Recombinant influenza virus protein, vaccine, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to a recombinant influenza virus protein, a vaccine, and a preparation method therefor and the use thereof. To overcome the defects of existing influenza vaccines with respect to safety and high toxic side effects, a recombinant influenza virus protein is provided, which has an amino acid sequence as shown in at least one of SEQ ID No. 1 to SEQ ID No. 6. Recombinant hemagglutinin proteins of H1N1 and H3N2 subtypes, and Victoria and Yamagata lineages are expressed and purified by means of an insect baculovirus expression system, and are mixed with an adjuvant to prepare a highly immunogenic recombinant bivalent, trivalent or quadrivalent recombinant influenza virus protein vaccine. The prepared vaccine can induce high levels of specific IgG antibodies and hemagglutination-inhibiting neutralizing antibodies, the addition of adjuvant WGa01 improves the immunogenicity of the vaccine, and the provided vaccine is suitable for people allergic to eggs and has high safety.

Description

Recombinant influenza virus proteins, vaccines, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to recombinant influenza virus protein, vaccines, their preparation methods, and applications. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus, with high morbidity and mortality rates. The influenza virus spreads rapidly and is highly contagious, primarily through airborne droplets, direct person-to-person contact, or contact with virus-contaminated objects. Influenza vaccination is an important strategy for influenza prevention.

[0003] Influenza viruses belong to the Orthomyxoviridae family and consist of a negative-sense, single-stranded, segmental RNA genome. Based on viral nucleoproteins and matrix proteins, they are classified into four types: influenza A (A), influenza B (B), influenza C (C), and influenza D (D). The vast majority of seasonal influenza is associated with influenza A and influenza B viruses. Based on the protein structure and genetic characteristics of viral surface hemagglutinin (HA) and neuraminidase (NA), influenza A (H1N1) viruses are divided into 18 HA subtypes and 11 NA subtypes; among them, H1, H2, H3, H5, and H7 antigenic subtypes can infect humans. Influenza B (H2N2) virus HA subtypes have differentiated into two serologically distinct lineages (Victoria and Yamagata). Influenza vaccines are typically developed targeting the HA protein. HA protein is the most important antigenic and immunogenic protein in influenza virus. On the one hand, it binds to the sialic acid receptor on the surface of host cells, thereby enabling the virus to attach to the cell and help the virus penetrate the host cell membrane to infect and reproduce. On the other hand, it can stimulate the body to produce corresponding humoral and cellular immunity, thereby enabling the body to resist the corresponding virus.

[0004] Current influenza vaccines are primarily inactivated vaccines produced using chicken embryos. However, this technology has many drawbacks, such as insufficient chicken embryo supply during outbreaks, limited vaccine production, local or systemic allergic reactions caused by residual egg components in the vaccine, and short duration of immune response. Therefore, a safe and effective vaccine to replace traditional inactivated vaccines is urgently needed. Unlike influenza virus vaccines based on chicken embryos or cells, recombinant influenza virus vaccines directly construct the antigen gene sequence into an expression vector and express it through recombinant protein technology. The production process does not require eggs or candidate vaccine viruses. Currently, the only commercially available recombinant protein influenza vaccine is Flublok, produced by Sanofi Pasteur in France, and this vaccine is only available to 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 shortcomings of existing influenza vaccines, such as narrow applicability, high safety and toxic side effects, this invention provides a recombinant influenza virus protein, a vaccine, its preparation method and application.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present invention provides a recombinant influenza virus protein for the prevention and / or treatment of 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] Further, the amino acid sequence of the recombinant protein 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 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 activities.

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

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

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

[0014] SEQ ID NO.4: H3N2 influenza virus HA amino acid sequence (A / District Of Columbia / 27 / 2023(H3N2))

[0015] SEQ ID NO.5: HA amino acid sequence of influenza B / Victoria lineage virus (B / Austria / 1359417 / 2021)

[0016] SEQ ID NO.6: B / Yamagata lineage influenza virus HA amino acid sequence (B / Phuket / 3073 / 2013)

[0017] Furthermore, the nucleic acid sequence encoding the amino acid sequence of the recombinant protein against 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.

