RU2294759C2 - Inactivated emulsion vaccine against foot-and-mouth disease of type a - Google Patents

Abstract

FIELD: veterinary virology, biotechnology.

SUBSTANCE: the suggested vaccine contains avirulent and purified antigenic material out of strain A (Georgia) being homologous to infection agent 1999/N1721 obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus. Moreover, the vaccine contains maintenance medium and butyric adjuvant in efficient ratios. The strain has been deposited in collection of FGU VGNKI under registration number - industrial culture strain of foot-and-mouth disease virus A (Georgia) 1999/N1721-DEP of serotype A. As maintenance medium it is necessary to apply serum-free Earle's solution at addition of FGMS, GBCS and antibiotics at pH being 7.4-7.6. Out of butyric adjuvants the vaccine contains butyric adjuvant of the All-Russia Research Institute of Animal Protection (VNIIZZH) or butyric adjuvant of Montanide ISA-70 or Montanide ISA-260 marks by "Seppic" (France). The vaccine provides efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.

EFFECT: higher efficiency.

11 cl, 1 dwg, 5 ex, 8 tbl

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of inactivated emulsion FMD vaccine type A.

Foot and mouth disease is an acute contagious viral disease of artiodactyl animals. It is characterized by a tendency to widespread and epizootic course. The disease is accompanied by large losses of milk, meat and other types of livestock products, complicates commercial operations and economic activity. 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, and vaccination prevention in areas of high risk. At present, in the Russian Federation and the CIS countries for the immunization of cattle and small cattle against foot and mouth disease, inactivated adsorbed vaccines are used, as a rule, and inactivated emulsion vaccines are used for immunization of pigs (1).

Published research results indicate the benefits of emulsion preparations, which are prepared using oil adjuvants (2, 3).

Emulsion preparations are obtained by mixing two mutually insoluble phases: oil (oil adjuvant) and water, containing antigen (antigens) in a support medium, by vigorous mixing (emulsification).

At the same time, depending on the composition of the oil adjuvant, emulsions of various types are obtained: “water in oil” (reverse), “oil in water” (direct) or “water-oil-water” (multiple). The reverse type of emulsion is most commonly used to immunize farm animals.

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 has requirements for 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 (4).

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 a natural strain are very likely to weaken specific immunity induced by a non-homologous antigen. They also cause difficulties in strain-specific diagnosis (4, 5).

As a result, it becomes necessary to create new diagnostic tools and specific immunoprophylaxis of foot and mouth disease.

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 A ₇ 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 and Other Animal Pathogens of FSI ARRIAH (1-6).

A type A inactivated emulsion vaccine against foot and mouth disease is known, containing the active substance in the form of avirulent and purified antigenic material from a foot and mouth disease virus virus homologous to the causative agent of the infection, obtained in a sensitive biological system (transplantable cell culture of pig kidney IB-RS-2 clone 3), and targeted additives in the form of a supporting medium and an oil adjuvant in an effective ratio (7).

A known inactivated emulsion vaccine against foot and mouth disease type A containing the active substance in the form of avirulent and purified antigenic material from a foot and mouth disease virus A ₂₂ No. 550 homologous to the pathogen, obtained in a sensitive biological system (newborn rabbits), and targeted additives in the form of a support medium and oil adjuvant in the effective ratio (8).

A known inactivated emulsion vaccine against foot and mouth disease type A containing the active substance in the form of avirulent and purified antigenic material from a foot and mouth disease virus A ₂₂ No. 550 homologous to the pathogen, obtained in a sensitive biological system (transplantable cell culture of BHK-21), and target additives in the form supporting medium and oil adjuvant in an effective ratio (9).

The main disadvantage of the known vaccines is that they have low immunogenic activity.

