Vaccine against fmd type o and the method of its manufacture

 

The invention relates to veterinary Virology and biotechnology. Vaccine against FMD type O contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type O1strain "seaside-2000-DEPT", adjuvant and supportive environment in effective ratios. The strain is deposited in the collection VGNKI under registration 1734 "seaside-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 of impurities 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 the adsorbate and emulsion vaccines use antigenic content of immunogenic (146S+75S) components of the virus, at least 4.0 µg/ml of the Vaccine has a high immunogenic activity, provides protection against FMD virus type O circulating in South-East Asia and the Far East. 2 C. and 18 h.p. f-crystals, 12 tab.

The invention relates Yves FMD type O.

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 use appropriate types of FMD virus and select strains with a wide antigenic collatwrali 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 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 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 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 using subtle methods of molecular analysis, and the emergence of entirely different mi [2, 3].

Known strains of FMD virus type O, which are used in Russia in the last 34-35 years. These include the following strains: O11618 "Checheno-Ingushetia", dedicated in 1966 in the Chechen-Ingush ASSR; O1194 "Volgograd", dedicated in 1967 in the Volgograd region, and O11182/83 "Armenia", dedicated in 1983 on the territory of Armenia [4, 5].

These strains were used as production in the manufacture of diagnostic products and inactivated FMD vaccines used in different regions of the country. However, after the elimination of FMD caused similar antigenically strains of the virus, they have been discontinued and are currently supported only in the Museum strains Institute for the protection of animals [1-5].

Closest to the proposed invention, the essential features is the vaccine against FMD type O containing avirulent and purified antigenic material of the immunogenic components of FMDV O1194 reproduced in sensitive biological system, adjuvant and supportive environment.

Strain O1194 "Volgograd" selected 24.02.64, Omsk biocarbonate and is used in the change throughout Russia and the CIS.

Strain 194 of FMDV O1has the characteristics presented in table 1.

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

As inactivant use formaldehyde, aminoethylethanolamine (AAAI) or binary ethylenimine (BI). Adjuvant is aluminium hydroxide (GOA) with saponin or oil adjuvant [6].

The lack of a vaccine prototype is that it does not provide satisfactory protection against FMD virus type O circulating in South-East Asia and the far East (Japan, Russia, Mongolia).

Known methods for the production of FMD GOA-formulatin of aphthous, cultural and lepidosirenidae virus type [2, 5].

There is a method of production of FMD vaccines from aphthous virus type Of cattle, comprising preparing vaccinated suspension, purification, concentration, inactivation and adsorption of the virus in GOA [7].

There is a method of production of FMD vaccines from lepidosirenidae virus type About, including infection of newborn rabbits and the reproduction of the virus preparation vaccinated suspension, sovenia FMD vaccines from lepidosirenidae virus type O including an infection of newborn rabbits reproduction of virus in them, getting vaccinated suspension, its treatment, sorption of virus in GOA and inactivation of the virus with formalin [9].

Known method of preparing a vaccine against FMD type O, 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 mixing [10].

There is a method of production of FMD vaccines from lepidosirenidae virus type About, including the cultivation of the virus, its purification, inactivation and subsequent emulsification of the resulting antigen with an oil adjuvant [11].

A method of obtaining FMD GOA-formulatin of lepidosirenidae virus type About, 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 [12].

A known method of manufacturing the FMD vaccinal virus in it, cleaning vaccinated suspension by centrifugation or chloroform or 3-chlorethylene, inactivation of the virus with formalin and subsequent emulsification of the resulting antigen with an oil adjuvant [13].

The main disadvantage of the known methods for the production of FMD vaccine of virus type About is that obtained in accordance with these vaccines have low immunogenic activity and do not provide satisfactory protection against FMD virus type O circulating in South-East Asia and the Far East.

The closest present invention, the set of essential characteristics is a method of making a vaccine against a virus, FMD type O, 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 [6].

