RU2563345C1 - Inactivated sorbed vaccine against fmd of type a - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: characterised vaccine is inactivated sorbed and comprises avirulent and purified antigenic material from the strain of FMD virus deposited in the collection of FSBI "All-Russian Research Institute for Animal Health" under the registration number VY A No. 2187/Cuti/2013 (production), adjuvants aluminium hydroxide with saponin and a supportive medium in effective proportions. The strain is obtained in the passaged cell culture of VNK-21 and represents a suspension containing mainly immunogenic components of FMD virus 146S and 75S.

EFFECT: vaccine is highly immunogenic and able to provide effective protection against homologous pathogen circulating in South-East Asian countries, in the Zabaykalsky Krai and in the Far East of the Russian Federation.

11 cl, 1 dwg, 8 tbl, 7 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of a vaccine inactivated adsorbed against foot and mouth disease type A.

Animal foot and mouth disease continues to be a global problem, to which the attention of veterinary services of most countries of the world and international organizations (OIE, FAO, EU, their commissions and committees) is riveted [1].

Foot and mouth disease is a highly contagious and rapidly spreading disease of artiodactyl animals, manifested by fever, vesicular lesions of the mucous membrane of the oral cavity, hairless areas of the scalp, udder, corolla, and inter-claw gap and accompanied by impaired movement. It is characterized by a tendency to widespread and epizootic course [2].

Control of foot and mouth disease is significantly complicated by the plurality of its pathogen. There are 7 serotypes of foot and mouth disease virus (VJ): A, O, C, Asia-1, SAT-1, SAT-2 and SAT-3. Within each serotype, numerous antigenic variants have been detected.

The reason for the antigenic diversity of VW is its extremely high variability. Antigenic differences between viruses arise as a result of spontaneous mutations in certain parts of the genome due to errors in viral RNA polymerase during viral RNA replication. Of all parts of the genome, the VP1 gene, which encodes the main immunogenic protein of the virus, differs in maximum variability [3].

To ensure sustainable well-being of the country by foot and mouth disease in the Russian Federation, a system of measures is being implemented, the priority of which is the prevention of the introduction of foot-and-mouth disease virus into the territory of the country, and vaccination prevention in areas of high risk. In the Russian Federation and the CIS countries, inactivated adsorbed vaccines are used for immunization of cattle and small cattle against foot and mouth disease, and inactivated emulsion vaccines are used for immunization of pigs [4].

The manufacturing technology of FMD vaccine from an inactivated virus begins with the selection of production strains based on an epizootological analysis of the dynamics of foot and mouth disease in the country and neighboring countries. When creating drugs for specific prophylaxis, appropriate types of foot-and-mouth disease virus are used and strains with a wide antigenic spectrum inside the type with pronounced cross-immunogenicity are selected. A strain with a wide range of immunogenicity and satisfying the requirements of the region is selected by testing it in a cross-protection reaction or more often in a cross-neutralization reaction. As a rule, a virus population is used as a production strain, which, together with the system and conditions of industrial cultivation, ensures a guaranteed and high accumulation of 146S and 75S virus components and the production of an immunogenic vaccine.

In addition, the production strain is required to maintain the stability of the virus during purification from tissue components and concentration, as well as the preservation of the virus during inactivation and long-term storage [5].

A well-known feature of the causative agent of foot and mouth disease is the antigenic variability of strains within the same serotype, occurring at different time intervals, in different territories, depending on the species composition of the susceptible livestock of animals, its immune status and many other factors.

It is believed that the main cause of antigenic variation is a change in the amino acid sequence in the polypeptides of the viral proteins that make up the virion capsid. In practice, such antigenic changes can be expressed both in insignificant differences between the strains, captured only using subtle molecular analysis methods, and in the appearance of completely different strains requiring the use of new means of specific prophylaxis of the disease caused by these strains [2,5].

Type A foot and mouth disease virus strains isolated in the USSR and used as production strains in the manufacture of FMD inactivated vaccines are known. These include: strain Au No. 103, isolated in 1962 in the Kuibyshev region; strain A _t No. 2, isolated in 1965 in the Tajik SSR; strain A No. 717/73, isolated in 1973 in the Stavropol Territory. After the elimination of foot and mouth disease caused by antigenically similar strains of the virus, they were discontinued and are currently only supported in the Collection of Epizootic Strains of Foot and Mouth Disease Virus and Other Animal Pathogens of FSBI ARRIAH [2, 5].

A known vaccine is inactivated sorbed against type A foot and mouth disease, containing the active substance in the form of avirulent and purified antigenic material from strain A ₂₂ No. 550, homologous to the pathogen, obtained in a sensitive biological system, and targeted additives in the form of an adjuvant and support medium in an effective ratio [6] .

Strain A ₂₂ No. 550 of foot-and-mouth disease virus was isolated in 1964 in Azerbaijan and used in the Russian Federation as an industrial one in the manufacture of specific prophylaxis and diagnostics that are used throughout Russia and the CIS countries.

Newborn rabbits are used as a sensitive biological system, and a phosphate-buffered solution is used as a supporting medium.

To clean the virus-containing suspension from ballast impurities, chloroform in 2% concentration and subsequent centrifugation are used.

Formaldehyde is used to inactivate the virus, and aluminum hydroxide (GOA) with saponin is used as adjuvant.

