CN114561364A

CN114561364A - Method and reagent for efficiently amplifying human influenza virus

Info

Publication number: CN114561364A
Application number: CN202210205040.4A
Authority: CN
Inventors: 朱绍荣; 喻志远; 吴熠潇
Original assignee: SHANGHAI RONGSHENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Current assignee: SHANGHAI RONGSHENG BIOLOGICAL PHARMACEUTICAL CO Ltd
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2022-05-31

Abstract

The invention provides a method and a reagent for efficiently amplifying human influenza virus. The present inventors have made extensive screening work to develop an improved maintenance culture solution and culture method that can be applied to efficiently amplify influenza virus, in view of the characteristics of influenza virus replication. The maintenance culture solution and the culture can obtain the virus with high titer in a short period, and the method has low cost, safety and stability.

Description

Method and reagent for efficiently amplifying human influenza virus

Technical Field

The invention belongs to the field of virology, and particularly relates to a method and a reagent for efficiently amplifying human influenza virus.

Background

The influenza virus belongs to the family of orthomyxoviridae, the influenza virus comprises a type A, a type B and a type C, the antigenic variability of the type A is strongest, the influenza virus infects human beings and other animals to cause moderate and severe diseases, attacks all age groups of people and often causes a worldwide pandemic. B-variability is weak, infects humans only, generally causes mild disease, mainly affects children, and can cause local outbreaks. The type C antigenicity is relatively stable, and only causes infant infection and adult sporadic cases. Influenza A viruses are divided into many subtypes according to the difference between H and N antigens, H can be divided into 15 subtypes (H1-H15), and N has 9 subtypes (N1-N9). Of these, only H1N1, H2N2, H3N2 primarily infect humans, and many other subtypes of natural hosts are diverse avian and animal species. Among them, the strains with the greatest harm to birds are subtype H5, H7 and H9 strains. Generally, avian influenza virus does not infect birds and animals other than pigs. Once the avian influenza viruses with high pathogenicity such as H5N1, H7N7, H9N2 and the like have the human-to-human transmission capability due to mutation, the avian influenza epidemics among people can be caused, and the avian influenza viruses are predicted to have great potential threat to human beings. If a mutation occurs in an epitope (epitope) specific to the hemagglutinin molecule, compared to the previously found strains, the new strains are considered to be heterogeneous variants of the previous strains, have epidemiological significance, and can cause influenza epidemics. The transmission route of influenza is mainly air droplet transmission, and then indirectly transmits through tea sets, tableware, towels and the like polluted by viruses, and is also one of the transmission routes of influenza by close contact.

The loss of influenza viruses to humans is not only an excessive mortality rate, but also involves various forms of direct or indirect economic loss. The best currently accepted method of preventing influenza is vaccination with influenza. For most countries and regions, safe and effective inactivated vaccines have been the basis for the prevention of influenza. Influenza vaccines have become one of the most appreciated and marketed vaccine varieties today. This requires the efficient and large-scale cultivation of influenza viruses for vaccine preparation.

Compared with the traditional chick embryo production process, the production process based on the cell culture technology can realize rapid large-scale culture in the pandemic of influenza so as to solve the problem of insufficient supply of chick embryos, and thus the production process gradually becomes a mainstream production mode. In recent years, the demand for influenza vaccines has been increasing with outbreaks of various epidemic diseases.

However, the productivity of the existing cell culture production process in the field is still relatively low, and the improvement of the expansion efficiency of influenza virus in cells is urgent. In contrast to the chick embryo method and other cell matrices for the production of influenza viruses, the MDCK cell suspension culture technology has come into play, and the use of culture solution in this technology is one of the cores to ensure cell growth and virus replication. However, in the current influenza virus production phase, most of the virus maintenance culture solution used is the same as the cell growth culture solution used in the cell growth phase, which causes a deficiency in the requirement of nutrients required for virus replication, and ultimately affects the intracellular amplification efficiency of the virus, resulting in a low virus yield. Therefore, it is necessary to design a maintenance medium for efficiently amplifying influenza virus with respect to the characteristics of influenza virus replication.

Disclosure of Invention

The invention aims to provide a method and a reagent for efficiently amplifying human influenza virus.

In a first aspect of the invention, there is provided a method for amplifying influenza virus, the method comprising: (1) providing an influenza virus producing cell cultured in a basic culture medium; (2) providing a serum-free maintenance medium comprising a supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate; (3) adding the culture solution of (2) to the basic culture solution of the virus-producing cells of (1); infecting the virus-producing cells with influenza virus, culturing the cells, and producing influenza virus.

In one or more embodiments, in (a), the sodium butyrate is present in the serum-free maintenance medium in an amount of: 20-200 mg/L, wherein the content of galactose in the serum-free maintenance culture solution is as follows: 5-80 mg/L.

In one or more embodiments, in (b), the sodium butyrate is present in the serum-free maintenance medium in an amount of: 20-200 mg/L.