[0018] SEQ ID NO.7: H1N1 influenza virus HA nucleic acid sequence (A / Wisconsin / 588 / 2019(H1N1)pdm09)

[0019] SEQ ID NO.8: H1N1 influenza virus HA nucleic acid sequence (A / Wisconsin / 67 / 2022(H1N1)pdm09)

[0020] SEQ ID NO.9: H3N2 influenza virus HA nucleic acid sequence (A / Darwin / 6 / 2021(H3N2))

[0021] SEQ ID NO.10: H3N2 influenza virus HA nucleic acid sequence (A / District Of Columbia / 27 / 2023(H3N2))

[0022] SEQ ID NO.11: B / Victoria lineage influenza virus HA nucleic acid sequence (B / Austria / 1359417 / 2021)

[0023] SEQ ID NO.12: B / Yamagata lineage influenza virus HA nucleic acid sequence (B / Phuket / 3073 / 2013)

[0024] Furthermore, the recombinant influenza virus protein is obtained by introducing the encoding gene of the target antigen protein into an expression vector, transfecting it into host cells to express the protein, and then purifying it.

[0025] Preferably, the expression vector is selected from at least one of insect baculovirus expression vectors, mammalian cell expression vectors, Escherichia coli expression vectors, or yeast expression vectors.

[0026] Preferably, the insect baculovirus expression vector is pFastBac1.

[0027] Preferably, the Escherichia coli expression vector is pET32a.

[0028] Preferably, the yeast expression vector is pPICZαA;

[0029] Preferably, the mammalian cell expression vector is the CHO cell expression vector pTT5 or FTP-002.

[0030] Preferably, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli, or yeast.

[0031] 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 from rhabdovirus contamination, with accession number CCTCC NO: C202246. WSK-Sf9 insect cells were published on September 12, 2023, with patent publication number CN116731953A.

[0032] More preferably, the mammalian cell is a CHO cell.

[0033] In a second aspect, the present invention provides a recombinant influenza virus protein vaccine for the prevention and / or treatment of influenza virus infection, which contains an antigen and a pharmaceutically acceptable auxiliary component, wherein the antigen is the aforementioned recombinant influenza virus protein.

[0034] The recombinant influenza virus protein used as the antigen can be a single recombinant influenza virus protein or 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.

[0035] It can also be a recombinant influenza virus protein, wherein the amino acid sequence of the protein 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.

[0036] Furthermore, the auxiliary component is an immune adjuvant.

[0037] Preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponins, plant polysaccharides, lipid monophosphate A (MPL), muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin (rCTB), GM-CSF cytokines, lipids, cationic liposome materials, and CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as the core sequence, artificially synthesized CpG).

[0038] Furthermore, the squalene oil-in-water emulsion is selected from at least one of WGa01, MF59, AS03, AF03, SE (Squalene Emulsion) or AddaVax (InvivoGen).

[0039] Further, the WGa01 adjuvant comprises: 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, with the balance being water.

[0040] Furthermore, the aluminum salt is selected from at least one of aluminum hydroxide and alum.

[0041] Furthermore, the calcium salt is tricalcium phosphate.

[0042] Furthermore, the plant saponin is QS-21 or ISCOM.

[0043] Furthermore, the plant polysaccharide is Astragalus polysaccharide (APS).

[0044] Furthermore, the lipid is selected from at least one of the following: phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), and dioleoylphosphatidylethanolamine (DOPE).

[0045] Furthermore, the cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), dimethyl-2,3-dioleenoyloxypropyl-2-(2-spermineformylamino)ethylammonium trifluoroacetate (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), and dimethylbisoctadecylammonium bromide (DDAB).

[0046] Furthermore, the dosage form of the vaccine includes injections, nasal drops, sprays, inhalers, or oral formulations.

[0047] Preferably, the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, or intradermal injection.

[0048] Thirdly, the present invention provides a pharmaceutical composition for the prevention and / or treatment of respiratory diseases, comprising the above-mentioned recombinant influenza virus protein or recombinant influenza virus protein vaccine, as well as other drugs for the prevention and / or treatment of influenza caused by vaccine-associated influenza strains.

[0049] Fourthly, the present invention provides the use of the above-mentioned recombinant influenza virus protein, vaccine and pharmaceutical composition thereof in the prevention and / or treatment of influenza caused by vaccine-associated influenza virus types.

[0050] Fifthly, 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 a host cell, and purifying it.

[0051] Furthermore, the expression vector is selected from at least one of insect baculovirus expression vectors, mammalian cell expression vectors, Escherichia coli expression vectors, or yeast expression vectors.

