Efficient screening method for influenza virus vaccine candidate strains and application thereof

Abstract

The application relates to a high-efficiency screening method of an influenza virus candidate vaccine strain and application thereof, and the high-efficiency screening method comprises the following steps: (1) diluting a mother generation influenza virus to be screened by using a virus maintenance liquid, adding the diluted virus into a culture plate containing dog kidney cells, adsorbing the influenza virus to the dog kidney cells, and discarding the virus liquid in the culture plate; (2) covering the cell surface by using a liquid-state maintenance culture medium, cooling and solidifying the maintenance culture medium, and inverting the culture medium in a culture box to culture the virus; and (3) picking virus spots and mixing the virus spots with a buffer, inoculating the virus spots into chicken embryos and / or dog kidney cells to culture, and obtaining the influenza virus candidate vaccine strain. The method provided by the application is simple and easy to operate, short in time consumption, low in cost, few in passage times, not easy to mutate, and capable of achieving the purpose of quickly and effectively screening the influenza virus vaccine candidate strain with excellent growth characteristics from the mother generation virus in a short time.

Description

Technical Field

[0001] This invention relates to the field of virology, and more particularly to a method for efficiently screening candidate influenza virus vaccine strains and its application. Background Technology

[0002] Influenza virus is a major respiratory pathogen that can cause high fever, fatigue, headache, and cough in humans, leading to severe respiratory illness and even death. It belongs to the genus *Influenza virus* of the family Orthomyxoviridae, and its genome is a single-stranded, negative-sense segmented RNA fragment. Based on the antigenic differences between its nucleoprotein (NP) and matrix protein (M), influenza viruses can be classified into four types: A, B, C, and D. Each year, the co-circulation of influenza A and B viruses worldwide causes seasonal pandemics, infecting millions and resulting in approximately 290,000 to 650,000 deaths.

[0003] Currently, vaccination remains the most effective way to prevent influenza virus infection. However, the high incidence of seasonal influenza and the incompatibility between vaccine strains and circulating strains due to viral antigenic drift, which renders seasonal vaccines ineffective, have always been major challenges in influenza prevention and control. Because influenza virus subtypes are numerous and mutate rapidly, countries around the world have failed to establish effective herd immunity, resulting in the resurgence of influenza every year and a continued threat to global public health security.

[0004] The surface of influenza virus particles is encapsulated by a lipid envelope derived from the host cell membrane. Embedded on this membrane are two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). These two glycoproteins are the main antigens on the surface of the influenza virus and are also the main effective components of current influenza vaccines. Various technical approaches exist for influenza vaccines, including inactivated vaccines, live attenuated vaccines, recombinant protein vaccines, and mRNA vaccines. Inactivated vaccines were the earliest developed and are currently the most widely used. In recent years, with the development of biotechnology, novel vaccination methods such as nasal spray immunization and microneedle administration have emerged. However, the mainstream influenza vaccine products currently on the market mainly use an inactivated lysis protocol, which involves culturing the influenza virus on chicken embryos or cell matrix, lysing it, and then purifying it to obtain hemagglutinin. The advantages of inactivated vaccines—low cost, stability, safety, reliability, and rapid production—remain irreplaceable. Therefore, the yield of the effective antigen of the influenza virus, hemagglutinin, during the culturing process is a crucial factor affecting vaccine yield and quality.

[0005] The production of influenza virus hemagglutinin is mainly influenced by both internal and external factors. The sequence of the viral genome determines the strength of the role of viral functional proteins during replication, thus determining the virus's growth characteristics. Viruses with strong growth characteristics and rapid replication rates produce relatively higher hemagglutinin yields within the same culture time. In addition, external factors such as environmental conditions (temperature, pH, dissolved oxygen ratio, etc.) also affect viral growth and replication. Under the premise of a fixed viral culture environment, the growth characteristics of the viral strain itself determine the production of influenza virus hemagglutinin per unit time.

[0006] CN116218791A discloses a rabies virus screening method based on in vivo-in vitro cross-selection, which increases the virus titer through multiple passages after in vivo and in vitro screening. CN105671002A discloses a method for constructing and screening high-yield avian influenza virus cell-based vaccine strains, which obtains high-yield vaccine strains through reverse genetics and site-directed mutagenesis. Other methods include limiting dilution methods involving continuous passage in cells or chicken embryos.

