Recombinant influenza virus protein, vaccine and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant influenza virus protein, a vaccine and a preparation method and application thereof. In order to overcome the defects of high safety and toxic and side effects of the existing influenza vaccine, the invention provides a recombinant influenza virus protein, the amino acid sequence of which is as shown in at least one of SEQ ID No.1-SEQ ID No.6. According to the invention, H1N1 subtype, H3N2 subtype, Victoria line and Yamagata line recombinant hemagglutinin proteins are expressed and purified through an insect baculovirus expression system, and are mixed with an adjuvant to prepare a recombinant divalent, trivalent or tetravalent recombinant influenza virus protein vaccine with high immunogenicity. The prepared vaccine can induce high-level specific IgG antibodies and hemagglutination inhibition neutralizing antibodies, the immunogenicity of the vaccine is improved by adding the adjuvant WGa01, and the vaccine is suitable for egg allergic people and high in safety.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to recombinant influenza virus protein, vaccine, and preparation method and application thereof. Background Art

[0002] Influenza is an acute respiratory infection caused by the influenza virus, associated with high morbidity and mortality. The virus is highly contagious and spreads rapidly through airborne droplets, direct contact between people, and contact with contaminated objects. Flu vaccination is a key preventative measure against the flu.

[0003] Influenza viruses belong to the Orthomyxoviridae family and consist of a negative-sense, single-stranded, segmented RNA genome. Based on the viral nucleoprotein and matrix proteins, influenza viruses are divided into four types: A (A), B (B), C (C), and D (D). Most seasonal influenza is associated with influenza A and B viruses. Based on the protein structure and genetic characteristics of the viral surface hemagglutinin (HA) and neuraminidase (NA), influenza A (H1) viruses are divided into 18 HA subtypes and 11 NA subtypes; of these, the H1, H2, H3, H5, and H7 antigenic subtypes can infect humans. The HA subtypes of influenza B (H1B) viruses have differentiated into two serologically distinct lineages (Victoria and Yamagata). Influenza vaccines are typically developed against the HA protein. HA protein is the most important antigenic protein and immunogenic protein in influenza virus. On the one hand, it binds to the sialic acid receptors on the surface of host cells, allowing the virus to attach to the cells, helping the virus to penetrate the host's cell membrane, infect cells and reproduce; on the other hand, it can stimulate the body to produce corresponding humoral immunity and cellular immunity, thereby enabling the body to resist the corresponding virus.

[0004] Current influenza vaccines primarily rely on inactivated vaccines produced using embryonated chicken eggs. However, this technology has numerous drawbacks, such as insufficient embryonated chicken supply during epidemics, limited vaccine production, local or systemic allergic reactions caused by residual egg components in the vaccine, and a short duration of immune responses. Therefore, there is an urgent need for a safe and effective alternative to traditional inactivated vaccines. Unlike embryonated or cell-based influenza virus vaccines, recombinant influenza virus vaccines directly construct the antigen gene sequence into an expression vector and express it using recombinant protein technology, eliminating the need for eggs or candidate vaccine viruses during production. Currently, the only recombinant protein influenza vaccine on the market is the Flublok vaccine produced by Sanofi Pasteur in France, and this vaccine can only be used in people over 18 years of age. Therefore, there is an urgent need to develop more safe, effective, and widely applicable recombinant influenza virus vaccines. Summary of the Invention

[0005] In order to overcome the defects of existing influenza vaccines such as narrow application range, safety and severe toxic side effects, the present invention provides a recombinant influenza virus protein, vaccine and preparation method and application thereof.

[0006] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0007] In a first aspect, the present invention provides a recombinant influenza virus protein for preventing and / or treating influenza virus infection, wherein the amino acid sequence of the recombinant protein is derived from the full-length or truncated sequence of the hemagglutinin protein of an influenza virus strain.

[0008] Furthermore, the influenza virus strain is selected from at least one of H1N1, H3N2, Victoria, Yamagata lineage, H5N1, H7N9 or H5N8.

[0009] Furthermore, the recombinant protein amino acid sequence is selected from at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 6;

[0010] Or at least one of the amino acid sequences that has more than 90% homology with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6 and has the same or similar biological activity.

[0011] SEQ ID No. 1: H1N1 influenza virus HA amino acid sequence (A / Wisconsin / 588 / 2019 (H1N1) pdm09)

[0012] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACPHAGAKSFYKNLIWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMERDAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLDSTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICISEQ ID No.2: Amino acid sequence of HA of H1N1 influenza virus (A / Wisconsin / 67 / 2022 (H1N1)pdm09)

[0013] DTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGQCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSDNGVTAACSHAGARSFYKNLIWLVKKGKSYPKINQTYINDKGKEVLVLWGIHHPPTITDQESLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQAGRMNYYWTLVEPGDKITFEATGNLVAPRYAFTMEKEAGSGIIISDTPVHDCNATCQTPEGAINTSLPFQNVHPITIGKCPKYVRSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNDQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDVWTYNAELLVLLENERTLDYHDSNVKNLYEKVRHQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLDSTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICISEQ ID NO.3: HA amino acid sequence of H3N2 influenza virus (A / Darwin / 6 / 2021 (H3N2))

[0014] QKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICGSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPDTDKNQISLFAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQKGNIRCNICI

[0015] SEQ ID NO.4: Amino acid sequence of HA of H3N2 influenza virus (A / District Of Columbia / 27 / 2023(H3N2))

