Influenza mRNA vaccine and preparation method therefor

Abstract

The present invention relates to an influenza virus mRNA vaccine, wherein the mRNA vaccine encodes a fusion protein containing an influenza virus hemagglutinin protein (HA) or a functional fragment thereof and an Fc or a variant thereof. The mRNA vaccine can improve the immunogenicity of an influenza virus HA antigen and the titer of a functional antibody by means of expressing the fusion protein containing the influenza hemagglutinin protein (HA) and the Fc or the variant thereof. The influenza virus mRNA vaccine prepared in the present invention can induce the body to generate a high-level and long-acting immune response at a low dose, thereby enabling a reduction in the clinical administration dose.

Description

Influenza mRNA vaccine and preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an influenza virus mRNA vaccine and application. BACKGROUND

[0002] Influenza virus is one of the most prevalent viruses in the world, which can infect both humans and domestic animals. Influenza causes significant economic burden, morbidity and even mortality. After the influenza virus infects the human body, it can induce cytokine storm in severe cases, leading to infection toxicosis, and thus cause multiple complications such as shock, encephalopathy and multiple organ dysfunction.

[0003] Influenza virus belongs to Orthomyxoviridae and is a single-stranded negative-sense segmented RNA virus, which can be divided into four types, i.e. Influenza A virus, Influenza B virus, Influenza C virus and Influenza D virus. The main viruses spreading in human population are Influenza A virus and Influenza B virus.

[0004] Influenza virus has two surface glycoproteins, i.e. hemagglutinin (HA) and neuraminidase (NA). According to the differences in HA and NA sequences, influenza virus can be divided into multiple subtypes, mainly centered on Influenza A virus. Currently, 18 hemagglutinins from H1 to H18 and 11 neuraminidases from N1 to N11 have been found. When the two are combined, theoretically, 198 subtypes of Influenza A virus can be generated. Influenza B virus is divided into Victoria and Yamagata two systems according to the antigen type.

[0005] Hemagdutinin (HA) is one of the major surface antigens of influenza virus, influenza virus initiates infection by binding to sialic acid receptors on the surface of respiratory epithelial cells through hemagglutinin (HA), enters host cells through endocytosis, and the viral genome is transcribed and replicated in the nucleus, replicating a large number of new progeny viruses and infecting other cells. The influenza virus HA protein is synthesized in the form of precursor HA0, which needs to be hydrolyzed by host proteases into HAl and HA2 two subunits, and connected by disulfide bond, and the virus becomes infectious (Figure 1C). When the influenza virus infects the host, the receptor binding site of the HAl head recognizes and binds to the polysaccharide receptor on the surface of the host cell membrane, and the virus particle is wrapped in the cell membrane to form a vesicle; the action of the M2 ion channel causes the pH in the vesicle to drop, which in turn triggers the structural change of HA2, and its N-terminal fusion peptide (Fusion peptide, FP) shifts and embeds into the target cell membrane, and finally the virus membrane and the target membrane fuse, opening the replication of the virus in the cell (Figure 1B). As can be seen, the prerequisite for influenza virus HA to cause membrane fusion is that HA must be converted into a low-pH-sensitive structural form—mature HA2 produced by protease cleavage at a specific site. Only mature HA that exposes FP can cause fusion of the virus and target cell membrane under low pH conditions, and this process is accompanied by a change in the structure of HA from pre-fusion to post-fusion conformation (Figure 1D).

[0006] The most effective and economical measure to prevent influenza is vaccination. Currently, the approved influenza vaccine routes on the market include inactivated influenza vaccine IIV, attenuated live vaccine RIV, and subunit influenza vaccine LAIV. Among them, inactivated influenza vaccine and attenuated live vaccine are prepared by culturing influenza virus in chicken embryos, which may cause the risk of decreased vaccine strain matching and reduced vaccine effectiveness due to adaptive mutation of the strain during production; although subunit vaccine is highly safe and has small side effects, it needs to increase the dose or add adjuvant to improve immunogenicity. Influenza virus mutates rapidly, which can easily lead to a decrease in the effectiveness of traditional vaccines. The rapid evolution of the HA protein of influenza virus leads to the continuous emergence of new virus strains, thereby causing the adaptive immune response of the host to have only partial protection against new infection. The biggest challenge in using traditional vaccines for the treatment and prevention of influenza and other infections is the limitation of the breadth of the vaccine, that is, providing protection only against closely related subtypes. In addition, the time required to complete the current standard influenza virus vaccine production process hinders the rapid development and production of suitable vaccines in the event of a pandemic.

[0007] Deoxyribonucleic acid (DNA) vaccination is a technique for stimulating both humoral and cellular immune responses to foreign antigens such as influenza antigens. Direct injection of genetically engineered DNA (e.g., naked plasmid DNA) into a living host results in a few of its cells directly producing the antigen, thereby generating a protective immune response. However, in the case of this technique, potential problems arise, including the potential for insertional mutagenesis, which can lead to activation of oncogenes or suppression of tumor suppressor genes.

[0008] Thus, there remains a need for effective influenza vaccines that provide robust protection against influenza viruses. In particular, there remains a need for influenza vaccines that protect individuals against heterologous influenza virus strains, including future influenza virus strains of both seasonal and pandemic evolution. RNA (e.g., messenger RNA (mRNA)) can safely direct the cellular machinery of the body to produce almost any protein of interest, from native proteins to antibodies and other completely novel protein constructs that can have therapeutic activity inside and outside of cells. RNA (e.g., mRNA) vaccines can be used in a variety of settings. RNA vaccines can be used to treat and / or prevent influenza viruses belonging to various genotypes, strains, and isolates. RNA vaccines generally have superior properties in that they produce much greater antibody titers and produce responses earlier than commercially available antiviral therapeutic treatments. While mRNA vaccines are better designed to produce proper protein conformation post-translation, unlike traditional vaccines that are manufactured ex vivo and can trigger unwanted cellular responses, mRNA vaccines are presented to the cellular system in a more natural way. Furthermore, mRNA vaccines have many advantages in preventing seasonal influenza, and the current large-scale COVID-19 mRNA application has demonstrated the strong power and high safety of mRNA. And mRNA vaccines can be iterated more quickly, with the ability to rapidly respond to enable specific immune responses within the short window of time for seasonal influenza, suitable for large-scale rapid production.

[0009] The disclosed RNA vaccines can be used to induce balanced immune responses against influenza viruses, which include both cellular immunity and humoral immunity, without the risk of insertional mutagenesis. By providing mRNA influenza vaccines encoding fusion proteins of HA and Fc, the mRNA influenza vaccines can elicit broadly neutralizing influenza antibodies. This approach aims to improve the immunogenicity of HA by designing the influenza virus HA antigen. At the same time, with the advantages of flexibility and rapidity of the mRNA technology platform, timely and effective influenza vaccines are developed. SUMMARY

[0010] The disclosed embodiments provide RNA (e.g., mRNA) vaccines comprising polynucleotides encoding influenza virus antigens. The influenza virus RNA vaccines as provided herein can be used to induce a balanced immune response, which includes both cellular immunity and humoral immunity, without many of the risks associated with DNA vaccination.

[0011] The present invention relates to an influenza virus mRNA vaccine comprising mRNA encoding an influenza virus hemagglutinin (HA) protein or functional fragment, a Th cell epitope, an immunoglobulin Fc region. The vaccine is capable of boosting the immunogenicity of the influenza virus HA antigen and the titer of neutralizing antibodies by expressing a HA and Fc fusion protein.

[0012] In some specific embodiments, the present invention selects the extracellular domain of HA, Foldon motif, PADRE epitope, Fc region of hlgGl to prepare mRNA vaccine.

[0013] The term "fusion protein" as used herein refers to an artificial protein constructed by chemically or genetically fusing different proteins.

[0014] The term "homology" as used herein refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity, as determined by alignment of matching residues, are said to be homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that describes the degree of sequence match between two sequences being compared. In some embodiments, polymeric molecules are considered "homologous" to each other if they are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" must refer to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical for at least a segment of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a segment of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a segment of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if they are at least 50%, 60%, 70%, 80%, or 90% identical for at least a segment of at least 20 amino acids.

[0015] The term "Fc region of an immunoglobulin" or "Fc fragment of an antibody" refers to an important fusion tag that can be used for co-expression of various viral proteins and vaccine preparation.

[0016] The term "PADRE epitope" refers to an epitope that assists the activation of Th cells, which is a short peptide containing 13 amino acids, can bind to class II MHC molecules to stimulate the proliferation and differentiation of CD4+ Th cells, and plays an immunoregulatory role.

[0017] The term "Foldon" refers to T4 fibritin Foldon, which is a protein trimerization motif, and serves as a linker peptide to reduce the mutual influence of HA and Fc domain protein folding.

[0018] The term "neutral lipid" refers to a lipid molecule that is uncharged and non-phosphoglyceride.

[0019] The term "polyethylene glycol (PEG)-lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

[0020] The term "lipid nanoparticle" refers to a particle having at least one size in the nanometer range, which comprises at least one lipid.

[0021] The term "vaccine" refers to a composition suitable for application to animals (including humans) that induces an immune response after administration, the strength of which is sufficient to minimally help prevent, improve or cure clinical diseases caused by microbial infection.

[0022] The term "N / P" refers to the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.

[0023] It has been found that the mRNA vaccines described herein are superior in several ways to existing vaccines. First, the present mRNA vaccines are based on mRNA nucleic acid molecules that encode a fusion protein of an influenza hemagglutinin (HA) protein and an immunoglobulin Fc region. By fusion of the HA to the Fc, the immunogenicity of the HA antigen is enhanced, and by the addition of a Th helper epitope, the immune response is enhanced. The present inventors have found that by adjusting the relative positions of the HA antigen, the Th helper epitope, the Fc, and the connecting peptide in the fusion protein, a more immunogenic fusion protein, and the mRNA nucleic acid molecule encoding it, and the vaccine formulation comprising the mRNA nucleic acid molecule, are obtained. The use of LNPs enables efficient delivery of chemically modified mRNA vaccines or unmodified mRNA vaccines. In addition, the present modified LNP-formulated mRNA vaccines are superior to both conventional vaccines and unmodified LNP-formulated mRNA vaccines to a significant degree. In some embodiments, the mRNA vaccines of the present invention are at least 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or 1,000-fold superior to conventional vaccines in terms of superiority.