[0007] The above methods can improve viral titer and viral yield to varying degrees, but several problems still exist in industrial applications: 1) Some methods involve multiple passages and long passage times, making it impossible to guarantee a single viral sequence after passage, which may lead to mutations and changes in antigenicity; 2) Reverse genetics technology has certain operational barriers and a low success rate. Generally, reverse genetics technology involves processes such as vector construction, homologous recombination, point mutation, and virus rescue, which are complex, time-consuming, and have a low success rate, making it unsuitable for technology promotion; 3) In vitro and in vivo cross-screening is costly and time-consuming, requiring animal experiments. Due to the role of the immune system in animals, viruses are usually subjected to stress screening after infection and are prone to mutations to adapt to the individual environment, which may lead to changes in antigenicity.

[0008] In summary, how to quickly screen influenza virus candidate vaccine strains with high titers, high hemagglutinin production, and no mutations, and apply them to the construction of influenza virus candidate vaccine libraries, has become one of the urgent problems to be solved in this field. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a highly efficient screening method for influenza virus candidate vaccine strains and its application, which can rapidly and effectively screen candidate vaccine strains with excellent growth characteristics and antigen yield from working seed batches of parent viruses with minimal passages.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for efficiently screening candidate influenza virus vaccine strains, the method comprising the following steps:

[0012] (1) Dilute the parent influenza virus to be screened with virus maintenance solution, and add it to a culture plate containing canine kidney cells. The influenza virus adsorbs the canine kidney cells, and the virus solution in the culture plate is discarded.

[0013] (2) Cover the cell surface with liquid maintenance medium, keep the medium cool and solidify, and invert the cell in an incubator to culture the virus.

[0014] (3) Pick up the virus spots, mix them with buffer solution, and inoculate them into chicken embryos and / or canine kidney cells for culture to obtain the influenza virus candidate vaccine strain.

[0015] To obtain highly antigenic and mutation-free influenza virus strains for influenza virus candidate vaccine library construction, it is necessary to reduce the number of passages and improve screening efficiency. This invention provides a screening method that can rapidly and effectively screen candidate vaccine strains with excellent growth characteristics and antigen yield from working seed batches of maternal viruses with minimal passages, applicable to the screening of most influenza viruses. The chicken embryos used in this invention are 8-10 day old SPF-grade chicken embryos; however, this invention is not limited to influenza virus vaccine candidate strains for chicken embryo vaccine production, but can also be used for candidate strains of influenza viruses cultured in cell matrix.

[0016] Preferably, the influenza virus includes any one of influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0017] Preferably, the virus maintenance medium in step (1) is a DMEM medium containing 0.2% to 1% (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) w / v bovine serum albumin and 1 to 5 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, or 5 μg / mL).

[0018] Preferably, the pancreatic enzyme includes TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme.

[0019] Preferably, the dilution using virus maintenance solution in step (1) specifically involves performing a 10-fold volumetric gradient dilution using virus maintenance solution, starting from 10... 3 Dilute to 10 times 8 Times, specifically 10 3 10 4 10 5 10 6 10 7 and 10 8 .

[0020] Preferably, the method for preparing the culture plate containing canine kidney cells in step (1) includes: digesting and counting canine kidney cells in the logarithmic growth phase and in good condition, and then culturing them at a density of 4 × 10⁻⁶ cells / mL. 5 ~6×10 5 One per hole (e.g., 4×10) 5 4.5×10 5 4.8×10 5 5×10 5 5.2×10 5 5.5×10 5 Or 6×10 5 (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C) are seeded into 6-well cell culture plates and incubated at 35–40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C) in a CO2 incubator containing 4%–6% (e.g., 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, or 6%) for 18–24 hours (e.g., 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours) until a dense monolayer of cells is formed. The culture medium is then discarded.

[0021] Preferably, the temperature at which the influenza virus adsorbs onto canine kidney cells in step (1) is 34–37°C (e.g., 34°C, 35°C, 35.5°C, 36°C, or 37°C, etc.), and the time is 1–2 hours (e.g., 1 hour, 1.2 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.8 hours, or 2 hours, etc.).

[0022] Preferably, step (1) after discarding the virus solution in the culture plate further includes washing the cells 2 to 3 times with PBS buffer.

[0023] Preferably, the maintenance medium in step (2) is a MEM medium containing 0.5% to 1% (e.g., 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) of agarose, 0.2% to 1% (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%) of bovine serum albumin (w / v) and 1 to 5 μg / mL (e.g., 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, or 5 μg / mL).