[0052] Preferably, the expression vector is pFastBac1.

[0053] Furthermore, the host cell is selected from at least one of insect cells, mammalian cells, Escherichia coli, or yeast.

[0054] 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 from rhabdovirus contamination, with the accession number CCTCC NO:C202246.

[0055] Further, 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.

[0056] Beneficial Effects: This invention utilizes genetic engineering technology, using the hemagglutinin (HA) protein of influenza virus strains published by the WHO as the antigen source, to design recombinant influenza virus proteins for the prevention of influenza virus infection. 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. Recombinant HA proteins of the H1N1 subtype, H3N2 subtype, Victoria lineage, and Yamagata lineage were expressed and purified using an insect baculovirus expression system, respectively. Any two or more proteins were then mixed with adjuvants to prepare recombinant bivalent, trivalent, or quadrivalent recombinant influenza virus protein vaccines with high purity of the active ingredient.

[0057] Animal experiments have demonstrated that the vaccine of this invention has the following advantages:

[0058] 1. The recombinant influenza virus protein vaccine prepared by this invention can induce high levels of specific IgG antibodies and hemagglutination inhibition (HI) neutralizing antibodies in mice and rats. The addition of adjuvants improves the immunogenicity of the vaccine and can induce balanced humoral and cellular immunity.

[0059] 2. Because the vaccine is derived directly from the HA gene sequence of the vaccine strain, there are no adaptive mutations, resulting in high vaccine stability.

[0060] 3. The vaccine of this invention does not contain egg protein, antibiotics or preservatives, will not cause allergies, has high safety, is also suitable for people with egg allergies, and has a wide range of applications.

[0061] 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.

[0062] 5. The vaccine of this invention has a short production cycle, only 2 to 3 months, does not depend on chicken embryos, and has low cost. Attached Figure Description

[0063] Figure 1 is an illustration of the composition of the recombinant HA protein of influenza virus in Example 1.

[0064] Figure 2 shows the enzyme digestion diagram of the HA expression vector plasmid represented by the H3N2 subtype in Example 1;

[0065] Figure 3 shows the PCR diagram of the HA gene, represented by the H3N2 subtype, in Example 1;

[0066] Figure 4 shows the preparation results of H1N1 subtype influenza virus HA protein (corresponding to SEQ ID No. 1) in Example 1;

[0067] Figure 5 shows the preparation results of H3N2 subtype influenza virus HA protein (corresponding to SEQ ID No. 3) in Example 1;

[0068] Figure 6 shows the preparation results of the HA protein (corresponding to SEQ ID No. 5) of the B / Victoria subtype influenza virus in Example 1;

[0069] Figure 7 shows the preparation results of the B / Yamagata subtype influenza virus HA protein (corresponding to SEQ ID No. 6) in Example 1;

[0070] Figure 8 shows the changes in specific IgG antibodies in mice at 14, 35, and 98 days after vaccination with different doses of recombinant trivalent influenza virus protein vaccine in Experiment Example 1.

[0071] Figure 9 shows the changes in hemagglutination inhibition neutralizing antibody titers against the three influenza strains in the recombinant trivalent influenza virus protein vaccine at 35 days and 98 days in Experiment Example 1.

[0072] Figure 10 shows the changes in specific IgG antibodies in rats after 14, 35, and 98 days following vaccination with different doses of recombinant trivalent influenza virus protein vaccine in Experiment Example 2.

[0073] Figure 11 shows the changes in hemagglutination inhibition neutralizing antibody titers against three influenza strains at 35 and 98 days in the recombinant trivalent influenza virus protein vaccine in Experiment Example 2.

[0074] Figure 12 shows the changes in body weight of mice infected with different strains in Experiment 3;

[0075] Figure 13 shows the viral load in the lungs and trachea of ​​mice infected with different strains 7 days after Experiment Example 3.

[0076] Figure 14 shows the pathological scores of lung tissue in mice infected with different strains 7 days after Experiment Example 3. Detailed Implementation

[0077] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0078] In one specific embodiment of the present invention, a baculovirus-insect cell expression system is used to prepare recombinant influenza virus protein. First, the influenza virus HA gene is introduced into an expression vector, preferably the pFastBac 1 vector, and then transposed into the baculovirus genome. Recombinant baculovirus is obtained by transfecting insect cells with the baculovirus. The insect cells rapidly produce HA antigen after being transfected with the recombinant baculovirus. 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. Therefore, the expressed HA antigen is genetically identical to the selected influenza virus strain.

