RU2236253C2 - Emulsion inactivated vaccine against pig reproductive-respiratory syndrome - Google Patents

Abstract

FIELD: veterinary virology.

SUBSTANCE: emulsion inactivated vaccine against pig reproductive-respiratory syndrome (PRRS) comprises antigenic material from the strain "BD" of homologous virus reproduced in transplanted culture of cells Marc-145, aminoethylethyleneimine as an inactivating agent and oily adjuvant in the effective ratio. The strain is deposited in collection of microorganisms VGNKI at registration number "BD-DEP". The strain "BD" is reproduced in transplanted culture of cells Marc-145 with biological activity, at least, 6.0 lg TCD₅₀/cm³. Vaccine represents emulsion of white or white color with pink tint. Vaccine is administrated to animals by intramuscular route behind of ear in the dose 2 cm³ being independently of their age. Immunity in vaccinated animals is developed on 21-30 day after vaccination and retains for up to 12 months. Vaccine induces high level of antibodies against PRRS, provides safe passive mother immunity in young animals before 20-25 day age that provides protecting animals against field virulent virus PRRS in early period of life.

EFFECT: enhanced effectiveness and valuable properties of vaccine.

10 cl, 5 tbl, 5 ex

Description

The invention relates to veterinary virology and biotechnology and can be used in the development and manufacture of means for the specific prevention of pig reproductive and respiratory syndrome (PRRS).

PRRS is a new disease of viral etiology, characterized by impaired reproductive functions in sows and repair gilts, pneumonia in piglets during rearing and an increase in their mortality in the pre-period. The disease was first registered and described in 1987 in the United States and Canada, and since 1990, the disease has been reported in Western Europe and Southeast Asia. According to official data of the OIE in 1996-1998. the disease was recorded in 30 countries. PRRS was established in Russia in 1993 and today this disease has been diagnosed in many regions and regions of Russia. For the first time, the PRRS virus was isolated and studied by scientists from the Central Veterinary Institute in the Netherlands in 1991. The virus was classified as a member of the Arteriviridae family and assigned to the genus Arterivirus. In animals, the virus multiplies in the alveolar macrophages of the lungs and subsequently, as a result of viremia, it accumulates in the uterus and other organs. When the virus circulates in the blood, it easily overcomes the transplacental barrier and affects the fetus. Depending on the gestational age of the sow or the immune response of the mother, the fetus dies or the birth of sick piglets that die in the first days of life. In some farms, the death of newborn piglets reaches 80-90%. An important anti-PRRS agent is its vaccine prophylaxis. Specific PRRS prophylaxis is carried out using live and inactivated vaccines.

Although the advantage of live vaccines due to manufacturability and ease of use is not in doubt, the immune response in pigs is not always stable when administered and in some cases causes complications after vaccination.

At present, emulsion inactivated vaccines prepared from various strains of the virus reproduced in sensitive biological systems are widely used in the global industrial pig breeding for specific prophylaxis of PRRS. Vaccination creates pronounced immunity in sows and prevents the transmission of the virus to embryos and fetuses (1, 2).

However, the problem of increasing the immunogenic activity and safety of the inactivated PRRS vaccine continues to be relevant and is one of the main areas of research to improve them.

Known emulsion inactivated vaccine against PRRS containing antigenic material from strain CNCM No. 1-1102 of a homologous virus reproduced in a sensitive biological system, inactivant and oil adjuvant in an effective ratio (3).

Known emulsion inactivated vaccine against PRRS containing antigenic material from strain CNCM No. 1-1140 homologous virus reproduced in a sensitive biological system, inactivant and oil adjuvant in an effective ratio (4).

A known inactivated emulsion vaccine against PRRS containing antigenic material from the CNCM strain No. 1-1153 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (5).

A known inactivated emulsion vaccine against PRRS containing antigenic material from strain CNCM No. 1-1387 or CNCM No. 1-1388 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (6).

