A method for preparing influenza virus hemagglutinin protein HA and its application

Abstract

This invention provides a method for preparing influenza virus hemagglutinin protein HA and its application, belonging to the field of biopharmaceutical technology. The method utilizes engineered cell chassis cells with glycosylation modification capabilities. After fermentation expression, the expressed protein from the engineered cells is extracted in a single step to obtain a crude extract containing the target antigen. The crude extract obtained from the engineered cells constructed in this invention, after induction, has the potential to serve as a candidate for a dual-route vaccine (intramuscular injection and mucosal immunization), providing a new technical approach for the multi-route immunization development of influenza vaccines.

Description

Technical Field

[0001] This invention belongs to the field of biological vaccine technology, specifically relating to a method for preparing and applying influenza virus hemagglutinin protein HA. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus. It is characterized by rapid transmission, wide spread, and high morbidity, seriously threatening human health and public health security. The hemagglutinin (HA) protein on the surface of the influenza virus is a key protein for viral invasion of host cells. It mediates the binding of the virus to the sialic acid receptors on the surface of host cells and is also the main target antigen for inducing the body to produce specific neutralizing antibodies. Therefore, it has become a core target for influenza vaccine development.

[0003] Currently available influenza vaccines mainly include whole-virus inactivated vaccines, split vaccines, and subunit vaccines. While whole-virus inactivated vaccines can induce a more comprehensive immune response, they have drawbacks such as the need for large-scale culture of live viruses during production, high biosafety risks, and a tendency to trigger allergic reactions. Split vaccines and subunit vaccines reduce some safety risks, but they typically require complex purification processes, which not only increase production costs but may also disrupt the native conformation of the HA protein during purification, leading to decreased immunogenicity. Furthermore, most mainstream influenza vaccines are produced using chicken embryo technology, which is limited by egg production and quality, making automated and large-scale production difficult. Additionally, some virus strains have extremely low yields in chicken embryos, failing to meet emergency prevention and control needs. Moreover, the mainstream administration method is intramuscular injection, requiring professional medical personnel to administer the vaccine. The process can cause pain, local redness, and swelling, resulting in low patient compliance, especially for vulnerable groups such as children and the elderly, making vaccination even more challenging.

[0004] In terms of vaccine production expression systems, traditional prokaryotic expression systems (such as E. coli) have advantages such as low culture cost and fast growth rate, but they cannot perform correct glycosylation modification of HA proteins. The expressed HA proteins are prone to forming inclusion bodies, and the refolding process is complex and inefficient, making it difficult to meet the requirements of vaccine protein activity and immunogenicity. Although mammalian cell expression systems can achieve correct glycosylation modification of HA proteins and produce vaccines with higher immunogenicity, they have drawbacks such as long culture cycles, high costs, and susceptibility to viral contamination. Even with optimized production processes using the suspension culture MDCK cell system, the high cost of large-scale culture and complex process control still exist, which is not conducive to large-scale industrial production.

[0005] Yeast expression systems combine the low cost and high yield advantages of prokaryotic expression systems with the protein folding and modification capabilities of eukaryotic expression systems, making them an ideal choice for recombinant protein expression. By genetically engineering elements such as promoters in yeast expression vectors, the expression efficiency of target proteins can be significantly improved; this approach has been validated in the production of recombinant proteins such as glycosyltransferases. However, conventional yeast expression systems suffer from excessive glycosylation modification, which can alter the immunogenicity of HA proteins and affect vaccine efficacy. Glycosyl engineered yeast, through genetic modification, optimizes the glycosylation pathway, mimicking the glycosylation pattern of mammalian cells to express HA proteins with native conformation and biological activity, providing a new technological direction for influenza vaccine development.

[0006] Current research on influenza vaccines based on yeast-expressed HA protein largely relies on complex protein purification processes to obtain high-purity HA protein, ensuring vaccine safety and efficacy. However, these complex purification steps not only significantly increase production costs and prolong the production cycle but may also lead to HA protein loss and reduced activity. Therefore, developing an influenza vaccine that does not require complex purification and can maintain good immunogenicity using crude HA protein extract expressed in glycosylated yeast is currently a mature technological solution and is of great significance for overcoming the current bottlenecks in influenza vaccine production technology. Summary of the Invention

[0007] This invention provides a method for preparing influenza virus hemagglutinin protein HA and its application. The supernatant extract obtained after expression can induce the body to produce a specific immune response and generate a certain level of neutralizing antibodies, exhibiting good immunogenicity.

[0008] This invention provides a method for preparing influenza virus hemagglutinin protein HA, comprising the following steps: inducing expression of engineered cells expressing influenza virus hemagglutinin protein HA, collecting the precipitate and lysing the cells, centrifuging and collecting the supernatant, wherein the supernatant contains the influenza virus hemagglutinin protein HA; The chassis cells of the engineered cells are cells capable of glycosylation modification.

[0009] In one specific embodiment of the present invention, the influenza virus includes influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0010] In one specific embodiment of the present invention, the serotype of the hemagglutinin protein HA derived from the influenza A virus includes H1, H3, H5 or H7.

