RU2330885C2 - Method of production of live culture influenza virus vaccines - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology. Method includes cultivation of transplantable cells MDCK in nutrient medium, production of virus-containing substances by cultivation vaccinating influenza virus strains in MDCK cells culture on the same nutrient medium, virus substance purification from ballast impurities, stabilising additives introduction to purified substance and drying of the finished vaccine form. Thus inoculation dose of transplantable cells MDCK is 100-600 thousand cells per 1 ml of environment. Vaccinating cold-adapted reasortants of influenza virus strains are used as virus strains with inoculation dose of infection multiplicity 0.01-0.0001 "ЭИД₅₀/_КЛ-". Serum-free nutrient medium added with soya hydrolysate concentrated 0.01-10% and proteolytic enzyme in amount 0.25-50.0 mkg/ml prior to introduction of influenza virus strains inoculation dose is used as nutrient medium for cultivation of MDCK cells and influenza virus strains on specified cells. Cultivation of MDCK cells cultures and cold-adapted influenza virus strains in specified cell culture is performed in suspensions on microcarriers brought in serum-free nutrient medium in concentration 1-6 g/l. Collection of virus-containing liquid is carried out after specific activity of influenza virus is not less than 8.0 lg "ЭИД"₅₀/ml. Saccharose and soya peptone in final concentration (5-10) and (3-8) mass % respectively are used as stabilising additives introduced into purified substance before drying.

EFFECT: method enables to produce vaccine with lower content of foreign animal protein components.

7 cl, 3 tbl, 7 ex

Description

Technical field

The invention relates to a technology for producing live influenza vaccine and can be used in the pharmaceutical industry for the production of vaccines and therapeutic drugs.

All over the world, inactivated, subunit forms of influenza vaccines prepared on chicken embryos are produced and used to protect the population against influenza, which, when used correctly, can protect 80% of those vaccinated against influenza. However, the high cost of such drugs, as well as the complexity of the production and use of inactivated vaccines make it difficult to use them for mass prevention of influenza. Despite the fact that inactivated vaccines form a higher level of general humoral immunity, these drugs are practically not able to induce local secretory immunity. The effectiveness of an inactivated influenza vaccine decreases dramatically even with small changes in the antigenic structure of the epidemic strain compared with the vaccine.

In Russia, a live vaccine is widely used that stimulates the development of asymptomatic infection with the formation of humoral, secretory and cellular immunity and immunological memory with intranasal administration. Sufficiently effective live vaccines have certain advantages over killed vaccines in terms of cost-effectiveness, ease of production and use, as well as a greater breadth of immunological effect. These benefits are associated with increased attention recently to the development of live influenza vaccine in the United States. Currently used influenza vaccines need to be adjusted annually, taking into account the constantly occurring changes in the antigenic composition of the surface glycoproteins of the virus. Every year, an unpredictable situation arises when in a very short period of time it is necessary to produce a sufficient amount of vaccine that is adequately effective against strains that are most likely to cause an influenza epidemic.

Live influenza vaccine induces a wide range of antibodies that can neutralize variants with altered antigenic specificity of hemagglutinin. In addition, a live vaccine is more economical because requires about 10 times less chicken embryos for production. The use of cell culture in the production of vaccines makes the vaccine even more economical, and the use of a serum-free culture medium completely eliminates the allergic reactions of eggs vaccinated on chicken proteins.

State of the art

A known method of producing an inactivated viral vaccine, including culturing influenza virus strains in chicken embryos for 72-96 hours, incubating the embryos overnight in a refrigerator, collecting allantoic fluid, inactivating the virus, purifying by filtration and / or centrifugation (Requirements For Inactivated Influenza Vaccine, World Health Organization Technical Report Series, 384 (1966).

The disadvantages of this method are the content in the vaccine of certain amounts of proteins of chicken embryos, which cause adverse reactions in individuals allergic to these proteins; a change in the antigenic structure of the virus during cultivation in chicken embryos due to the loss of certain antigenic domains; the possibility of contamination with extraneous viruses; the need (one-time) for a large number of chicken embryos during vaccine production. To receive a single dose of the vaccine, 1-2 chicken embryos are required, i.e. more than 1 million chicken embryos are needed to produce 1 million doses of influenza vaccine. In addition, each chicken embryo is a unique biological system and thus suggests a certain instability of the resulting vaccine preparation.

