Compositions comprising mRNA encoding influenza virus HA protein and uses thereof

Abstract

The present disclosure provides compositions comprising mRNA encoding an influenza virus HA protein and uses thereof. The present invention relates to multivalent vaccines and compositions against different influenza viruses. The present disclosure provides immunogenic compositions comprising one or more influenza virus surface hemagglutinin proteins (HA proteins) or mRNA encoding the same, and uses thereof for the prevention or treatment of diseases or conditions associated with influenza virus infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and vaccine technology, and in particular to an mRNA composition encoding influenza virus HA protein and an application thereof. Background Art

[0003] Vaccines are an effective means of preventing and controlling the spread of viruses. Since seasonal influenza viruses are highly variable and uncertain, the viruses contained in the corresponding vaccines must be updated regularly to optimize the immune effect of the vaccine. Compared with traditional vaccines, mRNA vaccines have a short development cycle and can be quickly updated and iterated for personalized treatment. The combined immunization strategy provides a new idea for preventing seasonal influenza infection, reducing the number of vaccine injections, and reducing adverse immune reactions. Summary of the invention

[0004] The present invention provides an mRNA vaccine targeting different seasonal influenza viruses at the same time, which can produce immunity to multiple influenza viruses after one vaccination, and the immunity to multiple influenza viruses is further enhanced after two vaccinations. The quadrivalent combined mRNA vaccine of the present invention can provide multiple protections against various popular human influenza viruses (influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Victoria lineage, and influenza B virus Yamagata lineage) at the same time.

[0005] Influenza virus particles are spherical and consist of an outer membrane and a nucleocapsid surrounded by it. There are two glycoprotein protrusions on the outer surface of the outer membrane: hemagglutinin protrusions (HA) and neuraminidase protrusions (NA). When the influenza virus enters the host cell, the function of the HA protein is to connect with the sialic acid on the cell surface, so that the virus adheres to the host surface and enters the host body through endocytosis. Under the induction of low pH, it eventually achieves the transition from the pre-fusion state to the post-fusion state, causing the fusion of the virus and the host cell membrane. After the new virus is propagated, the NA protein is responsible for releasing the new virus from the host cell, making it easier for the virus to adhere to the host cell membrane.

[0006] Seasonal influenza viruses are divided into three types: A, B, and C. Type A influenza HA and NA are prone to mutation, resulting in changes in immunogenicity, causing global pandemics many times, so they are divided into multiple subtypes. Currently, type A influenza HA can be divided into 18 subtypes, numbered H1 to H18, and NA can be divided into 11 subtypes, numbered N1 to N11. Type B influenza HA and NA have no subtypes, and currently mainly include Yamagata or Victoria strains. Type C influenza viruses rarely cause epidemics.

[0007] In a first aspect, the present invention provides an immunogenic composition comprising mRNA encoding the hemagglutinin protein (HA protein) of one or more influenza virus strains.

[0008] In some embodiments, the HA protein of the one or more influenza virus strains is selected from influenza A virus HA protein, influenza B virus HA protein or a combination thereof. In a preferred embodiment, the HA protein of the one or more influenza virus strains comprises influenza A virus HA protein and influenza B virus HA protein.

[0009] In some embodiments, the HA protein of the one or more influenza virus strains is selected from one or more of the following (1)-(6):

[0010] (1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0011] (2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus;

[0012] (3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0013] (4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage);

[0014] (5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus; and

[0015] (6) HA protein of type A / Massachusetts / 18 / 2022 (H3N2) virus.

[0016] In a preferred embodiment, the HA protein of the one or more influenza virus strains comprises:

[0017] (1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0018] (2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus;

[0019] (3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); and

[0020] (4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage).

[0021] In a preferred embodiment, the HA protein of the one or more influenza virus strains comprises:

[0022] (1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0023] (2) A / Darwin / 6 / 2021(H3N2)-like virus HA protein; and

[0024] (3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage).

[0025] In a preferred embodiment, the HA protein of the one or more influenza virus strains comprises:

[0026] (3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0027] (4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage);

[0028] (5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus; and

[0029] (6) HA protein of type A / Massachusetts / 18 / 2022 (H3N2) virus.

[0030] In a preferred embodiment, the HA protein of the one or more influenza virus strains comprises:

[0031] 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0032] 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus; and

[0033] 6) Type A / Massachusetts / 18 / 2022 (H3N2) virus HA protein.

[0034] In some embodiments, the immunogenic composition further comprises mRNA encoding neuraminidase-NA protein of one or more influenza virus strains. In some embodiments, the NA protein of the one or more influenza virus strains is selected from influenza A virus NA protein, influenza B virus NA protein or a combination thereof. In a preferred embodiment, the NA protein of the one or more influenza virus strains comprises influenza A virus NA protein and influenza B virus NA protein. In some embodiments, the NA protein of the one or more influenza virus strains is selected from: class B / Austria / 1359417 / 2021 virus (B / Victoria lineage) NA protein.

[0035] In some embodiments, the immunogenic composition comprises HA protein and NA protein, wherein the HA protein of the one or more influenza virus strains comprises HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6):

[0036] 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0037] 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus;

[0038] 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0039] 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage);

[0040] 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus;

[0041] 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus;

[0042] The NA protein includes one selected from: B / Austria / 1359417 / 2021-like virus (B / Victoria lineage) NA protein.

[0043] In some embodiments, the HA protein of the one or more influenza virus strains is selected from one or more of the amino acid sequences of SEQ ID NOs: 1-6; preferably four HA proteins having the amino acid sequences shown in SEQ ID NOs: 1-4, or three HA proteins having the amino acid sequences shown in SEQ ID NOs: 1-3, or four HA proteins having the amino acid sequences shown in SEQ ID NOs: 3-6, or three HA proteins having the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6.

[0044] In some preferred embodiments, the immunogenic composition further comprises the NA protein of an influenza virus strain, wherein the amino acid sequence of the NA protein of the influenza virus strain is shown in SEQ ID NO:7.

[0045] In some embodiments, each of the mRNAs comprises, from 5' to 3' direction, a cap structure, a 5'-UTR, an open reading frame (ORF), a 3'-UTR, and a polyA tail.