[0016] QKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGKICNSPHQILDGGNCTLIDALLGDPQCDGFQNKEWDLFVERSRANSSCYPYDVPDYASLRSLVASSGTLEFKDESFNWTGVKQNGTSSACKRGSSNSFFSRLNWLTSLNNIYPAQNVTMPNKEQFDKLYIWGVHHPDTDKNQFSLFAQSSGRITVSTKRSQQAVIPNIGSRPRVRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGECKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQISGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAMSCFLLCIALLGFIMWACQKGNIRCNICI

[0017] SEQ ID NO.5: Amino acid sequence of HA of B / Victoria lineage influenza virus (B / Austria / 1359417 / 2021)

[0018] DRICTGITSSNSPHVVKTATQGEVNVTGVIPLTTTPTKSHFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPIMHDRTKIRQLPNLLRGYEHVRLSTHNVINTEDAPGGPYEIGTSGSCLNITNGKGFFATMAWAVPKNKTATNPLTIEVPYICTEEEDQITVWGFHSDDETQMARLYGDSKPQKFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIVVDYMVQKSGKTGTITYQRGILLPQKVWCASGKSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWVKTPLKLANGTKYRPPAKLLKERGFFGAIAGFLEGGWEGMIAGWHGYTSHGAHGVAVAADLKSTQEAINKITKNLNSLSELEVKNLQRLSGAMDELHNEILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKKMLGPSAVEIGNGCFETKHKCNQTCLDRIAAGTFDAGEFSLPTFDSLNITAASLNDDGLDNHTILLYYSTAASSLAVTLMIAIFVVYMVSRDNVSCSICL

[0019] SEQ ID NO.6: Amino acid sequence of HA of B / Yamagata lineage influenza virus (B / Phuket / 3073 / 2013)

[0020] .

[0021] Furthermore, the nucleic acid sequence encoding the amino acid sequence of the above-mentioned recombinant protein for resisting influenza virus infection is selected from at least one of SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12.

[0022] SEQ ID NO.7: H1N1 influenza virus HA nucleic acid sequence (A / Wisconsin / 588 / 2019(H1N1)pdm09)

[0023]

[0024] SEQ ID NO.8: H1N1 influenza virus HA nucleic acid sequence (A / Wisconsin / 67 / 2022 (H1N1) pdm09)

[0025]

[0026] SEQ ID NO.9: H3N2 influenza virus HA nucleic acid sequence (A / Darwin / 6 / 2021 (H3N2))

[0027]

[0028] SEQ ID NO.10: H3N2 influenza virus HA nucleic acid sequence (A / District Of Columbia / 27 / 2023 (H3N2))

[0029]

[0030] SEQ ID NO.11: B / Victoria lineage influenza virus HA nucleic acid sequence (B / Austria / 1359417 / 2021)

[0031]

[0032] SEQ ID NO.12: B / Yamagata lineage influenza virus HA nucleic acid sequence (B / Phuket / 3073 / 2013)

[0033]

[0034] Furthermore, the recombinant influenza virus protein is prepared by introducing the coding gene of the target antigen protein into an expression vector, transferring the vector into a host cell to express the protein, and purifying the cell.

[0035] Preferably, the expression vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector or a yeast expression vector.

[0036] Preferably, the insect baculovirus expression vector is pFastBac1.

[0037] Preferably, the E. coli expression vector is pET32a.

[0038] Preferably, the yeast expression vector is pPICZαA;

[0039] Preferably, the mammalian cell expression vector is the CHO cell expression vector pTT5 or FTP-002.

[0040] Preferably, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli or yeast.

[0041] More preferably, the insect cells are selected from at least one of Sf9 cells, Sf21 cells, and Hi5 cells. Most preferably, the Sf9 cells are WSK-Sf9 insect cells, which are free of rhabdovirus contamination and have a CCTCC accession number of C202246. WSK-Sf9 insect cells were disclosed on September 12, 2023, under the patent publication number CN116731953A.

[0042] More preferably, the mammalian cells are CHO cells.

[0043] In a second aspect, the present invention provides a recombinant influenza virus protein vaccine for preventing and / or treating influenza virus infection, which comprises an antigen and pharmaceutically acceptable auxiliary components, wherein the antigen is the above-mentioned recombinant influenza virus protein.

[0044] Among them, the above-mentioned recombinant influenza virus protein used as the antigen can be a single recombinant influenza virus protein; or it can be a mixture of two or more recombinant influenza virus proteins; the protein amino acid sequence is selected from SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.

[0045] It can also be a recombinant influenza virus protein, wherein the protein amino acid sequence is a tandem combination of at least one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0046] Furthermore, the auxiliary component is an immune adjuvant.

[0047] Preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoryl lipid A (MPL), muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin (rCTB), GM-CSF cytokine, lipid, cationic liposome material, CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotide as the core sequence, artificially synthesized CpG).

[0048] Furthermore, the squalene oil-in-water emulsion is selected from at least one of WGa01, MF59, AS03, AF03, SE (Squalene Emulsion) or AddaVax (InvivoGen).

[0049] Furthermore, the WGa01 adjuvant comprises: squalene 4.3% (v / v), Tween 80 0.5% (v / v), Span 85 0.5% (v / v), sodium citrate buffer 10 mM, pH 6.5, and the balance is water.

[0050] Furthermore, the aluminum salt is selected from at least one of aluminum hydroxide and alum.

[0051] Furthermore, the calcium salt is tricalcium phosphate.

[0052] Furthermore, the plant saponin is QS-21 or ISCOM.

[0053] Furthermore, the plant polysaccharide is astragalus polysaccharide (APS).