[0024] Despite attempts to produce functional RNA vaccines, including mRNA vaccines and self-replicating RNA vaccines, the therapeutic efficacy of these RNA vaccines has not been fully determined. Surprisingly, in accordance with various aspects of the present invention, the present inventors have discovered a class of mRNA nucleic acid molecules and vaccine formulations thereof for the prevention or treatment of influenza virus infection that result in significantly enhanced immune responses, including enhanced antigen production and higher antigen titers. These results are achieved even when significantly lower doses of mRNA are administered. The vaccine formulations of the present invention have shown significantly unexpected in vivo immune responses sufficient to determine the efficacy of functional mRNA vaccines as prophylactic and therapeutic agents.

[0025] In some aspects, the present invention relates to the surprising discovery that lipid nanoparticle (LNP) formulations significantly enhance the effectiveness of mRNA vaccines, including chemically modified mRNA vaccines and unmodified mRNA vaccines. The efficacy of mRNA vaccines formulated in LNPs was examined in vivo using several different antigens. The results presented herein demonstrate that mRNA vaccines formulated in LNPs have unexpectedly superior efficacy over other commercially available vaccines.

[0026] In addition to providing enhanced immune responses, the formulations of the present invention also produce a more rapid immune response with less dose of antigen compared to other tested vaccines. The mRNA-LNP formulations of the present invention also produce a better immune response, both quantitatively and qualitatively, compared to vaccines formulated in different carriers.

[0027] The data described herein demonstrate that the formulations of the present application produce a significant and unexpected improvement over existing antigen vaccines. In addition, the mRNA-LNP formulations of the present application are superior to other vaccines even when the dose of mRNA is lower than other vaccines.

[0028] In a first aspect, the present application provides an mRNA nucleic acid molecule encoding a fusion protein, said fusion protein consisting of an influenza hemagglutinin (HA) protein or a functional fragment thereof, a Th cell helper epitope, an immunoglobulin Fc region, wherein the influenza hemagglutinin (HA) protein or a functional fragment thereof is not directly linked to the immunoglobulin Fc region, said influenza hemagglutinin (HA) protein being a wild type or a mutant engineered HA protein, said immunoglobulin Fc region being a wild type or a mutant engineered Fc region.

[0029] In some embodiments, the mRNA encodes an influenza hemagglutinin (HA) protein or a functional fragment thereof linked to the Fc region of an immunoglobulin via a linker peptide and / or the Th cell helper epitope.

[0030] In some embodiments, the mRNA encodes a fusion protein having the structure (linking order from N- to C-terminus) of HA-Fd-Fc, HA-Fd-PADRE-Fc6, HA-MutC-Fd-PADRE-Fc6, HA-2P-Fd-PADRE-Fc6, or HA-2P-MutC-Fd-PADRE-Fc6, wherein HA is the hemagglutinin ectodomain (2P stands for double Pro mutation), Fd stands for Foldon sequence, Fc stands for Fc segment of hlgGl, Fc6 stands for hexamerized Fc, HA-MutC stands for HA antigen with mutation of protease cleavage site, and PADRE stands for CD4 cell epitope PADRE.

[0031] In some embodiments, the mRNA encodes a fusion protein having the structure (linking order from N- to C-terminus) of HA-Fd-Fc6-PADRE, HA-MutC-Fd-Fc6-PADRE, HA-2P-Fd-Fc6-PADRE, HA-2P-MutC-Fd-Fc6-PADRE, wherein HA is the hemagglutinin ectodomain (2P stands for double Pro mutation), Fd stands for Foldon sequence, Fc stands for Fc segment of hlgGl, Fc6 stands for hexamerized Fc, HA-MutC stands for HA antigen with mutation of protease cleavage site, and PADRE stands for CD4 cell epitope PADRE.

[0032] In some embodiments, the structure of the mRNA-encoded fusion protein (order of linkage from N- to C-terminus) is PADRE-HA-Fd-Fc6, PADRE-HA-MutC-Fd-Fc6, PADRE-HA-2P-Fd-Fc6, or PADRE-HA-2P-MutC-Fd-Fc6, wherein HA is a hemagglutinin ectodomain (2P represents double Pro mutations), Fd represents a Foldon sequence, Fc represents an Fc segment of hlgGl, Fc6 represents a hexamerized Fc, HA-MutC represents a protease cleavage site mutated HA antigen, and PADRE represents a CD4 cell epitope PADRE.

[0033] In some embodiments, the structure of the mRNA-encoded fusion protein (order of linkage from N- to C-terminus) is Fc6-PADRE-HA-Fd, Fc6-PADRE-HA-MutC-Fd, Fc6-PADRE-HA-2P-Fd, Fc6-PADRE-HA-2P-MutC-Fd, wherein HA is a hemagglutinin ectodomain (2P represents double Pro mutations), Fd represents a Foldon sequence, Fc represents an Fc segment of hlgGl, Fc6 represents a hexamerized Fc, HA-MutC represents a protease cleavage site mutated HA antigen, and PADRE represents a CD4 cell epitope PADRE.

[0034] The present disclosure found that the position of each component in the above-mentioned fusion protein structure can result in different immune effects of the mRNA nucleic acid molecule and vaccine formulation.

[0035] It should be appreciated that the present disclosure is not intended to be limited to a particular influenza strain, and as such, the influenza strain used as provided herein can be any influenza strain. In some embodiments, the virus is an influenza A strain, an influenza B strain, an influenza C strain, an influenza D strain, or any combination thereof. In some embodiments, the influenza A strain or the influenza B strain is associated with avian, swine, equine, canine, human, or non-human primate. In some embodiments, the antigenic polypeptide encodes a hemagglutinin protein or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or an immunogenic fragment thereof. In some embodiments, the hemagglutinin protein does not comprise a head domain. In some embodiments, the hemagglutinin protein comprises a portion of a head domain. In some embodiments, the hemagglutinin protein does not comprise a cytoplasmic domain.

[0036] In some embodiments, the source strain of the influenza hemagglutinin (HA) protein or functional fragment thereof includes, but is not limited to:

[0037] B / Phuket / 3073 / 2013 (B / Yamagata), A / Wisconsin / 588 / 2019 (H1N1), A / Wisconsin / 67 / 2022 (H1N1), A / Sydney / 5 / 2021 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), A / Darwin / 6 / 2021 (H3N2), B / Austria / 1359417 / 2021 (B / Victoria), B / Washington / 02 / 2019 (B / Victoria).

[0038] In some embodiments, the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10; more preferably, the amino acid sequence of the influenza hemagglutinin protein is set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0039] In some embodiments, the influenza hemagglutinin (HA) protein or functional fragment thereof is mutated.

[0040] In some embodiments, the basic amino acid residues of the protease cleavage site between the HA1 and HA2 subunits of the influenza hemagglutinin (HA) protein are mutated.

[0041] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to SEQ ID NO: 1, or the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof is set forth in SEQ ID NO: 1, the lysine at position 359 of the influenza hemagglutinin (HA) protein is substituted with a glycine and the arginine at position 361 is substituted with an alanine.

[0042] In some embodiments, the amino acid sequence of the influenza hemagglutinin (HA) protein is set forth in SEQ ID NO: 2.

[0043] In some embodiments, the influenza hemagglutinin (HA) protein is mutated to stabilize the HA protein in a prefusion conformation.

[0044] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with SEQ ID NO: 1, or the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof is as set forth in SEQ ID NO: 1, the leucine at position 416 and the aspartic acid at position 432 of the influenza hemagglutinin (HA) protein are substituted with proline.

[0045] In some embodiments, the amino acid sequence of the influenza hemagglutinin (HA) protein is as set forth in SEQ ID NO: 3.

[0046] In some embodiments, the influenza hemagglutinin (HA) protein comprises a combination of all the above mutation modes.

[0047] In some embodiments, the Fc region of the immunoglobulin encoded by the mRNA nucleic acid molecule is selected from the constant region of antibody IgGl, IgG2, IgG3, and / or IgG4; the antibody IgGl, IgG2, IgG3, and / or IgG4 can be of human origin.

[0048] In some embodiments, the IgGl Fc region comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with the hIgGl amino acid sequence; preferably, the IgGl Fc region amino acid sequence is as set forth in SEQ ID NO: 11.

[0049] In some embodiments, the CH3 region of the hIgGl Fc region has a proline mutation to threonine near the C-terminus to reduce the formation of non-specific disulfide bonds; further, the hIgGl Fc region has two amino acid mutations at positions Cys249Ser and Pro474Thr; preferably, the amino acid sequence of the Fc region of the immunoglobulin is as set forth in SEQ ID NO: 12.

[0050] In some embodiments, the Fc region of the immunoglobulin is hexamerized.

[0051] In some embodiments, the end of the Fc region of the hlgG1 can be further linked to an IgM μtp tail piece short peptide to facilitate the formation of hexamerization of the IgG Fc region; preferably, the amino acid sequence of the IgM μtp tail piece short peptide is shown as SEQ ID NO: 13.

[0052] In some embodiments, the Th cell helper epitope is a PADRE epitope.

[0053] In some embodiments, the amino acid sequence of the PADRE epitope can be KFVAAWTLKAA, KYVAAWTLKAA or KXVAAWTLKAA, wherein X represents any standard amino acid.

[0054] In some embodiments, the amino acid sequence of the PADRE epitope is shown as SEQ ID NO: 14.

[0055] In some embodiments, the connecting peptide is a Foldon domain of T4 bacteriophage fiber protein.

[0056] In some embodiments, the amino acid sequence of the Foldon domain of T4 bacteriophage fiber protein is shown as SEQ ID NO: 15.

[0057] In some embodiments, the fusion protein encoded by the mRNA nucleic acid is BY223, BY224, BY225, BY226, BY227, BY228, BY229, BY230, BY231.

[0058] In some embodiments, the amino acid sequence of the fusion protein encoded by the mRNA nucleic acid is shown as SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0059] The mRNA nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and / or a poly-A tail. Preferably, the mRNA nucleic acid molecule is a natural or modified RNA, wherein the modified RNA comprises modification of the RNA by substitution of natural uridine with modified uridine partially or entirely.

[0060] In some embodiments, the mRNA nucleic acid molecule is a modified RNA, wherein the modified RNA is by substitution of natural uridine with 1-methyl-pseudouridine entirely.

[0061] Specifically, the application provides an mRNA nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises an influenza hemagglutinin (HA) protein or a functional fragment thereof and an immunoglobulin Fc region, the HA protein and the immunoglobulin Fc region are wild type or are mutated, and the Fc region is connected with a sequence facilitating hexamerization of the fusion protein, and the fusion protein encoded by the mRNA nucleic acid molecule is a hexameric fusion protein formed by hexamerization of the Fc region.

