QMC influenza virus seed as well as preparation method and application thereof

Abstract

The invention discloses a QMC influenza virus seed as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. According to the preparation method of the QMC virus seed, virus passage adaptation and virus seed preparation are carried out synchronously, and the preparation method comprises the following steps: inoculating an MDCK-XF06 cell with a chick embryo source influenza virus, and culturing to obtain a resuscitation generation virus seed; and then inoculating the resuscitation generation virus seeds on the MDCK cells in a manner of adjusting the virus inoculation infection complex number generation by generation, and culturing to obtain P2-generation main seed batch virus seeds and P3-generation working seed batch virus seeds. According to the preparation method disclosed by the invention, the QMC virus seed with remarkable advantages can be obtained, the virus activity is good, the virus titer is high, a large number of live viruses can be efficiently produced, the genetic stability is excellent, and the consistency and effectiveness of product quality are ensured; in addition, the virus seed completely eradicates tumorigenicity risks, is high in safety and lays a solid foundation for subsequent production of safe, efficient and stable vaccine products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a QMC influenza virus strain, its preparation method, and its application. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus. It can cause symptoms such as fever, runny nose, sore throat, cough, headache, muscle pain and general malaise. Severe cases can lead to various complications such as viral pneumonia, secondary bacterial pneumonia, acute respiratory distress syndrome, myocarditis, encephalitis or myositis and rhabdomyolysis, sepsis and multiple organ failure, and even death.

[0003] Currently available influenza vaccines mainly include trivalent, quadrivalent, trivalent adjuvanted, and quadrivalent adjuvanted influenza vaccines, as well as H1N1 influenza vaccines. Influenza viruses can proliferate in chicken embryos and passaged cells, but the choice between culturing in chicken embryos or cells should be based on the host source of the virus. Most avian viruses replicate very efficiently in the allantoic cavity of chicken embryos; however, some human and other mammalian influenza viruses replicate less efficiently in chicken embryos. Furthermore, passage of mammalian influenza viruses in chicken embryos may lead to mutations in receptor binding specificity and antigenicity. Therefore, it is best to proliferate mammalian-derived influenza viruses in mammalian cells. Currently, the production substrate for influenza vaccines is gradually shifting from chicken embryos to mammalian cells. Common cell lines used for influenza vaccine development include MDCK, Vero, insect cells SF9, and PER.C6. MDCK cells, as the most sensitive cell line for influenza viruses, have been widely used in influenza vaccine production.

[0004] Influenza vaccines produced and marketed abroad already use MDCK cells, insect SF9 cells, etc. as cell substrates, but the MDCK cells used to prepare the virus strains have tumorigenicity, posing a safety risk when used in vaccine production.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a QMC influenza virus strain, its preparation method, and its application. The QMC influenza virus strain prepared by this invention has the characteristics of better safety, shorter production cycle, higher yield, better immunogenicity, and higher genetic stability.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for preparing QMC influenza virus strain, wherein the preparation method involves simultaneous virus passage adaptation and strain preparation, comprising: The original influenza virus strain derived from chicken embryos was inoculated into MDCK cells, and after culture, a revived virus seed was obtained. Then, the revived virus seed was inoculated into MDCK cells in a manner that adjusted the virus multiplicity of infection in each generation. After culture, the master seed batch of P2 generation and the working seed batch of P3 generation were obtained. Among them, the MDCK cells were MDCK-XF06 cells, with the preservation number CCTCC NO: C2023328; the chicken embryo-derived influenza viruses included influenza A virus and influenza B virus.

[0008] Secondly, the present invention provides the QMC influenza virus strain obtained by the above preparation method.

[0009] Thirdly, the present invention also provides the application of the above-mentioned QMC influenza virus strain in the production of influenza vaccines.