[0011] In one specific embodiment of the present invention, the cell type is a eukaryotic cell.

[0012] In one specific embodiment of the present invention, the cell type includes yeast cells, mammalian cells, or insect cells.

[0013] In one specific embodiment of the present invention, the yeast cell is glycosyl engineered yeast GJK01.

[0014] In one specific embodiment of the present invention, methanol is used to induce the expression of the influenza virus hemagglutinin protein HA.

[0015] The present invention also provides an influenza virus hemagglutinin protein HA antigen extract prepared using the above preparation method.

[0016] The present invention also provides the application of the above-mentioned influenza virus hemagglutinin protein HA antigen extract in the preparation of influenza vaccines.

[0017] The present invention also provides an influenza vaccine, wherein the influenza vaccine uses the above-mentioned influenza virus hemagglutinin protein HA antigen extract as the core antigen component, and further comprises a pharmaceutically acceptable diluent and / or adjuvant.

[0018] Beneficial effects: This invention provides a method for producing influenza virus hemagglutinin protein HA. The method involves inducing expression of influenza virus hemagglutinin protein HA in engineered cells, collecting the precipitate, breaking the cells, centrifuging, and collecting the supernatant, which contains the influenza virus hemagglutinin protein HA. The chassis cells of the engineered cells are glycosylated cells, such as glycosylated engineered yeast GJK01 used in the example to express influenza virus hemagglutinin protein HA.

[0019] Animal experiments showed that intramuscular injection of the crude extract induced a specific immune response and generated a certain level of neutralizing antibodies, indicating its good immunogenicity. When administered via mucosal immunization (e.g., nebulized inhalation), the crude extract not only induced a systemic neutralizing antibody response but also induced the production of secretory immunoglobulin A (sIgA) in the respiratory tract, thus providing additional protection at the mucosal barrier level. Therefore, the crude extract obtained from the engineered cells constructed in this invention, after induction, has the potential to serve as a candidate for a dual-route vaccine (intramuscular injection and mucosal immunization), providing a new technical approach for the multi-route immunization development of influenza vaccines.

[0020] This invention also uses the clarified yeast lysate as the core antigen, preserving the correct conformation of the HA protein, glycosylation modifications, and natural auxiliary immune components in the yeast cells. It can elicit a strong immune response in the body without purification, with serum HA-specific IgG antibody titers reaching up to 1×10⁻⁶. 5.01 The highest titer of sIgA antibody in bronchoalveolar lavage fluid was 1×10⁻⁶. 3.20The serum hemagglutination inhibition titer reached 1:457, which can induce the body to produce neutralizing antibodies. At the same time, the antigen preparation process of HA lysis clarifier is extremely simple and low-cost. It is obtained by extraction in one step, requiring only two core operations: cell disruption and centrifugation to obtain the supernatant. There are no purification steps such as chromatography and precipitation, which greatly shortens the preparation cycle, reduces equipment investment and raw material loss, and is easy to mass-produce. It provides a low-cost and highly immunogenic technical means for the prevention of influenza virus infection. Attached Figure Description

[0021] Figure 1 A schematic diagram of the construction of the pPICZαA-H7 (2013) vector; Figure 2 This is a diagram showing the results of cloning screening for recombinant H7 expression strains. Figure 3 This is a Western Blot image of the H7N9-H7 lysate clarified antigen (X33CLS-H7), where X33CLS-H7 represents the H7N9-H7 lysate clarified antigen and rH7 represents the recombinant H7N9-H7 protein. Figure 4 Quantitative graph of hemagglutination test for H7N9-H7 lysed clarified solution antigen (X33CLS-H7); Figure 5 The titers of anti-H7 IgG antibodies and neutralizing antibodies in mouse serum three weeks after the first immunization and two weeks after the second immunization were determined by intramuscular injection of H7N9-H7 lysate antigen (X33CLS-H7). Figure 6 The titers of anti-H7 IgG antibodies in the serum of mice immunized with the H7N9-H7 lysate clarified antigen (X33CLS-H7) two weeks after the first, second, third, and fourth immunizations. Figure 7 The titers of anti-H7 IgA antibodies in the serum of mice immunized with the H7N9-H7 lysate clarified antigen (X33CLS-H7) two weeks after the first, second, third, and fourth immunizations. Figure 8 The titer of anti-H7 IgG antibodies in bronchoalveolar lavage fluid of mice two weeks after immunization with the H7N9-H7 lysate clarified antigen (X33CLS-H7) mucosal immunization. Figure 9 The titer of anti-H7 IgA antibody in bronchoalveolar lavage fluid of mice two weeks after immunization with the H7N9-H7 lysate clarified antigen (X33CLS-H7) mucosal immunization. Figure 10 The titers of neutralizing antibodies against H7 in the serum of mice immunized with the H7N9-H7 lysate clarified antigen (X33CLS-H7) two weeks after the first, second, third, and fourth immunizations. Figure 11 A schematic diagram of the construction of the pPICZαA-H5 (2014) vector; Figure 12 This is a diagram showing the results of cloning screening for recombinant H5 expression strains. Figure 13 The titers of anti-H5 IgG antibodies and neutralizing antibodies in mouse serum three weeks after the first immunization and two weeks after the second immunization were determined by intramuscular injection of H5N6-H5 lysate antigen (X33CLS-H5). Figure 14 This is a diagram showing the results of cloning screening for recombinant H3 expression strains. Figure 15 The titers of anti-H3 IgG antibodies and neutralizing antibodies in mouse serum were measured three weeks after the first immunization and two weeks after the second immunization, using the H3N2-H3 lysate clarified antigen (X33CLS-H3) injected intramuscularly. Detailed Implementation

