Virusvaktsinu against classical swine fever

 

(57) Abstract:

The invention relates to veterinary Virology and biotechnology. Virusvaktsinu classical swine fever (CSF) contains vaccinated material derived from an attenuated strain "SENLAC virus CoES and protective environment in effective ratios. The strain is deposited in the collection of microorganisms VGNKI under the registration name "SENLAC"-DEPT. The virus to reproduce in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/cm3. To obtain a protective environment use solutions of sucrose, gelatin and hydrolyzed lactalbumin (GLA) in the effective ratio. Effect: increase the antigenic and immunogenic activity of the drug. 6 C.p. f-crystals, 2 ill., table 1.

The invention relates to the field of veterinary Virology and biotechnology and can be used in the manufacture of tools for the specific prevention of classical swine fever (CSF).

CSF is a severe, often fatal, contagious viral disease of swine characterized by a variety of clinical forms and manifestations, namely fever, lesions of the blood and blood-forming systems (phenomena hemorrhagic septicemia), troponymy, belonging to the genus Pestivirus of the family Flaviviridae.

Currently CoES on the relevance is one of the first places in infectious diseases of pigs and considered for countries with intensive system hog one of the economically important diseases, veterinary and sanitary plan - one of the most difficult liquidated epizootics.

CoES long time registered in Russia. Over the past years, there had been several periods of activation of epizootic process of this disease with subsequent reduction of its intensity. There is a wide geographical spread of the disease. CoES is registered in almost all economic regions of Russia (from Kaliningrad to the Far East and from the Arkhangelsk region to Krasnodar Krai). Although transmission and infection is well known, practical experience shows that to implement complex security of the country and farms from the introduction of the pathogen CoES, and especially to eliminate the epidemic is extremely difficult.

For the specific prevention of pigs used live lepidosirenidae or culture vaccine derived from an attenuated virus strains CoES.

Known virusvaktsinu against HT is new in the body of the rabbit, and protective environment, taken in the effective number (1, 2).

Known virusvaktsinu against CSF containing vaccinated material derived from an attenuated strain of GM-94 virus CoES, reproduced in the body of a rabbit, and a protective environment, taken in the effective number (3).

Known virusvaktsinu against CSF containing vaccinated material from the attenuated strain of "LK-Vniivvim virus CoES, reproduced in primary culture cells of the testes of the lambs, and protective environment, taken in the effective number (4).

Known virusvaktsinu against CSF containing vaccinated material from the attenuated strain of "LK-virus CoES reproduced in transplantable cell cultures of RK-15, and a protective environment, taken in the effective number (5).

Known virusvaktsinu against CSF containing vaccinated material derived from an attenuated strain, "KS" virus CoES reproduced in sensitive biological system, and protective environment, taken in the effective number (6).

The General disadvantages of the known drugs are increased reactogenicity for vaccinated animals or low immunogenic activity.

Many vaccines HT is ractice vaccination of pigs against CSF (7).

Closest to the present invention, the set of essential characteristics is virusvaktsinu against CSF containing vaccinated material from the attenuated strain of "Chinese" virus CoES, reproduced in the body of the rabbit with the biological activity of 3.0-3.5 lg FIGHT50/cm3and protective environment, taken in an effective amount.

Strain "Chinese" of the CSF virus has the following characteristics:

- it has low reactogenicity for pigs of all ages, including newborn piglets and pregnant sows;

is not transferred to non-immune animals when sharing their content with immune and not reverses when passirovannye on the pigs (6-12 passages);

not persists in vaccinated pigs after experimental infection last virulent virus is not excreted from the body vaccinated animals;

- good breeds (up to 3.0-3.5 lg FIGHT50/cm3only in the tissues of the rabbit with the introduction of the virus in animals or in tissue cultures of kidney cells of the embryo rabbit (8).

