QMC influenza virus seed, and preparation method and application thereof

Abstract

The application discloses a QMC influenza virus seed, a preparation method and application thereof, and belongs to the technical field of biological medicines.The preparation method of the QMC virus seed is virus passage adaptation and virus seed preparation which are synchronously performed, and comprises the following steps: inoculating chicken embryo source influenza virus on MDCK-XF06 cells, and obtaining recovered passage virus seed after culture; then inoculating the recovered passage virus seed on MDCK cells according to a mode of adjusting virus inoculation infection multiplicity from generation to generation, and obtaining P2 generation master seed batch virus seed and P3 generation working seed batch virus seed after culture.The QMC virus seed obtained through the preparation method has the following advantages: good virus activity, high virus titer, efficient production of a large amount of live virus, excellent genetic stability, ensured consistency and effectiveness of product quality, completely eliminated tumorigenicity risk, high safety, and the like, and lays a solid foundation for subsequent production of safe, efficient and stable vaccine products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a QMC influenza virus seed, a preparation method and application thereof. BACKGROUND

[0002] Influenza is an acute respiratory infectious disease caused by influenza virus, which can cause fever, runny nose, sore throat, cough, headache, muscle pain and general malaise, etc. Severe cases can develop 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 marketed influenza vaccines mainly include trivalent influenza vaccine, quadrivalent influenza vaccine, trivalent adjuvant influenza vaccine, quadrivalent adjuvant influenza vaccine, H1N1 influenza vaccine, etc. Influenza virus can be propagated in chicken embryos and passaged cells, but should be selected according to the host source of the virus seed to culture in chicken embryos or cells. Most viruses from birds have high replication efficiency in the allantoic cavity of chicken embryos, however, some human and other mammalian influenza viruses have low replication efficiency in chicken embryos, and passaging of mammalian influenza viruses in chicken embryos can lead to mutations in receptor binding specificity and antigenicity, so it is best to propagate influenza viruses derived from mammals in mammalian cells. Currently, the production matrix of influenza vaccine is gradually transferred from chicken embryos to mammalian cells, and common cell lines used for influenza vaccine research and development include MDCK, Vero, insect cell SF9 and PER.C6, etc. MDCK cells, as the most sensitive cell strain of influenza virus, have been widely used in influenza vaccine production.

[0004] Foreign countries have used MDCK cells and insect cell SF9 as cell matrix to produce marketed influenza vaccines, but the MDCK cells used to prepare the virus seed have tumorigenicity, which has safety risks for vaccine production.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide a QMC influenza virus seed, a preparation method and application thereof. The QMC influenza virus seed prepared by the present application has the characteristics of better safety, short production cycle, high yield, good immunogenicity and high genetic stability.

[0007] The present application is realized as follows:

[0008] In a first aspect, the present application provides a preparation method of a QMC influenza virus seed, which is a simultaneous process of virus passage adaptation and virus seed preparation, comprising:

[0009] The original influenza virus strain from chicken embryo is inoculated on MDCK cells, and after culture, a recovered seed virus is obtained; then the recovered seed virus is inoculated on MDCK cells in a manner of adjusting the virus inoculation multiplicity of infection from generation to generation, and after culture, a P2 generation master seed batch virus and a P3 generation working seed batch virus are obtained;

[0010] In the formula, the MDCK cells are MDCK-XF06 cells, and the preservation number is CCTCC NO: C2023328; the influenza virus from chicken embryo includes influenza A virus and influenza B virus.

[0011] In a second aspect, the application provides a QMC influenza virus seed obtained by the above preparation method.

[0012] In a third aspect, the application further provides application of the above QMC influenza virus seed in influenza vaccine production.