[0022] This invention provides a method for preparing influenza virus hemagglutinin protein HA, comprising the following steps: inducing expression of engineered cells expressing influenza virus hemagglutinin protein HA, collecting the precipitate and lysing the cells, centrifuging and collecting the supernatant, wherein the supernatant contains the influenza virus hemagglutinin protein HA; The chassis cells of the engineered cells are cells capable of glycosylation modification.

[0023] The influenza virus described in this invention is a virus belonging to the genus *Influenza* of the family Orthomyxoviridae, which can cause influenza infection in humans or animals; including influenza A virus, influenza B virus, influenza C virus, and influenza D virus, and of course, variants of the above-mentioned influenza virus types. The variant is a protein or protein that has a conformational change compared to the wild-type protein, or an amino acid or nucleotide sequence with one or more sites of its sequence replaced, added, or deleted, or a truncated amino acid or nucleotide sequence.

[0024] The engineered cells described in this invention can exogenously express hemagglutinin protein HA derived from influenza virus, and there is no particular limitation on the serotype of the HA, such as H1, H3, H5, or H7 derived from influenza A virus, or HA derived from influenza B virus, etc. The HA described in this invention can maintain its binding activity with erythrocytes and mediate viral invasion of host cells through sialic acid receptors. In this invention, the HA can be the full-length HA spike protein (membrane protein) of influenza virus, or it can be truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus, or extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. Furthermore, when elongating, the elongated amino acid can be an amino acid from HA itself, or it can be an amino acid tag added to obtain a specific function, such as adding an amino acid sequence to the C-terminus for suitable protein precipitation.

[0025] This invention uses glycosylated cells as the basal cells or host cells of the engineered cells. Generally, this refers to individual cells, cell lines, or cell cultures that can or already contain nucleic acid molecules, plasmids, or vectors encoding the HA gene described in this invention and are capable of expressing the HA gene. Host cells can include progeny of a single host cell, as well as cells that differ morphologically or genomically from the original parent cell due to mutations, but are still capable of expressing the HA gene. The host cells described in this invention are cells capable of glycosylation modification. Specifically, these cells are eukaryotic cells, such as yeast cells, COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells. In the examples, recombinant HA protein (hereinafter referred to as HA glycoprotein) was prepared using glycosylated engineered yeast as the host cell. Specifically, Pichia pastoris GJK01, genetically modified via glycosylation, was obtained from the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 1853. This application does not limit the method of obtaining HA glycoprotein. HA glycoproteins that have the same structure and function as those of this invention and are obtained by any other means are also within the scope of protection of this invention.

[0026] The glycosylation modification described in this invention refers to protein glycosylation, a common post-translational modification of proteins. It involves the transfer of sugars to proteins and the formation of glycosidic bonds between specific amino acid residues on the protein under the action of glycosyltransferases. Protein glycosylation can be mainly divided into two types: N-glycosylation and O-glycosylation. "O-glycosylation modification" refers to the covalent linkage of an O-glycan to a free OH group of a serine or threonine residue in the protein. O-glycosylation sites do not have conserved sequences, and the glycan does not have a fixed core structure; it can be a monosaccharide or a large sulfonated polysaccharide. The "N-glycosylation modification" involved in this invention refers to the N-glycan being covalently linked to the amide nitrogen of an asparagine residue in the protein.

[0027] In this invention, the coding gene for influenza virus hemagglutinin protein HA is codon-optimized and ligated into an expression vector to construct a recombinant expression vector; the recombinant expression vector is then used to transform cells with glycosylation modifications to construct the engineered cells.

[0028] The expression vectors described in this invention include eukaryotic expression vectors, which are generally nucleic acid molecules capable of self-replication in a suitable eukaryotic host, transferring inserted nucleic acid molecules to host cells and / or between host cells. The eukaryotic expression vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors with any or more functions primarily for transcription and / or translation of DNA or RNA.

[0029] This invention involves inserting the coding gene for HA, a codon-optimized coding gene, or a homologous gene with more than 80% homology to the aforementioned coding gene into a suitable position in the eukaryotic expression vector, thereby constructing a recombinant expression vector. The eukaryotic expression vector can be pPIC9, pPIC9K, pPICZαA, pPICZαB, pPICZαB, pET series vectors, pGEX series vectors, pMAL series vectors, pQE series vectors, pBADmycHis series vectors, pTrcHis series vectors, pTXB series, T series vectors, or other vectors, as well as modified vectors of the above vectors. In one embodiment, the pPICZαA vector is used as an example, where the target gene is inserted between BstBⅠ and NotⅠ.