[0010] The present invention has the following beneficial effects: This invention presents a method for preparing QMC influenza virus strains based on MDCK-XF06 cells. This method successfully yields QMC virus strains with significant advantages, exhibiting not only excellent culture performance (high viral viability and viral titer) and efficient production of large quantities of live virus, but also outstanding stability, including good genetic stability, ensuring the consistency and effectiveness of product quality. Furthermore, this strain completely eliminates the risk of tumorigenesis, demonstrating high safety and laying a solid foundation for the subsequent production of safe, efficient, and stable vaccine products. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 Flowchart of the preparation process for QMC influenza virus strain; Figure 2 A schematic diagram of the immunogenicity GMT of the trivalent influenza virus split vaccine (MDCK cells); Figure 3 A schematic diagram of the immunogenicity GMT of a quadrivalent influenza virus split vaccine (MDCK cells); Figure 4 Schematic diagram of GMT immunogenicity of trivalent adjuvanted influenza virus split vaccine (MDCK cells) A1 type; Figure 5 Schematic diagram of GMT immunogenicity of trivalent adjuvanted influenza virus split vaccine (MDCK cells) A3 type; Figure 6 This is a schematic diagram of the immunogenicity GMT of the trivalent adjuvanted influenza virus split vaccine (MDCK cells) BV type. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] QMC (Quality Controlled Cell) virus seed is a set of initial virus seed systems that have undergone comprehensive identification, testing, and are under strict quality control management for the production of biological products (such as vaccines).

[0015] In modern biopharmaceutical manufacturing, to ensure the safety, efficacy, and consistent quality of every batch, global regulatory agencies (such as the FDA, EMA, and NMPA) mandate the use of a two-tiered cell bank system and a virus seed system. The cell bank acts as the "factory" for the virus, serving as the vector for its mass cultivation and replication. Cell banks are divided into two levels: the Master Cell Bank (MCB) and the Working Cell Bank (WCB). The MCB consists of a large quantity of homogeneous cells prepared and packaged in a single batch from original cells after expansion. It is the original source of cells for all production and the starting point of production. The WCB is prepared by further expanding and packaging cells from the MCB and is used directly in production. The MCB is properly stored, and cells are taken from the Working Cell Bank for each production run. This minimizes manipulation of the MCB and ensures its originality. A virus seed bank serves as a "template" for production. Viruses used in production require the establishment of a seed bank system, which mainly consists of a Master Virus Seed (MVS) and a Working Virus Seed (WVS). MVS is a fully identified and validated stock solution of viruses obtained by culturing on MCB or WCB cells. It is aliquoted and stored once and is the original source of all viruses used in production. WVS is prepared by amplifying MVS and is used directly for large-scale vaccine production.

[0016] Therefore, a very rigorous set of quality testing procedures are conducted on them to ensure their safety (no exogenous contaminants, no safety risks), consistency (stable generation, stable quality), and effectiveness (sufficient viral titer, high sensitivity to cells).

[0017] Currently, only CSL Company abroad has the capability to prepare QMC seed cells using MDCK cells, but the specific preparation process is not publicly available. There are also no reports on QMC seed preparation processes in China. Furthermore, even if CSL Company could prepare QMC seed cells, their tumorigenicity would pose safety concerns for vaccine production. To fill the gap in domestic QMC seed preparation technology and overcome the safety issues present in existing QMC seed preparation technologies, the inventors propose a method for preparing QMC seed cells.

[0018] Regarding the issue of tumorigenicity, this invention proposes using the first reported non-tumorigenic MDCK cells (MDCK-XF06 cells) as a culture medium to prepare QMC virus strains, thereby addressing safety concerns. However, since there are currently no relevant technical guidelines for QMC virus strain preparation, and MDCK-XF06 cells are a new cell line (MDCK cell lines from different sources, substrains, and with different domestication methods exhibit significant differences in culture methods, viral adaptability, final yield, and effectiveness when preparing influenza virus strains), there is no suitable reference method for preparing QMC virus strains using MDCK-XF06 cells. Therefore, through extensive experimental research, the inventors have developed a method for preparing QMC virus strains using MDCK-XF06 cells.

[0019] The method for preparing QMC virus seed proposed in this invention is as follows: Chicken embryo-derived influenza virus is inoculated into MDCK cells, and after culture, a revived generation of virus seed is obtained; then, the revived generation of virus seed is inoculated into MDCK cells in a manner that adjusts the virus inoculation multiplicity with each generation, and after culture, the master seed batch of P2 generation and the working seed batch of P3 generation are obtained.

[0020] Among them, the MDCK cells are MDCK-XF06 cells, which are deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2023328, and have been disclosed in Chinese patent 202410695674.1.