[0046] In some embodiments, the cap structure may have m 7 G5'ppp5'(2'-OMe)NpG, where m 7 G is N7-methylguanosine; p is phosphate; ppp is triphosphate; 2'-OMe is 2'-methoxy modification; N is any nucleoside, such as adenine nucleoside (A), guanosine nucleoside (G), cytidine nucleoside (C) and uridine nucleoside (U), or other naturally occurring nucleosides or modified nucleosides.

[0047] In some embodiments, the 5'-UTR may comprise a 5'-UTR of a gene selected from the group consisting of a β-globin (HBB) gene, a heat shock protein 70 (Hsp70) gene, a axial filament motor protein heavy chain 2 (DNAH2) gene, a 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene. For example, the variant sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 5'-UTR sequence of the corresponding gene. In some embodiments, the 5'-UTR comprises a 5'-UTR derived from a 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene, or a homolog, fragment or variant thereof. In some embodiments, the 5'-UTR comprises a sequence as shown in SEQ ID NO: 15.

[0048] In some embodiments, the 3'-UTR may comprise a 3'-UTR of a gene selected from the group consisting of an albumin (ALB) gene, an alpha-globin gene, a beta-globin (HBB) gene, a tyrosine hydroxylase gene, a heat shock protein 70 (Hsp70) gene, a lipoxygenase gene, and a collagen alpha gene. For example, the variant sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the wild-type 3'-UTR sequence of the corresponding gene. In some embodiments, the 3'-UTR comprises a 3'-UTR derived from an albumin (ALB) gene, or a homolog, fragment, or variant thereof. Preferably, the 3'-UTR comprises a sequence as shown in SEQ ID NO: 16.

[0049] In some embodiments, the length of the poly A tail can be 100-200 nucleotides, such as about 100 nucleotides, about 110 nucleotides, about 120 nucleotides, about 130 nucleotides, about 140 nucleotides, about 150 nucleotides, about 160 nucleotides, about 170 nucleotides, about 180 nucleotides, about 190 nucleotides, or about 200 nucleotides. In some embodiments, the length of the poly A tail can be 100-150 nucleotides. In some embodiments, the length of the poly A tail can be about 120 nucleotides.

[0050] In some embodiments, the mRNA encoding the HA protein of the one or more influenza virus strains is selected from one or more of SEQ ID NOs: 8-13, preferably four mRNAs having sequences as shown in SEQ ID NOs: 8-11, or three mRNAs having sequences as shown in SEQ ID NOs: 8-10, or four mRNAs having sequences as shown in SEQ ID NOs: 10-13, or three mRNAs having sequences as shown in SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13.

[0051] In some embodiments, the immunogenic composition comprises mRNA of the NA protein of an influenza virus strain, the sequence of which is selected from SEQ ID NO:14.

[0052] In some embodiments, one or more nucleotides in the mRNA can be modified. For example, one or more nucleotides (e.g., all nucleotides) in the mRNA can be independently replaced with naturally occurring nucleotide analogs or artificially synthesized nucleotide analogs.

[0053] In some embodiments, the naturally occurring nucleotide analogs are selected from pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine and N6-methyladenosine. In some embodiments, the artificially synthesized nucleotide analogs are selected from N1-methylpseudouridine and 5-ethynyluridine.

[0054] In some embodiments, one or more uridine triphosphates in the mRNA are each independently replaced with pseudouridine triphosphate, 2-thiouridine triphosphate, 5-methyluridine triphosphate, N1-methylpseudouridine triphosphate, or 5-ethynyluridine triphosphate, and / or one or more cytidine triphosphates are replaced with 5-methylcytidine triphosphate, and / or one or more adenosine triphosphates are replaced with N6-methyladenosine triphosphate.

[0055] In some embodiments, one or more uridine triphosphates in the mRNA are each independently replaced with pseudouridine triphosphate, 1-methyl-pseudouridine triphosphate, or 5-ethynyluridine triphosphate, and / or one or more cytidine triphosphates are replaced with 5-methylcytidine triphosphate.

[0056] In some embodiments, mRNA is encapsulated in a carrier material. In some embodiments, the carrier material can be selected from protamine, lipid nanoparticles (LNP), polymer materials and inorganic nanoparticles. In a preferred embodiment, the carrier material is LNP. In some embodiments, LNP can include one or more of ionized lipids, PEGylated lipids, cholesterol and derivatives and phospholipids. For example, LNP can include any one of ionized lipids, PEGylated lipids, cholesterol and derivatives and phospholipids, any two, any three or all four combinations. In a preferred embodiment, LNP includes 8-(3-hydroxypropyl) (9,12-dienyl-octadecyl-1)-amino-octanoic acid heptadecan-9-ol ester, distearoyl phosphatidylcholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) and cholesterol.

[0057] In some embodiments, the molar concentration of each mRNA in the composition is no more than 3 times, 2.5 times, 2 times or 1.5 times the molar concentration of any other mRNA, or the molar concentration of each mRNA in the composition is substantially the same. Preferably, the molar concentration of each mRNA is substantially the same.

[0058] In some embodiments, the immunogenic composition comprises mRNA encoding HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6):

[0059] 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0060] 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus;

[0061] 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0062] 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage);

[0063] 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus;

[0064] 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus;

[0065] Preferably, when the immunogenic composition comprises four mRNAs, the molar ratio of the mRNAs is 1-3:1-3:1-3:1-3, for example, 1-2.5:1-2.5:1-2.5:1-2.5, 1-2:1-2:1-2:1-2, 1-1.5:1-1.5:1-1.5:1-1.5, preferably 1:1:1:1.

[0066] In some embodiments, the immunogenic composition comprises mRNA encoding HA proteins selected from the following 1)-4), or 1)-3), or 3)-6), or 3), 5), and 6), and encoding NA proteins 7):

[0067] 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus;

[0068] 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus;

[0069] 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0070] 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage);

[0071] 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus;

[0072] 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus;

[0073] 7) NA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage);

[0074] Preferably, when the immunogenic composition comprises four mRNAs, the molar ratio of the mRNAs is 1-3:1-3:1-3:1-3, for example, 1-2.5:1-2.5:1-2.5:1-2.5, 1-2:1-2:1-2:1-2, 1-1.5:1-1.5:1-1.5:1-1.5, preferably 1:1:1:1.