[0054] Furthermore, the lipid is selected from at least one of the following: phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), and dioleoylphosphatidylethanolamine (DOPE).

[0055] Furthermore, the cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethyldioctadecylammonium bromide (DDAB).

[0056] Furthermore, the dosage form of the vaccine includes injection, nasal drops, spray, inhalation or oral preparation.

[0057] Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, and intradermal injection.

[0058] In a third aspect, the present invention provides a pharmaceutical composition for preventing and / or treating respiratory diseases, which contains the above-mentioned recombinant influenza virus protein or recombinant influenza virus protein vaccine, as well as other drugs for preventing and / or treating influenza caused by vaccine-related influenza strains.

[0059] In a fourth aspect, the present invention provides the use of the above-mentioned recombinant influenza virus protein, vaccine and pharmaceutical composition thereof in preventing and / or treating influenza caused by vaccine-associated influenza viruses.

[0060] In a fifth aspect, the present invention provides a method for preparing the above-mentioned recombinant influenza virus protein, comprising the following steps: constructing an expression vector containing the target gene, expressing the protein in host cells, and purifying the protein.

[0061] Furthermore, the expression vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector or a yeast expression vector.

[0062] Preferably, the expression vector is pFastBac1.

[0063] Furthermore, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli or yeast.

[0064] More preferably, the insect cells are selected from at least one of Sf9 cells, Sf21 cells, and Hi5 cells. Most preferably, the Sf9 cells are WSK-Sf9 insect cells, free of rhabdovirus contamination, and have a deposit number of CCTCC NO: C202246.

[0065] Furthermore, the nucleotide sequence of the target gene is selected from at least one of SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12.

[0066] Beneficial effects: The present invention uses genetic engineering technology to design recombinant influenza virus proteins for preventing influenza virus infection based on the hemagglutinin (HA) protein of the influenza virus strain published by the WHO as an antigen source. The amino acid sequences are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and / or SEQ ID No. 6. The recombinant HA proteins of the H1N1 subtype, H3N2 subtype, Victoria strain and Yamagata strain are expressed and purified respectively through an expression system, preferably an insect baculovirus expression system. After mixing any two or more proteins, they are mixed with an adjuvant to prepare a recombinant bivalent, trivalent or quadrivalent recombinant influenza virus protein vaccine with high purity of the vaccine active ingredient.

[0067] Animal experiments have shown that the vaccine of the present invention has the following advantages:

[0068] 1. The recombinant influenza virus protein vaccine prepared by the present invention can induce high levels of specific IgG antibodies and hemagglutination inhibition (HI) neutralizing antibodies in mice and rats. The addition of adjuvant improves the immunogenicity of the vaccine and can induce balanced humoral immunity and cellular immunity.

[0069] 2. Since the HA gene sequence from the vaccine strain is directly expressed, there is no adaptive mutation and the vaccine is highly stable.

[0070] 3. The vaccine of the present invention does not contain egg protein, antibiotics or preservatives, will not cause allergies in the body, is highly safe, is also suitable for people who are allergic to eggs, and has a wide range of applications.

[0071] 4. Compared with recombinant influenza vaccines without adjuvants, the vaccine of the present invention can reduce the amount of antigen used while maintaining effectiveness and can reduce adverse reactions.

[0072] 5. The vaccine of the present invention has a short production cycle of only 2 to 3 months, does not rely on chicken embryos, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 This is a diagram illustrating the composition of the recombinant HA protein of influenza virus in Example 1;

[0074] Figure 2This is the restriction enzyme digestion map of the HA expression vector plasmid represented by the H3N2 subtype in Example 1;

[0075] Figure 3 This is the PCR image of the HA gene represented by the H3N2 subtype in Example 1;

[0076] Figure 4 This is a diagram showing the preparation results of the H1N1 subtype influenza virus HA protein (corresponding to SEQ ID No. 1) in Example 1;

[0077] Figure 5 This is a diagram showing the preparation results of the H3N2 subtype influenza virus HA protein (corresponding to SEQ ID No. 3) in Example 1;

[0078] Figure 6 This is a diagram showing the preparation results of the B / Victoria subtype influenza virus HA protein (corresponding to SEQ ID No. 5) in Example 1;

[0079] Figure 7 This is a diagram showing the preparation results of the B / Yamagata subtype influenza virus HA protein (corresponding to SEQ ID No. 6) in Example 1;

[0080] Figure 8 This is a graph showing the changes in specific IgG antibodies in mice at 14, 35, and 98 days after inoculation with different doses of the recombinant trivalent influenza virus protein vaccine in Experimental Example 1;

[0081] Figure 9 This is a graph showing changes in hemagglutination inhibition neutralizing antibody titers of the recombinant trivalent influenza virus protein vaccine against three influenza strains at 35 days and 98 days in Experimental Example 1;

[0082] Figure 10 This is a graph showing the changes in specific IgG antibodies in rats 14, 35, and 98 days after inoculation with different doses of the recombinant trivalent influenza virus protein vaccine in Experimental Example 2;

[0083] Figure 11 This is a graph showing changes in hemagglutination inhibition neutralizing antibody titers against three influenza strains at 35 and 98 days for the recombinant trivalent influenza virus protein vaccine in Experimental Example 2;

[0084] Figure 12 This is a graph showing weight changes in mice after infection with different strains in Experimental Example 3;

[0085] Figure 13 Figure 3 shows the viral load in the lungs and trachea of ​​mice infected with different strains 7 days after infection;

[0086] Figure 14 This is a graph showing the lung tissue pathology scores of mice infected with different strains in Experimental Example 3 for 7 days. DETAILED DESCRIPTION

[0087] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clear, the application is further described in detail below in conjunction with the embodiments. Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by ordinary technicians in this field.

