The strain senlac virus pestis suum for the manufacture of vaccines

 

(57) Abstract:

The invention relates to veterinary Virology and biotechnology. Vaccinal strain "SENLAC virus CoES obtained by the alternating passages lepidosirenidae of CSF virus strain Chinese on cell cultures of RK-13, hybrids AND4WITH2and adult rabbits. The strain is deposited in the Collection of microorganisms VGNKI under the registration name "SENLAC"-DEPT. The strain SENLAC" differs from the production and from the previously selected epizootic strains of the virus in CSF. The virus strain SENLAC" well reproduces in the body of rabbits and transplantable cell cultures of RK-13 (up to 5.0 lg TCD50/cm3and hybrids AND4WITH2(5.5 lg TCD50/cm3). Preserves the original characteristics of the sequential passirovannye in sensitive biological systems for 9-10 passages (observation period). The strain has a broad antigenic spectrum of activity, high biological and immunogenic activity, suitable for the manufacture of effective vaccines. 2 Il., table 1.

The invention relates to the field of veterinary Virology and biotechnology and can be used in the manufacture of tools for specifice disease of pigs characterized by a variety of clinical forms and manifestations, namely fever, lesions of the blood and blood-forming systems (phenomena hemorrhagic septicemia), lobar pneumonia and lobar-differencesin inflammation of the intestine. The causative agent is a virus 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 the reservoirs and transmission routes and infection is well known, practical experience shows that implement a set of security is about.

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

Known lepidosirenidae strain "Hudson" Flavivirus suis "Hudson" virus CoES, which was used in the USSR in the 60-ies for manufacturing lepidosirenidae VirusWall against CSF (1,2).

The disadvantage of this strain is its increased reactogenicity for vaccinated animals.

Known strain GM-94" VGNKI virus CSF used for manufacturing lepidosirenidae VirusWall. The strain GM-94" VGNKI obtained from strain "Hudson" of the CSF virus by serial passages through the body of the rabbit and interspersed by serial passages in cell culture PPP and rabbits using the method of limiting dilutions with identification of the pathogen in the neutralization (PH) with specific serum against reference strains of the virus CoES, as well as a selection of options on the grounds of safety for piglets 1.5 to 3 months of age and intensity of its reproduction in the sensory system of the body of the rabbit. Evaluation of immunogenic activity of the obtained strain was carried out by inoculation of pigs with their subsequent infection control EP"ptx2">

However, many vaccines against CSF lies Chinese attenuated lepidosirenidae strain of the virus in CSF. It is widely used in the world practice of vaccination of pigs against CSF (4).

A significant disadvantage is the low immunogenic activity and low level of accumulation in human cell cultures.

A known strain "LK-Vniivvim virus CSF used for the manufacture of VirusWall. The virus propagated in primary culture cells of the testes of the lambs (5).

The disadvantages of this strain are unprofitable use of substandard (young) farm animals, unintended consequences of viral contamination of raw materials other pathogenic or opportunistic agents from the donor tissue, the seasonality of donor tissue (testes lambs, spring and summer), low immunogenicity, derived from this strain.

A known strain "LC-virus CoES for manufacturing culture VirusWall. Strain "LK-K" obtained from a strain of "LK-Vniivvim" cloning and selective sampling of the obtained clones on the basis of high accumulation in human cell cultures of RK-15 (6).

The disadvantage of anstamm "CS" for making VirusWall against CSF. The vaccine of this strain is administered to pigs in dysfunctional households repeatedly in large doses containing previfem volume 100000-1000000 Eid50(7).

The disadvantage of this strain is its low immunogenic activity.

The closest present invention, the set of essential characteristics is a strain of "Chinese" virus CoES used as production in the manufacture of dry lepidosirenidae VirusWall.

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 disadvantage is the low broadca creation of the present invention was to obtain a new production strain of the virus CoES, with a broad antigenic spectrum of activity, high biological and immunogenic activity and for producing effective VirusWall.

The technical result from the use of the invention is to obtain a new production of CSF virus strain with a broad spectrum of activity, high biological and immunogenic activity and suitable for the manufacture of dry lepidosirenidae VirusWall.

This technical result is achieved by obtaining strain SENLAC virus CSF. The strain SENLAC" is a new, previously unknown. The strain SENLAC" obtained by carrying out intermittent passages lepidosirenidae of CSF virus strain "Chinese" in cell cultures of RK-13, hybrids AND4WITH2and 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) 5 October 1999 under registrationmanager 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, where:

Fig. 1 shows the amino acid sequence of the region's major antigenic domains of the glycoprotein E2 (gp55) of CSF virus strain SENLAC";

Fig.2 shows a dendrogram reflecting the relationship between different strains of the virus CoES based on structural homology of their genomes.

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 passages lepidosirenidae of CSF virus strain "Chinese" in cell cultures of RK-13, hybrids AND4WITH2and 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 PAE is and cell cultures of RK-13, 25 passages on the hybrid cell cultures AND4WITH2and 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/O that expresses these characteristics: single-stranded linear RNA with a molecular weight of 410 Dalton, % 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.

Morphological and physical characteristics

When electron-microscopic study of the strain SENLAC" presents typical flaviviruses separate virions are spherical or round shape with a diameter of 40 to 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 antipathy research presented 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 AND4WITH2. Research results in PH showed the specificity of the strain SENLAC".

