Sorbed inactivated vaccine against foot-and-mouth disease of type a

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

 

The invention relates to the field of veterinary Virology and biotechnology and can be used in the development and manufacture of vaccines inactivated adsorbed against FMD type A.

Foot and mouth disease is an acute contagious viral disease of cloven-hoofed animals. It is characterized by a tendency to widespread epizootic. The disease is accompanied by large losses of milk, meat and other animal products, it is difficult for commercial transactions and economic activities. For sustainable well-being of the country for FMD in the Russian Federation implemented a system of measures, the priority of which is the prevention of introduction of FMD virus into the territory, and in areas of high risk vaccination. Currently in the Russian Federation and CIS countries for immunization of large and small cattle against FMD apply, as a rule, inactivated adsorbed and for immunization of pigs - inactivated emulsion vaccine (1).

Technology of production of FMD vaccine of inactivated virus production begins with the selection of strains based on epidemiological analysis of the dynamics of FMD in the country and neighboring States. When creating products for specific prophylaxis of IP is result appropriate types of FMD virus and select strains with a broad antigenic spectrum within the type with a pronounced cross-immunogenicity. Strain with a wide range of immunogenicity and satisfying the requirements of the region selected with the help of his trials in the reaction cross-protection, or more frequently in the reaction cross-neutralization. Generally, as the production strain is used, the population of the virus, which in conjunction with the system and the conditions of industrial cultivation provides guaranteed and high accumulation of 146S and 75S components of the virus and getting immunogenic vaccine.

In addition, the production strain demands the stability of the virus in the process of purification from tissue components and concentration, as well as the preservation of virus inactivation and its long-term storage (2).

A well-known feature of the causative agent of foot and mouth disease is antigenic variability of strains within the same serotype occurring in different time periods in different areas, depending on the species composition of the susceptible animal population, 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 of the polypeptides of viral proteins that form the capsid of the virion. Practically such antigenic change can be expressed as in minor differences between strains, captured only by OSU subtle methods of molecular analysis, and in the emergence of a completely different strains, requiring the use of new means of specific prophylaxis of disease caused by these strains (2,3).

Known strains of FMD virus type a, isolated on the territory of the USSR and used as production in the manufacture of inactivated FMD vaccines. These include strain And7No. 103, dedicated in 1962 in the Kuibyshev region; strain AndtNo. 2, dedicated in 1965 in the Tajik SSR; strain And No. 717/73 allocated in 1973 in the Stavropol region. After the elimination of FMD caused similar antigenically strains of the virus, they have been discontinued and are currently supported only in the Collection of epizootic strains of FMD virus and other pathogens of animals FGI ARRIAH (1-4).

Known vaccine inactivated adsorbed against FMD type a, containing the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain A22No. 550, obtained in a sensitive biological system, and targeted supplements adjuvant and supportive environment in effective relation (5).

Strain And22No. 550 of FMD virus isolated in 1964 in Azerbaijan and is used in the Russian Federation as production in the manufacture of tools specification is specific prevention and diagnosis, apply throughout the territory of Russia and CIS countries.

As a sensitive biological systems use newborn rabbits, and as a supportive environment - phosphate-buffer solution.

For cleaning vaccinated suspension from the ballast impurities using chloroform in a 2% concentration and subsequent centrifugation.

For virus inactivation using formaldehyde, and of the adjuvant is aluminium hydroxide (GOA) with saponin.

Closest to the present invention, the set of essential characteristics is inactivated vaccine adsorbed against FMD type a, containing the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain And No. 1707 "Armenia-98-DEPT", received in a sensitive biological system, and targeted supplements adjuvants GOA with saponin and supportive environment in the ratio, µg:

Antigenic material is not less than3,0
GOA1132,0-2108,0
Saponin750,0
Support the environmentTo 1000000,0 (5)

Strain A22No. 1707 "Armenia-98-DEPT" selected in 1998 in the Republic of Armenia and is used in the Russian Federation in the quality of the ve production in the manufacture of means of specific prophylaxis and diagnostics, apply throughout the territory of Russia and CIS countries.