There is another way to obtain a live viral vaccine (Pharmacopoeia article of the Ministry of Health of Russia No. 42-3569-98 "Flu vaccine allantoic live intranasal dry for adults"), including infection of chicken embryos with vaccine strains of the influenza virus, collection of virus-containing allantoic fluid, introduction of meat peptone, bottling into ampoules, lyophilization and sealing. The finished product is a dry porous mass with the smell of peptone.

However, purification from ballast impurities is not provided for in the technology, which adversely affects the allergological safety of the vaccine. In addition, there is a change in the antigenic structure of the influenza virus during cultivation in chicken embryos, and vaccine contamination with retroviruses and other avian viruses present in the embryos is not ruled out.

Another method is known (US 5698433, B1, 16.16.1997) for producing an inactivated influenza vaccine, including culturing influenza virus strains on primary cells of chicken embryos in the presence of trypsin in a laboratory spinner or two-liter fermenter, collecting virus-containing liquid, ultrafiltration, centrifugation, virus inactivation and the introduction of aluminum hydroxide as an adjuvant. The technology is simple to implement, it allows to obtain large volumes of virus-containing liquid with a high titer of virus activity (the output from cells obtained from 4 chicken embryos is 400-500 ml of virus-containing liquid for 96 hours or 100 ml of antigen, which is 14 times higher than on chicken embryos). The resulting vaccine does not contain chicken protein impurities.

However, this method has several disadvantages: a primary cell culture is used that does not have standard properties and is not sufficiently characterized; contamination of cell culture and vaccine with extraneous viruses, mycoplasmas, prions is possible; cell culture of chicken embryos is used, therefore, it is possible to change the antigenic structure of strains of the influenza virus, which occurs during cultivation on chicken embryos.

In addition to these shortcomings, all known technologies for the production of influenza vaccines, including the analogues described above, use animal products (bovine serum, trypsin, gelatose or human serum albumin as a stabilizer), which can lead to allergenization of immunized, and possible infection with extraneous viruses, mycoplasmas, prions contained in animal products.

The closest analogue (prototype) is a method for producing inactivated and live influenza vaccines (US 6344354, B1, 02/05/2002), including culturing highly productive strains of the influenza virus on certified transplantable cell lines (Vero, MDCK) in nutrient medium MEM Needle with the addition of 5- 10% fetal serum and trypsin in an amount (0.05-1.0 μg / ml), washing the cells after the formation of a monolayer, introducing bovine serum albumin, collecting virus-containing liquid, purification by centrifugation or chromatography and subsequent administration of chemotherapeutic components, adjuvants or polymers (polyethylene, polypropylene).

The disadvantages of this prototype method are monolayer cell cultivation in culture bottles, which cannot ensure the production of large volumes of virus-containing fluid; the use of MEM Needle for the cultivation of cells of the nutrient medium with the addition of 5-10% fetal serum, as well as the introduction of bovine serum albumin virus at the stage of production, which leads to the introduction of components of animal feed that can be a source of extraneous viruses, mycoplasmas, prions, and also cause additional allergization of the immunized organism.

Disclosure of invention

The technical result of the proposed method is the production on an industrial scale of a live cultural trivalent influenza vaccine based on cold-adapted vaccine strains of influenza A and B virus for intranasal and parenteral administration with a lower content of extraneous protein components of animal origin, which reduces the possibility of allergic reactions.

This result is achieved by the fact that in a method for producing a live influenza vaccine, comprising cultivating transplantable MDCK cells in a nutrient medium, obtaining a virus-containing substance by cultivating vaccine strains of the influenza virus in an MDCK cell culture on the same nutrient medium, purifying the viral substance from ballast impurities, introducing the purified substance of stabilizing additives and the drying of the finished vaccine form, according to the invention, cold-adapted vaccines reas are used as viral strains Orthants of influenza virus strains, as a nutrient medium in the cultivation of MDCK cells and influenza virus strains on these cells, use a serum-free culture medium with the addition of soy hydrolyzate in a concentration of 0.01-10% and a proteolytic enzyme in an amount of 0.25-50.0 μg / ml prior to the administration of a seed dose of influenza virus strains, and the cultivation of MDCK cell cultures and cold-adapted influenza virus strains in the indicated cell culture is carried out in suspension on microcarriers introduced into serum-free culture medium in concentration of 1-6 g / l.

As a culture of MDCK cells, a culture stored at liquid nitrogen temperature in the form of seed and working banks and certified for the absence of extraneous viruses, the absence of oncogenic potentials and species identity is used.