An emulsion inactivated PRRS vaccine is known containing antigenic material from the ATCC strain VR 2402 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (7).

A known inactivated emulsion vaccine against PRRS containing antigenic material from the ATCC strain VR 2509 homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (8).

A known inactivated emulsion vaccine against PRRS containing antigenic material from the ATCC strain VR-2525 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (9).

An emulsion inactivated PRRS vaccine is known containing antigenic material from strain ECACC V-93070108 from a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (10).

An oil adjuvant ARRIAH is also known, containing an oil base and an alkyl-modified polyoxyalkylene siloxane copolymer (APS) as an emulsifier at a ratio of these components (wt.%) 5: 10-90: 95 (11).

The closest to the invention according to the set of essential features is an inactivated emulsion PRRS vaccine containing antigenic material from CNCM strain No. 11102 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (3).

The disadvantages of the known emulsion inactivated vaccines, including the vaccine of the prototype, are that in their manufacture using virulent strains, which always create problems when meeting the requirements of veterinary safety.

In addition, the well-known emulsion inactivated vaccines have never been used in the territory of the Russian Federation, therefore, their effectiveness against Russian PRRS virus isolates has not been studied.

The objective of the present invention was to develop a highly immunogenic and harmless inactivated emulsion PRRS vaccine based on a new production strain of a homologous virus isolated in Russia, which has high biological, antigenic and immunogenic activity in its native form and after inactivation and is suitable for the manufacture of an emulsion inactivated vaccine preparation , creating intense and lasting immunity in vaccinated animals against circulating in the territory Russian Federation of epizootic isolates of PRRS virus.

The technical result from the use of the present invention is to expand the arsenal of inactivated emulsion vaccines against PRRS, which have high immunogenic activity and harmlessness and create intense and lasting immunity in vaccinated animals against epizootic isolates of PRRS virus circulating in Russia.

The specified technical result is achieved by the creation of an emulsion inactivated vaccine against PRRS, characterized by the following set of features:

1. Emulsion inactivated vaccine against PRRS.

2. Antigenic material from an infectious agent reproduced in a sensitive biological system.

3. As the causative agent of infection, the strain "DB" of the PRRS virus in an effective amount.

4. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145.

5. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145, with biological activity of at least 6.0 Ig TCD ₅₀ / cm ³ .

6. Inactivant.

7. Of the inactivants of aminoethylethyleneimine (AEEI).

8. AEEI in the form of a 1% aqueous solution.

9. AEEI at a concentration of 0.001-0.05%.

10. Oil adjuvant.

11. Of the oil adjuvants, the oil adjuvant “Montanide IZA-70”.

12. Antigenic material and oil adjuvant “Montanide IZA-70” in a ratio of at least 1: 2.

13. From oil adjuvants oil adjuvant ARRIAH.

14. Antigenic material and oil adjuvant ARRIAH in a ratio of at least 1: 1.

15. Antigenic material from the strain “DB” of the PRRS virus, AEI inactivant and oil adjuvant in the ratio, wt.%:

Antigenic material 33.3-50.0

AEEI (1% aqueous solution) 0.001-0.05

Oil adjuvant Up to 100.0

The present invention includes a combination of essential features that provide a technical result in all cases for which legal protection is sought:

1. Emulsion inactivated vaccine against PRRS.

2. Antigenic material from an infectious agent reproduced in a sensitive biological system.

3. As the causative agent of infection, the strain "DB" of the PRRS virus in an effective amount.

4. Inactivant.

5. Oil adjuvant.

The present invention is also characterized by other features expressing specific forms of execution or special conditions for its use:

1. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145.

2. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145, with a biological activity of at least 6.0 Ig TCD ₅₀ / cm ³ .