As a sensitive biological system using suspension culture cells KSS-21, and as a supportive environment - solution Earl without serum with the addition of FGMS, GBX and antibiotics at pH 7.4 and 7.6.

For cleaning vaccinated suspension from the ballast impurities using polyhexamethylene guanidine (phmg), and for inactivation of virus - aminoethylethanolamine (AAAI). To neutralize AEEI in the slurry, add sodium thiosulfate.

Avirulent and purified antigenic material from strain And No. 1707 "Armenia-98-DEPT" is a suspension mainly of 146S and 75S immunogenic components of FMD virus. As adjuvants use GOA with saponin.

The main disadvantage of vaccines, including vaccines of the prototype, is their lack of immunogenic activity. They do not provide reliable protection of susceptible animals from the epidemic of FMD virus type a, circulating in the Caucasus, Central Asia, Middle East.

In the task of creating the present invention was the development of a vaccine FMD inactivated adsorbed creating effective protection of susceptible animals against the epidemic of FMD virus type a, circulating in the Caucasus, the center of InEU Asia, Middle East.

The technical result from use of the present invention is to expand the Arsenal of vaccines inactivated adsorbed against FMD type A.

This technical result is achieved by the creation of a vaccine, inactivated adsorbed against FMD type a, characterized by the following set of features.

The proposed vaccine contains 1 ml of the preparation: the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT received preferably in suspension culture cells KSS-21, in the amount of not less than 3,0 µg and targeted supplements: GOA preferably in the amount of 1132,0-2108,0 mcg, saponin, preferably in the amount of 750,0 mcg and supportive environment for up to 1000000,0 mcg.

The original virus to obtain strain And (Georgia) 1999/No. 1721-DEPT allocated in 1999 in the Republic of Georgia. The strain obtained by multiple serial passages in susceptible hetero - and homologous cell cultures. Strain adapted to particination cells pork kidney and transplantable cell cultures KSS-21, IBRS-2 and PSGC-30.

For the manufacture of vaccines as a sensitive biological systems use preferably the suspension culture cells VNC, and as a supportive environment solution Earl without serum with the addition of FGMS, GBX and antibiotics at pH 7.4 and 7.6.

For virus inactivation using AAAI, which is added in vaccinated suspension to a concentration of 0.025 to 0.05%. After inactivation AAAI neutralized by introducing a suspension of sodium thiosulfate (7).

The resulting antigen purified from ballast impurities using pgmg, which is introduced into 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 FMD virus.

Quantitative and qualitative viral content of raw materials is determined by the method of turbidimetry (9).

For the preparation of vaccines using viral material, containing 1 ml of at least 0.5 µg 146S and 75S immunogenic components of FMD virus.

The necessary concentration of 146S and 75S immunogenic components of FMD virus in the vaccine, which is not less than 3 µg in 1 ml of the finished product, obtained by adding in avirulent and purified antigenic material estimated quantity of adjuvant-sorbent GOA. The optimum content of GOA in 1 ml of drug in the range from 1132,0 μg to 2108,0 mcg.

To the obtained concentrate, add an additional 10% aqueous rest the R saponin to a final concentration of 0.075%, which corresponds 750,0 µg of its content in 1 ml of the finished product.

The resulting vaccine is a liquid light yellow color with loose sediment sorbent, which is formed on the bottom of the vial during storage and can be easily broken in a homogeneous suspension with shaking.

The present invention includes the following set of essential features that provide technical result, in all cases, which sought legal protection.

1. The inactivated vaccine adsorbed against FMD type A.

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

3. Additives target.

Features of the invention, characterizing the proposed vaccine that matches the characteristics of the prototype, including a generic term that reflects the assignment are:

1. the inactivated vaccine adsorbed against FMD type A;

2. active substance;

3. additives target.

Compared with the vaccine prototype a distinctive feature of the proposed vaccine is that as the active substances it contains avirulent and purified antigenic material from strain A (Georgia) 1999/No. 1721-DEPT" of FMD virus type a in an effective amount.

1. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT received in a sensitive biological system representing a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A.

2. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell cultures of animal origin and represents a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A.

3. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A.

4. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type a in the amount of not less than 3,0 the kg in 1 ml of the finished product.

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

6. GOA preferably in the amount of 1132,0-2108,0 µg in 1 ml of the finished product.

7. As the target additive vaccine contains an adjuvant saponin.

8. Saponin, preferably in the amount of 750,0 µg in 1 ml of the finished product.

9. As the target additive vaccine contains supporting environment.

10. Supportive environment up 1000000,0 µg in 1 ml of the finished product.

11. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type a, GOA, saponin and supportive environment in the ratio, µg:

Antigenic material is not less than3,0
GOA1132,0-2108,0
Saponin750,0
Support the environmentTo 1000000,0

We offer the vaccine has a high immunogenic activity and provides reliable protection against FMD virus serotype And circulating in the Caucasus, Central Asia, Middle East.

The technical achievement is about result from use of the invention is achieved by that the composition of the proposed vaccine introduced as the active substance antigenic material from strain A (Georgia) 1999/No. 1721-DEPT of FMDV serotype A, which has high biological, antigenic and immunogenic activity in the native form and after inactivation and providing FMD vaccine, inactivated adsorbed creating effective protection of susceptible animals against FMD virus serotype And causing outbreaks in recent years in the Caucasus, Central Asia, Middle East.

Strain a (Georgia) 1999/No. 1721-DEPT is a new, previously unknown. The original virus to obtain strain And (Georgia) 1999/No. 1721-DEPT isolated in 1999 from sick cows in the private sector of the village Ude Adigeni district of the Republic of Georgia. The production strain derived from this isolate by serial passages in susceptible hetero - and homologous cell culture.

The resulting strain deposited on 19 August 2003, in the Russian state collection of strains of microorganisms used in veterinary medicine and animal production, Federal state institution "all-Russian state research Institute for control, standardisation and certification of veterinary preparations Center quality of veterinary drugs and feed the (FSI VGNKI) under registration number - production, cultural strain of FMD virus (Georgia) 1999/No. 1721-DEPT, serotype A.

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

Morphological properties

Strain a (Georgia) 1999/No. 1721-DEPT belongs to the family Picornaviridae, genus Aphtovirus, serotype a and has morphological features that are specific to the causative agent of foot and mouth disease: a form of icosahedral virion, size 23-25 nm. The virion consists of a molecule of RNA enclosed in a protein shell. The protein shell consists of 32 capsomeres located in the cubic symmetry.

Antigenic properties

According to their antigenic properties of the strain (Georgia)1999/No. 1721-DEPT of FMDV belongs to serotype A.

The virus is stable neutralized homologous anticorodal. The virus does not manifest hemagglutinine activity. The animals recover in serum antibodies are formed, which are identified in the RDP, ELISA and PH. When vaccination of cattle vaccine of inactivated virus induces the formation of specific antibodies detected in ELISA, RDP and PH. If hyperimmunization Guinea pigs concentrated inaktivirovannye virus induces the formation of virousspecificakih antibodies detected in the RAC at a dilution of 1:128-1:512 and in ELISA at a dilution of 1:6000-1:10000.

Antigenic relatedness of strain A (Georgia) 1999/No. 1721-DEPT with the corresponding p is izvodstvennye and previously isolated strains in Turkey and Iran were studied in RAC. The results obtained are presented in table 1.

As the data presented in table 1, strain a (Georgia) 1999/No. 1721-DEPT of FMD virus type a is different from both the production and the previously selected epizootic strains.

On the antigenic properties of a new strain of A (Georgia) 1999/No. 1721-DEPT of FMD virus type a is also different from the previously studied production strain And No. 1707/Armenia/98-Deptvinigonee relatedness (R) in the RAC between them is 20%.

The study of the antigenic relatedness of the selected strain was carried out in PH, where the immune serum was used convalescents cattle virus strains (Georgia) 1999/No. 1721-DEPT, AND Turkey/97, and A22No. 550. The results of these studies are presented in table 2.

