Inactivated emulsion 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 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

 

The invention relates to the field of veterinary Virology and biotechnology and can be used in the development and manufacture of vaccines inactivated emulsion 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).

Published studies indicate the advantages of emulsion preparations that are prepared with oil adjuvants (2, 3).

Emulsion products derived by mixing two mutually insoluble phases: oil (oil adjus the NTA) and water, containing the antigen (antigens) in a supportive environment, through their vigorous stirring (emulsification).

However, depending on the composition of the oil adjuvant receive emulsions of various types: "water in oil" (reverse), oil-in-water" (live) or "water-oil-water" (plural). For immunization of farm animals the most widely used reverse emulsion type.

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 use 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 product is the only strain demands the stability of the virus in the process of purification from tissue components and concentration, and save the virus inactivation and its long-term storage (4).

The causative agent of FMD has significant antigenic variability of strains within the same serotype, which is revealed in the various time periods and in different areas and depends on the species composition of the susceptible population, its immune status, and many other various factors.

Antigenic variability of the virus of foot and mouth disease caused by substitutions of amino acids in the polypeptide fragments (antigenic epitopes)exposed on the surface of the capsid proteins. The antigenic shifts the spectrum corresponding to the update of the structure of the new field strain can vary from minor, captured monoclonal antibodies to significant logged using conventional polyclonal immunoglobulins. Significant changes in the antigenic characteristics of the natural strain is likely to cause weakening of specific immunity induced by non-homologous antigen. They also cause difficulties statusbarisvisible diagnosis (4, 5).

In the result there is a need for new diagnostics and specific prophylaxis of FMD.

Known strains of FMD virus type a, isolated on the territory of the USSR and used as production is 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 (1-6).

Known vaccine inactivated emulsion against FMD type a, containing the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain of FMD virus obtained in a sensitive biological system (human cell culture porcine kidney IB-RS-2 clone 3), and the target additive in the form of a supportive environment and an oil adjuvant in effective relation (7).

Known vaccine inactivated emulsion against FMD type a, containing the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain And22No. 550 of FMD virus obtained in a sensitive biological system (newborn young rabbits), and targeted supplements in the form of a supportive environment and an oil adjuvant in effective relation (8).

Known vaccine in tigerbunny emulsion against FMD type a, contains the active substance in the form of avirulent and purified antigenic material from homologous pathogen strain A22No. 550 of FMD virus obtained in a sensitive biological system (human cell culture KSS-21), and the target additive in the form of a supportive environment and an oil adjuvant in effective relation (9).

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

Closest to the proposed invention, the essential features is the vaccine inactivated emulsion against FMD type a, containing the active substance in the form of avirulent and purified antigenic material from immunogenic 146S and 75S components of FMD virus type a strain And No. 1707 "Armenia-98", received in a sensitive biological system (human cell culture KSS-21), and the target additive in the form of a supportive environment and an oil adjuvant at the ratio, µg/ml pravilnoy dosage:

Antigenic material is not less than2,0
Support the environment299998,0-499998,0
Oil adjuvantto 1000000,0 (10).

The main disadvantage of a vaccine prototype is also in its lack of IME is Noynoy activity. It does 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 emulsion that creates an effective protection of susceptible animals against the epidemic of FMD virus type a, circulating in the Caucasus, Central Asia, Middle East.

The technical result from use of the present invention is to expand the Arsenal of vaccines inactivated emulsion against FMD type And providing effective protection of susceptible animals against the epidemic of FMD virus type a, circulating in the Caucasus, Central Asia, Middle East.

This technical result is achieved by the creation of a vaccine, inactivated emulsion against FMD 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 immunogenic 146S and 75S components of FMD virus type a strain (Georgia) 1999/No. 1721 (author's name), preferably obtained in suspension culture cells KSS-21, if the esto not less than 3,0 µg in one pravilnoy dose vaccines and targeted supplements, a supportive environment and an oil adjuvant in an amount to provide a tolerant presentation of antigen in the body immunized animal. The content of the target additives in final product calculated by considering the amount of pravilnoy doses of the vaccine for each species.