3. Of the inactivants AEEI.

4. AEEI in the form of a 1% aqueous solution.

5. AEEI at a concentration of 0.001-0.05%.

6. Of the oil adjuvants, the oil adjuvant “Montanide IZA-70”.

7. Antigenic material and oil adjuvant “Montanide IZA-70” in a ratio of at least 1: 2.

8. Of the oil adjuvants, the oil adjuvant ARRIAH.

9. Antigenic material and oil adjuvant ARRIAH in a ratio of at least 1: 1.

10. Antigenic material from the strain “DB” of the PRRS virus, inactivator AEI and oil adjuvant in the ratio, wt.%:

Antigenic material 33.3-50.0

AEEI (1% aqueous solution) 0.001-0.05

Oil adjuvant Up to 100.0

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. Emulsion inactivated vaccine against PRRS.

2. Antigenic material from an infectious agent reproduced in a sensitive biological system.

3. Inactivant.

4. Oil adjuvant.

Compared with the prototype vaccine, an essential distinguishing feature of the present invention is that the proposed vaccine contains antigenic material from the strain “DB” of the PRRS virus taken in an effective amount.

The present invention is also characterized by other distinctive features expressing specific forms of execution or special conditions for its use:

1. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145.

2. The strain "DB" of the PRRS virus, reproduced in transplanted cell culture Mars-145, with biological activity of at least 6.0 Ig TCD ₅₀ / cm ³ .

3. Of the inactivants AEEI.

4. AEEI in the form of a 1% aqueous solution.

5. AEEI at a concentration of 0.001-0.05%.

6. Of the oil adjuvants, the oil adjuvant “Montanide IZA-70”.

7. Antigenic material and oil adjuvant “Montanide IZA-70” in a ratio of at least 1: 2.

8. Of the oil adjuvants, the oil adjuvant ARRIAH.

9. Antigenic material and oil adjuvant ARRIAH in a ratio of at least 1: 1.

10. Antigenic material from the strain “DB” of the PRRS virus, inactivator AEI and oil adjuvant in the ratio, wt.%:

Antigenic material 33.3-50.0

AEEI (1% aqueous solution) 0.001-0.05

Oil adjuvant Up to 100.0

The proposed vaccine expands the arsenal of means of specific prevention of PRRS.

The achievement of the technical result from the use of the invention is explained by the fact that the composition of the emulsion inactivated vaccine against PRRS introduced antigenic material from a new strain of “DB” homologous virus with high biological, antigenic and immunogenic activity in its native form and after inactivation and providing a harmless vaccine, creating intense and lasting immunity in vaccinated pigs.

An additional technical result from the use of the invention is achieved due to the fact that of the inactivants, the vaccine contains AEEI, which can significantly reduce labor and energy costs in the manufacture of the drug and improve the quality of antigenic material.

An additional technical result from the use of the invention in terms of increasing the antigenic and immunogenic activity of the target product is also achieved through the use of the oil adjuvant “Montanide IZA-70” or the oil adjuvant ARRIAH in its composition.

The strain "DB" is a new, previously unknown strain of the PRRS virus, in its native form non-pathogenic.

The initial virus for obtaining the strain “DB” was isolated from the blood serum of pigs with clinical signs of PRRS obtained from the farm “Dawn” of the Kirovsky district of the Stavropol Territory in 1996. The production strain “DB” was obtained by repeated consecutive passages on a 3-day culture of Mars-145 cells.

The strain "DB" was deposited in the All-Russian state collection of strains of microorganisms used in veterinary medicine and livestock, the All-Russian State Research Institute of Control, Standardization and Certification of Veterinary Medicines (VGNKI) May 25, 1998 under the registration code "BD-DEP".

The strain "DB" is harmless and has a high biological antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the manufacture of vaccine preparations has been experimentally confirmed.

The strain "DB" of the PRRS virus is characterized by the following features and properties.

Morphological properties

The strain “DB” of the PRRS virus belongs to the family Arteriviridae, the genus Arterivirus, is RNA genome, has morphological characteristics characteristic of the PRRS virus: the shape of the virion is spherical, the size of the virion is 45-75 nm with a core filling 3/4 of the virion with a diameter of 25-35 nm surrounded by a lipid membrane.