In one or more embodiments, in (a), the sodium butyrate is present in the serum-free maintenance medium in an amount: 25 to 150mg/L, preferably 30 to 120mg/L, more preferably 40 to 100 mg/L; and the content of the galactose in the serum-free maintenance culture solution is as follows: 10 to 60mg/L, preferably 20 to 50 mg/L.

In one or more embodiments, in (b), the sodium butyrate is present in the serum-free maintenance medium in an amount of: 25-150 mg/L, preferably 30-120 mg/L, and more preferably 40-100 mg/L.

In one or more embodiments, the amount of sodium butyrate in the serum-free maintenance medium in (a) or (b) is, for example: 28mg/L, 48mg/L, 58mg/L, 68mg/L, 78mg/L, 88mg/L, 100mg/L, 150mg/L, and the like.

In one or more embodiments, in (a), the content of galactose in the serum-free maintenance medium is, for example: 8. 18, 28, 38, 48, 58, 68mg/L, etc.

In one or more embodiments, the serum-free maintenance medium comprises a medium selected from the group consisting of: Xeno-SFM maintenance medium, Xeno-CDM maintenance medium, Ex-Cell medium.

In one or more embodiments, the influenza virus-producing cell is a non-human mammalian cell; preferably MDCK cells; preferably, the cell culture solution in (1) is an MDCK cell culture solution.

In one or more embodiments, the influenza virus is a human influenza virus; preferably, the influenza virus is influenza a virus; preferably, the influenza A virus has H selected from H1-H15 and N selected from N1-N9 according to the difference of H antigen and N antigen; more preferably, the influenza a virus comprises a virus selected from the group consisting of: H1N1 virus, H9N2 virus, H5N1 virus, H7N9 virus.

In one or more embodiments, in (3), when the culture solution of (2) is added to the culture solution of influenza virus-producing cells of (1), the ratio of the culture solution of (2) to the cell culture solution of influenza virus-producing cells of (1) is 1:0.5 to 1:2.5, preferably 1:0.9 to 1:2.2, more preferably 1:0.9 to 1: 1.5.

In one or more embodiments, (3) the MOI is 0.001 to 0.1, more preferably 0.005 to 0.05 (further preferably about 0.01) when the virus-producing cells are infected with an influenza virus.

In one or more embodiments, (3) the culturing is suspension culturing.

In one or more embodiments, the ratio of the culture solution of (2) to the culture solution of the virus-producing cell of (1) is, for example: 1:0.95, 1:1, 1:1.2, 1:1.3, etc.

In one or more embodiments, TPCK-pancreatin is added after infection of the virus-producing cells with influenza virus; preferably, the TPCK-pancreatin is TPCK-pancreatin with a final concentration of 5 + -3 μ g/mL, preferably 5 + -2 μ g/mL, more preferably 5 + -1 μ g/mL.

In another aspect of the present invention, there is provided a method of producing an influenza virus vaccine, the method comprising: amplifying influenza virus by any of the methods described above; the obtained influenza virus is inactivated, attenuated or separated from the obtained influenza virus to obtain the influenza virus vaccine with immunogenicity.

In one or more embodiments, the virus obtained after step (3) may also be passaged, i.e., a new round of cell infection and expansion may be performed.

In another aspect of the present invention, there is provided a method for preparing a serum-free maintenance medium for amplifying influenza virus, comprising: adding a supplement to the serum-free maintenance medium, the supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate.

In one or more embodiments, in (a), the sodium butyrate is present in the serum-free maintenance medium in an amount: 20-200 mg/L, wherein the content of galactose in the serum-free maintenance culture solution is as follows: 5-80 mg/L.

In another aspect of the present invention, there is provided a Kit (Kit) for amplifying influenza virus, comprising: serum-free maintenance medium prepared as described above.

In one or more embodiments, the kit further comprises: influenza virus producing cells, cell culture fluid for influenza virus producing cells; wherein the influenza virus producing cells are independent of the culture medium or are cultured in the culture medium.

In one or more embodiments, the serum-free maintenance medium comprises a medium selected from the group consisting of: Xeno-SFM maintenance medium, Xeno-CDM maintenance medium.

In one or more embodiments, the influenza virus-producing cell is an MDCK cell; preferably, the cell culture solution in (i) is an MDCK cell culture solution.

In one or more embodiments, in the kit, the ratio of the serum-free maintenance medium containing the additive to the culture medium of the influenza virus production cells is 1:0.5 to 1:2.5, preferably 1:0.9 to 1:2.2, and more preferably 1:0.9 to 1: 1.5.

In another aspect of the invention, the use of said kit is provided for the amplification of influenza viruses.

In one or more embodiments, the influenza virus is a human influenza virus.

In one or more embodiments, the influenza virus is an influenza a virus.

In one or more embodiments, the influenza a virus differs from an H antigen, H being selected from H1-H15, and N being selected from N1-N9.

In one or more embodiments, the influenza a virus comprises a virus selected from the group consisting of: H1N1 virus, H9N2 virus, H5N1 virus, H7N9 virus.