[0021] MDCK-XF06 cells have been verified to be non-tumorigenic and sensitive to influenza virus. However, sensitivity to influenza virus means that the influenza virus can proliferate on these cells, but it does not necessarily mean that a dominant strain can be produced. In order to obtain a dominant strain, this invention obtains the desired dominant strain through specific technical means.

[0022] The original strains used in this invention to prepare QMC influenza virus strains are chicken embryo-derived influenza viruses introduced from authoritative institutions, including influenza A virus and influenza B virus.

[0023] When viral strains isolated from chicken embryos are inoculated into MDCK cells, their replication efficiency is not high. This is because the viruses have already adapted to the chicken embryo environment. When switched to the MDCK cell environment, the viruses need a readaptation process. This process typically involves inoculating chicken embryo-derived viruses into MDCK cells and waiting for them to replicate; then harvesting the culture and inoculating it into new MDCK cells, repeating this process continuously. During this process, viral strains that can more effectively bind to MDCK cell receptors and utilize their intracellular mechanisms for replication are selected and gradually become the dominant strain. After multiple generations of adaptation, viruses that can replicate efficiently in MDCK cells can be obtained for large-scale production. Therefore, the conventional method for preparing viral strains in this field is to first adapt and passage (about 10 generations) before establishing a viral strain library.

[0024] In this invention, the inventors adopted a virus strain library construction strategy that simultaneously carried out virus passage adaptation and strain preparation - adjusting the virus inoculation multiple of infection (MOI) generation by generation. The chicken embryo-derived virus was passed through cells for three generations, thus establishing the master seed batch (P2) and working seed batch (P3) for influenza vaccine production. This simultaneously achieved the proliferation adaptation of the chicken embryo-derived virus on MDCK-XF06 cells and strain preparation.

[0025] Passage is the process of transferring a virus from one culture environment to another. In this invention, P2 refers to the virus seed after two passages, and P3 refers to the virus seed after three passages. Compared to conventional passage at a fixed MOI, the vaccination method of this invention can shorten the number of passages, which reduces the probability of influenza virus mutation and ensures vaccine effectiveness. It can also produce more QMC influenza virus in the same amount of time, with the advantages of short production cycle and high yield.

[0026] Specifically, the preparation process of the QMC influenza virus strain provided by this invention is as follows: Figure 1 As shown, the operation steps are as follows: S1. Add the thawed working bank cells MDCK-XF06 to SFM-MDCK medium, centrifuge, collect the precipitate and add it to SFM-MDCK medium, repeatedly pipette to prepare a cell resuspension, and culture it in a shaker; take the well-grown cells and passage them at an appropriate density, and after culture, dilute the well-grown passaged cells and prepare a cell suspension; S2. Chicken embryo-derived influenza virus strains were inoculated into the cell suspension of MDCK cells according to the inoculation ratio, and then placed in a shaker for the first culture. Then, a culture medium with a hemagglutination titer ≥1:160 was selected, and the supernatant was collected by centrifugation to obtain the resuscitated virus strain. S3. The resuscitated virus seed was inoculated into the cell suspension of MDCK cells according to the inoculation ratio, and cultured for the second time. Then, a culture medium with a hemagglutination titer ≥1:160 was selected, and the supernatant was collected by centrifugation to obtain the master seed batch virus. S4. Inoculate the master seed batch virus into the cell suspension of MDCK cells according to the inoculation ratio, and carry out the third culture. Then select the culture medium with a hemagglutination titer ≥1:160, centrifuge and collect the supernatant to obtain the working seed batch virus.

[0027] In the above preparation process, the introduced original virus strain, resuscitated virus strain, and master seed batch virus strain were inoculated at a virus inoculation ratio of 10:1. -3 ~10 -8 The inoculation was carried out within a certain range, and the inoculation ratio showed a decreasing trend with each generation, decreasing by 1 to 2 exponential levels. Then, the culture medium with a hemagglutination titer ≥ 1:160 was selected for centrifugation and the supernatant was collected to obtain the ideal strain.

[0028] In some embodiments, the culture conditions of the cell resuspension in S1 are: temperature of 36~38℃, CO2 concentration of 4%~6%, rotation speed of 80~180rpm, and culture time of 72~120h.