[0088] In one embodiment of the present invention, a baculovirus-insect cell expression system is used to produce recombinant influenza virus proteins. The influenza virus HA gene is first introduced into an expression vector, preferably the pFastBac 1 vector, and then transposed into the baculovirus genome. The bacmid is then transfected into insect cells to generate the recombinant baculovirus, which rapidly produces HA antigen. Because the HA antigen is expressed directly from the genetic sequence, rather than derived from influenza virus replicated in chicken embryos or mammalian cells, potential egg-adaptive and cell-adaptive mutations during recombinant vaccine production are avoided. Consequently, the expressed HA antigen is genetically identical to the selected influenza strain.

[0089] In some specific embodiments of the present invention, the insect cells used are Sf9 cells, preferably WSK-Sf9 insect cells (deposit number: CCTCC NO: C202246). Compared with other Sf9 cells, WSK-Sf9 insect cells are not infected with rhabdovirus.

[0090] In some specific embodiments of the present invention, the influenza virus HA is selected from the trivalent or quadrivalent vaccine strains of the Northern Hemisphere or Southern Hemisphere published by the WHO, such as A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus, A / Wisconsin / 67 / 2022(H1N1)pdm09-like virus, A / Darwin / 6 / 2021(H3N2)-like virus, A / Massachusetts / 18 / 2022(H3N2)-like virus, A / District Of Columbia / 27 / 2023(H3N2)-like virus, B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus, and B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus.

[0091] In some specific embodiments of the present invention, the amino acid sequence of the expressed HA antigen is such as SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0092] In some specific embodiments of the present invention, the amino acid sequence of the expressed HA antigen is at least one of the amino acid sequences having more than 90% homology with SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 and having the same or similar biological activity.

[0093] The above 90% homology may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0094] One of the most effective strategies for improving vaccine efficacy against infection is the addition of adjuvants. In the context of influenza vaccines, adjuvants effectively enhance immunogenicity and reduce the amount of viral antigen and the number of vaccinations required for protection. Furthermore, adjuvants can broaden reactivity to antigenic variants and effectively counteract antigenic mismatches between vaccine strains and circulating viruses. Numerous adjuvants have been approved for use in influenza vaccines, including aluminum adjuvants, MF59, and AS03.

[0095] Existing literature indicates that oil-emulsion adjuvants can better enhance the immunogenicity of antigens compared to aluminum adjuvants. MF59 is a water-in-oil emulsion adjuvant composed of 4.3% (v / v) squalene, 0.5% (v / v) Tween 80, 0.5% (v / v) Span 85, 10 mM sodium citrate buffer, pH 6.5, and the balance water. Studies have shown that after entering the body, MF59 can enhance the uptake and presentation of antigens by antigen-presenting cells, activate myeloid cells such as macrophages and dendritic cells to secrete chemokines (CCL2, CCL4, CXCL8, etc.) and cytokines (IL-6, G-CSF, etc.), and recruit more immune cells such as monocytes and neutrophils to induce the production of DAMPs such as uric acid and ATP, induce immune cell apoptosis, enhance the movement of antigens to draining lymph nodes, and amplify humoral and cellular immunity. The prescription composition, preparation method and pharmaceutical properties of WGa01 adjuvant are consistent with those of MF59. Based on this, it can be inferred that the efficacy, mechanism and safety of WGa01 adjuvant-enhanced protein vaccine are also similar to those of MF59.

[0096] In some specific embodiments of the present invention, any two, three, or four combinations of prepared recombinant influenza virus proteins, such as subtype strain H1N1 subtype, H3N2 subtype, Victoria lineage, and Yamagata lineage HA proteins, are mixed with WGa01 adjuvant to prepare bivalent, trivalent, or quadrivalent recombinant influenza virus vaccines, and their immunogenicity and safety are verified.

[0097] Specific examples will be listed below to explain the scheme of the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific technology or conditions are not specified in the examples, they are carried out according to the technology or conditions described in the literature in this field or according to the product instructions. The reagents or instruments used are conventional products that can be obtained commercially if the manufacturer is not specified. WSK-Sf9 insect cells (deposit number is CCTCC NO: C202246) were published on September 12, 2023, and the patent publication number is CN116731953A.

[0098] Example 1: Preparation of recombinant influenza virus protein using insect baculovirus expression system

[0099] 1. Vector construction: The amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 6 were used to construct an expression vector for HA protein based on the pFastBac 1 vector. The BamHI and HindIII restriction sites were used to insert the vector into the pFastBac 1 vector. The gene synthesis sequence was codon-optimized based on insect cell expression. Figure 1 The signal peptide sequence of GP67 (SEQ ID NO.13: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) was added to the N-terminus of the protein to assist in the secretory expression of the protein. This signal peptide will be spontaneously removed by insect cells during the process of protein secretion.

[0100] 2. Amplification of Recombinant Baculovirus: Using the Bac-to-Bac expression system, which leverages the bacterial transposon principle and site-specific transposition via the Tn7 transposable element, recombinant bacmids were constructed within Escherichia coli DH10Bac. The recombinant bacmids were extracted and transfected into WSK-Sf9 insect cells (Deposit Number: CCTCC NO: C202246) to generate recombinant baculovirus expressing the target protein. Two to three days after transfection, P0 passage virus was harvested and amplified for recombinant protein expression.