The original virus to obtain strain And (Georgia) 1999/No. 1721 allocated in 1999 in the Republic of Georgia. The strain obtained by passage isolate the sensitive hetero - and homologous cell cultures. Strain adapted to the primary cells of the porcine kidney and transplantable cell cultures KSS-21, IB-RS-2 and PSGC-30.

For the manufacture of vaccines as a sensitive biological systems use preferably the suspension culture cells KSS-21, and as a supportive environment solution Earl without serum with the addition of enzymatic hydrolysate of dry muscle (FGMS), hydrolyzed proteins in the blood dry (GBX) and antibiotics at pH 7.4 and 7.6.

For virus inactivation using aminoethylethanolamine (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 (11).

The resulting antigen purified from ballast impurities using the guanidine (phmg), which is introduced into the suspension to a concentration of 0,005-0,007% (12).

Avirulent is hydrated and purified antigenic material from strain A (Georgia) 1999/No. 1721 is a suspension, containing mainly 146S and 75S immunogenic components of FMD virus.

Quantitative and qualitative viral content of raw materials is determined by the method of turbidimetry (13).

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 preparation is provided by the concentration of antigen flow ultrafiltration or precipitation with polyethylene glycol - 115 (PEG-115). Emulsion vaccine is produced by dispersion in colloidal mills concentrate yasunaga antigen and an oil adjuvant at a ratio of 3:7-1:1, respectively. From the oil adjuvants can be used with oil adjuvant ARRIAH (14), and oil adjuvant brand Montanide ISA 70 or Montanide ISA 206 by Seppic (France).

Content-aware environment and oil adjuvant in pravilnoy dose of vaccine for each species in the ratio 3:7-1:1 is optimal, as it provides a tolerant presentation of antigen in the body immunized animal.

For immunization of pigs (privigna dose 2 ml) the optimal is the following composition of the proposed vaccine, ug in pravilnoy the dose for pigs:

Avirulent and purified antigenic material
of immunogenic 146S and 75S components of FMDV
type a strain (Georgia) 1999/No. 1721 of not less than3,0
Support the environment599997,0-999997,0
Oil adjuvant1000000,0-1400000,0.

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

Avirulent and purified antigenic material
of immunogenic 146S and 75S components of FMDV
type a strain (Georgia) 1999/No. 1721
not less than3,0
Support the environment1499997,0-2499997,0
Oil adjuvant2500000,0-3500000,0.

For immunization of sheep (privigna dose 1 ml) the optimal is the following composition of the proposed vaccine, ug in pravilnoy dosage for sheep:

Avirulent and purified antigenic material
immunogenic 146S and 75S components of FMDV
type a strain (Georgia) 1999/No. 1721
not less than3,0
Support the environment299997,0-499997,0
Oil adjuvant500000,0-700000,0.

The resulting vaccine is a molokopodobnye liquid, insoluble in water.

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

1. Vaccine inactivated emulsion against FMD type A.

2. The active substance in the form of avirulent and purified antigenic material from immunogenic 146S and 75S components of FMD virus type a strain (Georgia) 1999/No. 1721 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. Vaccine inactivated emulsion against FMD type A.

2. The 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 antigen is the material of the strain (Georgia) 1999/No. 1721 of FMD virus type a in an effective amount.

The invention is also characterized other distinctive signs, expressing a particular form of execution or specific conditions of its use:

1. Avirulent and purified antigenic material from homologous pathogen strain (Georgia) 1999/No. 1721 obtained 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, 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, 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, preferably obtained in a transplantable cell culture KSS-21 and constituting a suspension containing predominantly 146S THE 75S immunogenic components of FMD virus type a, not less than 3,0 µg pravilnoy the dose for each animal species.

5. As the target additive vaccine contains supporting environment.

6. The vaccine contains a supportive environment in an amount to provide a tolerant presentation of antigen in the body immunized animal.

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

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

9. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999/No. 1721 of FMD virus type a, a supportive environment and an oil adjuvant at the ratio of ICG in pravilnoy the dose for pigs:

Antigenic material is not less than3,0
Support the environment599997,0-999997,0
Oil adjuvant1000000,0-1400000,0.

10. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999/No. 1721 of FMD virus type a, a supportive environment and an oil adjuvant at the ratio of ICG in pravilnoy the dose for cattle:

Antigenic material is not less than 3,0
Support the environment1499997,0-2499997,0
Oil adjuvant2500000,0-3500000,0.

11. The vaccine contains avirulent and purified antigenic material from strain A (Georgia) 1999/No. 1721 of FMD virus type a, a supportive environment and an oil adjuvant at the ratio of ICG in pravilnoy dosage for sheep:

Antigenic material is not less than3,0
Support the environment299997,0-499997,0
Oil adjuvant500000,0-700000,0.

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

Strain a (Georgia) 1999/No. 1721 is a new, previously unknown. The original virus to obtain strain And (Georgia) 1999/No. 1721 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 passage on sensitive 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" (FGI VGNKI) under the registration number and production, cultural strain of FMD virus (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 FMD virus type a with epidemic and vaccine strains of FMD virus type a (dendrogram based on the comparison of the complete nucleotide sequences of the gene VP1), and in the sequence listing, in which the om:

SEQ ID NO:1 is the nucleotide sequence of the gene VP1 strain (Georgia) 1999/No. 1721 of FMD virus type A;

SEQ ID NO:2 is the amino acid sequence of VP1 strain (Georgia) 1999/No. 1721 of FMD virus type A.

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

Morphological properties

Strain a (Georgia) 1999/No. 1721 belongs to the family Picomaviridae, 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 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 is in ELISA at a dilution of 1:6000-1:10000.

Antigenic relatedness of strain A (Georgia) 1999/No. 1721 with relevant industrial 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 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 of FMD virus type a is also different from the previously studied production strain And No. 1707/Armenia/98. Bilateral kinship (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, A/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 NO:1) strain And (Georgia) 1999/No. 1721 and out of the deduced amino acid sequence of VP1 protein (SEQ ID NO:2). Comparative analysistrading sequences showed that the primary structure of the gene and protein VP1 strain (Georgia) 1999/No. 1721 differs from all previously selected isolates of FMD virus, including 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 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 reproducerea in the monolayer culture of cells of the kidney pig (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), IB-RS-2 and KSS-21 and over 18-24 hours incubation accumulated from 6.0 to 7.5 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 accumulated from 6.0 to 7.5 Ig TCD50/ml. Preserves the original characteristics when the pass is the formation of sensitive biological systems within 10 passages.

Chemo - and geotectonically feature

Strain a (Georgia) 1999/No. 1721 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.

In turn, the precursors are decomposed with the formation of a more stable structural and non-structural proteins of the virus. Of the 8 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 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 inak is ivali virus. Sensitive to formaldehyde, 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 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, x is rasterizes signs, 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 essence of pre the proposed 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 FMD virus 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 cultures of primary 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 aphthous material (multiplicity of infection was 1-10 TCD50for 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°D. the appearance of the JRC of the 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 isolate 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 vesicular disease swine 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. the Isolate was assigned to the author's name strain (Georgia) 1999/No. 1721 of FMD virus type A.

Example 2.

Inactivated emulsion VA is the Qing against FMD type a, prepared from virus strain A (Georgia) No. 1999/No. 1721, 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 the infectivity of the virus is carried out in 12-24 hours at 36-37°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 is aktivacii possible contaminants. Flocculated ballast impurities is subjected to sedimentation, followed by decantation. The resulting antigen control for avirulence, content virousspecificakih protein, 146S and 75S components of the virus and sterility. Quantitative and qualitative viral content of raw materials is determined by the method of turbidimetry. The necessary concentration of 146S and 75S components in emulsion vaccine obtained by concentration of the antigen flow ultrafiltration or precipitation of antigen PEG-115.

For concentration of antigen flow ultrafiltration using ultrafilter BTU 0.5 to 2.

Filtering are under pressure of 1.5 ATM. The obtained concentrate antigen stored at 4-6°until use in the vaccine.