[0029] In some embodiments, the conditions for cell passage culture in S1 are: a seeding density of 60 × 10⁶ cells / year. 4 ~200×10 4 The cells / ml incubation period was 36-38℃, CO2 concentration was 4%-6%, rotation speed was 80-180 rpm, and incubation time was 60-84 h.

[0030] In some embodiments, the density of MDCK cells in the cell suspension prepared in S1 is 200 × 10⁻⁶. 4 ~500×10 4 The final concentration of TPCK trypsin was 1.0~5.0 μg / ml.

[0031] In some embodiments, the conditions for the first virus culture are: temperature of 32~36℃, CO2 concentration of 4%~6%, rotation speed of 60~180rpm, and culture time of 36~84h.

[0032] In some embodiments, the conditions for the second virus culture are: temperature of 32~36℃, CO2 concentration of 4%~6%, rotation speed of 60~180rpm, and culture time of 36~84h.

[0033] In some embodiments, the conditions for the third virus culture are: temperature of 32~36℃, CO2 concentration of 4%~6%, rotation speed of 60~180rpm, and culture time of 36~84h.

[0034] In some embodiments, influenza A viruses include H1N1 and H3N2 influenza viruses; influenza B viruses include BV and BY influenza viruses.

[0035] Furthermore, when preparing influenza virus seed using MDCK cells, selecting the optimal infection time is also one of the decisive factors in obtaining high viral titers. In this invention, the suitable infection period is the late logarithmic growth stage of the cells, specifically when the cell density is ≥500 × 10⁻⁶. 4 cells / ml, cell viability ≥90%.

[0036] The preparation method provided by this invention can obtain QMC influenza virus strains with better safety, shorter production cycle, higher yield, better immunogenicity, and higher genetic stability, and has good application prospects in the preparation of influenza virus vaccines.

[0037] Using the aforementioned QMC influenza virus strains, influenza vaccines that can be prepared include trivalent influenza vaccines, quadrivalent influenza vaccines, trivalent adjuvanted influenza vaccines, quadrivalent adjuvanted influenza vaccines, H1N1 vaccines, and other influenza-related vaccines.

[0038] Among them, the trivalent influenza vaccine contains antigens of three types: H1N1, H3N2, and BV, or antigens of three types: H1N1, H3N2, and BY; the quadrivalent influenza vaccine contains antigens of four types: H1N1, H3N2, BV, and BY; the trivalent and quadrivalent adjuvanted influenza vaccines are based on the trivalent and quadrivalent influenza vaccines with the addition of adjuvants, which are selected from any one or more of the following: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin, anionic liposome adjuvant containing saponin, CpG adjuvant, nanoemulsion, and adjuvant containing 3D-MPL.

[0039] In some embodiments, each dose unit of the influenza vaccine contains 3-65 μg of HA antigen protein. Experimental verification has shown that the influenza vaccine prepared from the QMC influenza virus strain of this invention has good immunogenicity.

[0040] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0041] In embodiments of the present invention, the strain used for the H1N1 influenza virus is 23 / 250 (NIBSC strain number) of the WHO recommended strains for 2024-2025; the strain used for the H3N2 influenza virus is 23 / 206 (NIBSC strain number) of the WHO recommended strains for 2024-2025; the strain used for the BV influenza virus is 22 / 204 (NIBSC strain number) of the WHO recommended strains for 2024-2025; and the strain used for the BY influenza virus is 21 / 132 (NIBSC strain number) of the WHO recommended strains for 2023-2024.

[0042] Example 1 This example demonstrates the preparation of the MDCK working cell bank, with the following specific steps: (1) Cell resuscitation Remove MDCK-XF06 master cell bank cells from the liquid nitrogen tank and thaw them in purified water at 37℃±2℃. In a clean bench, use a pipette / pipette to transfer the cells to centrifuge tubes containing cell culture medium. Tighten the caps and centrifuge at 1000 rpm for 10 min, discarding the supernatant. Add 40 ml of SFM-MDCK medium, mix well, and transfer to a shake flask for incubation at 37℃ with shaking for 4 days.