[0062] Further, the sequence facilitating hexamerization of the fusion protein is an IgM μtp tail piece short peptide.

[0063] In some embodiments, the amino acid sequence of the IgM μtp tail piece short peptide is shown in SEQ ID NO: 13.

[0064] Further, the influenza hemagglutinin (HA) protein or a functional fragment thereof and the immunoglobulin Fc region are not directly connected, and preferably, are sequentially connected in the order of 5'→3' by a connecting peptide.

[0065] In some embodiments, the connecting peptide is a Foldon domain; preferably, the connecting peptide is a Foldon domain of a T4 bacteriophage fiber protein; more preferably, the amino acid sequence of the Foldon domain of the T4 bacteriophage fiber protein is shown in SEQ ID NO: 15.

[0066] In some embodiments, the connecting peptide is sequentially connected in the order of 5'→3' by a Foldon domain and a Th cell helper epitope.

[0067] Further, the Foldon domain is a Foldon domain of a T4 bacteriophage fiber protein, and the Th cell helper epitope is a PADRE epitope.

[0068] Preferably, the amino acid sequence of the Foldon domain of the T4 bacteriophage fiber protein is shown in SEQ ID NO: 15, and the amino acid sequence of the PADRE epitope is shown in SEQ ID NO: 14.

[0069] In some embodiments, the connecting peptide is a repeating sequence with GGGGS as a basic unit, or the connecting peptide is a connecting peptide sequentially connected in the order of 5'→3' by a repeating sequence with GGGGS as a basic unit and a Th cell helper epitope.

[0070] Preferably, the repeating sequence with GGGGS as a basic unit is GGGGSGGGGSGGGGS.

[0071] Preferably, the Th cell helper epitope is PADRE.

[0072] In some embodiments, the source strain of the influenza hemagglutinin (HA) protein or functional fragment thereof includes, but is not limited to, B / Phuket / 3073 / 2013 (B / Yamagata), A / Wisconsin / 588 / 2019 (H1N1), A / Wisconsin / 67 / 2022 (H1N1), A / Sydney / 5 / 2021 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), A / Darwin / 6 / 2021 (H3N2), B / Austria / 1359417 / 2021 (B / Victoria), B / Washington / 02 / 2019 (B / Victoria), A / Vietnam / 1203 / 2004 (H5N1), A / Indonesia / 05 / 2005 (H5N1), A / turkey / Turkey / 1 / 2005 (H5N1), A / Vietnam / 1194 / 2004 (H5N1), NYMC X-179A (H1N1), A / Shanghai / 2 / 2013 (H7N9), A / SG / 1 / 1957 (H2N2), A / KR / 426 / 1968 (H2N2), A / chicken / Shanghai / F / 98 (H9N2), A / Chicken / Beijing / 1 / 94 (H9N2), A / chicken / Guangxi / 55 / 2005 (H9N2), A / Jiangxi-Donghu / 346 / 13 (H10N8).

[0073] In some embodiments, the influenza hemagglutinin (HA) protein or functional fragment thereof has an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homologous to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0074] Preferably, the amino acid sequence of the influenza hemagglutinin protein is as set forth in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0075] In some embodiments, the basic amino acid residues at the protease cleavage site between the HA1 and HA2 subunits of the influenza hemagglutinin (HA) protein are mutated; or the influenza hemagglutinin (HA) protein is mutated to stabilize the HA protein in a prefusion conformation.

[0076] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homology with SEQ ID NO: 1, the lysine at position 359 of the influenza hemagglutinin (HA) protein is substituted with a glycine and the arginine at position 361 is substituted with an alanine.

[0077] Preferably, the amino acid sequence of the influenza hemagglutinin (HA) protein is as set forth in SEQ ID NO: 2.

[0078] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homology with SEQ ID NO: 1, the leucine at position 416 of the influenza hemagglutinin (HA) protein is substituted with a proline and the aspartic acid at position 432 is substituted with a proline.

[0079] Preferably, the amino acid sequence of the influenza hemagglutinin (HA) protein is as set forth in SEQ ID NO: 3.

[0080] Alternatively, the Fc region of the encoded fusion protein is selected from the constant region of an antibody IgGl, IgG2, IgG3, and / or IgG4.

[0081] Preferably, the antibody IgGl, IgG2, IgG3, and / or IgG4 is of human origin. Preferably, the IgGl Fc region is the Fc region of hIgGl.

[0082] Preferably, the Fc region of the immunoglobulin has an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homologous to the amino acid sequence of SEQ ID NO: 11.

[0083] In some embodiments, the CH3 region of the hlgGl Fc region has a proline to threonine mutation proximal to the C-terminus, and / or the hlgGl Fc region has a proline to serine mutation at position 249.

[0084] In some embodiments, the hlgGl Fc region comprises mutations L234S / L235T / G236R, and / or the hlgGl Fc region comprises mutations L234F / L235E / P331S, and / or the hlgGl Fc region comprises mutations M252Y / S254T / T256E, and / or the hlgGl Fc region comprises mutations M428L, N434S.

[0085] Preferably, the Fc region of the immunoglobulin has an amino acid sequence as set forth in SEQ ID NO: 12.

[0086] The mRNA nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region, and / or a poly-A tail.

[0087] In particular, the present application provides a fusion protein encoded by any of the above-described mRNA nucleic acid molecules.

[0088] In some embodiments, the HA of the present application is derived from strain B / Yamagata, and the fusion protein constructed therefrom is BY222, BY223, BY224, BY225, BY226, BY227, BY228, BY229, BY230, BY231, BY232, BY233, BY234, BY235, BY236, BY237, BY238, BY239, the corresponding amino acid sequences of which are set forth in the sequence listing.

[0089] In some embodiments, the HA of the present application is derived from strain B / Victoria, and the fusion protein constructed therefrom is, for example, BV001, BV002, BV003, BV004, the corresponding amino acid sequences of which are set forth in the sequence listing.

[0090] In some embodiments, the HA of the present application is derived from strain H1N1, and the fusion protein constructed therefrom is, for example, A1001, A1002, A1003, A1004, the corresponding amino acid sequences of which are set forth in the sequence listing.

[0091] In some embodiments, the HA of the present application is derived from strain H3N2, and the resulting fusion protein is, for example, A3001, A3002, A3003, A3004, and the corresponding amino acid sequence is shown in the sequence listing.

[0092] In some embodiments, the HA of the present application is derived from strain H5N1, and the resulting fusion protein is, for example, A5001, A5002, A5003, A5004, and the corresponding amino acid sequence is shown in the sequence listing.

[0093] In particular, the present application provides an engineered nucleic acid encoding any of the above-described mRNA nucleic acid molecules.

[0094] In particular, the present application provides an expression vector comprising the above-described engineered nucleic acid.

[0095] In particular, the present application also provides a host cell comprising the above-described engineered nucleic acid or expression vector.

[0096] The present application provides a vaccine comprising any of the above-described mRNA nucleic acid molecules or fusion proteins.

[0097] The present application also provides a method of preparing a vaccine, which comprises dissolving a cationic lipid, a neutral phospholipid, a steroidal lipid, a polyethylene glycol (PEG)-lipid into a solvent, and mixing the resulting solution with any of the above-described mRNA nucleic acid molecules.

[0098] The present application also provides a use, i.e., any of the above-described mRNA nucleic acid molecules, fusion proteins, engineered nucleic acids, expression vectors, host cells in the preparation of a medicament or vaccine for preventing or treating Orthomyxoviridae virus infection.

[0099] The virus is an Orthomyxoviridae virus of the genus Influenza virus; preferably, the virus is an influenza virus of type A, B, C, or D.

[0100] The present application also provides a method of inducing an immune response against an influenza virus in a subject in need thereof, which comprises administering any of the above-described vaccines to the subject in need thereof.

[0101] Preferably, the subject is a mammal or a bird.

[0102] Preferably, the subject is a human, a chicken, a duck, a goose, a pig, a horse, a dog, or a cat.

[0103] The present application also provides a fusion protein encoded by any of the above-described mRNA nucleic acid molecules.

[0104] The present application also provides an engineered nucleic acid encoding any of the above-described mRNA nucleic acid molecules.

[0105] The present application also provides an expression vector comprising the engineered nucleic acid encoding any of the above mRNA nucleic acid molecules.

[0106] The present application also provides a host cell comprising the engineered nucleic acid or the expression vector.

[0107] In another aspect of the present application, an mRNA vaccine is provided, comprising an mRNA nucleic acid molecule encoding a fusion protein, the fusion protein comprising an influenza hemagglutinin (HA) protein or a functional fragment thereof, a Th cell helper epitope, and an immunoglobulin Fc region, wherein the influenza hemagglutinin protein is a wild type or a mutant protein, and the immunoglobulin Fc region is a wild type or a mutant protein, and wherein the influenza hemagglutinin protein or the functional fragment thereof is not directly linked to the immunoglobulin Fc region.

[0108] Preferably, the mRNA encoded influenza hemagglutinin (HA) protein or the functional fragment thereof is linked to the immunoglobulin Fc region via a linker peptide and / or the Th cell helper epitope.

[0109] In some embodiments, the mRNA encoded fusion protein has a structure comprising HA-Fd-Fc, HA-Fd-PADRE-Fc6, HA-MutC-Fd-PADRE-Fc6, HA-2P-Fd-PADRE-Fc6, or HA-2P-MutC-Fd-PADRE-Fc6, wherein HA is a hemagglutinin extracellular domain (2P represents double Pro mutation), Fd represents Foldon sequence, Fc represents Fc segment of hlgG1, Fc6 represents hexamerized Fc, HA-MutC represents HA antigen with protease cleavage site mutation, and PADRE represents CD4 cell epitope PADRE.

[0110] In some embodiments, the mRNA encoded fusion protein has a structure comprising HA-Fd-Fc6-PADRE, HA-MutC-Fd-Fc6-PADRE, HA-2P-Fd-Fc6-PADRE, or HA-2P-MutC-Fd-Fc6-PADRE, wherein HA is a hemagglutinin extracellular domain (2P represents double Pro mutation), Fd represents Foldon sequence, Fc represents Fc segment of hlgG1, Fc6 represents hexamerized Fc, HA-MutC represents HA antigen with protease cleavage site mutation, and PADRE represents CD4 cell epitope PADRE.

