RU2603003C1 - Inactivated sorptive vaccine to fmd types a, o, asia-1 - Google Patents

Abstract

FIELD: veterinary science; biotechnology.

SUBSTANCE: invention relates to veterinary virology and biotechnology. Vaccine contains active substance and target additives. As active substance, vaccine contains mixture of avirulent purified antigen material of foot FMD A strains No. 2171/Kabardino-Balkar/2013, A No. 2187/Kuti/2013, Asia-1 No. 1946/Shamir 3/89 and O No. 2123/South Ossetia/2011, obtained from transplantable cell culture VNK-21, representing suspension, containing mainly 146S and 75S immunogenic components of FMD, adjuvants: aluminium hydroxide with saponin and preserving medium in effective ratios.

EFFECT: vaccine exhibits high immunogenicity and is capable of providing effective protection against homologous infectious agents, circulating in Transcaucasus, Central Asia, middle and Near East.

16 cl, 4 dwg, 7 tbl, 5 ex

Description

The invention relates to the field of veterinary virology and biotechnology, relates to a vaccine inactivated adsorbed against foot and mouth disease types A, O, Asia-1 with bishtamm antigen of foot and mouth disease virus type A, which can be used for specific prophylaxis of artiodactyl animals against foot and mouth disease virus types A, O, Asia one.

The problem of the prevention of foot and mouth disease in animals and the reduction of economic damage from its occurrence and spread, which can cause emergency situations in animal husbandry with severe socio-economic consequences, is relevant for many countries of the world, including the Russian Federation.

Foot and mouth disease is a worldwide problem, as it is a highly contagious and rapidly spreading disease that is susceptible to the main agricultural animals: cattle (cattle), pigs, small cattle (MPC), and many wild animals, with more than 100 species. There are 7 different types of virus; vaccination against one type of foot-and-mouth disease virus does not protect against another type of foot-and-mouth disease virus [1].

For prophylactic vaccination, traditional mono- and polyvalent sorbed and emulsion vaccines made from a culture virus grown in a suspension of BHK-21 cells inactivated by aminoethylethyleneimine (AEEI) have found practical application [2].

Despite the measures taken, the epizootic situation of foot and mouth disease in the world, including in Asian countries, in 2013-2014. not improved. From the beginning of 2013 and subsequently in China, outbreaks of foot and mouth disease among cattle and pigs caused by the O and A virus were recorded in several provinces in China. In 2013, 4 outbreaks of foot and mouth disease were noted in 4 areas of the Trans-Baikal Territory bordering China, they remain unsuccessful to date time. According to the OIE World Reference Laboratory for foot and mouth disease, isolates isolated in the Trans-Baikal Territory and Amur Region of the Russian Federation, Kazakhstan and Mongolia are 99% identical to isolates A / QHXN-CHA-2013-B and A / GDMM-CHA-2013S. In 2013, foot and mouth disease among cattle was noted in the North Caucasus and in the Krasnodar Territory. The isolated isolates belonged to the genetic line A / Iran / 2005. Isolates of this genetic line in 2013 caused outbreaks of foot and mouth disease in the Middle East [3].

It should be noted that all isolated isolates differ in antigenicity from FMD virus strains of type A, used until recently for the manufacture of FMD vaccines [3].

Due to the systematic vaccination of animals and the control of the immune background of the livestock, the vast majority of the constituent entities of the Russian Federation, including the North Caucasus, have long been successful in foot and mouth disease. However, the unfavorable epizootic situation of foot and mouth disease in the world and the real possibility of introducing its causative agent into the territory of Russia dictate the need to strengthen anti-FMD measures, among which the main is the implementation of preventive universal vaccination of animals in areas of high risk of the occurrence and spread of foot and mouth disease [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].

The causative agent of foot and mouth disease has significant antigenic variability of strains within the same serotype, which is detected at different time intervals and in different territories and depends on the species composition of the susceptible population, its immune status and many other various factors.

The antigenic variability of foot and mouth disease virus is caused by amino acid substitutions in polypeptide fragments (antigenic epitopes) exposed on the surface of capsid proteins. The shifts of the antigenic spectrum corresponding to the renewal of the structure of a new field strain can vary from insignificant ones captured by monoclonal antibodies to significant ones recorded using traditional polyclonal immunoglobulins. Significant changes in the antigenic characteristics of the natural strain lead to the fact that vaccination of animals with a production vaccine strain does not guarantee protection of the livestock from antigenically different epizootic strains of foot and mouth disease [5, 6].

Foreign scientists showed that a mixture of two vaccines caused protection in guinea pigs against a heterologous strain, while each of them separately did not provide an immune response against a heterologous strain of foot and mouth disease [7].