Antigenic properties

The PRRS virus of the “DB” strain is stably neutralized by a homologous antiserum. The virus does not have hemagglutinating properties. When vaccinated, the virus induces the formation of specific antibodies detected in an enzyme-linked immunosorbent assay (ELISA). The method of polymerase chain reaction (PCR) revealed genomic RNA of the PRRS virus. The nucleotide sequencing of the strain “DB” of the PRCC virus showed its significant (about 40%) difference from the CNCM strain No. 1-1102 and the ATCC strain VR-2332, recognized as reference in Europe and North America, respectively. Identification and specificity of PRRS virus was determined in ELISA (IDEXX, USA) with specificity control. The result of the differential diagnosis was negative for Aujeszky's disease virus, swine parvovirus, cattle diarrhea virus and transmissible gastroenteritis virus.

Biotechnological characteristics

The strain "DB" exhibits high biological, antigenic and immunogenic activity. The strain "DB" is intended for the preparation of diagnostic and vaccine preparations. The strain “DB” of the PRRS virus is reproduced in the cytoplasm of cells of a 3-day culture of Mars-145 cells at a temperature of 37 ° C. The original suspension from the blood serum containing the virus was infected with a Marc-145 cell culture. After 7-10 passages within 48-72 hours of incubation, the virus accumulates to a titer of 4.0-4.5 Ig TCD ₅₀ / cm ³ . Virus infectivity was increased by serial, rapidly alternating passages, which by passage 41 reached 7.0-7.5 Ig TCD ₅₀ / cm ³ . The strain "DB" is stable and harmless.

Chemo- and genotaxonomic characterization

The strain "DB" is an RNA-containing virus. Nucleic acid contains one strand, the surface of the virus is covered with a layer of glycoproteins. The protein shell detects the following fragments:

M - transmembrane protein,

E - membrane protein

G _L - surface glycoprotein,

G _S - surface glycoprotein,

N is a nucleocapsid protein.

Physical properties

The mass of the virion is from 49 · 10 ³ Yes to 55 · 10 ³ Yes. The floating density in the gradient of cesium chloride is 1.18-1.2 g / cm ³ .

Resistance to external factors

The strain "DB" is not resistant to solvents (ether, chloroform) and detergents, sensitive to formaldehyde, ultraviolet radiation, gamma radiation and drying.

Additional features and properties

Immunogenic activity - 14 days after the immunization of pigs it causes them to form specific antibodies.

Reactogenicity is absent.

Pathogenicity is absent.

Virulence is absent.

Oncogenicity is absent.

There is no contagiousness.

The attenuation stability was established during 5 passages on sensitive gilts.

Based on the data obtained, it can be argued that the strain “DB” of the PRRS virus by antigenic and immunological spectra is an original, taxonomically new, previously unknown strain of the PRRS virus.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, allowed to establish that the applicant did not find sources characterized by signs that are identical (identical) to all the essential features of the invention. The definition from the list of identified analogues of the prototype as the closest in terms of the totality of the features of the analogue made it possible to establish the set of essential distinguishing features of the proposed inactivated vaccine relative to the applicant's technical result, as set forth in the independent claim.

Therefore, the claimed solution meets the condition of patentability “novelty”.

To verify the conformity of the proposed solution to the condition of patentability “inventive step”, an additional search of known solutions was carried out to identify features included in the distinctive part of the independent claim. The search results showed that the proposed solution does not follow explicitly for the specialist from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the present invention have been identified), and the effect of the transformations provided for by the essential features of the proposed invention has not been identified to achieve a technical result.

Therefore, the proposed vaccine meets the condition of patentability “inventive step”.

The essence of the invention is illustrated by examples of its execution, which do not limit the scope of the invention.