[0013] The application has the following beneficial effects:

[0014] The application designs a preparation method of a QMC influenza virus seed based on MDCK-XF06 cells, and successfully obtains a QMC virus seed with significant advantages, which not only exhibits excellent culture performance (good virus activity and high virus titer) and can efficiently produce a large amount of live virus, but also has excellent stability, including good genetic stability, ensuring the consistency and effectiveness of product quality; in addition, the virus seed completely eliminates the risk of tumorigenicity and has high safety, laying a solid foundation for subsequent production of safe, efficient and stable vaccine products. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0016] Figure 1 The figure is a process flow chart for preparation of a QMC influenza virus seed;

[0017] Figure 2 The figure is a schematic diagram of immunogenic GMT of trivalent influenza virus split vaccine (MDCK cell);

[0018] Figure 3 The figure is a schematic diagram of immunogenic GMT of tetravalent influenza virus split vaccine (MDCK cell);

[0019] Figure 4 The figure is a schematic diagram of immunogenic GMT of trivalent adjuvant influenza virus split vaccine (MDCK cell) A1 type; The figure is a schematic diagram of immunogenic GMT of tetravalent adjuvant influenza virus split vaccine (MDCK cell) A2 type.

[0020] Figure 5 Figure 6 shows a schematic diagram of immunogenic GMT for trivalent adjuvanted influenza split vaccine (MDCK cells) type A3.

[0021] Figure 6 Figure 7 shows a schematic diagram of immunogenic GMT for trivalent adjuvanted influenza split vaccine (MDCK cells) type BV. DETAILED DESCRIPTION

[0022] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are used. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased on the market are used.

[0023] QMC (Quality Controlled Cell) virus seed is a set of initial virus seed systems for the production of biological products (such as vaccines) under strict quality control management after comprehensive identification and inspection.

[0024] In modern biological product production, in order to ensure that each batch of products is safe, effective and of consistent quality, global regulatory agencies (such as FDA, EMA and NMPA) have compulsorily required the use of a “two-level cell bank system” and a “virus seed system”. The cell bank is the “factory” of the virus and is a carrier for culturing and replicating the virus in large quantities. The cell bank is divided into two levels: a master cell bank (MCB) and a working cell bank (WCB). The MCB is a large number of cells that are uniformly prepared by expanding the original cells and are once divided and packaged. It is the most original source of all production cells and is the starting point of production. The WCB is prepared by expanding the cells of the MCB and is directly used for production. The MCB is properly preserved, and each production is taken from the working cell bank, which can minimize the operation of the MCB and ensure its originality. The virus seed bank is the “template” for production. The virus used for production needs to establish a seed bank system, which mainly consists of a master virus seed (MVS) and a working virus seed (WVS). The MVS is a virus stock obtained by culturing the MCB or WCB cells, which has been fully identified and verified, and is once divided and preserved. It is the original source of all production viruses. The WVS is prepared by expanding the MVS and is directly used for large-scale production of vaccines.

[0025] Therefore, a series of extremely strict quality detection procedures are carried out on them to ensure their safety (free of exogenous contaminants, free of safety risks), consistency (stable generation, stable quality) and effectiveness (sufficient virus titer, high sensitivity to cells).

[0026] At present, only CSL company abroad can prepare QMC virus strains through MDCK cells, but the specific preparation process is not disclosed, and there is no related report on the preparation process of QMC virus strains in China at present. Moreover, even if the company can prepare QMC virus strains, the tumorigenicity will also bring safety problems to vaccine production. In order to fill the gap of QMC virus strain preparation technology in China and to overcome the safety problems existing in the existing QMC virus strain preparation technology, the inventors provide a preparation method of QMC virus strain.

[0027] For the tumorigenicity problem, the present application proposes that the first reported MDCK cell with no tumorigenicity (MDCK-XF06 cell) can be used as a culture medium to prepare QMC virus strain, which can solve the safety problem through the cell. However, since there is no any related technical guidance for the preparation of QMC virus strain at present, and the MDCK-XF06 cell is a new cell strain (MDCK cell strain with different sources, different sublines and different domestication methods, which has significant differences in culture method, virus adaptability, final yield and effect, etc. when preparing influenza virus strains), there is no suitable reference method for preparing QMC virus strain by using MDCK-XF06 cell. For this reason, the inventors have explored a method for preparing QMC virus strain by using MDCK-XF06 cell through a large number of experimental explorations.

[0028] The preparation method of QMC virus strain provided by the present application is as follows: inoculating chicken embryo-derived influenza virus on MDCK cells to obtain recovered passage virus strain after culture; then inoculating the recovered passage virus strain on MDCK cells according to the method of adjusting the multiplicity of infection of virus inoculation by generation, and obtaining P2 generation master seed batch virus strain and P3 generation working seed batch virus strain after culture.