Domestic scientists conducted studies comparing the immunogenic activity of mono- and multi-strain vaccines. From the obtained results it follows that the introduction of antigenically different strains of a virus of the type into the vaccine leads to an expansion of the antigenic spectrum of action, which increases the immunogenic activity of the multi-strain vaccine against homo- and heterologous strains of the virus of this type and does not decrease the activity against the production [8] .

The development of new diagnostic tools and specific immunoprophylaxis of foot and mouth disease is an urgent task.

Known vaccine is trivalent inactivated adsorbed against foot and mouth disease virus types A, O and Asia-1 RAKSHA TRIVALENT manufactured by INDIAN IMMUNOLOGICALS LIMITED RAKSHAPURAM, GACHIBOWLI HYDEREBAD, India [9].

Known vaccine inactivated purified against foot and mouth disease of ruminants "Aftovaxpur" (Aftovaxpur) types A, O and Asia-1, manufactured by "Merial Animal Health Ltd", France. The vaccine has a protective activity of ≥6.00 PD ₅₀ [10].

A known vaccine against foot and mouth disease sorbed trivalent (from a virus grown in BHK-21 cells) [11].

The main disadvantage of known vaccines is their low efficiency due to existing antigenic differences between strains of the same type of foot and mouth disease virus, as well as insufficient purity of antigenic materials.

The objective of the invention is to obtain a vaccine inactivated adsorbed against foot and mouth disease types A, O, Asia-1, with high immunogenic activity, with a wide spectrum of antigenicity when vaccinated against different epizootic strains of foot and mouth disease.

This object is achieved in that for the prevention and control of the disease, an inactivated sorbed vaccine was obtained against foot and mouth disease types A, O, Asia-1 with bishtamm antigen of foot and mouth disease virus type A, which creates effective protection of susceptible animals from infection with foot and mouth disease virus types A, O, Asia- 1 and having an expanded antigenic effect against FMD virus type A.

The technical result from the use of the present invention is to increase the immunogenic activity of the vaccine inactivated adsorbed against foot and mouth disease types A, O, Asia-1, by expanding the antigenic spectrum of activity by valency A.

The specified technical result was achieved by creating a vaccine inactivated adsorbed against foot and mouth disease types A, O and Asia-1 with a bishtamm antigen type A, characterized by the following set of features.

The proposed vaccine in 1 cm ^{3 of the} preparation as an active substance contains a mixture of avirulent and purified materials from FMD virus strains, containing mainly 146S and 75S immunogenic components: type A bishtamm antigen of FMD virus in the form of avirulent and purified materials from FMD virus strains A No. 2187 / Kuti / 2013 and A No. 2171 / Kabardino-Balkarian / 2013, avirulent and purified material from a foot-and-mouth disease strain O No. 2123 / South Ossetia / 2011 type O and avirulent and purified material from a foot-and-mouth virus strain Asia-1 No. 1946 / Shamir 3 / 89 type Asia-1, p preferably obtained in a transplanted culture of BHK-21 cells, in amounts of at least 3 μg of each strain, which provide antigenic activity in the animal’s body upon administration of the target drug and the target additives: GOA, saponin and a support medium in an amount that ensures tolerant presentation of antigen in the body of the immunized animal. The content of target additives in the finished product is calculated taking into account the volume of the vaccinated dose of the vaccine for each animal species.

The original virus for obtaining the strain of the virus of foot and mouth disease A No. 2187 / Kuti / 2013 was isolated in 2013 in the village of Kuti, Priargunsky district, Trans-Baikal Territory. 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.

The source virus to obtain the strain

And No. 2171 / Kabardino-Balkarsky / 2013 was allocated in 2013 in the village of Nizhny Kurkuzhin, Baksan district, Kabardino-Balkarian Republic. 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.

Asia-1 foot-and-mouth disease virus strain No. 1946 / Shamir 3/89 obtained from the Pirbright World Reference Laboratory.

The foot and mouth disease strain O No. 2123 / South Ossetia / 2011, was isolated in 2011 in the village of Styrfas, Dzau district of the Republic of South Ossetia. 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 the vaccine, a suspension culture of BHK-21 cells is preferably used as a sensitive biological system, and Serum without serum supplemented with enzymatic dry muscle hydrolysates (FSMS), dry blood protein hydrolysates (GBKS) and antibiotics at pH 7.4 is used as a sensitive biological system. ÷ 7.6.

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 [12].

The resulting antigen is purified from ballast impurities using polyhexamethylene guanidine hydrochloride (PHMG), which is added to the suspension to a concentration of 0.005–0.007% [13].

