Vaccine against fmd type asia-1 and the method of its manufacture

 

The invention relates to veterinary Virology and biotechnology. Inactivated vaccine against FMD type Asia-1 contains avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMD virus type Asia-1 strain 1737/Georgia/2000, adjuvant and supportive environment in effective ratios. The strain is deposited in the collection of strains of microorganisms VGNKI under registration 1737/Georgia/2000/DEPT. The virus reproduce in suspension culture cells KSS-21. As inactivant use aminoethylethanolamine at a concentration of 0.025 to 0.05%. To clean the suspension of the antigen from the ballast impurities used polyhexamethylene guanidine at a concentration of 0,005-0,007%. For the manufacture of the adsorbate-vaccine adjuvants using gel aluminium hydroxide, and optionally, saponin, and emulsion vaccine in oil adjuvant. For the manufacture of adsorbed vaccines use antigenic content of immunogenic (146S + 75S) components of FMD virus at least 2.0 µg/ml, and emulsion vaccine - 4.0 µg/ml Invention improves immunogenic activity of a vaccine against FMD virus type Asia-1 circulating in the countries of the Caucasus and the Middle East. 2 C. and 18 h.p. f-crystals, 10 and can be used in the manufacture of vaccines against FMD type Asia-1.

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 of other species of livestock production, 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 the virus into the territory, and in areas of high risk vaccination. Currently in the Russian Federation and CIS countries for immunization of 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 use appropriate types of FMD virus and select strains with a broad antigenic spectrum within the type is avanian region selected with the help of his trials in the reaction cross-protection (EBL) or more in the reaction cross-neutralization (RPN). 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 vaccines. 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 conservation of components of virus inactivation and 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 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 changes can be minor differences between strains, captured only by using subtle methods of molecular analysis to the rise of completely different strains, t is>Known strains of FMD virus type Asia-1, which were registered on the territory of the USSR: Armenia in 1973 and 1984, in Tajikistan in 1974, in Turkmenistan in 1978 and in Georgia in 1984 and 2000

Also known strain Asia 1 48 used for several years as production in the manufacture of diagnostic products and inactivated FMD vaccines used in different regions of the country (4).

Closest to the proposed invention, the essential features is the vaccine against FMD type Asia-1, containing avirulent and purified antigenic material of the immunogenic components of FMDV Asia 1 48 reproduced in sensitive biological system, adjuvant and supportive environment (5).

Strain Asia 1 48 dedicated 19.11.64, from cattle on the farm Lenin-Julia Dangara production Department of the Tajik SSR and used in the Russian Federation as production in the manufacture of means of specific prophylaxis applied on the whole territory of Russia and CIS countries.

Strain Asia 1 48 has the following characteristics shown in table 1.

For cleaning vaccinated suspension from ballistically formaldehyde, aminoethylethanolamine (AAAI) or binary ethylenimine (BI). Adjuvant is aluminium hydroxide (GOA) with saponin or oil adjuvant.

The lack of a vaccine prototype is that it does not provide satisfactory protection against FMD virus type Asia-1 circulating in the countries of the Caucasus and the Middle East.

Known methods for the production of FMD GOA-formulatin of aphthous, lepidosirenidae and cultural virus type Asia-1 (2).

There is a method of production of FMD vaccines from aphthous virus type Asia-1 cattle, including manufacturing vaccinated suspension, purification, concentration, inactivation and adsorption of the virus to GOA (6).

There is a method of production of FMD vaccines from lepidosirenidae virus type Asia-1, including infection of newborn rabbits and the reproduction of the virus preparation vaccinated suspension, sorption of virus in GOA, the inactivation of the virus with formalin and heat, adding saponin (7).

There is a method of production of FMD vaccines from lepidosirenidae virus type Asia-1, including infection of newborn rabbits, the reproduction of the virus, getting vaccinated suspension, it ocratic FMD type Asia-1, including the cultivation of the virus of FMD on experiencing tissue epithelium of the tongue of cattle with changing nutrient medium under continuous aeration and stirring (9).