Known vaccine is inactivated adsorbed against foot and mouth disease type A, containing the active substance in the form of avirulent and purified antigenic material from strain A ₂₂ Iraq 24/64, obtained in a sensitive biological system, and target additives in the form of adjuvant and support medium in an effective ratio.

Strain A ₂₂ Iraq 24/64 of the FMD virus was transmitted in the form of aphthous material from the Razi Institute (Iran) in 1967. In the Russian Federation it is used as an industrial one in the manufacture of specific prophylaxis and diagnostics, used throughout Russia and the CIS countries.

Newborn rabbits are used as a sensitive biological system, and a phosphate-buffered solution is used as a supporting medium.

To clean the virus-containing suspension from ballast impurities, chloroform in 2% concentration and subsequent centrifugation are used.

For inactivation of the virus, aminoethylethyleneimine (AEEI) is used, and from the adjuvants GOA with saponin.

The closest to the invention according to the set of essential features is an inactivated adsorbed vaccine against foot and mouth disease type A containing the active substance in the form of avirulent and purified antigenic material from strain A No. 2045 / Kyrgyzstan / 2007, obtained in a sensitive biological system, and targeted additives in the form of adjuvants and maintenance medium in the ratio, mcg [7]:

antigenic material not less 3.0 GOA 11000.0 ÷ 15000.0 saponin 500.0 ÷ 1500.0 support environment up to 1,000,000.0

The foot-and-mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007 was isolated in the Kyrgyz Republic from a foot-and-mouth disease heifer in 2007. It belongs to a genetic line called A Iran / 2005 [8]. It is used as a production strain in the manufacture of specific prophylaxis and diagnostics, used throughout Russia and the CIS countries.

A suspension culture of VNK-21 cells is used as a sensitive biological system, and Serum without serum with the addition of enzymatic dry muscle hydrolysates (FSMS), dry blood protein hydrolysates (GBKS) and antibiotics at pH 7.4 ÷ 7 are used as a supporting medium 6.

Polyhexamethylene guanidine (PHMG) is used to purify the virus-containing suspension from ballast impurities, and AEI is used to inactivate the virus. To neutralize AEEI, sodium thiosulfate is added to the suspension.

Avirulent and purified antigenic material from strain A No. 2045 / Kyrgyzstan / 2007 is a suspension mainly of 146S and 75S immunogenic components of foot and mouth disease virus. As adjuvants, GOA with saponin is used.

The main disadvantage of the known vaccines, including the prototype vaccine, is that they do not provide reliable protection for susceptible animals from the type A FMD epidemic virus circulating in the countries of Southeast Asia, the Trans-Baikal Territory and the Far East of the Russian Federation.

The task of creating the present invention was to develop a vaccine for FMD inactivated adsorbed, which creates effective protection of susceptible animals against the epizootic virus of foot and mouth disease type A circulating in countries of Southeast Asia, the Trans-Baikal Territory and the Far East of the Russian Federation.

The technical result from the use of the invention is to expand the arsenal of vaccines inactivated adsorbed against foot and mouth disease type A.

The specified technical result was achieved by the creation of a vaccine inactivated adsorbed against foot and mouth disease type A, characterized by the following set of features.

The proposed vaccine contains 1 cm ^{3 of the} preparation: the active substance in the form of avirulent and purified antigenic material from a foot and mouth disease virus strain A No. 2187 / Kuti / 2013, obtained preferably in a suspension culture of BHK-21 cells, in an amount of at least 3.0 μg, and target additives: GOA, preferably in an amount of 11000.0 ÷ 15000.0 μg, saponin, preferably in an amount of 500.0 ÷ 1500.0 μg and a support medium in an amount of up to 1,000,000.0 μg.

The source virus for obtaining the strain of the virus of foot and mouth disease A No. 2187 / Kuti / 2013 was isolated in 2013 in Russia. The strain was obtained by repeated consecutive passages on sensitive hetero- and homologous cell cultures. The strain is adapted to primary trypsinized pork kidney cells and transplantable cell cultures of BHK-21, IBRS-2 and PSGK-30.

For the manufacture of a vaccine, a suspension culture of BHK-21 cells is preferably used as a sensitive biological system, and Earle's serum-free solution with the addition of FGMS, GBKS, and antibiotics at pH 7.4–7.6 is used as a supporting medium.

For inactivation of the virus using AEEI, which is added to the virus-containing suspension to a concentration of 0.025 ÷ 0.05%. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to the suspension [9].

The resulting antigen is purified from ballast impurities using PHMG, which is introduced into the suspension to a concentration of 0.005 ÷ 0.007% [10].

Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013 is a suspension containing predominantly 146S and 758 immunogenic components of foot and mouth disease virus.

The quantitative and qualitative content of viral raw materials is determined by turbidimetry [11].

For the preparation of the vaccine, viral material is used that contains at least 0.5 μg of 146S and 75S immunogenic components of foot and mouth disease virus in 1 cm ³ .

The required concentration of 146S and 75S immunogenic components of foot and mouth disease virus in the proposed vaccine, comprising at least 3 μg in 1 cm ^{3 of the} finished product, is obtained by adding the calculated amount of GOA sorbent adjuvant to the avirulent and purified antigenic material. The optimum is the GOA content in 1 cm ^{3 of the} finished product in the range from 11000.0 μg to 15000.0 μg.

An additional 10% aqueous solution of saponin is added to the resulting concentrate to its content in 1 cm ^{3 of the} finished preparation in the range from 500.0 μg to 1500.0 μg.