Example 1

An inactivated PRRS vaccine is prepared from the mother virus of the “DB” strain grown in a transplanted 3-day old Mars-145 cell culture with a cell concentration of more than 100 thousand cells / cm ³ . A mother virus is considered suitable for producing viral raw materials if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Mars-145 cells 6.5 Ig TCD ₅₀ / cm ³ , pH 7.2-7.4 and in the absence of contamination. For the manufacture of vaccines take mattresses with a capacity of 1.5 DM ³ with cell culture. After the growth medium is drained and the FBI or nutrient medium is washed once, 5 cm ^{3 of} virus-containing material of the matrovy series are introduced into the mattresses. Virus adsorption is carried out at 37 ° C for an hour. After that, the needle medium is introduced into the mattresses in a volume of 200 cm ³ with the addition of 5% fetal cattle serum with antibiotics (gentamicin at a concentration of 50 μg / cm ³ or its analogues).

Cultivation is carried out at 37 ° C for 48-72 hours. For control, 3 mattresses are left uninfected, in which the medium is replaced. Virus propagation in the Mars-145 cell culture is determined by the nature of the CPD with the formation of clusters of spherical cells rising above the monolayer. There should not be any destructive cell changes in the control mattresses. Mattresses in which cytopathic changes are observed with damage to up to 60% of the cell monolayer (usually after 48-72 hours) are selected and frozen at -20 ° C. The virus for the manufacture of the vaccine is obtained by 2-fold freezing and thawing of infected cells, followed by discharge of the contents of the mattresses into one container, subject to sterile conditions. The resulting virus is freed from cell detritus by centrifugation at 1000 rpm for 10 minutes. The suspension purified from detritus should look like a clear pinkish-cherry liquid. Then, a sample is taken from the container for laboratory production control for infectious activity and sterility. A production series of PRRSV virus is considered suitable if it meets the following requirements: an infectivity titer after reproduction in a monolayer of Mars-145 cells, at least 6.0 Ig TCD ₅₀ / cm ³ , pH 7.2-7.4 and in the absence of contamination . The purified virus-containing suspension is subjected to inactivation. Inactivation of the accumulated viral raw materials is carried out using a 1% aqueous solution of AEI, introduced to a final concentration of 0.001-0.05%. For this, a suspension of AEEI is added to the suspension heated to 26-28 ° C with constant stirring and the pH value is set at 7.6-7.8 by adding 5% succinic acid solution to it. Inactivation is carried out in a thermostat at a temperature of (37 ± 2) ° C for 20-22 hours with periodic stirring. At the end of inactivation, the suspension is cooled to 4-6 ° C, its pH value is set in the range of 7.6-7.8, and antigen samples are taken to test for sterility, avirulence and immunogenic activity using methods known to those skilled in the art. An emulsion vaccine is prepared by dispersing a mixture of antigen-containing material and an oil adjuvant containing mineral oil with an emulsifier. From oil adjuvants use the preparation "Montanide IZA-70" company "Seppik" (France) or oil adjuvant ARRIAH. The oil adjuvant and inactivated antigen are mixed as follows:

1) when using “Montanida IZA-70” - homogenization for 4-5 minutes at a screw speed of 3000 rpm and a temperature of 10 ° C, a homogeneous white-pink emulsion of the “water-oil” type is obtained;

2) when using the oil adjuvant ARRIAH homogenization in tissue grinder RT-2 for 5 minutes at 5000 rpm The type of water-oil emulsion obtained.

In the manufacture of the vaccine, the antigen is combined with an oil adjuvant in the following ratio: with the Montanid IZA-70 adjuvant, at least 1: 2, and with the ARRIAH oil adjuvant, at least 1: 1. The finished emulsion vaccine is collected in a sterile container and Packed in sterile glass vials and controlled in accordance with the technical conditions. The vaccine is considered immunogenic if, in three gilts vaccinated in a dose of 2 cm ³ , specific antibodies are detected in the blood serum 30 days after the vaccination and no clinical signs of the disease are observed in all animals during control infection. A vaccine is considered harmless if, after subcutaneous administration of a whole vaccine to five white mice weighing 18-21 g in a volume of 0.5 cm ³ and when intramuscularly administered to two gilts weighing 20-25 kg in a volume of 5 cm ³ , it does not cause disease or death of laboratory animals within 10 days, and gilts within 15 days of observation. There should not be any changes in the subcutaneous tissue at the injection site.

