RU2294760C2 - Sorbed inactivated 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-DEP obtained in passaged cell culture VNK-21 being the suspension that contains, predominantly, 146S and 75S immunogenic components of foot-and-mouth disease virus, adjuvants aluminum hydroxide with saponin and maintenance medium in efficient ratios. The vaccine is of high immunogenicity and is capable to provide efficient protection against homologous infection agent circulating in Transcaucasian countries and those of Central Asia, Near and Middle East.

EFFECT: higher efficiency.

10 cl, 1 dwg, 4 ex, 10 tbl

Description

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

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. Currently in the Russian Federation and the CIS countries for the immunization of cattle and small cattle against foot and mouth disease, inactivated sorbed vaccines are used, as a rule, and inactivated emulsion vaccines are used for immunization of pigs (1).

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 subject to the requirements of the stability of the virus during the cleaning process from tissue components and concentration, as well as the preservation of the virus during inactivation and long-term storage (2).

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

It is believed that the main cause of antigenic variation is a change in the amino acid sequence in the polypeptides of the viral proteins that make up the virion capsid. In practice, such antigenic changes can be expressed both in insignificant differences between the strains, captured only using subtle molecular analysis methods, and in the appearance of completely different strains requiring the use of new means of specific prophylaxis of the disease caused by these strains (2,3).

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 Virus and Other Animal Pathogens of FSI ARRIAH (1-4).

There is a known vaccine inactivated adsorbed against type A foot and mouth disease, containing the active substance in the form of avirulent and purified antigenic material from strain A ₂₂ No. 550, homologous to the pathogen, obtained in a sensitive biological system, and targeted additives in the form of an adjuvant and support medium in an effective ratio (5) .

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

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

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

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

The closest to the invention according to the set of essential features is a vaccine inactivated adsorbed against foot and mouth disease type A, containing the active substance in the form of avirulent and purified antigenic material homologous to the pathogen of strain A No. 1707 "Armenia-98-DEPT" obtained in a sensitive biological system, and target additives in the form of GOA adjuvants with saponin and a supporting medium in the ratio, μg:

Antigenic material not less 3.0 GOA 1132.0-2108.0 Saponin 750.0 Support environment Up to 1,000,000.0 (5)

Strain A ₂₂ No. 1707 “Armenia-98-DEP” was isolated in 1998 in the Republic of Armenia and is used in the Russian Federation as an industrial one in the manufacture of specific prophylaxis and diagnostics that are used throughout Russia and the CIS countries.

A suspension culture of BHK-21 cells is used as a sensitive biological system, and Earle's solution without serum with the addition of FGMS, GBKS, and antibiotics at pH 7.4-7.6 is used as a supporting medium.

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

Avirulent and purified antigenic material from strain A No. 1707 “Armenia-98-DEPT” is a suspension mainly of 146S and 75S immunogenic components of foot and mouth disease virus. As adjuvants, GOA with saponin is used.

The main disadvantage of known vaccines, including the prototype vaccine, is their insufficient immunogenic activity. They do 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 the development of a vaccine for FMD inactivated adsorbed, which creates effective protection of susceptible animals against the epizootic virus of foot and mouth disease type A circulating in the countries of the Caucasus, Central Asia, the Middle and Middle East.

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

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

The proposed vaccine contains in 1 ml of the drug: the active substance in the form of avirulent and purified antigenic material from a strain A (Georgia) homologous to the pathogen infection (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a suspension culture of BHK-21 cells, in an amount of at least 3.0 μg and target additives: GOA, preferably in an amount of 1132.0-2108.0 μg, saponin, preferably in an amount of 750.0 μg and a support medium in an amount of up to 1,000,000.0 μg.

The original virus for obtaining strain A (Georgia) 1999 / No. 1721-DEPT was isolated in 1999 in the Republic of Georgia. 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 Earle's serum-free solution with the addition of FGMS, GBKS, and antibiotics at pH 7.4-7.6 is used as a supporting medium.

For inactivation of the virus, 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 (7).

The resulting antigen is purified from ballast impurities using PHMG, which is added to the suspension to a concentration of 0.005-0.007% (8).

Avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721-DEPT 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 (9).

