RU2770814C1 - Serum-free environment “vniizzh” for the cultivation of vnk-21 kidney cells of the syrian hamster and the production of immunogenic components of culture-based viruses of foot-and-mouth disease and rabies for the manufacture of vaccines - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, namely to the development of a serum-free environment "VNIIZZH" (Federal animal health protection center), used for the cultivation of kidney cells of the Syrian hamster VNK-21 and the production of immunogenic components of culture viruses of foot-and-mouth disease and rabies for the manufacture of vaccines. Adaptation of the transplanted suspension cell line VNK-21 to cultivation in the developed serum-free environment "VNIIZZH" is carried out with a decrease in the amount of blood serum of cattle from 5 to 0% for 6 consecutive passages. The cultivation of adapted cells of the VNK-21 line in the developed serum-free medium is carried out for 3 consecutive passages, while in 48 hours the cell concentration increases to 5.68-5.94 million/ml at a sowing concentration of 0.6-0.7 million/ml. Using the developed serum-free medium, cells of the VNK-21 line are cultured, which makes it possible to obtain FMD virus antigen with concentrations of 146S and 146S+75S in inactivated suspensions of 0.79-0.85 and 0.95-1.06, respectively, as well as rabies virus antigen with concentrations of ribonucleoprotein and glycoprotein 0.64-0.72 and 0.66-0.73, respectively, for the manufacture of safe and highly immunogenic anti-FMD and anti-rabies vaccines.EFFECT: expansion of the range of methods for treating animals.1 cl, 3 dwg, 19 tbl, 6 ex

Description

The invention relates to the field of biotechnology, namely to the development of a serum-free medium "ARRIAH", used for culturing cells of the Syrian hamster BNK-21 kidney and obtaining immunogenic components of cultured FMD and rabies viruses for the manufacture of vaccines.

FMDV remains an important pathogen of wild and domestic carnivores for more than 120 years after its discovery, with annual production losses and vaccination costs estimated at 5.3-17 billion euros per year. Control and eradication is difficult because foot-and-mouth disease is highly contagious to a wide variety of species and is genetically and antigenically diverse [1].

FMD is caused by an RNA-containing virus that belongs to the order Picornavirales, family Picornaviridae, genus Aphthovirus [2]. FMDV is characterized by high antigenic variability due to mutations in the genes of capsid proteins [1].

Cultivation of foot-and-mouth disease virus in a transplantable suspension cell line of Syrian hamster kidney (BHK-21), used for the manufacture of FMD vaccines, makes it possible to obtain four variants of structural components with different values of sedimentation constants:

1) 146S component (complete particles), consisting of one viral RNA molecule and 60 copies of the polypeptide, each of which is represented by a complex of proteins VP ₁ (1D), VP ₂ (1B), VP ₃ (1C), VP ₄ (1A); responsible for the immunogenic properties of the antigen;

2) 75S particles (“empty” capsids), devoid of RNA and including 60 copies, consisting of polypeptides VP ₀ (1AB), VP ₁ (1D), VP ₃ (1C); are responsible for the immunogenic properties of the antigen;

3) 12S particles (capsomeres) represented by structural proteins VP ₁ (1D), VP ₂ (1B), VP ₃ (1C);

4) 3.8S subunits represented by the nonstructural protein VP _g [1, 3].

The system of measures for the control and prevention of foot and mouth disease includes the vaccination of large and small cattle in the buffer and threatened zones of the Russian Federation [1]. This requires the production of FMD vaccines on an industrial scale. Routine vaccination against this disease is carried out in many countries or zones recognized as free from FMD with vaccination and in countries where the disease is endemic. In addition, many disease-free countries do not refuse to immunize animals and have their own strategic stocks of inactivated viral preparations containing high concentrations of 146S and 146S + 75S immunogenic components [2]. Such stocks of antigen allow the delivery of received vaccines in an emergency and in a short time. Based on this, the need for obtaining immunogenic components of FMDV is high for the manufacture of FMD vaccines and ensuring the epizootic safety of individual countries and entire regions.

Traditional inactivated FMD vaccines should include certain amounts of FMDV immunogenic components obtained as a result of virus reproduction in a susceptible cell line, with the addition of an adjuvant and additional compounds [2].

To obtain 146S and 75S immunogenic components of FMDV, a continuous suspension cell line from the kidney of the Syrian hamster BNK-21, free from contaminating microorganisms, is used [1, 2].

Rabies is a zoonotic infection with acute viral encephalitis, with almost 100% mortality, the highest among known infectious diseases [4].

The causative agent of this disease is the rabies virus of the order Mononegavirales, the family Rhabdoviridae, the genus Lyssavirus, the species Rabies lyssavirus [5].

The virus genome is represented by an unsegmented single-stranded negative RNA molecule about 12,000 bp long. Nucleic acid encodes 5 main proteins arranged in a conservative linear order: nucleoprotein (N-protein), phosphoprotein (P-protein), matrix protein (M-protein), glycoprotein (G-protein), RNA-dependent RNA polymerase (L -protein) [9]. The rabies virus virion is bullet-shaped, 130-250 nm long and 60-100 nm in diameter. It consists of the following functional structures: an RNP complex (ribonucleoprotein) located inside the virion and an external protein-lipid component. The RNP complex is an internal nucleocapsid that includes genomic RNA tightly bound to a nucleoprotein and a phosphoprotein, as well as a viral polymerase. Ribonucleoprotein is an important immunogenic component of rabies vaccine preparations. The RNP complex contains 4% RNA to 96% protein [2]. Outside the virion is a two-layer lipid membrane with glycoprotein "spikes". The matrix protein is located between the RNP complex and the external component of the virion, condenses the nucleoprotein, and interacts with the glycoprotein endodomain [4].

The proteins of the rabies virus are polyfunctional. The nucleoprotein protects the viral genome from cellular RNase activity and interacts with L- and P-proteins during transcription and replication. The phosphoprotein is responsible for the correct positioning of the L-protein and acts as a chaperone during nucleoprotein synthesis, forming an N-P complex that prevents the N-protein from self-aggregating and binding to cellular RNA. The matrix protein is a structural element that binds to the ribonucleoprotein and the cytoplasmic domain of the glycoprotein to facilitate the assembly of viral particles. The glycoprotein is the only viral protein located on the surface of the virion. It provides binding to host cell receptors, induces endocytosis, and interacts with the M-protein, facilitating virion morphogenesis. The L protein performs the functions required for transcription and genome replication [4].

Post-exposure prophylaxis can provide a near-complete cure for the disease when applied promptly and correctly, and has been available for over a century. However, this disease still leads to the death of tens of thousands of people and animals every year due to the poor availability of appropriate biological drugs, especially in developing countries in tropical Asia and Africa [2]. As with any infectious disease, effective rabies control programs rely on rigorous laboratory surveillance, reliable diagnostic tools, and highly effective biologics, including rabies vaccines [4].

The main immunogenic components of the inactivated rabies virus in rabies vaccines are specific ribonucleoprotein and glycoprotein [4].

Cell cultivation is carried out using various growth media containing nutrients, growth factors and hormones, as well as compounds that regulate the hydrogen index (pH) and osmotic pressure in cells [6]. Depending on the need to add blood serum of cattle (cattle), several types of media are distinguished:

- basic (basic) environments (basal media), requiring the addition of 10% blood serum of cattle;

- improved media (advanced media), requiring the addition of 1-5% of the blood serum of cattle;

- serum-free media (serum-free medium) that do not require the addition of serum [7, 8].

For the cultivation of the continuous suspension cell line BHK-21, predominantly improved media are used. The qualitative and quantitative composition of the improved environment is shown in table 1 (environment No. 1) (prototype) [6]. The presented medium includes proteins, peptides and lipids that are part of the blood serum of cattle (5.0%), blood protein hydrolyzate (15-20 ml/l) and Hottinger digest (2-10 ml/l), 8 proteinogenic amino acids, glucose, 8 vitamins and vitamin-like compounds, mineral salts.