The vaccine is an emulsion of a white or pink-white with a slightly viscous consistency. During storage, slight exfoliation of mineral oil on the surface of the emulsion is allowed. When shaken, the vaccine acquires a homogeneous homogeneous structure.

The resulting vaccine has an optimal component composition, wt.%:

Antigenic material from

strain "DB" of PRRS virus,

reproduced in

transplantable cell culture

Mars-145 with biological

activity at least

least 6.0 Ig TCD ₅₀ / cm ³ 33.3-50.0

AEEI (1% aqueous solution) 0.001-0.05

Oil adjuvant Up to 100.0

The content of antigen in the vaccine in the above range is its effective amount in the drug, ensuring the achievement of a technical result.

The resulting vaccine is used as a preventive measure in dysfunctional and threatened PRRS farms. Sows, pigs and boars-producers are inoculated twice with an interval of 15-20 days 3 weeks before mating (insemination). Subsequently, previously immunized sows are inoculated once 3 weeks before mating (insemination), and boars-producers are revaccinated every 6 months. The vaccine is administered intramuscularly behind the ear in a dose of 2.0 cm ³ regardless of the age of the animal. Immunity in vaccinated animals occurs on 21-30 days after vaccination and lasts up to 12 months. The vaccine induces a high level of antibodies against PRRS, provides reliable passive maternal immunity in young animals up to 20-25 days of age, which allows you to protect it from the field virulent PRRS virus in early life.

Example 2

Tests of the immunogenic activity of the inactivated emulsion vaccine against PRRS based on the strain “DB”, manufactured as described in example 1, and containing, wt.%:

Antigenic material from the strain “DB”

PRRS virus reproduced

on transplantable cell culture

Mars-145 with biological activity

at least 6.0 Ig TCD ₅₀ / cm ³ 33.3

AEEI (1% aqueous solution) 0.001

Oil adjuvant Up to 100.0

The immunogenic activity of the vaccine of this formulation was determined on gilts with a live weight of 20-25 kg of 6-8 weeks of age, free of antibodies to the PRRS virus. The vaccine was administered 2.0 cm ³ intramuscularly in the neck. In animals, before vaccination and 34 days after vaccination, blood samples were taken and examined in ELISA (IDEXX kit, USA) for the presence of specific antibodies. Their assessment was carried out by the value of s / p against the PRRS virus. The research results are shown in table 1.

Note:

“+” - clinical signs of the disease;

“-” - absence of clinical signs of the disease;

s / p <0.3 - negative result;

s / p <0.3 - negative result;

s / p = 0.3-0.4 - doubtful result;

s / p> 0.4 is a positive result.

From table 1 it is seen that in the blood serum of vaccinated animals appeared specific antibodies in high titers. The vaccinated animals did not become ill after their infection with the virulent reference PRRS virus, strain Lelystad.

Example 3

Tests of the immunogenic activity of the inactivated emulsion vaccine against PRRS based on the strain “DB”, manufactured as described in example 1, and containing, wt.%:

Antigenic material from the strain “DB”

PRRS virus reproduced

on transplantable cell culture

Mars-145 with biological activity,

at least 6.0 Ig TCD ₅₀ / cm ³ 40.0

AEEI (1% aqueous solution) 0.01

Oil adjuvant Up to 100.0

Studies to determine the immunogenic activity of the vaccine of this formulation were carried out as described in example 2. The research results are shown in table 2.

The data in table 2 indicate that the drug induces vaccine antibodies in high titer in vaccinated animals.