In table 2, the data confirm the antigenic difference between isolates from the production strain And22No. 550 and epizootic strains, isolated in Turkey in 1997.

Molecular-genetic characteristics

Method of nucleotide sequencing determined the primary structure of the gene VP1 (SEQ ID NO1) strain And (Georgia)1999/No. 1721 and out of the deduced amino acid sequence of VP1 protein (SEQ ID NO2). A comparative analysis of the set of sequences showed that the primary structure of the gene and protein VP1 strain (Georgia)1999/No. 1721 differs from all previously vydeleny the x isolates of FMD virus, including from strains And22No. 550 and No. 1707/Armenia/98, currently used for the production of vaccines against FMD type A. 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, reflected in the dendrogram.

Biotechnological characteristics

Strain a (Georgia) 1999/No. 1721-DEPT of FMDV type And exhibits a high biological, antigenic and immunogenic activity as in the native form and after inactivation. The strain intended for diagnostic sera and antigen preparations, and also for the manufacture of inactivated FMD vaccines. Strain a (Georgia) 1999/No. 1721-DEPT reproducerea in monolayer cultures particination cell culture kidney pig (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), IB-RS-2, KSS-21 and within 18-24 hours of incubation up to 6,0-8,0 Ig TCD50/ml. With a massive infection (1-100 TCD/cell) causes the JRC 4 hours. The results are shown in table 3.

After 3-4 passages incubation up to 6,0-8,0 Ig TCD50/ml. Preserves the original characteristics when passirovannye in sensitive biological systems within 10 passages.

Chemo - and g is nontaxonomic feature

Strain a (Georgia) 1999/No. 1721 - DEPT is an RNA-containing virus with a molecular mass of 7×106D.

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

The major antigenic protein VP1 is. The virion contains approximately 31.5% of RNA and 68.5% protein. Firiona RNA is infectious and is involved in the formation of protein precursors in infected cells. Predecessors, in turn, are split with the formation of more stable structural and non-structural proteins of the virus. Of the 6 non-structural polypeptides that accumulate in infected cells, one (VP3D) is an RNA-dependent RNA polymerase that is involved in the replication of RNA new virions.

Physical properties

The mass of the virion is 8.4×10-18, the sedimentation Coefficient 146S in the sucrose gradient. Floating density of 1.45 g/ml

Resistance to external factors

Strain a (Georgia) 1999/No. 1721-DEPT of FMDV type And resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.0 and 7.6. Changes of pH in acidic and in the alkaline side lead to inactivation of the virus. Sensitive to formaldehyde is, UV-irradiation, γ-radiation, and high temperatures.

Additional characteristics and properties

Immunogenic activity - immunogene comprising inactivated vaccine.

Reactogenicity - reactogenic properties is not.

The pathogenicity of the pathogenic for cloven-hoofed animals, newborn mice, Guinea pigs.

Virulence - virulent for naturally susceptible animals in contact, aerosol and parentelem infection.

Stability - maintains the original biological properties when passirovannye in sensitive biological systems within 10 passages.

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

To reduce the epidemic danger timely vaccination emerging foci of the disease, which requires highly immunogenic vaccine.

Conducted by the applicant's analysis of the prior art, including searching by the patent and scientific and technical information sources, and identify sources that contain information about the analogues of the present invention has allowed to establish that the applicant has not found the source, which is characterized by the signs of the, identical to all characteristics of the present invention. The definition from the list of identified unique prototype, as the most similar set of features analogue, has allowed to establish the essential towards perceived by the applicant to the technical result of the distinctive features of the proposed vaccine set forth in the independent claim.

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

To check compliance with the proposed vaccine condition of patentability "inventive step" conducted an additional search of the known solutions to identify topics included in the characterizing portion of the independent claim. The search results showed that the proposed solution does not follow for the expert in the obvious way from the prior art, as set out in the description section (not identified solutions that have the signs consistent with the distinctive features of the present invention), and revealed no effect provided the essential features of the proposed vaccine transformations to achieve a technical result.