[0043] (2) Cell passage Take well-grown resuscitation passages with a cell density of 500 × 10⁶ cells / year. 4 Cells with a density of 90% or higher and a cell viability of 90% or higher were used to confirm cell density and viability, and the concentration was determined at 100 × 10⁻⁶ cells / ml. 4 Subculture the cells / ml seeding density into appropriately sized shake flasks. After subculturing, tighten the caps and place the flasks in a shaker at 37±1℃, 5% CO2 concentration, and 120 rpm for 3 days. Continue subculturing to the desired volume.

[0044] (3) Cell cryopreservation Collect cells into centrifuge flasks, centrifuge at 1000 rpm for 10 min, discard the supernatant, add cryopreservation solution, mix well by pipetting, and aliquot into cryovials. Incubate at 2-8℃ for 2 h, then freeze overnight at -60℃ or below, and transfer to liquid nitrogen for storage to obtain the working cell bank MDCK-XF06.

[0045] Example 2 This example describes the preparation of the QMC influenza virus strain. The specific steps are as follows: (1) Preparation of MDCK cells MDCK cell resuscitation: Prepare purified water at 37℃±2℃ in a suitable container. Take out the working bank cells MDCK-XF06 from the liquid nitrogen tank. Immediately place the cell cryopreservation tube into warm water and quickly shake it in a water bath to thaw the cells. In a clean workbench, use a pipette / pipette to transfer the cells to centrifuge tubes containing cell culture medium. Tighten the centrifuge tube caps and centrifuge at 1000 rpm for 10 min. Discard the supernatant. Add 40 ml of SFM-MDCK culture medium to the collected cell pellet and repeatedly pipette to resuspend the cells. Transfer the cell suspension to a shake flask and place it in a shaker at 37±1℃, 5% CO2 concentration, and 120 rpm for 84 h.

[0046] MDCK cell passage: Harvest well-grown cells (cell density reaching 500 × 10⁶). 4 Cell density and cell viability were confirmed by measuring cells / ml or higher (cells with a viability of 90% or higher) and then 100 × 10⁻⁶ cells / ml.4 Subculture the cells / ml seeding density into appropriately sized shake flasks. After subculturing, tighten the caps and place the flasks in a shaker at 37±1℃, CO2 concentration of 4%~6%, and rotation speed of 80~180 rpm for 72 h. Continuously subculture to the required cell volume for virus preparation.

[0047] (2) Preparation of main seed batch of virus seed Virus inoculation: Harvest well-grown MDCK cells and confirm cell density and viability (cell density reaches 500 × 10⁶). 4 Cells / ml or higher, cell viability of 90% or higher), cells were packed at 400 × 10⁻⁶. 4 Dilute the virus at a density of cells / ml to a suitable size shake flask, and inoculate at a virus inoculation ratio of 10:1. -4 The virus was inoculated into MDCK cell suspension, and the final concentration of TPCK trypsin solution in the culture system was 1.0~5.0ug / ml.

[0048] Virus culture harvest: After inoculation with the virus, the cells were placed in a shaker at 34°C, CO2 concentration of 5%, and rotation speed of 120 rpm for 72 h. The culture medium with relatively high hemagglutination titer (hemagglutination titer ≥ 1:160) was selected, and the cells were centrifuged at 5000xg for no less than 10 min. The supernatant was collected and combined in a sterile storage bottle as the resuscitation substitute.

[0049] Passaging: Take the revived virus strain and administer it at a ratio of 10:10. -5 Inoculate into MDCK cells, culture the virus using the same method as above, and after harvest, package into batches to obtain the master seed virus.

[0050] (3) Batch preparation of working seeds Virus inoculation: Harvest well-grown MDCK cells and confirm cell density and viability (cell density reaches 500 × 10⁶). 4 Cells / ml or higher (cells with a viability of 90% or higher), cells were packed at 400 × 10⁻⁶. 4 Dilute the virus at a density of cells / ml to a suitable size shake flask, and inoculate the master seed batch with 10 at the appropriate ratio. -6 The final concentration of TPCK trypsin solution in the culture system was 3 μg / ml, added to the MDCK cell suspension.