[0101] 3. Protein expression: WSK-Sf9 insect cells were infected with the amplified recombinant baculovirus at a culture temperature of 27-28°C. Cell pellets were collected after 3-5 days of culture.

[0102] 4. Protein Purification: Cell pellets were collected by high-speed centrifugation, and lysis buffer (lysis buffer: 20-30 mmol / L PB, 20-50 mmol / L NaCl, 1-5% glycerol, 1-5% Tergitol NP-9, pH 7.0-7.5) was added to the cell pellet and stirred at room temperature to obtain a crude protein extract. The crude extract containing the target protein was pretreated: the first step was two-stage clarification filtration, the second step was ultrafiltration and liquid exchange, first by anion exchange chromatography for crude purification, then by affinity chromatography (lectin medium filler) and hydroxyapatite chromatography to purify the protein. The HA protein was purified by SDS-PAGE to obtain a purity ≥90%. The HA protein stock solution was obtained after sterile filtration. Figure 2 、 3 They are the restriction enzyme digestion map of the HA expression vector plasmid and the HA gene PCR map of the H3N2 subtype (SEQ ID NO.3).

[0103] By the above method, HA proteins of four subtypes of influenza virus strains, H1N1 subtype, H3N2 subtype, Victoria strain and Yamagata strain were prepared. The amino acid sequences of the recombinant influenza virus proteins obtained are shown in SEQ ID NO. 1 to 6. The protein preparation results are shown in Figures 4 to 7 After pretreatment, crude purification, and fine purification, a protein antigen with good purity was obtained, which can be used for subsequent immune protection research.

[0104] Example 2: Preparation of recombinant influenza virus protein vaccine

[0105] Prepare under sterile conditions. The recombinant HA protein antigen prepared in purification Example 1 was diluted to the desired concentration with phosphate buffer solution (20-30 mmol / L phosphate buffer, 150-300 mmol / L sodium chloride solution, pH 7.0-7.9). WGa01 adjuvant (consistent with the MF59 prescription composition, preparation method and formulation properties, defined as WGa01 adjuvant in the embodiments of the present invention) was added to the above-mentioned antigen solution, and the adjuvant and mixed stock volume ratio was (v / v) 1: 1, so that the final concentration of recombinant HA antigen in the mixed solution was 30-90 μg / subtype / mL. Stirring was turned on to produce a slight vortex in the medicinal liquid. After stirring, a secondary redundant filtration method was adopted, and a filtration system was connected. A 0.45 μm+0.22 μm capsule filter was used as a primary filter and a 0.22 μm capsule filter was used as a secondary filter. The solution was filtered into a sterilized filling solution bottle, which was a semi-finished vaccine product.

[0106] During this period, the adsorbed semi-finished vaccine product is characterized, including antigen content, pH, osmotic pressure, sterility testing, and bacterial endotoxin testing. Filling: The vaccine preparation is filled into 2mL sterile vials, with a fill volume of 0.65mL / vial, and the filling volume variation is controlled within ±5%. After filling, the vials are immediately capped, numbered, and labeled. Store at 2-8°C in the dark to obtain the recombinant influenza virus protein vaccine of the present invention.

[0107] The beneficial effects of the recombinant influenza virus protein vaccine prepared by the present invention were verified by biological experiments. The recombinant trivalent influenza virus protein vaccine used in the following experiments was prepared by mixing the recombinant influenza virus HA protein antigens of the H1N1 subtype (amino acid sequence SEQ ID NO. 1), H3N2 subtype (amino acid sequence SEQ ID NO. 3), and Victoria (amino acid sequence SEQ ID NO. 5) prepared in Example 1 in equal weight ratios and then adding an equal volume ratio of WGaO1 adjuvant. The split vaccine was a commercially available trivalent influenza split vaccine.

[0108] The detection methods used in the following test examples are as follows:

[0109] 1.ELISA

[0110] Reagents: coating buffer (50 mM carbonate buffer, pH 9.6), washing solution (PBS solution containing 0.05% Tween-20 (PBST)), sample diluent / sample diluent (1% BSA, PBST), color development solution, stop solution.

[0111] ELISA assay method:

[0112] 1) Dilute the antigen protein with coating solution, add 100 μL / well to the ELISA plate, cover with film, and coat overnight at 2-8°C.

[0113] 2) Wash the plate three times, add sample diluent, and incubate at 37°C for 1 hour.

[0114] 3) Sample Dilution: Dilute each sample with 1% BSA Sample Diluent (prepared in PBST) at the appropriate dilution factor before loading. Initial dilution factors are as follows: For pre-immune serum samples, an 800-fold dilution or other optimal dilution factor is recommended, without gradient dilution. For post-immune samples, set the optimal starting dilution factor, a 2-fold dilution method, and 11 concentration gradients.

[0115] 4) After dilution, add the sample to the ELISA plate at 100 μL / well, cover with the membrane, and incubate at 37°C for 1 hour.

[0116] 5) Wash the plate three times with washing buffer (0.05% PBST).

[0117] 6) Add detection reagent (secondary antibody incubation): 100 μL / well to the ELISA plate, cover with film, and incubate at 37°C for 1 hour.

[0118] 7) Wash the plate 5 times with washing solution (0.05% PBST).

[0119] 8) Add TMB substrate colorimetric solution and develop color at room temperature in the dark for 5-15 minutes.

[0120] 9) Add stop solution to terminate the reaction.

[0121] 10) Set the detection wavelength of the microplate reader to 450 nm (reference wavelength 630 nm) and read the OD value.