[0111] In some embodiments, the structure of the mRNA-encoded fusion protein comprises PADRE-HA-Fd-Fc6, PADRE-HA-MutC-Fd-Fc6, PADRE-HA-2P-Fd-Fc6, or PADRE-HA-2P-MutC-Fd-Fc6, wherein HA is a hemagglutinin ectodomain (2P stands for double Pro mutations), Fd stands for Foldon sequence, Fc stands for Fc segment of hlgGl, Fc6 stands for hexamerized Fc, HA-MutC stands for HA antigen with protease cleavage site mutations, and PADRE stands for CD4 cell epitope PADRE.

[0112] In some embodiments, the structure of the mRNA-encoded fusion protein comprises Fc6-PADRE-HA-Fd, Fc6-PADRE-HA-MutC-Fd, Fc6-PADRE-HA-2P-Fd, Fc6-PADRE-HA-2P-MutC-Fd, wherein HA is a hemagglutinin ectodomain (2P stands for double Pro mutations), Fd stands for Foldon sequence, Fc stands for Fc segment of hlgGl, Fc6 stands for hexamerized Fc, HA-MutC stands for HA antigen with protease cleavage site mutations, and PADRE stands for CD4 cell epitope PADRE.

[0113] The source strains of the influenza hemagglutinin (HA) protein or functional fragment thereof include, but are not limited to:

[0114] B / Phuket / 3073 / 2013 (B / Yamagata), A / Wisconsin / 588 / 2019 (H1N1), A / Wisconsin / 67 / 2022 (H1N1), A / Sydney / 5 / 2021 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), A / Darwin / 6 / 2021 (H3N2), B / Austria / 1359417 / 2021 (B / Victoria), B / Washington / 02 / 2019 (B / Victoria).

[0115] In some embodiments, the amino acid sequence of the HA protein or functional fragment thereof has at least 75% homology, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% homology to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0116] Preferably, the amino acid sequence of the HA protein or functional fragment thereof includes, but is not limited to, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and mutant sequences of each.

[0117] In some embodiments, the influenza hemagglutinin (HA) protein or functional fragment thereof is mutagenized.

[0118] In some embodiments, the basic amino acid residues of the protease cleavage site between the HA1 and HA2 subunits of the influenza hemagglutinin (HA) protein are mutagenized.

[0119] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to SEQ ID NO: 1, or the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof is set forth in SEQ ID NO: 1, the lysine at position 359 of the influenza hemagglutinin (HA) protein is substituted with a glycine and the arginine at position 361 is substituted with an alanine.

[0120] In some embodiments, the amino acid sequence of the influenza hemagglutinin (HA) protein is set forth in SEQ ID NO: 2.

[0121] In some embodiments, the influenza hemagglutinin (HA) protein is mutagenized to stabilize the HA protein in a prefusion conformation.

[0122] In some embodiments, when the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology to SEQ ID NO: 1, or the amino acid sequence of the influenza hemagglutinin (HA) protein or functional fragment thereof is set forth in SEQ ID NO: 1, the leucine at position 416 of the influenza hemagglutinin (HA) protein is substituted with a proline and the aspartic acid at position 432 is substituted with a proline.

[0123] In some embodiments, the amino acid sequence of the influenza hemagglutinin (HA) protein is set forth in SEQ ID NO: 3.

[0124] In some embodiments, the influenza hemagglutinin (HA) protein comprises a combination of all of the above mutational modes.

[0125] In some embodiments, the immunoglobulin Fc region is selected from the constant region of antibody IgGl, IgG2, IgG3 and / or IgG4; the antibody IgGl, IgG2, IgG3 and / or IgG4 can be of human origin; preferably, the IgGl Fc region comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homologous to the hIgGl amino acid sequence.

[0126] In some embodiments, the amino acid sequence of the immunoglobulin Fc region is as set forth in SEQ ID NO: 11.

[0127] In some embodiments, the CH3 region of the hIgGl Fc region is mutated from proline to threonine near the C-terminus to reduce the formation of non-specific disulfide bonds; further, the hIgGl Fc region has two amino acid mutations at positions Cys249Ser and Pro474Thr, respectively.

[0128] In some embodiments, the amino acid sequence of the immunoglobulin Fc region is as set forth in SEQ ID NO: 12.

[0129] In some embodiments, the Fc region of the immunoglobulin is hexamerized.

[0130] In some embodiments, the Fc region of the hIgGl can further be linked to an IgM μtp tail piece short peptide to facilitate the formation of hexamerization of the IgG Fc region; preferably, the amino acid sequence of the IgM μtp tail piece short peptide is as set forth in SEQ ID NO: 13.

[0131] In some embodiments, the Th cell helper epitope is a PADRE epitope; the amino acid sequence of the PADRE epitope can be KFVAAWTLKAA, KYVAAWTLKAA or KXVAAWTLKAA, wherein X represents any standard amino acid; preferably, the amino acid sequence of the PADRE epitope is as set forth in SEQ ID NO: 14.

[0132] The connecting peptide is the Foldon domain of T4 bacteriophage fiber protein; preferably, the amino acid sequence of the Foldon domain of T4 bacteriophage fiber protein is as set forth in SEQ ID NO: 15.

[0133] In some embodiments, the fusion protein encoded by the mRNA nucleic acid is BY223, BY224, BY225, BY226, BY227, BY228, BY229, BY230, BY231.

[0134] In some embodiments, the fusion protein encoded by the mRNA nucleic acid has an amino acid sequence as set forth in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0135] The mRNA nucleic acid molecule comprised in the vaccine further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region, and / or a poly-A tail.

[0136] Preferably, the mRNA nucleic acid molecule is a natural or modified RNA, wherein the modified RNA comprises modification of the RNA by substitution of natural uridine with modified uridine, partially or entirely.

[0137] Preferably, the mRNA nucleic acid molecule is a modified RNA, wherein the modified RNA is by substitution of natural uridine with 1-methyl-pseudouridine, entirely.

[0138] Further, the vaccine further comprises a delivery vehicle.

[0139] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP), wherein the mRNA nucleic acid molecule is encapsulated in the lipid nanoparticle (LNP), and the vaccine induces strong influenza virus-specific humoral and / or T cell immune responses after immunization, and can induce production of broadly neutralizing antibodies against influenza virus.

[0140] Preferably, the lipid nanoparticle comprises a cationic lipid, a neutral phospholipid, a sterol lipid, and a polyethylene glycol (PEG)-lipid.

[0141] Preferably, the cationic lipid has the following structure:

[0142] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0143] G1and G2are each independently an unsubstituted C6-C10alkylene;

[0144] G3is an unsubstituted C1-C12alkylene;

[0145] R1and R2are each independently a C6-C24alkyl or C6-C24alkenyl;

[0146] R3 is OR5, CN, -C(=0)OR4, -OC(=0)R4, or -NR5C(=0)R4;

[0147] R4 is a C1-C12 hydrocarbyl group; and

[0148] R5 is H or a C1-C6 hydrocarbyl group.

[0149] Preferably, the cationic lipid compound has the following structure:

[0150] Preferably, the PEG-lipid is selected from one or more of 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero- methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ammonium(polyethylene glycol)] (PEG-DSPE), PEG- disteraroylglycero (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG- diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG- DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), or DMG-PEG2000, preferably DMG-PEG2000.

[0151] Preferably, the neutral phospholipid is selected from one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG), oleoyl phosphatidyl choline (POPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), preferably DSPC.

[0152] Preferably, the sterol lipid is selected from the group consisting of avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, faveol, epicholesterol, ergosterol, fucosterol, hexahydroergosterol, hydroxycholesterol, and polypeptide-modified cholesterol; one or more of lanosterol, luminsterol, algal sterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid, preferably cholesterol.

[0153] Preferably, the polyethylene glycol (PEG)-lipid is DMG-PEG2000; the neutral phospholipid is DSPC; and the sterol lipid is cholesterol.

[0154] Preferably, the lipid nanoparticle is characterized in that the molar percentage of the cationic lipid in the lipid component is 20-60%, the molar percentage of the neutral phospholipid in the lipid component is 5-25%, the molar percentage of the sterol lipid in the lipid component is 25-55%, and the molar percentage of the PEG-lipid in the lipid component is 0.1-15%.

[0155] Preferably, the mass ratio (w / w) of total lipids to mRNA in the lipid nanoparticle (LNP) is between 10-30:1.

[0156] Preferably, the lipid nanoparticle is characterized in that the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: PEG-lipid is 30-60:1-20:20-50:0.1-10.

[0157] Preferably, the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid conjugate is 40-60:10-20:30-50:1-5.

[0158] More preferably, the molar ratio of the cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid is 45:10:43:2 or 40:10:48:2.

[0159] In some embodiments, the average particle size of the nanoparticles in the influenza virus mRNA vaccine is 50-200 nm, or the nanoparticles have a net neutral charge at neutral pH, or the nanoparticles have a polydispersity of less than 0.4.

[0160] In some embodiments, the influenza virus mRNA vaccine formulation further comprises other excipients, which are one or more of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

[0161] In some embodiments, the dosage form of the mRNA vaccine is an oral formulation, a muscle injection formulation, an intravenous injection formulation, an inhalation formulation, a liquid formulation, a lyophilized powder, a nebulized inhalation, or a dry powder inhalation.

[0162] The mRNA vaccines described herein can also comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a carrier, diluent, adjuvant or adjuvant-encoding nucleotide sequence, solubilizing agent, binding agent, lubricating agent, suspending agent, transfection facilitator, and the like. The transfection facilitator includes, but is not limited to, a surfactant such as immunostimulatory complex, Freunds incomplete adjuvant, LPS analog (e.g., monophosphoryl A), muramyl peptide, benzoquinone analog, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamate (LGS)), or nanoparticles or other known transfection facilitators. The adjuvant-encoding nucleotide sequence is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-g, Lymphotoxin-a, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0163] In another aspect of the present application, a preparation method of the influenza virus mRNA vaccine is provided, wherein the preparation method is prepared by dissolving cationic lipids, neutral phospholipids, steroidal lipids and PEG-lipids into a solvent, and then mixing the nucleic acid as described above.

[0164] Preferably, the preparation method of the influenza virus mRNA vaccine is prepared by dissolving cationic lipids, neutral phospholipids, steroidal lipids and PEG-lipids into ethanol, and then mixing the diluted mRNA diluent, followed by ultrafiltration, dilution and filtration; preferably, the cationic lipids, neutral phospholipids, steroidal lipids and PEG-lipids are dissolved into ethanol, and then mixed with the diluted mRNA diluent at a certain flow rate ratio, followed by ultrafiltration, dilution and filtration; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent mixing, laminar mixing or microfluidic mixing.