[0075] In some embodiments, the composition may be a vaccine composition, optionally further comprising one or more adjuvants.

[0076] As used herein, the term "vaccine composition" refers to a biological preparation that induces or improves immunity to a specific disease. Stimulating the immune system of an individual with a vaccine composition induces the formation and / or proliferation of immune cells that specifically recognize the compounds contained in the vaccine. At least a portion of the immune cells remain viable for a period of time, which can be extended to 10, 20 or 30 years after vaccination. If the individual's immune system encounters a pathogen from which a compound capable of eliciting an immune response is derived within the aforementioned time period, the immune cells generated by vaccination are reactivated and the immune response to the pathogen is enhanced compared to the immune response of an individual who has not yet been stimulated with a vaccine and has encountered the immunogenic compound of the pathogen for the first time.

[0077] As used herein, "vaccination", "immunization", "immunization" or "vaccination" refers to the administration of a vaccine to a subject with the purpose of preventing the subject from developing one or more symptoms of a disease. In principle, vaccination includes a primary vaccination and optionally one or more booster vaccinations. Primary vaccination or primary immunization is defined as a plan of initial administration of a composition or unit dose disclosed herein to establish a protective immune response. Booster vaccination or booster immunization refers to an administration or plan of administration after the primary vaccination, for example, at least one week, at least 2 weeks, at least one month, at least 6 months, at least 1 year or even 5 or 10 years after the last administration of the primary vaccination plan. Booster administration attempts to enhance or rebuild the immune response of the primary vaccination.

[0078] The immune response to the composition or vaccine composition of the present invention is the formation of a humoral and / or cellular immune response in a subject to the antigenic protein present in the composition. For the purposes of the present invention, a "humoral immune response" refers to an immune response mediated by antibody molecules, including secretory (IgA) or IgG molecules, while a "cellular immune response" refers to an immune response mediated by T-lymphocytes and / or other leukocytes. An important aspect of cellular immunity involves the antigen-specific response of cytolytic T cells ("CTL"). CTLs are specific for peptide antigens presented in combination with proteins encoded by the major histocompatibility complex (MHC) and expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms, or the lysis of cells infected by such microorganisms. Another aspect of cellular immunity involves the antigen-specific response of helper T cells. Helper T cells act to help stimulate function and focus the activity of nonspecific effector cells against cells that display peptide antigens bound to MHC molecules on their surfaces. The cellular immune response also involves the production of cytokines, chemokines and other such molecules produced by activated T cells and / or other leukocytes, including those derived from CD4+ and CD8+ T cells.

[0079] Therefore, the immune response can be a response that stimulates CTL, and / or helper T cell generation or activation. The generation of chemokines and / or cytokines can also be stimulated. The composition or vaccine composition of the present invention can also trigger an antibody-mediated immune response. Therefore, the immune response can include one or more of the following effects: antibody (e.g., IgA or IgG) generation of B cells; and / or activation of inhibitors, cytotoxic or helper T cells and / or T cells specific for proteins present in the vaccine. These responses can be used to neutralize infectivity, and / or mediate antibody-complement, or antibody-dependent cellular cytotoxicity (ADCC) to provide protection to immunized individuals. Such responses can be measured using standard immunoassays and neutralization assays known in the art.

[0080] As used herein, the term "adjuvant" refers to an agent that increases, stimulates, activates, strengthens or regulates the immune response to the active ingredient of the composition at the cellular level or the body fluid level, for example, an immune adjuvant stimulates the immune system to respond to the actual antigen but does not have an immune effect itself. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS and INF-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes or biodegradable microspheres), viral particles, bacterial adjuvants (e.g., monophosphoryl lipid A or cell wall peptides), synthetic adjuvants (e.g., nonionic block copolymers, cell wall peptide analogs or synthetic lipid A) or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine). Preferably, the adjuvant is selected from aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, alum), MF59, AS03, virosomes (e.g., hepatitis virus virosomes and influenza virus virosomes), AS04, thermoreversible oil-in-water emulsions, ISA51, Freund's adjuvant, IL-12, CpG motifs, mannose, or any combination thereof.

[0081] In a preferred embodiment, the adjuvant is selected from aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, alum), MF59, AS03, AS04, thermoreversible oil-in-water emulsions, ISA51, Freund's adjuvant, IL-12, CpG motifs and mannose.

[0082] In some embodiments, the compositions of the present invention further comprise one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0083] As used herein, the term "pharmaceutically acceptable" refers to a carrier, excipient or diluent that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0084] Exemplary carriers for compositions of the present invention include saline, buffered saline, glucose and water. Exemplary excipients for compositions of the present invention include fillers, adhesives, disintegrants, coating agents, adsorbents, antiadhesives, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers and tension regulators. It is known to those skilled in the art that suitable excipients are selected to prepare compositions of the present invention. Typically, the selection of suitable excipients depends especially on the desired dosage form of the active agent used, the disease to be treated and the compositions.

[0085] Depending on the active agent (e.g., mRNA) employed, the compositions of the present disclosure may be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or fluid extracts, or in a form particularly suitable for the desired method of administration. The processes known in the invention for producing drugs are shown in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.

[0086] In some embodiments, the composition further comprises one or more additional therapeutic agents. For example, the therapeutic agent can be selected from another antigenic protein or polypeptide, an antibody, a hormone or hormone analog, and a small molecule drug.

[0087] In a second aspect, the present invention provides a method for preventing and / or treating a disease or condition associated with influenza virus infection in a subject, comprising administering to the subject an effective amount of the immunogenic composition of the first aspect of the present invention.

[0088] As used herein, the terms "prevent" or "treat" refer to the reduction of the risk of acquiring or developing a disease or condition, i.e., preventing at least one of the clinical symptoms of a disease from developing in a subject susceptible to the disease, or not exposed to the pathogen prior to the onset of the disease. For example, treatment may include: (i) preventing the disease, disorder, and / or condition in a patient who may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed with the disease, disorder, and / or condition; (ii) inhibiting the disease, disorder, and / or condition, i.e., preventing its development; or (iii) alleviating the disease, disorder, and / or condition, i.e., causing regression of the disease, disorder, and / or condition.