A significant drawback of VirusWall of the prototype is its low antigenic and immunogenic activity because of the low level of accumulation of the virus in the tissues of rabbits is izvodstvennogo attenuated strain of the virus CoES, with a broad antigenic spectrum of activity, high biological and immunogenic activity and for producing effective VirusWall to protect animals from virus circulating on the territory of the Russian Federation.

The technical result from the use of the invention is to increase the antigenic and immunogenic activity VirusWall.

This technical result is achieved by creating VirusWall against CSF, characterized by the following set of features:

1) virusvaktsinu against CSF;

2) vaccinated material derived from an attenuated strain "SENLAC virus CoES;

3) vaccinated material derived from an attenuated strain "SENLAC virus CoES, reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/2 cm3;

4) vaccinated material derived from an attenuated strain "SENLAC virus CoES taken in an effective quantity;

5) vaccinated material derived from an attenuated strain "SENLAC virus CoES taken in an amount of 60-80 wt.%;

6) protective environment;

7) protective environment contains 50% sucrose solution, 10% solution of gelatin and 20% solution of the hydrolyzate lactalbumin/BR> 9) protection of the environment taken in an amount of 20-40 wt.%;

10) vaccinated material derived from an attenuated strain "SENLAC virus CoES and protective environment is taken in the effective value;

11) vaccinated material derived from an attenuated strain "SENLAC", reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/cm3and protective environment is taken in the ratio, wt.%:

Vaccinated material - 60-80

Protective environment 100

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

1) virusvaktsinu against CSF;

2) vaccinated material derived from an attenuated strain "SENLAC virus CoES;

3) vaccinated material derived from an attenuated strain "SENLAC virus CoES taken in an effective quantity;

4) protective environment;

5) vaccinated material derived from an attenuated strain "SENLAC virus CoES and protective environment is taken in the effective value.

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

1) of the IR with the biological activity of 4.5-5.0 lg EID50/2 cm3;

2) vaccinated material derived from an attenuated strain "SENLAC virus CoES taken in an amount of 60-80 wt.%;

3) protective environment contains 50% sucrose solution, 10% solution of gelatin and 20% solution GLA, taken in the ratio of 1:0,5:2,5, respectively;

4) protection of the environment taken in an amount of 20-40 wt.%;

5) vaccinated material derived from an attenuated strain "SENLAC virus CoES, reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/2 cm3and protective environment is taken in the ratio, wt.%:

Vaccinated material - 60-80

Protective environment 100

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) virusvaktsinu against CSF;

2) vaccinated material derived from an attenuated strain of the virus CoES;

3) protective of the environment.

Compared with venusvalley prototype the main feature of the proposed VirusWall is that it contains an effective amount vaccinated material derived from an attenuated strain "SENLAC virus CSF.

The present invention is characterized by the so the output:

1) vaccinated material of the strain SENLAC" of classical swine fever virus reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/cm3;

2) vaccinated material derived from an attenuated strain "SENLAC virus CoES taken in an amount of 60-80 wt.%;

3) protective environment in an effective quantity;

4) protective environment contains 50% sucrose solution, 10% solution of gelatin and 20% solution GLA, taken in the ratio of 1:0,5:2,5, respectively;

5) protective environment is taken in an amount of 20-40 wt.%;

6) vaccinated material derived from an attenuated strain "SENLAC virus CoES, reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/2 cm3and protective environment is taken in the ratio, wt.%:

Vaccinated material - 60-80

Protective environment 100

The proposed virusvaktsinu has a high antigenic and immunogenic activity and provides protection against CSF virus circulating on the territory of the Russian Federation.

The achievement of the technical result of the proposed use of VirusWall due to the fact that when it is used to make new strain "SENLAC virus CSF. The strain SENLAC" obtained by carrying out x2and adult rabbits. The strain SENLAC" consistently cause disease of rabbits with pronounced hyperthermia and is avirulent for pigs.

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) October 5, 1999 registration by name "SENLAC"-DEPT.