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 proposed vaccine, comprising at least 3 μg in 1 ml of the finished product, is obtained by adding the calculated amount of GOA adjuvant-sorbent to avirulent and purified antigenic material. The optimal GOA content in 1 ml of the finished product in the range from 1132.0 μg to 2108.0 μg.

An additional 10% aqueous solution of saponin is added to the resulting concentrate to a final concentration of 0.075%, which corresponds to 750.0 μg of its content in 1 ml of the finished preparation.

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

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

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

2. The active substance in the form of avirulent and purified antigenic material from a strain A homologous to the pathogen of infection (Georgia) 1999 / No. 1721-DEPT in an effective amount.

3. Targeted supplements.

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

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

2. active substance;

3. targeted supplements.

Compared to the prototype vaccine, an essential distinguishing feature of the proposed vaccine is that it contains an avirulent and purified antigenic material from strain A (Georgia) 1999 / No. 1721-DEPT "of type A foot and mouth disease virus in an effective 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 pathogen of strain A (Georgia) 1999 / No. 1721-DEPT, 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 a strain A (Georgia) homologous to the pathogen (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a transplanted cell culture of animal origin and consisting of a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

3. Avirulent and purified antigenic material from strain A (Georgia), homologous to the pathogen (Georgia) 1999 / No. 1721-DEPT, obtained preferably in the transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus A.

4. Avirulent and purified antigenic material from strain A (Georgia), homologous to the causative agent of the infection (Georgia) 1999 / No. 1721-DEPT, 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 in an amount not less than 3.0 mcg in 1 ml of the finished product.

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

6. GOA, preferably in an amount of 1132.0-2108.0 μg in 1 ml of the finished product.

7. The vaccine contains adjuvant saponin as a targeted supplement.

8. Saponin, preferably in an amount of 750.0 μg in 1 ml of the finished product.

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

10. Maintenance medium in an amount up to 1,000,000.0 mcg in 1 ml of the finished product.

11. Avirulent and purified antigenic material from strain A (Georgia) homologous to the pathogen (Georgia) 1999 / No. 1721-DEPT, 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, GOA , saponin and support medium in the ratio, mcg:

Antigenic material not less 3.0 GOA 1132.0-2108.0 Saponin 750.0 Support environment Up to 1,000,000.0

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 the antigenic material from strain A (Georgia) 1999 / No. 1721-DEP of the FMD virus serotype A, which has high biological, antigenic and immunogenic activity in its native form, is introduced into the composition of the proposed vaccine. after inactivation and providing a FMD vaccine adsorbed inactivated, which creates effective protection of susceptible animals against foot and mouth disease virus serotype A, causing outbreaks of I have in recent years in the Caucasus, Central Asia and the Middle East.

Strain A (Georgia) 1999 / No. 1721-DEPT is a new, previously unknown. The original virus for obtaining strain A (Georgia) 1999 / No. 1721-DEPT was isolated in 1999 from sick cows in the private sector of the village of Ude, Adigensky District of the Republic of Georgia. The production strain is obtained from this isolate by successive passages 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-DEPT, serotype A.

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

Morphological properties

Strain A (Georgia) 1999 / No. 1721-DEPT 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 (Georgia) 1999 / No. 1721-DEPT of 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-DEPT 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-DEPT of FMD virus type A differs from both production and previously isolated epizootic strains.

According to the antigenic properties, the new strain A (Georgia) 1999 / No.1721-DEPT of FMD virus of type A also differs from the previously studied production strain A # 1707 / Armenia / 98-DEPT. Bilateral relationship (R) in CSCs 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-DEPT, 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 NO1) of strain A (Georgia) 1999 / No.1721 was determined by nucleotide sequencing and the amino acid sequence of the VP1 protein (SEQ ID NO2) 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-DEPT of FMD virus type A 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-DEPT is reproduced in monolayer cultures of primary trypsinized culture of pig kidney cells (SP), transplanted cultures of kidney cells of the Siberian mountain ibex (PSGK-30), IB-RS-2, VNK-21 and for 18 -24 hours of incubation accumulates up to 6.0-8.0 Ig TCD ₅₀ / ml. 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, up to 6.0-8.0 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 - DEPT 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. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 6 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-DEPT of FMD virus type A 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-DEPT according to 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 invention is explained on a dendrogram that reflects the phylogenetic relationships of strain A (Georgia) 1999 / No. 1721-DEPH of the FMD virus of serological type A with epizootic and vaccine strains of FMD virus of type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

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

Example 1

Strain A (Georgia) 1999 / No. 1721-DEPT, 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 trypsinized 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 the 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 resulting 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 sera and serum stored in the Museum of Strains ARRIAH were used.