[0051] Virus culture harvest: After inoculation with the virus, the cells were placed in a shaker at 34℃, CO2 concentration of 5%, and rotation speed of 120 rpm for 72 h. The culture medium with relatively high hemagglutination titer (hemagglutination titer ≥ 1:160) was selected, and the cells were centrifuged at 5000xg for no less than 10 min. The supernatant was collected and combined in a sterile storage bottle and dispensed to obtain the working seed batch of virus.

[0052] Example 3 This example describes the production of a trivalent / quadrivalent influenza vaccine. The specific steps are as follows: 1. Production of raw materials Batch cell preparation: Resuscitate one or more working cell banks and passage them continuously to production scale.

[0053] Upstream production: The working seed batch of virus seeds is produced at an MOI of 2 × 10⁻⁶. -2 ~10 -10 Inoculate the cells and incubate at 34°C for 72 hours. Stop the culture and centrifuge to harvest the supernatant.

[0054] Downstream production: The supernatant is ultrafiltered according to conventional methods, and then subjected to enzymatic digestion, inactivation, purification, lysis, removal of lysis agent, sterilization and other process steps to obtain the monovalent stock solution.

[0055] 2. Formulation production Calculate the required volume of monovalent stock solutions for H1N1, H3N2, and BV, or H1N1, H3N2, and BY, or H1N1, H3N2, BV, and BY, based on a preparation point of 30-36 μg / ml. Transfer the stock solutions to a preparation container, add 10 mM PBS solution to the prepared volume, mix well, and dispense into the final product. The final product should be a colorless or slightly milky white liquid, free of foreign matter.

[0056] The amount of stock solution added = preparation point × preparation volume / stock solution hemagglutinin content.

[0057] Example 4 This example describes the production of a trivalent / quadrivalent adjuvanted influenza vaccine. The specific steps are as follows: 1. Production of raw materials Batch cell preparation: Resuscitate one or more working cell banks and passage them continuously to production scale.

[0058] Upstream production: The working seed batch of virus seeds is produced at an MOI of 2 × 10⁻⁶. -2 ~10 -10 Inoculate the cells and incubate at 34°C for 72 hours. Stop the culture and centrifuge to harvest the supernatant.

[0059] Downstream production: The supernatant is ultrafiltered using conventional methods, and then subjected to enzymatic digestion, inactivation, purification, lysis, removal of lysis agent, and sterilization to obtain the monovalent stock solution.

[0060] 2. Formulation production Calculate the required volume of monovalent stock solutions of H1N1, H3N2, BV (triple type), H1N1, H3N2, BY (triple type), or H1N1, H3N2, BV, BY (quadruple type). Transfer the stock solutions to a preparation container, add the standard dose of adjuvant (PolyI:C, XA301, XA302, XA401, and CPG1018), add 10 mM PBS solution to the prepared volume, mix well, and dispense into the final product. The final product should be a white, homogeneous liquid free of foreign matter.

[0061] The amount of stock solution added = preparation point × preparation volume / stock solution hemagglutinin content.

[0062] Example 5 This example illustrates the immunogenicity analysis of a trivalent (H1N1, H3N2, BV) influenza vaccine, as detailed below: The experimental animals were healthy SD rats aged 5-7 weeks, which were intramuscularly injected with trivalent influenza virus split vaccine (MDCK cells). The dosage design is shown in Table 1.

[0063] The drugs were administered once each on days 1 and 29 of the experiment, with a total observation period of 8 weeks. Blood samples were collected before administration on days 1 and 29 and on day 56 of the experiment for neutralizing antibody detection using the HA-HI method (hemagglutination and hemagglutination inhibition assay). On day 57 of the experiment, rats were dissected, and spleens from each group were collected for analysis of cellular immune indicators using flow cytometry.

[0064] Table 1 Dosage Design Table

[0065] like Figure 2 As shown, the GMT results of the immunogenicity of the trivalent influenza virus split vaccine were all greater than the WHO's 1:40 serological positivity standard.

[0066] Example 6 This example describes the immunogenicity analysis of a quadrivalent influenza vaccine, as detailed below: The experimental animals were healthy SD rats aged 5-7 weeks, which were injected intramuscularly with a quadrivalent influenza virus split vaccine (MDCK cells). The dosage design is shown in Table 2.