Compared with the strain of "Chinese" strain "SENLAC" has a higher biological, antigenic and immunogenic activity. Experimentally confirmed high immunological activity of VirusWall of strain SENLAC". The results of the extensive production tests VirusWall of strain SENLAC" in the areas of preventive vaccination, as well as foci of infection give grounds to confirm quite pronounced on the effectiveness of this drug.

The invention is explained in graphic materials:

in Fig.1 shows the amino acid sequence of the region's major antigenic domains of the glycoprotein E2 (t) of CSF virus strain SENLAC";

in Fig. 2 - dendrogram reflecting the/P> The strain SENLAC virus CSF is characterized by the following characteristics and properties.

Origin

The strain SENLAC virus CoES obtained in the research Institute of animal protection in 1995, interspersed with passages lepidosirenidae of CSF virus strain "Chinese" in cell cultures of RK-13, hybrids AND4x C2and adult rabbits. The resulting strain stably causes disease in rabbits with pronounced hyperthermia. The titer of virus in the blood of infected animals during the period of maximum increase of the body temperature reaches 4.5 to 5.0 lg EID50/2 ml Vaccine strain "SENLAC virus CSF was 20 passages in cell cultures of RK-13, 25 passages on the hybrid cell cultures AND4x2and 17 passages in adult rabbits.

Taxonomic characterization

The strain SENLAC" CSF virus belongs to the family Flaviviridae, genus Pestivirus. The cryptogram code taxonomic parameters of the CSF virus - has the form: R/1; 4/? ; S/S; V/0, which expresses the following characteristics: single-stranded linear RNA with a molecular mass of 4 x 10 daltons, % content of RNA in the virion is not installed (?), the virion and nucleocapsid spherical shape (S), the natural host are pigs (V vertebrates), is being distributed without vectors.

Mo is submitted to typical flaviviruses separate virions are spherical or rounded form, diameter 40-44 nm. In ultrathin sections, the magnitude of the nucleocapsid defined in 34-38 nm, and the thickness of the lipoprotein membrane 3 nm. The sedimentation coefficient equal 10418 on Swedberg, floating density of 1.16-1.20 g/cm3.

Antigenic properties

When injecting into the body of the pig and rabbit strain SENLAC" causes the formation of neutralizing antibodies in titer of 1:16-1:64.

To determine the antigenic properties of the used methods polymerase chain reaction (PCR) and nucleotide sequencing. The results are shown in Fig.1 and 2. PCR detected only genomic RNA of the virus in CSF. As a result of nucleotide sequence on the 5' end is determined that the strain SENLAC" refers to the group of "Chinese" and easily identified from the existing vaccine strains of the virus CSF (see Fig.2). The percentage of nucleotide homology with the strains of "Chinese" is 99,26. The specificity of serum obtained at the strain SENLAC virus CSF was determined with virulent virus strain "Shi-Ming" in the neutralization IgM microblades (RPM) on the hybrid cells AND4x2. Research results in PH showed the specificity of the strain SENLAC".

Of biotechnological is Noynoy activity. The strain intended for use as raw material in the manufacture of VirusWall against CSF. The strain SENLAC virus CoES well reproduces in the body of rabbits. For intravenous injection of the virus is most clearly react adult rabbits weighing 2.0 to 2.5 kg In these rabbits temperature increase observed after 26-30 hours after exposure and lasts up to 72-96 hours. The highest titer of virus in the blood of 5.0 lg EID50/2 cm3been through 44-50 hours after the injection of the material. For growing strain SENLAC" tested cell culture spew, VIRO, KSS-21, RK-15 and other transplantable cells. The greatest accumulation of virus observed in the cultures of RK-13 (up to 5.0 lg TCD50/cm3and hybrids AND4x C2(5.5 lg TCD50/cm3). The optimal conditions for the cultivation of strain SENLAC" was a 3-day transplantable cell cultures of RK-13 and hybrids AND4x2making in a supportive environment 5-10% fetal serum of cattle.