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 O, circulating in the countries of Sa O1194 did not provide satisfactory protection of immunized animals. In ARRIAH as a result of laboratory research aphthous material isolated from sick animals, was installed virus type About having antigenic differences from strain O1194 in the reaction of complement fixation and reaction seroneutralization virus. This indicates a discrepancy of tools specific FMD type O used Russia and CIS countries, epizootic virus circulating in South-East Asia and the Far East.

In the task of creating these inventions included the development of a highly immunogenic vaccine virus type About circulating in South-East Asia and the Far 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 O providing protection against FMD virus type O circulating in South-East Asia and the Far East.

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 ive FMD type O characterized by the following combination of characteristics: 1) the vaccine against FMD type O; 2) avirulent and purified antigenic material in the form of immunogenic components (146S+75S) virus O11734 "seaside-2000-DEPT"; 3) avirulent and purified antigenic material in the form of immunogenic components (146S+75S) of FMD virus type O1strain 1734 "seaside-2000-DEPT" taken in an effective quantity; 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 O11734 "seaside-2000-DEPT", adjuvants gel GOA and saponin, supportive environment taken in the ratio, µg/ml:
Antigenic material -4,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997138,0-998114,0
11) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT", oil adjuvant and supportive environment taken in the ratio, µg/ml:
Antigen the The present invention includes the following set of essential features, providing technical result, in all cases, which sought legal protection:
1) the vaccine against FMD type O;
2) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT";
3) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" 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 antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT", adjuvants gel GOA and saponin, supportive environment taken in the ratio, µg/ml:
Antigenic material -4,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997138,0-998114,0
5) avirulent and purified antigenic material in wausa environment taken in the ratio, ág/ml:
Antigenic material -4,0
Oil adjuvant - 500000-700000
Supportive environment - 299996,0-499996.

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 O;
2) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMD virus homologous strain;
3) 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 1734 "seaside-2000-DEPT" of FMD virus type O1taken 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 O11734 "seaside-2000-DEPT", chr/8805.gif">4,0
Gel GOA - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997138,0-998114,0
5) avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT", oil adjuvant and supportive environment taken in the ratio, µg/ml:
Antigenic material -4,0
Oil adjuvant - 500000-700000
Supportive environment - 299996,0-499996,0
We offer the vaccine has a high immunogenic activity and provides protection against FMD type O circulating in South-East Asia and the Far East.

Strain O11734 "seaside-2000-DEPT" is a new, previously unknown variant of the virus of FMD type O.

This technical result is also achieved by the creation of a method of making a vaccine against FMD type O is characterized by the following set of features:
1) a method of manufacturing a vaccine against FMD type O;
2) viral antigen reproduce in suspension culture cells KSS-21;
3) as a viral antigen using the FMD virus O11734 "seaside-2000-DEPT";
4) the FMD virus O11734 "seaside-2000-DEPT" cultivated in suspension culture cells KSS-21 for 10-15 hours the EP" with a title of no less than 7.0 lg TCD50/ml and the content of 146S+75S components not less than 0.5 μg/ml;
6) immediately after the end of the cycle of reproduction of the virus O11734 "seaside-2000-DEPT" subjected to inactivation;
7) the FMD virus strain O11734 "seaside-2000-DEPT" inactivate AEEI at a concentration of 0.025 to 0.05%;
8) the FMD virus O11734 "seaside-2000-DEPT" inactivate AAAI within 12-24 hours at 36-37oC and pH 7,2-7,6;
9) suspension of antigen purified from ballast impurities and possible contaminants, adding flocculant-antiseptic pgmg at a concentration of 0,005-0,007%;
10) ballast impurities are separated from the suspension of the 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 FMD virus11734 "seaside-2000-DEPT";
12) for the manufacture of the adsorbate-vaccine use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" at least 4.0 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 O11734 "seashore 14) for the manufacture of the adsorbate-vaccine to concentrate antigen add additional adjuvant saponin concentration, 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 O11734 "seaside-2000-DEPT" at least 4.0 µg/ml;
17) for the manufacture of emulsion vaccine concentrate antigen is combined with an oil adjuvant at a ratio of 3:7-1:1.