[0024] The present invention maintains cell viability for a certain period of time by maintaining bovine serum albumin in the culture medium. The MEM culture medium can use commonly used products in the field. After screening and comparison of various culture media, 2×MEM serum-free culture medium as the maintenance medium component can significantly promote the growth of influenza virus.

[0025] Preferably, the method for preparing the maintenance culture medium in step (2) includes: preparing solution A and solution B separately; heating and melting solution A before use; and mixing solution A and solution B at a volume ratio of 1:(0.8-1.2) when the temperature of solution A drops to 25-35℃ (e.g., 25℃, 27℃, 29℃, 30℃, 31℃, 33℃, or 35℃, etc.). The mixture is prepared and used immediately. Solution A contains 1%-2% (e.g., 1%, 1.2%, 1.4%, 1.5%) of the culture medium by mass. The solution B is a PBS buffer containing 1.6%, 1.8%, or 2% agarose, and the solution B is a MEM medium containing 0.4% to 2% (e.g., 0.4%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, or 2%) w / v bovine serum albumin and 2 to 10 μg / mL (e.g., 2 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, or 10 μg / mL).

[0026] The specific point values ​​for (0.8 to 1.2) can be selected from 0.8, 0.9, 1, 1.1 or 1.2, etc.

[0027] The maintenance culture medium with specific components provided by this invention is suitable for culturing most influenza viruses, and neither the maintenance culture medium nor the virus maintenance solution provided by this invention contains antibiotics. By preparing the components of the maintenance culture medium into separate solutions A and B, and using them fresh each time, this invention achieves thorough mixing of agarose and other components, thereby improving the uniformity of the maintenance culture medium and making it more conducive to the growth of influenza viruses.

[0028] Preferably, the agarose comprises low-melting-point agarose.

[0029] Preferably, the pancreatic enzyme includes TPCK-treated pancreatic enzyme and / or recombinant pancreatic enzyme.

[0030] Preferably, the conditions for culturing the virus in step (2) are as follows: culturing in a CO2 incubator containing 4% to 6% (e.g., 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, or 6%) at 33 to 37°C (e.g., 33°C, 34°C, 35°C, 36°C, or 37°C) for 48 to 120 hours (e.g., 48 hours, 60 hours, 80 hours, 84 hours, 90 hours, 100 hours, or 120 hours).

[0031] In this invention, the virus culture time is 48-120 hours. A culture time of more than 48 hours is required for multiple viral clones to show differences in growth characteristics and for the viral particles to replicate to a certain number. The culture time is determined based on the growth of the viral clones. The optimal time is when the diameter of different viral clones is significantly different, but the edges of adjacent clones do not touch. A culture time of less than 120 hours can ensure that the obtained viral clones have consistent sequences and are less prone to mutation.

[0032] Preferably, when the virus used for culturing the virus in step (2) is an influenza A virus, the culturing temperature is 35-37°C (for example, it can be 35°C, 35.5°C, 36°C, 36.5°C or 37°C, etc.).

[0033] Preferably, when the virus used for culturing the virus in step (2) is influenza B virus, the culturing temperature is 33-35℃ (for example, it can be 33℃, 33.5℃, 34℃, 34.5℃ or 35℃, etc.).

[0034] Preferably, step (3) of picking viral spots specifically includes: picking viral spots with a diameter of 1 to 4 mm (e.g., 1 mm, 2 mm, 2.5 mm, 3 mm or 4 mm, etc.) and clear edges, and vertically separating and picking out the solid maintenance culture medium along with the underlying cells.

[0035] This invention obtains influenza viruses with strong growth and replication capabilities by picking out virus spots with larger diameters, while removing all the culture in the vertical space to preserve the virus to the greatest extent.

[0036] Preferably, the buffer solution in step (3) includes PBS buffer.

[0037] Preferably, step (3) further includes a step of pre-cooling the buffer before mixing the virus spot with the buffer solution.

[0038] This invention uses a pre-cooled buffer solution to ensure the selected monoclonal virus particles have good activity and improve the selection success rate, as room temperature will cause the virus particles to be rapidly inactivated.

[0039] Preferably, the culture conditions in step (3) are as follows: cultured in a CO2 incubator containing 4% to 6% (e.g., 4%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, or 6%) at 33 to 37°C (e.g., 33°C, 34°C, 35°C, 36°C, or 37°C) for 48 to 96 hours (e.g., 48 hours, 50 hours, 60 hours, 70 hours, 72 hours, 75 hours, 80 hours, 90 hours, or 96 hours).