In one or more embodiments, the virus comprises: a strain-preserved virus, a primary isolated virus, or a virus isolated from an individual (e.g., a patient).

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows the results of culturing H1N1 influenza virus by adding sodium butyrate to the Xeno-SFM maintenance medium at different concentrations.

FIG. 2 shows the results of culturing H9N2 influenza virus by adding sodium butyrate to the Xeno-SFM maintenance medium at different concentrations.

FIG. 3 shows the results of culturing H1N1 influenza virus by adding sodium butyrate to the Xeno-CDM maintenance medium at different concentrations.

FIG. 4 shows the results of culturing H9N2 influenza virus by adding sodium butyrate to the Xeno-CDM maintenance medium at different concentrations.

FIG. 5 shows the results of culturing H1N1 influenza virus by adding 50mg/L sodium butyrate and various concentrations of galactose to the Xeno-SFM maintenance medium.

FIG. 6 shows the results of culturing H9N2 influenza virus by adding 50mg/L sodium butyrate and various concentrations of galactose to the Xeno-SFM maintenance medium.

Detailed Description

The present inventors have made extensive screening work in an effort to improve the efficiency of culturing and propagating influenza viruses and, as a result, have proposed an improved maintenance culture solution and culture method which can be applied to efficiently amplifying influenza viruses with respect to the characteristics of influenza virus replication. The culture solution and the method have the advantages of simple operation conditions, low cost, safety and stability, and can obtain high-titer viruses in a short period.

As used herein, unless otherwise indicated, the term "cell" or "virus-producing cell" refers to a cell suitable for expansion/propagation of influenza virus, which is cultured in an appropriate culture medium to provide an appropriate assembly environment for influenza virus after infection with influenza virus. The cells are preferably non-human mammalian cells, more preferably MDCK cells.

As used herein, the term "minimal medium" refers to a medium in which virus-producing cells are routinely grown, and is used in the present invention for the purpose of routinely culturing cells prior to viral infection. Commercial minimal media, more specifically, Driving-M media or media similar thereto, have been developed in the art for virus-producing cells.

As used herein, the term "serum-free maintenance medium" refers to a medium used for viral infection and propagation of cultured cells.

As used herein, "passaging" generally includes: allowing the influenza virus to replicate in cell culture; for example, collecting the replicated virus from the culture supernatant; and transferring the collected replication viruses to uninfected cell cultures. The process may be repeated.

As used herein, the terms "comprising," "having," or "including" include "comprising," "consisting essentially of … …," "consisting essentially of … …," and "consisting of … …"; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

As used herein, the phrase "substantially" does not exclude "completely," e.g., a composition that is "substantially free of Y may be completely free of Y. The word "substantially" may optionally be omitted from the definition of the invention.

Culture method

The present invention discloses a method for amplifying influenza virus, comprising: (1) providing an influenza virus producing cell cultured in a cell culture fluid; (2) providing a serum-free maintenance medium comprising a supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate; and, (3) adding the culture solution of (2) to the culture solution of the virus-producing cells of (1); infecting the virus-producing cells with an influenza virus, and culturing the cells to produce the influenza virus.

Examples of such virus-producing cells include, for example: kidney cells, fibroblasts, retinal cells, lung cells, and the like. More specifically, for example: canine cell lines, such as including CLDK and MDCK cell lines; hamster cell lines, such as including BHK21 or HKCC cell lines; monkey cell lines, e.g., including african green monkey cells (e.g., kidney cell Vero cell line).

In the present invention, the virus-producing cell is preferably not a human cell line. In some preferred embodiments, contamination of influenza virus by potentially other human viruses in the presence of such cells is avoided by not using human cells or cells from animals in close proximity to humans. In some preferred embodiments, the virus-producing cell is not a Vero cell line (monkey kidney cell line). A list of some cell lines is for example: MDCK, Vero, 293T, CHO, CLDK, HKCC, BHK, MRC5, PER. C6, FRhL2, WI-38.

In a preferred embodiment of the invention, the virus-producing cell used is a canine cell line, such as the MDCK cell line (Madin Darby canine kidney cell line). MDCK cells provide a good environment for the virus to replicate, but have a relatively low probability of contamination. Compared with the method for producing influenza virus by adopting a chick embryo method and other cell matrixes, the MDCK cell serum-free suspension culture is easy to realize the large-scale production of influenza vaccine because the production process is simple to operate and does not add serum.

The cultured influenza viruses suitable for use in the present invention may be viruses that have been established lines, or may be primary isolated viruses, viruses isolated from organisms (e.g., patients). Influenza viruses can be isolated from respiratory secretions, including but not limited to: direct suction, mouthwash, nasal wash, nasal swab, pharyngeal tube swab, pharyngeal swab, and the like. These samples are typically obtained from patients suspected of being infected with influenza virus, including patients with the novel influenza virus strain.