As vaccine cold-adapted reassortants of influenza virus, vaccine strains of type A / H3N2 are used; A / H1N1 and type B of this virus.

The seeding dose of transplanted MDCK cells is 100-600 thousand cells per 1 ml of medium, and the seeding dose of vaccine strains of influenza virus has a multiplicity of infection of 0.01-0.0001 EID ₅₀ / CL.

Papain is a proteolytic enzyme of plant origin as a proteolytic enzyme, and Cytodex 1 is used as a microcarrier.

The collection of virus-containing fluid is carried out after the specific activity of the influenza virus is not less than 8.0 lg EID ₅₀ / ml, and the removal of ballast impurities from the viral substance is carried out by filtration.

As stabilizing additives, sucrose and peptone from soybeans are used in a final concentration of (5-10) and (3-8) wt.%, Respectively.

The vaccine is dried by freezing it for 18 hours at a temperature not exceeding minus 40 ° С followed by lyophilization for 48 hours, and before sealing, the ampoules with the viral vaccine are filled with an inert gas, in particular argon.

Vaccine strains of a live influenza vaccine are reassortants obtained in Russia on the basis of cold-adapted mutants - attenuation donors A / Leningrad / 134/47/57 (H2N2) and B / USSR / 60/69. These donors are characterized by cold adaptation, i.e. the ability to reproduce at low temperatures, temperature sensitivity and harmlessness for laboratory animals and humans. A necessary condition for the safety and genetic stability of vaccine strains is considered to be the presence of 5-6 mutant genes encoding the internal proteins of the attenuation donor in their genome, while the hemagglutinin (HA) and neuraminidase (NA) genes always come from the current epidemic virus.

Strain A / 47 / Chandon / 93/1 (H1N1) was obtained by crossing the cold-adapted master strain A / Leningrad / 134/47/57 (H2N2) with the epidemic strain A / Chandon / 93/1 (H1N1).

The influenza virus strain A / 47 / Johannesburg / 94/2 (H3N2) was obtained by crossing the cold-adapted master strain A / Leningrad / 134/47/57 (H2N2) with the epidemic strain A / Johannesburg / 94/2 (H3N2).

Strain B / 60 / Petersburg / 95/20 was obtained by crossing a cold-adapted master strain B / USSR / 60/69 with the epidemic strain B / Petersburg / 95/20.

Vaccine strains of the influenza virus are stored at the State Research Institute for Standardization and Control of Medical Biological Products (GISK) named after L.A. Tarasevich, Moscow.

A significant advantage of transplanted cells is their stability, standardization, the possibility of obtaining a large number of producer cells in a fairly short time, long-term use of cells, and the relatively low cost of vaccine preparations. WHO-certified cell lines are free of contaminating agents, while chicken embryos often contain retroviruses and some other agents. When growing influenza viruses on transplantable cell lines that can be cultured using serum-free media, the possibility of allergic reactions to the vaccine in people with allergies to the components of chicken embryo cells and components of the serum is excluded, which is also especially important in the manufacture of live influenza vaccines that do not pass such thorough cleaning like inactivated flu vaccines. In addition, the hemagglutinin of human influenza virus strains isolated and cultured in chicken embryos may lose one of the antigenic domains, while the isolation and cultivation of the influenza virus on the MDCK cell line preserves all antigenic domains, and the hemagglutinin of such isolates is antigenically completely identical to hemagglutinin influenza virus that multiplies in the human body. There is also evidence that vaccines prepared on MDCK or Vero cells better protect vaccinated animals from infection with virulent strains of the influenza virus, and also induce more cross-reactive antibodies than vaccines obtained on chicken embryos, which stimulate antibodies specific for to the homologous antigen (Alymova I., Kodihalli S., Govorkova E. et al. Immunogenicity and protective efficacy in mice of influenza virus B vaccines grown in mammalian).

Among cell lines potentially suitable for the production of influenza vaccines, mainly the MDCK cell line derived from dog kidney cells and the Vero cell line derived from African green monkey kidney cells were examined. In comparative experiments (Merten O., Managuerra J., Hannoun C. et al. Production of influenza virus in cell cultures for vaccine preparation. In: Novel Strategies in Design and Production of Vaccines. Eds. Cohen S., Shafferman A., Plenum Press, N.-Y., 1996, pp. 141-150), it was shown that MDCK cells produce the same number of cells 2 times faster than Vero cells and 7 times faster than BHK cells. As for the production of hemagglutinin, the total amount was approximately the same in MDCK and Vero cells, however, in MDCK cells this amount was formed on the 6th day after infection, and in Vero cells on the 14th.