[0122] 2. Hemagglutination inhibition test

[0123] Reagents: Standard antisera (positive quality control serum): A(H1N1) subtype standard antiserum, A(H3N2) subtype standard antiserum, B Yamagata type standard antiserum, B Victoria type standard antiserum. 1% red blood cell suspension (chicken or guinea pig red blood cells). PBS buffer (0.01M, pH 7.4, sterilized at 121°C for 20 min). Physiological saline (0.85% NaCl).

[0124] Receptor destroying enzyme (RDE)

[0125] Consumables: 96-well microtiter plate.

[0126] Virus hemagglutination titer test:

[0127] Prepare a 1% chicken or guinea pig red blood cell suspension in the second column of a 96-well microplate. Add 50 μL of PBS buffer to the last column. Pipette 100 μL of the virus solution to be tested and add it to the corresponding well in the first column of the 96-well microplate. Add 100 μL of PBS to well H1 as a negative control. Serially dilute the virus solution in the microplate by two-fold. Discard 50 μL of the liquid from the last dilution well (each well in the last column). Pipette the 1% red blood cell suspension and add 50 μL / well of the diluted 96-well microplate. Tap the microplate gently to thoroughly mix the red blood cells and virus. Incubate at room temperature for 30 minutes for chicken red blood cells and 60 minutes for guinea pig red blood cells without disturbing the microplate. Observe for red blood cell agglutination and record the results.

[0128] HI detection:

[0129] Before the HI assay, follow the RDE manufacturer's instructions. Add 25 μL of PBS buffer to each well and load samples into row A in the following order: standard diagnostic serum as a positive control in A1, negative serum as a negative control in A2, and 25 μL of the test serum in A3-A12. Mark the sample loading layout (the initial dilution factor and loading layout can be adjusted according to actual conditions and recorded accurately). Use a dispenser to draw 25 μL from each well in row A and perform a 2-fold dilution from rows A to H. Discard the last 25 μL of liquid in row H. Add 25 μL of the test antigen corresponding to the test serum (4 agglutination units of antigen) to each well, mix thoroughly, and incubate at room temperature for 20 min. Add 50 μL of red blood cell suspension to each well to thoroughly mix the red blood cells and virus. Incubate at room temperature for 30 min, and observe the results of the red blood cell agglutination inhibition test. Guinea pig cells were tested for 1 h.

[0130] 3. qRT-PCR detection

[0131] Viral nucleic acid extraction kit, one-step RT-qPCR kit (purchased from Thermo, QuantiTectTM ProbeRT-PCR Kit), primers and probes.

[0132] Type A (Target: Matrix-protein)

[0133] SEQ ID NO.14: FluA-F: 5'-GGAATGGCTAAAGACAAGACCAAT-3';

[0134] SEQ ID NO.15: FluA-R: 5'-GGGCATTTTGGACAAAGCGTCTAC-3';

[0135] SEQ ID NO.16: FluA-Probe: FAM-AGTCCTCGCTCACTGGGCACGGTG-BHQ1

[0136] Type B (Target: Matrix-protein)

[0137] SEQ ID NO.17: FluB-F: CTCTGTGCTTTRTGCGARAAAC

[0138] SEQ ID NO.18: FluB-R: CCTTCYCATTCTTTTGACTTGC

[0139] SEQ ID NO.19: FluB-P: Cy5-TCAGCAATGAACACAGCAA-BHQ3

[0140] Experimental animals: All surviving animals that were challenged with the poison. If any experimental animals died before dissection, the actual number at the time of dissection shall prevail.

[0141] Experimental materials: The mice were killed after blood was collected, and the trachea, lung (right lung) and other tissues were taken, and subsequent operations were carried out according to the instructions for viral nucleic acid extraction.

[0142] Test method: For virus extraction, refer to the instructions of the relevant kit, and determine the viral load according to the influenza virus RT-qPCR test method.

[0143] Experimental Example 1: Effects of different doses of recombinant trivalent influenza virus protein vaccine on the immunogenicity of BALB / c female mice

[0144] Animal immunization experiment: BALB / c female mice were divided into 8 groups, with 6 mice in each group, as follows: (1) PBS group; (2) WGa01 adjuvant group; (3) H1N1 / H3N2 / Victoria antigen group (HA group) (3 μg / subtype / mouse); (4) recombinant trivalent influenza virus protein vaccine ultra-low dose group (0.1 μg / subtype / mouse) (VL-V104); (5) recombinant trivalent influenza virus protein vaccine low dose group (1 μg / subtype / mouse) (L-V104); (6) recombinant trivalent influenza virus protein vaccine medium dose group (3 μg / subtype / mouse) (M-V104); (7) recombinant trivalent influenza virus protein vaccine high dose group (9 μg / subtype / mouse) (H-V104); (8) split vaccine group (M-SV group) (3 μg / subtype / mouse). Mice were immunized by intramuscular injection on day 0 and day 21 (2 immunizations in total).