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

The entity from whom retene explained on the dendrogram, reflecting the phylogenetic relationships of strain A (Georgia) 1999/No. 1721-DEPT of FMD virus serological type And with epidemic and vaccine strains of FMD virus type A. the Dendrogram based on the comparison of the complete nucleotide sequences of the VP1 gene.

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

Example 1.

Strain a (Georgia) 1999/No. 1721-DEPT virus of FMD type a was isolated from field material received in ARRIAH in the epithelium of the aft from cattle suspected of disease foot and mouth, when the laboratory diagnosis of this disease and differentiate it from other vesicular diseases. When isolation of the virus used a complex of biological, virological and biochemical methods.

Biological and virological methods included the inoculation material field isolates of cattle and the subsequent adaptation of the virus to the cultures pericentromeric and transplantable cell lines. We used cell culture JV, PSGK-30, IB-RS-2 and KSS-21. Primary and transplantable cell culture for the production of assay were grown in appropriate nutrient media under steady-state conditions in bottles with a capacity of 50-100 ml, washed from the growth medium and used to infect 10% suspension Afton the th material (multiplicity of infection was 1-10 TCD 50for a cell), prepared in Hanks solution with 0.5% GLA and antibiotics according to the standard recipe. Removal of microflora and ballast cellular components, the suspension was treated with chloroform in the ratio of 1:10. After a 30 minute incubation at 37°With vials made in 5-10 ml of maintenance medium and incubated at 37°until JRS virus. In the presence of JRS (rounding of cells, increasing their optical density, degeneration and separation of the cells from the glass vials were subjected to freeze-thawing, cleaning the cell suspension chloroform and centrifugation at 3000 g for 15 minutes. Received vaccinated material used for the subsequent passages and research in RAC and ELISA for the presence of viral antigen, used a set of commercial typespecification sera and sera were stored in the Museum of strains ARRIAH.

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

The data in table 3 indicate a good adaptive activity of the strain (Georgia) 1999/No. 1721-DEPT of FMD virus type a used cell cultures.

Isolated using the above methods, the virus was investigated in RAC with a set of diagnostics on all types of the virus of foot and mouth disease, vesicular stomatitis, vesicular exanthema and vesico Arnau disease of pigs to identify typical facilities and control of purity. The results of typing of the virus in RSK are shown in table 4.

Table 4 presents the results suggest that the isolated virus is of type A.

Example 2.

Inactivated adsorbate-vaccine against FMD type a, prepared from virus strain A (Georgia) No. 1999/No. 1721-DEPT grown in suspension culture cells KSS-21. As a supportive environment using the solution of the Earl without serum with the addition of FGMS, GBX and antibiotics at pH 7.4 and 7.6. Culture of cells infected by a virus at the rate of 0,4-1,5 TCD50on the cell.

The virus cultivation is carried out at a temperature of 36-37°C. Through 11-13 hours of incubation shall count live and dead cells at colouring Trifanova blue. If the number of living cells is 15-20%, the incubation continued for another 2-3 hours. When you reach the number of dead cells 90-95% cultivation cease and vaccinated suspension control for sterility and content 146S and 75S components. The number 146S±75S components in the suspension should be at least 0.5 μg/ml at the end of the cycle of reproduction of the virus, without temperature control, in vaccinated suspension add 15-20% solution AAAI, acidified with glacial acetic acid to a pH of 8.0 to 8.5. The final concentration AEEI in vaccinated suspension must be equal 0,025-0,05%. Inactivation of info is czynnosci virus is carried out in 12-24 hours at 36-37° C and a pH of 7.2 and 7.6 with stirring over 5-6 hours for 3-5 minutes. The remainder AAAI neutralized by adding sodium thiosulfate.

Warm the suspension is added 10% solution of pgmg to the concentration of 0,005-0,007% for ballasted flocculation of impurities and inactivation of possible contaminants. Flocculated ballast impurities is subjected to sedimentation, followed by decantation. The resulting antigen control for avirulence, content virousspecificakih protein and 146S and 75S components of the virus and sterility. The necessary concentration of 146S and 75S components in 1 ml of adsorbed vaccines obtained by concentration of the antigen GOA.