When using this improved growth medium for growing cells of the VNK-21 line with an inoculation concentration of 0.6–0.7 million cells/ml, suspensions with a content of 4.0–4.3 million cells/ml are obtained in 48 hours [6, 7] .

For the reproduction of foot-and-mouth disease and rabies viruses, a supporting medium containing 2.0% of cattle blood serum is used. The qualitative and quantitative composition of this medium is presented in Table 2 [6, 7]. It includes proteins, peptides and fats that are part of the blood serum of cattle (2.0%), blood protein hydrolyzate (5.0 ml/l) and Hottinger digest (2 ml/l), 4 proteinogenic amino acids, glucose, 8 vitamins and vitamin-like compounds, as well as mineral salts. When using this medium for the reproduction of FMDV from a suspension containing 1 million BHK-21 cells, the following yield of 146S and 146S + 75S immunogenic components is obtained: 0.57-0.72 and 0.71-0.88 μg / ml, respectively [6, 7, 8]. When using this medium for the reproduction of the rabies virus from 1 million BHK-21 cells, the following yield of immunogenic ribonucleoprotein and glycoprotein is obtained: 0.45-0.50 and 0.50-0.52 μg/ml, respectively.

However, this medium (media containing bovine blood serum) is used both for cultivating BHK-21 cells and for reproduction of foot-and-mouth disease and rabies viruses in the presence of bovine blood serum, reveals a number of disadvantages:

- blood serum can be cytotoxic, since it contains the enzyme polyamine oxidase, which acts on polyamines (spermine, spermidine), which are secretion products of rapidly proliferating cells;

- significant variability in the composition of blood sera from different batches;

- serum may contain an insufficient amount of specific growth factors, which necessitates their additional introduction into the base medium;

- blood serum can be contaminated with viruses, which is an additional unknown factor that cannot be controlled;

- whey is the most expensive component of the nutrient medium [6, 7, 8].

All of these problems can be resolved by removing the blood serum of cattle and other products of animal origin with the transition to the use of serum-free media.

Over the past three decades, bovine sera have been gradually replaced by other additives or by the use of serum-free media with a specific chemical composition. A number of serum-free medium formulations have been described for mammalian and insect cell lines, as well as for primary cultures [7]. However, the transition to serum-free medium still requires a laborious review of the literature and the search for a suitable medium composition for culturing cell lines and virus reproduction. All commercially available serum free media currently available from major distributors (eg ATCC, ECACC and DMSZ) are included in the database [9].

стабильности состава среды; снижение риска заражения культуры вирусами, бактериями, грибами и микоплазмами; облегчение очистки продуктов клеточного метаболизма; снижение влияния дополнительных протеинов на результаты биологических исследований; отсутствие цитотоксического влияния сыворотки по причине ее отсутствия. Поэтому в последние 2-3 десятилетия учеными ведущих научных институтов ведутся интенсивные исследования по разработке бессывороточных питательных сред для культивирования клеточных культур и получению высокого урожая иммуногенных компонентов вирусов [10].Serum-free media have the following advantages over media containing bovine serum: improved reproducibility of cell culture harvest results and immunogenic components of viruses due to

stability of the medium composition; reducing the risk of infection of the culture with viruses, bacteria, fungi and mycoplasmas; facilitating the purification of cellular metabolic products; reducing the influence of additional proteins on the results of biological studies; lack of cytotoxic effect of serum due to its absence. Therefore, in the last 2–3 decades, scientists from leading research institutes have been conducting intensive research on the development of serum-free nutrient media for cultivating cell cultures and obtaining a high yield of immunogenic components of viruses [10].

At the same time, at present, universal nutrient media have not been developed for an objective reason: the presence of specific biological needs of each cell culture and viruses that reproduce in different cells and require a certain unique composition of media [4, 5].

To date, there are several methods for cultivating BHK-21 cells and obtaining immunogenic components of FMD and rabies viruses in it using serum-free media.

In 1975, the American researcher L. David Tomei registered an FMD vaccine [11], which was obtained by growing BHK-21 cells in a nutrient growth medium without L-glutamine and bovine blood serum. The qualitative and quantitative composition of this thermostable medium is shown in Table 3. This medium includes 17 proteinogenic amino acids, glucose, 10 vitamins and vitamin-like compounds, methylcellulose and phenol red.

The medium was sterilized by autoclaving for 30 minutes (120° C., 1 bar) at pH 4.3 and stored at 4° C. until use. The suspension of VNK-21 cells was grown at a temperature of 35°C and pH 7.0-7.2 in the specified medium.

When using this serum-free growth medium for growing cells of the BNK-21 line with an inoculation concentration of 0.6-0.7 million cells/cm ³ , 3.0-3.5 million cells/cm ³ were obtained in 48 hours (Table 7).

The dose of FMDV infection was 0.10 TCD ₅₀ /cell. The reproduction of the virus was carried out for 12 hours, after which the amount of immunogenic components of the FMD pathogen was determined. With 1 million cells, the concentration of the 146S component of the FMD virus was 0.41-0.65 μg/ml, 146S + 75S components - 0.64-0.73 μg/ml [11] (Table 7).

The dose of infection with rabies virus was 0.10 CCID ₅₀ /cell. Reproduction was carried out for 48 hours, after which the amount of immunogenic components of the rabies pathogen was determined. From 1 million cells, the concentration of rabies virus ribonucleoprotein was 0.40-0.44 µg/ml, glycoprotein - 0.42-0.46 µg/ml (Table 7).

In 1997, Motono Mitsuru, Yamamoto Ryohel, Takase Kozo, Miyahara Tokuji presented a serum-free medium [12] suitable for animal cells, including BHK-21, and for obtaining immunogenic components of FMD and rabies viruses on their basis. The composition of this medium is shown in Table 4, which shows that this medium includes fibroblast growth factor (0.5-20.0 ng/ml), insulin (0.1-20.0 μg/ml), 17 proteinogenic amino acids, glucose, 9 vitamins and vitamin-like compounds, inorganic salts and phenol red dye. Fibroblast growth factor is a protein that promotes the proliferation of a wide range of cells. The concentration of this component is from 0.5 to 20.0 ng/ml. Insulin is a protein hormone produced in the β-cells of the islets of Langerhans of the pancreas. It has a multifaceted effect on the metabolism in almost all tissues. The main action of insulin is to regulate carbohydrate metabolism, in particular, the utilization of glucose in the cell. The amount of insulin added to the medium is 0.1-20.0 μg/ml, but 10 μg/ml is also sufficient for active cell division and growth, as well as from an economic point of view.

The use of this serum-free growth medium for growing cells of the BNK-21 line with an inoculation concentration of 0.6-0.7 million cells/cm ³ in 48 hours makes it possible to obtain a yield of 3.8-4.2 million cells/cm ³ . With 1 million cells, the concentration of 146S and 146S + 75S of the immunogenic components of the foot and mouth disease virus is 0.50-0.75 and 0.70-0.87 μg / ml, respectively, and the concentration of ribonucleoprotein and glycoprotein of the rabies virus is 0.45-0, 55 and 0.43-0.57 µg/ml, respectively (Table 7).

In 2011, the authors Ren Zhang, Wenqing Chen, Jlanchao Wang registered a serum-free medium for growing BHK-21 cells and reproducing FMD and rabies viruses in them - CN 102115729-2011 (Wednesday No. 2) [13] (the closest prototype). This medium includes the main culture medium containing the necessary set of proteinogenic amino acids, mineral salts, glucose, vitamins, and additionally the following components in terms of 100 liters of medium: 100-200 g of soy protein, 100-200 g of vegetable protein of the legume family, 0-100 g potato protein, 0-200 g wheat gluten protein and 50-100 g rice protein (Table 5).

When using the presented serum-free growth medium for growing cells of the VNK-21 line with an inoculation concentration of 0.6-0.7 million cells/cm ³ , 4.0-4.3 million cells/cm ³ were obtained in 48 hours (Table 7). The cells grown in this medium had a high culture density, and the medium had a low cost compared to the serum-free medium reflected in US patents 4049494 (A) [11], JP H09154571 (A) [12].