The resulting vaccine is a light yellow liquid with a loose precipitate of sorbent, which is formed at the bottom of the vial during storage and is easily broken into a homogeneous suspension with shaking.

The present invention includes the following set of essential features, providing a technical result in all cases for which legal protection is requested.

1. The vaccine is inactivated adsorbed against foot and mouth disease type A.

2. The active substance in the form of avirulent and purified antigenic material from a strain of FMD virus A No. 2187 / Kuti / 2013 in an effective amount.

3. Targeted supplements.

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. vaccine inactivated adsorbed against foot and mouth disease type A;

2. active substance;

3. targeted supplements.

Compared with the prototype vaccine, an essential distinguishing feature of the proposed vaccine is that it contains the avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013 of type A foot and mouth disease virus in an active amount.

The present invention is also characterized by other distinctive features expressing specific forms of execution or special conditions for its use.

1. Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained in a sensitive biological system and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

2. Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of animal origin and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

3. Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

4. Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A in an amount of at least 3.0 μg in 1 cm ^{3 of the} finished product.

5. As a target additive, the vaccine contains an adjuvant-sorbent GOA.

6. GOA, preferably in an amount of 11000.0 ÷ 15000.0 μg in 1 cm ^{3 of the} finished product.

7. As a targeted supplement, the vaccine contains an adjuvant saponin.

8. Saponin, preferably in an amount of 500.0 ÷ 1500.0 μg in 1 cm ^{3 of the} finished product.

9. As a targeted supplement, the vaccine contains a supportive environment.

10. Supporting medium in an amount up to 1,000,000.0 mcg in 1 cm ^{3 of the} finished product.

11. Avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot-and-mouth disease virus type A, GOA, saponin and supporting medium in ratio, mcg:

antigenic material not less than 3.0 GOA 11000.0 ÷ 15000.0 saponin 500.0 ÷ 1500.0 support environment up to 1,000,000.0

The proposed vaccine has high immunogenic activity and provides reliable protection against the foot and mouth disease virus serotype A circulating in the countries of Southeast Asia, the Trans-Baikal Territory and the Far East of the Russian Federation.

The achievement of the technical result from the use of the invention is achieved by the fact that the composition of the proposed vaccine introduced as an active substance antigenic material from strain A No. 2187 / Kuti / 2013 of foot and mouth disease virus serotype A, which has high biological, antigenic and immunogenic activity in its native form and after inactivation, which provides Obtaining FMD vaccine adsorbed inactivated, which creates effective protection of susceptible animals against FMD virus serotype A, causing outbreaks of disease in the last dnie years in South-East Asia, in the Trans-Baikal region and the Far East of the Russian Federation.

The viral isolate, which served as a source for strain A No. 2187 / Kuti / 2013, was isolated in September 2013 from sick pigs in the village of Kuti, Priargunsky district, Trans-Baikal Territory (examination No. 2187). The production strain is obtained from this isolate by successive passages on sensitive hetero- and homologous cell cultures.

Strain A No. 2187 / Kuti / 2013, type A foot-and-mouth disease virus was deposited on January 22, 2014 to the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under the registration number (link): foot-and-mouth disease virus strain A No. 2187 / Kuti / 2013 (production).

The invention is explained on a dendrogram reflecting the phylogenetic relationship of FMD virus strains A No. 2187 / Kuti / 2013 with epizootic and vaccine strains of FMD virus of serological type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

The invention is explained in the list of sequences in which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain A No. 2187 / Kuti / 2013 of type A foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP1 protein gene of strain A No. 2187 / Kuti / 2013 of type A foot and mouth disease virus.

The strain of FMD virus A No. 2187 / Kuti / 2013 of the disease of foot and mouth disease type A is characterized by the following features and properties.

Morphological features

The foot and mouth disease virus strain A No. 2187 / Kuti / 2013 type A belongs to the family Picomaviridae, genus Aphtovirus, serotype A and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the form of the virion is icosahedral, size 23-25 nm. Virion consists of an RNA molecule enclosed in a protein coat. The protein shell consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, foot-and-mouth disease strain A No. 2187 / Kuti / 2013 belongs to serotype A. The virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity (GA activity). In sick animals, antibodies are formed in the blood serum that are detected in an enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN). When cattle are immunized with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA and RMN.

During hyperimmunization of guinea pigs, a concentrated antigen from inactivated foot and mouth disease virus type A No. 2187 / Kuti / 2013 induces the formation of virus-specific antibodies detected in CSC at a 1:40 dilution.

Using the method of nucleotide sequencing, the primary structure of the type 1 FMD virus of the FMD virus No. 2187 / Kuti / 2013 was determined and the primary structure of the VP1 protein was derived. A comparative analysis of the nucleotide sequences showed that the type A foot-and-mouth disease virus strain No. 2187 / Kuti / 2013 significantly differs from the production strains of type A. The degree of nucleotide differences in the sequences of strain A No. 2187 / Kuti / 2013 and the types of foot-and-mouth disease virus strains A was: A ₂₂ No. 550 - 21.49%, A ₂₂ / Iraq / 64 - 21.73%, A / Iran / 96 - 22.03%, A / Armenia / 98 - 22.39%, A / Turkey / Ob - 22.11 %, A / Iran / 05 - 22.32%.