Biotechnological characteristics

The strain SENLAC virus CSF shows high biological, antigenic and immunogenic 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 to 30 h after infection 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 h after centuries the so called cells. The greatest accumulation of virus observed in the cultures of RK-13 (up to 5.0 lg50/cm3and hybrids AND4WITH2(5,5 lg50/cm3). The optimal conditions for the cultivation of strain SENLAC" was a 3-day transplantable cell cultures of RK-13 and hybrids AND4C2making 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" of the CSF virus is an RNA-containing virus with mol. weight 4106D. the Percentage of RNA in the virion is not installed. The viral 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: gp44/48-gp33-gp55.

Data on the structural proteins of strain SENLAC" obtained through research by electrophoresis and SDS page. Purified in the gradient virions contain 3 ptid 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(gp55), E2(gp46) and C(R36). Polypeptide gp55 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 control was used 2 rabbits that were injected with phosphate buffer solution in the same volume. Records of the results of 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 for 10-14 days after nutrimix the 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 at 5-7 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 40.1-41oWith, which is held within 1-4 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.

Stability

Acquired properties (absence of reversion and weakened reactogenicity) strain "SENLAC virus 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 +4o

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 original, in taxonomic relation to new, previously unknown variant of the virus CSF.

According to the applicant, the proposed strain meets the conditions of patentability of "novelty" and "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 D. through 42-52 h 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./min for 20 min to release from membranes destructively erythrocytes and possible clots. The supernatant is collected in a bottle 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. 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 which is a dry amorphous mass of red-brown color, easily soluble adding saline for 1-2 minutes

The vaccine used in threatened and affected with CSF farms. Pigs subjected to immunization vaccine in the amount of 2 cm3containing 100 EID50for rabbits.

Received a vaccine against CSF has an optimal component composition, wt.%, see the table at the end of the description.

Content vaccinated material in the range of 70-80 (wt.%) represents the effective amount of antigen in the vaccine.

Example 2.

A series of VirusWall against CSF from strain SENLAC", manufactured as described in example 1, is subjected to the control 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 E. the 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. Monitoring the clinical condition of the animals are within 14 days.

During this period, the animals do not reveal abnormalities. The vaccine is recognized as harmless.

Check immunogenic

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 heads of each dilution. Two unvaccinated piglets serve as a control and contained in a separate box. All animals before vaccination and before infection investigate the serum content of neutralizing antibodies. Observation of the animals are in a period of 20 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 cm32(1:36-1:64), while before vaccination was less than 1: 2.

In control animals the rise in body temperature observed at 5-7 day. Later they refused to feed, not moving. Death came at 11-13 days 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 vaccine has a pronounced immunogenicity, because it stimulates the production of neutralizing antibodies 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 IPD50/2 cm3and biological activity in rabbits 4,0 lg IPD50/2 cm3.

Example 3.

Another series virusburst on 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 observed for General condition. Control gilts in number 4 goals contained the entire period of the experiment in one 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 vnutrenestey content 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 hence the strain SENLAC", which made the drug, possess significant biological (titer of the virus in rabbits 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.

The effectiveness of VirusWall against CSF from strain SENLAC" checked against epizootic isolate virus CoES "Moldova-98", visualruby on the pigs, as well as 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 was subjected vaccinated material of the strain SENLAC" and the vaccine, from which it was made. In the present experiment was used 10% suspension of the spleen, lymphocytes and blood of piglets infected with epizootic virus CoES "Moldova-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 which you want to make VirusWall against CSF from strain SENLAC", manufactured as described in example 1, tested on pigs in JSC "Kommunar" the Vladimir region. All pigs in JSC "Kommunar" is 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(Mm=4,750,32), fattening 3,835,76 log2(4,920,43), suckling piglets aged 32-40 days 2,834,24 log2(3,610,42).

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(6,330,52), in young 5,0-6,55 log2(5,810,28). 30 days after re-vaccination titer neutralizing antibodies in calves ranged from 5,75 to 7.0 log2(6,350,24).

After 60 days after vaccination, the level of specific collective against CSF from strain SENLAC" causes the formation of a busy immunity in pigs of different age groups. The level of antibodies against CSF virus indicates a highly active drug. In the process of testing the developed product is not noted any deviations in animals from the normal physiological state.

Example 6.

The effectiveness of VirusWall against CSF from strain SENLAC", manufactured as described in example 1, 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.

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 grafted 70000 heads is used vaccine in the number 110800 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:

a strain of "SENLAC virus CoES embodying the invention, intended for use in veterinary Virology and biotechnology;

for the present invention in the form as it is described in the independent claim, confirmed the possibility of its use are given in the description or known before the priority date tools and techniques;

a strain of "SENLAC virus CoES obtained in accordance with the invention, has a high antigenic, biological and immunogenic activity and suitable for the manufacture of dry lepidosirenidae VirusWall against CSF.

Therefore, the present invention souvenance into account when preparing the description of the invention the application for the grant of a patent of the Russian Federation on the "Strain "SENLAC virus CoES for making vaccines"

1. Hudson R. L. Peste porcine. L adaptation du virus au papin et I utilisation du virus adapte pour immuniser les porcs. 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. Sergeev, C. A. Viral vaccines. Kiev, "Harvest", 1993, 285-294.

5. RF patent 2064302, IPC6A 61 To 39/187, 27.07.96,

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

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

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

The virus strain Pestis suum, SEM. Flaviviridae, genus Pestivirus, collection WGNC, registration name "SENLAC"-the DEPOT, for the manufacture of vaccines.

 

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3 ex, 3 tbl

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