The estimated amount GOA 3% concentration is added to a cooled suspension of antigen when operating the mixer. Mixing lead within 30 minutes. After sedimentation GOA merge estimated volume remaining suspension. 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 the virus, at least a 3.0 µg/ml of this drug. Then in the slurry, add an additional 10% solution of saponin to a final concentration of 0.075%, which corresponds to 750,0 μg saponin in 1 ml of vaccine. The resulting vaccine is filled into glass vials and supervise its sterility in accordance with GOST 28085-89.

The avirulence and harmlessness VA who care to check on the 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. monitoring the clinical condition of the animals are in a period of 10 days. Avirulent, harmless and sterile vaccine tested for immunogenic activity in cattle or Guinea pigs.

Received the vaccine are liquid light amber with a loose white solid sorbent, which is formed on the bottom of the vial during storage and can be easily broken in a homogeneous suspension with shaking.

The optimal composition of the resulting adsorbate-FMD vaccine type And are given in table 5.

In tables 6, 7 and 8 shows the results of culturing strains (Georgia) 1999/No. 1721-DEPT, AND No. 1707 "Armenia-98-DEPT" and A22 No. 550 of FMD virus type a, from which it follows that cultural properties listed strains differ greatly in the two studied parameters:

1) strains (Georgia) 1999/No. 1721-DEPT and No. 1707 "Armenia-98-DEPT" have a longer period of reproduction (15-16 hours) compared with the strain And22No. 550 (12.5 hours);

2) strains (Georgia) 1999/No. 1721-DEPT and No. 1707 "Armenia-98-DEPT" give a lower percentage of output immunogenic components (61,5 and to 58.1%, respectively) compared with the strain And22No. 550 (89,9%).

Table 9 shows the results of studies on the effects of inactivation and purification of antigenic material is using AAAI and pgmg on immunogenic (146S and 75S) components of FMD virus strains (Georgia) 1999/No. 1721-DEPT and No. 1707 "Armenia-98-DEPT".

In table 9, the data suggest that the immunogenic components of the FMD virus strain A (Georgia) 1999/No. 1721-DEPT not inferior in resistance to inactivation and purification in the production conditions of the FMD virus strain And No. 1707 "Armenia-98-DEPT". Especially important is the fact that in the process of inactivation and purification of virus strain A (Georgia) 1999/No. 1721-DEPT no change has occurred in the number of 146S and 75S components obtained during the reproduction of the virus.

Example 3.

Tested the adsorbate-vaccines against FMD type a, produced as described in example 2 and containing, mcg:

Avirulent and cleared
antigenic material from strain
(Georgia) 1999/No. 1721-DEPT of FMD virus type a3,0
GOA1132,0
Saponin750,0
Support the environmentTo 1000000,0

The avirulence and safety of the vaccine has been tested to 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. monitoring the clinical condition of the animals was performed for 10 days.

At the end of the control avirulence and the safety of the vaccine was tested for immuno the military activity. The test was conducted on 5 heads of cattle. The adsorbate-the vaccine was administered subcutaneously at a dose of 2.0 ml the Results are shown in table 10.

In table 10, the data showed that all 5 heads of cattle were protected from the generalization process on day 21 after vaccination. The level of humoral immunity in vaccinated animals was 5,15±0,19 log2.

Example 4.

Tested the adsorbate-vaccines against FMD type a, produced as described in example 2 and containing, mcg:

Avirulent and cleared
antigenic material from strain
(Georgia)1999/No. 1721-DEPT of FMD virus type a3,0
GOA1740,0
Support the environmentTo 1000000,0

The avirulence and harmlessness derived vaccine has been tested to 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. monitoring the clinical condition of the animals was performed for 10 days.

At the end of the control avirulence and the safety of the vaccine was tested for immunogenic activity in cattle. The test was carried out on cattle, IPD50the drug was equal to 0.15 ml, i.e. in one ml of the vaccine contained 6,07 the D 50.

For comparison, the production series of the adsorbate-vaccine from strain And22No. 550 had IPD50for cattle, equal to 0.22±a 0.012 ml, P<0,001, i.e. in 1 ml of vaccine contained 4,55 PD50.