Thus, phylogenetic analysis showed that strain A No. 2187 / Kuti / 2013 belongs to the genetic line of Southeast Asia 97 (Sea-97) of the Asia topotype.

The antigenic affinity of strain VW A No. 2187 / Kuti / 2013 with existing production strains of VW type A was investigated by the serological method in a cross microneutralization reaction (RMN). Strain A No. 2187 / Kuti / 2013 antigenically differs from production strains A22 No. 550, A No. 2179 / Shchelkovo biological complex, A / Iran / 97, A / Kyrgyzstan / 07 (A / Iran / 05). Strain VIA A No. 2187 / Kuti / 2013 has a weak antigenic relationship with strain A / Turkey / 06 (A / Iran / 05).

The results of the studies in the RMN are presented in table 1. Antigenic compliance (r ₁ ) was for A ₂₂ No. 550 - 0.13, A No. 2179 / Shchelkovo Biocombine - 0.11, A / Iran / 97 - 0.004, A / Turkey / 06 - 0.37, A No. 2045 / Kyrgyzstan / 07 - 0.13. With a value of r ₁ ≥0.3, the field isolate and the production strain are closely related, and the vaccine from the production strain will protect against epizootic virus, with a value of r ₁ <0.3 the field isolate is different from the production strain, and the vaccine from this strain does not protect against epizootic virus [12, 13].

Biotechnological characteristics

Strain A No. 2187 / Kuti / 2013 is reproduced in a primary trypsinized monolayer culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), BHK-21 and IB-RS-2, and for 18 ÷ 24 hours of incubation of the virus in these cell cultures accumulates from 6.0 to 7.0 lg TCD ₅₀ / cm ³ . With a high multiplicity of infection (1 ÷ 10 TCD / cell), it causes CPD after 5 hours. Retains the original characteristics when passaged in cell cultures for 5 passages (observation period).

Genotaxonomic characteristic

Strain A No. 2187 / Kuti / 2013 of FMD virus type A is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ D.

Nucleic acid is a single-chain linear molecule with a molecular weight of 2.8 × 10 ⁶ D. The virion has a protein shell consisting of four basic proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The lipoprotein membrane is absent.

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Virionic RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VP _66a ) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

Physical properties

The virion mass is 8.4 × 10 ⁻¹⁸ g. A sedimentation coefficient of 146S in the sucrose gradient. The floating density of 1.45 g / cm ³ .

Resistance to external factors

Strain A No. 2187 / Kuti / 2013 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.2 ÷ 7.6. PH shifts to both the acidic and alkaline sides lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-radiation, high temperatures.

Additional features and properties

Immunogenic activity - immunogen in the composition of an inactivated vaccine.

Antigenic activity - administration of an inactivated antigen to guinea pigs induces the formation of virus-specific antibodies.

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals, newborn mice, guinea pigs.

Virulence - virulence for naturally susceptible animals with contact, aerosol and parenteral infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period).

Based on the data obtained, it can be argued that strain A No. 2187 / Kuti / 2013 in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMD virus type A, which has a higher protective and immunogenic activity.

To reduce its epizootic danger, timely vaccine prophylaxis is important to prevent the emergence of new foci of the disease, for which a highly immunogenic vaccine is needed.

A new inactivated adsorbed vaccine against foot and mouth disease from strain A No. 2187 / Kuti / 2013 was developed. The possibility of its use for the prophylactic immunization of animals has been experimentally confirmed.

Example 1

The viral isolate, which served as the source for strain A No. 2187 / Kuti / 2013, was isolated at the All-Russian Scientific Research Institute of Health and Beauty Research from samples of aphthous material obtained in September 2013 from pigs suspected of having foot-and-mouth disease from the village of Kuti, Priargunsky district, Trans-Baikal Territory (examination No. 2187 / 2013). Samples of aphthous material were received at the Federal State Budget Scientific Institution VNIIZZH on September 26, 2013.

When isolating a virus in order to obtain a homogeneous population with optimal biotechnological properties, a complex of biological, virological, and biochemical methods was used, provided for by guidelines for identifying and identifying FMD virus strains [14].

Biological and virological methods included the isolation of the virus, which was carried out on a culture of primary trypsinized SP cells, transplanted PSGK-30, IB-RS-2 cell lines, followed by adaptation (3 passages). For bioassays on primary and transplantable cell cultures, they were grown on appropriate nutrient media, under stationary conditions in bottles with a surface area of 25 cm ² , washed from the growth medium and infected with a 10% suspension of aphthous material (the multiplicity of infection was 1 ÷ 10 TCD ₅₀ per cell ) prepared in a Hanks solution with 0.5% lactalbumin hydrolyzate (GLA) and antibiotics according to a standard formulation. To remove microflora and ballast cell components, the suspension was treated with 10% chloroform. After 30 minutes of contact at 37 ° C, 5 cm ^{3 of the} support medium was added to the vials and incubated at 37 ° C until the appearance of the virus CPD. In the presence of CPD (rounding of cells, increasing their optical density, degeneration and separation of cells from glass), the bottles were subjected to freezing, thawing, purification of the cell suspension with chloroform and centrifugation at 3000 g for 15 min. The resulting virus-containing material was used for subsequent passages and studies in the CSC for the presence of a viral antigen, using commercial type-specific sera stored in the Museum of strains of the Federal State Budget Scientific Institution VNIIZZH. The virus was considered adapted to cell cultures if, within 18–24 hours, (%) 90–100 CPDs appeared in the monolayer.