With 1 million cells, the concentrations of 146S and 146S + 75S of the immunogenic components of the foot-and-mouth disease virus were 0.51-0.75 and 0.74-0.89 μg / ml, respectively, and the concentration of ribonucleoprotein and glycoprotein of the rabies virus was 0.46-0, 52 and 0.49-0.58 µg/ml, respectively (Table 7).

In 2013, Zhang Shu and L.V. Hongliang registered a method for obtaining a purified FMD vaccine CN 103374547 using a serum-free medium [14]. The qualitative and quantitative composition of the medium is shown in Table 6 and includes 9 proteinogenic amino acids, sucrose, 4 salt variants, and phenol red dye.

When using the presented serum-free growth medium for growing cells of the VNK-21 line with an inoculation concentration of 0.6-0.7 million cells/cm ³ , 3.5-4.0 million cells/cm ³ were obtained in 48 hours (Table 7).

BHK-21 cells were infected with FMDV at a dose of 0.10-0.50 TCD ₅₀ /cell. With 1 million cells, the concentration of 146S and 146S + 75S of the immunogenic components of the foot-and-mouth disease virus was 0.43-0.54 and 0.65-0.75 μg / ml, respectively, and the concentration of ribonucleoprotein and glycoprotein of the rabies virus was 0.41-0, 49 and 0.40-0.45 µg/ml, respectively (Table 7).

The presented serum-free media for obtaining immunogenic components of foot-and-mouth disease and rabies viruses have the following disadvantages: 1) for 48 hours of cultivation of BHK-21 cells with an inoculation concentration of 0.6-0.7 million cells/ml, a suspension is obtained with a concentration of not more than 4.3 million cells/ml; 2) the concentration of 146S and 146S+75S immunogenic components of FMDV when using these media with 1 million cells is not more than 0.75 and 0.89 μg/ml, respectively; 3) the concentration of ribonucleoprotein and glycoprotein of the rabies virus when using these media with 1 million cells is not more than 0.52 and 0.58 µg/ml, respectively. In this regard, it is advisable to develop such a composition of a serum-free medium that: 1) will allow for 48 hours with a seeding cell concentration of 0.6-0.7 million cells/ml to obtain a yield of VNK-21 cells of more than 4.3 million cells/ml, 2) will make it possible to accumulate during the reproduction of the FMD virus 146S and 146S + 75S immunogenic components with amounts of more than 0.75 and 0.89 μg / ml, respectively, from 1 million cells in the manufacture of FMD vaccines; 3) will make it possible to accumulate during the reproduction of the rabies virus ribonucleoprotein and glycoprotein with amounts of more than 0.52 and 0.58 μg / ml, respectively, from 1 million cells in the manufacture of anti-rabies vaccines.

The problem was the lack of a serum-free medium for the manufacture of FMD and rabies vaccines, which would eliminate the above disadvantages.

To date, this problem has been solved due to the development of a serum-free medium "ARRIAH" with a new composition of the composition, which will increase the yield of cells of the transplanted suspension line VNK-21 without reducing the cell size, increase the accumulation of 146S and 146S + 75S immunogenic components of foot-and-mouth disease virus, increase the yield of ribonucleoprotein and glycoprotein compared to the serum-free media presented above.

The essence of the invention lies in the composition of the serum-free medium "ARRIAH" for obtaining immunogenic components of foot and mouth disease and rabies viruses necessary for the production of foot and mouth disease and rabies vaccines. The composition of the proposed serum-free medium is characterized by a special composition of components in certain concentrations, which allows to obtain high yields of the BHK-21 cell line (more than 4.3 million cells/ml); carry out the reproduction of the cultural FMD virus with the accumulation of 146S and 146S + 75S components of more than 0.75 and 0.89 μg / ml, respectively, from 1 million cells and reproduce the cultural rabies virus with the accumulation of ribonucleoprotein and glycoprotein with a concentration of more than 0.52 and 0 .58 µg/ml, respectively, from 1 million cells.

The improved medium (medium no. 1) and the closest prototype serum-free medium CN 102115729-2011 (medium no. 2) were used to cultivate BNK-21 cells and obtain immunogenic components of foot and mouth disease and rabies viruses [13]. Compared with the prototypes, the serum-free medium "ARRIAH" (medium No. 3) for obtaining immunogenic components of the cultured FMD and rabies viruses for the manufacture of vaccines allows: 7 million cells/ml to obtain a yield of BHK-21 cells from 5.68 to 5.94 million cells/ml; 2) to accumulate during the reproduction of the FMD virus 146S and 146S+75S immunogenic components with quantities of 0.79-0.85 and 0.95-1.06 μg/ml, respectively, from 1 million cells in the manufacture of FMD vaccines; 3) during reproduction of the rabies virus, accumulate ribonucleoprotein and glycoprotein at concentrations of 0.64-0.72 and 0.66-0.73 μg/ml, respectively, from 1 million cells in the manufacture of anti-rabies vaccines.

The key element of the claimed composition of the ARRIAH serum-free medium for obtaining immunogenic components of foot-and-mouth disease and rabies culture viruses for the manufacture of vaccines is the adaptation of the continuous suspension cell line VNK-21 to cultivation in the presented serum-free medium for 6 consecutive passages with a decrease in the amount of cattle blood serum from 5 to 0%, cultivation of cells of this line in the developed serum-free medium, reproduction of foot-and-mouth disease virus in an adapted BHK-21 cell culture and production of immunogenic components of the virus in high concentrations for the manufacture of FMD vaccines, reproduction of the rabies virus in an adapted cell culture of BHK-21 and production of immunogenic components of the virus in high concentrations for the manufacture of rabies vaccines.

Comparative analysis with the prototype allows us to conclude that the novelty and inventive step of the claimed invention lies in: 1) adaptation of the cells of the suspension transplantable line VNK-21 to cultivation in the developed serum-free medium to obtain immunogenic components of the cultured FMD and rabies viruses for the manufacture of vaccines, 2) reproduction foot-and-mouth disease virus in a cell culture adapted to this serum-free medium to obtain higher concentrations of 146S and 146S+75S immunogenic components used for the manufacture of foot-and-mouth disease vaccines; 3) reproduction of the rabies virus in a cell culture adapted to this serum-free medium to obtain higher concentrations of the ribonucleoprotein and glycoprotein of the virus used for the manufacture of rabies vaccines.

The technical result from the use of the present invention is to increase the amounts of 146S and 146S+75S immunogenic components of the foot-and-mouth disease virus, ribonucleoprotein and glycoprotein of the immunogenic components of the rabies virus in the production of FMD and rabies vaccines.

The essence of the invention is reflected in the graphics:

Fig. 1 - Cell concentrations of the continuous suspension cell line VNK-21 during adaptation for 6 consecutive passages to cultivation in a serum-free medium "ARRIAH" to obtain immunogenic components of cultured FMD and rabies viruses for the manufacture of vaccines.

Fig. 2 - Concentrations of 146S and 146S+75S immunogenic components of the cultured FMDV in non-inactivated (A) and inactivated (B) suspensions after reproduction in VNK-21 cells adapted for cultivation in the developed serum-free medium "ARRIAH".

Fig. 3 - Concentrations of ribonucleoprotein and glycoprotein of the cultural rabies virus in suspensions after reproduction in VNK-21 cells adapted for cultivation in the developed serum-free medium "ARRIAH".