[0145] Serum samples were collected from mice on days 14, 35, and 98 of the experiment, and specific IgG antibodies were detected by indirect ELISA. The results were as follows: Figure 8 Serum samples were collected on the 35th and 98th day to test the hemagglutination inhibition (HI) neutralizing antibody titer. The results are as follows: Figure 9 .from Figure 8The experiment showed that the recombinant trivalent influenza virus protein vaccine of the present invention can quickly stimulate a high level of immune response. On the 14th day of the experiment, the specific IgG antibody levels of each dose group of the recombinant trivalent influenza virus protein vaccine increased, showing a certain dose dependence, and the titer level of the antibody produced was also better than that of the M-SV group and the HA group. On the 35th day of the experiment, the specific IgG antibody and HI neutralizing antibody titers of each dose group of the recombinant trivalent influenza virus protein vaccine reached a peak, and were much higher than those of the HA group and the M-SV group. On the 98th day of the experiment, the specific IgG antibody and HI neutralizing antibody levels of each test group of the recombinant trivalent influenza virus protein vaccine decreased, but still maintained at a high level, and were higher than those of the HA group and the M-SV group. In short, the recombinant trivalent influenza virus protein vaccine prepared by the present invention showed good immunogenicity by intramuscular injection of BALB / c mice, and there was little difference between different dose groups. The medium dose (3μg / subtype / mouse) (M-V104) was selected as the subsequent research dose based on the comprehensive specific IgG antibody and HI neutralizing antibody data.

[0146] Experimental Example 2: Effects of different doses of recombinant trivalent influenza virus protein vaccine on the immunogenicity of SD female rats

[0147] Animal immunization experiment: SD female rats were divided into 7 groups, with 6 mice in each group, as follows: (1) WGa01 adjuvant group; (2) H1N1 / H3N2 / Victoria antigen group (HA group) (15μg / subtype / mouse); (3) recombinant trivalent influenza virus protein vaccine ultra-low dose group (3μg / subtype / mouse) (VL-V104); (4) recombinant trivalent influenza virus protein vaccine low dose group (15μg / subtype / mouse) (L-V104); (5) recombinant trivalent influenza virus protein vaccine medium dose group (30μg / subtype / mouse) (M-V104); (6) recombinant trivalent influenza virus protein vaccine high dose group (45μg / subtype / mouse) (H-V104); (7) split vaccine group (M-SV group) (15μg / subtype / mouse). Mice were immunized by intramuscular injection on days 0 and 21 (a total of 2 immunizations).

[0148] Rat serum samples were collected on the 14th, 35th, and 98th day after the immune test and specific IgG antibodies were detected by indirect ELISA. Figure 10 Serum samples were collected on the 35th and 98th day to test the HI neutralizing antibody titer through hemagglutination inhibition test. The results are as follows: Figure 11. The results showed that on the 14th day of the experiment, the specific IgG level of the HA (15μg / subtype / individual) group was low. In comparison, the antibody levels of each dose group of the recombinant trivalent influenza virus protein vaccine were significantly higher. Except for the high-dose group (45μg / subtype), the specific IgG antibody levels of the remaining dose groups were higher than those of the HA antigen group and the M-SV group. On the 35th day of the experiment, the antibody titer of each dose group of the recombinant trivalent influenza virus protein vaccine reached a peak and was much higher than that of the HA group and the M-SV group. Among them, the antibody level of the medium-dose group (30μg / subtype) increased significantly, showing the strongest immune response. The results of HI neutralizing antibodies showed that the low-dose group (15μg / subtype), the medium-dose group (30μg / subtype) and the high-dose group (45μg / subtype) produced high levels of HI neutralizing antibodies against H3N2, H1N1 and B / Victoria strains, and the antibody levels did not differ much, with a difference of no more than 2 times. On the 98th day of the experiment, the levels of specific IgG antibodies and HI neutralizing antibodies in each dose group of the recombinant trivalent influenza virus protein vaccine decreased, but still remained at a high level. In terms of specific IgG antibodies, the antibody levels of the 3-45μg dose groups were similar. In terms of HI neutralizing antibodies, the antibody levels of the 15, 30 and 45μg dose groups still maintained a high level, and were much higher than the HA and M-SV groups. The results of this study showed that the recombinant trivalent influenza virus protein vaccine (Sf9 cells) vaccine showed good immunogenicity after intramuscular injection of SD rats. There was no significant difference in the specific IgG antibodies and HI neutralizing antibodies induced by the three dose groups of 15, 30 and 45μg, and they were able to maintain a high level for a long time. At the same time, the different dose groups of the recombinant trivalent influenza virus protein (Sf9 cells) vaccine were superior to the split vaccine group (M-SV group) in terms of the intensity and long-term maintenance of the immune response.

[0149] Experimental Example 3: Immunogenicity experiment of recombinant trivalent influenza virus protein vaccine injected intramuscularly into mice - challenge experiment

[0150] Animal Immunization Experiment: BALB / c female mice were divided into 4 groups, with 6 mice in each group, as follows: (1) WGa01 adjuvant group; (2) H1N1 / H3N2 / Victoria antigen group (HA group) (3 μg / subtype / mouse); (3) recombinant trivalent influenza virus protein vaccine low-dose group (3 μg / subtype / mouse) (L-V104); (4) recombinant trivalent influenza virus protein vaccine high-dose group (9 μg / subtype / mouse) (H-V104). Mice were immunized by intramuscular injection on days 0 and 21 (a total of 2 immunizations).