The present invention includes the following set of essential features that provide technical result, in all cases, which sought legal protection:
1) a method of manufacturing a vaccine against FMD type O;
2) viral antigen reproduce in a sensitive biological system;
3) as a viral antigen using the FMD virus O11734 "seaside-2000-DEPT" 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 the specific form made pensionnoy cell culture KSS-21;
2) the FMD virus O11734 "seaside-2000-DEPT" cultivated in suspension culture cells KSS-21 for 10-15 hours at 36-37o;
3) immediately after the end of the cycle of reproduction of the virus O11734 "seaside-2000-DEPT" subjected to inactivation;
4) the FMD virus O11734 "seaside-2000-DEPT" inactivate AEEI at a concentration of 0.025 to 0.05%;
5) the FMD virus O11734 "seaside-2000-DEPT" inactivate within 12-24 hours at 36-37oC 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 the antigen by sedimentation 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 purified antigenic content of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" at least 4.0 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 emulsionable;
12) for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" 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 FMD type O;
2) viral antigen reproduce in a sensitive biological system;
3) cleaning virusanderica 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 1734 "seaside-2000-DEPT" of FMD virus type O1;
2) the FMD virus O11734 "seaside-2000-DEPT" is used in an effective amount.

The invention is also characterized other distinctive mstitelnoy biological systems use suspension culture cells KSS-21;
2) the FMD virus O11734 "seaside-2000-DEPT" cultivated in suspension culture cells KSS-21 for 10-15 hours at 36-37o;
3) immediately after the end of the cycle of reproduction of the virus O11734 "seaside-2000-DEPT" subjected to inactivation;
4) the FMD virus O11734 "seaside-2000-DEPT" inactivate AEEI at a concentration of 0.025 to 0.05%;
5) the FMD virus O11734 "seaside-2000-DEPT" inactivate within 12-24 hours at 36-37oC 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 the antigen by sedimentation followed by decantation;
8) for the manufacture of the adsorbate-vaccine use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" at least 4.0 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 emulsionable;
12) for the manufacture of emulsion vaccines use avirulent and purified antigenic content of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" 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 O circulating in South-East Asia and the Far East.

The achievement of the technical result from the use of the proposed method due to the fact that in the manufacture of vaccines use a new strain 1734 "seaside-2000-DEPT" of FMD virus type O1.

Additional technical result from the use of the proposed method is due to the fact that 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 flocs is possible contaminants [14].

The original virus to obtain strain 1734 "seaside-2000-DEPT" of FMD virus type O1isolated from sick pigs in the farm village Elite Ussuri region of Primorsky Krai of the Russian Federation. Production strain O11734 "seaside-2000-DEPT" obtained by multiple serial passages in susceptible hetero - and homologous cell cultures. Strain O11734 according to the RAC, PH and sequencing of regions of the genome differs significantly from the production strain O1194 (R amounted to 40-50%, respectively, and the degree of nucleotide differences - 12%), as well as from other strains of FMD virus type O, available in the collection ARRIAH. The resulting strain deposited in the Collection of microorganisms of the all-Russian scientific research Institute of control, standardization and certification of veterinary preparations Ministry of agriculture of the Russian Federation (VGNKI) 22.03.2001, under registration 1734 "seaside-2000-DEPT". Strain O11734 "seaside-2000-DEPT" 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 Viracocha.

Strain 1734 "seaside-2000-DEPT" of FMD virus type O1characterized by the following features and properties.

Morphological properties
Strain O11734 "seaside-2000-DEPT" belongs to the family Picornaviridae, genus Aphtovirus, mind Aphtae, serotype O 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 strain O11734 "seaside-2000-DEPOT" refers to serotype O. the Virus is stable neutralized homologous anticorodal. The virus does not manifest hemagglutinine activity. The animals recover in the blood serum of antibodies are formed, which are identified in the RDP, ELISA, PH. When vaccination of swine vaccine of inactivated virus induces the formation of specific antibodies detected in ELISA, RDP, PH.