[0089] As used herein, the term "effective amount" means an amount of a compound that, when administered to a subject for treatment or prevention of a disease, is sufficient to achieve such treatment or prevention. The "effective amount" may vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. A "therapeutically effective amount" refers to an effective amount for therapeutic treatment. A "prophylactically effective amount" refers to an effective amount for prophylactic treatment.

[0090] The term "administering" as used herein refers to the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0091] As used herein, the terms "subject", "individual", and "patient" are well known in the art and are used interchangeably herein to refer to any subject, particularly a mammalian subject, in need of treatment. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species. The terms individual, subject, and patient do not themselves denote a particular age, sex, race, etc.

[0092] In an embodiment of the method of the present invention, the disease or condition is a disease or condition caused by one or more infections of influenza A virus and influenza B virus. In some embodiments, the disease or condition is a disease or condition caused by one or more of the following: influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Victoria lineage, influenza B virus Yamagata lineage. In a preferred embodiment, the disease or condition is a disease or condition caused by one or more of the following: A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, B / Austria / 1359417 / 2021-like virus (B / Victoria lineage), B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage), A / Victoria / 2570 / 2019(H1N1) virus, A / Cambodia / E0826360 / 2021(H3N2) virus, A / California / 04 / 2009(H1N1) virus, B / Florida / 4 / 2006 virus.

[0093] In some embodiments, the method comprises administering the immunogenic composition of the first aspect of the invention to the subject using a prime-boost vaccination regimen. "A prime-boost vaccination regimen refers to repeated administration of an immunogenic composition or vaccine composition of the invention directed against a specific virus or group of viruses. In one embodiment, a prime-boost vaccination regimen involves at least two administrations of an immunogenic composition or vaccine composition directed against a specific virus or group of viruses. The first administration of a vaccine is referred to as "primary immunization," and any subsequent administrations of the same vaccine or a vaccine directed against the same virus or group of viruses as the first vaccine are referred to as "boost immunizations." Therefore, in a further embodiment of the invention, a prime-boost vaccination regimen involves one administration of a vaccine for eliciting an immune response and at least one subsequent administration for boosting the immune response. It should be understood that the present invention also encompasses 2, 3, 4 or even 5 administrations for boosting the immune response.

[0094] The time period between the primary immunization and the booster immunization is optionally 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks. More specifically, it is 4 weeks or 8 weeks. If more than one boost is implemented, subsequent boost is applied 1 week, 2 weeks, 4 weeks, 6 weeks or 8 weeks after the previous boost. For example, the time interval between any two boosts is 4 weeks or 8 weeks.

[0095] In some embodiments, the immunogenic compositions of the invention are administered via intranasal, intramuscular, subcutaneous, intradermal, intragastric, oral, or topical routes.

[0096] "Intranasal administration" is the administration of the vector of the present invention to the mucous membranes of the entire respiratory tract, including the lungs. More specifically, the composition is administered to the nasal mucosa. In one embodiment, intranasal administration is achieved by instillation, spraying or aerosol. In another embodiment, the administration does not involve perforation of the mucosa by mechanical means (such as a needle).

[0097] The term "intramuscular administration" refers to the injection of the vector into any muscle of a subject. Exemplary intramuscular injections are administration into the deltoid, vastus lateralis, ventrogluteal, and dorsogluteal regions.

[0098] The term "subcutaneous administration" refers to injection of the vector into the subcutaneous tissue.

[0099] The term "intradermal administration" refers to injection of the vector into the dermis between the skin layers.

[0100] The term "oral administration" refers to administration of a vector to the gastric system via the mouth.

[0101] "Topical application" is the application of the vector to any part of the skin without penetrating the skin with a needle or equivalent device. The vector can also be applied topically to the mucous membranes of the mouth, nose, genital area and rectum.

[0102] In some embodiments, the immune response is elicited by intranasal administration and the immune response is boosted by at least one intramuscular administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one subcutaneous administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one intradermal administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one intragastric administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one oral administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one topical administration; the immune response is elicited by intranasal administration and the immune response is boosted by at least one intranasal administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one intramuscular administration intramuscular administration and boosted; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one subcutaneous administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one intradermal administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one intragastric administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one oral administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one topical administration; the immune response is elicited by intramuscular administration and the immune response is boosted by at least one intranasal administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one intramuscular administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one subcutaneous administration administration boost; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one intradermal administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one intragastric administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one oral administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one topical administration; the immune response is elicited by subcutaneous administration and the immune response is boosted by at least one intranasal administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one intramuscular administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one subcutaneous administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one intradermal administration boost; the immune response is elicited by intradermal administration and the immune response is boosted by at least one intragastric administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one oral administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one topical administration; the immune response is elicited by intradermal administration and the immune response is boosted by at least one intranasal administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one intramuscular administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one subcutaneous administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one intradermal administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one intragastric administration;The immune response is elicited by intragastric administration and the immune response is boosted by at least one oral administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one topical administration; the immune response is elicited by intragastric administration and the immune response is boosted by at least one intranasal administration; the immune response is elicited by oral administration and the immune response is boosted by at least one intramuscular administration; the immune response is elicited by oral administration and the immune response is boosted by at least one subcutaneous administration; the immune response is elicited by oral administration and the immune response is boosted by at least one intradermal administration; the immune response is elicited by oral administration and the immune response is boosted by at least one intragastric administration; the immune response is elicited by oral administration and the immune response is boosted by at least one oral administration; the immune response is elicited by oral administration and the immune response is boosted by at least one oral administration The immune response is triggered by at least one local administration and is strengthened by at least one intranasal administration; the immune response is triggered by oral administration and is strengthened by at least one intranasal administration; the immune response is triggered by local administration and is strengthened by at least one intramuscular administration; the immune response is triggered by local administration and is strengthened by at least one subcutaneous administration; the immune response is triggered by local administration and is strengthened by at least one intradermal administration; the immune response is triggered by local administration and is strengthened by at least one intragastric administration; the immune response is triggered by local administration and is strengthened by at least one oral administration; the immune response is triggered by local administration and is strengthened by at least one local administration; the immune response is triggered by local administration and is strengthened by at least one intranasal administration. In one embodiment, the immune response is triggered by intranasal administration and is strengthened by at least one intramuscular administration. In another embodiment, the immune response is triggered by intranasal administration and is strengthened by at least one intranasal administration. In yet another embodiment, the immune response is triggered by intramuscular administration and is strengthened by at least one intramuscular administration. ;

[0103] In a third aspect, the present invention provides use of the immunogenic composition of the first aspect of the present invention in the preparation of a medicament for preventing and / or treating a disease or condition associated with influenza virus infection in a subject.