Serum-free medium "ARRIAH" for obtaining immunogenic components of foot and mouth disease and rabies culture viruses for the manufacture of vaccines is a light gray fine powder that dissolves well in purified water without precipitation. The qualitative and quantitative composition of the developed serum-free medium is shown in Table 8, from the data of which it can be seen that, compared with the serum-free medium CN 102115729-2011 [13] (the closest prototype), the total amounts of amino acids are higher. They consisted of the content of free amino acids added as an independent component to the medium, as well as from plant proteins that underwent enzymatic hydrolysis. Concentrations of proteinogenic amino acids L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-valine, L-histidine, L-glycine, L-glutamine, L-glutamic acid, L-isoleucine, L-leucine, L -lysine, L-tryptophan, L-methionine, L-proline, L-serine, L-tyrosine, L-threonine, L-phenylalanine, L-cystine in the developed medium was 134, 65, 69, 56, 80, 85 , 66, 73, 216, 81, 484, 39, 25,. 25, 82, 55, 99, 39, 111, 18 mg/l more, respectively, compared with the prototype. The content of vitamins and vitamin-like substances was increased, namely, in the developed serum-free medium "ARRIAH", the concentrations of inositol, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, folic acid and choline chloride by 0.2; 0.3; 0.3; 0.5; 0.05; 0.2; 0.5; 0.5 mg/l more, respectively, than in the prototype. The mitogen bovine insulin was added to the developed medium at a concentration of 3.0 mg/L. This component does not exist in the prototyping environment. The concentration of glucose medical and mineral salts is the same for two serum-free media, with the exception of iron (II) chloride (0.05 mg/l), copper (II) chloride (0.05 mg/l), chromium (III) chloride (0 05 mg/l), which are present only in the serum-free medium "ARRIAH" and are necessary for the full functioning of the enzyme link of the antioxidant system of Syrian hamster kidney cells [7]. To maintain a certain degree of viscosity, which is created by the blood serum of cattle in improved nutrient media, the developed serum-free medium "ARRIAH" includes a safe polymeric organic component Pluronic F-68 (0.13%).

The most important building components of the developed serum-free medium "ARRIAH" for cells and structures of foot-and-mouth disease and rabies viruses are amino acids, which play a significant role in vital processes [17, 18].

The composition of the developed serum-free medium also included bovine insulin, which regulates the absorption, storage and utilization of glucose, amino acids and fatty acids, and inhibits the breakdown of proteins and lipids. Bovine insulin is a hormone, has the form of a double-chain polypeptide chain, is synthesized by β-cells of the pancreatic islets. Molecular weight 5.8 kDa, α- and β-chains are connected by two interchain disulfide bonds. The α-chain contains an intrachain disulfide bridge. Bovine insulin is used as a growth factor in the cultivation of a wide range of mammalian cells. Bovine insulin is used to differentiate mesenchymal stem cells (MSCs) into adipocytes [19].

It has been shown that the proposed composition of the ARRIAH serum-free medium developed at the Federal Center for Animal Health (FSBI ARRIAH) is optimal for cultivating and maintaining the VNK-21 cell line used for the production of viral vaccines, is suitable for the expression of foot and mouth disease viruses and rabies and accumulation of their immunogenic components in high concentrations. When the BHK-21 cell line reaches the required concentration, the virus can be added directly to the bioreactor without changing the nutrient medium, thereby achieving high efficiency in the production of viral particles.

At the first stage of work, a solution of the developed serum-free medium "ARRIAH" is prepared. To do this, 200 g of powder of the developed serum-free medium "ARRIAH" is slowly added to 9.5 liters of purified water (Milli-Q®) with slow stirring (300 rpm) for 10 minutes. The pH value is measured, which is equal to 6.95±0.02 and the pH is adjusted to 7.10-7.20 with a 7.5% sodium bicarbonate solution. The final volume is adjusted to 10 liters. The resulting medium is subjected to sterilization filtration using a Millipore Express® Plus membrane filter (0.22 μm, polyethersulfone).

объема разработанной бессывороточной среды «ВНИИЗЖ» и в течение 5 пассажей (240 ч) концентрация сыворотки крови КРС уменьшается по схеме: 5,0%→2,5%→1,25%→0,625%→0,3125%. Затем проводят осаждение клеток при 1000 об./мин для того, чтобы их освободить от среды с сывороткой крови КРС и полностью перевести в разработанную бессывороточную среду «ВНИИЗЖ» без добавления сыворотки (шестой пассаж). Иными словами, осуществляют полный перевод клеток на выращивание в бессывороточной среде «ВНИИЗЖ».The continuous suspension cell line VNK-21 is adapted for cultivation in the developed serum-free medium "ARRIAH" for 6 successive passages in Erlenmeyer flasks before scaling in a bioreactor. For adaptation, a suspension of VNK-21 cells of the 1st passage after cryofreezing is used. The inoculum concentration of cells of the continuous suspension cell line VNK-21 is 0.6-0.7 million cells/ml. As a growth medium, an improved medium is used, the composition of which is shown in table 1. Daily, an improved medium (prototype) is added

volume of the developed serum-free medium "ARRIAH" and within 5 passages (240 h) the concentration of blood serum of cattle decreases according to the scheme: 5.0%→2.5%→1.25%→0.625%→0.3125%. Then the cells are sedimented at 1000 rpm in order to free them from the medium with the blood serum of cattle and completely transfer them to the developed serum-free medium "ARRIAH" without the addition of serum (sixth passage). In other words, complete transfer of cells to cultivation in a serum-free medium "ARRIAH" is carried out.

The transfer of cells to serum-free culture conditions affects the biochemical and morphological characteristics of cell lines. During the study, after 48 hours for 6 consecutive passages, the concentration of cells was assessed and their sizes were analyzed using light microscopy.

VNK-21 cells adapted to a serum-free medium, the composition of which is presented in Table 5 (the closest prototype), were obtained earlier and were stored in the FGBI ARRIAH bank.

At the next stage, cells of the VNK-21 line are cultivated with serum-free medium "ARRIAH" at pH 7.10-7.20 and moderate enrichment of the medium with purified air during bubbling. The inoculum concentration of cells is 0.6-0.7 million/ml. The duration of each passage is 48 hours. After a specified period of time, the final concentration of cells is determined and compared with the control. As a control, cells of the continuous suspension line VNK-21, grown according to the "Industrial Regulations for the Production of Vaccines against Foot and Mouth Disease of Various Types" using an improved medium, the composition of which is shown in table 1 (prototype), as well as cells adapted to a serum-free medium, the composition of which presented in table 5 (closest prototype).

At the next stage of the work, reproduction of the foot-and-mouth disease virus is carried out in a continuous suspension cell line VNK-21 (1 medium (prototype), 2 medium CN 102115729-2011 (the closest prototype) and medium 3 - serum-free medium "ARRIAH")). The dose of infection of each medium with foot-and-mouth disease virus is 0.05 TCD ₅₀ /cell. The cultivation of the virus is carried out in these nutrient media at pH 7.5-7.7. The pH is adjusted with a sodium bicarbonate solution. The reproduction of foot-and-mouth disease virus is estimated by the number of cells subjected to specific destruction, and expressed in%.

After cultivating FMDV in reverse transcription and polymerase chain reaction (RT-PCR), strain specificity is confirmed [2].

In reverse transcription and real-time polymerase chain reaction (RT-PCR-RT), the obtained suspensions of foot-and-mouth disease virus are examined and the concentration of the 146S immunogenic component is determined [15]. The amount of 146S+75S immunogenic components is determined in the complement fixation test (CFR) [20].

Carry out the process of inactivation of the FMD virus antigen. As a first-order inactivant, 1,2-aminoethylethyleneimine (AEEI) is used at a concentration of 0.025-0.050%. The inactivation is carried out for 12 hours at a temperature of 37°C.

Evaluate the completeness of the process of inactivation of the FMDV antigen in accordance with the requirements of the OIE (OIE) [2].

The concentrations of 146S and 146S+75S of the FMDV immunogenic components in the inactivated suspension are determined using the complement fixation test (CFR), which is carried out in accordance with the requirements of the OIE (OIE) [2, 20]. The concentration of immunogenic components is recalculated from 1 million cells.

For the manufacture of an emulsion vaccine, antigenic material is used with a content of 146S immunogenic components of FMDV of at least 3.0 μg/ml. The required concentration of immunogenic components in the emulsion vaccine is obtained by concentrating the antigen by flow ultrafiltration. The emulsion vaccine is prepared by dispersing in colloid mills the concentrate of foot and mouth disease virus antigen obtained on three different media (3 different vaccines) and oil adjuvant Montanide ISA-206 VG in a ratio of 50/50 by weight, respectively.