A virus adapted to IB-RS-2 cell cultures was used to produce antigen for CSC.

Epizootic isolate A No. 2187 / Kuti / 2013, adapted to PSGK-30 cell culture, was used to infect a suspension of BHK-21 cells in order to obtain antigen for the manufacture of an experimental vaccine series, as well as for hyperimmunization of guinea pigs.

The results of the adaptation of the virus to various cell cultures are presented in table 2.

The data shown in table 2 indicate the high adaptive ability of strain A No. 2187 / Kuti / 2013 of the disease of foot and mouth disease type A to the used cell cultures.

Isolated using the above methods, the viral preparation was studied in the CSC in order to identify its type affiliation (table 3). The strain was tested for the absence of contamination with extraneous viruses in PCR and RT-PCR.

The results shown in table 3 indicate that in the aphthous test material No. 2187 / Kuti / 2013 an FMD virus antigen of type A was detected at a 1: 2 dilution in CSC. No contamination with bacterial and fungal microflora, mycoplasmas and extraneous viruses was found.

The resulting strain of FMD virus type A is assigned the copyright name: strain A No. 2187 / Kuti / 2013.

Example 2

Table 4 shows the results of the cultivation of strain A # 2187 / Kuti / 2013 of foot and mouth disease virus type A in a suspension of BHK-21 cells, from which it follows:

1) strain A No. 2187 / Kuti / 2013 has a reproduction time of 12.60 ± 0.47 hours;

2) the strain And No. 2187 / Kuti / 2013 observed the accumulation of virus-specific protein in the range of 2.98 ± 0.13 μg / cm ³ ;

3) the accumulation of immunogenic components (146S + 75S) in strain A No. 2187 / Kuti / 2013 - 2.54 ± 0.14 μg / cm ³ .

Example 3

An inactivated sorbed vaccine against FMD type A is prepared from a foot and mouth disease virus strain A # 2187 / Kuti / 2013 grown in a suspension culture of BHK-21 cells. As a supporting medium, an Earle solution without serum is used with the addition of FGMS, GBKS and antibiotics at a pH of 7.4–7.6. The cell culture is infected with a virus at the rate of 0.005 ÷ 0.2 TCD ₅₀ per cell.

Immediately after the end of the virus reproduction cycle, without stopping thermostating, a 15–20% AEEI solution, acidified with glacial acetic acid to pH 8.0–8.5, is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to 0.025 ÷ 0.05%. Inactivation of the infectiousness of the virus is carried out for 12 ÷ 24 hours at 36 ÷ 37 ° C and a pH of 7.2 ÷ 7.6 with stirring after 5 ÷ 6 hours for 3 ÷ 5 minutes. The remainder of the AEEI is neutralized by the addition of sodium thiosulfate.

A 10% solution of PHMG is added to the warm suspension to a concentration of 0.005–0.007% for flocculation of ballast impurities and inactivation of possible contaminants. Flocculated ballast impurities are subjected to sedimentation followed by decantation.

Table 5 shows the results of studies on the effect of inactivation and purification of antigenic material using AEI and PHMG on the immunogenic (146S and 75S) components of foot and mouth disease virus of strains A No. 2187 / Kuti / 2013 and A No. 2045 / Kyrgyzstan / 2007.

From the data shown in table 5, it is seen that the components of the foot-and-mouth disease virus of strain A No. 2187 / Kuti / 2013 are retained after inactivation and purification under production conditions. Especially important is the fact that in the process of inactivation and purification of the virus from strain A No. 2187 / Kuti / 2013, there were no changes in the amounts of 146S and 75S components obtained during virus reproduction.

Example 4

The resulting antigen is monitored for avirulence, virus-specific protein content, 146S and 75S virus components, and sterility. The required concentration of 146S and 75S components in 1 cm ^{3 of the} adsorbed vaccine is obtained by concentration of GOA antigen.

The calculated GOA volume of 3% concentration is added to the cooled antigen suspension with the stirrer operating. Stirring is carried out for 30 minutes. After sedimentation of GOA, the calculated volume of the remaining suspension is drained. The final concentration of GOA should be in the range of 1.28 ± 0.22 p <0.001 mg / cm ³ n = 10, and the concentration of 146S and 75S components of the FMD virus is at least 3.0 μg / cm ^{3 of the} finished product. Then, an additional 10% saponin solution is added to the suspension to a final concentration of 0.05 ÷ 0.15%, which corresponds to 500.0 ÷ 1500.0 μg of saponin in 1 cm ^{3 of the} finished vaccine. The vaccine is packaged in glass or plastic bottles and its sterility is monitored in accordance with GOST 28085-89.

Example 5

The virulence and harmlessness of the vaccine is checked on 5 heads of cattle, introducing the vaccine first under the mucous membrane of the tongue at a dose of 2.0 cm ³ and then subcutaneously at a dose of 10.0 cm ³ . Observation of the clinical condition of the animals is carried out for 10 days. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

The vaccine is a light yellow liquid with a loose white precipitate of sorbent that forms at the bottom of the vial during storage and easily breaks up into a homogeneous suspension with shaking.

The optimal component composition of the resulting sorbed vaccine against foot and mouth disease type A are shown in table 6.

Example 6

The antigenic activity of the sorbed vaccine from the production of foot and mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007 against the heterologous strain A No. 2187 / Kuti / 2013 was studied. The level of humoral immunity was evaluated against vaccine strain A No. 2045 / Kyrgyzstan / 2007 and against strain A No. 2187 / Kuti / 2013.