Influenza viruses include influenza a, influenza b, and influenza c. In a preferred embodiment of the present invention, the influenza virus is human influenza virus. In a preferred embodiment of the present invention, the influenza virus is influenza a virus. The influenza A virus has H1-H15 and N1-N9 based on the difference between H antigen and N antigen.

The inventor finds that the method of the invention has a particularly remarkable effect on promoting amplification of influenza A viruses H1N1 and H9N2, which are illustrated in the examples of the invention by detailed examples.

The virus can infect and expand on adherent or suspension cultured cells, and microcarrier can be used to culture cells. In a preferred embodiment of the present invention, influenza virus-producing cells are cultured in a suspension culture. As a preferred embodiment of the present invention, influenza virus-producing cells are cultured in a serum-free suspension culture (without serum additives derived from human or animal sources).

The MDCK cell is easy to be cultured in a suspension way, has good sensitivity to different types and subtypes of influenza viruses, and is suitable for the amplification of the influenza viruses.

In the method of the invention, commercial serum-free culture solution can be used, and key additives are added on the basis of the commercial serum-free culture solution to promote the amplification of the virus. The commercial serum-free culture solution comprises: serum-free medium Xeno-CDM or serum-free medium Xeno-SFM. Xeno-SFM maintenance medium: obtained from the company Kyoto Biotech, Ltd under the name Xeno^TM-S001S, cat # FG 0100403. Xeno-CDM maintenance medium: available from Beizhi Biotechnology Limited under the trade name Xeno^TMCD001S, cat # FG 0104103.

The present inventors have also found that the amount of the additive added in the method of the present invention is particularly critical to the effect of promoting viral amplification. When sodium butyrate and galactose are used in combination, the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 25 to 150mg/L, preferably 30 to 120mg/L, more preferably 40 to 100 mg/L; and the content of the galactose in the serum-free maintenance culture solution is as follows: 10 to 60mg/L, preferably 20 to 50 mg/L. When sodium butyrate is used alone, the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 25 to 150mg/L, preferably 30 to 120mg/L, and more preferably 40 to 100 mg/L. It is clear that the more additives are used, the better the appropriate amount is, and the key to successful efficient amplification of the virus.

The amplification time may be, for example, 0.5 to 6 days, 1 to 5 days, 1.5 to 4 days, 1.5 to 3.5 days after virus inoculation (virus infection) to harvest the virus. Specifically, the number of days is 1 day, 2 days, 3 days, 4 days or 5 days. As a preferred mode of the present invention, the amplification time is 1.5 to 4 days, preferably 1.5 to 3.5 days, more preferably 2 to 3 days.

Viral particles can be harvested from the virus-containing liquid by a variety of methods. The purification method may include zonal centrifugation with a linear sucrose gradient solution (containing detergent to disrupt the viral particles). After optional dilution, the antigen can be purified by diafiltration.

In addition, the influenza virus obtained by the method can be frozen, recovered, passaged and maintained and cultured for a long time.

Culture solution and Kit (Kit)

Among the various key operational factors of the process for producing influenza virus based on serum-free suspension culture of MDCK cells, the development and optimization of culture media is one of the cores to ensure cell growth and virus replication. Influenza viruses utilize host cells and various raw materials in culture fluid to synthesize viral genetic materials, proteins, lipids and other essential materials required for replication and amplification, and thus the culture fluid is also one of the key factors in the production stage of influenza vaccines. At present, in the production stage of influenza viruses, most of used virus maintenance culture solution is the same as cell growth culture solution used in the cell growth stage, so that the deficiency of nutrient substance requirements required by virus replication is caused, the amplification efficiency of the viruses in cells is influenced finally, and the virus yield is low.

The invention provides a maintenance culture solution capable of efficiently amplifying human influenza virus. Comprising adding sodium butyrate or a combination of sodium butyrate and galactose to a commercial maintenance medium for viral amplification. Compared with a culture solution without adding sodium butyrate or a sodium butyrate/galactose combination, in different influenza virus subtype viruses, the highest 1.5-2 times of virus yield improvement can be obtained by using the maintenance culture solution added with sodium butyrate, and the highest 2-3 times of virus yield improvement can be obtained by using the maintenance culture solution added with the sodium butyrate and the galactose combination, so that the problem of low amplification efficiency of the influenza viruses cultured based on MDCK cells at present is effectively overcome, and the effect of improving the virus yield of different types or subtypes of influenza viruses is achieved.

The influenza virus-producing cells are cultured in a basic culture solution (basic culture solution) before the addition of the additive. It will be appreciated that the basic cell culture solution suitable for use with the cells used in the present invention may be one well known to those skilled in the art. For example, MDCK cells are known as a cell, and suitable culture media are also known in the art. Therefore, the basic culture solution may not be limited to those exemplified in the present invention.