The strain SENLAC" retains its original characteristics in consecutive passirovannye in sensitive biological systems for 9-10 passages (observation period).

Chemo - and geotectonically feature

The strain SENLAC virus CoES ablany genome is a single-stranded plus-RNA, consisting of 12284 nucleotides with a single long open reading frame corresponding to polyprotein long 3898 amino acid residues. In the genome of the virus CSF genes are from T-end-to - end in the following sequence: Tr/48-Dr-Dr.

Data on the structural proteins of strain SENLAC" obtained through research by electrophoresis and SDS page. Purified in the gradient virions contain 3 main types virousspecificakih proteins, mol. weight 54-C, 45-C and 35 C. The two most large polypeptide are glycoproteins located on the surface of the virion. The viral origin of these polypeptides was confirmed by the method of radioimmune precipitation. The strain SENLAC" has the polypeptides El(t), E2(t) and C(R36). Polypeptide Dr contains major immunoreactive dominants involved in the neutralization of the infectivity of the virus in CSF.

Biological activity

The biological activity of strain SENLAC" was determined by titration on rabbits weighing 2.0 to 2.5 kg were Prepared by 10-fold dilution of the virus from 10-3-10-5. A suspension of each dilution, starting with the largest, was injected intravenously in a volume of 2 cm34 rabbits. As a con the titration was carried out for 7 days in the presence of hyperthermia. The biological activity of vaccinated suspension was 4,67 lg EID50/2 cm3.

Immunogenic properties

The strain SENLAC" of the CSF virus in injecting adult rabbits and pigs stimulates the formation of a busy immunity and resistance of pigs to the disease when infected with CSF virus strain "Shi-Ming". In experiments with piglets that on the 10-14th day after intramuscular injection of a suspension of the virus during the study RPMB appear antibody titer of 1: 16 with a subsequent increase to 21 days, up to 1:32-1:64. When infection control 21 days after immunization with virulent strain of "Shi-Ming" in a dose of 104,5LD50vaccinated pigs did not get sick. In control animals the fever came on day 3 after infection, and on the 5th-9th day they fell.

Reactogenic properties

After injection of strain SENLAC virus CoES pigs at a dose of 100-1000 EID50in about 20-25% of vaccinated animals there is an increase in body temperature to 40.1 40.7 inoWith, which is held within 1-3 days without suppression of the General condition. The average weight gain of animals (per month) after injection of strain is reduced (compared to control) of only 0.6 to 1.2 kg.

When irusa CoES stores within 9-10 serial passages on the pigs.

Persistence

The strain SENLAC virus CoES well stored at -70oWith the native or dried form in vials under hermetically sealed tubes and metal caps. The titer of the virus within 12 months of storage is practically not reduced, and immunogenic activity was not changed. However, the native storage of the virus within one month at +4oWith substantially reduces its contagious.

For more information about the strain

Vaccinal strain "SENLAC is contagious. With the introduction of the seronegative gilts does not cause signs of disease animals CoES.

Free from viral, bacterial and mycoplasmal contaminants.

Safety and immunogenicity of the strain SENLAC" fully meets the requirements of O. I. E. (OIE).

Commission trials have confirmed full compliance with strain SENLAC" the requirements and was the basis for its Deposit in the Collection of microorganisms VGNKI veterinary preparations under the registration name "SENLAC"-DEPT.

On the basis of the received information, it can be argued that the strain SENLAC virus CoES on antigenic and immunological spectra is the origin of isoetaceae danger CoES timely vaccination of animals offered by the vaccine on the basis of strain SENLAC" in threatened and affected by this disease farms.

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, characterized by signs, identical to all the essential features 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 VirusWall set forth in the independent claim.

