RU2259844C1 - Virus vaccine against newcastle bird disease - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: vaccine has antigenic material produced from La-Sota strain reproduced in 9-10 days old SPF-hen embryos having infectious activity of at least 9.7 lg EID₅₀/cm³ and hemagglutination activity equal to at least 1:512, protective medium and adjuvant of thymogen taken in effective proportions. The protective medium comprises 20% lactalbumin hydrolyzate solution and degreased milk in 1:5 proportion. The vaccine is produced by mixing its ingredients and with following preparation lyophilization. Ready vaccine is dry porous mass of light yellow color. The vaccine is applicable for carrying out specific prophylaxis. The vaccine is introduced as spray at a dose of 1 cm³/head.

EFFECT: high antigenic and immunogenic activity; no adverse side effects.

7 cl, 8 tbl

Description

The invention relates to the field of veterinary virology and biotechnology, namely, vaccines against Newcastle disease (NB) birds, and can be used both in industrial poultry and in small livestock farms (farms and ornamental poultry farming) for the prevention of the above disease.

Newcastle disease (pseudo-plague of birds) is the most common highly contagious poultry viral infection characterized by pneumonia, encephalitis, multiple renal hemorrhages and damage to internal organs. The disease causes enormous economic damage to poultry farming. An indispensable means of preventing this disease is vaccination.

For specific prophylaxis of pseudo-plague of birds, two types of vaccines are used: live and inactivated [1].

The most widely used vaccines are from vaccine strains of La Sota and Bor-74 VGNKI, which are evaporated with water, administered intranasally or aerosol [2-11].

However, known virus vaccines do not sufficiently stimulate the necessary level of antibodies to create a tense immunity.

It was established that one of the complicating factors in this case is the presence of maternal antibodies in vaccinated chickens that inhibit the reproduction of the vaccine virus in the body and thereby prevent the formation of intense immunity. The content of maternal antibodies in different batches of day old chickens varies significantly (from 1: 2 to 1: 128), and the bird is not able to fully respond to live vaccines with a titer of maternal antibodies of 1: 8 and higher.

If immunization is carried out at the age of 1-3 weeks, when the level of maternal antibodies is significantly reduced, the aforementioned virus-inhibiting effect will be less pronounced.

However, with an aerosol administration form, chickens that do not have maternal antibodies may exhibit an acute reaction to vaccination in the form of respiratory symptoms (cough, runny nose, catarrh, etc.).

It is possible that the immunization of older chickens activates latent infections, inhibits the growth of chickens, which can lead to significant losses during mass breeding. In addition, with this method of immunization, the chickens of the first days of life are left without protection.

One of the directions for increasing the immunogenic activity of vaccine preparations is known, based on the use of adjuvants in their composition.

Adjuvants are substances that add to the antigen and which non-specifically enhance the immune response resulting from antigen administration. The stimulating effect of adjuvants, different in chemical structure, is the result of slowing down the hydrolysis and prolonging the action of the antigen, more efficient presentation of the antigen to immunocompetent cells and the intensification of immunogenesis mechanisms (stimulation of macrophages, T and B lymphocytes, increased synthesis of immunoglobulins).

Adjuvants primarily stimulate the most fundamental and universal mechanisms of nonspecific defense of the body, and on their basis a more productive reaction of effective immune systems to the introduced antigen is formed. A higher level of functioning of non-specific defense mechanisms is transformed (under the action of antigen) into a more productive reaction of specific protection [12].

Known virus vaccine against NB birds containing antigenic material from strain P / 77/8 of a homologous virus, a protective environment and a pharmaceutically acceptable carrier in an effective ratio [13].

Known virus vaccine against NB birds containing antigenic material from a La Sota strain of a homologous virus, a protective environment and an oil adjuvant in an effective ratio [14].

Known virus vaccine against NB birds containing antigenic material from a La Sota strain of homologous virus, a protective environment and an adjuvant in an effective ratio [15].

Known virus vaccine against NB birds containing antigenic material from the ATCC strain No. VK-2239 of a homologous virus, a protective environment and an adjuvant in an effective ratio [16].

Known virus vaccine against NB birds containing antigenic material from CNCM strain No. 1-781 of a homologous virus, a protective environment and a pharmaceutically acceptable carrier in an effective ratio [17].

Known virus vaccine against NB birds containing antigenic material from a La Sota strain of a homologous virus, a protective environment and an adjuvant based on mineral or vegetable oil in an effective ratio [18].

Known virus vaccine against NB birds containing antigenic material from a strain of Hitchner B1 homologous virus, a protective environment and a pharmaceutically acceptable carrier in an effective ratio [19].