When determining antigenic matching strain O11734 "seaside-2000-DEPT" with production strains and field isolates of FMDV type O, a dedicated during 1966-2000, on the territory of CIS countries and other goodcase characteristics
Strain O11734 "seaside-2000-DEPT" shows high biological, antigenic and immunogenic activity as in the native form and after inactivation. The strain intended for use as raw materials for diagnostic sera and antigen preparations, and also the manufacture of inactivated FMD vaccines. Virus11734 reproducerea in monolayer cultures of primary trypsinization cell culture kidney pig (SP), transplantable cell cultures kidney Siberian mountain goat PSGC-30, IB-RS-2, KSS-21 and for 10-18 hours of incubation up to 7,0-8,0 lg TCD50/ml. With a massive infection causes JRS 2-3 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 description
Strain O11734 "seaside-2000-DEPT" is an RNA-containing virus with mol. mass 7106. Nucleic acid represented by single-stranded linear molecule mol. mass of 2.6106D. the virion has a protein shell composed of look no further than what I VP1. The virion contains approximately 31.5% of RNA and 68.5% protein. 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 O11734 "seaside-2000-DEPT" 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, UV-irradiation, y-irradiation, high temperatures.

Additional characteristics and properties
Immunogenic activity - immunogene comprising inactivated vaccine.

Reactogenicity - reactogenic properties is not.

The pathogenicity of the pathogenic for vospreimchev 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 O11734 "seaside-2000-DEPT" antigenic and immunological spectra is original, in taxonomic relation to new, previously unknown variant of the virus of FMD type O. To reduce its epizootic danger timely vaccination emerging foci of the disease, which requires high-level 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 proposed invention, has allowed to establish that the applicant has not found the source, characterized by signs, identical to all the essential features of the proposed invention. The definition 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 technical is 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 "inventive step".

The essence of the proposed invention illustrated by examples of their execution.

Example 1
For the manufacture of the adsorbate-vaccine virus O11734 "seaside-2000-DEPT" reproduce in suspension culture cells KSS-21. As a supportive environment use a solution Earl is 0,005-0,1 TCD50on the cell. The virus cultivation is carried out at a temperature of 36-37oC. After 8-10 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+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 36-37oC and a pH of 7.2 and 7.6 with stirring over 5-6 hours for 3-5 minutes. After that, the antigen suspension is cooled to 4-8oC. To the cooled suspension, add 10% solution 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 with 46S+75S components of the virus and sterility. The necessary concentration of 146S+75S components in pravilnoy dose adsorbed vaccine is obtained by concentration of the antigen by gel GOA.

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 of the gel GOA merge estimated volume remaining suspension. The final concentration of GOA should be in the range of 1.620,488 P<0.01 mg/ml, n= 10, and the concentration 146S+75S components of FMD virus at least 4.0 µg/ml Then in the slurry, add an additional 10% solution of saponin to a final concentration of at least 0,075%. Received the vaccine Packed in glass bottles and they control the sterility [15].

The resulting vaccine is a 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-vaccines against FMD type O are shown in table 2. In tables 3, 4, 5 and 6 show the results of cultivation of FMD virus type O11734 "seaside-2000-DEPT" and of FMDV O1194 in qulckly, what caused the need for selection of the optimal dose of infection.

A second difference of strain O11734 is a lower percentage of output immunogenic (146S+75S) components on the background of comparable values virousspecificakih protein (BWA).

Table 7 summarizes the results of studies on the effects of inactivation and purification of antigenic material using AAAI and pgmg on immunogenic viral components O11734, and of FMDV O1194.

In table 7, the data suggest that the immunogenic components of a new strain of FMDV O11734 are not inferior in resistance to inactivation and purification in the production of FMDV O1194. Especially important is the fact that in the process of inactivation and purification of virus O11734 there was no change in the number of 146S+75S components obtained during the reproduction of the virus (see tab. 7).

Example 2
Tested the adsorbate-vaccines against FMD type O, manufactured as described in example 1 and containing, ug/ml:
Avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" - 4,0
Gel GOA - 1740,0
Saponin - 750,0
Supporting sredstvo language at a dose of 2.0 ml, 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 immunogenic activity in cattle or Guinea pigs.