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 claim. The search results showed that the proposed solution does not follow for professionals explicitly of the world is s solutions have signs consistent with the distinctive features of the present invention), and revealed no effect provided the essential features of the invention transformations to achieve a technical result.

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

The invention is illustrated by the following examples which do not limit the scope of the invention.

Example 1.

The strain SENLAC" is used for the manufacture lepidosirenidae VirusWall against CSF. To do this, the rabbits weighing 2.5 to 3.0 kg intravenously injecting a suspension of strain SENLAC" in a dose of 100 ID50in the amount of 2.0 cm3. In animals daily measure body temperature. Rabbits, reacting through 42-52 hour temperature rise to 40.5-41,8oWith that Deplete totally. From each animal blood is taken in a separate sterile vial containing 1000 IU of heparin. The blood is then poured into one container, mix, select a sample of 10 cm3to control the titer of the virus, cooled to a temperature of 4-8oWith and observing sterile conditions, centrifuged at 3000 rpm for 20 min for the liberation the and add antibiotics in an effective amount. Received vaccinated material combine with medium drying in the ratio required for obtaining vaccines with a given immunogenic activity. Components of a protective environment using commonly used for these purposes, ingredients: sucrose, gelatin and hydrolyzed lactalbumin. For the preparation of a protective environment take 50% sucrose solution, 10% solution of gelatin and 20% solution of hydrolyzed lactalbumin and connect them in the ratio of 1:0,5:2,5 respectively. The mixture of antigen and protective environment is thoroughly mixed, if necessary, bring the pH of the mixture to 7.2 to 7.4, poured into sterile vials (ampoules) 2-4 cm3and subjected to freeze-drying. After drying, the vials closed with rubber stoppers and running aluminum caps, ensuring the integrity of the bottles. Ready biologic is a dry amorphous mass of red-brown color, easily soluble adding saline for 1-2 minutes

The vaccine is used for prophylactic purpose, as well as internally in threatened and affected with CSF farms. Pigs subjected to immunization vaccine in the amount of 2 cm3containing 100 EID50for rabbits.

Polucen the obsession vaccinated material in the range of 70-80 wt.% represents the effective amount of antigen in the vaccine.

Example 2.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 4.5 lg EID50/2 cm3- 60

Protective environment 100

Received vaccine inspected for sterility, safety, biological and immunogenic activity.

Check sterility

Control vaccine in the absence of bacterial and fungal contamination is subjected to the average sample of the vaccine of five vials. The test is carried out in accordance with GOST 28085-89. The vaccine is recognized sterile.

Verification of the safety of vaccines

Test harmlessness spend two piglets 3-4 months of age weighing 30-35 kg, obtained from a well-plague economy and have not been previously vaccinated against this infection.

The average sample prepared from 10 vials of vaccine. In the following of the average sample of whole vaccine in the amount of 2 cm3(tenfold privigna dose) injected pigs intramuscularly. Before the introduction of vaccines and for the next 7 days the animals measure body temperature. Observed the t deviations from the norm. The vaccine is recognized as harmless.

Verification of biological activity in rabbits

To determine the biological activity using adult rabbits weighing 2.0 to 2.5 kg Of the average sample is prepared by mixing digidratirovannogo content of 10 vials of vaccine, prepared dilution in saline solution of 10-2up to 10-5.

Of each dilution, starting with the largest, administered to rabbits intravenously in a volume of 2 cm34 heads of each dilution. The controls are 2 of the rabbit, which is administered saline in the same volume. All animals three times daily measure body temperature. The number of rabbits with hyperthermia calculate the titer of the virus in the vaccine, the value of which expresses the biological activity of the vaccine. She is 4.5 lg EID50/2 cm3.