Avirulent and purified antigenic material from FMD virus strains: A No. 2187 / Kuti / 2013, A No. 2171 / Kabardino-Balkarian / 2013, Asia-1 No. 1946 / Shamir 3/89 and No. No. 2123 / South Ossetia / 2011, is a suspension containing predominantly 146S and 75S immunogenic components of foot and mouth disease virus.

The quantitative and qualitative content of viral raw materials is determined by turbidimetry [14].

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 of each strain 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 adjuvant-sorbent to the avirulent and purified antigenic material. The optimal content is YEAR in 1 cm ^{3 of the} finished product in the range of 24000.0 ÷ 36000.0 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 that provide a technical result in all cases for which legal protection is sought:

1. Inactivated adsorbed vaccine against foot and mouth disease types A, O and Asia-1 with bishtamm FMD virus antigen type A.

2. The active substance in the form of a mixture of avirulent purified antigenic materials from immunogenic 146S and 75S components of foot and mouth disease virus of strains A No. 2187 / Kuti / 2013 type A, A No. 2171 / Kabardino-Balkarian / 2013 type A, Asia-1 No. 1946 / Shamir 3 / 89 type Asia-1 and 0№2123 / South Ossetia / 2011 type O, obtained preferably in transplantable cell culture of BHK-21 in an effective amount.

3. Targeted supplements.

The salient features of the proposed vaccine are that as an active substance it contains a mixture of avirulent purified antigenic materials from FMD virus strains A No. 2187 / Kuti / 2013, A No. 2171 / Kabardino-Balkarsky / 2013, Asia-1 No. 1946 / Shamir 3/89 and O No. 2123 / South Ossetia / 2011.

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 of type A foot and mouth disease virus, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease.

2. 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 predominantly 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.

3. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplanted 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. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease virus type A, b the amount of not less than 3.0 μg in 1 cm ^{3 of the} finished product.

5. Avirulent and purified antigenic material from strain O No. 2123 / South Ossetia / 2011, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type O.

6. Avirulent and purified antigenic material from strain O No. 2123 / South Ossetia / 2011, 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 O, in an amount of at least 3, 0 mcg in 1 cm ^{3 of the} finished product.

7. Avirulent and purified antigenic material from strain Asia-1 No. 1946 / Shamir 3/89, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease type Asia-1.

8. Avirulent and purified antigenic material from strain Asia-1 No. 1946 / Shamir 3/89, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease type Asia-1, in an amount not less than 3.0 mcg in 1 cm ^{3 of the} finished product.

9. Of the targeted additives, the vaccine contains the adjuvant-sorbent GOA.

10. GOA, preferably in an amount of 24000.0 ÷ 36000.0 μg in 1 cm ^{3 of the} finished product.

11. Of the target additives, the vaccine contains adjuvant saponin.

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

13. Of the targeted supplements, the vaccine contains a supportive environment.

14. The vaccine contains a support medium in an amount of up to 1,000,000.0 μg in 1 cm ^{3 of the} finished product.

15. Avirulent and purified antigenic material from foot and mouth disease virus strains A No. 2187 / Kuti / 2013 type A, A No. 2171 / Kabardino-Balkarian / 2013 type A, Asia-1 No. 1946 / Shamir 3/89 type Asia-1 and O No. 2123 / South Ossetia / 2011 type O, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot-and-mouth disease virus, GOA, saponin and support medium in the ratio, μg / cm ^{3 of the} finished preparation:

The proposed vaccine has high immunogenic activity and provides reliable protection against FMD virus of serotypes A, O and Asia-1 circulating in the countries of the Caucasus, Central Asia, the Middle and Far East.

The achievement of the technical result from the use of the invention is achieved by the fact that the antigenic material of foot and mouth disease virus from strains A No. 2187 / Kuti / 2013 type A, A No. 2171 / Kabardino-Balkarian / 2013 type A, Asia-1 is introduced into the composition of the proposed vaccine No. 194b / Shamir 3/89 type Asia-1 and O No. 2123 / South Ossetia / 2011 type O, having high immunogenic activity, creating effective protection of susceptible animals against foot-and-mouth disease virus of serotypes A, O and Asia-1, causing outbreaks of the disease in recent years in the countries of Transcaucasia, Central Asia, Middle and Far East.

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 dendrogram reflects the phylogenetic relationship of the strain of foot and mouth disease virus No. 2187 / Kuti / 2013 with epizootic and vaccine strains of foot and mouth disease virus of serological type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene (Fig. 1).