[0165] Preferably, the diluent in the preparation method of the influenza virus mRNA vaccine can be acetate buffer, citrate buffer, phosphate buffer or tris buffer.

[0166] Preferably, the preparation method of the influenza virus mRNA vaccine has a buffer pH of 3-6 and a concentration of 6.25-200 mM.

[0167] Preferably, the preparation method of the influenza virus mRNA vaccine has an N / P of 2-10 when the mRNA is encapsulated by lipids, and preferably an N / P of 3-9.

[0168] Preferably, the method prepares the vaccine into an oral preparation, a muscle injection preparation, an intravenous injection preparation, an inhalation preparation, a liquid preparation, a freeze-dried powder, an aerosol inhalation or a dry powder inhalation.

[0169] The method of making the influenza virus mRNA vaccine further comprises the step of adding a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be a carrier, diluent, adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizing agent, a binding agent, a lubricating agent, a suspending agent, a transfection facilitator, and the like. The transfection facilitator includes, but is not limited to, a surfactant such as immunostimulatory complex, Freunds incomplete adjuvant, LPS analogs (e.g., monophosphoryl A), muramyl peptide, benzoquinone analogs, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamate (LGS)), or nanoparticles or other known transfection facilitators. The nucleotide sequence encoding an adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-g, Lymphotoxin-a, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2, and functional fragments thereof.

[0170] One aspect of the present application relates to the use of the mRNA nucleic acid molecule, the influenza virus mRNA vaccine in the preparation of a medicament or vaccine for preventing or treating viral infection, preferably the virus is a virus of the family Orthomyxoviridae, further, the virus belongs to the genus of the family Orthomyxoviridae, further, the virus can be influenza A virus, influenza B virus, influenza C virus, influenza D virus.

[0171] The mRNA vaccine of the present application can deliver bioactive substances by oral, inhalation or injection.

[0172] In some embodiments, the dosage form of the vaccine of the present application is oral preparation, intramuscular injection preparation, intravenous injection preparation, inhalation preparation, liquid preparation, freeze-dried powder, aerosol inhalation or dry powder inhalation.

[0173] Another aspect of the present application provides a method for inducing an immune response against influenza virus in a subject in need thereof, the method comprises administering the mRNA vaccine to a subject in need thereof, the subject is a mammal or a bird; preferably, the subject is a human, a chicken, a duck, a goose, a pig, a horse, a dog, a cat.

[0174] In some embodiments, an effective amount of an influenza mRNA vaccine is a dose equal to at least 1 / 2 of a standard of care dose of a recombinant or purified influenza protein vaccine. For example, an effective amount of an influenza mRNA vaccine can be a dose equal to at least 1 / 3, at least 1 / 4, at least 1 / 5, at least 1 / 6, at least 1 / 7, at least 1 / 8, at least 1 / 9, or at least 1 / 10 of a standard of care dose of a recombinant or purified influenza protein vaccine. In some embodiments, an effective amount of an influenza mRNA vaccine is a dose equal to at least 1 / 100, at least 1 / 500, or at least 1 / 1000 of a standard of care dose of a recombinant or purified influenza protein vaccine. In some embodiments, an effective amount of an influenza mRNA vaccine is a dose equal to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 250, 1 / 500, or 1 / 1000 of a standard of care dose of a recombinant or purified influenza protein vaccine. In some embodiments, an anti-influenza antigenic polypeptide antibody titer produced in a subject administered an effective amount of an influenza mRNA vaccine is equal to an anti-influenza antigenic polypeptide antibody titer produced in a control subject administered a standard of care dose of a recombinant or protein influenza protein vaccine or live attenuated or inactivated influenza vaccine. In some embodiments, an effective amount of an influenza mRNA vaccine is a dose equal to 1 / 2 to 1 / 1000 (e.g., 1 / 2 to 1 / 100, 1 / 10 to 1 / 1000) of a standard of care dose of a recombinant or purified influenza protein vaccine, wherein an anti-influenza antigenic polypeptide antibody titer produced in a subject is equal to an anti-influenza antigenic polypeptide antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified influenza protein vaccine or live attenuated or inactivated influenza vaccine.