Check immunogenic in pigs

To control immunogenic vaccines use 14 piglets at the age of 3-4 months, weighing 30-45 kg Of average samples prepared by mixing registrationpage content of 10 vials of vaccine, prepared in saline solution breeding with 10-2up to 10-4that the amount of 2 cm3enter the pigs intramuscularly at a rate of 4 goal of all animals before vaccination and before infection investigate the serum content of neutralizing antibodies. Observing animals are within 14 days with daily changes in body temperature during the first 7 days. On day 14, all vaccinated and control group animals infect the CSF virus strain "Shi-Ming" by intramuscular injection of 1 cm3with 104LD100. For infected animals are observed for 15 days with daily thermometry. It is established that all vaccinated animals after control of infection did not show deviations from the norm and remained healthy the entire period of observation. Activity of blood serum in their pH ranged from 5,23 to 6.0 log2(1:36-1:64), while before vaccination was less than 1:2.

In control animals the rise in body temperature observed at 5-7-th day. Later they refused to feed, not moving. Death came on 11-13 day after infection. When pathologoanatomic study proved characteristic of the plague changes in the skin, spleen, lymph nodes, bleeding heart, kidney, large intestine.

Thus, dry lepidosirenidae virusvaktsinu against CSF from strain SENLAC" sterile, harmless, reactogenic. The biological activity of the vaccine in rabbits is 4.5 neutralizing antibody 14 days after inoculation in the credits from 5,23 to 6.0 log2and provides protection from the control of infection of vaccinated animals. Immunogenic activity of the vaccine in pigs amounted to 4.5 lg EID50/2 cm3.

Example 3.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 4.5 lg EID50/2 cm3- 70

Protective environment 100

The resulting virusvaktsinu tested for immunogenic activity in seronegative gilts weighing 16-25 kg not previously been vaccinated against CSF. To do this, the contents of 10 vials of the drug rehydratable adding 2 cm3the saline solution in each and mixed in the same container. For vaccination of pigs from the initial mixture was prepared by ten-fold dilution with a final concentration of 1000, 100, 10 and 1 ID50/2 cm3for rabbits, respectively, is 10; 1; 0.1, and 0.01 prilivnykh doses for pigs. Each dilution was injected 3 gilts of 2 cm3intramuscularly behind the ear. Before vaccination and within 14 days after vaccination and 14 days after infection the animals was measured daily body temperature and led viewers the bottom box from immunized animals. In animals prior to vaccination and before infection was obtained blood samples for research neutralizing activity in serum RPM. After 21 days, all vaccinated and 2 control animals were infected with CSF virus strain "Shi-Ming". Source vaccinated suspension after thawing and centrifugation was mixed with a phosphate buffer solution (FBI) in the ratio of 1:30 and was administered to animals in a volume of 1 cm3intramuscularly with the inner side of the thigh. The monitoring was conducted within 14 days.

During the titration of the vaccine in rabbits from the original content of the mixture 10 bottles prepared tenfold diluted to 10-5, which was injected intravenously in a volume of 2 cm3. Before vaccination in animals was measured body temperature and monitoring process within 7 days measurement was performed three times daily.

As a result of the research showed that the CSF virus strain SENLAC and vaccine made from vaccinated this material is not contagious. Intact pigs in confinement for 21 days in one box with grafted did not show signs of disease CSF and serum of these animals have been detected neutralizing antibodies to the virus CSF. The vaccine, and sledovatelno 4,5 lg EID50/2 cm3and immunogenic activity. In piglets immunized with the vaccine with the content of 10; 1; 0.1, and 0.01 pravilnoy dose for pigs, the titers of neutralizing antibodies 21 days after vaccination ranged from a 5.25 to 6,33 log2and the animals were resistant to experimental infection with CSF virus strain "Shi-Ming".

Example 4.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 5.0 lg EID50/2 cm3- 80

Protective environment 100

The efficiency obtained VirusWall checked against epizootic isolate virus CoES "Moldova-98", causing disease of pigs in Moldova. Epizootic isolate received at the research Institute of animal production assay in the pig, as well as by PCR and nucleotide sequencing of part of the genome was diagnosed as a virus CSF. At the same time for more research in PCR and nucleotide sequencing were subjected vaccinated material of the strain SENLAC" and the vaccine, from which it was made. In this experiment it is ldova-98", passed 2 of the passage of the pigs. To test the effectiveness of VirusWall against CSF from strain SENLAC" was carried out as described in example 2.