[0079] In some specific embodiments of the present invention, the insect cells used are Sf9 cells, preferably WSK-Sf9 insect cells (preservation number CCTCC NO:C202246). Compared with other Sf9 cells, WSK-Sf9 insect cells are free from rhabdovirus infection.

[0080] In some specific embodiments of the present invention, the selected influenza virus HA is derived from the trivalent or quadrivalent vaccine strains published by the WHO for the Northern or Southern Hemisphere. Examples include 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.

[0081] In some specific embodiments of the present invention, the amino acid sequence of the expressed HA antigen is as follows: 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.

[0082] 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 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 activities.

[0083] The above 90% homology can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0084] One of the most effective strategies for improving the efficacy of vaccines against infection is the addition of adjuvants. In the case of influenza vaccines, adjuvants effectively enhance immunogenicity and reduce the amount of viral antigen and number of vaccinations required for protection. Furthermore, adjuvants broaden responsiveness to antigenic variants and effectively combat antigenic mismatches between vaccine strains and circulating viruses. Many adjuvants have been approved for use in influenza vaccines, including aluminum adjuvants, MF59, and AS03.

[0085] Existing literature indicates that oil-in-oil adjuvants are more effective at enhancing the immunogenicity of antigens compared to aluminum adjuvants. MF59 is an oil-in-water 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, with the balance being 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.), recruit more immune cells such as monocytes and neutrophils to induce the production of DAMPs such as uric acid and ATP, and induce immune cell apoptosis to enhance antigen migration to draining lymph nodes, thus amplifying humoral and cellular immunity. The formulation, preparation method, and pharmacological properties of the WGa01 adjuvant are consistent with those of MF59. Based on this, it can be inferred that the efficacy, mechanism, and safety of the WGa01 adjuvant-enhanced protein vaccine are also similar to those of MF59.

[0086] In some specific embodiments of the present invention, the prepared recombinant influenza virus proteins, such as subtypes H1N1, H3N2, Victoria lineage and Yamagata lineage HA proteins, are combined in any two, three or four combinations and mixed with WGa01 adjuvant to prepare bivalent, trivalent or quadrivalent recombinant influenza virus vaccines, which are then used to verify their immunogenicity and safety.

[0087] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially. WSK-Sf9 insect cells (accession number CCTCC NO: C202246) were published on September 12, 2023, patent publication number CN116731953A.

[0088] Example 1: Preparation of recombinant influenza virus protein using an insect baculovirus expression system

[0089] 1. Vector Construction: The amino acid sequences shown in SEQ ID No. 1 to SEQ ID No. 6 were used. An expression vector for the HA protein was constructed based on the pFastBac1 vector. BamHI and HindIII restriction sites were inserted into the pFastBac1 vector. The gene synthesis sequence was codon-optimized based on insect cell expression, as shown in Figure 1. A GP67 signal peptide sequence (SEQ ID No. 13: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA) was added to the N-terminus of the protein to assist in secretory expression. This signal peptide will be spontaneously cleaved by the insect cell during protein secretion.

[0090] 2. Amplification of Recombinant Baculovirus: Using the Bac-to-Bac expression system, site-specific transposons were generated via the Tn7 transposon element to construct recombinant bacmids within *E. coli* DH10Bac. The recombinant bacmids were extracted and transfected into WSK-Sf9 insect cells (CCTCC NO: C202246) to produce recombinant baculovirus expressing the target protein. P0 generation virus was collected 2–3 days after transfection, and the recombinant baculovirus was amplified to express the recombinant protein.

[0091] 3. Protein expression: WSK-Sf9 insect cells were infected with amplified recombinant baculovirus at 27-28°C and the cell pellet was collected after 3-5 days of culture.

[0092] 4. Protein Purification: Cell pellet was collected by high-speed centrifugation. 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 crude protein extract. The crude extract containing the target protein underwent pretreatment: first, two-stage clarification filtration; second, ultrafiltration with medium replacement. The protein was first crudely purified by anion exchange chromatography, then purified further by affinity chromatography (lectin media) and hydroxyapatite chromatography. SDS-PAGE analysis confirmed the purification of HA protein with a purity ≥90%. After sterile filtration, the HA protein stock solution was obtained. Figures 2 and 3 show the restriction enzyme digestion diagram of the HA expression vector plasmid of the H3N2 subtype (SEQ ID NO.3) and the PCR diagram of the HA gene, respectively.