The closest to the invention according to the set of essential features is an inactivated emulsion vaccine against foot and mouth disease type A containing the active substance in the form of avirulent and purified antigenic material from the immunogenic 146S and 75S components of foot and mouth disease virus type A strain A No. 1707 "Armenia-98", obtained in a sensitive biological system (transplantable cell culture of BHK-21), and target additives in the form of a support medium and an oil adjuvant in the ratio, μg / ml of the vaccine dose:

Antigenic material not less 2.0 Support environment 299998.0-499998.0 Oil adjuvant up to 1,000,000.0 (10).

The main disadvantage of the prototype vaccine is also its lack of immunogenic activity. It does not provide reliable protection of susceptible animals from the epizootic virus of type A foot and mouth disease circulating in the countries of Transcaucasia, Central Asia, the Middle and Near East.

The task of creating the present invention was to develop a vaccine against FMD inactivated emulsion, which creates effective protection of susceptible animals against the epizootic virus of foot and mouth disease type A circulating in the countries of the Caucasus, Central Asia, the Middle and Middle East.

The technical result from the use of the present invention is to expand the arsenal of inactivated emulsion vaccines against foot and mouth disease type A, which provide effective protection of susceptible animals against the epizootic virus of foot and mouth disease type A circulating in the countries of the Caucasus, Central Asia, the Middle and Middle East.

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

The proposed vaccine contains the active substance in the form of avirulent and purified antigenic material from the immunogenic 146S and 75S components of foot-and-mouth disease virus type A strain A (Georgia) 1999 / No. 1721 (copyright), obtained preferably in a suspension culture of BHK-21 cells, in an amount of not less than 3.0 μg in a single vaccine dose of the vaccine and targeted additives, supporting medium and oil adjuvant in an amount that provides a tolerant presentation of antigen in the body of an 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 source virus for obtaining strain A (Georgia) 1999 / No. 1721 was isolated in 1999 in the Republic of Georgia. The strain was obtained by passaging the isolate on sensitive hetero- and homologous cell cultures. The strain is adapted to the primary cells of the pig kidney and transplantable cell cultures of BHK-21, IB-RS-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 Earle's serum-free solution with the addition of dry muscle enzymatic hydrolyzate (FSHMS), dry blood protein hydrolyzate (GBKS) and antibiotics at pH 7.4 is used as a sensitive biological system. -7.6.

To inactivate the virus, aminoethylethyleneimine (AEEI) is used, 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 (11).

The resulting antigen is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.005-0.007% (12).

Avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721 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 (13).

To prepare 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 ml.

The required concentration of 146S and 75S immunogenic components of foot and mouth disease virus in the vaccine preparation is provided by concentration of the antigen by flow ultrafiltration or precipitation with polyethylene glycol - 115 (PEG-115). An emulsion vaccine is obtained by dispersing in colloidal mills a concentrate of foot-and-mouth disease antigen and oil adjuvant in a ratio of 3: 7-1: 1, respectively. Of the oil adjuvants, the VNIIZH oil adjuvant can be used (14), as well as the Montanide ISA-70 or Montanide ISA-206 brand oil adjuvant manufactured by Seppic (France).

The content of the supporting medium and the oil adjuvant in the vaccine dose of the vaccine for each animal in the ratio of 3: 7-1: 1 is optimal, as it provides a tolerant presentation of the antigen in the body of the immunized animal.

For the immunization of pigs (vaccination dose of 2 ml), the following composition of the proposed vaccine, mcg in the vaccination dose of the drug for pigs, is optimal:

Avirulent and purified antigenic material from immunogenic 146S and 75S components of foot and mouth disease virus type A strain A (Georgia) 1999 / No.1721 no less than 3.0 Support environment 599997.0-999997.0 Oil adjuvant 1000000.0-1400000.0.

For immunization of cattle (vaccine dose of 5 ml), the following composition of the proposed vaccine is optimal, mcg in the vaccine dose of the drug for cattle:

Avirulent and purified antigenic material from immunogenic 146S and 75S components of foot and mouth disease virus type A strain A (Georgia) 1999 / No. 1721 no less 3.0 Support environment 1499997.0-2499997.0 Oil adjuvant 2500000.0-3500000.0.