[0175] In some embodiments, the effective amount of the influenza mRNA vaccine is 1 / 2 to 1 / 1000, 1 / 2 to 1 / 900, 1 / 2 to 1 / 800, 1 / 2 to 1 / 700, 1 / 2 to 1 / 600, 1 / 2 to 1 / 500, 1 / 2 to 1 / 400, 1 / 2 to 1 / 300, 1 / 2 to 1 / 200, 1 / 2 to 1 / 100, 1 / 2 to 1 / 90, 1 / 2 to 1 / 80, 1 / 2 to 1 / 70, 1 / 2 to 1 / 60, 1 / 2 to 1 / 50, 1 / 2 to 1 / 40, 1 / 2 to 1 / 30, 1 / 2 to 1 / 20, 1 / 2 to 1 / 10, 1 / 2 to 1 / 9, 1 / 2 to 1 / 8, 1 / 2 to 1 / 7, 1 / 2 to 1 / 6, 1 / 2 to 1 / 5, 1 / 2 to 1 / 4, 1 / 2 to 1 / 3, 1 / 3 to 1 / 1000, 1 / 3 to 1 / 900, 1 / 3 to 1 / 800, 1 / 3 to 1 / 700, 1 / 3 to 1 / 600, 1 / 3 to 1 / 500, 1 / 3 to 1 / 400, 1 / 3 to 1 / 300, 1 / 3 to 1 / 200, 1 / 3 to 1 / 100, 1 / 3 to 1 / 90, 1 / 3 to 1 / 80, 1 / 3 to 1 / 70, 1 / 3 to 1 / 60, 1 / 3 to 1 / 50, 1 / 3 to 1 / 40, 1 / 3 to 1 / 30, 1 / 3 to 1 / 20, 1 / 3 to 1 / 10, 1 / 3 to 1 / 9, 1 / 3 to 1 / 8, 1 / 3 to 1 / 7, 1 / 3 to 1 / 6, 1 / 3 to 1 / 5, 1 / 3 to 1 / 4, 1 / 4 to 1 / 1000, 1 / 4 to 1 / 900, 1 / 4 to 1 / 800, 1 / 4 to 1 / 700, 1 / 4 to 1 / 600, 1 / 4 to 1 / 500, 1 / 4 to 1 / 400, 1 / 4 to 1 / 400, 1 / 4 to 1 / 200, 1 / 4 to 1 / 100, 1 / 4 to 1 / 90, 1 / 4 to 1 / 80, 1 / 4 to 1 / 70, 1 / 4 to 1 / 60, 1 / 4 to 1 / 50, 1 / 4 to 1 / 40, 1 / 4 to 1 / 30, 1 / 4 to 1 / 20, 1 / 4 to 1 / 10, 1 / 4 to 1 / 9, 1 / 4 to 1 / 8, 1 / 4 to 1 / 7, 1 / 4 to 1 / 6, 1 / 4 to 1 / 5, 1 / 4 to 1 / 4, 1 / 5 to 1 / 1 / 5 to 1 / 900, 1 / 5 to 1 / 800, 1 / 5 to 1 / 700, 1 / 5 to 1 / 600, 1 / 5 to 1 / 500, 1 / 5 to 1 / 400, 1 / 5 to 1 / 300, 1 / 5 to 1 / 200, 1 / 5 to 1 / 100, 1 / 5 to 1 / 90, 1 / 5 to 1 / 80, 1 / 5 to 1 / 70, 1 / 5 to 1 / 60, 1 / 5 to 1 / 50, 1 / 5 to 1 / 40, 1 / 5 to 1 / 30, 1 / 5 to 1 / 20, 1 / 5 to 1 / 10, 1 / 5 to 1 / 9, 1 / 5 to 1 / 8, 1 / 5 to 1 / 7, 1 / 5 to 1 / 6, 1 / 6 to 1 / 1000, 1 / 6 to 1 / 900, 1 / 6 to 1 / 800, 1 / 6 to 1 / 700,1 / 6 to 1 / 600, 1 / 6 to 1 / 500, 1 / 6 to 1 / 400, 1 / 6 to 1 / 300, 1 / 6 to 1 / 200, 1 / 6 to 1 / 100, 1 / 6 to 1 / 90, 1 / 6 to 1 / 80, 1 / 6 to 1 / 70, 1 / 6 to 1 / 60, 1 / 6 to 1 / 50, 1 / 6 to 1 / 40, 1 / 6 to 1 / 30, 1 / 6 to 1 / 20, 1 / 6 to 1 / 10, 1 / 6 to 1 / 9, 1 / 6 to 1 / 8, 1 / 6 to 1 / 7, 1 / 7 to 1 / 1000, 1 / 7 to 1 / 900, 1 / 7 to 1 / 800, 1 / 7 to 1 / 700, 1 / 7 to 1 / 600, 1 / 7 to 1 / 500, 1 / 7 to 1 / 400, 1 / 7 to 1 / 300, 1 / 7 to 1 / 200, 1 / 7 to 1 / 100, 1 / 7 to 1 / 90, 1 / 7 to 1 / 80, 1 / 7 to 1 / 70, 1 / 7 to 1 / 60, 1 / 7 to 1 / 50, 1 / 7 to 1 / 40, 1 / 7 to 1 / 30, 1 / 7 to 1 / 20, 1 / 7 to 1 / 10, 1 / 7 to 1 / 9, 1 / 7 to 1 / 8, 1 / 8 to 1 / 1000, 1 / 8 to 1 / 900, 1 / 8 to 1 / 800, 1 / 8 to 1 / 700, 1 / 8 to 1 / 600, 1 / 8 to 1 / 500, 1 / 8 to 1 / 400, 1 / 8 to 1 / 300, 1 / 8 to 1 / 200, 1 / 8 to 1 / 100, 1 / 8 to 1 / 90, 1 / 8 to 1 / 80, 1 / 8 to 1 / 70, 1 / 8 to 1 / 60, 1 / 8 to 1 / 50, 1 / 8 to 1 / 40, 1 / 8 to 1 / 30, 1 / 8 to 1 / 20, 1 / 8 to 1 / 10, 1 / 8 to 1 / 9, 1 / 9 to 1 / 1000, 1 / 9 to 1 / 900, 1 / 9 to 1 / 800, 1 / 9 to 1 / 700, 1 / 9 to 1 / 600, 1 / 9 to 1 / 500, 1 / 9 to 1 / 400, 1 / 9 to 1 / 300, 1 / 9 to 1 / 200, 1 / 9 to 1 / 100, 1 / 9 to 1 / 90, 1 / 9 to 1 / 80, 1 / 9 to 1 / 70, 1 / 9 to 1 / 60, 1 / 9 to 1 / 50, 1 / 9 to 1 / 40, 1 / 9 to 1 / 30, 1 / 9 to 1 / 20, 1 / 9 to 1 / 10, 1 / 10 to 1 / 1000, 1 / 10 to 1 / 900, 1 / 10 to 1 / 800, 1 / 10 to 1 / 700, 1 / 10 to 1 / 600, 1 / 10 to 1 / 500, 1 / 10 to 1 / 400, 1 / 10 to 1 / 300, 1 / 10 to 1 / 200, 1 / 10 to 1 / 100, 1 / 10 to 1 / 90, 1 / 10 to 1 / 80, 1 / 10 to 1 / 70, 1 / 10 to 1 / 60, 1 / 10 to 1 / 50, 1 / 10 to 1 / 40, 1 / 10 to 1 / 30, 1 / 10 to 1 / 20, 1 / 20 to 1 / 1000, 1 / 20 to 1 / 900, 1 / 20 to 1 / 800, 1 / 20 to 1 / 700, 1 / 20 to 1 / 600, 1 / 20 to 1 / 500,1 / 20 to 1 / 400, 1 / 20 to 1 / 300, 1 / 20 to 1 / 200, 1 / 20 to 1 / 100, 1 / 20 to 1 / 90, 1 / 20 to 1 / 80, 1 / 20 to 1 / 70, 1 / 20 to 1 / 60, 1 / 20 to 1 / 50, 1 / 20 to 1 / 40, 1 / 20 to 1 / 30, 1 / 30 to 1 / 1000, 1 / 30 to 1 / 900, 1 / 30 to 1 / 800, 1 / 30 to 1 / 700, 1 / 30 to 1 / 600, 1 / 30 to 1 / 500, 1 / 30 to 1 / 400, 1 / 30 to 1 / 300, 1 / 30 to 1 / 200, 1 / 30 to 1 / 100, 1 / 30 to 1 / 90, 1 / 30 to 1 / 80, 1 / 30 to 1 / 70, 1 / 30 to 1 / 60, 1 / 30 to 1 / 50, 1 / 30 to 1 / 40, 1 / 40 to 1 / 1000, 1 / 40 to 1 / 900, 1 / 40 to 1 / 800, 1 / 40 to 1 / 700, 1 / 40 to 1 / 600, 1 / 40 to 1 / 500, 1 / 40 to 1 / 400, 1 / 40 to 1 / 300, 1 / 40 to 1 / 200, 1 / 40 to 1 / 100, 1 / 40 to 1 / 90, 1 / 40 to 1 / 80, 1 / 40 to 1 / 70, 1 / 40 to 1 / 60, 1 / 40 to 1 / 50, 1 / 50 to 1 / 1000, 1 / 50 to 1 / 900, 1 / 50 to 1 / 800, 1 / 50 to 1 / 700, 1 / 50 to 1 / 600, 1 / 50 to 1 / 500, 1 / 50 to 1 / 400, 1 / 50 to 1 / 300, 1 / 50 to 1 / 200, 1 / 50 to 1 / 100, 1 / 50 to 1 / 90, 1 / 50 to 1 / 80, 1 / 50 to 1 / 70, 1 / 50 to 1 / 60, 1 / 60 to 1 / 1000, 1 / 60 to 1 / 900, 1 / 60 to 1 / 800, 1 / 60 to 1 / 700, 1 / 60 to 1 / 600, 1 / 60 to 1 / 500, 1 / 60 to 1 / 400, 1 / 60 to 1 / 300, 1 / 60 to 1 / 200, 1 / 60 to 1 / 100, 1 / 60 to 1 / 90, 1 / 60 to 1 / 80, 1 / 60 to 1 / 70, 1 / 70 to 1 / 1000, 1 / 70 to 1 / 900, 1 / 70 to 1 / 800, 1 / 70 to 1 / 700, 1 / 70 to 1 / 600, 1 / 70 to 1 / 500, 1 / 70 to 1 / 400, 1 / 70 to 1 / 300, 1 / 70 to 1 / 200, 1 / 70 to 1 / 100, 1 / 70 to 1 / 90, 1 / 70 to 1 / 80, 1 / 80 to 1 / 1000, 1 / 80 to 1 / 900, 1 / 80 to 1 / 800, 1 / 80 to 1 / 700, 1 / 80 to 1 / 600, 1 / 80 to 1 / 500, 1 / 80 to 1 / 400, 1 / 80 to 1 / 300, 1 / 80 to 1 / 200, 1 / 80 to 1 / 100, 1 / 80 to 1 / 90,1 / 90 to 1 / 1000, 1 / 90 to 1 / 900, 1 / 90 to 1 / 800, 1 / 90 to 1 / 700, 1 / 90 to 1 / 600, 1 / 90 to 1 / 500, 1 / 90 to 1 / 400, 1 / 90 to 1 / 300, 1 / 90 to 1 / 200, 1 / 90 to 1 / 100, 1 / 100 to 1 / 1000, 1 / 100 to 1 / 900, 1 / 100 to 1 / 800, 1 / 100 to 1 / 700, 1 / 100 to 1 / 600, 1 / 100 to 1 / 500, 1 / 100 to 1 / 400, 1 / 100 to 1 / 300, 1 / 100 to 1 / 200, 1 / 200 to 1 / 1000, 1 / 200 to 1 / 900, 1 / 200 to 1 / 800, 1 / 200 to 1 / 700, 1 / 200 to 1 / 600, 1 / 200 to 1 / 500, 1 / 200 to 1 / 400, 1 / 200 to 1 / 300, 1 / 300 to 1 / 1000, 1 / 300 to 1 / 900, 1 / 300 to 1 / 800, 1 / 300 to 1 / 700, 1 / 300 to 1 / 600, 1 / 300 to 1 / 500, 1 / 300 to 1 / 400, 1 / 400 to 1 / 1000, 1 / 400 to 1 / 900, 1 / 400 to 1 / 800, 1 / 400 to 1 / 700, 1 / 400 to 1 / 600, 1 / 400 to 1 / 500, 1 / 500 to 1 / 1000, 1 / 500 to 1 / 900, 1 / 500 to 1 / 800, 1 / 500 to 1 / 700, 1 / 500 to 1 / 600, 1 / 600 to 1 / 1000, 1 / 600 to 1 / 900, 1 / 600 to 1 / 800, 1 / 600 to 1 / 700, 1 / 700 to 1 / 1000, 1 / 700 to 1 / 900, 1 / 700 to 1 / 800, 1 / 800 to 1 / 1000, 1 / 800 to 1 / 900, 1 / or 900 to 1 / 1000.

[0176] In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is equal to the anti-antigenic polypeptide antibody titer produced in a control subject administered a standard-of-care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated influenza vaccine. In some embodiments, the effective amount is a dose equal to (at least) 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 110, 1 / 120, 1 / 130, 1 / 140, 1 / 150, 1 / 160, 1 / 170, 1 / 1280, 1 / 190, 1 / 200, 1 / 210, 1 / 220, 1 / 230, 1 / 240, 1 / 250, 1 / 260, 1 / 270, 1 / 280, 1 / 290, 1 / 300, 1 / 310, 1 / 320, 1 / 330, 1 / 340, 1 / 350, 1 / 360, 1 / 370, 1 / 380, 1 / 390, 1 / 400, 1 / 410, 1 / 420, 1 / 430, 1 / 440, 1 / 450, 1 / 4360, 1 / 470, 1 / 480, 1 / 490, 1 / 500, 1 / 510, 1 / 520, 1 / 530, 1 / 540, 1 / 550, 1 / 560, 1 / 5760, 1 / 580, 1 / 590, 1 / 600, 1 / 610, 1 / 620, 1 / 630, 1 / 640, 1 / 650, 1 / 660, 1 / 670, 1 / 680, 1 / 690, 1 / 700, 1 / 710, 1 / 720, 1 / 730, 1 / 740, 1 / 750, 1 / 760, 1 / 770, 1 / 780, 1 / 790, 1 / 800, 1 / 810, 1 / 820, 1 / 830, 1 / 840, 1 / 850, 1 / 860, 1 / 870, 1 / 880, 1 / 890, 1 / 900, 1 / 910, 1 / 920, 1 / 930, 1 / 940, 1 / 950, 1 / 960, 1 / 970, 1 / 980, 1 / 990, or 1 / 1000 of a standard-of-care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated influenza vaccine. In some embodiments, the anti-antigenic polypeptide antibody titer produced in the subject is equal to the anti-antigenic polypeptide antibody titer produced in a control subject administered a standard-of-care dose of a recombinant or purified influenza protein vaccine or a live attenuated or inactivated influenza vaccine.

[0177] In some embodiments, an effective amount of an influenza mRNA vaccine is a total dose of 50-1000 pg. In some embodiments, an effective amount of an influenza mRNA vaccine is a total dose of 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900, 600-900, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or 900-1000 pg. In some embodiments, an effective amount of an influenza mRNA vaccine is a total dose of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 pg. In some embodiments, an effective amount is a dose of 25-500 pg administered to a subject a total of two times.In some embodiments, the effective amount of the influenza mRNA vaccine is a dose of 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400, 150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400, 250-300, 300-500, 300-400, 350-500, 350-400, 400-500, or 450-500 μg administered to the subject a total of two times. In some embodiments, the effective amount of the influenza mRNA vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg administered to the subject a total of two times.

[0178] The present application has the following advantages over the prior art:

[0179] I. The mRNA vaccine formulation of the present application has good stability. In vivo expression of mRNA can avoid contamination from foreign factors such as viruses and proteins, and has less side effects.

[0180] II. The mRNA composition and mRNA vaccine of the present application have high immunogenicity. The present application improves the immunogenicity of HA by constructing a hexamerized form of HA-Fc fusion protein, and enhances the immune response by adding Th cell epitopes. HAI experiments show that the mRNA vaccine encoding the Fc fusion protein of the extracellular domain of HA induces HAI antibody titers 1-fold higher than wild type (160:80), significantly improving the immunogenicity of HA. At very low doses and dosing regimens, high levels of neutralizing antibodies against influenza virus can be induced, and antigen-specific immune responses against the encoded protein can be very effectively induced, providing better antiviral effects than existing influenza vaccines.