The resulting vaccine preparations are examined for safety and sterility in accordance with the requirements of the OIE (OIE) [2].

The method of enzyme-linked immunosorbent assay (ELISA) in the blocking variant evaluates the absence of non-structural proteins of the foot-and-mouth disease virus in the final product [21]. To do this, Landrace pigs are immunized, 3 heads for each of the three vaccines. On the 14th day after immunization, blood is taken from the animals, sera are obtained and they are tested using the test system for ELISA "PrioCHECK ^® FMDV NS ELISA for in vitro detection of antibodies against Foot and Mouth Disease Virus in serum of cattle, sheep and pigs" ( Prionics Lelystad BV, The Netherlands). For the control and test samples of the vaccine, after the reaction on the spectrophotometer-reader, the values of optical densities are measured and converted to the percentage of inhibition using the formula:

PI=(1-OP _S /OP _KO )*100,

where, PI is the percentage of inhibition (%),

OD _S - optical density of the studied blood serum,

OD _KO - optical density of the negative control.

The blood serum of pigs in relation to the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus should be considered positive if the value of PI≥50%, negative - with PI<50%. In this case, the value of the optical density of the negative control should be >1.000. The percentage of inhibition of the weakly positive control should be >50.00%, of the positive control - >70.00% [22].

Vaccine formulations are tested in animals to assess safety. To test vaccines for safety, 6 heads of pigs (2 per vaccine) are injected intramuscularly into the region of the upper third of the neck at a dose of 6.0 cm 3 (three times the dose) and the clinical condition of the animal is monitored for 6 days. The vaccine is recognized as harmless if all animals remain clinically healthy according to the results of observation, and tissue necrosis does not occur at the injection site.

Before immunization and 21 days after the last injection of FMD vaccines, blood is taken from pigs and sera are obtained, which are examined for the presence of virus neutralizing antibodies (VNA) using the microneutralization reaction (RMN) of the virus according to the standard procedure [2].

Vaccines are also examined for immunogenicity and the protective properties of the final product are evaluated. The immunogenic dose (IMD ₅₀ ) is calculated using the Kerber formula:

ImD ₅₀ in cm ³ \u003d 10 ^{lgD-lgd ⋅ (ΣI / N-0.5)}

where, D is the dose of the vaccine,

d - breeding step (for pigs - 5),

I - number of protected animals,

N is the number of infected animals tested for one breeding.

The number of protective doses (PD ₅₀ ) is calculated by the formula:

PD ₅₀ = inoculation dose / IMD ₅₀ [2].

At the next stage, the rabies virus is reproduced in the cells of the transplanted suspension line VNK-21, (1 medium (prototype), 2 medium CN 102115729-2011 (closest prototype) and medium 3 - serum-free medium "ARRIAH"). The virus infection dose of each medium is 0.05 CCID ₅₀ /cell. The cultivation of the virus is carried out in these media at pH 7.4-7.6. The pH is adjusted with a sodium bicarbonate solution. The cultivation of the virus is carried out for 48 hours.

After cultivation of the rabies virus in reverse transcription and polymerase chain reaction (RT-PCR), strain specificity is confirmed [2].

In the reaction of immunofluorescence (RIF) determine the titer of infectious activity of the virus, which should not be lower than 6.0 lg KKID ₅₀ /ml.

Carry out the process of inactivation of the rabies virus antigen. As a first-order inactivant, 1,2-aminoethylethyleneimine (AEEI) is used at a concentration of 0.05% (by volume). The inactivation is carried out for 12 hours at a temperature of 37°C. Assess the completeness of the process of inactivation of the rabies virus antigen in accordance with the requirements of the OIE (OIE) [2].

In reverse transcription and real-time polymerase chain reaction (RT-PCR-RT), the obtained suspensions of the rabies virus are examined and the concentration of ribonucleoprotein is determined [23]. The amount of rabies virus glycoprotein is estimated by ELISA [21]. The concentration of immunogenic components is recalculated from 1 million cells.

For the manufacture of anti-rabies emulsion vaccine, antigenic material is used with a content of ribonucleoprotein and rabies virus glycoprotein of at least 3.0 μg/ml each. The required concentration of immunogenic components in the emulsion vaccine is obtained by concentrating the antigen by flow ultrafiltration. An emulsion vaccine is prepared by colloid milling the virus antigen concentrate and Montanide ISA-61 VG oil adjuvant in a ratio of 40/60 by weight, respectively.

The resulting vaccine preparation is examined for areactogenicity, safety and sterility in accordance with the requirements of the OIE (OIE) [2].

The presence of post-vaccination antibodies is examined after immunization of cattle with the obtained vaccines. Tests are carried out on ten heads of cattle at the age of 1 year with an average weight of 200 kg. Animals are vaccinated with a vaccine at a dose of 2.0 cm ³ intramuscularly once. To assess the intensity of post-vaccination anti-rabies immunity in animals, in accordance with the recommendations of the OIE (OIE), a neutralization reaction is used in a monolayer BHK-21 cell culture (reaction modification - FAVN (Fluorescent antibody virus neutralization)), expressing the titer of anti-rabies antibodies in IU/cm ³ [2]. For analysis, a positive serum control standard diluted to an immunogenicity of 0.5 IU/mL and a negative serum control standard with an immunogenicity of <0.1 IU/mL were used [2].

Evaluation of protective properties is not carried out for ethical reasons, so as not to expose animals to a deadly disease.

Statistical data processing was carried out using the mean values and standard deviation, taking into account the Student's coefficient [24].

Example 1. Adaptation of the continuous suspension cell line VNK-21 to cultivation in the developed serum-free medium "ARRIAH".

At the first stage of the work, a solution of the developed serum-free medium "ARRIAH" was prepared. To do this, 200 g of the developed serum-free nutrient medium powder was slowly added to 9.5 liters of purified water (Milli-Q®) with slow stirring (300 rpm) for 10 minutes. The pH value was measured, which is equal to 6.95±0.02, and the pH was adjusted to 7.10-7.20 with a 7.5% sodium bicarbonate solution. The final volume was adjusted to 10 liters. The resulting medium was subjected to sterilization filtration using a Millipore Express® Plus membrane filter (0.22 μm, polyethersulfone).

объема бессывороточной среды «ВНИИЗЖ» и в результате в течение 5 последовательных пассажей концентрация сыворотки крови КРС уменьшалась по схеме: 5,0%→2,5%→1,25%→0,625%→0,3125%. Затем проводили осаждение клеток при 1000 об./мин, чтобы их освободить от среды с сывороткой крови КРС и полностью перевести в бессывороточную среду «ВНИИЗЖ». В качестве контроля использовали клетки линии ВНК-21, выращенные с применением улучшенной среды (среда №1), состав которой отражен в таблице 1 (прототип), а также клетки линии ВНК-21, адаптированные к выращиваю в бессывороточной среде CN 102115729-2011 (среда №3 - наиболее близкий прототип). В ходе исследования через каждые 48 ч в течение 5 последовательных пассажей оценивали концентрацию клеток и анализировали их размеры с помощью световой микроскопии (таблица 9, фиг. 1).The transplanted suspension cell line VNK-21 was adapted for cultivation in the developed serum-free medium "ARRIAH" for 6 successive passages in Erlenmeyer flasks. For adaptation, a suspension of BNK-21 cells of the first passage after cryofreezing was used. The cell seed concentration of the transplanted suspension cell line was 0.70 million cells/ml. Added daily to the improved medium (Wednesday No. 1)

volume of serum-free medium "ARRIAH" and as a result, during 5 consecutive passages, the concentration of blood serum of cattle decreased according to the scheme: 5.0%→2.5%→1.25%→0.625%→0.3125%. Then the cells were sedimented at 1000 rpm in order to free them from the medium with bovine blood serum and completely transfer them to the ARRIAH serum-free medium. As a control, VNK-21 cells grown using an improved medium (medium No. 1), the composition of which is shown in table 1 (prototype), as well as VNK-21 cells adapted to growing in a serum-free medium CN 102115729-2011 ( Wednesday No. 3 - the closest prototype). During the study, every 48 hours for 5 consecutive passages, the concentration of cells was assessed and their sizes were analyzed using light microscopy (Table 9, Fig. 1).