There is a method of production of FMD vaccines from lepidosirenidae virus type Asia-1, including the cultivation of the virus, its purification, inactivation and subsequent emulsification of the resulting antigen with an oil adjuvant (10).

A method of obtaining FMD GOA-formulatin of lepidosirenidae virus type Asia-1, including infection of newborn rabbits and the reproduction of the virus preparation vaccinated suspension in isotonic saline Hanks, Earl, Tirade or Dulbecco with the addition of 0.5% lactalbumin hydrolysate or casein, cleaning vaccinated suspension 5-10% of chloroform, the sorption of virus in GOA, the inactivation of the virus with formaldehyde and subsequent addition of saponin (11).

There is a method of production of FMD vaccines of the cultural virus type Asia-1, including contamination of transplantable cell line pork kidney JB-RS-2 clone 3 and the reproduction of the virus, clean vaccinated suspension by centrifugation or chloroform or 3-chlorethylene, inactions disadvantage of the known methods for the production of FMD vaccine of virus type Asia-1, received in accordance with these vaccines have low immunogenic activity and do not provide satisfactory protection against FMD virus type Asia-1 circulating in the Caucasus and the Middle East (Iran).

The closest present invention, the set of essential characteristics is a method of making a vaccine against FMD virus type Asia-1, including the reproduction of viral antigen in a sensitive biological system, cleaning vaccinated suspension from the ballast impurities, the inactivation of the virus, the concentration of the obtained antigen and subsequent connection of the concentrate antigen with adjuvant (5).

The disadvantage of the prototype method is that obtained in accordance with the vaccine also has low immunogenic activity and does not provide satisfactory protection against FMD virus type Asia-1 circulating in the countries of the Caucasus and the Middle East.

When there is disease vaccine production strain Asia 1 48 did not provide satisfactory protection of immunized animals. In ARRIAH as a result of laboratory research aphthous material isolated from sick animals, was ustanavlivaetsya virus. This indicates a mismatch of funds specific FMD type Asia-1, used in Russia and CIS countries, epizootic virus circulating in the countries of the Caucasus and the Middle East.

In the task of creating these inventions included the development of a highly immunogenic vaccine against FMD virus type Asia-1 circulating in the Caucasus and the Middle East, and method of its manufacture.

The technical result of the proposed use of the invention is to increase the immunogenic vaccines against FMD type Asia-1, providing protection against FMD virus type Asia-1 circulating in the Caucasus and the Middle East (Iran).

This technical result is achieved by the creation of a group of inventions to form a single inventive concept. Included in the group of inventions, one of which is intended to receive the other.

This technical result is achieved by the creation of a vaccine against FMD type Asia-1, characterized by the following combination of characteristics: 1) the vaccine against FMD type Asia-1; 2) avirulent and purified antigenic material in the form of immunogenic components (146S+75S) of FMDV Asia 1 1737/Georgia/2000;Georgia/2000 taken in an effective amount; 4) of inactivated AAAI; 5) polyhexamethylene guanidine (phmg) as a means to clean the suspension from the ballast impurities; 6) of adjuvants gel GOA; 7) saponin as an additional adjuvant; 8) from oil adjuvants adjuvant; 9) support environment;
10) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, adjuvants gel GOA and saponin, supportive environment taken in the ratio, µg/ml:
Antigenic material is at least 2,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997140,0-998116,0;
11) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, oil adjuvant and supportive environment taken in the ratio, µg/ml:
Antigenic material is At least about 4.0
Oil adjuvant - 500000,0-700000,0
Supportive environment - 299996,0-499996,0
The present invention includes the following set of essential features that provide technical result, in all cases, which sought legal protection:
1) the vaccine against FMD type Asia-1;
2) avirulent and purified material in the form of immunogenic (146S+75S) components of the in of FMDV Asia 1 1737/Georgia/2000 taken in an effective quantity;
4) adjuvant;
5) supportive environment.