[0181] III. The immune effect of the mRNA composition and mRNA vaccine of the present application has long-lasting persistence. By modifying the mRNA nucleic acid molecule and using appropriate delivery systems, the expression and in vivo half-life of the mRNA can be effectively regulated, thereby achieving high immunogenicity while maintaining long-lasting persistence of the immune effect.

[0182] Fourthly, the mRNA vaccine of the present application has rapid reaction capability. In the face of fast mutation of influenza viruses, the traditional vaccine protection effect decreases, while the rapid iteration of the mRNA vaccine can well solve this problem. In addition, the rapid reaction capability of the mRNA vaccine can realize specific immune response in the short time window of seasonal influenza, rapidly supply vaccines when pandemic influenza breaks out, and is suitable for large-scale rapid production. BRIEF DESCRIPTION OF DRAWINGS

[0183] Figure 1: Influenza virus invasion process and HA protein conformation change: (A) schematic diagram of the structure of influenza virus; (B) when influenza virus infects host, the receptor binding site of HA1 head recognizes and binds to the polysaccharide receptor on the surface of host cell membrane, and the virus particle is wrapped by the cell membrane to form a vesicle; the action of M2 ion channel makes the pH in the vesicle decrease, thereby triggering the structure change of HA2, the N-terminal fusion peptide shifts and embeds into the target cell membrane, and finally the virus membrane and the target membrane fuse, opening the replication of the virus in the cell; (C) influenza virus HA protein is synthesized in the form of precursor HA0, which is hydrolyzed by host protease into two subunits HAl and HA2, and is connected by disulfide bond, and the virus acquires infectivity; (D) low pH-induced HA pre-fusion to post-fusion conformational transition.

[0184] Figure 2: HA mRNA and the fusion protein encoded thereby: (A) HA extracellular domain-Fc fusion protein; (B) HA extracellular domain-Fc fusion protein hexamer.

[0185] Figures 3-10: results of integrity detection of part of mRNA stock solutions: BY222, BY223, BY224, BY225, BY226, BY227, BY228 and BY229 in turn;

[0186] Figures 11-18: results of integrity detection of part of mRNA-LNP intermediate products: BY222, BY223, BY224, BY225, BY226, BY227, BY228 and BY229 in turn;

[0187] Figure 19: HAI titers of 14-day and 28-day sera of mice immunized with fusion antigens;

[0188] Figure 20: HAI titers of 28-day sera of mice immunized with fusion antigens;

[0189] Figure 21: HAI titers of 28-day sera of mice immunized with fusion antigens. DETAILED DESCRIPTION

[0190] In order to better understand the present application, the beneficial effects of the present application are further illustrated by the following examples, but not as a limitation to the present application.

[0191] Example 1: HA fusion protein design

[0192] The influenza mRNA vaccines of the present application select HA as the antigen, and the sources of the HA used include but are not limited to:

[0193] B / Phuket / 3073 / 2013 (B / Yamagata), A / Wisconsin / 588 / 2019 (H1N1), A / Wisconsin / 67 / 2022 (H1N1), A / Sydney / 5 / 2021 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), A / Darwin / 6 / 2021 (H3N2), B / Austria / 1359417 / 2021 (B / Victoria), B / Washington / 02 / 2019 (B / Victoria), A / Vietnam / 1203 / 2004 (H5N1), A / Indonesia / 05 / 2005 (H5N1), A / turkey / Turkey / 1 / 2005 (H5N1), A / Vietnam / 1194 / 2004 (H5N1), NYMCX-179A (H1N1), A / Shanghai / 2 / 2013 (H7N9), A / SG / 1 / 1957 (H2N2), A / KR / 426 / 1968 (H2N2), A / chicken / Shanghai / F / 98 (H9N2), A / Chicken / Beijing / 1 / 94 (H9N2), A / chicken / Guangxi / 55 / 2005 (H9N2), A / JiangxiDonghu / 346 / 13 (H10N8).

[0194] In this scheme, the HA proteins of seasonal influenza B / Yamagata, B / Victoria, H1N1, H3N2 and pandemic influenza H5N1 strains are designed as antigens, and the specific design methods are shown in Table 1:

[0195] Table 1 Fusion antigen design

[0196] Note: HA in the table is hemagglutinin extracellular domain; 2P represents double Pro mutation; Linker 1: Fd, Fd represents Foldon sequence; Linker 2: Fd-PADRE, PADRE represents CD4 cell epitope PADRE; Linker 3: GGGGS*3; Linker 4: GGGGS*3-PADRE; Fc represents the Fc segment of hlgG1; Fc6 represents hexamerized Fc. MutC represents the protease cleavage site of mutant HA; BY232, BY233, BY234 and BY235 are functional mutations of Fc added on the basis of BY225.

[0197] Example 2: Preparation of mRNA-LNP samples

[0198] 1. Preparation of plasmid and mRNA stock solution

[0199] In this experiment, the selected HA is influenza B / Yamagata subtype, and the specific strain is B / Phuket / 3073 / 2013. First, the target gene sequences of HA, Foldon+Fc, Foldon+PADRE+Fc6 were optimized and outsourced for synthesis; then the primers for point mutation and homologous recombination (as shown in Table 2) were designed, the target band was obtained by PCR amplification, and the target gene was inserted into the vector by homologous recombination; finally, the correct plasmid containing the target gene was obtained by sanger sequencing. Take the glycerol bacteria containing the recombinant plasmid, inoculate 200 ml of LB medium at a 1% inoculation amount, culture at 37°C, 220pm for 16h, then perform plasmid extraction, and the OD600 value of the culture after incubation is in the range of 5.0-6.0, collect the bacteria, lyse, and extract the plasmid. The extracted plasmid is subjected to BspQI single enzyme digestion and purification to obtain linearized plasmid as a template for subsequent transcription experiments. Take 20ug of linearized plasmid, prepare 400ul of in vitro transcription reaction system, after transcription, DNase I digestion of the transcription template, proteinase K digestion, purification, obtain the mRNA stock solution, and use capillary electrophoresis method to detect the integrity of the mRNA, the results are shown in Table 3 and Figure 3. Among them, the mRNA containing 5' cap and 3' poly A tail is obtained by co-transcription method in this experiment.

[0200] Table 2 Primer Table

[0201] Table 3 mRNA Stock Solution Integrity

[0202] 2. mRNA-LNP preparation

[0203] The lipid mixture solution for encapsulating mRNA was configured according to Table 4, and the mRNA stock solution was encapsulated by a microfluidic method to form an mRNA-LNP encapsulation solution; the encapsulation solution was then diluted, ultrafiltered, and concentrated using a 50 mM sodium acetate buffer containing 435 mg / ml sucrose to obtain an LNP intermediate product, and the mRNA concentration and encapsulation rate of the LNP intermediate product were detected, and the results are shown in Table 5. The intermediate product was diluted to 30 ng / ul and 150 ng / ul LNP finished product using a 20 mM Tris, 10.7 mM sodium acetate, 87 mg / ml sucrose solution, and the concentration, encapsulation rate, particle size, and potential of the mRNA in the LNP finished product were measured, and the results are shown in Tables 6, 7, 8, respectively. At the same time, the integrity of the mRNA in the LNP finished product was detected again by capillary electrophoresis, and the results are shown in Table 9 and Figure 4.

[0204] Table 4 Lipid formulation

[0205] Table 5 LNP intermediate product measurement concentration and encapsulation rate

[0206] Table 6 LNP finished product concentration and encapsulation rate (30 ng / ul)

[0207] Table 7 LNP finished product concentration and encapsulation rate (150 ng / ul)

[0208] Table 8 LNP intermediate product particle size and potential

[0209] Table 9 Intermediate product integrity

[0210] Example 3: Immunogenicity detection of influenza mRNA vaccine

[0211] 1. Animal experiment grouping and immunization plan

[0212] In order to verify the immunogenicity of the immunogen designed for the HA protein of the B / Yamagata strain in this scheme, 6-8 week old female BALB / c mice were randomly grouped according to the table below, and immunized by intramuscular injection of the hind leg. BY222 (wild type HA) was used as a positive control, and 0.9% NaCl solution was used as a negative control. Two different dose groups were set for each antigen design, 3 pg and 15 pg per mouse. The immunization program was single immunization, and the blood was collected on the 14th and 28th day after immunization to detect the HAI antibody titer of the mouse serum against B / Puget Sound (BY222). The animal experiment grouping and immunization program are shown in Table 10:

[0213] Table 10 Animal grouping and immunization procedure

[0214] To verify the immunogenicity of the immunogens designed against B / Victoria, H1N1 and H3N2 strain HA proteins in this scheme, 6-8 week old female BALB / c mice were randomly grouped according to Table 11, and immunized by intramuscular injection of the hind leg. Flublok quadrivalent recombinant influenza vaccine was used as a positive control, and 0.9% NaCl solution was used as a negative control. Groups 1-14 were immunized with 1 / 10 human dose (4.5 ug / antigen / dose) of Flublok for the primary immunization, and the mRNA vaccine dose was 2 ug / dose for the secondary immunization. The positive control vaccine Flublok was used at 1 / 3 human dose (15 ug / antigen / dose). Blood samples were collected on day 28 post-immunization and HAI antibody titers in mouse sera were detected. The animal experiment grouping and immunization procedure are shown in Table 11:

[0215] Table 11 Animal grouping and immunization procedure

[0216] To verify the immunogenicity of the immunogens designed against pandemic influenza strain H5N1 HA protein in this scheme, 6-8 week old female BALB / c mice were randomly grouped according to Table 12, and immunized by intramuscular injection of the hind leg. Blood samples were collected on day 28 post-immunization and HAI antibody titers in mouse sera were detected. The animal experiment grouping and immunization procedure are shown in Table 12:

[0217] Table 12 Animal grouping and immunization procedure

[0218] 2. HAI antibody titer detection

[0219] Hemagglutination inhibition (HAI) titers, which measure neutralizing antibody responses to the variable region of the HA head, are a common method for evaluating the effectiveness of influenza vaccines. Currently, HAI titers are the only measure of protection that regulatory agencies accept as a correlate of protection for licensed seasonal influenza vaccines. In this animal experiment, two dose groups were designed for each HA antigen, 3 ug and 15 ug, respectively. Sera collected at 14 days and 28 days post-immunization were used to detect the level of antibodies induced by influenza HA antigens through HAI experiments. The HAI antibody titer data of mouse sera immunized with different designs of HA antigens are shown in Table 13:

[0220] Table 13 Summary of mouse serum HAI antibody titer data

[0221] The mouse immune serum data showed that the addition of the Fc hexamerization motif can significantly improve the immunogenicity of the HA protein; in addition, the use of linker 2 to connect HA and Fc6 can induce a better immune response; through the comparison of BY225, BY235, BY236, BY237, BY238 and BY239, the Fc6 L234A / L235A / P329G functional mutation designed in BY235 can improve the immune response of the HA protein, the Fc6 mutations (L234S / L235T / G236R and L234F / L235E / P331S) in BY236 and BY237 do not have a negative impact on the immunogenicity of the HA antigen, while the Fc6 mutations (M252Y / S254T / T256E and M428L, N434S) in BY238 and BY239 reduce the immune response of the HA protein; in addition, compared with BY225, the HAI antibody titer level stimulated by BY227 is significantly reduced, indicating that the protease cleavage site mutation reduces the immunogenicity of HA; compared with BY225, BY228 cannot stimulate the production of HAI antibodies, indicating that the double Pro mutation completely destroys the HAI antibody response of the HA protein. The above data show that the addition of the Fc hexamerization motif and the use of linker 2 can improve the immunogenicity of the HA protein. On this basis, the Fc fragment L234A / L235A / P329G functional mutation can further improve the immunogenicity of the HA protein. In addition, the introduction of the Fc6 functional mutation can remove the ADCC, ADCP and CDC functions of Fc without affecting the immunogenicity of the HA antigen, reducing the potential safety risk.