[0030] In gene description, homology as used in this invention refers to the identity of nucleotide sequences. In some embodiments, the identity of nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search for the identity of a pair of nucleotide sequences to calculate the identity value (%), the identity value can then be obtained.

[0031] The present invention utilizes the recombinant expression vector to transform host cells to obtain the engineered cells. The transformation method is not particularly limited in the present invention. Any method in the art can be used to express the target protein or a variant of the target protein in the host cells.

[0032] This invention involves expanding the culture of engineered cells and then inoculating them into a fermentation medium for fermentation. After a period of fermentation, methanol is used to induce the expression of HA glycoprotein. After fermentation, the cell pellet is collected, lysed, and the supernatant is collected. The supernatant can be used as a crude extract, which exhibits good immunogenicity through injection and mucosal immunization, achieving one-step antigen extraction. In one embodiment of this invention, a glycosyl engineered yeast strain highly expressing HA protein is inoculated into YPD liquid medium (containing 100 μg / ml Zeocin) and cultured at 25°C and 200 rpm until OD200. 600 The inoculum was 15-20, and the cells were transferred to BMGY medium at a 5% (V / V) inoculum. After fermentation at 25°C and 200 rpm for 36 hours, 1% (v / v) methanol was added to induce HA expression. Induction was repeated every 24 hours. After 48 hours of induction, the cell pellet was collected by centrifugation at 4°C and 8000-10000 rpm for 15-20 min. 5 mM EDTA buffer was added at a mass-to-volume ratio of 1:5-1:10 and the pellet was thoroughly stirred and resuspended. The pellet was homogenized 3-4 times at 700-900 bar. Finally, the lysate was centrifuged at 4°C and 8000-10000 rpm for 15-20 min to remove the pellet. The supernatant was collected to obtain the HA glycoprotein antigen. This process does not involve any purification steps and achieves a "one-step" extraction of the antigen.

[0033] The present invention also provides an influenza virus hemagglutinin protein HA antigen extract prepared using the above preparation method.

[0034] This invention uses the aforementioned yeast lysate (X33CLS-H7) as the core antigen. This antigen is directly derived from glycosylated engineered yeast cells, preserving the correct conformation of the HA protein, glycosylation modifications, and natural auxiliary immune components in the yeast cells. It can elicit a strong immune response in the body without purification, with serum HA-specific IgG antibody titers reaching up to 1×10⁻⁶. 5.01 The highest titer of sIgA antibody in bronchoalveolar lavage fluid was 1×10⁻⁶. 3.20 The serum hemagglutination inhibition titer reached 1:457, which can induce the body to produce neutralizing antibodies.

[0035] The present invention also provides the application of the influenza virus hemagglutinin protein HA antigen extract prepared by the above preparation method in the preparation of influenza vaccines.

[0036] The present invention also provides an influenza vaccine, wherein the influenza vaccine uses the above-mentioned influenza virus hemagglutinin protein HA antigen extract as the core antigen component, and further comprises a pharmaceutically acceptable diluent and / or adjuvant.

[0037] The influenza vaccine of this invention can be administered via injection or via mucosal route. When different routes of administration are used, the diluent and / or adjuvant of the influenza vaccine may differ. In one embodiment of this invention, the diluent is physiological saline.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an engineered cell expressing influenza virus hemagglutinin protein HA, its expression method, and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] In the embodiments of this invention, unless otherwise specified, all materials used are conventional commercially available materials in the art, such as pPICZαA and GS115 Pichia pastoris products from Invitrogen. The Pichia pastoris strain GJK01 is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 1853 and is disclosed in Chinese patent CN101195809A.

[0040] The Q5 enzyme, Taq enzyme, dNTPs, restriction endonucleases, and T4 ligase used in the experiment were purchased from NEB. Pfu enzyme, kits, and DH5α competent cells were products of Beijing TransGen Biotech Co., Ltd. Whole-genome synthesis, nucleotide synthesis, primer synthesis, and sequencing were provided by Beijing Qingke Biotechnology Co., Ltd.

[0041] The various HA antibodies (catalog numbers: H1, H3, H5, H7, beta-HA antibody, etc.) are products of Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.; the HIS antibody and goat anti-rabbit IgG secondary antibody (SAB3700885) are products of Sigma-Aldrich; and the goat anti-mouse IgG secondary antibody (ab205719) is a product of Abcam. Bgl II. Restriction endonucleases are products of NEB Corporation.

[0042] The specific formulation of YPD liquid medium containing 100 μg / ml Zeocin is as follows: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 100 μg / ml Zeocin. Zeocin is a product of Invitrogen, catalog number ant-zn-1.