As a result of the research showed the following. Virusvaktsinu against CSF from strain SENLAC" is specific, as the serum of vaccinated pigs will neutralize in RPM antigen CSF virus strain "Shi-Ming". The titer of antibody in immunized animals ranged from 5.17 to to 5.75 log2and not depended on pravilnoy dose.

Virusvaktsinu against CSF from strain SENLAC" has sufficient immunogenicity and against epizootic virus, when administered to pigs 1 or 10 prilivnykh doses of 2 cm3stimulates the production of specific antibodies in titers higher than 5.0 log2and protects animals from disease during experimental infection with isolate "Moldova-98".

Example 5.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 4.5 lg EID50/2 cm3- 70

Protective environment 100

The efficiency obtained VirusWall dvigaetsa vaccination against CSF virus. The last time the animals were immunized with venusvalley against CSF from strain "LK-Vniivvim". Prior to testing in animals of different ages were selected blood with the aim of studying the level of neutralizing antibody to the virus CSF. It was found that the titer of antibodies in sows amounted to 4.23-5.33 log2(M m = 4,75 0,32), fattening 3,83 5,76 log2(4,92 0,43), suckling piglets aged 32-40 days 2,83 4,24 log2(3,61 0,42).

Vaccination subjected 74 sows, 115 heads of pigs at the age of 35-40 days. Animals were vaccinated according to the following scheme: sows for 3-40 days prior to mating, pigs at the age of 35-40 days, followed by a booster 3-4 weeks. The level of neutralizing antibodies 21 days after vaccination was in sows 5,76-7,17 log2(6,33 0,52), in young 5,0-6,55 log2(5,81 0,28). 30 days after re-vaccination titer neutralizing antibodies in calves ranged from 5,75 to 7.0 log2(6,35 0,24).

After 60 days after vaccination, the level of specific antibodies in sows to the virus classical swine fever was 7.5 - 8,23 log2.

Thus, virusvaktsinu against CSF from strain SENLAC" causes the formation of a busy immobilisation the drug. In the process of testing the developed product is not noted any deviations in animals from the normal physiological state.

Example 6.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 5.0 lg EID50/2 cm3- 60

Protective environment 100

The efficiency obtained VirusWall tested on pigs in disadvantaged by CoES JSC "Syzran" Radischevskaya district of the Ulyanovsk region. In the seat of the disease once and ten doses of vaccine were inoculated 3840 pigs of different age groups, including piglets from 7 days of age. 10 days after the vaccine disease in the outbreak ceased.

The quarantine management was removed. The use of vaccines in single and ten doses had the same positive result.

Example 7.

Tests VirusWall against CSF, produced as described in example 1 and containing, wt.%:

Vaccinated material of the strain SENLAC", reproducido is 100

The effectiveness of VirusWall against CSF from strain SENLAC", manufactured as described in example 1, tested on pigs on a good CoES the pig farm "Pornoroliki" suburban district of Sverdlovsk region. The vaccine is used for prophylactic vaccination. Was vaccinated 70 000 pigs, including 19 980 sows basic and repair, as well as 50 020 heads of young animals. For immunization of pigs used vaccine in the amount of 110 800 doses. After drug complications in vaccinated animals have been detected. Periodically examined serum from vaccinated animals in RPM on the intensity of the immune system. Just was studied 53 samples. Antibody titers were in the range from 5.0 to 6.0 log2, i.e., at a sufficiently high level. Agriculture remained prosperous for CoES.