For immunization of sheep (vaccination dose of 1 ml), the following composition of the proposed vaccine is optimal, μg in the vaccination dose of the drug for sheep:

Avirulent and purified antigenic material from immunogenic 146S and 75S components of foot and mouth disease virus type A strain A (Georgia) 1999 / No. 1721 no less 3.0 Support environment 299997.0-499997.0 Oil adjuvant 500000.0-700000.0.

The resulting vaccine is a milk-like liquid, insoluble in water.

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 emulsion vaccine against foot and mouth disease type A.

2. The active substance in the form of an avirulent and purified antigenic material from the immunogenic 146S and 75S components of FMD virus type A strain A (Georgia) 1999 / No. 1721 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. Inactivated emulsion vaccine against foot and mouth disease type A.

2. The 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 (Georgia) 1999 / No. 1721 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 homologous to the causative agent of infection of strain A (Georgia) 1999 / No. 1721, 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 (Georgia), homologous to the causative agent of the infection (Georgia) 1999 / No. 1721, obtained preferably in a transplanted cell culture of animal origin and consisting of a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A.

3. Avirulent and purified antigenic material from strain A (Georgia), homologous to the causative agent of the infection (Georgia) 1999 / No. 1721, obtained preferably in the 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 (Georgia), homologous to the pathogen (Georgia) 1999 / No. 1721, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A, in an amount not less than 3.0 micrograms per vaccine dose for each animal species.

5. As a targeted supplement, the vaccine contains a support medium.

6. The vaccine contains a support medium in an amount that provides a tolerant presentation of the antigen in the body of the immunized animal.

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

8. The vaccine contains an oil adjuvant in an amount providing a tolerant presentation of the antigen in the body of the immunized animal.

9. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus, supporting medium and oil adjuvant in the ratio, μg in the vaccine dose for pigs:

Antigenic material not less 3.0 Support environment 599997.0-999997.0 Oil adjuvant 1000000.0-1400000.0.

10. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus, supporting medium and oil adjuvant in the ratio, μg in the vaccine dose for cattle:

Antigenic material not less 3.0 Support environment 1499997.0-2499997.0 Oil adjuvant 2500000.0-3500000.0.

11. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus, supporting medium and oil adjuvant in the ratio, μg in the vaccine dose for the sheep:

Antigenic material not less 3.0 Support environment 299997.0-499997.0 Oil adjuvant 500000.0-700000.0.

The proposed vaccine has a high immunogenic activity and provides reliable protection against the FMD virus serotype A circulating in the countries of the Caucasus, Central Asia, the Middle and Middle East.

The achievement of the technical result from the use of the invention is achieved by the fact that antigenic material from strain A (Georgia) 1999 / No. 1721 of foot and mouth disease virus serotype A, which has high biological, antigenic and immunogenic activity in its native form and after inactivation, is introduced into the composition of the proposed vaccine. and providing a FMD vaccine inactivated emulsion, which creates effective protection of susceptible animals against FMD virus serotype A, causing an outbreak of the disease in recent years in the countries of Transcaucasia, Central Asia, the Middle and Middle East.

Strain A (Georgia) 1999 / No. 1721 is new, previously unknown. The original virus for obtaining strain A (Georgia) 1999 / No. 1721 was isolated in 1999 from sick cows in the private sector of the village of Ude, Adigen region of the Republic of Georgia. A production strain is obtained from this isolate by passaging on sensitive hetero- and homologous cell cultures.

The resulting strain was deposited on August 19, 2003 in the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, Federal State Institution "All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines - Center for Quality of Veterinary Medicines and Feed" (FGU VGNKI) under registration number - production, culture strain of foot and mouth disease virus A (Georgia) 1999 / No. 1721, serotype A.

The invention is explained on the dendrogram reflecting the phylogenetic relationships of strain A (Georgia) 1999 / No. 1721 of type A foot-and-mouth disease virus with epizootic and vaccine strains of type A foot-and-mouth disease virus (dendrogram based on a comparison of the complete nucleotide sequences of the VP1 gene), and in the list of sequences in which :

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 gene of strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus;

SEQ ID NO: 2 — amino acid sequence of VP1 strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus.