The test was conducted on 10 heads of cattle. Sorbed vaccine was administered subcutaneously at a dose of 2.0 cm ³ . The research results are presented in table 7.

From the data shown in table 7, it can be seen that the sorbed vaccine stimulated the production of virus-neutralizing antibodies against the homologous strain A No. 2045 / Kyrgyzstan / 2007 in cattle in the amount of 4.80 ÷ 5.40 log ₂ . Against the heterologous strain A # 2187 / Kuti / 2013, the titer of neutralizing antibodies was 3.7–4.4 times less and amounted to 2.90–3.25 log ₂ . The difference in the titers of neutralizing antibodies is significant and significant.

On the 4th day after cattle revaccination, the number of virus-neutralizing antibodies against vaccine strain A No. 2045 / Kyrgyzstan / 2007 increased by 2 times. The number of antibodies against foot and mouth disease virus strain A No. 2187 / Kuti / 2013 increased slightly.

From experience it is clear that the vaccine from production strain A No. 2045 / Kyrgyzstan / 2007 does not stimulate the production of humoral immunity to protect animals from heterologous strain A No. 2187 / Kuti / 2013.

According to the recommendations of the OIE, titers of virus-neutralizing antibodies in the blood serum of animals immunized with a whole dose of the vaccine should be at least 5.00 log ₂ [12].

Example 7

Tests of the sorbed vaccine against foot and mouth disease type A, made as described in examples 3 and 4, and containing, μg:

avirulent and purified

strain antigenic material

A No. 2187 / Kuti / 2013, type A foot and mouth disease virus 3.0 GOA 13000.0 saponin 750.0 support environment up to 1,000,000.0

The virulence and safety of the obtained vaccine was tested on 5 heads of cattle. The drug was administered to each animal under the mucous membrane of the tongue at a dose of 2.0 cm ³ and then subcutaneously at a dose of 10.0 cm ³ . Observation of the clinical condition of the animals was carried out for 10 days. Throughout the entire observation period, deviations in the clinical condition of the animals were not detected, which means that the vaccine administered is avirulent and harmless.

At the end of the control for avirulence and safety, the vaccine was tested for immunogenic activity for cattle. The test was conducted on 15 heads of cattle. The results are presented in table 8. ImD _{50 of the} drug was equal to 0.25 cm ³ , i.e. the vaccine dose contained 8 PD ₅₀ .

According to the recommendations of the OIE, the vaccine against foot and mouth disease should contain at least 6 PD ₅₀ in the vaccine volume for cattle [12].

It follows that the vaccine made from a foot and mouth disease virus strain A No. 2187 / Kuti / 2013 has a high immunogenic activity.

Thus, the above information indicates that the vaccine is sorbed inactivated against FMD type A, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology; confirmed the possibility of carrying out the invention; the vaccine made from strain A No. 2187 / Kuti / 2013 in accordance with the invention has high immunogenic activity and is able to provide effective protection of susceptible animals against the type A FMD virus circulating in Southeast Asia, the Trans-Baikal Territory and the Far East.

Table 1 Antigenic compliance (r ₁ ) in the RMN of strain A No. 2187 / Kuti / 2013 of type A foot and mouth disease virus with production type A foot and mouth disease virus strains Strain Exponent r ₁ A22 No. 550 0.13 A No. 2179 / Schelkovo Biocombine 0.11 A / Iran / 97 0.004 A / Turkey / 06 (A / Iran / 05) 0.37 A No. 2045 / Kyrgyzstan / 2007 (A / Iran / 05) 0.13 r ₁ ≥0.3 - the field isolate is antigenically related to the production strain, the vaccine from the production strain can be used if a more related strain is not found and provided that the animals are immunized more than 1 time;
r ₁ <0.3 - the field isolate differs from the production strain, the vaccine from which is not acceptable for protection against infection by the field isolate

table 2 Biological properties of the virus of epizootic strain A No. 2187 / Kuti / 2013 of type A foot and mouth disease virus Virus reproduction system The time of manifestation of biological activity (hours) Number of adaptation passages Adapted material characteristics Activity in RSK The titer of infectious activity (lg TCD ₅₀ / cm ³ ) PSGK-30 twenty 5 1: 2 6.0 IB-RS-2 24 5 1: 4 6.23 VNK-21 eighteen 5 1: 2 7.0

Table 3 Determination of the type of virus in a 33% suspension of aphthous material in the CSC (examination No. 2187 / Kuti / 2013) Hyperimmune sera in working dilution Examination No. 2187 The control whole 1: 2 1: 4 1: 8 to / a A / Turkey / 06 A ₂₂ / Iraq / 64 O panasia2 Asia-1 Shamir 3/89 b / a A / Turkey / About + - - - four four - - - A ₂₂ / Iraq / 64 four four - - four - - - O panasia2 - - - - - - four - - Asia-1 Shamir 3/89 - - - - - - four - b / s - - - - - - - - - b / c four four four four four four four four four Note: 4 - 100% delayed hemolysis; "-" complete hemolysis; b / s - without serum; b / a - without antigen; b / c - no complement