[0040] Preferably, when the culture in step (3) is for culturing influenza A virus, the culture temperature is 35-37°C (e.g., 35°C, 35.5°C, 36°C, 36.5°C, or 37°C).

[0041] Preferably, when the culture in step (3) is for culturing influenza B virus, the culture temperature is 33-35℃ (e.g., 33℃, 33.5℃, 34℃, 34.5℃ or 35℃, etc.).

[0042] Preferably, step (3) further includes sequencing to confirm the HA and NA sequences of the virus, and selecting strains without amino acid mutations as candidate influenza virus vaccine strains for library construction.

[0043] Secondly, the present invention provides the application of the efficient screening method for influenza virus candidate vaccine strains as described in the first aspect in screening highly antigenic influenza viruses.

[0044] The influenza virus candidate vaccine strains obtained by screening using the method provided in this invention have high antigenicity, high hemagglutinin yield, and high influenza virus antigenicity.

[0045] Preferably, the highly antigenic influenza virus exhibits a high hemagglutinin production.

[0046] Thirdly, the present invention provides the application of the efficient screening method for influenza virus candidate vaccine strains as described in the first aspect in the preparation of influenza virus vaccines.

[0047] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) The method provided by the present invention is simple and easy to operate, short in time, low in cost, has few passages, and is not prone to mutation. Applying the plaque test for determining virus titer to the field of isolating monoclonal vaccine candidate strains and increasing the effective antigen components (i.e., hemagglutinin production) can achieve the goal of rapidly and effectively screening influenza virus vaccine candidate strains with excellent growth characteristics from the parent strain in a short time.

[0050] (2) Compared with the reverse genetics method commonly used in the field of influenza virus research to modify viruses, the method provided by this invention is time-saving, low-cost, and does not require steps such as constructing expression vectors and rescuing recombinant viruses. It only takes 5 to 7 days to complete a round of screening and obtain preliminary screening results.

[0051] (3) Compared with screening influenza viruses on chicken embryos, the virus screened by the method provided by the present invention has the characteristics of single sequence and not easy to mutate. Since chicken embryos have natural screening pressure, the virus has a certain selective adaptability on chicken embryos, and its hemagglutinin sequence is very easy to mutate.

[0052] (4) Compared with the commonly used limiting dilution method for virus screening, the method provided by the present invention can directly observe the differences in the growth characteristics of the virus, and the screened virus strains are all monoclonal strains. A single spot is a collection formed by a single virus particle infecting and replicating the surrounding healthy cells, which has the characteristics of being less prone to mutation and having a single sequence. Attached Figure Description

[0053] Figure 1 The graph shows the results of hemagglutinin content determination in a 250mL shake flask before and after screening H1N1 strains using the method in Example 1.

[0054] Figure 2 The graph shows the results of the hemagglutinin content determination in a 250mL shake flask after screening H1N1 strains using the methods of Example 1 and Comparative Example 1.

[0055] Figure 3 The graph shows the results of hemagglutinin content determination in the harvested liquid cultured in a 10L bioreactor before and after screening for H1N1 strains using the method in Example 1.

[0056] Figure 4 The image shows the empty plaques formed on canine kidney cells before and after screening for H1N1 strains using the method in Example 1. Detailed Implementation

[0057] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] The reagents used in the following examples:

[0060] Bovine serum albumin (BSA): purchased from MP Biomedicals;

[0061] TPCK-treated trypsin: purchased from Sigma;

[0062] Recombinant trypsin: purchased from Beijing Kangshi;

[0063] DMEM medium: purchased from Gibco;

[0064] Low melting point agarose: purchased from Invitrogen;

[0065] 2xMEM culture medium: purchased from Gibco, catalog number 11935046.

[0066] Example 1

[0067] This embodiment provides a highly efficient method for screening candidate influenza virus vaccine strains, including the following steps:

[0068] (1) Digest and count canine kidney cells in the logarithmic growth phase and in good condition, using a density of 5 × 10⁻⁶. 5 Cells were seeded per well in 6-well cell culture plates and cultured at 38°C in a 5% CO2 incubator for 20 hours until a dense monolayer of cells was formed. The culture medium was then discarded.

[0069] (2) The parent influenza A virus to be screened was serially diluted 10-fold using virus maintenance solution, from 10... 3 Dilute to 10 times 8 The virus solution was diluted and added to a culture plate containing canine kidney cells. The influenza virus was incubated at 35°C for 1.5 hours to adsorb onto the canine kidney cells. The virus solution in the culture plate was then discarded, and the cells were washed 2-3 times with PBS buffer. The virus maintenance medium was DMEM medium containing 0.6% w / v bovine serum albumin and 3 μg / mL TPCK-treated trypsin.