Example 4

Tests of the immunogenic activity of the inactivated emulsion vaccine against PRRS based on the strain “DB”, manufactured as described in example 1, and containing, wt.%:

Antigenic material from the strain “DB”

PRRS virus reproduced

on transplantable cell culture

Mars-145 with biological activity,

at least 6.0 Ig TCD ₅₀ / cm ³ 50.0

AEEI (1% aqueous solution of 0.05

Oil adjuvant Up to 100.0

Studies to determine the immunogenic activity of the vaccine of this formulation were carried out as described in example 2. The research results are shown in table 3 and indicate a high immunogenic activity of the obtained vaccine.

Example 5

Tests of the emulsion inactivated vaccine against PRRS on the basis of the strain “DB”, manufactured as described in example 1, and containing, wt.%.

Antigenic material from the strain “DB”

PRRS virus reproduced

on transplantable cell culture

Mars-145 with biological activity,

at least 6.0 Ig TCD ₅₀ / cm ³ 50.0

AEEI (1% aqueous solution) 0.01

Oil adjuvant Up to 100.0

Vaccine samples were tested for safety. For this purpose, five white mice weighing 18-21 g subcutaneously and two gilts were injected intramuscularly with 0.5 cm ³ and 5 cm ^{3 of the} vaccine, respectively. The mice were monitored for 10 days, with gilts 15 days. During the observation period, all mice remained alive and healthy. In gilts, the body temperature remained within normal limits, a slight swelling was observed at the injection site, and at the autopsy, when examining the injection site, a lot of small encapsulated foci 1-3 mm in diameter were noted, the incision of which contained vaccine contents.

Then, the obtained vaccine was tested for immunogenicity in five seronegative pigs of 2.5-3 months of age. The vaccine was administered once at a dose of 2.0 cm ³ intramuscularly in the middle third of the neck (behind the ear). The animals were monitored for 45 days and blood samples were taken periodically. Blood serum was tested in ELISA for the presence of specific antibodies to PRRS virus. The research results are presented in table 4.

Note: n.i. - the sample has not been investigated; in IFA (ARRIAH) value of 1: 100 and higher - the result is positive; for ELISA (IDEXX) s / p value of 0.4 and higher - the result is positive.

As can be seen from table 4, specific antibodies to PRRS virus in blood serum at the diagnostic level appeared by 21 days after vaccination and averaged 1: 200 ± 56 in the group. In the subsequent selection periods (30, 45 days), the level of specific antibodies increased and amounted to 1: 280 ± 50 and 1: 440 ± 99, respectively. The presence of a high level of antibodies in the blood serum of piglets after vaccination was also confirmed in a commercial ELISA kit from IDEXX (USA).

The second stage of the test was to determine the safety and immunogenicity of the vaccine in two pregnant sows (47-50 days of gestation). The vaccine was administered once in a dose of 2.0 cm ³ intramuscularly (behind the ear). 42 days after the vaccine was introduced (90-93 days of gestation), the sows were intranasally infected with virulent European PRRS virus, strain Lelystad, in a volume of 10 cm ^{3 of} virus-containing liquid with a titer of 10 ^4.0 Ig TCD ₅₀ / cm ³ . The animals were clinically observed for 4 months. 14, 21, 37, 66-69 days after vaccination, blood samples were taken and serums were examined in ELISA (ARRIAH, HIPRA, IDEXX) for the presence of antibodies against PRRS virus. After vaccination and control infection in pregnant sows, no deviations from the physiological norm were noted. The body temperature of the animals remained within the normative indicators, the appetite remained, the animals actively reacted to the environment. There was no local response to vaccine administration.

The results of studies of blood serum of sows are presented in table 5.

Note: n.i. - the sample has not been investigated; in IFA (ARRIAH) value of 1: 100 and higher - the result is positive; for ELISA (HIPRA), the value is greater than 20 - the result is positive; for ELISA (IDEXX) the value s / p> 0.3 - the result is negative; 0.3-0.39 - doubtful; 0.4 and higher - the result is positive.