[0043] BALB / c (01011) is a product of Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0044] In the following examples, unless otherwise specified, the first position of each nucleotide sequence is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0045] Example 1: Construction of a glycosyl engineered yeast strain expressing recombinant H7 1. Construction of pPICZαA-H7 (2013) plasmid Based on the amino acid sequence of the HA gene (GenBenk number: kc853766.1) of influenza A / Hangzhou / 1 / 2013 (H7N9) virus published in GenBank, the HA gene was synthesized using GenScript (GenScript, Nanjing) with codon optimization. The synthesized HA gene fragment was then inserted between the BstBI and NotI restriction sites of the pPICZαA vector to obtain... Figure 1 The expression plasmid shown is pPICZαA-H7 (2013).

[0046] 2. Construction of glycosyl engineered yeast expression strains pPICZαA-H7 (2013) plasmid for use Bgl After II enzyme digestion, the linearized expression vector was transformed into glycosyl engineered yeast GJK01 by electroporation. Positive clones were screened using YPD / Zeocin plates, inoculated into BMGY medium for induction, and then Western blot was used to analyze the expression level of recombinant hemagglutinin. Finally, glycosyl engineered yeast strains expressing recombinant H7 were obtained. Figure 2 ).

[0047] Example 2: Preparation of influenza virus hemagglutinin H7 yeast nanoparticle membrane protein antigen 1. Strains culture The strain from Example 1 was selected and inoculated into 200 mL of YPD seed medium (containing 100 μg / mL Zeocin), and cultured at 25°C with shaking at 210 rpm until OD200. 600 The inoculum was 15-20; it was transferred to BMGY medium at an inoculum of 5% (V / V), fermented at 25°C and 200 rpm for 36 hours, and then 1% (v / v) methanol was added to induce the expression of influenza virus hemagglutinin H7 yeast nanoparticle protein. The induction was repeated every 24 hours. After 48 hours of induction, the mixture was centrifuged at 4°C and 8500 rpm for 20 minutes, the supernatant was discarded, and the cell pellet was collected.

[0048] 2. Antigen extraction from H7N9-H7 lysate clarified solution Add 5 mM pH 7.0 EDTA buffer (1 g: 5 mL) to the bacterial cell pellet and stir magnetically for 12 min until the cells are completely resuspended. Transfer the resuspended solution to a high-pressure homogenizer and homogenize three times at 800 bar. Centrifuge the homogenized lysate at 4°C and 8500 rpm for 20 min, discard the pellet, and collect the supernatant, which is the H7N9-H7 lysate antigen. Western blot analysis was performed, and the results are as follows: Figure 3 As shown, a specific band appears at a relative molecular mass of approximately 75 kDa (consistent with the theoretical molecular mass of the H7 protein), proving the successful preparation of the H7 yeast nanoparticle membrane protein antigen.

[0049] 3. Experimental grouping and mouse immunization experiment The immunization method is as follows: Thirty female BALB / c mice aged 6-8 weeks were randomly divided into the following 5 groups: Group 1 immunization: 200 μL of vaccine was nebulized on days 0, 14, 28, and 42. The vaccine used was 36 μg of H7 membrane protein antigen.

[0050] Group 2 immunizations: 800 μL of vaccine was nebulized on days 0, 14, 28, and 42. The vaccine used contained 144 μg of H7 membrane protein antigen.

[0051] Group 3 immunized: 100 μL of vaccine was injected intramuscularly on days 0 and 14. The vaccine used was 18 μg of H7 membrane protein antigen.

[0052] Group 4 immunizations: 200 μL of vaccine was nebulized on days 0, 14, 28, and 42. The vaccine used was 0 μg H7 membrane protein antigen.

[0053] Group 5: 100 μL of vaccine was administered intramuscularly on days 0 and 14. The vaccine used was the X33 host bacterium lysate clarification.

[0054] Immunization schedule: A second immunization was administered 14 days after the first immunization, and the booster immunization cycle was 14 days. The nebulized group received a total of 4 immunizations, while the intramuscular injection group received 2 immunizations.

[0055] Blood samples were collected from the orbital cavity of immunized mice before immunization and two weeks after the first, second, third, and fourth immunizations.

[0056] The titer of anti-HA antibodies in the serum of mice in each group was measured by ELISA. The procedure is described in the "Concise Guide to Molecular Biology Experiments" [M]. Science Press, 2008. Fourteen days post-immunization, five mice were randomly selected from each group, and serum (blood from the eyeball) and bronchoalveolar lavage fluid (recovered after intratracheal injection of PBS buffer) were collected. The titer of serum H7-specific IgG antibodies (coated with H7N9-H7 lysate antigen) and bronchoalveolar lavage fluid sIgA antibodies were detected by indirect ELISA. The neutralizing activity of serum against H7 was detected by hemagglutination inhibition assay.

[0057] Antibody titer such as Figures 5 to 8 As shown: Serum IgG titers in the blank control group were all <1×10⁻⁶ 2 The sIgA titers in bronchoalveolar lavage fluid were all <1×10⁻⁶. 1 The highest serum IgG titer in the high-dose nebulized group of the experimental group reached 1×10⁻⁶. 5.01 The sIgA titer of bronchoalveolar lavage fluid was 1×10⁻⁶. 3.20 Blood coagulation inhibition titer: all in the blank control group were <1:10; the secondary immune serum titer in the intramuscular injection group reached 1:1621, and the tertiary immune serum titer in the nebulized high-dose group was 1:457.