[0104] In a fourth aspect, the present invention provides an immunogenic composition according to the first aspect of the present invention for use in preventing and / or treating a disease or condition associated with influenza virus infection in a subject.

[0105] In an embodiment of the use of the present invention, the disease or condition is a disease or condition caused by one or more infections of influenza A virus and influenza B virus. In some embodiments, the disease or condition is a disease or condition caused by one or more of the following: influenza A virus H1N1 subtype, influenza A virus H3N2 subtype, influenza B virus Victoria lineage, influenza B virus Yamagata lineage. In a preferred embodiment, the disease or condition is a disease or condition caused by one or more of the following: A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, B / Austria / 1359417 / 2021-like virus (B / Victoria lineage), B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage), A / Victoria / 2570 / 2019(H1N1) virus, A / Cambodia / E0826360 / 2021(H3N2) virus, A / California / 04 / 2009(H1N1) virus, B / Florida / 4 / 2006 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0107] Figure 1 The graph shows the results of Western blotting of HA proteins expressed by RBMRNA-01, RBMRNA-02, RBMRNA-03, and RBMRNA-04 of the present invention.

[0108] Figure 2 A graph showing the results of IgG antibody levels against HA protein in mouse sera after one dose of quadrivalent vaccine.

[0109] Figure 3 The graph shows the results of IgG antibody levels against HA protein in mouse sera after two doses of quadrivalent vaccine.

[0110] Figure 4 A graph showing the results of hemagglutination inhibition (HI) antibody levels in mouse sera after two doses of quadrivalent vaccine.

[0111] Figure 5 The graph shows the results of cytokine secretion by mouse spleen lymphocytes stimulated with different HA proteins induced by the quadrivalent vaccine of the present invention. DETAILED DESCRIPTION

[0112] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0113] Example 1 Preparation of mRNA

[0114] Based on the sequence of the HA protein of the class A / Wisconsin / 588 / 2019 (H1N1) pdm09 virus, a DNA coding sequence was designed. After adding, for example, 5'-UTR, 3'-UTR, and polyA sequences on the basis of the DNA coding sequence, the sequences were inserted into the pT7TS plasmid by homologous recombination to form a recombinant vector pT7TS-2.0, and the final recombinant plasmid was named RBMRNA-01 plasmid. The elements contained in the RBMRNA-01 plasmid include a start sequence (SEQ ID NO: 17), a T7 promoter sequence (SEQ ID NO: 18), a 5'-UTR sequence (SEQ ID NO: 15), a 3'-UTR sequence (SEQ ID NO: 16), a 3'-end polyadenylic acid (poly A) sequence, an ampicillin resistance gene promoter, and a kanamycin sulfate resistance gene. These non-coding structures are used to regulate the stability, translation efficiency, and immunogenicity of the mRNA transcribed from the RBMRNA-01 plasmid.

[0115] The coding region and polyA region of the RBMRNA-01 plasmid were sequenced, and the inserted target gene sequencing was completely consistent with the reference sequence. The RBMRNA-01 plasmid was transcribed in vitro to obtain mRNA (named RBMRNA-01), and the mRNA was translated to obtain protein (named RBMRNA-01 protein, the sequence is shown in SEQ ID NO: 1).

[0116] Referring to the above method, a plasmid targeting HA protein or NA protein of other influenza viruses is designed, and the corresponding mRNA is obtained after in vitro transcription, and the mRNA is translated to obtain the corresponding protein.

[0117] Table 1. Plasmids designed from different influenza virus strains and their encoded mRNA and proteins

[0118]

[0119]

[0120] Example 2 mRNA expression verification

[0121] Referring to the instruction manual of Lipofectamine Messager MAX (ThermoFisher Scientific), 293T cells were transfected with 5 μg of RBMRNA-01, RBMRNA-02, RBMRNA-03, and RBMRNA-04 obtained in Example 1, and cells not transfected with mRNA were used as negative controls (NC). 24 hours after transfection, the expression of HA protein was detected by western blotting. The primary antibodies used included: Influenza A H1N1 (A / California / 04 / 2009) Hemagglutinin / HA antibody (Sino Biological, Cat#11055-T62), Influenza A H3N2 Hemagglutinin / HA antibody (Sino Biological, Cat#11056-MM03), Influenza B virus (B / Florida / 4 / 2006) Hemagglutinin / HA antibody (Sino Biological, Cat#11053-T62), Influenza B Hemagglutinin / HA antibody (Sino Biological, Cat#11053-MM06), and were labeled with goat anti-rabbit-HRP secondary antibody or goat anti-mouse-HRP secondary antibody. The results are shown in Figure 2. Figure 1 As shown, the four antigen proteins were significantly expressed in the cells.

[0122] Example 3 Evaluation of IgG and HI antibody titers induced by quadrivalent mRNA vaccines

[0123] Use RBMRNA-01, RBMRNA-02, RBMRNA-03, and RBMRNA-04 in Example 1 to prepare mRNA vaccines, which are respectively called RBMRNA-01 vaccine, RBMRNA-02 vaccine, RBMRNA-03 vaccine, and RBMRNA-04 vaccine.

[0124] mRNA is encapsulated using lipid nanoparticles comprising the following components: 8-(3-hydroxypropyl)(9,12-dienyl-octadecyl-1)-amino-octanoic acid heptadecan-9-ol ester, distearoylphosphatidylcholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and cholesterol. The preparation method includes dissolving the above components in an ethanol solution, mixing the lipid ethanol solution with an mRNA aqueous solution by a microfluidic mixer to obtain lipid nanoparticles, and dialyzing, ultrafiltration and micron membrane filtration of the mixture to obtain an mRNA LNP vaccine preparation. For a specific vaccine preparation method, see patent application CN112480217A.