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 strain of foot and mouth disease virus A No. 2187 / Kuti / 2013 type A belongs to the family Picornaviridae, 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, strain A No. 2187 / Kuti / 2013 of the foot and mouth disease virus belongs to serotype A. The virus is stably neutralized by a 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 was significantly different from the type A production strains. 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 A22 No. 550 - 21.49%, A22 / Iraq / 64 - 21.73%, A / Iran / 96 - 22.03%, A / Armenia / 98 - 22.39%, A / Turkey / 06 - 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 [15, 16].

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).

Gene and chemotaxonomic characteristics

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).

Strain A No. 2171 / Kabardino-Balkarian / 2013, type A foot-and-mouth disease virus was deposited on January 28, 2014 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under the registration number (link): virus strain foot and mouth disease A No. 2171 / Kabardino-Balkarian / 2013 (production).

The dendrogram reflects the phylogenetic relationships of the FMD virus strain A No. 2171 / Kabardino-Balkarsky / 2013 with the 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 (Fig. 2).

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

SEQ ID NO: 3 represents the nucleotide sequence of the VP1 protein gene of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus;

SEQ ID NO: 4 represents the amino acid sequence of the VP1 protein gene of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus.

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus is characterized by the following features and properties. Morphological features:

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus belongs to the family Picornaviridae, genus Aphtovirus, serotype A and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the shape 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, the strain

A No. 2171 / Kabardino-Balkarian / 2013 of the FMD virus is of type A. The virus is stably neutralized by a 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, the concentrated antigen from inactivated foot and mouth disease virus type A No. 2171 / Kabardino-Balkarsky / 2013 induces the formation of virus-specific antibodies detected in CSC at a dilution of 1:30.

Using the method of nucleotide sequencing, the primary structure of the type 1 FMD virus of the FMD virus No. 2171 / Kabardino-Balkarsky / 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. 2171 / Kabardino-Balkarsky / 2013 is significantly different from the production strains of type A. The degree of nucleotide differences in the sequences of strain A No. 2171 / Kabardino-Balkarian / 2013 sequences with foot-and-mouth disease virus strains of serological type A was : A22№550 - 22.65%, A22 / Iraq / 64 - 21.66%, A / Iran / 96 - 21.43%, A / Armenia / 98 - 21.98%, A / Turkey / 06 - 8 , 23%, A / Iran / 05 - 8.75%.

Thus, phylogenetic analysis showed that strain A No. 2171 / Kabardino-Balkarsky / 2013 belongs to the Iran 2005 genetic line of the Asia topotype of FMD virus of serological type A and is antigenically different from existing production strains of VJ type A.

The antigenic affinity of strain VYA A No. 2171 / Kabardino-Balkarsky / 2013 with existing production strains of VYA A was investigated by the serological method in the cross microneutralization reaction (RMN). Strain A No. 2171 / Kabardino-Balkarian / 2013 antigenically differs from production strains A ₂₂ No. 550, A ₂₂ Iraq / 64, A / Iran / 97, A / Turkey / 06 (A / Iran / 05), A / Kyrgyzstan / 07 (A / Iran / 05).

The results of studies at the Russian Medical Library are presented in Table 2. Antigenic compliance (r ₁ ) for A22 No. 550 was 0.013, A ₂₂ Iraq / 64 - 0.02, A / Iran / 97 - 0.03, A / Turkey / 06 - 0 , 19 and A / Kyrgyzstan / 2007 - 0.03. 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 [15, 16].

Biotechnological characteristics

Strain A No. 2171 / Kabardino-Balkarsky / 2013 is reproduced in monolayer cell cultures: a primary trypsinized cell culture of pig kidneys (SP), transplantable cultures of kidney cells of the Siberian mountain ibex (PSGK-30), BHK-21 and IB-RS-2. Within 18 ÷ 24 hours of incubation, the virus harvest in these cultures reaches values from 6.00 to 7.50 lg TCD ₅₀ / cm ³ . With a high multiplicity of infection (1-10 TCD / cell), the virus causes CPD after 5 hours. The virus retains its original characteristics when passaged in cell cultures for 5 passages (observation period).

Gene and chemotaxonomic characteristics

Strain A No. 2171 / Kabardino-Balkarian / 2013, type A foot and mouth disease virus 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. 2171 / Kabardino-Balkarsky / 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.

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 10 passages (observation period).

Strain O No. 2123 / South Ossetia / 2011, type O foot and mouth disease virus was deposited on April 25, 2013 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under registration number (link): foot and mouth disease virus strain About No. 2123 / South Ossetia / 2011 (industrial).

The dendrogram reflects the phylogenetic relationship of a strain of foot and mouth disease virus No. 2123 / South Ossetia / 2011 with epizootic and vaccine strains of foot and mouth disease virus of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene (Fig. 3).