[0058] 4. Blood coagulation inhibition test 1) Sample pretreatment: Mix the serum to be tested with receptor-destroying enzyme (RDE) at a ratio of 1:3, incubate at 37°C for 16-18 hours, and then heat-inactivate at 56°C for 30 minutes to inactivate RDE.

[0059] 2) In a 96-well V-bottom microplate, the serum was serially diluted twice at a starting ratio of 1:10. 20 μL of serum treated with LDE and 30 μL of PBS (i.e., 1:10 serum dilution) were added to the first well. 25 μL of PBS and an equal volume of serum were added to each of the remaining wells. 25 μL of the serum was then taken from the first well and serially diluted twice.

[0060] 3) Add 25 μL of purified rH7 protein containing 4 HAU (published in the article: Liu, B.; Shi, P.; Wang, T.; Zhao, Y.; Lu, S.; Li, X.; Luo, S.; Chang, S.; Wang, S.; Sun, P.; et al. Recombinant H7 Hemagglutinin Expressed in Glycoengineered Pichia PastorisForms Nanoparticles That Protect Mice from Challenge with H7N9 InfluenzaVirus. Vaccine 2020, 38, 7938–7948, doi:10.1016 / j.vaccine.2020.10.061.) antigen to each well, mix gently, and incubate at room temperature for 40 minutes to allow the specific antibodies in the serum to fully bind to the purified rH7 protein.

[0061] 4) Add 25 μL of 1% chicken red blood cell suspension to each well, mix gently, and let stand at room temperature for 30 minutes.

[0062] 5) After incubation, observe the morphology of red blood cells at the bottom of the well: tight precipitation (button-shaped) indicates that blood coagulation is inhibited; uniform spreading of red blood cells indicates that blood coagulation has occurred.

[0063] Quantitative results of blood coagulation test as follows Figure 4 As shown, X33CLS-H7 can produce a clear erythrocyte agglutination phenomenon, and its agglutination pattern is similar to that of the standard rH7 protein. Finally, the equivalent protein activity of rH7 in the X33CLS-H7 antigen was calibrated to 180 μg / mL for antigen dosage conversion and standardization in subsequent animal immunization experiments.

[0064] The above results indicate that mucosal vaccines using H7N9-H7 lysate as antigen can effectively induce humoral immunity. Figure 9 ) and mucosal immune response ( Figure 10 Furthermore, it has been demonstrated that the antigen possesses good immunogenicity.

[0065] Example 3: Construction of a glycosyl engineered yeast strain expressing recombinant H5 1. Construction of pPICZαA-H5 (2014) plasmid Based on the amino acid sequence of the HA gene (GenBenk number: KM873638.1) of influenza A / cat / Sichuan / SC18 / 2014 (H5N6) virus published in GenBank, the HA gene was synthesized using GenScript (GenScript, Nanjing) with codon optimization. The synthesized HA gene fragment was then inserted into the pPICZαA vector. Xho I and Not Between the I restriction sites, we obtain Figure 11 The expression plasmid pPICZαA-H5 (2014) is shown.

[0066] 2. Construction of glycosyl engineered yeast expression strains pPICZαA-H5 (2014) plasmid for use Bgl After II enzyme digestion, the linearized expression vector was transformed into glycosyl engineered yeast GJK01 by electroporation. Positive clones were screened using YPD / Zeocin plates, inoculated into BMGY medium for induction, and then Western blot was used to analyze the expression level of recombinant hemagglutinin. Finally, glycosyl engineered yeast strains expressing recombinant H5 were obtained. Figure 12 ).

[0067] Example 4: Preparation of influenza virus hemagglutinin H5 yeast nanoparticle membrane protein antigen 1. Strains culture The strain from Example 3 was selected and inoculated into 200 mL of YPD seed culture medium (containing 100 μg / mL Zeocin), and cultured at 25°C with shaking at 210 rpm until OD200. 600 The inoculum was 15-20; it was transferred to BMGY medium at an inoculum of 5% (V / V), fermented at 25°C and 200 rpm for 36 hours, and then 1% (v / v) methanol was added to induce the expression of influenza virus hemagglutinin H5 yeast nanoparticle protein. The induction was repeated every 24 hours. After 48 hours of induction, the mixture was centrifuged at 4°C and 8500 rpm for 20 minutes, the supernatant was discarded, and the cell pellet was collected.

[0068] 2. Antigen extraction from H5N6-H5 lysate clarified solution Add 5 mM pH 7.0 EDTA buffer (1 g: 5 mL) to the bacterial cell pellet and stir magnetically for 12 min until the bacterial cells are completely resuspended. Transfer the resuspended solution to a high-pressure homogenizer and homogenize it three times at 800 bar. Centrifuge the homogenized lysate at 4 °C and 8500 rpm for 20 min, discard the pellet, and collect the supernatant, which is the H5N6-H5 lysate clarified antigen.