Strain A (Georgia) 1999 / No. 1721 of the FMD virus type A is characterized by the following features and properties.

Morphological properties

Strain A (Georgia) 1999 / No. 1721 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, strain A (Georgia) 1999 / No. 1721 of the FMD virus belongs to serotype A.

The virus is stably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. In sick animals, antibodies are formed in the blood serum that are detected in RDP, ELISA and pH. When vaccinating cattle with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA, RDP and pH. During hyperimmunization of guinea pigs with a concentrated inactivated virus, it induces the formation of virus-specific antibodies detected in CSC at a dilution of 1: 128-1: 512 and in ELISA at a dilution of 1: 6000-1: 10000.

The antigenic relationship of strain A (Georgia) 1999 / No.1721 with the corresponding production and previously isolated strains in Turkey and Iran was studied in the RSK. The results are presented in table 1.

As shown by the data presented in table 1, strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus differs from both production and previously isolated epizootic strains.

According to the antigenic properties, the new strain A (Georgia) 1999 / No.1721 of the type A foot and mouth disease virus also differs from the previously studied production strain A # 1707 / Armenia / 98. Bilateral relationship (R) in the CSC between them is 20%.

A study of the antigenic relationship of the isolated strain was carried out in the PH, where cattle of convalescents of cattle for viruses of strains A (Georgia) 1999 / No.1721, A / Turkey / 97 and A ₂₂ No.550 were used as immune. The results of these studies are presented in table 2.

The data in table 2 data confirm the antigenic difference of the isolated strain from the production strain A ₂₂ No. 550 and the epizootic strain isolated in Turkey in 1997.

Molecular genetic characterization

The primary structure of the VP1 gene (SEQ ID NO: 1) of strain A (Georgia) 1999 / No.1721 was determined by nucleotide sequencing and the amino acid sequence of the VP1 protein (SEQ ID NO: 2) was derived from it. A comparative analysis of the established sequences showed that according to the primary structure of the VP1 gene and protein, strain A (Georgia) 1999 / No.1721 differs from all previously isolated FMD virus isolates, including strains A ₂₂ No.550 and A / No.1707 / Armenia / 98, currently used for the production of vaccines against FMD type A. The phylogenetic relationships of strain A (Georgia) 1999 / No.1721 with the previously studied isolates of FMD virus type A isolated in different regions of the world over the past four decades are reflected in the dendrogram.

Biotechnological characteristics

Strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus exhibits high biological, antigenic and immunogenic activity both in its native form and after inactivation. The strain is intended for the production of diagnostic sera and antigenic preparations, as well as for the manufacture of inactivated FMD vaccines. Strain A (Georgia) 1999 / No. 1721 is reproduced in a monolayer culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), IB-RS-2 and BHK-21 and during 18-24 hours of incubation from 6.0 to 7.5 Ig TCD ₅₀ / ml accumulates. With massive infection (1-100 TCD / cell), it causes a CPD after 4 hours. The research results are presented in table 3.

After 3-4 incubation passages, from 6.0 to 7.5 Ig TCD ₅₀ / ml accumulates. It retains its original characteristics when passaged in sensitive biological systems for 10 passages.

Chemo- and genotaxonomic characterization

Strain A (Georgia) 1999 / No. 1721 is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ D.

Nucleic acid is represented by a single-stranded linear molecule with a molecular weight of 2.8 × 10 MD. The virion has a protein shell consisting of four basic proteins VP1, VP2, VP3 and VP4. The lipoprotein membrane is absent.

The main antigenic protein is VP1. 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.

In turn, the precursors cleave to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VP3D) 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. Floating density 1.45 g / ml.

Resistance to external factors

Strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.0-7.6. 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.

Based on the data obtained, it can be argued that strain A (Georgia) 1999 / No. 1721 in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMD virus type A.