[0125] The above four vaccines were mixed at an mRNA molar ratio of 1:1 to prepare a quadrivalent mRNA vaccine (quadrivalent vaccine) for BALB / c mouse immunization test. SPF-level healthy BALB / c mice, 6-8 weeks old, female, were used for immunization. The experiment was randomly divided into a solvent control (PBS) group and an immunization group according to the weight balance of the mice, 5 mice / group. The immunization group was given a quadrivalent vaccine by intramuscular injection at a dose of 20 μg / mouse, and the solvent control group was given an equal volume of PBS. They were immunized twice on day 0 (D0) and day 21 (D21) for primary immunization and booster immunization. Serum was collected 14 days after the primary immunization and booster immunization, and the IgG antibody titer against influenza virus HA protein in the serum was detected by indirect ELISA, and the hemagglutination inhibition antibody titer in the serum was detected by hemagglutination inhibition (HI) test.

[0126] The antigen proteins involved in the indirect ELISA method are: Influenza AH1N1 (A / Wisconsin / 588 / 2019) / (A / Victoria / 2570 / 2019) HA protein (Sino Biological Cat#40787-V08H1), Influenza AH3N2 (A / Darwin / 6 / 2021) HA protein (Sino Biological Cat#40868-V08H), Influenza B (B / PHUKET / 3073 / 2013) HA protein (Sino Biological Cat#40498-V08B), Influenza B (B / Austria / 1359417 / 2021) HA protein (Sino Biological Cat#40862-V08H).

[0127] The specific method of the hemagglutination inhibition test is as follows:

[0128] (1) Preparation of 4HAU hemagglutinin working solution: The HA titer of the influenza virus solution used was detected by hemagglutination experiment, and the HA titer of the influenza virus solution used was made to be exactly 4 hemagglutination units by diluting with PBS.

[0129] (2) Nonspecific hemagglutination inhibition activity in mouse serum treated with receptor destroying enzyme (RDE): First, 1 volume of mouse serum was mixed with 3 volumes of RDE and incubated in a 37°C water bath for 16 h, and then incubated at 56°C for 1 h to inactivate RDE activity.

[0130] (3) HI test: Add 25 μL PBS to wells 2 to 9 of a V-shaped 96-well hemagglutination plate, add 25 μL RDE-treated serum to wells 1 and 2 of each row, and dilute the serum to be tested in 2-fold series starting from well 2 to well 9. Pipette 25 μL and discard, and the dilution is complete. Add 25 μL PBS to well 10 as a virus control well, and add 50 μL PBS to well 11 as an erythrocyte control well. Add 25 μL of 4HAU antigen to wells 1 to 10 and shake thoroughly to mix. Let stand at room temperature for at least 20 minutes. Add 25 μL 1% chicken erythrocyte suspension to wells 1 to 11 and mix thoroughly. Let stand at room temperature for 20 to 30 minutes, and then judge the erythrocyte agglutination results.

[0131] (4) Result determination: The highest serum dilution factor that completely inhibits 4HAU antigen is determined as the HI antibody titer of the serum.

[0132] The IgG antibody titers against HA protein in the serum of mice after one and two doses of quadrivalent vaccine were as follows: Figure 2 and Figure 3 The results showed that compared with the mice in the solvent control group, after one dose of quadrivalent vaccine, the IgG antibody levels against HA of influenza A virus H1N1 and H3N2, influenza B Yamagata virus and influenza B Victoria virus HA in the mouse serum were significantly increased, with an average titer of more than 4Log10 ( Figure 2 After two doses of quadrivalent vaccine, the serum HA IgG antibody levels of mice were higher than those after one dose, and the average titer reached more than 6Log10 ( Figure 3 ).

[0133] The HI antibody titer in the mouse serum after two doses of quadrivalent vaccine was as follows Figure 4 As shown. The results showed that after two doses of quadrivalent vaccine, the HI titers against A / Victoria / 2570 / 2019 (H1N1) strain and B / Austria / 1359417 / 2021 (B-Victoria) strain reached 2304 and 1408 respectively, and HI antibodies were produced against multiple H3N2 subtypes and multiple influenza B strains, including A / Cambodia / E0826360 / 2021 (H3N2), ADawin / 6 / 2021 (H3N2), A / HongKong / 2671 / 2019 (H3N2)45040, B / Washington / 02 / 2019 (B / Victoria) and B / Phuket / 3073 / 2013 (B / Yamagata). Figure 4It can be seen that even for A / HongKong / 2671 / 2019(H3N2)45040 and B / Washington / 02 / 2019(B / Victoria), the HI titer reached 1:80-1:160, and it is generally recognized in the art that a HI titer of 1:40 can produce a 50% protection effect. The above results show that the quadrivalent vaccine can induce a strong humoral immune response and stimulate a broad immune response against antigenically diverse influenza A and B viruses.

[0134] Example 4: Th1-biased cellular immune response induced by quadrivalent mRNA vaccine

[0135] The BALB / c mouse immunization test was carried out using the quadrivalent vaccine prepared in Example 3. SPF-grade healthy BALB / c mice, 6 to 8 weeks old, female, were used for immunization. The mice were randomly divided into a solvent control (PBS) group and an immunization group according to the weight balance of the mice, 3 mice / group. The immunization group was given a quadrivalent vaccine by intramuscular injection at an injection dose of 20 μg / mouse, and the solvent control group was given an equal volume of PBS, and immunized twice on the 0th day and the 21st day for primary immunization and booster immunization, respectively. 14 days after the booster immunization, the spleen of the mice was taken to isolate spleen lymphocytes, and the levels of Th1 cytokine INFγ and Th2 cytokine IL4 were detected by ELISPOT method. The specific operation of detecting INF-γ and IL-4 was carried out according to the instructions of the Mouse IFN-γ / IL-4 ELISPOT kit. No stimulant was added to the negative control wells, 10 μL of positive stimulant was added to the positive control wells (refer to the instructions of the kit), and 1 μg of Influenza A H1N1 (A / Wisconsin / 588 / 2019) / (A / Victoria / 2570 / 2019) HA protein (Sino Biological Cat#40787-V08H1), Influenza AH3N2 (A / Darwin / 6 / 2021) HA protein (Sino Biological Cat#40868-V08H), Influenza B (B / PHUKET / 3073 / 2013) HA protein (Sino Biological Cat#40498-V08B), Influenza B(B / Austria / 1359417 / 2021)HA protein (Sino Biological Cat#40862-V08H) stimulated lymphocytes. Figure 5 shown.