Soy hydrolyzate is obtained by partial hydrolysis of Maisol soy isolate (Germany) in a 0.1 normal solution of hydrochloric acid under two excess atmospheres in an autoclave. The soy hydrolyzate at the indicated concentrations is an additional nutritional component for MDCK cell cultures.

Papain, a plant proteolytic enzyme of the hydrolase class, exhibits broad substrate specificity and is manufactured by ICN, USA. Papain provides in a nutrient medium at the indicated concentrations the guaranteed penetration of the influenza virus into MDCK cells during their infection with the indicated virus.

Microcarriers provide at the indicated concentrations higher production of cells and virus and a higher yield of virus-containing material. In the course of experimental studies, it was precisely Sigma Cytodex 1 microcarriers that were selected that were 1 mm in size, on the surface of which stable monolayers of MDCK cells are formed, which are capable of producing the influenza virus without losing their physiological functions.

The serum-free nutrient medium MDSS2 is manufactured by Axcevir, France.

Embodiments of the invention

The following are examples of a specific implementation of the method for producing a live culture influenza vaccine.

Example No. 1. Certification of cell culture MDCK

For work using a culture of MDCK cells from the seed and working banks. Certification of cell culture MDCK carried out in accordance with the requirements of the WHO for transplantable cell lines.

The cytomorphology of culture in native and fixed preparations in a light microscope was studied. It was shown that the MDCK cell culture consists of epithelial-like cells. The nuclei are rounded with chromatin grit. Nuclei rounded, from one to several in the nucleus. The cytoplasm is granular. There are giant and multinucleated cells, the number of which increases during cultivation. Cells form a monolayer on the 2nd day of cultivation, at the same time, a peak in mitotic activity is noted, pathological mitoses are not observed.

During the control of the culture fluid by the method of seeding on a thioglycol medium, it was found that the seed and working banks of MDCK cells are sterile.

Mycoplasma was not detected by direct inoculation of the culture fluid of the inoculum and working banks of MDCK cells on selective nutrient media and by DNA staining with intercalating fluorochromes. For the PCR analysis system, two pairs of primers were used (GPO-1 and MGSO-for the first cycle; GPO-2 and MGSO-for the second cycle), which make it possible to identify all known types of mycoplasmas. Mycoplasma was not detected by PCR analysis.

A control was conducted to detect foreign viruses in cell culture, chicken embryos and animals. During the control of the inoculum and working banks on the cell culture, the state of the monolayer of intact cells of the inoculating and working banks was studied for 14 days. No cytopathic effect (CPP) was found. After this period, samples of the culture fluid were examined in a hemagglutination reaction, and the monolayer in the hemadsor sion reaction with guinea pig erythrocytes. The reactions are negative. Culture fluid samples were examined in three types of cell cultures: MDCK (homologous), L-68 (diploid culture), Vero, for 14 days. No CPC detected. After this period, samples of the culture fluid were examined in a hemagglutination reaction, and the monolayer was studied in a hemadsorption reaction with guinea pig erythrocytes. The reactions are negative. In the seed and working banks of the MDCK line, no cytopathogenic, hemadsorbing and hemagglutinating viruses were detected.

To control the seed and working banks on chicken embryos, the studied material in the amount of 10 ⁶ cells was introduced into the yolk sac, allantoic cavity and the chorionallantoic membrane of chicken embryos (10 embryos per point), the embryos were incubated for 7-10 days, after which the viability was determined embryos and put a hemagglutination reaction with allantoic fluid. No foreign viruses were detected in the seed and working banks of the MDCK line.

To control the seed and working banks in laboratory animals, the studied material was intramuscularly administered to sucker mice at the age of 24 hours (28 heads of sucker mice, 10 ⁷ cells per group), adult mice (10 goals, 10 ⁷ cells per group of mice), and guinea pigs (5 animals, 10 ⁷ cells per group), rabbits (5 animals, 10 ⁷ cells per animal group), the animals were monitored for 28 days, after which autonasia and internal organs were examined. All animals in the experiment were alive, no changes in internal organs were noted. No extraneous viruses were found in the seed and working banks of MDCK cells.