[0069] 3. Experimental grouping and mouse immunization experiment The immunization method is as follows: Twelve female BALB / c mice aged 6-8 weeks were randomly divided into the following two groups: Group 1: 100 μL of vaccine was administered intramuscularly on days 0 and 14. The vaccine used was 18 μg of H5 membrane protein antigen.

[0070] Group 2: 100 μL of vaccine was administered intramuscularly on days 0 and 14. The vaccine used was the X33 host bacterium lysate clarification.

[0071] Immunization schedule: A second immunization was administered 14 days after the first immunization, and the booster immunization cycle was 14 days. The intramuscular injection group received two immunizations.

[0072] Blood was collected from the orbital cavity of immunized mice before immunization and two weeks after the first immunization and two weeks after the second immunization.

[0073] The titer of anti-HA antibodies in the serum of mice in each group was measured using ELISA. The procedure is described in the "Concise Guide to Molecular Biology Experiments" [M]. Science Press, 2008. Fourteen days post-immunization, five mice from each group were randomly selected, and serum was collected (blood from the eye). The titer of serum H5-specific IgG antibodies (coated with purified H5 protein) was detected using an indirect ELISA method. The neutralizing activity of serum against H5 was detected using a hemagglutination inhibition assay.

[0074] Antibody titer such as Figure 13 As shown: Serum IgG titers in the blank control group were all <1×10⁻⁶ 2 The highest serum IgG titer in the experimental group after intramuscular injection of secondary immunoglobulin reached 1×10⁻⁶. 5.51 Blood coagulation inhibition titer: all in the blank control group were <1:10; the secondary immune serum titer in the intramuscular injection group reached 1:112.

[0075] 4. Blood coagulation inhibition test 1) Sample pretreatment: Mix the serum to be tested with receptor-destroying enzyme (RDE) at a ratio of 1:3, incubate at 37°C for 16-18 hours, and then heat-inactivate at 56°C for 30 minutes to inactivate RDE.

[0076] 2) In a 96-well V-bottom microplate, the serum was serially diluted twice at a starting ratio of 1:10. 20 μL of serum treated with LDE and 30 μL of PBS (i.e., 1:10 serum dilution) were added to the first well. 25 μL of PBS and an equal volume of serum were added to each of the remaining wells. 25 μL of the serum was then taken from the first well and serially diluted twice.

[0077] 3) Add 25 μL of rH5 purified protein antigen containing 4 HAU to each well, mix gently, and let stand at room temperature for 40 minutes to allow the specific antibodies in the serum to fully bind to the rH5 purified protein.

[0078] 4) Add 25 μL of 1% chicken red blood cell suspension to each well, mix gently, and let stand at room temperature for 30 minutes.

[0079] 5) After incubation, observe the morphology of red blood cells at the bottom of the well: tight precipitation (button-shaped) indicates that blood coagulation is inhibited; uniform spreading of red blood cells indicates that blood coagulation has occurred.

[0080] The above results indicate that using the H5N6-H5 lysate as an antigen can effectively induce a humoral immune response, and demonstrate that the antigen possesses good immunogenicity.

[0081] Example 5: Construction of a glycosyl engineered yeast strain expressing recombinant H3 1. Construction of pPICZαA-H3 (2012) plasmid Based on the amino acid sequence of the HA gene (GenBenk number: AGB97282.1) of influenza A / Kyrgyzstan / 3051 / 2012 (H3N2) virus published in GenBank, the HA gene was synthesized using GenScript (GenScript, Nanjing) with codon optimization. The synthesized HA gene fragment was then inserted into the pPICZαA vector. Xho I and Not Between the I restriction sites, the expression plasmid pPICZαA-H3 (2012) was obtained.

[0082] 2. Construction of glycosyl engineered yeast expression strains pPICZαA-H3 (2012) plasmid for use Bgl After II enzyme digestion, the linearized expression vector was transformed into glycosyl engineered yeast GJK01 by electroporation. Positive clones were screened using YPD / Zeocin plates, inoculated into BMGY medium for induction, and then Western blot was used to analyze the expression level of recombinant hemagglutinin. Finally, glycosyl engineered yeast strains expressing recombinant H3 were obtained. Figure 14 ).

[0083] Example 6: Preparation of influenza virus hemagglutinin H3 yeast nanoparticle membrane protein antigen 1. Strains culture The strain from Example 5 was selected and inoculated into 200 mL of YPD seed medium (containing 100 μg / mL Zeocin), and cultured at 25°C with shaking at 210 rpm until OD200. 600The inoculum was 15-20; it was transferred to BMGY medium at an inoculum of 5% (V / V), fermented at 25°C and 200 rpm for 36 hours, and then 1% (v / v) methanol was added to induce the expression of influenza virus hemagglutinin H3 yeast nanoparticle protein. The induction was repeated every 24 hours. After 48 hours of induction, the mixture was centrifuged at 4°C and 8500 rpm for 20 minutes, the supernatant was discarded, and the bacterial precipitate was collected.