[0136] The results showed that after booster immunization, stimulating spleen lymphocytes with HA protein antigen again could produce specific cellular immune responses against four antigens, among which the cellular response against influenza A virus H3N2 HA protein was the strongest. In addition, the frequency of T lymphocytes secreting Th1 cytokine INF-γ was significantly higher than that of T lymphocytes secreting Th2 cytokine IL-4, indicating that the immune response induced by the quadrivalent vaccine was a Th1-biased immune response.

[0137] Example 5 Evaluation of the protective effect of quadrivalent and pentavalent vaccines against influenza virus challenge

[0138] Referring to the method of Example 3, mRNA vaccines were prepared using RBMRNA-01, RBMRNA-02, RBMRNA-03, RBMRNA-04, and RBMRNA-05 in Example 1, respectively, and were called RBMRNA-01 vaccine, RBMRNA-02 vaccine, RBMRNA-03 vaccine, RBMRNA-04, and RBMRNA-05 vaccine, respectively.

[0139] The above RBMRNA-01, RBMRNA-02, RBMRNA-03, and RBMRNA-04 were mixed at an mRNA molar ratio of 1:1 to prepare a quadrivalent vaccine; RBMRNA-01, RBMRNA-02, RBMRNA-03, RBMRNA-04, and RBMRNA-05 were mixed at an mRNA molar ratio of 1:1 to prepare a pentavalent vaccine. Both the quadrivalent and pentavalent vaccines were used in BALB / c mouse immunization tests. The cross-neutralization activity of the vaccine was evaluated by detecting neutralizing antibodies against different strains, and the virus titer in the lung tissue of mice was detected by TCID50 experiments to evaluate the protective effects of the quadrivalent and pentavalent vaccines against influenza A and B viruses.

[0140] BALB / c mice were used in the experiment. For each virus challenge protection test, a challenge control group, a quadrivalent vaccine group and a pentavalent vaccine group were set up, with 4 mice / group. The quadrivalent vaccine group and the pentavalent vaccine group were given 20μg / mouse of quadrivalent vaccine and pentavalent vaccine by intramuscular injection, respectively, and the challenge control group was given an equal volume of normal saline. Immunization was performed twice on day 0 (as the initial immunization) and day 21 (as the booster immunization). Blood was collected 2 weeks after the booster immunization, and the serum was separated to detect neutralizing antibodies. After blood collection, the challenge was carried out: the challenge control group (4 mice / group), the quadrivalent vaccine group (4 mice / group) and the pentavalent vaccine group (4 mice / group) of the influenza A virus challenge protection test were intranasally infected with A / California / 04 / 2009 (H1N1) virus, and the infection dose was 2×10 4TCID50 / animal; Influenza B virus challenge test The challenge control group (4 / group), quadrivalent vaccine group (4 / group) and pentavalent vaccine group (4 / group) were infected with B / Austria / 1359417 / 2021 virus by intranasal drip, and the infection dose was 6.5×10 5 TCID50 / mouse. All mice were dissected 4 days after infection, and the right lung tissue was taken to detect the virus titer by TCID50 test. The detection limit was 10TCID50 / mL.

[0141] The specific method for neutralizing antibody detection is as follows:

[0142] (1) Add 100 μL of MDCK cells to each well of a 96-well cell culture plate so that each well has 1×10 cells. 4 Place in a 37°C, 5% carbon dioxide incubator and culture horizontally. When the cells grow to a monolayer, perform a micro-neutralization test.

[0143] (2) Inactivate the serum in a 56°C water bath for 30 min, centrifuge at 6000 g for 3 min, and transfer the supernatant to a 1.5 mL centrifuge tube.

[0144] (3) The inactivated serum was diluted 2-fold with virus maintenance medium.

[0145] (4) Dilute the virus to the working concentration (200TCID50 / 100μL) with virus maintenance solution, add it to the diluted serum at a ratio of 1:1, mix well, and place in a 37°C incubator for 1 hour.

[0146] (5) Discard the cell culture medium from the 96-well culture plate filled with cell monolayers and wash twice with PBS.

[0147] (6) Sequentially add the neutralized mixed solution to the above monolayer cells and culture in a cell culture incubator at 37°C and 5% CO2 for 5-7 days.

[0148] (7) Observe and record the number of cytopathic lesions daily. The final experimental result is when the number of cytopathic lesions no longer changes.

[0149] (8) The highest serum dilution factor that can protect 50% of the cell wells from virus infection is used as the neutralizing antibody titer of the serum.

[0150] As shown in Table 2, virus replication was detected in the lung tissues of mice in the challenge control group 4 days after infection with A / California / 04 / 2009(H1N1) or B / Austria / 1359417 / 2021, while no virus was detected in the lung tissues of mice in the quadrivalent vaccine group and the pentavalent vaccine group. The results show that both the quadrivalent vaccine and the pentavalent vaccine can effectively protect mice from infection with A / California / 04 / 2009(H1N1) and B / Florida / 4 / 2006 viruses, and have a protective effect against influenza A and B viruses.

[0151] The neutralizing antibody results are shown in Table 3. Both the quadrivalent vaccine and the pentavalent vaccine can produce neutralizing antibodies against influenza A and B viruses. The pentavalent vaccine has stronger cross-neutralizing activity against different virus strains than the quadrivalent vaccine.

[0152] Table 2. Virus titer in right lung tissue (TCID50 / ml)

[0153]

[0155] Table 3. Neutralizing antibody titer of mixed serum (n=4)

[0156]

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An immunogenic composition comprising mRNA encoding the hemagglutinin protein (HA) of one or more influenza virus strains.

2. The immunogenic composition according to claim 1, wherein the HA protein of the one or more influenza virus strains is selected from the group consisting of influenza A virus HA protein and influenza B virus HA protein.

3. The immunogenic composition according to claim 2, wherein the HA protein of the one or more influenza virus strains is selected from one or more of the following 1)-6): 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus; 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus; 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage); 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus; and 6) Type A / Massachusetts / 18 / 2022 (H3N2) virus HA protein.

4. The immunogenic composition according to any one of claims 1 to 3, further comprising mRNA encoding the neuraminidase-NA protein of one or more influenza virus strains.