[0067] The drugs were administered once each on days 1 and 29 of the experiment, with a total observation period of 8 weeks. Blood samples were collected before administration on days 1 and 29 and on day 56 of the experiment for neutralizing antibody detection using the HA-HI method (hemagglutination and hemagglutination inhibition assay). On day 57 of the experiment, rats were dissected, and spleens from each group were collected for analysis of cellular immune indicators using flow cytometry.

[0068] Table 2 Dosage Design Table

[0069] like Figure 3As shown, the immunogenicity of the quadrivalent influenza virus split vaccine met the GMT standard.

[0070] Example 7 This example illustrates the immunogenicity analysis of a trivalent (H1N1, H3N2, BV) adjuvanted influenza vaccine, as detailed below: The experimental animals were healthy SD rats aged 5-7 weeks. They were injected intramuscularly with trivalent adjuvanted influenza virus split vaccine (MDCK cell), trivalent influenza virus split vaccine (MDCK cell), and commercially available trivalent influenza virus split vaccine. The dosage design is shown in Table 3.

[0071] The drugs were administered once each on days 1 and 29 of the experiment, with a total observation period of 8 weeks. Blood samples were collected before administration on days 1 and 29 and on day 56 of the experiment for neutralizing antibody detection using the HA-HI method (hemagglutination and hemagglutination inhibition assay). On day 57 of the experiment, rats were dissected, and spleens from each group were collected for analysis of cellular immune indicators using flow cytometry.

[0072] Table 3 Dosage Design Table

[0073] like Figure 4 , 5 As shown in Figures 6 and 7, the trivalent adjuvanted influenza virus split vaccine is more effective than the influenza vaccine without adjuvant.

[0074] Example 7 This example is a study on the genetic stability of the QMC strain, as detailed below: Four types of QMC virus strains were inoculated into MDCK cells and passaged for 11 generations. The nucleic acid sequences corresponding to the HA and NA proteins of the 1st, 2nd, 3rd, 6th and 11th generations were determined to analyze the stability of the amino acid sequences of the HA and NA proteins of the QMC virus strains.

[0075] Table 4. Amino acid mutation analysis of HA and NA proteins

[0076] As shown in Table 4, the QMC strain prepared by this invention has good genetic stability in subsequent generations.

[0077] Experimental Example 1 To verify the differences in genetic stability of influenza virus strains obtained by different preparation methods, this experiment compared the mutations of chicken embryo virus strains obtained by conventional methods in chicken embryos and MDCK-XF06 cells after continuous passage, and the mutations of QMC virus strains prepared in Example 2 in MDCK-XF06 cells after continuous passage. The results are shown in Tables 5-8: Table 5. Statistical Table of Genetic Stability Mutations in H1N1 Influenza Viruses

[0078] Table 6. Statistical Table of Genetic Stability Mutations in H3N2 Influenza Viruses

[0079] Table 7. Statistical Table of Genetic Stability Mutations in Influenza V Virus Strains

[0080] Table 8. Statistical table of genetic stability mutations in BY-type influenza virus strains.

[0081] Note: "-" indicates no amino acid mutation.

[0082] As can be seen from the table above, after continuous passage of chicken embryo virus strains in chicken embryos and cells, the mutation sites of H1N1 and H3N2 influenza virus strains increase with the number of passages, while no mutations are found in the four types of QMC virus strains after 10 consecutive passages in cells.

[0083] Experiment Example 2 To verify the adaptability of virus strains obtained by different preparation methods in influenza vaccine production, this experiment compared the hemagglutinin content (μg / mL) of the virus harvest fluid from chicken embryo virus strains prepared by conventional methods and cell virus strains prepared in Example 2. The results are shown in Table 9. Table 9 Hemagglutinin content in harvested fluid

[0084] As shown in Table 9, using cell-prepared virus strains for the production of cell matrix influenza vaccines results in higher yields and more controllable quality.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a QMC influenza virus strain, characterized in that, The preparation method involves simultaneously performing virus passage adaptation and preparing the viral strain, and includes: The original influenza virus derived from chicken embryos was inoculated into MDCK cells, and after culture, a revived virus seed was obtained. Then, the revived virus seed was inoculated into MDCK cells in a manner that adjusted the virus inoculation multiplicity in each generation. After culture, the master seed batch of P2 generation and the working seed batch of P3 generation were obtained. The MDCK cells are MDCK-XF06 cells, with the accession number CCTCC NO: C2023328; the viruses include influenza A virus and influenza B virus.