The invention is also characterized by other symptoms, expressing a particular form of execution or specific conditions of its use:
1) of the adjuvant vaccine contains gel GOA;
2) of the adjuvant vaccine contains additional saponin;
3) of the adjuvant, the vaccine contains an oil adjuvant;
4) avirulent and purified material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, adjuvants gel GOA and saponin, supportive environment taken in the ratio, µg/ml:
Antigenic material is At least 2,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997140,0-998116,0
5) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, oil adjuvant and supportive environment taken in the ratio, µg/ml:
Antigenic material is At least about 4.0
Oil adjuvant - 500000,0-700000,0
Supportive environment - 299996,0-499996,0
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 vaccine against FMD type Asia-1;
2) avirulent and adjuvant;
4) supportive environment.

Compared with the vaccine prototype the main feature of the invention is that of the strains the vaccine contains a strain 1737/Georgia/2000 FMD virus type Asia-1, taken 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) of the adjuvant vaccine contains gel GOA;
2) of the adjuvant vaccine contains additional saponin;
3) of the adjuvant, the vaccine contains an oil adjuvant;
4) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, adjuvants gel GOA and saponin, supportive environment taken in the ratio, µg/ml:
Antigenic material is At least 2,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997140,0-998116,0
5) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, oil adjuvant and supportive environment taken in the ratio, µg/ml:
Antigenic material is At least about 4.0
Oil adjuvant - 500000,0-700000,0
Supportive environment - 299996,0-499996,0
Y-1, circulating in the Caucasus and the Middle East (Iran).

Strain Asia 1 1737/Georgia/2000 is a new, previously unknown variant of FMD virus type Asia-1.

This technical result is also achieved by the creation of a method of making a vaccine against FMD type Asia-1, characterized by the following set of features:
1) a method of manufacturing a vaccine against FMD type Asia-1;
2) viral antigen reproduce in suspension culture cells KSS-21;
3) as a viral antigen using the FMD virus Asia 1 1737/Georgia/2000;
4) the virus of FMD Asia 1 1737/Georgia/2000 cultivated in suspension culture cells KSS-21 for 9-14 hours at 35-36,5o;
5) for the manufacture of vaccines use a suspension of FMDV Asia 1 1737/Georgia/2000 titre not less than 7,0 lg50/ml and the content of 146S+75S components is not less than 0.5 μg/ml;
6) immediately after the end of the cycle of reproduction of the virus of FMD Asia 1 1737/Georgia/2000 is subjected to inactivation;
7) the virus of FMD Asia 1 1737/Georgia/2000 inactivate AEEI at a concentration of 0.025 to 0.05%;
8) the FMD virus Asia 1 1737/Georgia/2000 inactivate AAAI within 12-24 h at 36-37,5oC and pH 7,2-7,6;
9) suspension of antigen purified from ballast impurities and possible contaminantfocus antigen by sedimentation followed by decantation;
11) for the manufacture of vaccines use avirulent and purified antigenic material in the form of an effective amount of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000;
12) for the manufacture of the adsorbate-vaccine use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 at least 2.0 µg/ml;
13) for the manufacture of the adsorbate-vaccine avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 concentrate adsorption on the gel GOA, which is added to the suspension of the antigen at a concentration of 2-5%;
14) for the manufacture of the adsorbate-vaccine to concentrate antigen add additional adjuvant saponin at a concentration of at least 0,075%;
15) for the manufacture of emulsion vaccine antigen concentrate flow ultrafiltration or polyethylene glycol;
16) for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 at least 4.0 µg/ml;
17) for the manufacture of emulsion vaccine concentrate antigen is combined with oil the hell the characters, providing technical result, in all cases, which sought legal protection:
1) a method of manufacturing a vaccine against FMD type Asia-1;
2) viral antigen reproduce in a sensitive biological system;
3) as a viral antigen using the FMD virus Asia 1 1737/Georgia/2000 in an effective quantity;
4) inactivation of the virus;
5) purification of the antigen from the ballast impurities;
6) the concentration of the antigen;
7) the connection of the concentrate antigen with adjuvant.