To reduce its epizootic danger, timely vaccine prophylaxis of newly emerging foci of the disease is necessary, for which a highly immunogenic vaccine is needed.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information, and the identification of sources containing information about analogues of the invention, it was established that the applicant did not find sources characterized by signs that are identical (identical) to all signs of the invention. The determination from the list of identified analogues of the prototype, as the closest in the totality of the features of the analogue, made it possible to establish a set of significant distinctive features of the proposed vaccine relative to the applicant's technical result, as set forth in the independent claim.

Therefore, the claimed vaccine corresponds to the level of patentability "novelty."

To verify the conformity of the proposed vaccine with the patentability condition “inventive step”, an additional search for known solutions was carried out to identify features included in the distinctive part of the independent claim. The search results showed that the proposed solution does not follow explicitly for the specialist from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the present invention were identified), and the effect of the transformations provided for by the essential features of the proposed vaccine was not identified to achieve a technical result.

Therefore, the proposed vaccine meets the condition of patentability "inventive step".

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

Example 1

Strain A (Georgia) 1999 / No. 1721 of type A foot-and-mouth disease virus was isolated from field material received at ARRIAH as an aphthous aphthous epithelium from cattle suspected of foot-and-mouth disease during laboratory diagnosis of this disease and its differentiation from other vesicular diseases. When isolating the virus, a complex of biological, virological and biochemical methods was used.

Biological and virological methods included inoculation of cattle field isolate material and subsequent adaptation of the virus to cultures of primary and transplanted cell lines. Cell cultures of SP, PSGK-30, IB-RS-2 and BHK-21 were used. Primary and transplantable cell cultures for bioprobe production were grown on appropriate nutrient media under stationary conditions in 50-100 ml vials, 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 Hanks solution with 0.5% GLA and antibiotics according to the standard formulation. To remove microflora and ballast cell components, the suspension was treated with chloroform in a ratio of 1:10. After 30 minutes of incubation at 37 ° C, 5–10 ml of the supporting 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 minutes. The obtained virus-containing material was used for subsequent passages and studies in CSC and ELISA for the presence of a viral antigen, and a set of commercial type-specific serums and serums were used, which were stored in the Museum of Strains ARRIAH.

The results of the adaptation of the virus to various cell cultures are presented in table 3.

The data shown in table 3 indicate a good adaptive activity of the isolate to the used cell cultures.

The virus isolated using the above methods was studied in CSC with a set of diagnostics for all types of foot and mouth disease virus, vesicular stomatitis, vesicular exanthema and porcine vesicular disease in order to identify the type of accessory and control for cleanliness. The results of virus typing in CSCs are presented in table 4.

The results shown in table 4 indicate that the isolated virus belongs to type A. The isolate was assigned the copyright name of strain A (Georgia) 1999 / No. 1721 of FMD virus type A.

Example 2

Inactivated emulsion vaccine against foot and mouth disease type A is prepared from virus strain A (Georgia) No. 1999 / No. 1721 grown in a suspension culture of BHK-21 cells. Serum without serum is used as the supporting medium 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 a rate of 0.4-1.5 TCD ₅₀ per cell.

The cultivation of the virus is carried out at a temperature of 36-37 ° C. After 11-13 hours of incubation, live and dead cells are counted with trypan blue staining. If the number of living cells is 15-20%, then incubation continues 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 / ml. 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. The resulting antigen is monitored for avirulence, virus-specific protein content, 146S and 75S virus components, and sterility. The quantitative and qualitative content of viral raw materials is determined by turbidimetry. The required concentration of 146S and 75S components in the emulsion vaccine is obtained by concentration of the antigen by flow ultrafiltration or precipitation of the PEG-115 antigen.

BTU 0.5-2 ultrafilters are used to concentrate antigen by flow ultrafiltration.

Filtering is carried out under a pressure of 1.5 atm. The resulting antigen concentrate is stored at 4-6 ° C until used in the vaccine.