The research of the invention shows that the addition of sodium butyrate with proper concentration can enable more glucose and glutamine to enter tricarboxylic acid cycle by improving the metabolism of cells so as to improve the metabolic rate and utilization efficiency of related nutrients, and the sodium butyrate can also be converted into a precursor acetyl coenzyme A of TCA cycle and lipid synthesis, so that the TCA cycle and the lipid synthesis can be accelerated. In addition, the physiological state of the cells can be changed by the sodium butyrate, on one hand, the cell cycle is regulated, more cells are in the G0/G1 stage, and the adsorption infection of the cells is facilitated due to the fact that the content of sialic acid on the surfaces of the cells in the G0/G1 stage is higher; on the other hand, the method delays the cell aging and apoptosis, and ensures that the cells have sufficient time to utilize intracellular resources to proliferate, thereby improving the yield of the influenza virus.

The research of the invention also shows that the addition of galactose with proper concentration can change the cell metabolism, stimulate the absorption of glutamine and synthesize energy substances required by the replication of influenza virus; in addition, galactose is a key raw material for the glycosylation synthesis of virus protein, and the virus protein glycosylation can influence the virulence of the virus, thereby influencing the proliferation condition of progeny viruses.

Therefore, appropriate concentrations of sodium butyrate and galactose can alter cell metabolism, cell physiological state, and provide material raw materials; moreover, it is even more important and surprising that these changes can be matched just effectively with the replication of influenza virus, increasing the intracellular replication efficiency of influenza virus and thus the virus yield.

In a preferred embodiment of the invention, the maintenance medium comprises adding 20-200 mg/L sodium butyrate to two commercial serum-free media (Xeno-CDM and Xeno-SFM), adding the sodium butyrate to the cell culture medium at a ratio of 1: 1-1: 2 (maintenance medium: original medium) before virus infection, diluting the cell density while increasing the culture volume, increasing nutrients required for virus amplification and diluting metabolic byproducts in the culture medium, and is used for amplifying H1N1 and H9N2 influenza viruses of different subtypes. Then, the corresponding H1N1 or H9N2 influenza virus was added at an MOI of 0.01, and TPCK-pancreatin was added at a final concentration of 5 μ g/mL, and the change in HA titer in the culture solution was measured every 24 hours.

In a preferred embodiment of the invention, HA titers of subtype H1N1 up to 512 HAU/50. mu.L and 192 HAU/50. mu.L, and HA titers of subtype H9N2 up to 1024 HAU/50. mu.L and 512 HAU/50. mu.L, respectively, were obtained using Xeno-SFM and Xeno-CDM maintenance medium supplemented with sodium butyrate. Compared with the maintenance culture solution without adding sodium butyrate, the maintenance culture solution with sodium butyrate can obtain 1.5-2 times of virus yield improvement aiming at different subtype influenza viruses. In which HAU is an agglutination Unit (Hemagglutinating Unit).

In a preferred embodiment of the invention, HA titres of subtype H1N1 up to 1024 HAU/50. mu.L and HA titres of subtype H9N2 up to 1536 HAU/50. mu.L are obtained using Xeno-SFM maintenance medium supplemented with a combination of appropriate amount of sodium butyrate and appropriate amount of galactose. Compared with the maintenance culture solution without adding the sodium butyrate/galactose combination, the maintenance culture solution with the sodium butyrate/galactose combination can obtain 2-3 times of virus yield improvement aiming at different subtype influenza viruses.

The invention also provides a Kit (Kit) comprising a serum-free maintenance medium according to the invention comprising a supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate. Preferably, the kit further comprises instructions for use, thereby facilitating the use of the kit in research or in production by a person skilled in the art.

Applications of

The virus prepared by the invention can be applied to preparing influenza virus vaccines. Vaccines are usually based on live or inactivated viruses. Inactivated vaccines can be based on whole virus particles, "split" virus particles, or on purified surface antigens. Influenza antigens may also be presented in virion form. The viruses produced by the methods of the present invention can be used in the manufacture of any of these types of vaccines.

Where inactivated virus is employed, the vaccine may comprise whole virus particles, split virus particles or purified surface antigens (including hemagglutinin and often also neuraminidase). Chemical means for inactivating viruses include treatment with an effective amount of one or more of the following agents: detergent, formaldehyde, beta-propiolactone, methylene blue, psoralen, carboxyfullerene (C60), diethylamine (binarylethylamine), acetylethyleneimine, or a combination thereof. Non-chemical methods of viral inactivation are known in the art, such as UV or gamma radiation.

Another form of inactivated influenza antigen is a virion. Virosomes can be prepared by solubilizing the influenza virus with a detergent, followed by removal of the nucleocapsid and reconstitution of the membrane containing the viral glycoproteins. Another method for preparing a virosome comprises: viral membrane glycoproteins are added to excess phospholipids, resulting in liposomes with viral proteins in the membrane.

Influenza viruses may be attenuated. The influenza virus may be temperature sensitive. The influenza virus may be a cold-adapted virus. These three features are particularly useful when using live viruses as antigens.

HA is the main immunogen in inactivated influenza vaccines, and vaccine doses are generally normalized by reference to HA levels by SRID assays.

Live viruses are used for example to prepare vaccines by: the virus is cultured and then the virus particles are purified from the fluid containing the virus particles. For example, the fluid may be separated by centrifugation and stabilized with a buffer (e.g., containing sucrose, potassium phosphate, and monosodium glutamate).