[0070] (3) Cover the cell surface with liquid maintenance medium to maintain the medium cooling and solidification, and invert the cell in an incubator to culture the virus. The conditions for culturing the virus are: culturing at 36°C in an incubator containing 5% CO2 for 80 hours.

[0071] The preparation method of the maintenance culture medium includes: preparing solution A and solution B separately; heating and melting solution A before use; and mixing solution A and solution B at a volume ratio of 1:1 when the temperature of solution A drops to 30°C. Solution A is a PBS buffer containing 1.5% low-melting-point agarose, and solution B is a MEM medium containing 1.2% w / v bovine serum albumin and 6 μg / mL TPCK-treated trypsin.

[0072] (4) Pick out virus spots with a diameter of 1-4 mm and clear edges, vertically separate the solid maintenance culture medium along with the bottom cells, mix with pre-cooled PBS buffer, inoculate into SPF grade chicken embryos, and incubate at 36°C in a 5% CO2 incubator for 70 h.

[0073] (5) Sequencing confirmed the HA and NA sequences of the virus, and selected strains without amino acid mutations as candidate vaccine strains for influenza A virus library construction.

[0074] Example 2

[0075] This embodiment provides a highly efficient method for screening candidate influenza virus vaccine strains, including the following steps:

[0076] (1) Digest and count canine kidney cells in the logarithmic growth phase and in good condition, using a density of 4 × 10⁻⁶. 5 Cells were seeded per well in 6-well cell culture plates and cultured at 40°C in a 4% CO2 incubator for 18 hours until a dense monolayer of cells was formed. The culture medium was then discarded.

[0077] (2) The parent influenza A virus to be screened was serially diluted 10-fold using virus maintenance solution, from 10... 3 Dilute to 10 times 8 The virus solution was diluted and added to a culture plate containing canine kidney cells. The influenza virus was incubated at 37°C for 1 hour to adsorb onto the canine kidney cells. The virus solution in the culture plate was then discarded, and the cells were washed 2-3 times with PBS buffer. The virus maintenance medium was DMEM medium containing 0.2% w / v bovine serum albumin and 5 μg / mL recombinant trypsin.

[0078] (3) Cover the cell surface with liquid maintenance medium to maintain the medium cooling and solidification, and invert the cell in an incubator to culture the virus. The conditions for culturing the virus are: culturing at 37°C in an incubator containing 6% CO2 for 48 hours.

[0079] The method for preparing the maintenance culture medium includes: preparing solution A and solution B separately; heating and melting solution A before use; and mixing solution A and solution B at a volume ratio of 1:0.8 when the temperature of solution A drops to 35°C. Solution A is a PBS buffer containing 1% low-melting-point agarose, and solution B is a MEM medium containing 2% w / v bovine serum albumin and 2 μg / mL recombinant trypsin.

[0080] (4) Pick out virus spots with a diameter of 1-4 mm and clear edges, vertically separate the solid maintenance culture medium along with the bottom cells, mix with pre-cooled PBS buffer, inoculate into canine kidney cells, and incubate at 37°C in a 6% CO2 incubator for 48 h.

[0081] (5) Sequencing confirmed the HA and NA sequences of the virus, and selected strains without amino acid mutations as candidate vaccine strains for influenza A virus library construction.

[0082] Example 3

[0083] This embodiment provides a highly efficient method for screening candidate influenza virus vaccine strains, including the following steps:

[0084] (1) Digest and count canine kidney cells in the logarithmic growth phase and in good condition, using a density of 6 × 10⁻⁶. 5 Cells were seeded per well in 6-well cell culture plates and cultured at 35°C in a 6% CO2 incubator for 24 hours until a dense monolayer of cells was formed. The culture medium was then discarded.

[0085] (2) The parent influenza A virus to be screened was serially diluted 10-fold using virus maintenance solution, from 10... 3 Dilute to 10 times 8 The virus solution was diluted and added to a culture plate containing canine kidney cells. The influenza virus was incubated at 34°C for 2 hours to adsorb onto the canine kidney cells. The virus solution in the culture plate was then discarded, and the cells were washed 2-3 times with PBS buffer. The virus maintenance medium was DMEM medium containing 1% w / v bovine serum albumin and 1 μg / mL TPCK-treated trypsin.