An emulsion vaccine is obtained by dispersing antigen concentrate and oil adjuvant in a ratio of 3: 7-1: 1, respectively, in colloidal mills. Of the oil adjuvants, the VNIIZH oil adjuvant can be used (14), as well as the Montanide ISA-70 or Montanide ISA-206 brand oil adjuvant manufactured by Seppic (France).

The result is an inactivated emulsion vaccine against foot and mouth disease type A, which is a milk-like liquid insoluble in water.

The resulting vaccine has an optimal component composition:

1) avirulent and purified antigenic material in the form of immunogenic 146S and 75S components of foot-and-mouth disease virus type A strain A (Georgia) 1999 / No. 1721 in an amount of at least 3.0 μg in a single vaccine dose of the vaccine;

2) maintenance medium in an amount of 299997.0-499997.0 μg in 1 ml of the vaccine dose, i.e. in an amount that provides a tolerant presentation of antigen in the body of an immunized animal;

3) an oil adjuvant in an amount of 500,000.0-700000.0 μg in 1 ml of the vaccine dose, i.e. in an amount that provides a tolerant presentation of antigen in the body of an immunized animal.

The vaccine readily dissolves from the injection site, does not cause abscesses, a general reaction in the form of a temperature increase and has a pronounced immunogenic activity for pigs in a vaccine dose of 2 ml, for cattle in a vaccine dose of 5 ml and for sheep in a vaccine dose of 1 ml 21 days after administration . The vaccine is administered intramuscularly to pigs, and cattle and sheep subcutaneously. Inoculated volume should contain at least 3 μg of 146S and 75S components of foot and mouth disease virus.

Example 3

Tests of the immunogenic activity of the vaccine inactivated emulsion against foot and mouth disease type A, made as described in example 2, and containing, mcg:

Avirulent and purified antigenic material as immunogenic 146S and 75S components of FMD virus type A strain A (Georgia) 1999 / No. 1721 7.0 Support environment 499,993.0 Oil adjuvant 500,000.0.

The immunogenic activity of this vaccine was tested on gilts weighing 35-40 kg. The research results are presented in table 5.

The vaccine was diluted with an oil adjuvant 3 and 9 times and was administered to the gilts intramuscularly at a dose of 2 ml.

Control infection was performed on day 21 after vaccination (DPV) with a homologous strain of foot and mouth disease virus.

The vaccinated gilts survived the challenge, and the unvaccinated became ill with a generalization of the process. The vaccine dose of the tested vaccine contained 15 PD ₅₀ .

The data presented in table 6 show that the number of virus-neutralizing antibodies in the blood serum of the gilts induced by the vaccine increased by 2 times to 10 DVPs, by 14-16 times to 21 DVPs, their number increased by 23-30 times by 55 DVPs, which testifies to the effectiveness of the tested vaccine.

Example 4

Tests of the immunogenic activity of the vaccine inactivated emulsion against foot and mouth disease type A, made as described in example 2, and containing, mcg:

Avirulent and purified antigenic material as immunogenic 146S and 75S components of FMD virus type A strain A (Georgia) 1999 / No. 1721 3.0 Support environment 499,997.0 Oil adjuvant 500,000.0.

The immunogenic activity of this vaccine was tested in pigs. Its IMD ₅₀ is 0.18 ml, i.e. the vaccine contains 11 PD ₅₀ in a vaccine dose of a vaccine of 2 ml. The research results are presented in table 7. The volume of the vaccine dose of the vaccine should contain at least 7 PD ₅₀ .

Example 5

Tests of the immunogenic activity of the vaccine inactivated emulsion against foot and mouth disease type A, made as described in example 2, and containing, mcg:

Avirulent and purified antigenic material as immunogenic 146S and 75S components of FMD virus type A strain A (Georgia) 1999 / No. 1721 3.0 Support environment 499,997.0 Oil adjuvant 500,000.0.

The immunogenic activity of the vaccine was tested on pigs weighing 35-50 kg. The research results are presented in table 8. The vaccine dose of the vaccine with a volume of 1 ml contains 7.1 PD ₅₀ for pigs.