As a form, purified virus particles are treated with a detergent (e.g., diethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, triton X-100, triton N101, cetyltrimethylammonium bromide, tergitol NP9, etc.) to obtain split virus particles, thereby producing a subviral particle formulation. Methods for splitting influenza viruses are well known in the art. The virus is typically split using a disrupting concentration of a splitting agent to disrupt or fragment the intact virus, regardless of the presence or absence of infectivity of the virus. This disruption results in complete or partial solubilization of viral proteins, altering the integrity of the virus.

The purified surface antigen vaccine comprises the influenza surface antigen hemagglutinin and typically also neuraminidase. Methods for preparing these proteins in purified form are well known in the art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, or according to the manufacturer's recommendations.

The present inventors have conducted intensive studies and screening in order to promote the culture efficiency of influenza virus. Based on various chemical substances, biological growth factors and the like, the inventors performed a great number of experimental analyses, and as a result, they showed that sodium butyrate and galactose have a significant effect of promoting the culture efficiency of influenza virus. The following examples illustrate the function of the preferred compounds.

Example 1 acceleration of influenza Virus amplification by sodium butyrate

In this example, an influenza virus amplification experiment was performed using a maintenance culture solution containing sodium butyrate at different concentrations, and the effect of sodium butyrate was observed.

1. Experimental setup

Virus-producing cells: MDCK cells, which were cultured in Driving-M medium before infection with virus.

The preparation method of the Driving-M culture solution comprises the following steps:

(1) weighing 100% of the final culture medium preparation volume of water for injection to the culture medium preparation container, wherein the water temperature is controlled at 28-32 deg.C.

(2) 22.21g/L dry powder (Dry-M dry powder) is accurately weighed and added into a preparation container, and the mixture is fully stirred for 20 to 30 minutes.

(3) Slowly dropwise adding 5mol/L sodium hydroxide solution into the solution prepared in the step (2), adjusting the pH value to 6.2-6.7, and fully stirring for 10-20 minutes.

(4) Accurately weighing 2.00g/L sodium bicarbonate powder, adding into a preparation container, and stirring for 10-20 minutes.

(5) The culture medium solution was sterile filtered through a sterile filter with a pore size of 0.22 μm.

Xeno-SFM maintenance medium: available from Beizhi Biotechnology Limited under the trade name Xeno^TM-S001S, cat # FG 0100403.

Xeno-CDM maintenance medium: obtained from the company Kyoto Biotech, Ltd under the name Xeno^TMCD001S, cat # FG 0104103.

Xeno-SFM and Xeno-CDM maintenance culture solutions containing sodium butyrate with different concentrations of 0-400 mg/L are prepared according to a preparation method, and an equal volume of maintenance culture solution is added into the culture solution at the moment of virus infection, so that the cell density of MDCK is diluted to half.

H9N2 or H1N1 influenza virus and TPCK pancreatin at a final concentration of 5. mu.g/mL were added at an MOI of 0.01, and virus culture was carried out at 33 ℃.

2. Experimental data and results

(1) Xeno-SFM maintenance culture solution for culturing H1N1

The results of culturing H1N1 influenza virus by adding sodium butyrate at different concentrations to the Xeno-SFM maintenance medium are shown in Table 1 and FIG. 1, where hpi is the number of hours after inoculation (hours-inoculation).

TABLE 1

According to Table 1 and FIG. 1, the addition of 48mg/L to 200mg/L of sodium butyrate significantly promoted viral amplification compared to the commercial maintenance medium alone, wherein a high viral titer was maintained for a longer period of time, which is relatively ideal when 48mg/L to 100mg/L of sodium butyrate was added; when the sodium butyrate with the dosage of about 78-100mg/L is used, the virus needs to be harvested within a shorter time (such as about 48 hours) through amplification, and does not need to be harvested within a longer time (such as about 72 hours). Meanwhile, the addition of an excessively high concentration of sodium butyrate inhibits the amplification of the virus.

(2) Xeno-SFM maintenance culture solution for culturing H9N2

The results of culturing H9N2 influenza virus by adding sodium butyrate to the Xeno-SFM maintenance medium at different concentrations are shown in Table 2 and FIG. 2.

TABLE 2

According to table 2 and fig. 2, the addition of 48mg/L to 100mg/L of sodium butyrate can significantly promote the amplification of virus, compared with the mere use of commercial maintenance medium, wherein the promotion effect is relatively ideal when about 48-100mg/L of sodium butyrate is added, and the high virus titer is maintained for a longer time; when the sodium butyrate is used at the dosage of about 48-100mg/L, the virus is harvested after a short amplification time (such as about 48 hours), and the titer is possibly reduced although the higher virus titer can be basically maintained when the virus is amplified for a longer amplification time (such as about 72 hours). Meanwhile, the addition of an excessively high concentration of sodium butyrate inhibits the amplification of the virus.