[0029] Among them, the MDCK cell is MDCK-XF06 cell, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: C2023328 and has been disclosed in Chinese patent 202410695674.1.

[0030] The MDCK-XF06 cell has been verified to be non-tumorigenic and sensitive to influenza virus, but the sensitivity to influenza virus means that the influenza virus can proliferate on the cell, and it does not necessarily mean that the dominant virus strain can be produced. In order to obtain the dominant virus strain, the present application obtains the required dominant virus strain through specific technical means.

[0031] The original virus for preparing the QMC influenza virus seed in the present application is an influenza virus from a chicken embryo source introduced by an authoritative agency, including an influenza A virus and an influenza B virus.

[0032] When the virus strain isolated from a chicken embryo is inoculated into MDCK cells, the replication efficiency is not high because the virus has been adapted to the chicken embryo environment, and when switching to the MDCK cell environment, the virus needs a re-adaptation process, which is usually to inoculate the virus from a chicken embryo source into MDCK cells, wait for its replication, then collect the culture, inoculate into new MDCK cells, and so on, in this process, those virus strains in the virus population that can more effectively bind to the receptors of MDCK cells and utilize the intracellular mechanisms for replication are screened out and gradually become the dominant population; after multiple generations of adaptation, a virus that can efficiently replicate in MDCK cells can be obtained for large-scale production. Therefore, the conventional virus seed preparation method in the art is to adapt and pass (about 10 generations) first, and then establish a virus seed bank.

[0033] In the present application, the inventors adopt a virus seed bank establishment strategy of synchronously performing virus passage adaptation and virus seed preparation- adjusting the virus inoculation multiplicity of infection (MOI) generation by generation, the virus from a chicken embryo source is passed on cells for three generations, that is, a master seed batch (P2) and a working seed batch (P3) for influenza vaccine production are established, and the proliferation adaptation of the virus seed from a chicken embryo source on MDCK-XF06 cells and the virus seed preparation are simultaneously realized.

[0034] Passage is the process of transferring virus from one culture environment to another new environment, in the present application, P2 refers to the virus seed after two passages, and P3 refers to the virus seed after three passages. Compared with the conventional virus seed passage with a fixed MOI, the inoculation method of the present application can shorten the passage number, and shortening the passage number can reduce the probability of influenza virus mutation and ensure the effectiveness of the vaccine; also, more QMC influenza virus seeds can be produced in the same time, having the advantages of short production cycle and high yield.

[0035] Specifically, the preparation process of the QMC influenza virus seed provided by the present application is as shown in Figure 1 The operation steps are as follows:

[0036] S1. Add the thawed working bank cells MDCK-XF06 into SFM-MDCK culture medium, centrifuge to collect the precipitate and add it into SFM-MDCK culture medium, repeatedly blow and beat to prepare a cell resuspension, and place it in a shaking bed for culture; take well-grown cells for subculture at an appropriate density, dilute the well-grown subculture cells after culture and prepare a cell suspension;

[0037] S2. The chicken embryo-derived influenza virus strain is inoculated into the cell suspension of MDCK cells according to the inoculation ratio, and is placed in a shaker for the first time of culture. Then, the culture solution with a hemagglutination titer of ≥1:160 is selected, and the supernatant is obtained by centrifugation to obtain the recovered seed virus;

[0038] S3. The recovered seed virus is inoculated into the cell suspension of MDCK cells according to the inoculation ratio, and is subjected to the second time of culture. Then, the culture solution with a hemagglutination titer of ≥1:160 is selected, and the supernatant is obtained by centrifugation to obtain the master seed virus.

[0039] S4. The master seed virus is inoculated into the cell suspension of MDCK cells according to the inoculation ratio, and is subjected to the third time of culture. Then, the culture solution with a hemagglutination titer of ≥1:160 is selected, and the supernatant is obtained by centrifugation to obtain the working seed virus.

[0040] In the above preparation process, the original virus seed, the recovered seed virus and the master seed virus are inoculated according to the virus inoculation ratio in the range of 10 -3 ~10 -8 , and the inoculation ratio shows a decreasing trend by 1~2 exponential levels per generation. Then, the culture solution with a hemagglutination titer of ≥1:160 is selected for centrifugation and collection of the supernatant to obtain the ideal virus seed.