From the data in Table 9 and FIG. Table 1 shows that the control cells of the VNK-21 line, grown in an improved medium (medium No. 1), with an inoculation concentration of 0.70 million cells/ml after 48, 96, 144, 192, 240, 288 hours with a decrease in the amount of blood serum of cattle from 5 to 0% had a final concentration of 4.20; 2.84; 2.21; 1.48; 0.95; 0.62 million cells/ml. Data on the adaptation of VNK-21 cells to the CN 102115729-2011 medium (medium No. 2) were also obtained and were: 4.20; 4.25; 4.31; 4.35; 4.52; 4.70 million cells/ml. VNK-21 cells with a seed concentration of 0.70 million cells/ml after 48, 96, 144, 192, 240, 288 hours with a decrease in the amount of bovine blood serum from 5 to 0% and replacing it with a serum-free medium "ARRIAH" (medium No. 3) had a concentration of 4.25; 4.32; 4.68; 5.02; 5.39; 5.65 million cells / ml, which is 1.01; 1.52; 2.12; 3.39; 5.67; 9.11 times higher than the prototype (Wednesday No. 1) and 1.01; 1.02; 1.08; 1.15; 1.19; 1.20 times higher than the closest prototype (Wednesday #2). The results of the studies also showed that cells grown in the developed serum-free medium "ARRIAH" - medium No. 3 (the amount of serum 0% after 6 passages) had a size of 10-14 microns (not less than in controls). Thus, the VNK-21 cell line was adapted to growing in the developed serum-free medium "ARRIAH" for 6 consecutive passages (288 h).

Example 2. The process of culturing cells of the continuous suspension cell line VNK-21 in the developed serum-free medium "ARRIAH".

After adaptation of VNK-21 cells to the serum-free ARRIAH medium, the cells were cultivated in 6-liter bioreactors for 3 successive passages, each of which lasted 48 hours. purified air during the bubbling process. The inoculum concentration of cells was 0.70 million cells/ml. Every 48 hours, the final concentration of cells was determined and compared with the control. As a control, cells of the VNK-21 line grown using an improved medium, the composition of which is reflected in table 1 (5% of cattle blood serum) (prototype) and cells adapted to a serum-free medium CN 102115729-2011 (the closest prototype), were used. The studies were carried out in triplicate. The results obtained are presented in table 10.

From the data shown in table 10, it can be seen that the cells of the VNK-21 line, adapted to the serum-free medium "ARRIAH", for 3 consecutive passages compared with the prototype and the closest prototype had a higher concentration on average by 35.0 and 21, 5%, respectively.

Example 3. Obtaining immunogenic components of the cultural foot-and-mouth disease virus of the Asia-1/Shamir/89 strain using a continuous suspension cell line VNK-21 adapted for cultivation in the developed serum-free medium "ARRIAH".

FMDV strain Asia-1/Shamir/89 was reproduced in bioreactors with a volume of 40 l in the obtained cells of the VNK-21 line, adapted for cultivation in a serum-free medium "ARRIAH". The virus challenge dose was 0.05 TCD ₅₀ /cell. The cultivation of the virus was carried out at pH 7.5-7.7. The pH was adjusted with sodium bicarbonate solution. The cytopathic effect (CPE) was assessed by the number of cells subjected to specific destruction. The study was carried out in three repetitions. Cultivation was carried out up to 95% cytopathic effect. As a control, VNK-21 cells grown in an improved medium, the composition of which is presented in Table 1 (medium No. 1 - prototype), as well as in serum-free medium CN 102115729-2011 (medium No. 2 - the closest prototype), were used. The reproduction time of the FMD virus for all cases was 10.5-11.0 hours.

After cultivation of FMDV, strain specificity was confirmed by RT-PCR [2]. As a result of the study in suspension, the presence of only FMD virus strain Asia-1/Shamir/89 was confirmed.

The resulting FMDV suspensions were studied and the concentration of 146S in reverse transcription and real-time polymerase chain reaction (RT-PCR-RT) and 146S+75S immunogenic components in CSCs was determined (Fig. 2A) [15].

Spent the process of inactivation of the FMD virus antigen. As a first-order inactivant, 1,2-aminoethylethyleneimine (AEEI) was used at a concentration of 0.050%. Inactivation was carried out for 12 hours.

The completeness of the process of inactivation of the FMDV antigen during the inoculation of a continuous monolayer cell line from the pig kidney IB-RS-2 was evaluated in accordance with the requirements of the OIE (OIE) [2].

After confirming the completeness of inactivation of the FMDV antigen, the concentrations of 146S and 146S+75S of the immunogenic components of the FMDV in the inactivated suspension were determined using RSC, which was carried out in accordance with the requirements of the OIE (OIE) [2, 16]. The concentration of immunogenic components was recalculated from 1 million cells (Fig. 2B).

The results of determining the concentration of 146S and 146S+75S components of FMDV are shown in Table 11 and in FIG. 2, from which it follows that the number of 146S particles in a non-inactivated suspension obtained using the developed serum-free medium is 18-38 and 13-55% more compared to the prototype and the closest prototype, respectively. The concentration of 146S particles in the inactivated suspension obtained using the developed environment "ARRIAH", more than the prototype and the closest prototype by 20-36 and 13-49%, respectively. The accumulation of 146S+75S particles in a non-inactivated suspension, obtained using the developed environment "ARRIAH", is greater than the prototype and the closest prototype by 20-34 and 19-28%, respectively. The concentration of 146S+75S immunogenic components in the inactivated suspension, obtained using the developed serum-free medium, is greater than the prototype and the closest prototype by 23-32 and 26-30%, respectively. In other words, the developed serum-free medium "ARRIAH" and cells of the VNK-21 line, adapted to it, can significantly increase the accumulation of 146S and 146S+75S immunogenic particles of foot-and-mouth disease virus compared to the prototype variants.

Example 4. Manufacture and study of FMD inactivated emulsion vaccine from the Asia-1/Shamir/89 strain, the production of immunogenic components of which was carried out using a continuous suspension cell line VNK-21 adapted for cultivation in the developed serum-free medium "ARRIAH".

Spent receiving three FMD inactivated emulsion vaccines from strain Asia-1/Shamir/89: 1) from the antigen obtained in cells of the VNK-21 line, grown in an improved medium with 5% serum of cattle (prototype); 2) from the antigen obtained in BHK-21 cells grown in serum-free medium CN 102115729-2011 (closest prototype); 3) from the antigen obtained in VNK-21 cells grown in the developed serum-free medium "ARRIAH". For the manufacture of three vaccine preparations, inactivated antigens of the foot-and-mouth disease virus of the Asia-1/Shamir/89 strain, obtained in example 3, were used. was 0.71; 0.75 and 0.85 μg/ml, respectively, the concentration of 146S+75S components corresponded to the values of 0.86; 0.84 and 1.06 µg/ml. The requirement for the antigen in the manufacture of FMD vaccine is the concentration of 146S immunogenic components of at least 3.0 µg/ml. After concentration by 10 times, the content of 146S components for vaccines No. 1, 2, 3 was 7.10; 7.50 and 8.50 µg/ml, respectively. After this process, the concentration of 146S+75S components for vaccines No. 1, 2, 3 was 8.60; 8.40 and 10.60 µg/ml, respectively.

Montanide ISA-206 (Seppic) was used as an oil adjuvant in the amount of 500,000.0-575,000.0. Emulsion vaccines were prepared by colloid mill dispersion of FMDV antigen concentrate and Montanide ISA-206 VG oil adjuvant in a 50/50 weight ratio, respectively.