Table 4 Cultivation of foot and mouth disease virus strain A No. 2187 / Kuti / 2013 in a suspension of BHK-21 cells n = 10 No. p / p Cell concentration, mln / cm3 Dose of infection, TCD ₅₀ / cl Duration of reproduction, hours Virus infectivity titer, lg TCD ₅₀ / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ % yield of 146S + 75S components one 3,5 0.17 13.5 8.00 3.42 3.24 94.7 2 3.4 0.10 13.5 7.75 3.37 2.78 82.5 3 4.1 0.10 fifteen 7.75 3.20 2.67 83,4 four 3,7 0.10 13 6.50 2.63 2,37 90.1 5 3,7 0.10 13 7.00 2,36 2.17 91.9 6 3.4 0.007 eleven 7.25 2,52 2.16 85.7 7 3.4 0.006 10.5 7.75 2.64 2,30 87.1 8 4.2 0.005 eleven 8.25 3.44 3.22 93.6 9 3.8 0.006 12 8.00 3.06 1.86 60.8 10 4.3 0.05 fourteen 7.75 3.17 2.63 83.0 M ± m 3.75 ± 0.11 p <0.001 0.064 ± 0.02 p <0.01 12.6 ± 0.47 p <0.001 7.60 ± 0.17 p <0.001 2.98 ± 0.13 p <0.001 2.54 ± 0.14 p <0.001 85.3 ± 3.06 p <0.001

Table 5 Comparative data on the resistance of foot and mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007 and strain A No. 2187 / Kuti / 2013 to inactivation by aminoethylethyleneimine and sterilizing purification with polyhexamethylene guanidine No. p / p A No. 2045 / Kyrgyzstan / 2007 A No. 2187 / Kuti / 2013 cultivation termination after inactivation and cleaning cultivation termination after inactivation and cleaning VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ one 2.02 1.89 2.14 2.00 3.17 2.63 3.01 2.60 2 3.24 2.48 2.95 2.44 3.06 1.86 2,50 1.77 3 2.59 1.99 2.69 2.16 2,52 2.16 2.85 2,39 four 1.87 1,60 2.17 1.98 2.64 2,30 2,57 2,35 5 3.30 2.73 2.72 2.42 2,36 1.88 2.63 1.81 6 2.72 2.47 2.91 2.65 3.20 2.81 2.96 2.66 M ± m 2.62 ± 0.24 p <0.001 2.19 ± 0.18 p <0.001 2.60 ± 0.15 p <0.001 2.28 ± 0.11 p <0.001 2.82 ± 0.15 p <0.001 2.27 ± 0.16 p <0.001 2.75 ± 0.09 p <0.001 2.2b ± 0.16 p <0.001

Table 6 The optimal formulation of FMD inactivated sorbed vaccine from a foot and mouth disease virus strain No. 2187 / Kuti / 2013 type A (μg in 1 cm ^{3 of the} finished vaccine) Vaccine components Minimum Average Maximum antigenic material 3.0 > 3.0 > 3.0 GOA 11000.0 13000.0 15000.0 saponin 500,0 750.0 1,500.0 support environment up to 1,000,000.0 up to 1,000,000.0 up to 1,000,000.0

Table 7 The study of humoral immunity in cattle grafted with a sorbed vaccine from the culture of foot and mouth disease virus A No. 2045 / Kyrgyzstan / 2007 against heterologous strain A No. 2187 / Kuti / 2013 No. p / p Vaccine characterization Vaccination rate Animal number The number of virus-neutralizing antibodies on 21 days after vaccination, (092 A No. 2045 / Kyrgyzstan / 2007 A No. 2187 / Kuti / 2013 one Sorbed PD - 2 cm ³ 1468 + 758-7.4 mcg A 2045 / Kyrgyzstan / 2007 one one 5.50 2.00 2 5.50 3,50 3 5.25 2,50 four 5.50 4.75 5 5.25 3,50 M ± m 5.40 ± 0.06 p <0.001 3.25 ± 0.44 p <0.001 2 Sorbed PD - 2 cm ³ 1468 + 758-7.05 μg A 2045 / Kyrgyzstan / 2007 one one 4.00 2.25 2 4,50 3.00 3 5.00 3.00 four 5.25 4.00 5 5.25 2.25 M ± m 4.80 ± 0.24 p <0.001 2.90 ± 0.32 p <0.001 2 4 days after revaccination 4 days after revaccination one 5.00 2.75 2 5.25 3.00 3 5.75 4.00 four 7.50 4,50 5 5.50 3.00 M ± m 5.80 ± 0.44 p <0.001 3.3 ± 0.27 p <0.001

Table 8 Monitoring the immunogenic and protective activity of an inactivated sorbed foot and mouth disease vaccine from strain A No. 2187 / Kuti / 2013 №№ Breeding Generalization availability Antibody titers on the 19th day after vaccination against FMD virus A No. 2187 / Kuti / 2013, log ₂ BMI ₅₀ , cm ³ PD ₅₀ one c - 6.25 0.25 8 2 - 6.50 3 - 7.00 four - 5.50 5 - 5.75 6.20 ± 0.27 p <0.001 6 1: 4 - 4.00 7 + 3.00 8 - 6.00 9 - 4.75 10 - 4.75 4.50 ± 0.49 p <0.001 eleven 1:16 - 3.25 12 + 1.75 13 + 2,50 fourteen + 2.25 fifteen + 2.75 2.50 ± 0.25 p <0.001 K1 + K2 +

Information sources

1. Rakhmanov A.M. The program of joint actions of the CIS member states on prevention and control of foot and mouth disease and its implementation // Tr. Federal Center for Animal Health. - Vladimir, 2011 .-- T.9. - S. 29-46.