5. The immunogenic composition according to claim 4, wherein the NA protein of the one or more influenza virus strains is selected from the group consisting of influenza A virus NA protein and influenza B virus NA protein.

6. The immunogenic composition of claim 5, wherein the NA protein of the one or more influenza virus strains is selected from: NA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage).

7. The immunogenic composition according to claim 4, wherein the HA proteins of the one or more influenza virus strains include HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6): 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus; 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus; 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage); 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus; 6) Type A / Massachusetts / 18 / 2022 (H3N2) virus HA protein.

8. The immunogenic composition according to claim 6, wherein the HA proteins of the one or more influenza virus strains include HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6): 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus; 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus; 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage); 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus; 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus; The NA protein includes one selected from: B / Austria / 1359417 / 2021-like virus (B / Victoria lineage) NA protein.

9. The immunogenic composition according to claim 3, 7 or 8, wherein the HA protein of the one or more influenza virus strains is selected from one or more of the amino acid sequences such as SEQ ID NOs: 1-6; Preferably, four HA proteins having the amino acid sequences shown in SEQ ID NOs: 1-4, or three HA proteins having the amino acid sequences shown in SEQ ID NOs: 1-3, or four HA proteins having the amino acid sequences shown in SEQ ID NOs: 3-6, or three HA proteins having the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO:

6.

10. The immunogenic composition according to any one of claims 6 or 8, wherein the amino acid sequence of the NA protein of the influenza virus strain is shown in SEQ ID NO:

7.

11. The immunogenic composition according to any one of claims 1 to 10, wherein each of the mRNAs comprises a cap structure, a 5'-UTR, an open reading frame, a 3'-UTR and a polyA tail from the 5' to the 3' direction.

12. The immunogenic composition according to claim 11, wherein the 5'-UTR comprises a 5'-UTR derived from the 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) gene or a homologue, fragment or variant thereof, preferably, the 5'-UTR comprises the sequence shown in SEQ ID NO: 15; wherein the 3'-UTR comprises a 3'-UTR derived from an albumin ALB gene or a homologue, fragment or variant thereof, preferably, the 3'-UTR comprises a sequence as shown in SEQ ID NO: 16; and / or The length of the polyA tail is 100-150 nucleotides.

13. The immunogenic composition according to any one of claims 1-12, wherein the mRNA encoding the HA protein of the one or more influenza virus strains is selected from one or more of SEQ ID NOs: 8-13; Preferred are four mRNAs having sequences as shown in SEQ ID NOs: 8-11, or three mRNAs having sequences as shown in SEQ ID NOs: 8-10, or four mRNAs having sequences as shown in SEQ ID NOs: 10-13, or three mRNAs having sequences as shown in SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO:

13.

14. The immunogenic composition according to any one of claims 1 to 13, wherein the mRNA encoding the NA protein of the influenza virus strain is selected from SEQ ID NO:

14.

15. The immunogenic composition according to any one of claims 1 to 14, wherein one or more uridine triphosphates in the mRNA are each independently replaced by pseudouridine triphosphate, 2-thiouridine triphosphate, 5-methyluridine triphosphate, N1-methylpseudouridine triphosphate or 5-ethynyluridine triphosphate, and / or one or more cytidine triphosphates are replaced by 5-methylcytidine triphosphate, and / or one or more adenosine triphosphates are replaced by N6-methyladenosine triphosphate; Preferably, one or more uridine triphosphates in the mRNA are each independently replaced by pseudouridine triphosphate, 1-methyl-pseudouridine triphosphate or 5-ethynyluridine triphosphate, and / or one or more cytidine triphosphates are replaced by 5-methylcytidine triphosphate.

16. The immunogenic composition of any one of claims 1-15, wherein the mRNA is encapsulated in a carrier material.

17. The immunogenic composition according to claim 16, wherein the carrier material is selected from lipid nanoparticles-LNP, protamine, polymer materials and inorganic nanoparticles.

18. The immunogenic composition of any one of claims 1-17, wherein the molar concentration of each mRNA in the immunogenic composition is no more than 3 times the molar concentration of any other mRNA; Preferably, the molar concentration of each mRNA is substantially the same.

19. The immunogenic composition according to claim 18, wherein the immunogenic composition comprises mRNA encoding HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6): 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus; 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus; 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage); 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus; 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus; Preferably, when the immunogenic composition comprises four mRNAs, the molar ratio of the mRNAs is 1-3:1-3:1-3:1-3, preferably 1:1:1:

1.

20. The immunogenic composition according to claim 19, wherein the immunogenic composition comprises mRNA encoding HA proteins selected from the following four types 1)-4), or three types 1)-3), or four types 3)-6), or three types 3), 5), and 6), and mRNA encoding NA proteins 7): 1) HA protein of A / Wisconsin / 588 / 2019(H1N1)pdm09 virus; 2) HA protein of A / Darwin / 6 / 2021(H3N2)-like virus; 3) HA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); 4) HA protein of B / Phuket / 3073 / 2013-like virus (B / Yamagata lineage); 5) HA protein of A / Wisconsin / 67 / 2022(H1N1)pdm09 virus; 6) HA protein of A / Massachusetts / 18 / 2022(H3N2) virus; 7) NA protein of B / Austria / 1359417 / 2021-like virus (B / Victoria lineage); Preferably, when the immunogenic composition comprises four mRNAs, the molar ratio of the mRNAs is 1-3:1-3:1-3:1-3, preferably 1:1:1:

1.

21. The immunogenic composition of any one of claims 1-19, wherein the immunogenic composition further comprises an adjuvant.

22. The immunogenic composition according to claim 21, wherein the adjuvant is selected from any one or more of the following: aluminum adjuvant, MF59, AS03, AS04, thermoreversible oil-in-water emulsion, ISA51, Freund's adjuvant, IL-12, CpG motif and mannose; Preferably, the aluminum adjuvant is selected from any one or more of the following: aluminum hydroxide, aluminum phosphate, aluminum sulfate or alum.

23. Use of the immunogenic composition of any one of claims 1 to 22 in the preparation of a vaccine for preventing and / or treating diseases or conditions associated with influenza virus infection.

24. The use according to claim 23, characterized in that The disease or disorder is a disease or disorder associated with influenza A virus and / or influenza B virus infection.

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