[0041] In some embodiments, the culture conditions of the cell resuspension in S1 are as follows: the temperature is 36~38℃, the CO2 concentration is 4%~6%, the rotation speed is 80~180rpm, and the culture time is 72~120h.

[0042] In some embodiments, the conditions of the subculture of the cells in S1 are as follows: the inoculation density is 60×10 4 ~200×10 4 cells / ml, the temperature is 36~38℃, the CO2 concentration is 4%~6%, the rotation speed is 80~180rpm, and the culture time is 60~84h.

[0043] In some embodiments, the density of the MDCK cells in the cell suspension prepared in S1 is 200×10 4 ~500×10 4 cells / ml, and the final concentration of TPCK trypsin is 1.0~5.0μg / ml.

[0044] In some embodiments, the conditions of the first time of virus culture are as follows: the temperature is 32~36℃, the CO2 concentration is 4%~6%, the rotation speed is 60~180rpm, and the culture time is 36~84h.

[0045] In some embodiments, the conditions of the second time of virus culture are as follows: the temperature is 32~36℃, the CO2 concentration is 4%~6%, the rotation speed is 60~180rpm, and the culture time is 36~84h.

[0046] In some embodiments, the third virus culture is performed at a temperature of 32-36℃, a CO2 concentration of 4%-6%, a rotation speed of 60-180 rpm, and a culture time of 36-84 h.

[0047] In some embodiments, the influenza A virus comprises H1N1 and H3N2 influenza viruses; and the influenza B virus comprises BV and BY influenza viruses.

[0048] In addition, when the MDCK cells are used to prepare the influenza virus seed, the selection of the optimal infection timing is also one of the decisive factors for obtaining a high virus titer. In the present application, the appropriate infection timing is the late logarithmic growth phase of the cells, specifically, the cell density is ≥ 500 × 10 4 cells / ml, and the cell viability is ≥ 90%.

[0049] The QMC influenza virus seed prepared by the preparation method provided in the present application has better safety, a short production cycle, high yield, good immunogenicity, and high genetic stability, and has a good application prospect when applied to the preparation of influenza virus vaccines.

[0050] The QMC influenza virus seed can be used to prepare influenza vaccines, including trivalent influenza vaccines, tetravalent influenza vaccines, trivalent adjuvant influenza vaccines, tetravalent adjuvant influenza vaccines, and H1N1 vaccines.

[0051] The trivalent influenza vaccine contains H1N1, H3N2, and BV antigens of three types, or H1N1, H3N2, and BY antigens of three types; the tetravalent influenza vaccine contains H1N1, H3N2, BV, and BY antigens of four types; the trivalent adjuvant influenza vaccine and the tetravalent adjuvant influenza vaccine are prepared by adding an adjuvant to the trivalent influenza vaccine and the tetravalent influenza vaccine. The adjuvant is selected from any one or more of aluminum hydroxide adjuvants, aluminum phosphate adjuvants, neutral liposome adjuvants containing saponin, cationic liposome adjuvants containing saponin, anionic liposome adjuvants containing saponin, CpG adjuvants, nanoemulsions, and 3D-MPL adjuvants.

[0052] In some embodiments, each dose unit of the influenza vaccine contains 3-65 μg of HA antigen protein. It has been verified by experiments that the influenza vaccine prepared from the QMC influenza virus seed of the present application has good immunogenicity.

[0053] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0054] In the embodiment of the present application, the strain of H1N1 influenza virus used is 23 / 250 (NIBSC strain number) among the WHO recommended strains in 2024-2025; the strain of H3N2 influenza virus used is 23 / 206 (NIBSC strain number) among the WHO recommended strains in 2024-2025; the BV influenza virus uses 22 / 204 (NIBSC strain number) among the WHO recommended strains in 2024-2025; and the BY influenza virus uses 21 / 132 (NIBSC strain number) among the WHO recommended strains in 2023-2024.