2. Foot and mouth disease: monograph / A.N. Burdov, A.I. Dudnikov, P.V. Malyarets [et al.]. - M .: Agropromizdat, 1990 .-- 320 p.

3. Antigenic heterogeneity of a foot-and-mouth disease virus serotype in the field is mediated by very limited sequence variation at several antigenic sites / M. G. Mateu, J. Hernandez, M. A. Martinez [et al.] / / J. Virol. - 1994. - Vol. 68. - P.1407-1417.

4. Viral diseases of animals / V.N. Syurin, A.Ya. Samuilenko, B.V. Soloviev [et al.]. - M.: VNIITIBP, 1998 .-- S.532-548.

5. Rerer X. Foot and mouth disease: Per. with him. / GA. Surkova; under the editorship of and with the foreword. P.V. Malyarets. - M .: Kolos, 1971. - 432 p.

6. Temporary instructions for the manufacture and control of FMD concentrated aluminum hydroxide formol vaccine from lapinized A22 virus. Approved by the GUV of the USSR on March 25, 1971

7. Application 2012134084 Russian Federation, IPC A61K 39/135, Inactivated type A foot and mouth disease vaccine / D.A. Lozovoi, V.V. Mikhalishin, D.V. Mikhalishin, V.A. Starikov [et al.]; FSBI "ARRIAH" - Decl. 08/09/2012; published. declared 02/20/2014

8. The study of the biological properties of the FMD virus line A Iran / 05 production strains of the type Ag2 / M.V. Zhiltsova, S.R. Kremenchug, A.I. Egorova, V.V. Borisov // Russian Veterinary Journal - 2008. - September. - S.15-16.

9. Pat. 594771 Russian Federation, IPC A61K 39/12, Tool for inactivation of viruses in the manufacture of antiviral drugs / N.A. Ulupov, A.I. Dudnikov, P.A. Gembitsky, D.S. Beetle; VNIIA - Decl. 05/07/1973; publ. 07/07/93

10. Pat.2054039 Russian Federation, IPC A61K 39/135, Method for cleaning and sterilizing a culture of foot and mouth disease virus / T.N. Lezova, N.A. Ulupov, V.V. Borisov, V.V. Mikhalishin, A.I. Dudnikov, P.A. Gembitsky; VNIIA - Decl. 02/07/1992; publ. 02/10/1996

11. Auth. testimonial. 784335 USSR, C12Q 1/02, Method for determining the quality of viral raw materials / A.F. Bondarenko; VNIIA - Decl. 07/04/1979; publ. 03/20/2000

12. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. - 7th Ed. - Paris, 2012 .-- Vol. 1, Chapter 2.1.5. - P.166-169.

13. Selection of foot-and-mouth disease vaccine strains a review / D.J. Paton, J.F. Valacher, J. Bergman [et al.] // Rev. Sci. Tech. OIE. - 2005 .-- Vol.24 (3). - P.981-983.

14. Guidelines for the identification and identification of strains of foot and mouth disease virus / Gusev AA, Zakharov VM, Shazhko Zh.A. [and etc.]. Vladimir, 2002, 31 p.

Claims (11)

1. The vaccine is inactivated adsorbed against foot and mouth disease type A, containing the active substance and the target additives, characterized in that as the active substance it contains avirulent and purified antigenic material from a foot and mouth disease virus Aphtae epizooticae, fam. Picornaviridae, genus Aphtovirus, serotype A, deposited in the collection of FSBI ARRIAH under registration number strain VIA A No. 2187 / Kuti / 2013 (production), in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain VYA A No. 2187 / Kuti / 2013, obtained in a sensitive biological system and consisting of a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease virus type BUT. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from strain VYA A No. 2187 / Kuti / 2013, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly immunogenic 146S and 75S components of FMD virus type A. 4. The vaccine according to p. 3, characterized in that it contains avirulent and purified antigenic material from strain VYA A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing mainly immunogenic 146S and 75S components of FMD virus type A in an amount of at least 3.0 μg in 1 cm ^{3 of the} finished product. 5. The vaccine according to claim 1, characterized in that as the target additive it contains an adjuvant-sorbent aluminum hydroxide (GOA). 6. The vaccine according to claim 5, characterized in that it contains GOA, preferably in an amount of 11000.0-15000.0 μg in 1 cm ^{3 of the} finished product. 7. The vaccine according to claim 1, characterized in that it contains saponin adjuvant as a target additive. 8. The vaccine according to claim 7, characterized in that it contains an adjuvant saponin, preferably in an amount of 500.0 - 1500.0 μg in 1 cm ^{3 of the} finished product. 9. The vaccine according to claim 1, characterized in that it contains a supportive medium as a target additive. 10. The vaccine according to claim 9, characterized in that it contains a support medium in an amount up to 1,000,000.0 μg in 1 cm ^{3 of the} finished product. 11. The vaccine according to any one of paragraphs. 1-10, characterized in that it contains avirulent and purified antigenic material from strain VYA A No. 2187 / Kuti / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease virus type A, GOA, saponin and support medium in the ratio, μg:
antigenic material not less than 3.0 GOA 11000.0 - 15000.0 saponin 500.0 - 1500.0 support environment up to 1,000,000.0

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