The oncogenic potencies of the cells of the seed and working banks were determined in vivo. In the control, the method of heterotransplantation of cells to immunosuppressive animals was used in comparative experiments with the Hela reference cell line, which causes progressive tumor growth. The animals were observed for 21 days. In this case, all animals were alive, with a macroscopic examination for the presence of a tumor in the lymph nodes, liver, kidneys, lungs and at the injection site, pathological changes were not detected. When monitoring in vivo, it was found that the MDCK cells of the seed and working banks do not possess oncogenic potentials.

The species identity of MDCK line cells was verified by karyological analysis and polyacrylamide gel electrophoresis to determine isoenzymes. A karyological analysis of MDCK cells revealed that the karyotype is a derivative of the normal dog karyotype, Canis familiaris, 2n = 78. MDCK cells are characterized by the presence of normal (not rearranged) autosomes; trisomy is noted for some autosomes (pairs 29, 30, 38). One of the X chromosomes is also not rearranged. In addition, MDCK cells are characterized by the presence of marker chromosomes resulting from a variety of translocations of chromosome regions (presumably X, 3, 4, 21, 23, 24, 28). No obvious polyploids were noted. The cells of the seed and working banks of MDCK have a fairly stable karyotype, characterized by clear morphological characteristics. An analysis of the electrophoretic mobility of the G-6-FDG isoenzyme was carried out. The MDCK cells of the seed and worker banks have G-6-FDG characteristic of dog cells.

Example No. 2. Preparation of the Substance of Live Culture Influenza Vaccine (VHF)

A culture vessel of a 1- or 10-liter fermenter is filled with serum-free culture medium. A certified MDCK cell culture (in accordance with Example 1) is taken from the collection cell bank of the State Scientific Center of the WB “Vector” and introduced into the indicated culture vessel at the rate of 100-600 thousand cells per ml of culture medium. Additionally, the microcarrier Cytodex I is introduced into the culture vessel at a concentration of 1-6 g / l. Cell deposition on a microcarrier is carried out for 12 hours at a temperature of 35-37 ° C (the filling volume of the culture vessel is 25-30%). Cell planting mode: 5 min stirring, 10 min break. The specified cultivation parameters: the stirring device rotational speed of 50-80 rpm, surface aeration with a blowing rate from 0 to 10 l / h. The pH is maintained between 6.9 and 7.2 with a dosed supply of a 7.5% sodium carbonate solution. The cultivation temperature is 35-37 ° C. Culture medium perfusion is started at a cell concentration of 600-900 thousand / ml. The perfusion rate depends on the concentration of cells. The cell cultivation time is 96-144 hours. After the accumulation of the cells, 50% of the medium is removed, one of the vaccine cold-adapted reassortants of the influenza virus, for example A / 47 / Johannesburg / 94/2 (H3N2), with a multiplicity of infection of 0.01-0.0001 EID ₅₀ / cell, and supporting serum-free culture medium containing the proteolytic enzyme papain in an amount of 0.25-50.0 μg / ml or trypsin in an amount of 2.0-20.0 μg / ml and a soy hydrolyzate in a concentration of 0.01-10%. Then the virus is cultured at a temperature of (34 ± 1) ° C. When the cytopathic effect of the virus on the cells appears (2-5 days), a virus-containing liquid is collected with a specific activity of at least 8.0 lg EID ₅₀ / ml, which is released by filtration from cellular detritus. A sterile stabilizer solution (sucrose and peptone from soybeans at a final concentration of 10% and 5%, respectively) is added to the virus-containing suspension to obtain a liquid semi-finished product of strain A / 47 / Johannesburg / 94/2 (H3N2) of live influenza vaccine (GHF).

In the same way, in parallel or sequentially, a liquid semi-finished product of vaccine strains of cold-adapted reassortants of the influenza virus A / 47 / Chandon / 93/1 (H1N1) and B / 60 / Petersburg / 95/20 is produced.

Note: EID is an embryonic infectious dose.

Example No. 3. Obtaining the finished form of ZhGV

To obtain a trivalent vaccine before bottling, liquid semi-finished products obtained from the virus-containing liquid of vaccine strains A / 47 / Johannesburg / 94/2 (H3N2), A / 47 / Chandon / 93/1 (H1N1) and B / 60 / Petersburg / 95/20 influenza virus, are combined in equal proportions. To obtain a single vaccine, a liquid semi-finished product of one of the above strains of the influenza virus is used. The resulting liquid form of the vaccine is poured into ampoules of 0.6 ml each and frozen at a temperature not exceeding minus 40 ° C for 18 hours. The product is freeze-dried for 48 hours under sterile conditions. After drying, the ampoules are filled with an inert gas (argon) and sealed.