[0055] Example 1

[0056] This embodiment is the preparation of MDCK working cell bank, and the specific steps are as follows:

[0057] (1) Cell recovery

[0058] The MDCK-XF06 master cell bank cells were taken out from the liquid nitrogen tank, thawed in 37℃±2℃ purified water, and transferred to a centrifuge tube containing cell culture medium using a pipette / syringe in a clean bench. The centrifuge tube cap was tightened, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. SFM-MDCK medium 40 ml was added, mixed, and transferred to a shake flask for 37℃ shaking culture for 4d.

[0059] (2) Cell passage

[0060] The well-grown recovery generation was taken, and the cell density reached 500×10 4 cells / ml, and the cell viability reached 90% and above. The cell density and cell viability were confirmed, and the cells were passaged into a suitable size of shake flask at a seeding density of 100×10 4 cells / ml. After passage, the bottle cap was tightened and placed in a shaking bed at a temperature of 37±1℃, a CO2 concentration of 5%, and a rotation speed of 120 rpm for 3d. Continuous passage was carried out to the desired volume.

[0061] (3) Cell cryopreservation

[0062] The cells were collected into a centrifuge bottle, centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. Cryopreservation solution was added and mixed evenly, and then divided into cryopreservation tubes. The tubes were placed at 2-8℃ for 2h, then frozen at-60℃ or below overnight, and then transferred to a liquid nitrogen tank for storage. The working cell bank MDCK-XF06 was obtained.

[0063] Example 2

[0064] This embodiment is the preparation of QMC influenza virus seed, and the specific steps are as follows:

[0065] (1) Preparation of MDCK cells

[0066] MDCK cell recovery: Take appropriate containers to prepare 37℃±2℃ purified water, take the working bank cell MDCK-XF06 from the liquid nitrogen tank, immediately put the cell cryopreservation tube into the warm water, quickly water bath shake to thaw the cells in the clean bench, transfer the cells to the centrifuge tube with added cell culture medium with a pipette / syringe, tighten the centrifuge tube cap, centrifuge at 1000 rpm for 10 min, discard the supernatant, add 40 ml SFM-MDCK medium to the collected cell pellet, repeatedly blow with a pipette, resuspend to prepare a cell suspension, transfer the cell suspension to a shake flask, place it in a shaker at a temperature of 37±1℃, a CO2 concentration of 5%, and a rotation speed of 120 rpm, and culture for 84 h.

[0067] MDCK cell passage: Take well-grown cells (cell density reaches 500×10 4 cells / ml, cell viability reaches 90% and above), confirm the cell density and cell viability, and passage to appropriate size shake flasks at a seeding density of 100×10 4 cells / ml. After passage, tighten the bottle cap and place it in a shaker at a temperature of 37±1℃, a CO2 concentration of 4%-6%, and a rotation speed of 80-180 rpm, and culture for 72 h. Continuously passage to the required amount of cells for virus preparation.

[0068] (2) Master seed batch virus preparation

[0069] Virus inoculation: Take well-grown MDCK cells, confirm the cell density and cell viability (cell density reaches 500×10 4 cells / ml, cell viability reaches 90% and above), dilute the cells to appropriate size shake flasks at a density of 400×10 4 cells / ml, inoculate the virus at a ratio of 10 -4 cells / ml, and inoculate the virus into the MDCK cell suspension, with a final concentration of 1.0-5.0 ug / ml of TPCK trypsin solution in the culture system.

[0070] Virus culture harvest: Place the cells inoculated with the virus in a shaker at 34℃, a CO2 concentration of 5%, and a rotation speed of 120 rpm, and culture for 72 h. Select the culture fluid with relatively high hemagglutination titer (hemagglutination titer ≥1:160), centrifuge at 5000xg for not less than 10 min, and collect the supernatant in a sterile stock bottle as the recovery seed virus.

[0071] Passage: Take the recovered seed virus, inoculate it into MDCK cells at a ratio of 10 -5 , and culture the virus as above. After harvest, aliquot it, which is the master seed batch virus.

[0072] (3) Working seed batch preparation

[0073] Virus inoculation: Take well-grown MDCK cells, confirm cell density and cell viability (cell density is 500 x 10 4 cells / ml, cell viability is 90% or more), dilute the cells to a density of 400 x 10 4 cells / ml in a suitable size of a shake flask, inoculate the master seed batch with virus into the MDCK cell suspension at a ratio of 10 -6 to the final concentration of 3 ug / ml in the culture system.