(3) Culture of H1N1 in Xeno-CDM maintenance culture solution

The results of culturing H1N1 influenza virus by adding sodium butyrate to Xeno-CDM maintenance medium at various concentrations are shown in Table 3 and FIG. 3.

TABLE 3

According to Table 3 and FIG. 3, the addition of 48mg/L to 88mg/L of sodium butyrate promoted the amplification of virus, compared with the case of using only commercial maintenance culture medium, and it is preferable to use 48 to 58 mg/L. Meanwhile, the addition of an excessively high concentration of sodium butyrate inhibits the amplification of the virus.

(4) Culture of H9N2 in Xeno-CDM maintenance culture solution

The results of culturing H9N2 influenza virus by adding sodium butyrate to Xeno-CDM maintenance medium at various concentrations are shown in Table 4 and FIG. 4.

TABLE 4

From Table 4 and FIG. 4, the addition of 48mg/L to 100mg/L of sodium butyrate promoted viral amplification as compared to the case of using only commercial maintenance medium. Meanwhile, the addition of an excessively high concentration of sodium butyrate rather inhibits the amplification of the virus.

3. Summary of the invention

According to the results of this example, the titer of influenza virus was improved in the range of about 48 to 100mg/L of sodium butyrate by using the Xeno-SFM maintenance medium to which 0 to 400mg/L of sodium butyrate was added. When the culture time is up to 72 hours, the control HA titers of the H1N1 and H9N2 of the group without adding sodium butyrate are 256HAU/50 muL and 512HAU/50 muL, and the highest Xeno-SFM added with 48-100mg/L of sodium butyrate can obtain the HA titers of 512HAU/50 muL and 1024HAU/50 muL, so that the yield is improved by about 2 times.

The use of a Xeno-CDM maintenance culture solution to which 0 to 400mg/L of sodium butyrate is added has been found to improve the titer of influenza viruses even at a concentration of 48 to 100mg/L of sodium butyrate. When the culture time reaches 72 hours, the control HA titers of the H1N1 and H9N2 of the groups without adding sodium butyrate are 128HAU/50 muL and 256HAU/50 muL, and the highest Xeno-CDM added with 48-100mg/L of sodium butyrate can obtain HA titers of 192HAU/50 muL and 512HAU/50 muL, and yield increase of 1.5 times and 2 times can be respectively obtained.

The results of this example also show that the use of appropriate sodium butyrate concentrations is critical, whereas too high concentrations are detrimental to viral amplification.

Example 2 acceleration of influenza Virus amplification by galactose in combination with sodium butyrate

The research results of the inventor also show that the promoting effect of the sodium butyrate on the virus amplification still presents a certain degree of instability or limitation for different types of commercial culture solutions or viruses, and the search for further useful substances for promoting the effect is also necessary.

In this example, an influenza virus amplification experiment was performed using a maintenance medium containing a combination of sodium butyrate and different concentrations of galactose, and the acceleration effect of galactose in combination with sodium butyrate on influenza virus amplification was observed.

1. Experimental setup

Preparing Xeno-SFM maintenance culture solution containing 50mg/L of sodium butyrate and galactose with different concentrations of 0-80 mg/L according to a preparation method, and adding equal volume of maintenance culture solution into the culture solution at the moment of virus infection to dilute the cell density to half.

H9N2 or H1N1 influenza virus and TPCK pancreatin at a final concentration of 5. mu.g/mL were added at an MOI of 0.01, and virus culture was performed at 33 ℃.

2. Experimental data and results

(1) Xeno-SFM maintenance culture solution for culturing H1N1

The results of culturing H1N1 influenza virus by adding 50mg/L sodium butyrate and different concentrations of galactose to the Xeno-SFM maintenance medium are shown in Table 5 and FIG. 5.

TABLE 5

According to the table 5 and fig. 5, compared with the commercial maintenance culture solution, the combined addition of galactose and sodium butyrate (especially 28-48mg/L) significantly increases the amplification result of the H1N1 influenza virus in different time periods, and the synergistic effect of galactose with proper concentration is very obvious.

(2) Xeno-SFM maintenance culture solution for culturing H9N2

The results of culturing H9N2 influenza virus by adding 50mg/L sodium butyrate and different concentrations of galactose to the Xeno-SFM maintenance medium are shown in Table 6 and FIG. 6.

TABLE 6

According to the table 6 and fig. 6, compared with the commercial maintenance culture solution, the combined addition of galactose and sodium butyrate (especially 28-48mg/L) significantly increases the amplification result of the H9N2 influenza virus in different time periods, and the synergistic effect of galactose with proper concentration is very obvious.

3. Summary of the invention

The results of this example show that the addition of a combination of appropriate concentrations of sodium butyrate and appropriate concentrations of galactose can further enhance influenza virus production over the addition of sodium butyrate alone. Wherein, the control HA titers of the H1N1 and H9N2 of the additive-free group at 72 hours are 256HAU/50 muL and 512HAU/50 muL, and the highest Xeno-SFM added with 50mg/L sodium butyrate and 28-48mg/L galactose can obtain the HA titers of 1024HAU/50 muL and 1536HAU/50 muL, and the yield is respectively improved by 2 times and 3 times. The results of this example are sufficient to support the synergistic effect of galactose at the appropriate concentration.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims. Also, all references cited herein are incorporated by reference in this application as if each reference were individually incorporated by reference.

