza B virus subtypes, preferably an immunogenic fragment of hemagglutinin HA of B / Yamagata subtype.

13. The composition of claim 11 or 12, further comprising: mRNA encoding an immunogenic fragment of influenza virus matrix protein M1, influenza virus ion channel protein M2, and / or influenza virus nucleoprotein NP.

14. A composition as claimed in any one of claims 11 to 13, comprising: A first mRNA selected from any one of claims 1 to 10, A second mRNA encoding a second immunogenic polypeptide, wherein the second immunogenic polypeptide comprises a fragment of H3N2 hemagglutinin HA. immunogenic fragments; A third mRNA encoding a third immunogenic polypeptide, wherein the third immunogenic polypeptide comprises an immunogenic fragment of hemagglutinin HA of H7N9; and A fourth mRNA encoding a fourth immunogenic polypeptide comprising an immunogenic fragment of hemagglutinin HA of B / Yamagata.

15. The composition of claim 14, wherein The content ratio of the first mRNA, the second mRNA, the third mRNA and the fourth mRNA is in the range of (2-5):(2-5):(2-5):(0.5-5) by mass, preferably 5:5:2:0.5 or 5:5:2:2.5 or 5:2:2:0.5 or 5:5:5:

5.

16. The composition of claim 14, further comprising: A fifth mRNA encoding a fifth immunogenic polypeptide, wherein the fifth immunogenic polypeptide comprises an immunogenic fragment of influenza virus ion channel protein M2.

17. The composition of claim 16, wherein The content ratio of the first mRNA, the second mRNA, the third mRNA, the fourth mRNA and the fifth mRNA is in the range of (2-5):(2-5):(2-5):(0.5-5):(2-5) by mass, and can be preferably selected from 5:5:5:5:5, 5:5:2:2:2, 5:2:2:2:2, 5:2:2:0.5:0.5, 5:5:2:0.5:2.5 or 5:2:2:0.5:

3.

18. The composition of any one of claims 11 to 17, wherein the hemagglutinin antigen is recommended or selected according to standardized criteria used by the World Health Organization's Global Influenza Surveillance and Response System (GISRS).

19. The composition according to any one of claims 11 to 18, wherein The immunogenic fragment sequence of the hemagglutinin HA of H3N2 comprises the amino acid sequence shown in SEQ ID NO:14 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:14, the immunogenic fragment sequence of the hemagglutinin HA of H7N9 comprises the amino acid sequence shown in SEQ ID NO:16 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:16, the immunogenic fragment sequence of the hemagglutinin HA of B / Yamagata comprises the amino acid sequence shown in SEQ ID NO:18 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:18, and / or the immunogenic fragment sequence of the influenza virus ion channel protein M2 comprises the amino acid sequence shown in SEQ ID NO:20 or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

20.

20. The composition of any one of claims 11 to 19, wherein The first mRNA comprises the nucleotide sequence as shown in SEQ ID NO: 11, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 11; The second mRNA comprises the nucleotide sequence as shown in SEQ ID NO: 13, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 13; The third mRNA comprises the nucleotide sequence as shown in SEQ ID NO: 15, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 15; The fourth mRNA comprises the nucleotide sequence as shown in SEQ ID NO: 17, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 17; and / or The fifth mRNA comprises the nucleotide sequence shown in SEQ ID NO:19, or comprises a sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO:

19.

21. The composition according to any one of claims 11 to 20, wherein The mRNA also contains regulatory elements such as 5'UTR, 3'UTR, poly A tail, etc. that are operably connected to the coding polynucleotide sequence. Preferably, the 5'UTR comprises a sequence as shown in SEQ ID NO:23 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:23, the 3'UTR comprises a sequence as shown in SEQ ID NO:24 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:24, and the poly A tail comprises a sequence as shown in SEQ ID NO:25 or a sequence having at least 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:

25.

22. The composition of any one of claims 11 to 21, further comprising a 5' cap, preferably m7G(5')ppp(5')(2'-OMeA)pG.

23. The composition of any one of claims 11 to 22, wherein The mRNA further comprises a chemical modification, and the chemical modification is preferably modification of all or part of the uridine nucleotides to 1-methylpseudouridine.

24. The composition of any one of claims 11 to 23, further comprising a pharmaceutically acceptable carrier, preferably a lipid nanoparticle, wherein the mRNA is encapsulated in one or more lipid nanoparticles.

25. The composition of any one of claims 11 to 24, wherein The individual particles in the lipid nanoparticles are encapsulated with the first to fourth mRNAs in substantially the same ratio, or the individual particles are respectively encapsulated with the first to fourth mRNAs in different ratios, or the individual particles are respectively encapsulated with any one of the first, second, third or fourth mRNAs.

26. The composition of any one of claims 11 to 25, wherein The individual particles in the lipid nanoparticles are encapsulated with the first to fifth mRNAs in substantially the same ratio, or the individual particles are respectively encapsulated with the first to fifth mRNAs in different ratios, or the individual particles are respectively encapsulated with any one of the first, second, third, fourth or fifth mRNAs.

27. The composition of any one of claims 11 to 26, wherein The lipid nanoparticles contain 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 preferably (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 preferably (40-55): (5-15): (30-50): (1-3).

28. The composition according to claim 27, wherein The phospholipids are selected from one or more of the following compounds: dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DUPC), palmitoyl oleoyl phosphatidylcholine (POPC), 1,2-di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterol dimethylsuccinate ... Choline (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-divinyl-sn-glycero-3-phosphocholine, 1,2-diaryl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-divinyl alcohol-sn-glycero-3-phosphoethanolamine, 1,2-divinyl-sn-glycero-3-phosphoethanolamine, 1,2-diaryl-sn-glycero-3-phosphoethanolamine, 1,2-dithiohexaenoic acid-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-(1-glycerol) sodium salt (DOPG), or sphingomyelin, preferably DSPC; The structural lipid is selected from one or more of cholesterol, coprostanol, 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.

29. The composition of any one of claims 11 to 28 which is a vaccine.

30. A nucleic acid molecule encoding the mRNA according to any one of claims 1 to 10, wherein the nucleic acid molecule is preferably DNA, preferably a DNA plasmid.

31. A fusion protein for preventing or treating influenza virus infection, comprising an amino acid sequence encoded by the mRNA according to any one of claims 1 to 10.

32. A composition for preventing or treating influenza virus infection, comprising the fusion protein according to claim 31.

33. A method of inducing an immune response to influenza virus in a subject, comprising administering to the subject an effective dose of the mRNA of any one of claims 1 to 10, the composition of any one of claims 11 to 29 or 32, the nucleic acid molecule of claim 30, or the fusion protein of claim 31; Preferably, the method comprises administering to the subject two or three times.

34. Use of the mRNA according to any one of claims 1 to 10, the composition according to any one of claims 11 to 29 or 32, the nucleic acid molecule according to claim 30 or the fusion protein according to claim 31 in the preparation of a medicament for preventing or treating influenza virus infection.

35. Use of the mRNA of any one of claims 1 to 10, the composition of any one of claims 11 to 29 or 32, the nucleic acid molecule of claim 30 or the fusion protein of claim 31 in preventing or treating influenza virus infection.

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