[0093] Using the above method, HA proteins from four subtypes of influenza virus strains—H1N1, H3N2, Victoria lineage, and Yamagata lineage—were prepared. The amino acid sequences of the final recombinant influenza virus proteins are shown in SEQ ID NOs 1–6, and the protein preparation results are shown in Figures 4–7. After purification through pretreatment, crude purification, and fine purification, protein antigens with good purity, suitable for subsequent immunoprotective studies, were obtained.

[0094] Example 2: Preparation of Recombinant Influenza Virus Protein Vaccine

[0095] Preparation was carried out under aseptic conditions. The recombinant HA protein antigen prepared in Example 1 was diluted and purified to the desired concentration using phosphate-buffered saline solution (20–30 mmol / L phosphate buffer, 150–300 mmol / L sodium chloride solution, pH 7.0–7.9). WGa01 adjuvant (consistent with the formulation, preparation method, and pharmaceutical properties of MF59, defined as WGa01 adjuvant in this embodiment) was added to the antigen solution at a volume ratio of adjuvant to the mixed stock solution of 1:1 (v / v), resulting in a final concentration of recombinant HA antigen of 30–90 μg / subtype / mL in the mixture. Stirring was initiated to create a slight vortex. After stirring, a two-stage redundant filtration system was used, connecting a filtration system with a 0.45 μm + 0.22 μm capsule filter as the primary filter and a 0.22 μm capsule filter as the secondary filter. The solution was filtered into sterile filling bottles to obtain the vaccine semi-finished product.

[0096] During the process, the adsorbed vaccine semi-finished product was characterized, including antigen content, pH value, osmotic pressure, sterility test, and bacterial endotoxin test. Filling: The vaccine preparation was filled into 2mL sterile vials, with a filling volume of 0.65mL / vial, and the volume variation was controlled within ±5%. Immediately after filling, the vials were sealed, labeled with a number, and stored at 2–8℃ protected from light to obtain the recombinant influenza virus protein vaccine of this invention.

[0097] The following biological experiments verify the beneficial effects of the recombinant influenza virus protein vaccine prepared according to this invention. The recombinant trivalent influenza virus protein vaccine used in the following experiments refers to the vaccine prepared by mixing the H1N1 subtype (amino acid sequence SEQ ID NO.1), H3N2 subtype (amino acid sequence SEQ ID NO.3), and Victoria lineage (amino acid sequence SEQ ID NO.5) recombinant influenza virus HA protein antigens in equal mass ratios and adding an equal volume ratio of WGa01 adjuvant. The split vaccine is a commercially available trivalent influenza split vaccine.

[0098] The detection methods used in the following test examples are as follows:

[0099] 1. ELISA method

[0100] Reagents: Coating buffer (50mM carbonate buffer, pH 9.6), washing buffer (PBS solution containing 0.05% Tween-20 (PBST)), sample diluent / sample diluent (1% BSA, PBST), chromogenic solution, stop solution.

[0101] ELISA assay method:

[0102] 1) Dilute the antigen protein with coating buffer, add 100 μL / well to the ELISA plate, cover with a membrane, and coat overnight at 2-8℃.

[0103] 2) Wash the plate 3 times, add sample diluent, and incubate at 37°C for 1 hour.

[0104] 3) Sample dilution: Dilute each sample with 1% BSA sample diluent (prepared with PBST) according to the corresponding dilution factor before adding the sample; the initial dilution factor is as follows: for serum samples before immunization, it is recommended to use 800 times or other optimal dilution factors, without gradient dilution; for samples after immunization, set the optimal initial dilution factor, 2-fold dilution method, 11 concentration gradients.

[0105] 4) After dilution, add 100 μL of sample to each well of the microplate, cover with a membrane, and incubate at 37°C for 1 hour.

[0106] 5) Wash the plate 3 times with washing buffer (0.05% PBST).

[0107] 6) Add detection reagent (secondary antibody incubation): 100 μL / well, add to the microplate, cover with membrane, and incubate at 37℃ for 1 h.

[0108] 7) Wash the plate 5 times with washing buffer (0.05% PBST).

[0109] 8) Add TMB substrate developing solution and develop at room temperature in the dark for 5-15 minutes.

[0110] 9) Add the stop solution to terminate the reaction.