2. The preparation method according to claim 1, characterized in that, The method for adjusting the multiplicity of infection through vaccination generation by generation is as follows: at 10 -3 ~10 -8 The infection multiplicity decreases with each generation as the vaccination rate decreases.

3. The preparation method according to claim 1, characterized in that, The influenza A viruses mentioned include H1N1 influenza virus and H3N2 influenza virus.

4. The preparation method according to claim 1, characterized in that, The influenza B virus includes both BV and BY influenza viruses.

5. The preparation method according to claim 1, characterized in that, The method for preparing the resuscitation progeny includes: Thawed working bank cells MDCK-XF06 were added to SFM-MDCK medium, centrifuged, and the precipitate was collected and added to SFM-MDCK medium. The cells were repeatedly pipetted to prepare a cell resuspension and cultured in a shaker. Well-grown cells were passaged at an appropriate density. After culture, the well-grown passaged cells were diluted and prepared into a cell suspension. Chicken embryo-derived influenza virus strains were inoculated into the cell suspension of the MDCK cells according to the inoculation ratio, and then placed in a shaker for the first virus culture. Then, a culture medium with a hemagglutination titer ≥1:160 was selected, and the supernatant was collected by centrifugation to obtain the resuscitated progeny. The culture conditions for the cell resuspension are: temperature 36~38℃, CO2 concentration 4%~6%, rotation speed 80~180rpm, and culture time 72~120h; The conditions for the subculture were: an inoculation density of 60 × 10⁶. 4 ~200×10 4 The cells / ml ratio was set at a temperature of 36-38℃, a CO2 concentration of 4%-6%, a rotation speed of 80-180 rpm, and a culture time of 60-84 h. The cell density of the passaged cells is ≥500×10⁻⁶. 4 cells / ml, cell viability ≥90%; The density of MDCK cells in the cell suspension was 200 × 10⁻⁶. 4 ~500×10 4 cells / ml, TPCK trypsin final concentration is 1.0~5.0μg / ml; The conditions for the first virus culture were: temperature 32~36℃, CO2 concentration 4%~6%, rotation speed 60~180rpm, and culture time 36~84h.

6. The preparation method according to claim 5, characterized in that, The method for preparing the master seed batch virus includes: inoculating the resuscitated progenitor virus into the cell suspension of the MDCK cells according to the inoculation ratio, performing a second virus culture, then selecting a culture medium with a hemagglutination titer ≥1:160, centrifuging and collecting the supernatant to obtain the master seed batch virus; The conditions for the second virus culture were: temperature 32~36℃, CO2 concentration 4%~6%, rotation speed 60~180rpm, and culture time 36~84h.

7. The preparation method according to claim 6, characterized in that, The method for preparing the working seed batch virus includes: inoculating the master seed batch virus into the cell suspension of the MDCK cells according to the inoculation ratio, performing a third virus culture, then selecting a culture medium with a hemagglutination titer ≥1:160, centrifuging and collecting the supernatant to obtain the working seed batch virus; The conditions for the third virus culture were: temperature 32~36℃, CO2 concentration 4%~6%, rotation speed 60~180rpm, and culture time 36~84h.

8. The QMC influenza virus strain obtained by the preparation method according to any one of claims 1 to 7.

9. The application of the QMC influenza virus strain as described in claim 8 in the production of influenza vaccines.

10. The application according to claim 9, characterized in that, The influenza vaccines include trivalent influenza vaccines, quadrivalent influenza vaccines, trivalent adjuvanted influenza vaccines, and quadrivalent adjuvanted influenza vaccines; The adjuvants in the trivalent and quadrivalent adjuvant influenza vaccines are selected from any one or more of the following: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin, anionic liposome adjuvant containing saponin, CpG adjuvant, nanoemulsion, and adjuvant containing 3D-MPL. Each dose unit of the influenza vaccine contains 3-65 μg of HA antigen protein.

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