As can be seen from table 5, on 14, 21, 37 days after vaccination, specific antibodies to the PRRS virus were detected in the blood serum of animals and by 26-28 days after infection (4-5 days after farrowing) their level increased significantly (to s / p 2.23 and 2.1 in IFA - IDEXX and 89.7, 69.5 in IFA - HIPRA). These sows brought 16 piglets, including 14 alive and healthy and 2 stillborn. The safety among living and healthy piglets during the suction period was 100%.

Blood samples were taken from suckling piglets and serum was examined for the presence of antibodies to PRRS virus. 4-7 days after farrowing of the blood serum of newborn piglets had high titers of antibodies to the PRRS virus (s / p 1.7-1.96 in ELISA - IDEXX), which persisted until 36 days of age.

Thus, the inactivated emulsion vaccine against PRRS is a harmless immunogenic preparation and protects sows from the control of infection with virulent virus PRRS, strain Lelystad.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:

- emulsion inactivated vaccine against PRRS, embodying the invention, is intended for use in agriculture, namely in veterinary medicine and biotechnology;

- for the proposed invention in the form described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed;

- when using the proposed emulsion inactivated vaccine against PRRS achieved the technical result provided for by the task of creating the invention.

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

Sources of information taken into account when drawing up the description of the invention

1. Wensvoort G. et. al. Mystery Swine Disease in the Netherlands: The isolation of Lelystad virus. Vet. Quarterly, 1991, 13, 3, 121-130.

2. Syurin V.N. and other viral diseases of animals. Moscow, VNITIBP, 1998, 552-558.

3. PCT No. 92/21375; A 61 K 39/12, G 01 N 33/569, C 12 N 7/00; 12/10/92, (prototype).

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11. Pat. Russia №2108111; A 61 K 39/39; A 61 K 39/00, 39/102, 39/12, 39/135; 04/10/98

Claims (10)

1. Inactivated emulsion vaccine against pig reproductive and respiratory syndrome, containing antigenic material from an infectious agent reproduced in a sensitive biological system, an inactivant and an oil adjuvant, characterized in that it contains a strain of pig reproductive and respiratory syndrome virus, a collection of VGNKI “BD-DEP”, in an effective amount. 2. The inactivated emulsion vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BD-DEPT" of the virus of reproductive and respiratory syndrome of pigs reproduced in transplanted cell culture Marc-145. 3. The inactivated emulsion vaccine according to claim 1 or 2, characterized in that it contains antigenic material from the strain “BD-DEP” of the virus of reproductive and respiratory syndrome of pigs with biological activity of at least 6.0 Ig TCD ₅₀ / cm ³ . 4. The inactivated emulsion vaccine according to claim 1, characterized in that it contains aminoethylethyleneimine from the inactivants. 5. The inactivated emulsion vaccine according to claim 4, characterized in that it contains aminoethylethyleneimine in a concentration of 0.001-0.05%. 6. The inactivated emulsion vaccine according to claim 1, characterized in that of the oil adjuvants it contains an oil adjuvant “Montanide IZA-70”. 7. Inactivated emulsion vaccine according to any one of claims 1 to 6, characterized in that it contains antigenic material from the strain BD-DEP "virus of the reproductive and respiratory syndrome of pigs and oil adjuvant" Montanide IZA-70, in the ratio of at least , 1: 2. 8. The inactivated emulsion vaccine according to claim 1, characterized in that of the oil adjuvants it contains an oil adjuvant ARRIAH. 9. Inactivated emulsion vaccine according to any one of claims 1 to 5 and 8, characterized in that it contains antigenic material from the strain “BD-DEP” of the virus of reproductive and respiratory syndrome of pigs and oil adjuvant ARRIAH in the ratio of at least 1: 1. 10. Inactivated emulsion vaccine according to any one of claims 1 to 9, characterized in that it contains antigenic material from the strain “BD-DEP” of the virus of reproductive and respiratory syndrome of pigs, aminoethylethyleneimine and oil adjuvant in the ratio, wt.%: Antigenic material 33.3-50.0 Aminoethylethyleneimine 0.001-0.05 Oil Adjuvant Up to 100.0