[0074] Virus culture harvest: Place the cells inoculated with virus in a 34°C, 5% CO2, 120 rpm shaker for 72 hours. Select the culture fluid with relatively high hemagglutination titer (hemagglutination titer ≥ 1:160), centrifuge at 5000 x g for not less than 10 min, and then collect the supernatant in a sterile stock bottle. The aliquot is the working seed batch.

[0075] Example 3

[0076] This example is for the production of trivalent / tetravalent influenza vaccine, and the specific steps are as follows:

[0077] 1. Bulk production

[0078] Cell batch preparation: Thaw one or more working cell bank cells and continuously subculture to the production scale.

[0079] Upstream production: inoculate the working seed batch with virus at a MOI of 2 x 10 -2 ~10 -10 on the cells, incubate at 34°C for 72 hours, stop the culture, and centrifuge to harvest the supernatant.

[0080] Downstream production: ultrafiltrate the supernatant in a conventional manner, and then perform enzyme digestion, inactivation, purification, lysis, de-lysing agent removal, and sterilization to obtain the monovalent bulk.

[0081] 2. Formulation production

[0082] According to the formulation point of 30-36 ug / ml, calculate the amount of H1N1, H3N2, BV, or H1N1, H3N2, BY, or H1N1, H3N2, BV, BY four type monovalent bulk, take the bulk to the preparation container, add 10 mM PBS solution to the preparation volume, mix well, and aliquot to obtain the finished product. The finished product should be a colorless or slightly milky white liquid without foreign matter.

[0083] Bulk increase = formulation point x formulation volume / hemagglutinin content of bulk.

[0084] Example 4

[0085] The embodiment is the production of trivalent / quadrivalent adjuvant influenza vaccine, and the specific steps are as follows:

[0086] 1. Bulk production

[0087] Cell batch preparation: one or more working cell bank cells are recovered and continuously subcultured to a production scale.

[0088] Upstream production: the working seed batch is inoculated into cells at a MOI of 2x10 -2 ~10 -10 Inoculate into cells and incubate at 34°C for 72h, stop the culture, centrifuge and harvest the supernatant.

[0089] Downstream production: the supernatant is ultrafiltered according to the conventional method, and the process steps of enzyme cutting, inactivation, purification, lysis, de-lysing agent, sterilization, etc. are carried out to obtain the monovalent bulk.

[0090] 2. Preparation production

[0091] According to the preparation point of 30-36 μg / ml, calculate the amount of H1N1, H3N2, BV three types or H1N1, H3N2, BY three types or H1N1, H3N2, BV, BY four types of monovalent bulk, take the bulk to the preparation container, add the conventional dose of adjuvant (Poly I:C, XA301, XA302, XA401 and CPG1018), add 10 mM PBS solution to the preparation amount, mix well, and distribute, which is the finished product. The finished product should be a white and uniform liquid without foreign matter.

[0092] Bulk increase = preparation point x preparation volume / bulk hemagglutinin content.

[0093] Example 5

[0094] The embodiment is the immunogenicity analysis of trivalent (H1N1, H3N2, BV) influenza vaccine, and the details are as follows:

[0095] The test animals are 5-7 week old healthy SD rats, and the trivalent influenza virus split vaccine (MDCK cells) is injected intramuscularly. The dose design is shown in Table 1.

[0096] Each administration is given once on the 1st and 29th day of the test, and the observation is carried out for 8 weeks. Blood is collected before administration on the 1st and 29th day of the test and on the 56th day of the test, and neutralizing antibody detection is carried out by HA-HI method (hemagglutination and hemagglutination inhibition test). The rats are dissected on the 57th day of the test, and the spleens of the rats in each group are collected. The cell immune index is analyzed by flow cytometry.

[0097] Table 1 Dose design table

[0098]

[0099] As Figure 2As shown in Table 5, the trivalent influenza virus split vaccine immunogenicity GMT results were all greater than the WHO specified 1:40 serum positive standard.