Thus, the above information shows the implementation of the use of the present invention, the following cumulative conditions:

- virusvaktsinu against CSF-based strain SENLAC", embodying the invention, intended for use in veterinary Virology and biotechnology;

for the present invention including usuwania with contained in the description or known before the priority date tools and techniques;

- virusvaktsinu against CSF-based strain SENLAC", received in accordance with the invention, has a high antigenic and immunogenic activity and can provide reliable protection against CSF virus circulating on the territory of the Russian Federation.

Therefore, the present invention meets the condition of patentability "industrial applicability".

Sources of information

1. Hudson R. L. Peste porcine. L adaptation du virus au papin et 'utilisation du virus adapte pour immuniser les pors. Bull. Off. Epis., 1953, 40, 60-73.

2. Likhachev N. In. Advances in viral biological products and vaccines for their further improvement. Proceedings of the VGNKI veterinary preparations, 1972, 18, 22-29.

3. RF patent 2096454; IPC612 N 7/00, a 61 K 39/187; 20.11.97,

4. RF patent 2064302, IPC6A 61 K 39/187, 27.07.96,

5. RF patent 2057805; IPC6C 12 N 7/00, a 61 K 39/187; 10.04.96,

6. RF patent 2129443; IPC6A 61 K 39/187, C 12 N 7/00, 27.04.99,

7. Sergeev, C. A. Viral vaccines, Kiev, "Harvest", 1993, 285-294.

8. Kojnok F. , Palatka Z et.al. Requirements of rabbit-adapted swine fever vaccine. Arch. Exp. Veterinarmed., 1980, 34(1), 67-72 (the prototype).

1. Virusvaktsinu against classical swine containing vaccinated material from attenuated rushkin contains a virus strain Pestis suum, SEM. Flaviviridae, genus Pestivirus, collection VGNKI, registration name "SENLAC"-the DEPOT, in an effective amount.

2. Virusvaktsinu under item 1, characterized in that it contains vaccinated material derived from an attenuated strain "SENLAC" of the causative agent, reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/2 cm3.

3. Virusvaktsinu under item 2, characterized in that it contains vaccinated material derived from an attenuated strain "SENLAC" in the amount of 60-80 wt. %.

4. Virusvaktsinu under item 1, characterized in that it contains a protective environment in an effective amount.

5. Virusvaktsinu under item 1 or 4, characterized in that it contains a protective medium containing 50% sucrose solution, 10% solution of gelatin and 20% solution of hydrolyzed lactalbumin in the ratio of 1: 0,5: 2,5 respectively.

6. Virusvaktsinu according to any one of paragraphs. 1, 4 and 5, characterized in that it contains a protective environment in the amount of 20-40 wt. %.

7. Virusvaktsinu according to any one of paragraphs. 1-6, characterized in that it contains vaccinated material of the strain SENLAC", reproduced in the body of the rabbit with the biological activity of 4.5-5.0 lg EID50/2 cm3and protective environment in the next soothes the

 

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SUBSTANCE: the suggested vaccine contains suspension of viable spores of anthracic vaccinal strain "55-VNIIBB&M" at initial concentration of 500-700 mln. spores/cu.cm, cultural virus-containing raw material of vaccinal virus of cattle plague of "LT" strain at activity of not less than 7.0 lg TCD50/cu. cm, lactosopeptonic stabilizing agent and distilled water at the following content of components,%: spores of anthracic strain "55-VNIIVV&M" -6.2 - 10.0; cultural raw material of cattle plague virus of "LT" strain -25.0 - 30.0; lactosopeptonic stabilizing agent -48.0 - 50.0; distilled water - the rest. One vaccinal dosage contains about 20-25 mln. anthracic spores and about 4.5-5.5 lg TCD50 of cattle plague virus The suggested vaccine is of high immunogenicity, develops tense immunity in once vaccinated cattle that lasts for 12 mo, not less, moreover, it is areactogenous, safe and stable at storage.

EFFECT: higher efficiency.

3 ex, 3 tbl

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