Also known is the drug thymogen, used as a means to increase the growth and preservation of animals [20].

Timogen, a synthetic thymus peptide glutamyl tryptophan (C ₁₆ H ₂ ON ₃ O ₅ ), has a regulatory effect on the reactions of cellular and humoral immunity, stimulates regeneration processes in the event of inhibition, and increases the body's resistance [21].

The closest to the invention according to the set of essential features is a virus vaccine against NB birds containing antigenic material from a La Sota strain of a homologous virus, a protective environment and an adjuvant based on aluminum hydroxide or aluminum phosphate, mineral or vegetable oil in an effective ratio [22].

The main disadvantage of the above vaccines, including the prototype virus vaccine, is the lack of study of their effectiveness against Russian epizootic isolates of the NB virus, since they have never been used in the Russian Federation.

The objective of the present invention was to search for new adjuvants and to develop using them a vaccine against NB birds based on the La Sota strain, which creates intense and lasting immunity in a vaccinated bird against the epizootic NB virus circulating in Russia.

The technical result from the use of the present invention is to expand the arsenal of virus vaccines against NB birds based on the La Sota strain of a homologous virus, creating intense and prolonged immunity in a vaccinated bird against the epizootic virus of bird NB circulating in Russia.

The specified technical result was achieved by creating a virus vaccine against NB birds, characterized by the following set of features:

1. Virus vaccine against NB birds.

2. Antigenic material from the La Sota strain of the NB virus of birds.

3. Antigenic material from the La Sota strain of the NB virus of birds reproduced in a sensitive biological system.

4. For reproduction of the NB virus of birds from the La Sota strain, 9-10-day-old SPF chicken embryos are used.

5. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and hemagglutinating activity (HA activity), at least 1: 512, in an effective amount.

6. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF-chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, in the amount of 49.9999999-49.99999 wt.%.

7. Protective environment.

8. The protective medium contains a 20% solution of lactolbumin hydrolyzate (GLA) and skim milk in a ratio of 1: 5.

9. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, in an effective amount.

10. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, in an amount of 50.0 wt.%.

11. Adjuvant.

12. Thymogen as adjuvant.

13. Thymogen in an effective amount.

14. Thymogen in an amount of 0.0000001-0.00001 wt.%.

15. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, a protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, and thymogen adjuvant in a ratio, wt.%:

Antigenic material 49,9999999-49,99999 Protective environment 50,0 Adjuvant thymogen 0.0000001-0.00001.

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

1. Virus vaccine against NB birds.

2. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, in an effective amount.

3. A protective environment in an effective amount.

4. Adjuvant thymogen in an effective amount.

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

1. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10 day old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512.

2. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF-chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least , 1: 512, in an amount of 49.9999999-49.99999 wt.%.

3. A protective environment containing 20% GLA solution and skim milk.

4. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5.

5. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, in an amount of 50.0 wt.%.

6. Thymogen in an amount of 0.0000001-0.00001 wt.%.

7. Antigenic material from the La Sota strain of avian NB virus reproduced in 9-10-day-old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, a protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, and thymogen adjuvant in a ratio, wt.%:

Antigenic material 49,9999999-49,99999 Protective environment 50,0 Adjuvant thymogen 0.0000001-0.00001.

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

1. Virus vaccine against NB birds.

2. Antigenic material from the La Sota strain of the NB virus of birds reproduced in a sensitive biological system.

3. Protective environment.

4. Adjuvant.

Compared with the prototype virus vaccine, an essential distinguishing feature of the invention is that the vaccine contains thymogen in an effective amount as an adjuvant.

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

1. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10 day old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512.

2. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, in the amount of 49.9999999-49.99999 wt.%.

3. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5.

4. A protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, in an amount of 50 wt.%.

5. Thymogen in an amount of 0.0000001-0.00001 wt.%.

6. Antigenic material from the La Sota strain of the NB virus of birds reproduced in 9-10-day-old embryos of SPF-chickens with infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ and HA activity of at least 1: 512, a protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5, and thymogen adjuvant in a ratio, wt.%:

Antigenic material 49,9999999-49,99999 Protective environment 50,0 Adjuvant thymogen 0.0000001-0.00001.

The proposed virus vaccine expands the arsenal of vaccines against NB birds based on the La Sota strain, creating intense and lasting immunity in a vaccinated bird against the epizootic NB virus circulating in Russia.

The achievement of the technical result from the use of the proposed vaccine is explained by the fact that it contains thymogen as an adjuvant in a concentration that ensures the production of a highly immunogenic and harmless drug.