[0111] 10) Set the detection wavelength of the microplate reader to 450nm (reference wavelength 630nm) and read the OD value.

[0112] 2. Hemagglutination inhibition test

[0113] Reagents: Standard antiserum (positive control serum): A(H1N1) subtype standard antiserum, A(H3N2) subtype standard antiserum, B-type Yamagata strain standard antiserum, B-type Victoria strain standard antiserum. 1% red blood cell suspension (chicken and guinea pig red blood cells). PBS buffer (0.01M, pH 7.4, sterilized at 121℃ for 20 min). Physiological saline (0.85% NaCl). Receptor-degrading enzyme RDE.

[0114] Consumables: 96-well microplate.

[0115] Viral hemagglutination titer detection:

[0116] Prepare a 1% chicken or guinea pig erythrocyte suspension in the second column of a 96-well microplate. Add 50 μL of PBS buffer to the last column. Add 100 μL of the virus solution to the corresponding well in the first column of the 96-well microplate. Add 100 μL of PBS to the H1 well as a negative control. Perform serial 2-fold dilutions of the virus solution from the microplate. Discard 50 μL of the final dilution well (each well in the last column). Add 50 μL of the 1% erythrocyte suspension to each well of the diluted 96-well microplate. Gently tap the microplate to mix the erythrocytes and virus thoroughly. Incubate at room temperature: 30 min for chicken erythrocytes and 60 min for guinea pig erythrocytes, without disturbing the microplate. Observe and record the erythrocyte agglutination.

[0117] HI testing:

[0118] Before performing the HI assay, follow the RDE manufacturer's instructions. Add 25 μL of PBS buffer to each well and add samples in the following order in row A: A1 contains standard diagnostic serum as a positive control, A2 contains negative serum as a negative control, and A3-A12 contain 25 μL of the serum to be tested. Label the sample layout (initial dilution and sample layout can be adjusted according to actual conditions and recorded accurately). Use a pipette to draw 25 μL from each well in row A and perform a 2-fold dilution from row A to row H. Discard the last 25 μL of liquid in row H. Add 25 μL of the corresponding detection antigen (4 agglutination units) to each well, mix well, 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 with the virus, and incubate at room temperature for 30 min. Observe the results of the red blood cell agglutination inhibition test. Guinea pig cells for 1 hour.

[0119] 3. qRT-PCR detection

[0120] Viral nucleic acid extraction kit, one-step RT-qPCR kit (purchased from Thermo, QuantiTect™ Probe RT-PCR Kit), primers and probes.

[0121] Type A (Target: Matrix-protein)

[0122] SEQ ID NO.14: FluA-F: 5'-GGAATGGCTAAAGACAAGACCAAT-3';

[0123] SEQ ID NO.15: FluA-R: 5'-GGGCATTTTGGACAAAGCGTCTAC-3';

[0124] SEQ ID NO.16: FluA-Probe: FAM-AGTCCTCGCTCACTGGGCACGGTG-BHQ1

[0125] Type B (Target: Matrix-protein)

[0126] SEQ ID NO.17: FluB-F: CTCTGTGCTTTRTGCGARAAAC

[0127] SEQ ID NO.18: FluB-R: CCTTCYCATTCTTTTGACTTGC

[0128] SEQ ID NO.19: FluB-P: Cy5-TCAGCAATGAACACAGCAA-BHQ3

[0129] Experimental animals: All surviving challenge animals. If any experimental animals die before dissection, the number at the time of actual dissection shall prevail.

[0130] Experimental material collection: Mice were euthanized after blood collection, and trachea, lung (right lung), and other tissues were collected. Subsequent operations were carried out in accordance with the viral nucleic acid extraction instructions.

[0131] Experimental method: Virus extraction was performed according to the relevant kit instructions, and viral load was determined using the influenza virus RT-qPCR assay.

[0132] Experimental Example 1: Effects of different doses of recombinant trivalent influenza virus protein vaccine on the immunogenicity of female BALB / c mice.