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

SEQ ID NO: 5 represents the nucleotide sequence of the VP1 protein gene of strain O No. 2123 / South Ossetia / 2011, type O foot and mouth disease virus;

SEQ ID NO: 6 represents the amino acid sequence of the VP1 protein gene of strain O No. 2123 / South Ossetia / 2011, type O foot and mouth disease virus.

The strain of foot and mouth disease virus O No. 2123 / South Ossetia / 2011, the disease of foot and mouth disease type O is characterized by the following features and properties.

Morphological features

The strain of foot and mouth disease virus No. 2123 / South Ossetia / 2011 type O belongs to the family Picornaviridae, genus Aphtovirus, serotype O 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, strain O No. 2123 / South Ossetia / 2011 of the foot and mouth disease virus belongs to the O serotype. 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 O No. 2123 / South Ossetia / 2011 induces the formation of virus-specific antibodies detected in CSC at a dilution of 1:30.

Phylogenetic analysis showed that strain O No. 2123 / South Ossetia / 2011 belongs to the genetic line O PanAsia 2 of the topotype Middle East - South Asia.

The antigenic affinity of strain VYA O No. 2123 / South Ossetia / 2011 with existing production strains of VYA type O was studied by the serological method in the cross microneutralization reaction (RMN). Strain O No. 2123 / South Ossetia / 2011 is antigenically related to production strains Ο ₁ Manisa, O No. 1734 / Primorsky / 2000 (PanAsia), O PanAsia-2.

The results of studies at the Russian Medical Library are presented in Table 3. Antigenic compliance (r ₁ ) was 0.74 for Ο ₁ Manisa, O No. 1734 / Primorsky 72000 (PanAsia) - 1.0, O PanAsia-2 - 1.0. 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 [15, 16].

Biotechnological characteristics

Strain O No. 2123 / South Ossetia / 2011 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), VNK-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. It retains its original characteristics when passaged in cell cultures for 10 passages (observation period).

Gene and chemotaxonomic characteristics

Strain O No. 2123 / South Ossetia / 2011, type O foot and mouth disease virus 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 O No. 2123 / South Ossetia / 2011 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 10 passages (observation period).

Strain Asia-1 No. 1946 / Shamir 3/89 of the FMD virus of type Asia-1 was deposited on November 24, 2014 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under registration number (link) : foot and mouth disease virus Asia-1 No. 1946 / Shamir 3/89 (production).

The dendrogram reflects the phylogenetic relationship of the strain of foot and mouth disease Asia-1 No. 1946 / Shamir 3/89 with epizootic and vaccine strains of foot-and-mouth disease virus of serological type Asia-1. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene (Fig. 4).

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

SEQ ID NO: 7 represents the nucleotide sequence of the VP1 protein gene of strain Asia-1 No. 1946 / Shamir 3/89 of the foot and mouth disease virus type Asia-1;

SEQ ID NO: 8 represents the amino acid sequence of the VP1 protein gene of strain Asia-1 No. 1946 / Shamir 3/89 of the Asia-1 FMD virus.

The strain of foot and mouth disease virus Asia-1 No. 1946 / Shamir 3/89 virus of foot and mouth disease type Asia-1 is characterized by the following features and properties.

Morphological features

The strain of foot and mouth disease virus Asia-1 No. 1946 / Shamir 3/89 of type Asia-1 belongs to the family Picornaviridae, genus Aphtovirus, serotype Asia-1 and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the icosahedral form of the virion, 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, the strain Asia-1 No. 1946 / Shamir 3/89 of the FMD virus belongs to the serotype Asia-1. 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, the concentrated antigen from inactivated foot-and-mouth disease virus of type Asia-1 No. 1946 / Shamir 3/89 induces the formation of virus-specific antibodies detected in CSC at a dilution of 1: 120.

Using the nucleotide sequencing method, the primary structure of the VP1 gene of foot and mouth disease virus type Asia-1 No. 1946 / Shamir 3/89 was determined and the primary structure of the VP1 protein was derived. A comparative analysis of the nucleotide sequences showed that the strain of foot and mouth disease virus type Asia-1 No. 1946 / Shamir 3/89 is significantly different from production strains of type Asia-1. The degree of nucleotide differences in the sequences of strain Asia-1 No. 1946 / Shamir 3/89 with strains of FMD virus of serological type Asia-1 was: Asia-1 No. 1987 / Amursky / 2005 - 26%, Asia-1 / Tajikistan / 2011 - 21.73 %, Asia-1 / Pakistan 29/09 - 22.03%.