Example No. 4. Determination of the specific activity of live influenza vaccine preparations

The specific activity of the virus-containing material is determined in monovaccines to the information in the trivalent vaccine obtained simultaneously with the trivaccine, by titration on chicken embryos according to the method described in the Methodological guidelines “Methods of control of medical immunobiological drugs administered to people”. MUK 4.1 / 4.2.588-96. The specific activity of the vaccine after lyophilization is not less than 6.0-6.5 lg EID ₅₀ / 0.2 ml.

Example No. 5. The study of the dynamics of the multiplication of influenza virus with different multiplicity of infection on the example of strain A / 47 / Chandon / 93/1 (H1N1) of the influenza virus

An MDCK cell culture is infected with influenza strain A / 47 / Chandon / 93/1 (H1N1) at different infection rates (0.001; 0.005, 0.0005 and 0.0001 EID ₅₀ / cell) and cultured in MEM needle or serum-free medium in the presence of 1-6 μg / ml trypsin. With a multiplicity of infection of 0.005 and 0.001 EID ₅₀ / cell, the maximum titer of the virus when using the Needle MEM medium (when using 5% fetal serum as a part of the growth medium for planting cells) is reached already for 2-3 days, and in the case of serum-free medium - only by 3 -4 days, but the titers were higher by 1-1.5 lg (table 1). A decrease in the multiplicity to 0.0005 EID ₅₀ / cell leads to a shift in the maximum titers by 1 day (for strain A / 47 / Chandon / 93/1 (H1N1) when using serum for planting cells, the maximum titer was on the 4th day and amounted to 10 ^10.5 EID ₅₀ / ml, when using serum-free medium on the 5th day, the titer is 10 ^9.5 EID ₅₀ / ml). A further decrease in the multiplicity of infection does not contribute to an increase in infectious titers within 5 days of the experiment.

Table 1 The dynamics of the accumulation of influenza A / 47 / Chandon / 93/1 (H1N1) in the MDCK cell culture with different multiplicity of infection Multiplicity of infection (EID ₅₀ / cell) Days of Accounting Virus titer in lg EID ₅₀ / ml Medium + 5% Fetal Serum Serum-free medium 0.005 one 4,5 4,5 2 7.5 7.5 3 8.0 8.5 four 8.0 9.5 5 7.7 7.5 0.001 one 2,5 0 2 7.5 5.5 3 7.5 6.5 four 75 8.5 5 7.5 8.5 0,0005 four 10.5 9.0 5 8.0 9.5 0.0001 four 8.3 8.0

Example No. 6. The study of the stability of ts ^- ₄₀ and ca ⁺ ₂₅ genetic traits

The stability of ts ^- ₄₀ (inability to reproduce at 40 ° C) and ca ⁺ ₂₅ (ability to reproduce at 25 ° C) genetic traits for 5 passages in monolayer MDCK cell cultures at 34 ° C in the presence of all three cold-adapted strains of the influenza virus were studied 1-6 μg / ml trypsin. Verification of these signs was carried out by parallel titration on chicken embryos of influenza virus strains after each of the five passages on an MDCK cell culture. The variants obtained after 5 passages in MDCK cells retained high reproductive performance in chicken embryos at the optimum temperature (34 ° C), as well as plaque-forming ability in the MDCK cell system, which is characteristic of the initial strains. All three strains in all passages, including the fifth, retained the ability to reproduce at 25 ° C, i.e. CA ⁺ ₂₅ sign, and did not breed at 40 ° C, i.e. saved ts ^- ₄₀ sign (Table 2).

table 2 Preservation of the ca- and ts-phenotype of reassortants of the influenza virus for 5 consecutive passages in the MDCK cell culture Flu strain Passage Number Reproduction of the virus in lg EID / ml at A / 47 / Chandon / 93/1 (H1N1) 40 ° C 34 ° C 25 ° C one 1,0 9.0 5,0 2 1,0 9.5 5,0 3 <1.0 9.3 4,5 four 1,0 9.5 5,0 5 1,5 10.5 5.5 Allantoic virus <1.0 9.5 5.5 A / 47 / Johannesburg / 94/2 H3N2 one <1.0 8.8 4,5 2 <1.0 8.5 4.0 3 <1.0 8.5 4.3 four <1.0 8.3 4.0 5 <1.0 8.7 4.8 Allantoic virus <1.0 8.7 4,5 B / 60 / Petersburg / 95/20 one <1.0 7.5 3.8 2 <1.0 7.3 3,5 3 <1.0 7.3 3.2 four <1.0 7.3 3.3 5 <1.0 7.5 3,5 Allantoic virus <1.0 7.4 3.6