The invention is also characterized by other symptoms, expressing a particular form of execution or specific conditions of its use:
1) as a sensitive biological system using suspension culture cells KSS-21;
2) the virus of FMD Asia 1 1737/Georgia/2000 cultivated in suspension culture cells KSS-21 for 9-14 hours at 35-36,5o;
3) immediately after the end of the cycle of reproduction of the virus Asia 1 1737/Georgia/2000 is subjected to inactivation;
4) the virus of FMD Asia 1 1737 /Georgia/2000 inactivate AEEI at a concentration of 0.025 to 0.05%;
5) the virus of FMD Asia 1 1737 /Georgia/2000 inactivate within 12-24 h at 36-37,5oC and pH 7,2-7,6;
6) suspension of antigen purified from ballast n the documentation, followed by decantation;
8) for the manufacture of the adsorbate-vaccine avirulent and purified antigenic material concentrated by adsorption on the gel GOA, which is added to the suspension at a concentration of 2-5%;
9) for the manufacture of the adsorbate-vaccine use avirulent and cleared the content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 at least 2.0 µg/ml;
10) for the manufacture of the adsorbate-vaccine to concentrate antigen add additional adjuvant saponin at a concentration of at least 0,075%;
11) for the manufacture of emulsion vaccine avirulent and purified antigenic material concentrate flow ultrafiltration or polyethylene glycol;
12) for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 at least 4.0 µg/ml;
13) for the manufacture of emulsion vaccine concentrate antigen is combined with an oil adjuvant at a ratio of 3:7-1:1.

Features of the invention, describing the proposed method and the matching with the characteristics of the prototype, including a generic term that reflects the assignment are:
1) a method of manufacturing a vaccine against the vaccinated and suspension from the ballast impurities;
4) inactivation of the virus;
5) the concentration of the obtained antigen;
6) the connection of the concentrate antigen with adjuvant. Compared with the method of the prototype of the salient features of the invention are:
1) as a viral antigen using strain 1737/Georgia/2000 FMD virus type Asia-1;
2) the virus of FMD Asia 1 1737/Georgia/2000 is used 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) as a sensitive system of cultivation using suspension culture cells KSS-21;
2) the virus of FMD Asia 1 1737/Georgia/2000 cultivated in suspension culture cells KSS-21 for 9-14 hours at 35-36,5o;
3) immediately after the end of the cycle of reproduction of the virus of FMD Asia 1 1737/Georgia/2000 is subjected to inactivation;
4) the virus of FMD Asia 1 1737/Georgia/2000 inactivate AEEI at a concentration of 0.025 to 0.05%;
5) the virus of FMD Asia 1 1737/Georgia/2000 inactivate within 12-24 h at 36-37,5oC and pH 7,2-7,6;
6) suspension of antigen purified from ballast impurities by adding pgmg at a concentration of 0,005-0,007%;
7) ballast impurities are separated from the suspension of antig the th and purified antigenic content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000, at least 2.0 µg/ml;
9) for the manufacture of the adsorbate-vaccine avirulent and purified antigenic material concentrated by adsorption on the gel GOA, which is added to the suspension at a concentration of 2-5%;
10) for the manufacture of the adsorbate-vaccine to concentrate antigen add additional adjuvant saponin at a concentration of at least 0,075%;
11) for the manufacture of emulsion vaccine avirulent purified antigenic material concentrate flow ultrafiltration or polyethylene glycol;
12) for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV Asia 1 1737/Georgia/2000 at least 4.0 µg/ml;
13) for the manufacture of emulsion vaccine concentrate antigen is combined with an oil adjuvant at a ratio of 3:7-1:1.

The vaccine obtained by the proposed method, has a high immunogenic activity and protects animals against FMD virus type Asia-1 circulating in the Caucasus and the Middle East (Iran).