The test results obtained vaccines immunogenic activity in cattle is shown in table 8. In table 8 the results showed that all six heads of cattle were protected from the generalization process control infection through day 21.

The immune response of cattle to the introduction of the adsorbate-vaccine yasunaga antigen O11734 checked also on 5 heads of cattle weighing 200-300 kg the Results are shown in table 9. In table 9 suggests that FMD adsorbate-vaccine from strain O11734 induced a good immune response, starting 21 days after vaccination. The level of neutralizing antibody (VNA) to 61 days increased by 5.07 log2compared to 21 days after vaccination. Thus, the task of creating the adsorbate-vaccines on the basis of a new strain of FMDV O11734 "seaside-2000-DEPT solved.

Example 3
For the manufacture of emulsion vaccine virus O11734 "seaside-2000-DEPT" reproduce in suspension culture cells Eanie virousspecificakih protein, 146S+75S components of the virus and sterility as described in examples 1 and 2.

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 colloidal mills concentrate antigen and an oil adjuvant at a ratio of 3:7-1:1, respectively.

The result is inactivated emulsion vaccine against FMD type O, which is a molokopodobnye liquid, insoluble in water. The vaccine has a liquid consistency, easily resolved with the introduction, does not cause formation of abscesses, the overall reaction in the form of a high temperature and has a pronounced immunogenicity for pigs at a dose of 2 ml through 1-21 days post-vaccination for cattle at a dose of 5 ml through 3-21 days post-vaccination for 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 CONV table 10.

Example 4
Emulsion vaccine against FMD O11734 "seaside-2000-DEPT" is prepared as described in examples 1, 2, and 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 O, manufactured as described in example 3 and containing, ug/ml:
Avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" - 1,56
Oil adjuvant - 500000,0
Supportive environment - 499998,44
Immunogenic activity of this vaccine tested on pigs weighing 35-50 kg the Results are shown in table 11 (p. 4). IPD50=0,18 ml previfem volume of 2 ml containing 11,2 IPD50for pigs. Presented in table 11 data demonstrate the level of humoral immunity in pigs vaccinated with vaccines containing different levels of immunogenic component.

Table 12 shows that the emulsion vaccine of FMD virus type O1strain 194 induces in the body of the pigs in 2-3 times fewer VNA against heterologous strain VIR the Eden test emulsion vaccine against FMD type O manufactured as described in example 4 and containing, ug/ml:
Avirulent and purified antigenic material in the form of immunogenic (146S+75S) components of FMDV O11734 "seaside-2000-DEPT" - 6,0
Oil adjuvant - 500000,0
Supportive environment - 499994,0
Immunogenic activity of this vaccine tested in pigs. Its IPD50equal 0,043 ml, i.e., the vaccine contains 46,50 IPD50dose of vaccine (2 ml). The results are shown in table 11 (p. 5). Volume pravilnoy doses of the vaccine should contain at least 7 IPD50.

Therefore, the task of creating an emulsion vaccines on the basis of FMDV O11734 "seaside-2000-DEPT solved.

Thus, the above information shows the implementation of the use group of the proposed invention the following cumulative conditions:
vaccine against FMD type O 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, which they described in the independent claims, confirmed the possibility of their implementation using Optina against FMD type O and the method of its manufacture is achieved technical result provided the task of creating inventions.

Sources of information
1. Syurin Century. N. and other Viral diseases of animals. - M: UNITEMP, 1998, S. 544-547.

2. Burdov A. N., Dudnikov A. I., Malaret P. C. and other FMD /Ed. by A. N. Burdova. - Moscow: Agropromizdat, 1990, S. 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. - 432 S.

4. Kruglikov B. A., Stefan M. K., and Tarasenko So Ya the Study of infectious properties of strains of FMDV type O, allocated in different periods of the epidemic. - In the book: Act. Probl. wet. virologist. Vladimir, 1988, h 1, 77-78.

5. Auth. the certificate of the USSR 1739559 And 61 To 39/135, 10.04.2000.

6. 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).

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

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

9. 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.