[0133] Animal immunization experiments: BALB / c female mice were used and divided into 8 groups of 6 mice each. The groups were: (1) PBS group; (2) WGa01 adjuvant group; (3) H1N1 / H3N2 / Victoria antigen group (HA group) (3μg / subtype / mouse); (4) recombinant trivalent influenza virus protein vaccine ultra-low dose group (0.1μg / subtype / mouse) (VL-V104); (5) recombinant trivalent influenza virus protein vaccine low dose group (1μg / subtype / mouse) (L-V104); (6) recombinant trivalent influenza virus protein vaccine medium dose group (3μg / subtype / mouse) (M-V104); (7) recombinant trivalent influenza virus protein vaccine high dose group (9μg / subtype / mouse) (H-V104); (8) split vaccine group (M-SV group) (3μg / subtype / mouse). Mice were immunized via intramuscular injection on days 0 and 21 (two immunizations in total).

[0134] Serum samples were collected from mice on days 14, 35, and 98 of the experiment, and specific IgG antibodies were detected by indirect ELISA, as shown in Figure 8. Serum samples collected on days 35 and 98 were used to detect hemagglutination inhibition (HI) neutralizing antibody titers by hemagglutination inhibition assay, as shown in Figure 9. Figure 8 shows that the recombinant trivalent influenza virus protein vaccine of this invention can rapidly elicit a high-level immune response. On day 14, the levels of specific IgG antibodies in all dose groups of the recombinant trivalent influenza virus protein vaccine increased, showing a certain dose-dependent effect, and the antibody titer levels were also superior to those in the M-SV and HA groups. On day 35, the specific IgG antibody and HI neutralizing antibody titers in all dose groups of the recombinant trivalent influenza virus protein vaccine reached their peak values, significantly higher than those in the HA and M-SV groups. On day 98, the levels of specific IgG antibodies and HI neutralizing antibodies in all experimental groups of the recombinant trivalent influenza virus protein vaccine decreased somewhat, but remained at a high level, all higher than those in the HA and M-SV groups. In summary, the recombinant trivalent influenza virus protein vaccine prepared in this invention showed good immunogenicity when immunized with BALB / c mice via intramuscular injection, with little difference between different dose groups. Based on the data of specific IgG antibodies and HI neutralizing antibodies, the medium dose (3 μg / subtype / mouse) (M-V104) was selected as the dose for subsequent studies.

[0135] Experimental Example 2: Effects of different doses of recombinant trivalent influenza virus protein vaccine on the immunogenicity of female SD rats.

[0136] Animal Immunization Experiment: Female SD rats were used and divided into 7 groups of 6 mice each. The groups were: (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 (two immunizations).

[0137] Serum samples were collected from rats on days 14, 35, and 98 after the immunization experiment. Specific IgG antibody levels were detected using an indirect ELISA method, as shown in Figure 10. Serum samples collected on days 35 and 98 were used to detect HI neutralizing antibody titers using a hemagglutination inhibition assay, as shown in Figure 11. The results showed that on day 14, the specific IgG level in the HA (15 μg / subtype / rat) group was low. In contrast, the antibody levels in all dose groups of the recombinant trivalent influenza virus protein vaccine were significantly higher. Except for the high-dose group (45 μg / subtype), the specific IgG antibody levels in the other dose groups were higher than those in the HA antigen group and the M-SV group. On day 35, the antibody titers in all dose groups of the recombinant trivalent influenza virus protein vaccine reached their peak values, significantly higher than those in the HA and M-SV groups. The medium-dose group (30 μg / subtype) showed a significant increase in antibody levels, indicating the strongest immune response. HI neutralizing antibody results showed that the low-dose group (15 μg / subtype), medium-dose group (30 μg / subtype), and high-dose group (45 μg / subtype) produced high levels of HI neutralizing antibodies against H3N2, H1N1, and B / Victoria strains, with little difference in antibody levels, not exceeding 2-fold. On day 98 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 remained at a high level. Regarding specific IgG antibodies, the antibody levels in the 3–45 μg dose groups were similar. Regarding HI neutralizing antibodies, the antibody levels in the 15, 30, and 45 μg dose groups remained high, significantly higher than those in the HA and M-SV groups. These results indicate that the recombinant trivalent influenza virus protein vaccine (Sf9 cell) immunized SD rats via intramuscular injection exhibits good immunogenicity; there were no significant differences in the specific IgG antibodies and HI neutralizing antibodies induced by the three dose groups (15, 30, and 45 μg), and these levels remained high for a long period. Meanwhile, different dose groups of the recombinant trivalent influenza virus protein (Sf9 cell) vaccine showed superior immune response intensity and long-term maintenance ability compared to the split vaccine group (M-SV group).