Thus, the above information indicates the fulfillment of the following set of conditions when using the proposed vaccine:

- vaccine inactivated emulsion against foot and mouth disease type A, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form described in the independent claim, the possibility of its implementation using the means and methods provided in the application or known prior to the priority date has been confirmed;

- inactivated emulsion vaccine against foot and mouth disease type A made from strain A (Georgia) 1999 / No. 1721 in accordance with the invention, has high immunogenic activity and is able to provide effective protection of susceptible animals against epizootic virus of foot and mouth disease type A circulating in the countries of the Caucasus, Central Asia, Middle and Middle East.

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Inactivated emulsion vaccine against foot and mouth disease type A".

1. Bojko A.A. Déclaration sur l'apparition en URSS d'un type virus aphteux différent des souches de type A antérieurement étudiéés dans le Pays. BOIE, 1965, 64, V.2. - P.1075-1077.

2. De Mello P.A. The use of oil adjuvanted foot-and-mouth disease vaccine in endemic areas. - Bol. CPFA, 1982, 45-46, 33-42.

3. Bahnemann H.G. Large scale application of oil adjuvanted foot-and-mouth disease vaccine. In .: Europ. Comm. Control FMD. Rio de Janeiro, Brasil, 5-10 Oct. 1985, Rome 1986, 132-135.

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

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

6. Syurin V.N., Samuilenko A.Ya., Soloviev B.V., Fomina N.V. Viral diseases of animals. - M .: VNITIBP, 1998 .-- S.532-548.

7. French patent No. 2088038; A 61 K 27/00, C 12 K 5/00; 01/07/1972

8. Auth. testimonial. USSR No. 411131, C 12 K 5/00, 01/15/1974

9. Instructions for the manufacture and control of a monovalent emulsion vaccine against foot and mouth disease type A, type O, type C, type Asia-1 (from a virus grown in BHK-21 cells). The GUV of the State Commission of the Council of Ministers of the USSR for Food and Purchases was approved on January 24, 1991.

10. RF patent No. 2143921, A 61 K 39/135, C 07 K 14/09, C 12 N 7/00, 7/04; January 10, 2000 (prototype).

11. RF patent No. 594771, A 61 K 39/12, 07/07/93.

12. RF patent No. 2054039; C 12 N 7/02, A 61 K 39/35; 02/10/1996.

13. Auth. testimonial. USSR №784335, C 12 Q 1/02, 03/20/2000

14. RF patent No. 2108111; A 61 K 39/39 // A 61 K 39/00, 39/102, 39/12, 39/135; 04/10/1998

Table 1
Antigenic spectrum of strain A (Georgia) 1999 / No. 1721 of type A foot and mouth disease virus (according to CSC) n = 3 Compare strains Indicators r ₁ r ₂ R% A (Georgia) 1999 / No.1721 and A / Iran / 96 0.28 0.9 49 A (Georgia) 1999 / No.1721 and A / Kyrgyzstan / 93 0.1 0.24 fifteen A (Georgia) 1999 / No.1721 and A / Armenia / 98 0.32 0.12 twenty A (Georgia) 1999 / No.1721 and A / Turkey / 97 0.23 1,0 47 A (Georgia) 1999/1721 and A ₂₂ No. 550 0.1 0.32 eighteen A (Georgia) 1999 No. 1721 and A / Turkey / 99 0.47 0.69 57

table 2
Antigenic relationship of strain A (Georgia) 1999 / No.1721 with known strains of FMD virus type A (according to PH) n = 3 Compare strains Indicators r ₁ r ₂ R% A (Georgia) 1999 / No.1721 and A ₂₂ No.550 0.38 0.4 41 A (Georgia) 1999 / No.1721 and A / Turkey / 97 0.44 0.58 47