[0086] (3) Cover the cell surface with liquid maintenance medium to maintain the medium cooling and solidification, and invert the cell in an incubator to culture the virus. The conditions for culturing the virus are: culturing at 35°C in an incubator containing 4% CO2 for 120 hours.

[0087] The preparation method of the maintenance culture medium includes: preparing solution A and solution B separately; heating and melting solution A before use; and mixing solution A and solution B at a volume ratio of 1:1.2 when the temperature of solution A drops to 25°C. Solution A is a PBS buffer containing 2% low-melting-point agarose and solution B is a MEM culture medium containing 0.4% w / v bovine serum albumin and 10 μg / mL recombinant trypsin.

[0088] (4) Pick out virus spots with a diameter of 1-4 mm and clear edges, vertically separate the solid maintenance culture medium along with the bottom cells, mix with pre-cooled PBS buffer, inoculate into canine kidney cells, and incubate at 35°C in a 4% CO2 incubator for 120 h.

[0089] (5) Sequencing confirmed the HA and NA sequences of the virus, and selected strains without amino acid mutations as candidate vaccine strains for influenza A virus library construction.

[0090] Example 4

[0091] This embodiment provides a highly efficient screening method for candidate influenza virus vaccine strains. The only difference from Embodiment 1 is that in step (3), the culture medium does not contain bovine serum albumin, and its fraction is allocated proportionally by other components.

[0092] Example 5

[0093] This embodiment provides a highly efficient screening method for candidate influenza virus vaccine strains. The only difference from Example 1 is that the maintenance culture medium in step (3) is prepared by directly mixing low melting point agarose, bovine serum albumin, TPCK-treated trypsin and MEM culture medium to obtain MEM culture medium containing 0.75% low melting point agarose, 0.6% w / v bovine serum albumin and 3 μg / mL TPCK-treated trypsin.

[0094] Example 6

[0095] This embodiment provides a highly efficient screening method for influenza virus candidate vaccine strains. The only difference from Embodiment 1 is that the virus culture time in step (3) is 40 hours.

[0096] Example 7

[0097] This embodiment provides a highly efficient screening method for candidate influenza virus vaccine strains. The only difference from Embodiment 1 is that the virus culture time in step (3) is 130 hours.

[0098] Comparative Example 1

[0099] This comparative example provides a method for screening candidate influenza virus vaccine strains. The parental influenza A virus was passaged several times in chicken embryos and several times in cells to obtain candidate influenza virus vaccine strains. The cell passage and chicken embryo passage steps were performed in accordance with the "Standard Operating Procedures of the National Influenza Center".

[0100] Test Example 1

[0101] This test case used the methods provided in Examples 1-7 to screen a working seed batch of an H1N1 candidate vaccine strain A / Victoria / 4897 / 2022 (IVR-238, from the World Health Organization) from the 2023–2024 Northern Hemisphere epidemic season, obtaining a candidate influenza virus vaccine strain. The viral titers (median tissue infectious dose, TCID) of the candidate strain and IVR-238 were determined using the Reed-Muench method. 50 The virus titration and sequencing results were compared by Shanghai Sangon Biotech Co., Ltd. using first-generation sequencing (Sanger method) of the candidate strain and the HA and NA sequences of the IVR-238 genome before screening. The results are shown in Table 1.

[0102] In addition, in this test case, the screened virus strain and IVR-238 were inoculated at a density of 3.5 × 10⁻⁶. 6Canine kidney cells were suspended in 250 mL cell shake flasks at a concentration of 3 μg / mL. Recombinant trypsin was added at a final concentration of 3 μg / mL. The culture environment was a suspension incubator at 34℃, 5% CO2, and 120 rpm. Hemagglutinin content was measured at 48 h and 72 h post-inoculation. The results are shown in Table 1. The comparison of hemagglutinin content among SX1204, SX1205, SX1206, SX1207, SX1208, SX1211, SX1212 (after selection), and IVR-238 (before selection) is shown in Table 1. Figure 1 As shown. Among them, Examples 2-7 only list one candidate strain, which can be used as a representative of the virus after screening in the corresponding experimental group, and represents the average level of the corresponding experimental group.