The obtained vaccine preparations were examined for avirulence to confirm the absence of infectivity of the antigen. The vaccines were destroyed and the antigen was separated, which was inoculated with a continuous monolayer cell line from the pig kidney IB-RS-2. It was found that according to the results of 3 consecutive passages in the IB-RS-2 cell culture, the virulent foot and mouth disease virus was not detected, in other words, the preparations are harmless.

Sterility was assessed by plating on solid and liquid nutrient media. The use of soybean-casein agar (SKA), soybean-casein broth (SKB), thioglycol medium (TGS) confirmed the sterility of all vaccine preparations.

Enzyme-linked immunosorbent assay (ELISA) [17, 18] was used to evaluate the absence of antibodies to non-structural proteins of foot-and-mouth disease virus in the final product. Pigs were immunized with three vaccines, and on the 14th day after inoculation, blood was taken, sera were obtained and examined by the indicated method. The results of the study are presented in table 12. From which it follows that in the study of sera after the administration of vaccine No. 1 to animals, the average optical density is 1.080 ± 0.007, the percentage of inhibition (PI) is 3.282 ± 0.609% (RK 50%); for vaccine No. 2, the average optical density is 1.020±0.003, the percentage of inhibition (PI) is 8.674±0.284% (PI<50%); for vaccine No. 3, the average optical density is 1.070±0.003, the percentage of inhibition (PI) is 4.238±0.288% (PI<50%). In other words, the absence of non-structural proteins of FMDV in the obtained FMD inactivated emulsion vaccines was proved.

To test each of the three vaccines for safety, 5 head of cattle intramuscularly in the area of the upper third of the neck at a dose of 6.0 cm ³ (three times the dose) were immunized with the preparations of these vaccines, and the clinical condition of the animals was observed for 6 days. The vaccines were recognized as harmless, since all animals remained clinically healthy according to the results of the observation, and no tissue necrosis was noted at the injection site.

The immunogenicity of each of the three vaccines was studied on 17 pigs by a quantitative method with control infection against the homologous strain Asia-1/Shamir/89 (genetic line A/ASIA/Shamir) of the foot-and-mouth disease virus. For immunization of animals, 3 samples of each of the vaccines were prepared: 1) FMD emulsion vaccine without dilution; 2) a vaccine diluted with "Placebo" in a ratio of 1:5; 3) vaccine diluted with "Placebo" in a ratio of 1:25. The prepared samples were injected intramuscularly into the upper third of the neck at a dose of 2.0 cm ³ . Pigs with serial numbers from 1 to 5 were inoculated with the vaccine without dilution, animals with numbers from 6 to 10 were injected with the vaccine diluted 1:5, animals with numbers from 11 to 15 - the vaccine diluted 1:25, animals from numbers 16 and 17 - not immunized (control group). 21 days after immunization, blood was taken from the vaccinated animals and the resulting sera were analyzed in the PMN to determine the level of virus neutralizing antibodies (VNA). The results of the study are shown in tables 13-15, from which it follows that with the introduction of a whole dose of vaccine No. 1, as well as in dilutions of 1/5 and 1/25, the average antibody titer was 5.40±0.14; 3.80±0.21 and 2.65±0.22 log ₂ , respectively, for vaccine No. 2 in whole form, in dilutions of 1/5 and 1/25 antibody titers were 5.88±0.21; 3.81±0.14 and 3.00±0.38 log ₂ , respectively, for vaccine No. 3 in whole form and in dilutions of 1/5 and 1/25 antibody titers were 6.81±0.25; 4.38±0.27 and 3.13±0.42 log ₂ , respectively.

Pigs were infected with a homologous strain Asia-1/Shamir/89 in the mucous membrane of the tongue at a dose of 10 ^4.0 ID ₅₀ /0.20 cm ³ (in two points of 0.10±0.05 cm ³ ). Within 7 days after infection, the animals were observed and the temperature was measured daily. 7 days after infection, all animals were euthanized and postmortem examination was performed. Animals were considered protected from foot-and-mouth disease, in which there were no lesions on the limbs characteristic of foot-and-mouth disease.

Based on the results of the presence/absence of generalized signs of foot and mouth disease in infected animals, the immunogenic dose and the number of protective doses were determined. The immunogenic dose (IMD ₅₀ ) and the number of protective doses (PD ₅₀ ) for vaccine No. 1 were 0.13 and 15.43, for vaccine No. 2 - 0.09 and 21.28, for vaccine No. 3 - 0.07 and 29 .37. Therefore, vaccine preparation No. 3 had the highest immunogenicity. This fact is evidence that the developed serum-free medium "ARRIAH" allows you to obtain an inactivated FMD vaccine with high values of immunogenic activity.

Thus, using the developed serum-free medium "ARRIAH" it is possible to obtain an inactivated FMD vaccine that is harmless, sterile, free from non-structural proteins and highly immunogenic.

Example 5. Obtaining immunogenic components of the cultural rabies virus of the ARRIAH strain using a transplantable suspension cell line VNK-21 adapted for cultivation in the developed serum-free medium "ARRIAH".

In the obtained cells of the VNK-21 line, adapted for cultivation in a serum-free medium "ARRIAH", reproduction of the rabies virus of the ARRIAH strain was carried out in bioreactors with a volume of 6 liters. The virus infection dose was 0.05 CCID ₅₀ /cell. The cultivation of the virus was carried out at pH 7.4-7.6. The pH was adjusted with sodium bicarbonate solution. The study was carried out in three repetitions. Cultivation was carried out for 48 hours. As a control, VNK-21 cells grown in an improved medium, the composition of which is presented in Table 1 (medium No. 1 - prototype) and in serum-free medium CN 102115729-2011 (medium No. 2 - the closest prototype).

After cultivation of the rabies virus, strain specificity was confirmed by RT-PCR [2]. As a result of the study in suspension, the presence of only the rabies virus of the ARRIAH strain was confirmed.

Spent the process of inactivation of the antigen of the rabies virus. As a first-order inactivator, 1,2-aminoethylethyleneimine (AEEI) was used at a concentration of 0.05%. Inactivation was carried out for 12 hours at 37°C.

The completeness of the process of inactivation of the rabies virus antigen in RIF was determined in accordance with the requirements of the OIE (OIE) [2].

The obtained suspensions of the rabies virus were studied and the concentrations of specific ribonucleoprotein (in reverse transcription and real-time polymerase chain reaction (RT-PCR-RT)) [23] and glycoprotein (in ELISA) [15] were determined. The concentration of immunogenic components was recalculated from 1 million cells.

The results of determining the concentration of immunogenic components of the rabies virus are shown in Table 16 and in FIG. 3, from which it follows that the amount of ribonucleoprotein in the viral suspension, obtained using the developed serum-free medium "ARRIAH", is greater than the prototype and the closest prototype by 42-44 and 38-39%, respectively. Accumulation of rabies virus glycoprotein in the viral suspension obtained using the proposed environment, more than the prototype and the closest prototype by 32-40 and 26-35%, respectively. Therefore, the developed serum-free medium "ARRIAH" and cells of the VNK-21 line, adapted to it, can significantly increase the accumulation of immunogenic components of the rabies virus compared to the prototype variants.

Example 6. Manufacture and study of an anti-rabies inactivated emulsion vaccine from the ARRIAH strain, the production of immunogenic components of which was carried out using a continuous suspension cell line VNK-21 adapted for cultivation in the developed serum-free medium "ARRIAH".