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

The data shown in table 3, indicate good adaptive activity of strain A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A 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, the results indicate that the selected virus belongs to type A.

Example 2

Inactivated adsorbate vaccine against foot and mouth disease type A is prepared from virus strain A (Georgia) No. 1999 / No. 1721-DEPT grown in a suspension cell culture of BHK-21. 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 and 146S and 75S virus components and sterility. The required concentration of 146S and 75S components in 1 ml of adsorbed vaccine is obtained by concentration of GOA antigen.

The calculated GOA volume of 3% concentration is added to the cooled antigen suspension with the stirrer operating. Stirring is carried out for 30 minutes. After sedimentation of GOA, the calculated volume of the remaining suspension is drained. The final concentration of GOA should be in the range of 1.62 ± 0.488 P <0.01 mg / ml n = 10, and the concentration of 146S and 75S components of FMD virus is at least 3.0 μg / ml of the finished product. Then, an additional 10% saponin solution is added to the suspension to a final concentration of 0.075%, which corresponds to 750.0 μg saponin in 1 ml of the finished vaccine. The resulting vaccine is packaged in glass bottles and its sterility is monitored in accordance with GOST 28085-89.

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 ml, and then subcutaneously at a dose of 10.0 ml. Observation of the clinical condition of the animals is carried out for 10 days. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

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

The optimal component composition of the obtained adsorbate vaccine against foot and mouth disease type A are shown in table 5.

Tables 6, 7 and 8 show the results of the cultivation of strains A (Georgia) 1999 / No. 1721-DEPT, A No. 1707 “Armenia-98-DEPT” and A22 No. 550 of FMD virus type A, from which it follows that the cultural properties of the listed strains significantly differ in two studied parameters:

1) strains A (Georgia) 1999 / No. 1721-DEPT and A No. 1707 “Armenia-98-DEP” have a longer reproduction period (15-16 hours) compared with strain A ₂₂ No. 550 (12.5 hours);

2) strains A (Georgia) 1999 / No. 1721-DEPT and A No. 1707 “Armenia-98-DEP” give a lower percentage of the output of immunogenic components (61.5 and 58.1%, respectively) compared with strain A ₂₂ No. 550 (89.9%).

Table 9 shows the results of studies on the effect of inactivation and purification of antigenic material using AEI and PHMG on the immunogenic (146S and 75S) components of foot-and-mouth disease virus of strains A (Georgia) 1999 / No. 1721-DEPT and A No. 1707 “Armenia-98-DEP ".

The data presented in table 9 indicate that the immunogenic components of the foot and mouth disease virus of strain A (Georgia) 1999 / No. 1721-DEPT are not inferior in resistance to inactivation and purification in the conditions of production of foot-and-mouth disease virus of strain A No. 1707 “Armenia-98-DEPT”. Especially important is the fact that in the process of inactivation and purification of the virus from strain A (Georgia) 1999 / No. 1721-DEPT, there were no changes in the number of 146S and 75S components obtained during virus reproduction.

Example 3

Tests of an adsorbate vaccine against foot and mouth disease type A, made as described in example 2, and containing, mcg:

Avirulent and purified strain antigenic material A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A 3.0 GOA 1132.0 Saponin 750.0 Support environment Up to 1,000,000.0

The virulence and safety of the 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 ml, and then subcutaneously at a dose of 10.0 ml. Observation of the clinical condition of the animals was carried out for 10 days.

At the end of the control for avirulence and safety, the vaccine was tested for immunogenic activity. The test was carried out on 5 heads of cattle. The adsorbate vaccine was administered subcutaneously at a dose of 2.0 ml. The research results are presented in table 10.

The data presented in table 10 showed that all 5 heads of cattle were protected from the generalization of the process for 21 days after vaccination. The level of humoral immunity in vaccinated animals was 5.15 ± 0.19 log ₂ .