Table 3
Biological properties of strain A (Georgia) 1999 / No. 1721 of FMD virus type A Cell culture Time has shown. CPD (hour) Adaptation Passages Adaptive Virus Characterization Activity in RSK Ig titre TCD ₅₀ / ml Joint venture eighteen 3 whole 10 ^6.0-6.5 PSGK-30 16 2 1: 2 10 ^6.0-7.0 VNK-21 18-20 3 1: 2-1: 4 10 ^6.5 - ^7.5 IB-RS-2 eighteen one 1: 2-1: 4 10 ^6.0-7.0

Table 4
Typing of a 33% suspension of foot-and-mouth disease aphthous material (examination No. 1721) Hyperim. serum in the working title Antigen (No. 1721) in dilutions whole 1: 2 1: 4 1: 8 1:12 1:24 Antigen free A ₂₂ No. 550 four 3 - - - - - A / Armenia / 98 four four 2 - - - - O ₁ NQ194 - - - - - - - C ₁ NQ564 - - - - - - - Asia-1 №48 - - - - - - - CAT-1 No. 96 - - - - - - - CAT-2 K183 / 74 - - - - - - - CAT-3 Bech - - - - - - - Serum free - - - - - - - Note: the percentage of hemolysis delay is expressed in crosses:
4-100% delay;
3-75% delay;
2-50% delay;
1-25% delay;
- negative reaction (complete hemolysis).

Table 5
The results of the test FMD inactivated emulsion vaccine prepared on the basis of strain A (Georgia) 1999 / No. 1721 The number of vaccinated alive Grafting Dose (ml) Vaccine dilution The content of immunogenic components in the dilution (mcg) Control infection results at 21 DPV Imd ₅₀ primary aphthae generalization four 2 1: 3 2,33 4/4 0/4 ≤0.13 ml four 2 1: 9 0.78 4/4 0/4 The control - - - 2/2 2/2 -

Claims (12)

1. Inactivated emulsion vaccine against foot and mouth disease type A, containing the active substance and target additives, characterized in that as the active substance it contains avirulent and purified antigenic material from the virus strain Aphtae epizooticae, fam. Picornaviridae, genus Aphtovirus, serotype A, collection of FGU VGNKI A (Georgia) 1999 / No. 1721-DEPT, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from a strain A (Georgia) homologous to the pathogen of infection (Georgia) 1999 / No. 1721-DEPT, obtained in a sensitive biological system and consisting of a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from a strain A (Georgia) homologous to the pathogen of infection (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a transplanted cell culture of animal origin and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from a strain A (Georgia) homologous to the pathogen of infection (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing predominantly 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 a strain A (Georgia) homologous to the pathogen of infection (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A, in an amount of at least 3.0 μg in the vaccine dose of the drug for each animal species. 6. The vaccine according to claim 1, characterized in that as the target additive, it contains a support medium. 7. The vaccine according to claim 6, characterized in that it contains a support medium in an amount providing a tolerant presentation of the antigen in the body of the immunized animal. 8. The vaccine according to claim 1, characterized in that it contains an oil adjuvant as a target additive. 9. The vaccine according to claim 1, characterized in that, as a target additive, it contains an oil adjuvant in an amount providing a tolerant presentation of the antigen in the body of the immunized animal. 10. The vaccine according to any one of claims 1 to 9, characterized in that it contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A, supporting medium and oil adjuvant in the ratio, μg in vaccine dose for pigs: Antigenic material Not less than 3.0 Support environment 599997.0-999997.0 Oil adjuvant 1000000.0-1400000.0 11. The vaccine according to any one of claims 1 to 9, characterized in that it contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A, supporting medium and oil adjuvant in the ratio, μg in vaccine dose for cattle: Antigenic material Not less than 3.0 Support environment 1499997.0-2499997.0 Oil adjuvant 2500000.0-3500000.0 12. The vaccine according to any one of claims 1 to 9, characterized in that it contains avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A, supporting medium and oil adjuvant in the ratio, μg in vaccine dose for sheep: Antigenic material Not less than 3.0 Support environment 299997.0-499997.0 Oil adjuvant 500000.0-700000.0