Thus, the above information shows the implementation of the use of the proposed vaccine following cumulative conditions:

vaccine inactivated adsorbed against FMD type And embodying the present invention, intended for use in agriculture, namely in veterinary Virology and biotechnology;

for the present invention in the form as it is described in the independent claim, confirmed the possibility of its implementation with the help provided in the application or known before the priority date tools and methods;

vaccine inactivated adsorbed against FMD type a, produced from strain A (Georgia) 1999/No. 1721-DEPT in accordance with the invention, has a high immunogenic activity and is able to ensure effective protection of susceptible animals against the epidemic of FMD virus type a, circulating in the Caucasus, Central Asia, Middle East.

Sources of information taken into account when preparing the description of the invention the application for the grant of a patent of the Russian Federation on from Britanie "Vaccine inactivated adsorbed against FMD type a".

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

2. Rarer X. FMD / Translation with it. Gasartobi. Ed. and Annot. Kida. wet. Sciences Pivalate. - M.: Kolos, 1971. - 432 S.

3. Burdov A.N., Dudnikov A.I., Malaret PV and other FMD / edited Angov. - M.: Agropromizdat, 1990. - S-250.

4. Syurin V.N., Samuyilenko YA, Soloviev BV, Fomin, NV Viral diseases of animals. - M.: UNITEMP, 1998. - S-548.

5. Temporary instruction of the manufacture and control of FMD concentrated hydroxide aluminum formulatin of lepidosirenidae virus a22. Approved BS USSR 25.03.1971,

6. RF patent №2143921 And 61 To 39/135, From 07 To 14/09, With 12 N 7/00, 7/04; 10.01.2000, (prototyp).

7. RF patent №594771, a 61 K 39/12, 07.07.93,

8. RF patent №2054039, C 12 N 7/02, And 61 To 39/35, 10.02.1996.

9. Auth. mon. The USSR №784335, C 12 Q 1/02, 20.03.2000,

Table 1

Antigenic spectrum of strain A (Georgia) 1999/No. 1721-DEPT of FMD virus type a (according to the RAC)
n=3
Compare strainsIndicators
r1r2R%
(Georgia) 1999/No. 1721-DEPT and a/Iran/960,280,949
(Georgia) 1999/No. 121-DEPT and/Kyrgyzstan/93 0,10,2415
(Georgia) 1999/No. 1721-DEPT and/Armenia/980,320,1220
(Georgia) 1999/No. 1721-DEPT and a/Turkey/970,231,047
(Georgia) 1999/1721-DEPT and A22No. 5500,10,3218
(Georgia) 1999 No. 1721-DEPT and a/Turkey/990,470,6957

Table 2

Antigenic relatedness of strain A (Georgia)1999/No. 1721-DEPT of known strains of FMD virus type a (according to PH)
n=3
Compare strainsIndicators
r1r2R%
(Georgia)1999/No. 1721-DEPT and a22No. 5500,380,441
(Georgia)1999/No. 1721-DEPT and Turkey/970,440,5847

Table 3

Biological properties of the strain (Georgia) 1999/No. 1721-DEPT of FMD virus type a
Cell cultureTime prowl. JRC (h) Qty adaptation passagesCharacteristics of the adapted virus
Activity in RACThe titer of Ig TCD50/ml
SP183one piece.106,0-6,5
PSGC-301621:2106,0-7,0
KSS-2118-2031:2-1:4106,5-7,5
IB-RS-21811:2-1:4106,07,0

Table 4

Typing 33%suspension yasunaga aphthous material (examination No. 1721)
Hyperim. serum in your titleAntigen (No. 1721) in dilutions
one piece.1:21:41:81:121:24Without antigen
And22No. 55043-----
And/Armenia/98442----
O1No. 194- ------
C1No. 564-------
Asia-1 no 48-------
CAT-1 No. 96-------
CAT-2 K183/74-------
CAT-3 Been 1/65-------
Without serum-------
Note: the percentage of delayed hemolysis 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 inactivated FMD adsorbate-vaccines FMD virus type a strain (Georgia) 1999/No. 1721-DEPT (µg in 1 ml g the postal vaccine)
IngredientsMinimumAverageMax
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
GOA1132,01620,02108,0
Saponin750,0750,0750,0
Support the environmentTo 1000000,0To 1000000,0To 1000000,0