Obtain a technical result from the use of the proposed method due to the fact that in the manufacture of vaccines use a new strain 1737/Gr is thetsa by that the inactivation of the virus AAAI carried out immediately after the end of its cycle of reproduction, which can significantly reduce labor and energy costs for manufacturing vaccines without reducing the quality of inactivation of the virus.

Additional technical result from the use of the proposed method is also achieved through the use of flocculant antiseptic pgmg to clean the suspension of the antigen from the ballast impurities and simultaneous inactivation of possible contaminants (13).

The original virus to obtain strain 1737/Georgia/2000 FMD virus type Asia-1 isolated from sick cows in Bogdanovsky district of Georgia in July 2000.

Production strain Asia 1 1737/Georgia/2000 obtained by multiple serial passages in susceptible hetero - and homologous cell cultures. Strain Asia 1 1737/Georgia/2000 according to the RAC, PH and sequencing of regions of the genome differs significantly from the production strain Asia 1 48, as well as from other strains of FMD virus type Asia-1, available in the collection ARRIAH. The resulting strain deposited at the Russian state collection of strains of microorganisms, used in veterinary and animal husbandry all-Russian scientific-research the economy of the Russian Federation on 26 January 2001 under registration 1737/Georgia/2000/DEPT.

Strain Asia 1 1737/Georgia/2000 has a high biological, antigenic and immunogenic activity in the native form and after inactivation and provides inactivated FMD vaccines that creates the protection of susceptible animals against FMD virus type Asia-1, causing outbreaks of disease in the countries of the Caucasus and the Middle East.

Strain 1737/Georgia/2000 FMD virus type Asia-1 is characterized by the following characteristics and properties.

Morphological properties
Strain Asia 1 1737/Georgia/2000 belongs to the family Picornaviridae, genus Aphtovirus, mind Aphtae, serotype Asia-1 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 Asia 1 1737/Georgia/2000 belongs to serotype Asia-1. The virus is stable neutralized homologous anticorodal. The virus does not manifest hemagglutinin activity (HA activity). The animals recover in the blood serum of antibodies are formed, which are identified in the RDP, ELISA and PH. Immunization of cattle VA is p>

When determining antigenic matching strain Asia 1 1737/Georgia/2000 with the production strain and field isolates of FMDV type Asia-1 allocated during 1964-2000, on the territory of CIS countries and other countries in the world, was a comparative study of the primary structure of the gene and protein VP1.

Biotechnological characteristics
Strain Asia 1 1737/Georgia/2000 shows 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 the manufacture of inactivated FMD vaccines. Virus Asia 1 1737/Georgia/2000 reproducerea in the monolayer of primary trypsinization culture of kidney cells pigs (SP), transplantable cell cultures kidney Siberian mountain goat (PSGC-30), IB-RS-2, KSS-21 and over 11-16 h incubation up to 7,0-8,0 lg TCD50/ml. With a massive infection causes JRS after 6-8 hours After 3-4 passages incubation up to 6,0-8,0 lg TCD50/ml. Preserves the original characteristics when passirovannye in sensitive biological systems within 10 passages.

Chemo - and geotectonically har is mg>106D. Nucleic acid represented by single-stranded linear molecule mol. weight 2,8106D. 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 (VP66a) is an RNA-dependent RNA polymerase that is involved in the replication of RNA new virions.

Physical properties
The mass of the virion - 8,410-18, the sedimentation Coefficient 146S in the sucrose gradient. Floating density of 1.45 g/ml

Resistance to external factors
Virus Asia 1 1737/Georgia/2000 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. Chu the 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 parenteral 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 the strain Asia 1 1737 /Georgia/2000 antigenic and immunological spectra is original, in taxonomic relation to new, previously unknown variant of FMD virus type Asia-1. To reduce the epidemic danger timely vaccination emerging foci of the disease, which requires a highly effective vaccine.

Conducted by the applicant's analysis of the prior art, including the search for patents and scientific and technical information sources, and identify sources that contain information about the analogues of the proposed invention, has allowed to establish that the applicant has not found the source, characterizes the tion of the list of identified unique prototypes, as the most similar set of features analogues, has allowed to establish the essential towards perceived by the applicant to the technical result of the distinctive features of the proposed vaccine and method of manufacture set forth in the independent claims.