Example No. 7. Study of the influence of the papain enzyme on the cultivation of cold-adapted vaccine strains of influenza virus

In order to replace the components of animal origin in the technology for producing live influenza vaccine with herbal preparations, studies have been carried out on the use of the enzyme papain of plant origin in the production of influenza virus strains. To do this, papain is added to the support medium used in the cultivation of influenza virus strains. The data are shown in table 3.

Table 3 The dynamics of the accumulation of influenza A / H1N1 virus in an MDCK cell culture in the presence of papain Papain concentration, mcg / ml Virus titer in lg EID ₅₀ / ml 3 days 4 days 0.25 7.7 7.5 0.5 7.4 7.5 1,0 7.7 6.7 2.0 7.5 7.7 2,5 8.5 8.0 4.0 7.8 7.7 5,0 7.7 7.3 7.5 7.5 6.5 10.0 7.4 6.3 15.0 7.3 7.5 20,0 7.7 6.7 30,0 6.5 6.7 40,0 6.7 6.7 50,0 6.7 6.5 Papain free (control) 6.5 6.7

The analysis of table 3 shows that at a concentration of papain in a support medium of 0.25-20.0 μg / ml, the titer of the influenza virus for 3-4 days of cultivation was 7.3-8.5 lg EID ₅₀ / ml, while while in the control experiment (without introducing the enzyme into the supporting medium) the titer of the influenza virus was 6.5-6.7 lg EID ₅₀ / ml, which confirms the effectiveness of this enzyme.

Claims (7)

1. A method of obtaining a live culture vaccine against influenza virus, including cultivating transplantable MDCK cells in a nutrient medium, obtaining a virus-containing substance by culturing vaccine strains of influenza virus in an MDCK cell culture on the same nutrient medium, purifying the viral substance from ballast impurities, introducing into a purified substance stabilizing additives and drying the finished form of the vaccine, characterized in that the seeding dose of transplantable MDCK cells is 100-600 thousand cells per 1 ml of medium, as viral of the strains using cold-adapted vaccine reassortants of influenza virus strains with a seed dose having a multiplicity of infection of 0.01-0.0001 EID ₅₀ / _KL , serum-free culture medium supplemented with a hydrolyzate is used as a culture medium for culturing MDCK cells and influenza virus strains on these cells soybeans at a concentration of 0.01-10% and a proteolytic enzyme in an amount of 0.25-50.0 μg / ml before the introduction of a seed dose of influenza virus strains, and the cultivation of MDCK cell cultures and cold-adapted virus strains flu in the specified cell culture is carried out in suspension on microcarriers introduced into serum-free nutrient medium at a concentration of 1-6 g / l, the collection of virus-containing fluid is carried out after reaching a specific activity of the influenza virus of at least 8.0 lg EID ₅₀ / ml, and as stabilizing additives introduced into the purified substance before drying, use sucrose and peptone from soy in a final concentration of (5-10) and (3-8) wt.%, respectively. 2. The method according to claim 1, characterized in that as a culture of MDCK cells, a culture stored at liquid nitrogen temperature in the form of a seed and working banks and certified for the absence of extraneous viruses, oncogenic potency and species identity is used. 3. The method according to claim 1, characterized in that vaccine strains of the A / H3N2 type are used as cold-adapted influenza virus reassortants; A / H1N1 and type B of this virus. 4. The method according to claim 1, characterized in that the proteolytic enzyme of plant origin papain is used as a proteolytic enzyme. 5. The method according to claim 1, characterized in that Cytodex 1 is used as a microcarrier. 6. The method according to claim 1, characterized in that the removal of ballast impurities from the viral substance is carried out by filtration. 7. The method according to claim 1, characterized in that the vaccine is dried by freezing it for 18 hours at a temperature not exceeding minus 40 ° C, followed by lyophilization for 48 hours, and before sealing, the ampoules with the viral vaccine are filled with an inert gas, particular argon.