[0100] Example 6

[0101] This example is a tetravalent influenza vaccine immunogenicity analysis, as follows:

[0102] The test animals were 5-7 week old healthy SD rats, and the tetravalent influenza virus split vaccine (MDCK cells) was injected intramuscularly. The dosage design is shown in Table 2.

[0103] The animals were dosed once on days 1 and 29, and observed for 8 weeks. Blood samples were collected before dosing on days 1 and 29 and on day 56, and neutralizing antibody detection was performed using the HA-HI method (hemagglutination and hemagglutination inhibition test). The rats were dissected on day 57, and the spleens of the rats in each group were collected for analysis of cellular immune indicators using a flow cytometer.

[0104] Table 2 Dosage design table

[0105]

[0106] As shown in Table 5, the trivalent influenza virus split vaccine immunogenicity GMT results were all greater than the WHO specified 1:40 serum positive standard. Figure 3 Example 7

[0107] This example is a trivalent (H1N1, H3N2, BV) adjuvant influenza vaccine immunogenicity analysis, as follows:

[0108] The test animals were 5-7 week old healthy SD rats, and the trivalent adjuvant influenza virus split vaccine (MDCK cells) was injected intramuscularly. The trivalent influenza virus split vaccine (MDCK cells) was injected intramuscularly. The trivalent influenza virus split vaccine (MDCK cells) was injected intramuscularly. The dosage design is shown in Table 3.

[0109] The animals were dosed once on days 1 and 29, and observed for 8 weeks. Blood samples were collected before dosing on days 1 and 29 and on day 56, and neutralizing antibody detection was performed using the HA-HI method (hemagglutination and hemagglutination inhibition test). The rats were dissected on day 57, and the spleens of the rats in each group were collected for analysis of cellular immune indicators using a flow cytometer.

[0110] Table 3 Dosage design table

[0111]

[0112] As shown in Table 5, the trivalent influenza virus split vaccine immunogenicity GMT results were all greater than the WHO specified 1:40 serum positive standard.

[0113] Figure 4 , 5 , 6, the effect of the trivalent adjuvant influenza virus split vaccine was better than that of the influenza vaccine without the addition of adjuvant.

[0114] Example 7​

[0115] The present embodiment is a genetic stability study of the QMC virus, and is specifically as follows:

[0116] The type 4 QMC virus was inoculated on MDCK cells, and was continuously passed for 11 generations. The HA and NA protein corresponding nucleic acid sequences of the 1st, 2nd, 3rd, 6th and 11th generations were determined, and the stability of the HA and NA protein amino acid sequences of the QMC virus was analyzed.

[0117] Table 4 HA and NA protein amino acid mutation analysis table

[0118]

[0119] As shown in Table 4, the QMC virus prepared by the present application has good genetic stability in subsequent passages.

[0120] Experimental Example 1

[0121] In order to verify the genetic stability difference of the influenza viruses obtained by different preparation methods, the mutations of the chicken embryo viruses obtained by the conventional method which were continuously passed on chicken embryos and MDCK-XF06 cells, and the QMC viruses prepared by the embodiment 2 which were continuously passed on MDCK-XF06 cells were compared, and the results are shown in Tables 5-8:

[0122] Table 5 H1N1 type influenza virus genetic stability mutation statistics table

[0123]

[0124] Table 6 H3N2 type influenza virus genetic stability mutation statistics table

[0125]

[0126] Table 7 BV type influenza virus genetic stability mutation statistics table

[0127]

[0128] Table 8 BY type influenza virus genetic stability mutation statistics table

[0129]

[0130] Note: "-" indicates no amino acid mutation.

[0131] As can be seen from the above table, after the chicken embryo viruses were continuously passed on chicken embryos and cells, the mutation sites of the H1N1 type and H3N2 type influenza viruses increased with the increase of the number of passages, while the QMC viruses of the four types were continuously passed on cells for 10 times, and there was no mutation.

[0132] Experimental Example 2

[0133] The present experiment was to verify the adaptability of the virus strains obtained by different preparation methods in the production of influenza vaccine. The hemagglutinin content (μg / mL) of the virus harvest liquid of the chicken embryo virus strain prepared by the conventional method and the cell virus strain prepared by Example 2 was compared, and the results are shown in Table 9:

[0134] Table 9 Hemagglutinin content of harvest liquid

[0135]

[0136] From the results in Table 9, it can be seen that the virus strain prepared by cells is used for the production of cell matrix influenza vaccine, and the yield is high and the quality is more controllable.