Therefore, the claimed invention meets the condition of patentability "novelty."

To check whether a proposed solution to the condition of patentability "inventive step" conducted an additional search of the known solutions to identify topics included in the characterizing portion of the independent claims. The search results showed that the proposed solution does not flow to 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 proposed inventions), revealed no effect provided the essential features of the proposed invention transformations to achieve a technical result.

Therefore, the proposed vaccine and method of its manufacture consistent with the condition of patentability "izobretatelskikh adsorbate-vaccine virus Asia 1 1737/Georgia/2000 reproduce 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 from the calculation of 0.01-0.1 TCD50on the cell. The virus cultivation is carried out at a temperature of 35-36,5oC. After 8-10 h 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 1-3 hours When the number of dead cells 90-95% cultivation cease and vaccinated suspension control for sterility and content 146S+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 h at 36-37,5oC and a pH of 7.2 and 7.6 with stirring over 5-6 hours for 3-5 minutes after the inactivation of the antigen suspension is cooled to 4-8oC. To the cooled suspension is added 10% solution of pgmg to conseillante impurities is subjected to sedimentation, followed by decantation.

The resulting antigen control for avirulence, content virousspecificakih protein and 146S+75S components of the virus, sterility. The necessary concentration of 146S+75S components in pravilnoy dose adsorbed vaccine is obtained by concentration of the antigen by gel YEAR.

The estimated amount of gel GOA 3% concentration is added to a cooled suspension of antigen when operating the mixer. Mixing lead within 30 minutes After sedimentation gel GOA merge estimated volume of the supernatant liquid or measure the volume of the remaining suspension. The final concentration of GOA should be in the range of 1.620,488 mg/ml, p<0,01, n=10, a concentration 146S+75S components of FMD virus at least 2.0 µg/ml Then in the slurry, add an additional 10% solution of saponin to a final concentration of at least 0,075%. The resulting vaccine is filled into glass vials and conduct monitoring sterility according to GOST 28085-89.

The avirulence and safety of the vaccine tested for 5 heads of cattle, introducing the vaccine first, under the mucous membrane of the tongue at a dose of 2 ml, and then subcutaneously at a dose of 10 ml of monitoring the clinical condition of the animals are 10 days. Avirulent, harmless and sterile vaccine tested for immuno what color with a friable 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 adsorbate-vaccines against FMD type Asia-1 are shown in table 2. In tables 3, 4, 5 and 6 shows the results of cultivation of FMDV Asia 1 1737/Georgia/2000 and virus Asia 1 48 cultivators KS-40 KM -2000, from which it follows that the strain of FMDV Asia 1 1737/Georgia/2000 has a shorter period of reproduction, which necessitated the selection of the optimal conditions for the cultivation of the virus and multiple doses of infection.

Table 7 summarizes the results of studies of the effect of inactivation and purification of antigenic material using AAAI and pgmg on immunogenic components of FMDV Asia 1 1737/Georgia/2000, of FMDV Asia 1 48. In table 7, the data suggest that the immunogenic components of a new strain of FMDV Asia 1 1737 /Georgia/2000 is not inferior in the resistance to inactivation and purification in the conditions of production of the virus of FMD Asia 1 48. Especially important is the fact that in the process of inactivation and purification of virus Asia 1 1737/Georgia/2000 no change has occurred in the number of 146S+75S components obtained during the reproduction of the virus.

Example 2
The conducted tests is e 8. From the results shown in table 8, we can conclude that the adsorbate-vaccine from strain 1737/Georgia/2000 FMD virus type Asia-1 induces a sufficient immune response to protect animals from infection control. In addition, it should be noted the increase in the number of BHA in the serum of cattle already at 10 DPA.

Thus, the task of creating the adsorbate-vaccines on the basis of a new strain of FMDV Asia 1 1737/Georgia/2000 solved.