Emulsion vaccine is produced by dispersion in colloidal mills concentrate antigen and an oil adjuvant at a ratio of 3:7-1:1, respectively. From the oil adjuvants can be used with oil adjuvant ARRIAH (14), and oil adjuvant brand Montanide ISA 70 or Montanide ISA 206 by Seppic (France).

As a result of receiving the vaccine inactivated emulsion against FMD type a, which is a molokopodobnye liquid, insoluble in water.

Received the vaccine has an optimal component composition:

1) avirulent and purified antigen is the first material in the form of immunogenic 146S and 75S components of FMD virus type a strain (Georgia) 1999/No. 1721 in an amount not less than 3,0 µg in one pravilnoy dose of vaccine;

2) supportive environment in quantity 299997,0-499997,0 µg in 1 ml pravilnoy doses of the vaccine, i.e. in an amount to provide a tolerant presentation of antigen in the body immunized animal;

3) oil adjuvant in the number 500000,0-700000,0 µg in 1 ml pravilnoy doses of the vaccine, i.e. in an amount to provide a tolerant presentation of antigen in the body immunized animal.

The vaccine is easily resolved with the introduction, does not cause abscesses, the overall reaction in the form of temperature rise and has expressed immunogenic activity for pigs in pravilnoy a dose of 2 ml, for cattle in pravilnoy dose of 5 ml and for sheep in pravilnoy dose of 1 ml 21 days after injection. The vaccine is administered to pigs intramuscularly, and cattle and sheep subcutaneously. In previfem volume must contain at least 3 µg 146S and 75S components of FMD virus.

Example 3.

Tested immunogenic vaccine inactivated emulsion against FMD type a, produced as described in example 2 and containing, mcg:

Avirulent and purified antigenic material
as immunogenic 146S and 75S components
the FMD virus type a strain (Georgia) 1999/No. 17217,0
Maintaining the environment I 499993,0
Oil adjuvant500000,0.

Immunogenic activity of this vaccine tested on pigs weighing 35-40 kg the Results are shown in table 5.

The vaccine was diluted with an oil adjuvant at 3 and 9 times, and entered the pigs intramuscularly at a dose of 2 ml

Controlling infection was performed on day 21 after vaccination (WPV) homologous strain of FMD virus.

Vaccinated pigs survived infection control and unvaccinated sick with the generalization process. In pravilnoy dose tested vaccine contained 15 PD50.

Presented in table 6 the data show that the number of virus-neutralizing antibodies in the serum of pigs, vaccine-induced increased to 10 fiberboard 2 times, 21 fiberboard in 14-16 times on 55 DVP their number has increased from 23 to 30 times, which testifies to the effectiveness of the tested vaccine.

Example 4.

Tested immunogenic vaccine inactivated emulsion against FMD type a, produced as described in example 2 and containing, mcg:

Avirulent and purified antigenic material
as immunogenic 146S and 75S components
virus ASU is and type a strain (Georgia) 1999/No. 1721 3,0
Support the environment499997,0
Oil adjuvant500000,0.

Immunogenic activity of this vaccine tested in pigs. Its IPD50equal to 0.18 ml, i.e. the vaccine contains 11 PD50in pravilnoy the vaccine dose volume of 2 ml the Results are shown in table 7. Volume pravilnoy doses of the vaccine should contain at least 7 PD50.

Example 5.

Tested immunogenic vaccine inactivated emulsion against FMD type a, produced as described in example 2 and containing, mcg:

Avirulent and purified antigenic material
as immunogenic 146S and 75S components
the FMD virus type a strain (Georgia) 1999/No. 17213,0
Support the environment499997,0
Oil adjuvant500000,0.

Immunogenic activity of the vaccine tested in pigs weighing 35-50 kg the Results are shown in table 8. In pravilnoy the vaccine dose volume of 1 ml containing 7,1 PD50for pigs.

Thus, the above information indicates the implementation of the use we offer the vaccine the following cumulative conditions:

vaccine inactivated emulsion 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 emulsion against FMD type a, produced from strain A (Georgia) 1999/No. 1721 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 for invention "Vaccine inactivated emulsion against FMD type a".