Table 8

The cultivation of FMD virus type a, strain And22No. 550, the cell suspension KSS-21
№№ p/pThe cell concentration (million/ml)The dose of infection (TCD50/eng)The duration of reproduction (hours)The titer of virus (Ig TCD50/ml)BWA (m kg/ml)146S+75S components (µg/ml)% output 146S+75S
1.2,51.5138,002,482,1988,3
2.2,61,0127,251,171,04and 88.8
3.2,71,011of 7.752,001,83to 91.6
4.2,40,8148,001,781,4889,3
5.2,40,5127,50of 1.341,2694,3
6.3,00,05137,25to 2.061,8087,2
M+m2,60±0,090,81+0,2012,5±0,43a 7.62+0,141,80±0,201,60+0,1789,9+1,1
p<0,001p<0,025p<0,001p<0,001p<0,001p<0,001P<0,001

Table 9

Comparative data on the resistance of the strain of FMD virus And No. 1707 "Armenia-98-DEPT and strain And (GRU is Oia) 1999/No. 1721-DEPT to inactivation AAAI and sterilizing cleaning pgmg
№№ p/pAnd No. 1707 "Armenia-98-DEPT"(Georgia) 1999/No. 1721-DEPT
after cultivationafter inactivation and purificationafter cultivationafter inactivation and purification
BWA mg/ml146S+75S mg/mlBWA mg/ml146S+75S mg/mlBWA mg/ml146S+75S mg/mlBWA mg/ml146S+75S mg/ml
1.2,541,762,722,052,381,272,141,18
2.2,311,191,891,272,711,832,271,61
3.2,342,032,56to 1.862,931,722,501,48
4.1,170,941,250,753,121,802,691,93
5.to 2.06of 1.342,15 the 1.44
2,09±0,311,48±0,252,10+0,341,48±0,302,64+0,191,59±0,22,35±0,111,53+0,12
p<0,001p<0,01p<0,001p<0,025p<0,001p<0,001p<0,001p<0,005

Table 10

The results of tests of the adsorbate-FMD vaccine type And strain (Georgia) 1999/No. 1721-DEPT
Qty grafted alive xPrivigna dose (ml)Qty 146S+75S components in pravilnoy doseThe results of the control of infection on day 21 after vaccinationThe number of BHA (log) in the serum on day 21 after vaccination
primary attygeneralization
526,04/50/55,15±0,19 p<0,001
Control-2--2/22/2

1. The inactivated vaccine adsorbed is th against FMD type a, containing the active substance and the target additives, characterized in that the active substance it contains avirulent and purified antigenic material from a strain of the virus Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, serotype A, collection UHF VGNKI (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 homologous pathogen strain (Georgia) 1999/No. 1721-DEPT received in a sensitive biological system representing a suspension containing predominantly 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 homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting 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 homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S and 75S immunogenic components VIR is CA FMD type a in the 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 additives it contains adjuvant-sorbent aluminium hydroxide (GOA).

6. The vaccine according to claim 5, characterized in that it contains GOA preferably in the 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 additives it contains saponin adjuvant.

8. The vaccine according to claim 7, characterized in that it contains saponin adjuvant is preferably in the amount of 750,0 µg in 1 ml of the finished product.

9. The vaccine according to claim 1, characterized in that as the target additives it contains a supporting environment.

10. The vaccine according to claim 9, characterized in that it contains a supportive environment in an amount up to 1000000,0 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 homologous pathogen strain (Georgia) 1999/No. 1721-DEPT, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type a, GOA, saponin and supportive environment in the ratio, µg:

Antigenic materialNot less than 3,0
GOA132,0-2108,0
Sa is onini 50,0
Support the environmentTo 1000000,0



 

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