Example 3
For the manufacture of emulsion vaccine FMD virus type Asia-1 strain 1737/Georgia/2000 reproduce in suspension culture cells KSS-21, inactivate, clear of ballast impurities and control the resulting antigen to avirulence, content virousspecificakih protein, 146S+75S components of the virus and sterility as described in example 1.

The necessary concentration of 146S+75S components in pravilnoy dose of emulsion vaccine obtained by concentration of the antigen flow ultrafiltration. To do this, use ultrafilter BTU 0.5 to 2. Filtering are under pressure of 1.5 ATM.

The obtained concentrate antigen stored at 4-6oWith until use in the vaccine. Emulsion vaccine is produced by dispersion in a colloid mill, the new emulsion vaccine against FMD type Asia-1, which is a molokopodobnye liquid, insoluble in water. The vaccine has a liquid consistency that is easily absorbed in the introduction, does not cause formation of abscesses, does not cause the overall reaction in the form of a high temperature and has a pronounced immunogenicity for pigs at a dose of 1 ml, for cattle, in the dose of 5 ml and sheep at a dose of 1 ml through 3-21 day after injection. The vaccine is administered to pigs intramuscularly, and cattle and sheep subcutaneously. In previfem volume must contain at least 4 µg 146S+75S components.

The obtained emulsion vaccine against FMD type Asia-1 is the optimal component composition (μg/ml) are presented in table 9.

Example 4
Emulsion vaccine against FMD Asia 1 31737/Georgia/2000 prepared as described in examples 1, 2, 3. The difference is that the necessary concentration of 146S+75S components in pravilnoy dose of emulsion vaccine obtained by concentration of antigen deposition of PEG-115.

Example 5
Tested emulsion vaccine against FMD type Asia-1, manufactured as described in example 3 and containing of 6.8 µg immunogenic components in pravilnoy dose.

Presented in table 10 data demonstrate the ability of the proposed vaccine is g2. Volume pravilnoy doses of the vaccine should contain at least 7 IPD50. Experienced vaccine induced a high level of humoral immunity in pigs and has achieved 40 IPD50in previfem volume.

Therefore, the task of creating an emulsion vaccines on the basis of FMDV Asia 1 1737/Georgia/2000 made.

Thus, the above information shows the implementation of the use group of the proposed invention the following cumulative conditions:
vaccine against FMD type Asia-1 and the method of its manufacture, embodying the present invention are intended for use in agriculture, namely, in veterinary Virology and biotechnology;
- for a group of proposed inventions, as they are described in the independent claims, confirmed the possibility of their implementation using the steps described in the application or known before the priority date tools and techniques;
- when using the proposed vaccine against FMD type Asia-1 and the method of its manufacture is achieved technical result provided by the task of creating inventions.

Consequently, the group proposed invention meets the condition of esni animals. -M-UNITEMP, 1998.-c. 544-547.

2. Burdov A. N. , Dudnikov A. I., Malaret P. C. and other FMD/Ed. by A. N. Burdova. -M.: Agropromizdat, 1990.-c.231-250.

3. Rarer X. FMD /Lane. with it. G. A. Surkova. Ed. and Annot. Kida. wet. Sciences P. C. of Malarze. -M.: Kolos, 1971.-432s.

4. Sobko, A. I. Identification of strains of FMD virus in complex epidemic events. Prof. Diss., Cover, 1972

5. 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 24.01.91, (prototype).