Example 4

Tests of an adsorbate vaccine against foot and mouth disease type A, made as described in example 2, and containing, mcg:

Avirulent and purified strain antigenic material A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A 3.0 GOA 1740.0 Support environment Up to 1,000,000.0

The virulence and safety of the obtained vaccine was tested on 5 heads of cattle. The drug was administered to each animal under the mucous membrane of the tongue at a dose of 2.0 ml, and then subcutaneously at a dose of 10.0 ml. Observation of the clinical condition of the animals was carried out for 10 days.

At the end of the control for avirulence and safety, the vaccine was tested for immunogenic activity in cattle. The test was performed on cattle, ImD _{50 of the} drug was 0.15 ml, i.e. one ml of the vaccine contained 6.07 PD ₅₀ .

For comparison, the production series of the adsorbate vaccine from strain A ₂₂ No. 550 had an ImD ₅₀ for cattle of 0.22 ± 0.012 ml P <0.001, i.e. in 1 ml the vaccine contained 4.55 PD ₅₀ .

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

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

- for the proposed invention in the form as 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 is confirmed;

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

1. Bojko A.A. Declaration sur l'apparition en URSS d'un type virus aphteux different des souches de type A anterieurement etudiees dans le Pays. BOIE, 1965, 64, V.2. - P.1075-1077.

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

3. 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.

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

5. Temporary instructions for the manufacture and control of FMD concentrated aluminum hydroxide formol vaccine from lapinized virus A ₂₂ . Approved by the GUV of the USSR on March 25, 1971

6. RF patent No. 2143921, A 61 K 39/135, C 07 K 14/09, C 12 N 7/00, 7/04; 01/10/2000 (prototype).

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

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

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

Table 1
Antigenic spectrum of strain A (Georgia) 1999 / No. 1721-DEPT of FMD virus type A (according to CSC) n = 3 Compare strains Indicators r1 r2 R% A (Georgia) 1999 / No.1721-DEP and A / Iran / 96 0.28 0.9 49 A (Georgia) 1999 / No.1721-DEP and A / Kyrgyzstan / 93 0.1 0.24 fifteen A (Georgia) 1999 / No.1721-DEP and A / Armenia / 98 0.32 0.12 twenty A (Georgia) 1999 / No.1721-DEP and A / Turkey / 97 0.23 1,0 47 A (Georgia) 1999/1721 -DEP and A ₂₂ No. 550 0.1 0.32 eighteen A (Georgia) 1999 No. 1721-DEP and A / Turkey / 99 0.47 0.69 57

table 2
Antigenic relationship of strain A (Georgia) 1999 / No. 1721-DEPT with known strains of FMD virus type A (according to pH) n = 3 Compare strains Indicators r1 r2 R% A (Georgia) 1999 / No.1721-DEP and A ₂₂ No.550 0.38 0.4 41 A (Georgia) 1999 / No.1721-DEP and A Turkey / 97 0.44 0.58 47

Table 3
Biological properties of strain A (Georgia) 1999 / No. 1721-DEPT 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 ^6.07.0

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 No. 194 - - - - - - - C ₁ No. 564 - - - - - - - Asia-1 №48 - - - - - - - CAT-1 No. 96 - - - - - - - CAT-2 K183 / 74 - - - - - - - CAT-3 Been 1/65 - - - - - - - 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 optimal formulation of FMD inactivated adsorbate vaccine from FMD virus type A, strain A (Georgia) 1999 / No. 1721-DEPT (μg in 1 ml of the finished vaccine) Ingredients Minimum Average Maximum Avirulent and purified antigenic material in the form of immunogenic (146S and 75S) components of FMD virus type A,
strain A (Georgia) 1999 / No. 1721-DEPT





3.0





> 3.0





> 3.0 GOA 1132.0 1620.0 2108,0 Saponin 750.0 750.0 750.0 Support environment Up to 1,000,000.0 Up to 1,000,000.0 Up to 1,000,000.0