Thus, phylogenetic analysis showed that strain Asia-1 No. 1946 / Shamir 3/89 belongs to the topotype Asia-1.

The antigenic affinity of the strain VYA Asia-1 No. 1946 / Shamir 3/89 with the existing production strains of VYA type Asia-1 was studied by the serological method in the cross microneutralization reaction (RMN). Strain Asia-1 No. 1946 / Shamir 3/89 is antigenically different from production strains Asia-1 No. 1987 / Amursky / 2005, Asia-1 / Tajikistan / 2011, Asia-1 / Pakistan 29/09, Asia-1 / Iran 58 / 99.

The results of the studies at the Russian Medical Library are shown in Table 4. Antigenic compliance (r ₁ ) was 0.20 for Asia-1 / Amur / 2005 - 0.20, Asia-1 / Tajikistan / 2011 - 0.21, Asia-1 / Pakistan 29 / 09 - 0.14.

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 [15, 16].

Biotechnological characteristics

Strain Asia-1 No. 1946 / Shamir 3/89 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), VNK-21 and IB-RS-2, and during 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. It retains its original characteristics when passaged in cell cultures for 10 passages (observation period).

Gene and chemotaxonomic characteristics

Strain Asia-1 No. 1946 / Shamir 3/89 of the FMD virus of type Asia-1 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 Asia-1 No. 1946 / Shamir 3/89 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 10 passages (observation period).

The essence of the invention is illustrated by examples of its implementation and use, which do not limit the scope of legal protection of the invention.

Example 1

An inactivated vaccine adsorbed against foot and mouth disease of types A, O, Asia-1 with a bishtamm antigen of type A is prepared from FMD virus strains A No. 2187 / Kuti / 2013, A No. 2171 / Kabardino-Balkarsky / 2013, Asia-1 No. 1946 / Shamir 3 / 89 and O No. 2123 / South Ossetia / 2011.

FMD strains are cultured in separate bioreactors.

The cultivation of foot and mouth disease virus is carried out at a temperature of 36-37 ° C. After 6–7 hours of incubation, living and dead cells are counted with trypan blue staining. If the number of living cells is 15–20%, then incubation is continued for another 2–3 hours. When the number of dead cells reaches 90 ÷ 95%, the cultivation is stopped and the virus-containing suspension is checked for sterility and the content of 146S and 75S components. The amount of 146S + 75S components in the suspension should be at least 0.5 μg / cm ³ . 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.

Example 2

The resulting antigen is monitored for avirulence in pork kidney cell culture, virus-specific protein content, 146S and 75S virus components, and sterility.

The obtained avirulent and purified antigenic material of the four strains, as described in example 1, are combined depending on the content of 146S and 75S virus components.

The required concentration of 146S and 75S components of each strain in 1 cm ^{3 of the} adsorbed vaccine is achieved 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 2.13 ± 0.26 P <0.01 mg / cm ³ n = 10, and the concentration of 146S and 75S components of each strain of foot-and-mouth disease virus, at least 3.0 μg / cm ³ finished the drug. Then, an additional 10% saponin solution is added to the suspension to a final concentration of 0.050-0.150%, which corresponds to 500 ÷ 1500 μg of saponin in 1 cm ^{3 of the} finished vaccine.

The resulting vaccine is packaged in glass or plastic bottles and its sterility is monitored in accordance with GOST 28085-89.

The optimal component composition of the vaccine inactivated adsorbed against foot and mouth disease of types A, O, Asia-1 with bishtamm antigen of type A is presented in table 5.

The vaccine easily resolves at the injection site, does not cause abscesses, a general reaction in the form of a rise in temperature. The vaccine has a pronounced immunogenic activity for cattle in a vaccine dose of 2 ÷ 3 cm ³ for MPC in a vaccine dose of 1 ÷ 1.5 cm ³ 21 days after the vaccine is administered. The vaccine is injected cattle subcutaneously into the middle third of the neck, and the cattle subcutaneously from the inside of the thigh.

Example 3

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.

Example 4

Tests of the immunogenic activity of the vaccine inactivated adsorbed against foot and mouth disease types A, O and Asia-1 with a bishtamm antigen of type A. The vaccine was manufactured as described in examples 1 and 2 with a content of, mcg in 1 cm ³ :

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.

After the end of the control of avirulence and harmlessness, we examined the humoral immunity of cattle grafted with a foot and mouth disease vaccine with bishtamm antigen type A. The vaccine was injected subcutaneously in an amount of 2.0 cm ³ . The research results are presented in table 6.