6. Pat. Germany 1138509; 30h, 6; 16.05.63,

7. Pat. Germany 1161387; 30h, 6; 08.05.69,

8. THE 10-09-123-91 "Universal mono - and polyvalent adsorbed vaccine against foot and mouth disease types A, O and Asia-1.- Approved BS Ministry of agriculture of the USSR, 16.06.91,

9. Pat. The USSR 352441, a 61 K 23/00, 21.09.72,

10. Auth. mon. The USSR 411131, With 12 To 5/00, 15.01.74,

11. Auth. mon. The USSR 561732, With 12 To 5/00, And 61 To 39/26; 15.06.77,

12. Pat. France 2088038; a 61 K 27/00, With 12 To 5/00; 07.01.72,

13. Pat. RF 2054039; 12 N 7/02, And 61 To 39/135; 10.02.96,


Claims

1. Vaccine against FMD type Asia-1, containing avirulent and purified antigenic material in the form of immunogenic , the fact of the strains the vaccine contains a strain of the virus Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, type Aphtae, type Asia-1, collection VGNKI No. 1737/Georgia/2000/DEPT, in an effective amount.

2. The vaccine under item 1, characterized in that it contains adjuvants adsorbent gel aluminium hydroxide.

3. The vaccine under item 1, characterized in that it contains adjuvants additionally saponin.

4. The vaccine under item 1, characterized in that it contains adjuvants oil adjuvant.

5. The vaccine according to any one of paragraphs.1-4, characterized in that it contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type Asia-1 strain No. 1737/Georgia/2000, gel aluminium hydroxide, saponin and supportive environment in the ratio, µg/ml:

Antigenic material At least 2,0

Gel aluminium hydroxide 1132,0 - 2108,0

Saponin 750,0

Supportive environment 997140,0 -998116,0

6. The vaccine under item 1 or 4, characterized in that it contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type Asia-1 strain No. 1737/Georgia/2000, oil adjuvant and supportive environment in the ratio, µg/ml:

Antigenic material At least 4,0

Oil adjuvant 500000,0 - 700000,0

Support with antigen in a sensitive biological system, cleaning vaccinated suspension from the ballast impurities, the inactivation of the virus, the concentration of the obtained antigen and subsequent connection of the concentrate antigen with adjuvant, characterized in that as the viral antigen used a strain of the virus Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, type Aphtae, type Asia-1, collection VGNKI No. 1737/Georgia/2000, in an effective amount.

8. The method according to p. 7, characterized in that as a sensitive biological system using suspension culture cells KSS-21.

9. The method according to p. 7 or p. 8, characterized in that the FMD virus type Asia-1 No. 1737/Georgia/2000 cultivated in suspension culture cells KSS-21 for 8-14 hours at 35-36,5C.

10. The method according to any of paragraphs.7-9, characterized in that immediately after the end of the cycle of reproduction of the virus is subjected to inactivation.

11. The method according to p. 10, characterized in that for the inactivation of FMD virus using aminoethylethanolamine at a concentration of 0.025 to 0.05%.

12. The method according to p. 10 or 11, characterized in that the FMD virus inactivating within 12-24 h at 36-37,5C and pH 7,2-7,6.

13. The method according to p. 7, characterized in that the cleaning suspension of the antigen from the ballast impurities using polyhexyl the C suspension of antigen by sedimentation followed by decantation.

15. The method according to any of paragraphs.7 to 14, characterized in that for the manufacture of the adsorbate-vaccine avirulent and purified antigenic material concentrated by adsorption on the gel of aluminium hydroxide at a concentration of 2-5%.

16. The method according to p. 15, characterized in that for producing the adsorbent-vaccine use avirulent and purified antigenic content of immunogenic components of the virus Asia 1 strain No. 1737/Georgia/2000 at least 2.0 µg/ml.

17. The method according to p. 16, characterized in that for the manufacture of the adsorbate-vaccine to concentrate antigen add additional adjuvant is a saponin at a concentration of at least 0,075%.

18. The method according to any of paragraphs.7 to 14, characterized in that for the manufacture of emulsion vaccine avirulent and purified antigenic material concentrate flow ultrafiltration or polyethylene glycol.

19. The method according to p. 18, characterized in that for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic components of FMDV Asia 1 strain No. 1737/Georgia/2000 at least 4.0 µg/ml.

20. The method according to p. 7 or 19, characterized in that for the manufacture of emulsion vaccine against FMD type Asia-1 concentrate anti-Christ.

 

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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

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