10. Pat. THE USSR 352441, A 61 K 23/00, 21.09.72.

11. Auth. mon. THE USSR 411131, WITH 12 TO 5/00, 15.01.74.

12. Auth. mon. THE USSR 561732, WITH 12 TO 5/00, AND 61 TO 39/26; 15.06.77.

13. Pat. France 2;">
Claims

1. Vaccine against FMD type O containing avirulent and purified antigenic material in the form of immunogenic components of FMD virus type O homologous strain, adjuvant and supportive environment, characterized in that the strains the vaccine contains a strain of the virus Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, type htae, type O1the collection VGNKI 1734 "seaside-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. 1-3, characterized in that it contains avirulent and purified antigenic material in the form of immunogenic components of FMD virus type O1strain 1734 "seaside-2000-DEPT", gel aluminium hydroxide, saponin and supportive environment in the ratio, µg/ml:
Antigenic material -4,0
Gel aluminium hydroxide - 1132,0-2108,0
Saponin - 750,0
Supportive environment - 997138,0-998114,0
6. The vaccine under item 1 or 4, characterized in that it contains avirulent-2000-DEPT", oil adjuvant and supportive environment in the ratio, µg/ml:
Antigenic material -4,0
Oil adjuvant - 500000-700000
Supportive environment - 299996,0-499996,0
7. A method of manufacturing a vaccine against FMD type O, 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, characterized in that as the viral antigen used a strain of the virus Aphtae epizooticae, SEM. Picornaviridae, genus Aphtovirus, type Aphtae, type O1the collection VGNKI 1734 "seaside-2000-DEPT" 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 8, characterized in that the FMD virus type O11734 "seaside-2000-DEPT" cultivated in suspension culture cells KSS-21 for 10-15 h at 36-37oC.

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 in the audiusa fact, that inactivate FMD virus within 12-24 h at 36-37oC 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 polyhexamethylene guanidine at a concentration of 0,005-0,007%.

14. The method according to p. 13, characterized in that the ballast impurities are separated from the suspension of the 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 the manufacture of the adsorbate-vaccine use avirulent and purified antigenic content of immunogenic components of FMDV strain O11734 "seaside-2000-DEPT" at least 4 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 polietilene and purified antigenic content of immunogenic components of FMDV strain O11734 "seaside-2000-DEPT" 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 O concentrate the antigen is combined with an oil adjuvant at a ratio of 3: 7-1: 1.

 

Same patents:

The invention relates to the field of veterinary Virology

The invention relates to veterinary Virology and biotechnology

The invention relates to medicine and relates to chimeric live attenuated infectious virus, which is used to create chimeric vaccines flavivirus

The invention relates to biotechnology, in particular to methods of producing virus-like particles (the VLP) human papillomavirus (HPV) that is used to create vaccines

The invention relates to biotechnology, in particular to methods of producing virus-like particles (the VLP) human papillomavirus (HPV) that is used to create vaccines

The invention relates to biotechnology, in particular to methods of producing virus-like particles (the VLP) human papillomavirus (HPV) that is used to create vaccines

The invention relates to veterinary Virology and biotechnology

The invention relates to medicine, namely to Virology, and can be used for virological diagnosis of arboviral infections, particularly tick-borne encephalitis

The invention relates to veterinary Virology and biotechnology

The invention relates to biotechnology

The invention relates to the field of veterinary Virology and biotechnology and can be used in the manufacture of tools for specific prophylaxis and diagnosis of FMD type A
The invention relates to the field of veterinary Virology and involves identification of immunogenic FMD (universal) vaccines

Adjuvant // 2108111
The invention relates to the field of biotechnology, veterinary Virology and Microbiology, and more particularly to an oil adjuvant, and can be used in the development and production of immunizing preparations for the diagnosis and prevention of infectious diseases in different species of farm animals

Adjuvant // 2058154

The invention relates to the field of veterinary medicine, in particular, to the production and application of biological preparations for vaccination of farm animals and is intended for the simultaneous specific prophylaxis of anthrax and foot and mouth disease

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

Up!