Table 8
Cultivation of FMD virus type A, strain A ₂₂ No. 550, in a suspension of cells of BHK-21 №№ Cell concentration (million / ml) Dose of infection (TCD ₅₀ / cell) Duration of reproduction, (hours) Virus titer (Ig TCD ₅₀ / ml) VSB (m kg / ml) 146S + 75S components (mcg / ml) % yield 146S + 75S one. 2,5 1.5 13 8.00 2.48 2.19 88.3 2. 2.6 1,0 12 7.25 1.17 1,04 88.8 3. 2.7 1,0 eleven 7.75 2.00 1.83 91.6 four. 2,4 0.8 fourteen 8.00 1.78 1.48 89.3 5. 2,4 0.5 12 7.50 1.34 1.26 94.3 6. 3.0 0.05 13 7.25 2.06 1.80 87.2 M + m 2.60 ± 0.09 0.81 + 0.20 12.5 ± 0.43 7.62 + 0.14 1.80 ± 0.20 1,60 + 0,17 89.9 + 1.1 p <0.001 p <0,025 p <0.001 p <0.001 p <0.001 p <0.001 P <0.001

Table 9
Comparative data on the resistance of the strain of foot and mouth disease virus A No. 1707 “Armenia-98-DEPT” and strain A (Georgia) 1999 / No. 1721-DEPT to inactivate AEEI and sterilizing purification of PHMG №№ A No. 1707 “Armenia-98-DEP” A (Georgia) 1999 / No. 1721-DEP after cultivation after inactivation and cleaning after cultivation after inactivation and cleaning VSB mcg / ml 146S + 75S mcg / ml VSB mcg / ml 146S + 75S mcg / ml VSB mcg / ml 146S + 75S mcg / ml VSB mcg / ml 146S + 75S mcg / ml one. 2.54 1.76 2.72 2.05 2,38 1.27 2.14 1.18 2. 2,31 1.19 1.89 1.27 2.71 1.83 2.27 1,61 3. 2,34 2.03 2,56 1.86 2.93 1.72 2,50 1.48 four. 1.17 0.94 1.25 0.75 3.12 1.80 2.69 1.93 5. 2.06 1.34 2.15 1.44 2.09 ± 0.31 1.48 ± 0.25 2.10 + 0.34 1.48 ± 0.30 2.64 + 0.19 1.59 ± 0.2 2.35 ± 0.11 1.53 + 0.12 p <0.001 p <0.01 p <0.001 p <0,025 p <0.001 p <0.001 p <0.001 p <0.005

Table 10
Test results of type A adsorbate vaccine against foot and mouth disease from strain A (Georgia) 1999 / No. 1721-DEP The number of vaccinated alive Grafting Dose (ml) Quantity of 146S + 75S components in a graft dose The results of the control infection at 21 days after vaccination The amount of VNA (log2) in serum at 21 days after vaccination primary aphthae generalization 5 2 6.0 4/5 0/5 5.15 ± 0.19 p <0.001 Control-2 - - 2/2 2/2

Claims (11)

1. The vaccine is inactivated adsorbed 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 strain of the virus 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 transplantable cell culture of BHK-21 and which is a suspension containing predominantly 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 infection pathogen (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 not less than 3.0 μg in 1 ml of the finished product. 5. The vaccine according to claim 1, characterized in that as the target additive it contains an adjuvant-sorbent aluminum hydroxide (GOA). 6. The vaccine according to claim 5, characterized in that it contains GOA, preferably in an amount of 1132.0-2108.0 μg in 1 ml of the finished product. 7. The vaccine according to claim 1, characterized in that as the target additive it contains an adjuvant saponin. 8. The vaccine according to claim 7, characterized in that it contains an adjuvant saponin, preferably in an amount of 750.0 μg in 1 ml of the finished product. 9. The vaccine according to claim 1, characterized in that, as a target additive, it contains a support medium. 10. The vaccine according to claim 9, characterized in that it contains a support medium in an amount of up to 1,000,000.0 μg in 1 ml of the finished product. 11. The vaccine according to any one of claims 1 to 10, characterized in that it contains avirulent and purified antigenic material from a strain A (Georgia) homologous to the pathogen infection (Georgia) 1999 / No. 1721-DEPT, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease virus type A, GOA, saponin and supporting medium in the ratio, μg: Antigenic material Not less than 3.0 GOA 132.0-2108.0 Saponin 50,0 Support environment Up to 1,000,000.0

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