[0138] Experiment Example 3: Immunogenicity Experiment of Recombinant Trivalent Influenza Virus Protein Vaccine in Mice via Intramuscular Injection—Challenge Experiment

[0139] Animal immunization experiments: BALB / c female mice were divided into 4 groups of 6 mice each. The groups were: (1) WGa01 adjuvant group; (2) H1N1 / H3N2 / Victoria antigen group (HA group) (3μg / subtype / mouse); (3) low-dose recombinant trivalent influenza virus protein vaccine group (3μg / subtype / mouse) (L-V104); (4) high-dose recombinant trivalent influenza virus protein vaccine group (9μg / subtype / mouse) (H-V104). Mice were immunized by intramuscular injection on days 0 and 21 (two immunizations in total).

[0140] On day 14 (day 35 of the experiment) after the completion of the immunization program, mice were challenged with three influenza strains (H1N1, H3N2, and B / Victoria) via nasal drops. The protective efficacy of the recombinant trivalent influenza virus protein vaccine against viral infection was evaluated by monitoring changes in body weight (results shown in Figure 12), viral load, and lung tissue pathological examination. Viral load was detected by quantitative PCR using collected tracheal and lung tissues from mice (results shown in Figure 13), and pathological evaluation was performed using HE staining and pathological scoring (results shown in Figure 14). The results showed that during days 1–7 (dpi), the body weight of mice challenged with the H1N1 strain significantly decreased in the WGa01 adjuvant group (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 in body weight were observed in the H3N2 and B / Victoria strain challenge groups (P>0.05). In infections with 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 1–5 log10 values ​​compared to the adjuvant group, especially for the B / Victoria strain, where viral load was undetectable in the vaccine group. In H1N1, H3N2, and B / Victoria strain infections, the pathological score of lung tissue in the recombinant trivalent influenza virus protein vaccine group was significantly lower than that in the adjuvant group (P<0.05). This study demonstrates that both low and high doses of the recombinant trivalent influenza virus protein vaccine exhibit significant protective effects in infection models of H1N1, H3N2, and B / Victoria influenza strains. The vaccine group showed superior results compared to the WGa01 adjuvant group in terms of weight change, viral load, and pathological examination. In viral infection, the recombinant trivalent influenza virus protein vaccine effectively inhibited viral replication and significantly reduced pathological damage to lung tissue, demonstrating the protective efficacy of the recombinant trivalent influenza virus protein vaccine against vaccine-associated influenza viruses.

[0141] In summary, the recombinant trivalent influenza virus protein (Sf9 cell) vaccine prepared in this invention exhibits good immunogenicity and safety in both mice and rats, making it a highly promising recombinant influenza virus vaccine.

Claims

1. A recombinant influenza virus protein for the prevention and / or treatment of 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 influenza virus strains.

2. The protein according to claim 1, characterized in that: The influenza virus strain is selected from at least one of the following: 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 activities.

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 obtained by introducing the encoding gene of the target antigen protein into an expression vector, transfecting it into host cells to express the protein, and then purifying it.

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

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 either 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 mentioned are selected from at least one of Sf9 cells, Sf21 cells, and Hi5 cells; Preferably, the Sf9 cells are WSK-Sf9 insect cells with the accession number CCTCC NO:C202246; The mammalian cells mentioned are CHO cells.

10. A recombinant influenza virus protein vaccine for the prevention and / or treatment of influenza virus infection, characterized in that: It contains an antigen and a pharmaceutically acceptable auxiliary component, 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 saponins, plant polysaccharides, monophosphate lipid A, muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin, GM-CSF cytokines, lipids, cationic liposome materials, and 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 the adjuvants WGa01, MF59, AS03, AF03, SE, or AddaVax. The aluminum salt is selected from at least one of aluminum hydroxide and alum; The calcium salt mentioned is tricalcium phosphate; The plant saponins mentioned are QS-21 or ISCOM; The plant polysaccharide mentioned is Astragalus polysaccharide; The lipids are 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-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, trifluoroacetic acid dimethyl-2,3-dioleenoyloxypropyl-2-(2-spermineformylamino)ethylammonium, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, and dimethylbisoctadecylammonium bromide.

13. The vaccine according to any one of claims 10 to 12, characterized in that, The vaccine may be available in injection, nasal drops, spray, inhaler, or oral formulations.

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

15. The 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 in the prevention and / or treatment of influenza caused by vaccine-associated influenza strains.

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

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