[0222] According to the above data, the HA antigens for B / Victoria, H1N1 and H3N2 seasonal influenza subtypes and pandemic influenza strain H5N1 were designed, and the universality of the design for different influenza virus strains was further verified on mice. After immunizing the mice, the serum HAI titer was detected, as shown in Table 14 and Table 15.

[0223] Table 14 Mouse serum HAI antibody titer against other subtype seasonal influenza

[0224] Table 15 Mouse serum HAI antibody titer against H5N1

[0225] The results show that the addition of Fc hexamerization motif, the use of linker 2 connection and Fc fragment L234A / L235A / P329G functional mutation can also improve the immunogenicity of HA protein for different seasonal influenza strains B / Victoria, H1N1 and H3N2 and pandemic strain H5N1, among which in seasonal influenza, the mRNA vaccine designed based on HA-Linker2-Fc6 and HA-Linker2-Fc6(L234A / L235A / P329G) induces higher HAI antibody titers at a dose of 2ug / antigen / dose than or not worse than the 15ug / antigen / dose Flublok marketed four-valent recombinant vaccine. In addition, the HA-Linker2-Fc6(L234A / L235A / P329G) design can induce the strongest immune response, and due to the functional mutation of Fc, it has better safety.

[0226] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-described embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0227] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

Claims

1. An mRNA nucleic acid molecule encoding a fusion protein, characterized in that: The fusion protein includes an influenza hemagglutinin (HA) protein or a functional fragment thereof and an immunoglobulin Fc region, wherein the HA protein and the immunoglobulin Fc region are wild type or mutated, and a sequence that promotes hexamerization of the fusion protein is connected to the end of the Fc region. The fusion protein encoded by the mRNA nucleic acid molecule is a hexameric fusion protein, and the hexameric fusion protein is formed by hexamerization of the Fc region.

2. The mRNA nucleic acid molecule according to claim 1, wherein The fragment that promotes hexamerization of the fusion protein is a short peptide of the tail piece of IgMμtp; preferably, the amino acid sequence of the short peptide of the tail piece of IgMμtp is shown in SEQ ID NO:

13.

3. The mRNA nucleic acid molecule according to claim 1, wherein The influenza hemagglutinin (HA) protein or its functional fragment and the immunoglobulin Fc region are not directly connected; preferably, they are sequentially connected in the 5'→3' direction via a connecting peptide.

4. The mRNA nucleic acid molecule according to claim 3, wherein The connecting peptide is a Foldon domain; preferably, the connecting peptide is the Foldon domain of T4 phage fiber protein; more preferably, the amino acid sequence of the Foldon domain of T4 phage fiber protein is shown in SEQ ID NO:

15.

5. The mRNA nucleic acid molecule according to claim 1, wherein The connecting peptide is a Foldon domain and a Th cell helper epitope connected sequentially in the 5'→3' direction; preferably, the Foldon domain is the Foldon domain of T4 phage fibrin, and the Th cell helper epitope is a PADRE epitope; more preferably, the amino acid sequence of the Foldon domain of T4 phage fibrin is shown in SEQ ID NO: 15, and the amino acid sequence of the PADRE epitope is shown in SEQ ID NO:

14.

6. The mRNA nucleic acid molecule according to claim 1, characterized in that The connecting peptide is a repeating sequence with GGGGS as the basic unit, or the connecting peptide is a connecting peptide formed by sequentially connecting a repeating sequence with GGGGS as the basic unit and a Th cell helper epitope; preferably, the repeating sequence with GGGGS as the basic unit is GGGGSGGGGSGGGGS.

7. The mRNA nucleic acid molecule according to claim 6, characterized in that The Th cell helper epitope is PADRE.

8. The mRNA nucleic acid molecule according to claim 1, wherein The source strains of the influenza hemagglutinin (HA) protein or its functional fragment include but are not limited to B / Phuket / 3073 / 2013 (B / Yamagata), A / Wisconsin / 588 / 2019 (H1N1), A / Wisconsin / 67 / 2022 (H1N1), A / Sydney / 5 / 2021 (H1N1), A / Cambodia / e0826360 / 2020 (H3N2), A / Darwin / 6 / 2021 (H3N2), B / Austria / 1359417 / 2021 (B / Victoria), B / Washington / 02 / 2019 (B / Victoria), A / Vietnam / 1203 / 2004 (H5N1) ,A / Indonesia / 05 / 2005(H5N1),A / turkey / Turkey / 1 / 2005(H5N1),A / Vietnam / 1194 / 2004(H5N1),NYMCX-179A(H1N1),A / Shanghai / 2 / 2013(H7N9),A / SG / 1 / 1957(H2N2), A / KR / 426 / 1968(H2N2),A / chicken / Shanghai / F / 98(H9N2),A / Chicken / Beijing / 1 / 94(H9N2),A / chicken / Guangxi / 55 / 2005(H9N2),A / Jiangxi-Donghu / 346 / 13(H10N8).

9. The mRNA nucleic acid molecule according to claim 1, wherein The amino acid sequence of the influenza hemagglutinin (HA) protein or its functional fragment is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% homologous to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; more preferably, the amino acid sequence of the influenza hemagglutinin protein is as shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

10. The mRNA nucleic acid molecule according to claim 1, wherein The basic amino acid residues of the protease cleavage site between the HA1 and HA2 subunits of the influenza hemagglutinin (HA) are mutated; or the influenza hemagglutinin (HA) protein is mutated to stabilize the HA protein in a pre-fusion conformation.

11. The mRNA nucleic acid molecule according to claim 10, characterized in that When the amino acid sequence of the influenza hemagglutinin (HA) protein or a functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homology to SEQ ID NO: 1, the lysine at position 359 of the influenza hemagglutinin (HA) protein is substituted with glycine and the arginine at position 361 is substituted with alanine; preferably, the amino acid sequence of the influenza hemagglutinin (HA) protein is as shown in SEQ ID NO:

2.

12. The mRNA nucleic acid molecule according to claim 10, characterized in that When the amino acid sequence of the influenza hemagglutinin (HA) protein or a functional fragment thereof has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homology to SEQ ID NO: 1, the leucine at position 416 of the influenza hemagglutinin (HA) protein is substituted with proline and the aspartic acid at position 432 is substituted with proline; preferably, the amino acid sequence of the influenza hemagglutinin (HA) protein is as shown in SEQ ID NO:

3.

13. The mRNA nucleic acid molecule according to claim 1, wherein The Fc region of the encoded fusion protein is selected from the constant region of antibodies IgG1, IgG2, IgG3 and / or IgG4; preferably, the antibodies IgG1, IgG2, IgG3 and / or IgG4 are of human origin; preferably, the IgG1 Fc region is the Fc region of hIgG1; preferably, the amino acid sequence of the Fc region of the immunoglobulin has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homology to the amino acid sequence of SEQ ID NO:

11.

14. The mRNA nucleic acid molecule according to claim 13, characterized in that The proline near the C-terminus of the CH3 region of the hIgG1 Fc region is mutated to threonine, and / or the proline at position 249 of the hIgG1 Fc region is mutated to serine, and / or the hIgG1 Fc region comprises the mutations L234A / L235A / P329G, and / or the hIgG1 Fc region comprises the mutations L234S, L235T and G236R, and / or the hIgG1 Fc region comprises the mutations L234F, L235E and P331S, and / or the hIgG1 Fc region comprises the mutations M252Y, S254T and T256E, and / or the hIgG1 Fc region comprises the mutations M428L and N434S; preferably, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:

12.

15. The mRNA nucleic acid molecule according to claim 1, wherein The mRNA nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and / or a polyadenylation tail.

16. A fusion protein encoded by the mRNA nucleic acid molecule according to any one of claims 1 to 15.

17. An engineered nucleic acid, characterized in that It encodes the mRNA nucleic acid molecule according to any one of claims 1 to 15.

18. An expression vector, characterized in that It comprises the engineered nucleic acid of claim 17.

19. A host cell, characterized in that The host cell comprises the engineered nucleic acid of claim 17 or the expression vector of claim 18.

20. An influenza vaccine, characterized in that The vaccine comprises the mRNA nucleic acid molecule according to any one of claims 1 to 15 or the fusion protein according to claim 16.

21. A method for preparing an influenza mRNA vaccine, characterized in that: The preparation method is prepared by dissolving cationic lipids, neutral phospholipids, steroid lipids, and polyethylene glycol (PEG)-lipids in a solvent and then mixing them with the mRNA nucleic acid molecule according to any one of claims 1 to 15.

22. Use of the mRNA nucleic acid molecule according to any one of claims 1 to 15, the fusion protein according to claim 16, the engineered nucleic acid according to claim 17, the expression vector according to claim 18, the host cell according to claim 19, or in the preparation of a drug or vaccine for preventing or treating an Orthomyxoviridae virus infection.

23. The use according to claim 22, characterized in that The virus is an influenza virus of the family Orthomyxoviridae; preferably, the virus is influenza virus A, B, C or D.

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