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 the totality of the features of the analogue allowed us to establish a set of essential distinguishing features of the proposed vaccine, as set forth in the independent claim, as set forth in the independent claim.

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

To verify the compliance of the proposed solution with the patentability condition "inventive step", the applicant conducted an additional search for known solutions to identify features included in the distinctive part of the independent claim.

Virus vaccines against NB of birds based on the La Sota strain are known. Information about them is given in the section "prior art" description of the present invention. An essential distinguishing feature of the proposed virus vaccine is thymogen, introduced into its composition in an effective amount as an adjuvant.

The indicated symptom is functionally independent.

The use of thymogen as a means to increase the growth and preservation of animals is known [20].

The authors of the invention discovered a new property of thymogen in the claimed concentration range to increase the level of antibodies when it is introduced as a part of a virus vaccine against NB into the bird's body, providing it with intense and long-lasting immunity to this disease and remaining harmless to the vaccinated bird at the same time.

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 totality of distinctive features of the present invention have been identified), and the effect of the essential features of the proposed inventions of transformations to achieve a technical result.

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

The effectiveness of the proposed virus vaccine is illustrated by examples of its manufacture and use, which does not limit the scope of the invention.

Example 1

To obtain the proposed vaccine as a seed virus, virus-containing material from the La Sota strain is used with titers of infectious and GA activity of at least 8.0 Ig EID ₅₀ / cm ³ and 1: 1024, respectively. Dilution of the virus with a titer of 4.5 Ig EID ₅₀ / cm ³ infects well-developed 9-10-day-old embryos of SPF-chickens. Embryos are incubated for 96-120 hours at 36-37 ° C and relative humidity 60-70%. At the same time, the laying of uninfected embryos from the same batch is performed to control the presence of hemi-absorbing viruses. Viewing infected embryos is carried out once a day. All embryos that die before 96 hours of incubation are discarded.

Only live embryos are used to make the vaccine series. Embryonic fluid from every 20-30 embryos is collected in sterile bottles with a capacity of 200-500 cm ³ . Before collecting allantois-amniotic fluid, benzylpenicillin sodium salt 200 IU / cm ³ and streptomycin sulfate 200 μg / cm ³ are added to sterile vials. Extraembryonic fluid contact with antibiotics is carried out for 2 hours at a temperature of 24 ± 6 ° C.

Then the extraembryonic fluid is centrifuged at 1000 rpm for 10-15 minutes to remove red blood cells. After centrifugation, the virus-containing material is poured into 15-20 liter bottles. From each bottle a sample is taken in an amount of 1.0 cm ³ to determine the hemagglutinin titer and the infectious activity of the NB virus.

For the manufacture of the vaccine using sterile antigenic material with titers of GA activity of at least 1: 512 and infectious activity of at least 9.7 Ig EID ₅₀ / cm ³ .

Thymogen adjuvant is added to the obtained antigenic material at the rate of 0.1-10 ml of 0.01% solution per 1 liter of virus-containing suspension, which is thoroughly mixed. One nasal dose of the vaccine should contain at least 0.0001 μg of thymogen, which corresponds to its content in the vaccine of 0.0000001 wt.%.

Then the antigenic material is combined with a protective medium containing a 20% GLA solution and skim milk in a ratio of 1: 5. Antigenic material is combined with a protective medium in a ratio of 1: 1. The mixture is thoroughly mixed, Packed in penicillin vials and subjected to freeze drying.

The finished vaccine is a dry porous mass of light yellow color.

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

Antigenic material from a strain of La Sota virus NB birds, reproduced in 9-10 day old embryos SPF chickens with infectious activity of at least at least 9.7 Ig EID ₅₀ / cm ³ and GA activity, at least 1: 512 49,9999999-49,99999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.0000001-0.00001,

The live dry vaccine against NB birds is intended for specific prophylaxis of infection in dysfunctional and threatened farms for this disease.

Before use, the vaccine is dissolved in water or phosphate-buffered saline.

After that, the number of doses in the vial is determined. One inoculated nasal dose contains at least 6.0 Ig EID ₅₀ / cm ³ .

The introduction of the vaccine is carried out by the spray method. The resulting vaccine is storage stable.

The finished vaccine is controlled according to the indicators established by the technical conditions.

The appearance and color of the drug is determined visually. The vaccine should be a porous mass of light yellow color.

The sterility of the vaccine is determined in accordance with GOST 28085-89 "Biological preparations. Methods of biological control of sterility."

The essence of the method lies in the fact that in the crops from vaccine samples on culture media there should be no growth of bacterial and fungal microflora.