1. Bojko A.A. Déclaration sur l apparition en URSS d'un virus type aphteux différent des souches de type A antérieurement étudiéés dans le Pays. BOIE, 1965, 64, V.2. - R-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 MD. Rio de Janeiro, Brasil, 5-10 Oct. 1985, Rome, 1986, 132-135.

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

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

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

7. French patent No. 2088038; a 61 K 27/00, With 12 To 5/00; 07.01.1972,

8. Auth. mon. The USSR №411131, With 12 To 5/00, 15.01.1974,

9. Instruction for production and control of vaccines monovalent emulsion against FMD type a, type O, type C, type Asia-1 (from virus grown in cell KSS-21). Approved BS of the state Commission of the USSR food and procurement 24.01.1991,

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

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

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

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

14. RF patent №2108111; a 61 K 39/39 // a 61 K 39/00, 39/102, 39/12, 39/135; 10.04.1998,

Table 1

Antigenic spectrum of strain A (Georgia) 1999/No. 1721 of FMD virus type a (according to the RAC) n=3
Compare strainsIndicators
r1r2R%
(Georgia) 1999/No. 1721 and Iran/96 0,280,949
(Georgia) 1999/No. 1721 and/Kyrgyzstan/930,10,2415
(Georgia) 1999/No. 1721 and/Armenia/980,320,1220
(Georgia) 1999/No. 1721 and a/Turkey/970,231,047
(Georgia) 1999/1721 and a22No. 5500,10,3218
(Georgia) 1999 No. 1721 and a/Turkey/990,470,6957

Table 2

Antigenic relatedness of strain A (Georgia) 1999/No. 1721 with known strains of FMD virus type a (according to PH) n=3
Compare strainsIndicators
r1r2R%
(Georgia) 1999/No. 1721 and A22No. 5500,380,441
(Georgia) 1999/No. 1721 and a/Turkey/970,440,5847

Table 3

Biological properties of the strain (Georgia) 1999/No. 1721 of FMD virus type a
Cell culturethe time 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-21811:2-1:4106,0-7,0

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----
1NQ194-------
C1NQ564-------
Asia-1 no 48-------
CAT-1 No. 96-------
CAT-2 K183/74-------
CAT-3 Bech1/65-------
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 results of tests of FMD inactivated emulsion vaccine prepared on the basis of the e strain (Georgia) 1999/No. 1721
Qty grafted alive xPrivigna dose (ml)Breeding vaccinesThe content of immunogenic components in dilution (µg)The results of the control of infection at 21 DPAIPD50
primary attygeneralization
421:32,334/40/4≤0,13 ml
421:90,784/40/4
Control---2/22/2-

1. Vaccine inactivated emulsion 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 the 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 cultures of animal origin and represents 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 of FMD virus type A.

5. The vaccine according to claim 4, 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, in the amount of not less than 3,0 µg pravilnoy dosage for each species alive is the shaft.

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

7. The vaccine according to claim 6, characterized in that it contains a supportive environment in an amount to provide a tolerant presentation of antigen in the body immunized animal.

8. The vaccine according to claim 1, characterized in that as the target additives it contains an oil adjuvant.

9. The vaccine according to claim 1, characterized in that as the target additives it contains oil adjuvant in an amount to provide a tolerant presentation of antigen in the body 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, a supportive environment and an oil adjuvant at the ratio of ICG in pravilnoy the dose for pigs:

Antigenic materialNot less than 3,0
Support the environment599997,0-999997,0
Oil adjuvant1000000,0-1400000,0

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, a supportive environment and an oil adjuvant in with the attitude, mcg in pravilnoy the dose for cattle:

Antigenic materialNot less than 3,0
Support the environment1499997,0-2499997,0
Oil adjuvant2500000,0-3500000,0

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, a supportive environment and an oil adjuvant at the ratio of ICG in pravilnoy dosage for sheep:

Antigenic materialNot less than 3,0
Support the environment299997,0-499997,0
Oil adjuvant500000,0-700000,0



 

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