[0151] On the 14th day after the immunization program (the 35th day of the experiment), the three influenza strains H1N1, H3N2 and B / Victoria were challenged by nasal drops. Figure 12), viral load detection and lung tissue pathology examination to evaluate the protective effect of recombinant trivalent influenza virus protein vaccine against viral infection. Viral load was detected by quantitative PCR by collecting mouse trachea and lung tissue (the results are shown in Figure 13 ), and pathological evaluation was performed by HE staining and pathological scoring (results as shown Figure 14 ). The results showed that during the period of 1 to 7 dpi, the body weight of the WGa01 adjuvant group decreased significantly during the H1N1 strain challenge (P<0.0001), while the body weight of the recombinant trivalent influenza virus protein vaccine group and the HA group remained stable. No significant changes were observed in the body weight of mice challenged with the H3N2 and B / Victoria strains (P>0.05). In the infection of H1N1, H3N2 and B / Victoria strains, the viral load in the lung tissue and trachea of ​​mice in the recombinant trivalent influenza virus protein vaccine group was significantly lower than that in the WGa01 adjuvant group (P<0.0001). The viral load in the vaccine group decreased by 1 to 5 log10 values ​​compared with the adjuvant group, especially for the B / Victoria strain, where the viral load was not even detected in the vaccine group. In the infection of H1N1, H3N2 and B / Victoria strains, the lung tissue pathology score of the recombinant trivalent influenza virus protein vaccine group was significantly lower than that of the adjuvant group (P<0.05). This study demonstrated that both low and high doses of the recombinant trivalent influenza virus protein vaccine demonstrated significant protective effects in infection models with three influenza strains: H1N1, H3N2, and B / Victoria. The vaccine group demonstrated superior body weight change, viral load, and pathological examinations compared to the WGa01 adjuvant group. During viral infection, the recombinant trivalent influenza virus protein vaccine effectively inhibited viral replication and significantly reduced pathological damage in lung tissue, demonstrating the protective efficacy of the recombinant trivalent influenza virus protein vaccine against vaccine-associated influenza viruses.

[0152] In summary, the recombinant trivalent influenza virus protein (Sf9 cell) vaccine prepared by the present invention has good immunogenicity in both mice and rats and good safety, and is a recombinant influenza virus vaccine with great potential.

Claims

1. A recombinant influenza virus protein for preventing and / or treating influenza virus infection, characterized in that: The amino acid sequence of the recombinant protein is derived from the full-length or truncated sequence of the hemagglutinin protein of the influenza virus strain.

2. The protein according to claim 1, characterized in that: The influenza virus strain is selected from at least one of H1N1, H3N2, Victoria, Yamagata lineage, H5N1, H7N9 or H5N8.

3. The protein according to claim 2, characterized in that: The recombinant protein amino acid sequence is selected from at least one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6; or at least one of amino acid sequences that have more than 90% homology with SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6 and have the same or similar biological activity.

4. The protein according to claim 2, characterized in that: The nucleic acid sequence encoding the recombinant protein is selected from at least one of SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No.

12.

5. The protein according to any one of claims 1 to 4, characterized in that: The recombinant influenza virus protein is prepared by introducing the coding gene of the target antigen protein into an expression vector, transferring the gene into a host cell to express the protein, and then purifying the cell.

6. The protein according to claim 5, characterized in that: The expression vector is selected from at least one of an insect baculovirus expression vector, a mammalian cell expression vector, an Escherichia coli expression vector or a yeast expression vector.

7. The protein according to claim 6, characterized in that Meet any of the following: The insect baculovirus expression vector is pFastBac1; The Escherichia coli expression vector is pET32a; The yeast expression vector is pPICZαA; The mammalian cell expression vector is a CHO cell expression vector pTT5 or FTP-002.

8. The protein according to claim 5, characterized in that: The host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli or yeast.

9. The protein according to claim 8, characterized in that Meet any of the following: The insect cells are selected from at least one of Sf9 cells, Sf21 cells, and Hi5 cells; Preferably, the Sf9 cell is a WSK-Sf9 insect cell, with a deposit number of CCTCC NO: C202246; The mammalian cells are CHO cells.

10. A recombinant influenza virus protein vaccine for preventing and / or treating influenza virus infection, characterized in that: The invention comprises an antigen and pharmaceutically acceptable auxiliary components, wherein the antigen is the recombinant influenza virus protein according to any one of claims 1 to 9.

11. The vaccine according to claim 10, characterized in that: The auxiliary component is an immune adjuvant; the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponin, plant polysaccharide, monophosphoric acid lipid A, muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin, GM-CSF cytokine, lipid, cationic liposome material, CpG ODN.

12. The vaccine according to claim 11, characterized in that Meet at least one of the following: The squalene oil-in-water emulsion is selected from at least one of WGa01, MF59, AS03, AF03, SE or AddaVax adjuvants; The aluminum salt is selected from at least one of aluminum hydroxide and alum; The calcium salt is tricalcium phosphate; The plant saponin is QS-21 or ISCOM; The plant polysaccharide is astragalus polysaccharide; The lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine; The cationic liposome material is selected from at least one of the following: (2,3-dioleyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoyl chloride)propyl]-N,N,N-trimethylammonium chloride, cationic cholesterol, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, and dimethyldioctadecylammonium bromide.

13. The vaccine according to any one of claims 10 to 12, characterized in that The dosage forms of the vaccine include injection, nasal drops, spray, inhalation or oral administration.

14. A pharmaceutical composition for preventing and / or treating respiratory diseases, comprising the recombinant influenza virus protein according to any one of claims 1 to 9 or the recombinant influenza virus protein vaccine according to any one of claims 10 to 13, and other drugs for preventing and / or treating influenza caused by vaccine-associated influenza strains.

15. Use of the recombinant influenza virus protein according to any one of claims 1 to 9, the recombinant influenza virus protein vaccine according to any one of claims 10 to 13, or the pharmaceutical composition according to claim 14 for preventing and / or treating influenza caused by vaccine-associated influenza strains.

16. The method for preparing the recombinant influenza virus protein according to any one of claims 1 to 9, characterized in that: The following steps are involved: Construct an expression vector containing the target gene, introduce it into the host cell to express the protein, and purify it.

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