[0137] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the preparation of a QMC influenza virus seed, characterized in that, The preparation method is carried out synchronously with virus passage adaptation and preparation of virus seed, and comprises the following steps: The original influenza virus from chicken embryo is inoculated on MDCK cells, and a recovered passage virus seed is obtained after culture; then the recovered passage virus seed is inoculated on MDCK cells in a manner of adjusting the virus inoculation multiplicity of infection from generation to generation, and a P2 generation master seed batch virus and a P3 generation working seed batch virus are obtained after culture; The MDCK cells are MDCK-XF06 cells with a preservation number of CCTCC NO: C2023328; and the virus comprises influenza A virus and influenza B virus; The way to adjust the multiplicity of infection with virus inoculation from generation to generation is to decrease the multiplicity of infection from generation to generation at a virus inoculation ratio of 10 -3 ~10 -8 .

2. The production method according to claim 1, characterized by, The influenza A virus comprises H1N1 influenza virus and H3N2 influenza virus.

3. The preparation method according to claim 1, characterized in that, The influenza B virus comprises BV influenza virus and BY influenza virus.

4. The method of claim 1, wherein, The preparation method of the recovered passage virus seed comprises the following steps: The thawed working library cell MDCK-XF06 is added into SFM-MDCK culture medium, and the precipitate is collected after centrifugation and added into SFM-MDCK culture medium, repeatedly blown and beaten to prepare a cell resuspension liquid, and placed in a shaking bed for culture; the well-grown cells are subcultured according to a suitable density, and the well-grown subcultured cells are diluted and prepared into a cell suspension after culture; The chicken embryo-derived influenza virus strain is inoculated into the cell suspension of the MDCK cells according to an inoculation ratio, and placed in a shaking bed for first-time virus culture, and then the culture liquid with a hemagglutination titer of not less than 1:160 is selected, and the supernatant is obtained after centrifugation to obtain the recovered passage virus seed; The culture conditions of the cell resuspension liquid are as follows: the temperature is 36-38℃, the CO2 concentration is 4%-6%, the rotation speed is 80-180rpm, and the culture time is 72-120h; The conditions of the subculture are as follows: the inoculation density is 60×10 4 4 cells / ml, the temperature is 36~38℃, the CO2 concentration is 4%~6%, the rotation speed is 80~180rpm, and the culture time is 60~84h.​ The cell density of the passaged cells is ≥500×10 4 cells / ml, and the cell viability is ≥90%. The density of MDCK cells in the cell suspension is 200 x 10 4 500 x 10 4 cells / ml, and the final concentration of TPCK trypsin is 1.0-5.0 μg / ml. The culture conditions of the first-time virus culture are as follows: the temperature is 32-36℃, the CO2 concentration is 4%-6%, the rotation speed is 60-180rpm, and the culture time is 36-84h.

5. The preparation method according to claim 4, characterized in that, The preparation method of the master seed batch virus comprises the following steps: the recovered passage virus seed is inoculated into the cell suspension of the MDCK cells according to an inoculation ratio for second-time virus culture, and then the culture liquid with a hemagglutination titer of not less than 1:160 is selected, and the supernatant is obtained after centrifugation to obtain the master seed batch virus; The culture conditions of the second-time virus culture are as follows: the temperature is 32-36℃, the CO2 concentration is 4%-6%, the rotation speed is 60-180rpm, and the culture time is 36-84h.

6. The production method according to claim 5, characterized by, The preparation method of the working seed batch virus comprises the following steps: the master seed batch virus is inoculated into the cell suspension of the MDCK cells according to an inoculation ratio for third-time virus culture, and then the culture liquid with a hemagglutination titer of not less than 1:160 is selected, and the supernatant is obtained after centrifugation to obtain the working seed batch virus; The culture conditions of the third-time virus culture are as follows: the temperature is 32-36℃, the CO2 concentration is 4%-6%, the rotation speed is 60-180rpm, and the culture time is 36-84h.

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