Claims

1. A method for amplifying influenza virus, the method comprising:

(1) providing an influenza virus producing cell cultured in a basic culture medium;

(2) providing a serum-free maintenance medium comprising a supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate;

(3) adding the culture solution of (2) to the basic culture solution of the virus-producing cells of (1); infecting the virus-producing cells with influenza virus, culturing the cells, and producing influenza virus.

2. The method of claim 1, wherein the sodium butyrate is present in the serum-free maintenance medium in (a) in an amount that: 20-200 mg/L, wherein the content of galactose in the serum-free maintenance culture solution is as follows: 5-80 mg/L; and/or

(b) Wherein the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 20-200 mg/L.

3. The method of claim 2, wherein the sodium butyrate is present in the serum-free maintenance medium in (a) in an amount that: 25 to 150mg/L, preferably 30 to 120mg/L, more preferably 40 to 100 mg/L; and the content of the galactose in the serum-free maintenance culture solution is as follows: 10-60 mg/L, preferably 20-50 mg/L; and/or

(b) Wherein the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 25 to 150mg/L, preferably 30 to 120mg/L, and more preferably 40 to 100 mg/L.

4. The method of claim 1, wherein said serum-free maintenance medium comprises a medium selected from the group consisting of: Xeno-SFM maintenance medium, Xeno-CDM maintenance medium, Ex-Cell medium; or

The influenza virus producing cell is a non-human mammalian cell; preferably MDCK cells; preferably, the cell culture solution in (1) is an MDCK cell culture solution; or

The influenza virus is human influenza virus; preferably, the influenza virus is influenza a virus; preferably, the influenza A virus is different from an H antigen and an N antigen, wherein H is selected from H1-H15, and N is selected from N1-N9; more preferably, the influenza a virus comprises a virus selected from the group consisting of: H1N1 virus, H9N2 virus, H5N1 virus, H7N9 virus.

5. The method according to claim 1, wherein in (3), when the culture solution of (2) is added to the culture solution of the influenza virus-producing cells of (1), the ratio of the culture solution of (2) to the cell culture solution of the influenza virus-producing cells of (1) is 1:0.5 to 1:2.5, preferably 1:0.9 to 1:2.2, more preferably 1:0.9 to 1: 1.5; and/or

(3) Wherein when the virus-producing cell is infected with an influenza virus, the MOI is 0.001 to 0.1, more preferably 0.005 to 0.05; and/or

(3) The culture is suspension culture.

6. A method of producing an influenza virus vaccine, comprising: amplifying influenza virus by the method of any one of claims 1 to 5; the obtained influenza virus is inactivated, attenuated or separated from the obtained influenza virus to obtain the influenza virus vaccine with immunogenicity.

7. A method of preparing a serum-free maintenance medium for amplifying influenza virus, comprising: adding a supplement to the serum-free maintenance medium, the supplement selected from the group consisting of: (a) sodium butyrate and galactose, or (b) sodium butyrate;

preferably, in (a), the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 20-200 mg/L, wherein the content of galactose in the serum-free maintenance culture solution is as follows: 5-80 mg/L;

preferably, in (b), the content of the sodium butyrate in the serum-free maintenance culture solution is as follows: 20-200 mg/L.

8. A kit for amplifying influenza virus, comprising: a serum-free maintenance medium prepared by the method of claim 7;

preferably, the kit further comprises: influenza virus producing cells, cell culture fluid for influenza virus producing cells; wherein the influenza virus producing cells are independent of the culture medium or are cultured in the culture medium.

9. The kit of claim 8, wherein said serum-free maintenance medium comprises a medium selected from the group consisting of: Xeno-SFM maintenance culture solution and Xeno-CDM maintenance culture solution; and/or

The influenza virus production cell is an MDCK cell; preferably, the cell culture solution in (i) is an MDCK cell culture solution; and/or

In the kit, the ratio of the serum-free maintenance culture solution containing the additive to the culture solution of the influenza virus production cells is 1: 0.5-1: 2.5, preferably 1: 0.9-1: 2.2, and more preferably 1: 0.9-1: 1.5.

10. Use of the kit of claim 8 for amplifying influenza virus; preferably, the influenza virus is human influenza virus; preferably, the influenza virus is influenza a virus; preferably, the influenza A virus has H selected from H1-H15 and N selected from N1-N9 according to the difference of H antigen and N antigen; more preferably, the influenza a virus comprises a virus selected from the group consisting of: H1N1 virus, H9N2 virus, H5N1 virus, H7N9 virus; preferably, the virus comprises: a strain-preserved virus, a primary isolated virus, or a virus isolated from an individual.