Three rabies inactivated emulsion vaccines were obtained from the ARRIAH strain: vaccine No. 1 - from the antigen obtained in VNK-21 cells grown in an improved medium with 5% bovine serum (medium No. 1 - prototype); vaccine No. 2 - from an antigen obtained in VNK-21 cells grown in a serum-free medium CN 102115729-2011 (medium No. 2 is the closest prototype); vaccine No. 3 - from the antigen obtained in VNK-21 cells grown in the developed serum-free medium "ARRIAH" (medium No. 3). For the manufacture of vaccine preparations, inactivated antigens of the rabies virus of the ARRIAH strain obtained in example 5 were used. The concentrations of ribonucleoprotein and glycoprotein of the rabies virus are presented in table 16. For vaccines No. 1, 2, 3, the concentration of ribonucleoprotein was equal to 0.50; 0.52 and 0.72 µg/ml, respectively, the glycoprotein concentration corresponded to the values of 0.52; 0.58 and 0.73 µg/ml. The requirement for the antigen in the manufacture of rabies vaccine is the concentration of ribonucleoprotein and glycoprotein of at least 3.0 µg/ml each. After concentration by 8 times, the content of rabies virus ribonucleoprotein for vaccines No. 1, 2, 3 was 4.00; 4.16 and 5.76 µg/ml, respectively. After this process, the concentration of rabies virus glycoprotein for vaccines No. 1, 2, 3 was 4.16; 4.64 and 5.84 µg/ml, respectively.

Montanide ISA-61 VG (Seppic) was used as an oil adjuvant. Emulsion vaccines were prepared by dispersing rabies virus antigen concentrate and Montanide ISA-61 VG oil adjuvant in a 40/60 weight ratio, respectively, on a Silverson L5 instrument.

Sterility was assessed by plating on solid and liquid nutrient media. The use of soybean-casein agar (SKA), soybean-casein broth (SKB), thioglycol medium (TGS) confirmed the sterility of all vaccine preparations.

The evaluation of the areactogenicity and safety of each rabies inactivated emulsion culture vaccine was carried out on cattle, small cattle, pigs, dogs, cats and rabbits.

Vaccines were administered to all animal species intramuscularly at the dose recommended for each animal species. A vaccination dose of 2.0 cm ³ is intended for cattle, 1.0 cm ³ - for sheep and goats, adult dogs of large and medium breeds, 0.5 cm ³ - for cats, puppies from 2 months of age of all breeds and for adults small breed dogs (lapdogs, dachshunds, etc.). The animals were clinically observed. Body temperature was measured within 7 days after injection, then 10 and 14 days after vaccination. The criterion for evaluating clinical observation was the development or absence of a general and local response to the administration of the test drugs. According to the results of the study, all the test animals were clinically healthy, no general and local reactions to the administration of drugs were detected. A slight increase in body temperature (0.2-0.5°C) in animals was observed on days 1-2 after the introduction of vaccine No. 3. During this observation time, the temperature difference (ΔT=T _max -T _{on day 0 after vaccination} ) in cattle was 0.3-0.5°C, in MRS - 0.3-0.4°C, in pigs - 0 ,2-0.5°С, in dogs - 0.2-0.5°С, in cats - 0.3-0.5°С. Starting from day 3, the temperature of the animals was normal. Local reaction (edema, pain, lameness) was not observed in the target animal species. Thus, according to the results of studies, three rabies inactivated emulsion culture vaccines are harmless and arectogenic preparations for target animal species.

Conducted a study of humoral immunity of the three obtained emulsion inactivated rabies vaccines. Tests of the presented vaccines were carried out on ten black-and-white cattle at the age of 1 year with an average weight of 200 kg. Animals were vaccinated with vaccines at a dose of 2.0 cm ³ intramuscularly once, dividing the cattle into groups of 5 animals: the first was inoculated with a whole dose of vaccines, the second - by diluting the vaccines in 1/15 M PBS solution by 4 times, the third - by diluting the vaccines by 1/15 M PBS solution 16 times. To assess the intensity of post-vaccination anti-rabies immunity in animals, in accordance with the recommendations of the OIE (OIE), a neutralization reaction was used in a monolayer culture of BHK-21 cells (reaction modification - FAVN (Fluorescent antibody virus neutralization)), expressing the values of the titer of anti-rabies antibodies in IU/ml [2]. A positive serum control standard diluted to an immunogenicity of 0.5 IU/cm ³ and a negative serum control standard with an immunogenicity of <0.1 IU/cm ³ were used for the analysis [2].

In accordance with international requirements, the level of virus neutralizing antibodies (VNA) against the rabies virus ≥0.50 IU/cm ³ is protective [2]. In the neutralization reaction, blood sera from cattle were evaluated before and on the 21st day after vaccination.

Before immunization, anti-rabies antibodies were not detected in the blood of animals. The level of anti-rabies neutralizing antibodies in cattle after the introduction of a single dose (2.0 cm ³ ) of the resulting vaccines is shown in tables 17-19, from which it follows that with the introduction of vaccine No. 1 - whole and in dilutions of 1/4 and 1/16, the average titer antibodies amounted to 24.14±1.84; 7.22 ± 0.61 and 2.3 ± 0.30 IU / ml, respectively, for vaccine No. 2 in whole form, in dilutions of 1/4 and 1/16, antibody titers were slightly higher compared to the immune response to the first vaccine and amounted to 24.88±1.86; 7.60 ± 0.50 and 2.92 ± 0.18 IU / ml, respectively, for vaccine No. 3 in whole form and in dilutions of 1/4 and 1/16, antibody titers were 30.82 ± 1.10; 10.44±0.73 and 4.10±0.32 IU/ml, respectively.

It should be noted that vaccine No. 3, for the manufacture of which the rabies virus antigen was obtained using the developed serum-free medium "ARRIAH", made it possible to induce a higher immune response on day 21 after inoculation (on average, 5.94 IU/ml higher for the whole vaccine , by 2.84 IU/ml for 1/4 dilution of the vaccine and by 1.18 IU/ml for 1/16 dilution of the vaccine). Thus, vaccine preparation No. 3 had the highest immunogenicity. This fact is evidence that the developed serum-free medium "ARRIAH" makes it possible to obtain an anti-rabies inactivated vaccine with high values of immunogenic activity.

Thus, using the developed serum-free medium "ARRIAH" it is possible to obtain an inactivated anti-rabies vaccine, which is harmless, sterile, areactogenic and highly immunogenic.

The main advantage of the invention is the use of the serum-free medium "ARRIAH" for adapting the cells of the continuous suspension line VNK-21 to growing in this medium without the presence of animal blood serum, for cultivating cells of the VNK-21 line in order to obtain suspensions with a higher concentration of cells, for the reproduction of foot-and-mouth disease viruses and rabies in order to increase the quantities of immunogenic components for the production of safe and highly immunogenic FMD and rabies vaccines.

The present invention makes it possible to obtain FMDV antigen with concentrations of 146S immunogenic component compared to prototypes greater by 18-39 and 13-55%, respectively, and 146S+75S immunogenic component - by 20-34 and 19-28%, respectively. The present invention makes it possible to obtain a rabies virus antigen with concentrations of ribonucleoprotein in comparison with the prototypes greater by 42-44 and 38-39%, respectively, and glycoprotein - by 32-40 and 26-35%, respectively.

Sources of information taken into account when compiling the description of the invention to the application for the issuance of a patent of the Russian Federation for the invention "serum-free medium "ARRIAH" for cultivating Syrian hamster kidney cells VNK-21 and obtaining immunogenic components of cultured FMD and rabies viruses for the manufacture of vaccines":

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Claims (1)

Serum-free medium for cultivating Syrian hamster kidney cells VNK-21 and obtaining immunogenic components of cultured FMD and rabies viruses for the manufacture of vaccines, characterized in that it contains the following components: a set of plant proteins, including soy protein, legume protein, potato protein, wheat gluten, rice and corn protein in a total concentration of 3000 mg/l; 21 proteinogenic amino acids in a total concentration of 3431 mg/l, vitamins and vitamin-like substances, including inositol 6.2 mg/l, nicotinamide 3.3 mg/l, calcium pantetonate 3.3 mg/l, pyridoxal hydrochloride 3.5 mg/l , riboflavin 0.35 mg/l, thiamine hydrochloride 3.2 mg/l, folic acid 3.5 mg/l, choline chloride 3.5 mg/l; mitogen - bovine insulin 3 mg/l; glucose 3500 mg/l, minerals, including inorganic water-soluble salts of potassium, sodium, magnesium and calcium with a total concentration of 9350 mg/l, iron (II) chloride 1 mg/l.

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