[0103] Table 1

[0104]

[0105]

[0106] As shown in Table 1, the viral titer of the candidate viral vaccine strains after screening can be increased from 7.5 lg TCID. 50 The maximum increase was 9.1 lg TCID / mL. 50 / mL. Under the same culture conditions and inoculation amount, the hemagglutinin content of the virus harvest fluid of the vaccine candidate strain in 250mL suspension cell shake flasks after screening and culture for 48h could be increased from 11.7μg / mL to a maximum of 28.2μg / mL, and after culture for 72h, the hemagglutinin content of the virus harvest fluid could be increased from 15.4μg / mL to a maximum of 38.4μg / mL, showing a significant increase in hemagglutinin content.

[0107] Comparing Example 1 with Example 4 shows that bovine serum albumin in the maintenance culture medium plays an important role in virus growth; comparing Example 1 with Example 5 shows that the maintenance culture medium prepared by the present invention using the freshly prepared method can improve the homogeneity of the culture medium and help virus growth; comparing Example 1 with Examples 6 and 7 shows that both too short and too long virus culture time will lead to a decrease in the yield of viral hemagglutinin obtained by screening.

[0108] In addition, in this test case, SX1204, SX1211, and the strains SX1 (passed twice with chicken embryo adaptation and once with cell adaptation), SX2 (passed twice with chicken embryo adaptation and once with cell adaptation), SX3 (passed once with chicken embryo adaptation and four times with cell adaptation), SX4 (passed once with chicken embryo adaptation and five times with cell adaptation), and SX7 (passed twice with chicken embryo adaptation) screened by the method provided in Comparative Example 1 were sampled at 72h, 96h, and 120h after inoculation to detect hemagglutinin content, following the aforementioned method. The results are shown in Table 2 and... Figure 2 As shown, where Figure 2 The hemagglutinin content of each time group, from left to right, represents SX1204, SX1211, SX1, SX2, SX3, SX4, and SX7. The influenza virus vaccine candidate strains obtained by the screening method provided in this invention have higher hemagglutinin content than those obtained using other methods.

[0109] Table 2

[0110]

[0111]

[0112] Test Example 2

[0113] In this test case, the screened virus strain SX1204 was inoculated into a 10L bioreactor at an MOI of 0.001. Unscreened IVR-238 was inoculated into 10L bioreactors at MOIs of 0.001 and 0.01, respectively, with a cell density of 3.5 × 10⁻⁶ cells / mL. 6 The recombinant trypsin concentration was set at 3 μg / mL, the culture temperature was controlled at 34℃, the stirring speed at 60 rpm, the pH at 7.0, and the dissolved oxygen ratio at 50%. Samples were taken at 72 h, 96 h, and 120 h post-inoculation. The hemagglutinin content in the supernatant was measured after centrifuging the virus harvest fluid at 1000 rpm for 5 min. The results are shown in Table 3. Figure 3 As shown, where Figure 3 The time groups from left to right are SX1204 (0.001 MOI), IVR-238 (0.001 MOI), and IVR-238 (0.01 MOI).

[0114] Table 3

[0115]

[0116] As shown in Table 3, under the same culture conditions in a 10L bioreactor, the hemagglutinin content of the virus harvested fluid of the screened strain can be increased from 22.5 μg / mL to 39.0 μg / mL, and the hemagglutinin yield is significantly improved.

[0117] Test Example 3

[0118] In this test case, the screened virus strain SX1204 and the unscreened IVR-238 were cultured on adherent canine kidney cells under the same conditions and time, and immunostaining was performed. Figure 4 As shown, the left image is IVR-238 and the right image is SX1204. The diameter of the vacuoles formed by SX1204 is larger than that of IVR-238, indicating that SX1204 has a stronger growth and replication ability.

[0119] In summary, the screening method provided by this invention is simple and easy to operate, time-saving, low-cost, requires fewer passages, and is less prone to mutation. Applying the plaque assay for determining viral titer to the field of isolating monoclonal vaccine candidate strains and increasing the effective antigenic component (i.e., hemagglutinin production) can achieve the goal of rapidly and effectively screening influenza virus vaccine candidate strains with excellent growth characteristics from the parent strain in a short period of time.