The data in table 6 confirm that the vaccine from production strains of foot and mouth disease virus A No. 2187 / Kuti / 2013, A No. 2171 / Kabardino-Balkarsky / 2013, Asia-1 No. 1946 / Shamir 3/89 and About No. 2123 / South Ossetia / 2011 stimulated production of antibodies against foot and mouth disease virus types A, O, Asia-1 more than 5.00 log ₂ .

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 [15].

Example 5

Tests of the immunogenic activity of the vaccine inactivated adsorbed against foot and mouth disease types A, O and Asia-1 with a bishtamm antigen of type A. The vaccine was manufactured as described in examples 1 and 2 with a content of, mcg in 1 cm ³ :

At the end of the control for avirulence and safety, the vaccine was tested for immunogenic activity for cattle. The tests were carried out on 5 heads of cattle. The results are presented in table 7. According to the results of the experiment, it is evident that the vaccine contributed to the production of virus-neutralizing antibodies in an amount sufficient to protect animals from the generalized form of foot and mouth disease.

Thus, the vaccine manufactured inactivated adsorbed against foot and mouth disease types A, O, Asia-1 with a bishtamm antigen of type A has a high immunogenic activity.

Information sources

1. Gulenkin V.M. The economic efficiency of preventive vaccination of animals against foot and mouth disease in the Russian Federation / Tr. Federal Center for Animal Health. - Vladimir. 2012.- T. 10.- S. 31-41.

2. Dudnikov A.I. Vaccination strategy for the elimination of foot and mouth disease / Tr. Federal Center for Animal Health. - Vladimir. 2006. - T. 4. - S. 81-90.

3. Mishchenko A.V., Kremenchugskaya S.R., Rakhmanov A.M. Aggravation of the epizootic situation of animal foot and mouth disease in Asia and Russia // Innovative processes in the agricultural sector: Sat. Articles of the 6th International scientific and practical. conf. teachers, young scientists, graduate students and students. - M., 2014 .-- S. 168-171.

4. Rakhmanov A.M., Mishchenko A.V., Fomina C.H. Epizootic situation of foot and mouth disease in the North Caucasus // Bulletin of Veterinary Medicine. - 2014. - T. 69, No. 2. - S. 11-14.

5. Rerer X. Foot and mouth disease / Translation with it. G.A. Surkova. Ed. and with the foreword. Cand. vet. sciences P.V. Malyarets. - M .: Kolos, 1971. - 432 p.

6. Burdov A.N., Dudnikov A.I., Malyarets P.V. and other foot and mouth disease / Ed. A.N. Burdova. - M .: Agropromizdat, 1990 .-- S. 231-250.

7. Etudi immulogique comporative sur bovis de deuxsouches de virus aphteux de type O. O Flanders et O Espangne 64 / J. Fontaine [et al.] // Bull. Off. Intern. Epiz. - 1966. - Vol. 65, No. 11-12. - P. 2051-2062. French Patent No. 2088038; A61K 27/00, C12K 5/00; 01/07/1972

8. A comparative study of the immunogenic activity of mono- and multi-strain vaccines from foot and mouth disease virus type Asia-1 / T.Yu. Chernyaeva, A.F. Bondarenko, Ε.V. Belik, L.A. Dudnikov // Actual issues. vet. Virology. - Vladimir, 1990 .-- S. 83-84.

9. Raksha (FOOT AND MOUTH DISEASE VACCINE BP (Vet)) - URL: https://www.indimmune.com/livestock-vaccines.pclf (accessed 02.15.2015).

10. Link to vaccine inactivated adsorbed against foot and mouth disease manufactured by Merial Animal Health Ltd, France - URL: ec.europa.eu/health/.../anx_128466_en.pdf (accessed 02.15.2015).

11. The official website of FSBI ARRIAH - URL: http://www.arriah.ru/main/production/price-vaccines/117 (accessed 02.15.2015).

12. Pat. RF 594771, IPC A61K 39/12. Means 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

13. Pat. RF 2054039, IPC A61K 39/135. The method of purification and sterilization of culture virus of foot and mouth disease / 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

14. Auth. testimonial. USSR 784335, C12Q 1/02. A method for determining the quality of viral raw materials / A.F. Bondarenko; ARRIAH. - Declared. 07/04/1979; publ. 03/20/2000

15. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 7th Ed. -, Paris, 2012 - Vol. 1, Chapter 2.1.5. - P. 166-169.

16. 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.