[0084] 2. Antigen extraction from H3N2-H3 lysate clarified solution Add 5 mM pH 7.0 EDTA buffer (1 g: 5 mL) to the bacterial cell pellet and stir magnetically for 12 min until the bacterial cells are completely resuspended. Transfer the resuspended solution to a high-pressure homogenizer and homogenize it three times at 800 bar. Centrifuge the homogenized lysate at 4 °C and 8500 rpm for 20 min, discard the pellet, and collect the supernatant, which is the H3N2-H3 lysate clarified antigen.

[0085] 3. Experimental grouping and mouse immunization experiment The immunization method is as follows: Twelve female BALB / c mice aged 6-8 weeks were randomly divided into the following two groups: Group 1 immunization: 100 μL of vaccine was injected intramuscularly on days 0 and 14. The vaccine used was 18 μg of H3 membrane protein antigen.

[0086] Group 2: 100 μL of vaccine was administered intramuscularly on days 0 and 14. The vaccine used was the X33 host bacterium lysate clarification.

[0087] Immunization schedule: A second immunization was administered 14 days after the first immunization, and the booster immunization cycle was 14 days. The intramuscular injection group received two immunizations.

[0088] Blood was collected from the orbital cavity of immunized mice before immunization and two weeks after the first immunization and two weeks after the second immunization.

[0089] The titer of anti-HA antibodies in the serum of mice in each group was measured using ELISA. The procedure is described in the "Concise Guide to Molecular Biology Experiments" [M]. Science Press, 2008. Fourteen days post-immunization, five mice from each group were randomly selected, and serum was collected (blood from the eye). The titer of serum H3-specific IgG antibodies (coated with purified H3 protein) was detected using indirect ELISA. The neutralizing activity of serum against H3 was detected using a hemagglutination inhibition assay.

[0090] Antibody titer such as Figure 15 As shown: Serum IgG titers in the blank control group were all <1×10⁻⁶ 2 The highest serum IgG titer in the experimental group after intramuscular injection of secondary immunoglobulin reached 1×10⁻⁶. 4.86 Blood coagulation inhibition titer: all in the blank control group were <1:10; the secondary immune serum titer in the intramuscular injection group reached 1:45.

[0091] 4. Blood coagulation inhibition test 1) Sample pretreatment: Mix the serum to be tested with receptor-destroying enzyme (RDE) at a ratio of 1:3, incubate at 37°C for 16-18 hours, and then heat-inactivate at 56°C for 30 minutes to inactivate RDE.

[0092] 2) In a 96-well V-bottom microplate, the serum was serially diluted twice at a starting ratio of 1:10. 20 μL of serum treated with LDE and 30 μL of PBS (i.e., 1:10 serum dilution) were added to the first well. 25 μL of PBS and an equal volume of serum were added to each of the remaining wells. 25 μL of the serum was then taken from the first well and serially diluted twice.

[0093] 3) Add 25 μL of rH3 purified protein antigen containing 4 HAU to each well, mix gently, and let stand at room temperature for 40 minutes to allow the specific antibodies in the serum to fully bind to the rH3 purified protein.

[0094] 4) Add 25 μL of 1% chicken red blood cell suspension to each well, mix gently, and let stand at room temperature for 30 minutes.

[0095] 5) After incubation, observe the morphology of red blood cells at the bottom of the well: tight precipitation (button-shaped) indicates that blood coagulation is inhibited; uniform spreading of red blood cells indicates that blood coagulation has occurred.

[0096] The above results indicate that using the H3N2-H3 lysate as an antigen can effectively induce a humoral immune response, and demonstrate that the antigen possesses good immunogenicity.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing influenza virus hemagglutinin protein HA, characterized in that, The process includes the following steps: inducing the expression of the influenza virus hemagglutinin protein HA in engineered cells, collecting the precipitate and lysing the cells, centrifuging and collecting the supernatant, which contains the influenza virus hemagglutinin protein HA. The chassis cells of the engineered cells are cells capable of glycosylation modification.

2. The preparation method according to claim 1, characterized in that, The influenza viruses mentioned include influenza A, influenza B, influenza C, and influenza D viruses.

3. The preparation method according to claim 2, characterized in that, The serotypes of the hemagglutinin protein HA derived from the influenza A virus include H1, H3, H5, or H7.

4. The preparation method according to claim 1, characterized in that, The cell type is a eukaryotic cell.

5. The preparation method according to claim 4, characterized in that, The eukaryotic cells include yeast cells, mammalian cells, or insect cells.

6. The preparation method according to claim 5, characterized in that, The yeast cell package is glycosyl engineered yeast GJK01.

7. The preparation method according to claim 6, characterized in that, The expression of the influenza virus hemagglutinin protein HA was induced using methanol.

8. An influenza virus hemagglutinin protein HA antigen extract prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The antigen extract is the supernatant obtained after the engineered cells are broken and centrifuged.

9. The use of the influenza virus hemagglutinin protein HA antigen extract as a vaccine antigen in the preparation of influenza vaccines according to claim 8.

10. An influenza vaccine, characterized in that, The influenza vaccine uses the influenza virus hemagglutinin protein HA antigen extract as the core antigen component as described in claim 8, and also contains pharmaceutically acceptable diluents and / or adjuvants.

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