[0120] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A highly efficient screening method for influenza virus candidate vaccine strains, characterized in that, The efficient screening method includes the following steps: (1) Dilute the parent influenza virus to be screened with virus maintenance solution, and add it to a culture plate containing canine kidney cells. The influenza virus adsorbs the canine kidney cells, and the virus solution in the culture plate is discarded. (2) Cover the cell surface with liquid maintenance medium to keep the medium cool and solidify, and invert the cell in an incubator to culture the virus; (3) Pick up the virus spots, mix them with buffer solution, and inoculate them into chicken embryos and / or canine kidney cells for culture to obtain the influenza virus candidate vaccine strain; The virus maintenance medium in step (1) is DMEM medium containing 0.2%~1% w / v bovine serum albumin and 1~5 μg / mL trypsin; The maintenance medium in step (2) is MEM medium containing 0.5%~1% agarose, 0.2%~1% w / v bovine serum albumin and 1~5 μg / mL trypsin; The pancreatic enzymes include TPCK-treated pancreatic enzymes and / or recombinant pancreatic enzymes; Step (3) after cultivation also includes sequencing to confirm the HA and NA sequences of the virus, and selecting strains without amino acid mutations as candidate influenza virus vaccine strains for library construction.

2. The efficient screening method according to claim 1, characterized in that, The influenza virus includes any one of influenza A, influenza B, influenza C, or influenza D viruses.

3. The efficient screening method according to claim 1, characterized in that, Step (1) specifically involves diluting the virus using a virus maintenance solution: performing a 10-fold volumetric gradient dilution using the virus maintenance solution, starting from 10... 3 Dilute to 10 times 8 times.

4. The efficient screening method according to claim 1, characterized in that, The method for preparing the culture plate containing canine kidney cells in step (1) includes: digesting canine kidney cells in the logarithmic growth phase and in good condition, counting them, and then arranging them at a density of 4 × 10⁻⁶ cells / mL. 5 ~6×10 5 Seeds were planted per well in 6-well cell culture plates and cultured at 35-40°C in an incubator containing 4%-6% CO2 for 18-24 h until a dense monolayer of cells was formed. The culture medium was then discarded.

5. The efficient screening method according to claim 1, characterized in that, In step (1), the influenza virus adsorbs onto canine kidney cells at a temperature of 34-37°C for 1-2 hours.

6. The efficient screening method according to claim 1, characterized in that, Step (1) after discarding the virus solution in the culture plate also includes washing the cells 2-3 times with PBS buffer.

7. The efficient screening method according to claim 1, characterized in that, The preparation method of the maintenance culture medium in step (2) includes: preparing solution A and solution B respectively. Before use, solution A is heated and melted. When the temperature of solution A drops to 25~35℃, solution A and solution B are mixed at a volume ratio of 1:(0.8~1.2). The solution is prepared and used immediately. Solution A is a PBS buffer containing 1%~2% agarose by mass, and solution B is a 2xMEM culture medium containing 0.4%~2% w / v bovine serum albumin and 2~10 μg / mL trypsin.

8. The efficient screening method according to claim 1, characterized in that, The agarose includes low-melting-point agarose.

9. The efficient screening method according to claim 1, characterized in that, The conditions for culturing the virus in step (2) are: culturing at 33~37℃ in an incubator containing 4%~6% CO2 for 48~120 h.

10. The efficient screening method according to claim 1, characterized in that, When the virus used for culturing the virus in step (2) is influenza A virus, the culturing temperature is 35~37℃.

11. The efficient screening method according to claim 1, characterized in that, When the virus used for culturing the virus in step (2) is influenza B virus, the culturing temperature is 33~35℃.

12. The efficient screening method according to claim 1, characterized in that, Step (3) specifically includes: picking out virus spots with a diameter of 1-4 mm and clear edges, and vertically separating the solid maintenance culture medium along with the underlying cells.

13. The efficient screening method according to claim 1, characterized in that, The buffer solution in step (3) includes PBS buffer.

14. The efficient screening method according to claim 1, characterized in that, Step (3) includes a step of pre-cooling the buffer before picking up the virus spots and mixing them with the buffer solution.

15. The efficient screening method according to claim 1, characterized in that, The cultivation conditions described in step (3) are: incubation at 33~37℃ in an incubator containing 4%~6% CO2 for 48~96 h.

16. The efficient screening method according to claim 1, characterized in that, When the culture described in step (3) is for culturing influenza A virus, the culture temperature is 35~37℃.

17. The efficient screening method according to claim 1, characterized in that, When the culture described in step (3) is for culturing influenza B virus, the culture temperature is 33~35℃.

18. The application of the efficient screening method for influenza virus candidate vaccine strains as described in any one of claims 1 to 17 in screening highly antigenic influenza viruses.

19. The application according to claim 18, characterized in that, The highly antigenic influenza virus exhibits a high hemagglutinin production.

20. The application of the efficient screening method for influenza virus candidate vaccine strains as described in any one of claims 1 to 17 in the preparation of influenza virus vaccines.

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