Table 7 The study of the immunogenic activity of the vaccine inactivated adsorbed against foot and mouth disease types A, O and Asia-1 Animal number Generalization availability Antibody titers on day 21 after vaccination against foot and mouth disease virus, log ₂ A No. 2187 /
Kuti / 2013 A No. 2171 /
Kabardino-Balkarian /
2013 About No. 2123 /
South Ossetia/
2011 Asia-1 No. 1946 /
Shamir /
3/89 one - 5.75 5.75 5.25 5.25 2 - 6.00 5.50 5.00 5.50 3 - 5.25 5.00 5.75 5.75 four - 5.50 6.00 5.50 4.75 5 - 5.75 5.75 4.75 5.00 M ± m 5.65 ± 0.13 5.60 ± 0.17 5.25 ± 0.18 5.25 ± 0.18 the control +

Claims (16)

1. The vaccine is inactivated adsorbed against foot and mouth disease types A, O, Asia-1 with a bishtamm type A virus antigen, containing the active substance and target additives, characterized in that it contains a mixture of the avirulent purified antigenic material from the Aphtae epizooticae virus strain , family Picornaviridae, genus Aphtovirus, serotype A, deposited in the collection of FSBI ARRIAH under registration number A No. 2187 / Kuti / 2013 (production), avirulent purified antigenic material from a virus strain Aphtae epizooticae, family P icornaviridae, genus Aphtovirus, serotype A, deposited in the collection of FSBI ARRIAH under registration number A No. 2171 / Kabardino-Balkarsky / 2013 (production), from avirulent purified antigenic material from the virus strain Aphtae epizooticae, family Picornaviridae, serotype Aphtov deposited in the collection of FSBI ARRIAH under registration number O No. 2123 / South Ossetia / 2011 (production), from avirulent purified antigenic material from the Aphtae epizooticae virus strain, fam. Picornaviridae, genus Aphtovirus, serotype Asia-1, deposited in the collection of FSBI ARRIAH under registration number Asia-1 No. 1946 / Shamir 3/89, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains an avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components FMD virus type A. 3. The vaccine according to p. 2, characterized in that it contains avirulent and purified antigenic material from strain A No. 2187 / Kuti / 2013, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components type A foot and mouth disease virus, in an amount of at least 3.0 μg in 1 cm ^{3 of the} finished product. 4. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A. 5. The vaccine according to claim 4, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplanted cell culture of BHK-21 and which is 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. 6. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain O No. 2123 / South Ossetia / 2011, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of FMD virus type O. 7. The vaccine according to p. 6, characterized in that it contains avirulent and purified antigenic material from strain O No. 2123 / South Ossetia / 2011, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of FMD virus type O, in an amount of at least 3.0 μg in 1 cm ^{3 of the} finished product. 8. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain Asia-1 No. 1946 / Shamir 3/89, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of the foot and mouth disease virus type Asia-1. 9. The vaccine according to claim 8, characterized in that it contains avirulent and purified antigenic material from strain Asia-1 No. 1946 / Shamir 3/89, obtained preferably in a transplanted cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of foot-and-mouth disease virus type Asia-1, in an amount of not less than 3.0 μg in 1 cm ^{3 of the} finished product. 10. The vaccine according to claim 1, characterized in that of the target additives it contains an adjuvant-sorbent aluminum hydroxide (GOA). 11. The vaccine according to p. 10, characterized in that it contains GOA, preferably in an amount of 24000.0 ÷ 36000.0 μg in 1 cm ^{3 of the} finished product. 12. The vaccine according to claim 1, characterized in that it contains saponin adjuvant from the target additives. 13. The vaccine according to p. 12, 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. 14. The vaccine according to claim 1, characterized in that of the target additives it contains a supportive environment. 15. The vaccine according to claim 14, 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. 16. The vaccine according to any one of paragraphs. 1-15, characterized in that as the active substance it contains a mixture of avirulent and purified antigenic materials from strains A No. 2187 / Kuti / 2013, A No. 2171 / Kabardino-Balkarsky / 2013, Asia-1 No. 1946 / Shamir 3/89 , About No. 2123 / South Ossetia / 2011, foot and mouth disease virus, obtained preferably in the transplanted cell culture of BHK-21, and target additives: GOA, saponin, supporting medium in an amount, μg / cm 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 predominantly 146S and 75S immunogenic components of FMD virus type A in an amount of at least 3.0;
Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A in an amount of at least 3.0;
Avirulent and purified antigenic material from strain O No. 2123 / South Ossetia / 2011, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type O in an amount of at least 3.0;
Avirulent and purified antigenic material from strain Asia-1 No. 1946 / Shamir 3/89, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing predominantly 146S and 75S immunogenic components of foot and mouth disease type Asia-1 in an amount of at least 3 0;
GOA 24000.0 ÷ 36000.0;
Saponin 500.0 ÷ 1500.0;
Supporting environment up to 1,000,000.0.

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