To determine the safety of the drug, a dose of 1.0 cm ³ is administered to 10 chickens of 20 days of age intramuscularly in the pectoral muscle. The bird is monitored for 21 days.

The vaccine is considered harmless if all the chickens remain alive for 21 days, without clinical signs of the disease. When opening a bird at the injection site, there should not be a pronounced inflammatory reaction.

To test the antigenic activity of the vaccine, experimental and control groups of chickens of 12-14 days old, 10 animals each, are formed.

The chickens of the experimental group are injected with a vaccine in a volume of 0.1 cm ³ intranasally. 10 intact chickens are left as a control.

Blood serum is obtained from all chickens of the experimental and control groups before vaccination and 21 days after vaccination to detect specific antibodies. For this, 2.5-3.0 cm ^{3 of} blood is taken from a porch vein from each bird into a separate tube. Serum is separated by a conventional method. Then the serum is placed in a thermostat or heated in a water bath at 56 ° C for 30 minutes to remove thermolabile inhibitors.

Evaluation of the antigenic activity of the vaccine by the NB component is carried out in RTGA or ELISA according to the instructions for using the appropriate kit.

A vaccine is considered antigenically active (immunogenic) if, after 21 days after vaccination, at least 80% of the vaccinated chickens have an antibody titer of NB virus of not lower than 3.0 log ₂ in RTGA or not lower than 1: 500 in ELISA in one dilution.

The vaccine is packaged in 4 cm ³ in sterile bottles, closed with rubber stoppers, rolled up with aluminum caps and labeled.

Example 2

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Virus-containing material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos SPF chickens with an infectious activity of 10.2 Ig EID ₅₀ / cm ³ and hemagglutinating activity 1: 512 49,999999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.000001.

In the experiment used three groups of chickens with 15 goals in each at the age of 14 days. The first and second groups are experienced, and the third served as control.

The first group of chickens was immunized with a commercial series of anti-NB virus vaccines from the La Sota strain, made according to TU 10-19-212-86, intranasally at a dose of 6.7 Ig EID ₅₀ / cm ³ per chicken. The second group of chickens was immunized with the proposed vaccine spray method at a dose of 1 cm ³ / goal., Containing thymogen adjuvant in a dilution of 10 ^-4 (the original preparation in the form of a 0.01% solution), which corresponds to its amount of 0.001 μg in one nasal dose and 0 , 000001 wt.% The content of thymogen in the proposed vaccine.

After 14 days, blood samples were taken from all chickens and serum was examined in RTGA. The results are presented in table 1.

According to table 1, the average antibody titer in the first group of chickens was 5.2 log ₂ 21 days after vaccination, the average antibody titer in the second group was 6.5 log ₂ after the same time after vaccination. The average antibody titer in the second group was 1.25 times higher than in the first group.

Example 3

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 10.2 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,999999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.000001.

In the experiment used 70 chickens, aged 17 days. Chickens were divided into 3 groups: a control in the amount of 30 animals and 2 experimental animals with 20 animals each. Background indicators of antihemagglutinins in 10 chickens of the control group were preliminarily determined.

Immunization of birds was carried out according to the following scheme:

1) a placebo control group (isotonic sodium chloride solution);

2) the first experimental group was vaccinated with a commercial series of vaccines in accordance with the instructions for its use;

3) the second experimental group, the proposed vaccine spray method at a dose of 1 cm ³ / goal., Containing thymogen adjuvant in a dilution of 10 ^-4 (the original drug in the form of a 0.01% solution), which corresponds to its amount of 0.001 μg in one nasal dose and 0.000001 wt.% the content of thymogen in the proposed vaccine.

The duration of the experiment is 28 days. Immunity was monitored in rtga, anti-hemagglutinin titers were determined on 7, 14, 21 and 28 days after vaccination. Blood was taken from 5 chickens from each group by decapitation. Along with this, the phagocytic activity of pseudo-eosinophils, a leukogram, an HCT test, T and B lymphocytes were determined.

Chickens were considered protected from infection by a virulent strain of the NB virus if the serum antibody titer in rtga was equal to or greater than 3.0 log ₂ . The research results are presented in table 2. Analysis of the results in table 2 indicates a pronounced antibody-stimulating effect of thymogen when used in a dilution of 10 ^-4 in the composition of the virus vaccine from La Sota strain. The observation period is 28 days.

Similar results were obtained when testing natural resistance and cellular immunity.

Example 4

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 10.2 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,9999999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.0000001.

Studies performed as described in Example 3. The difference is that the second experimental group of chickens immunized with the vaccine of the proposed spray method at a dose of 1 cm ³ / finish. Containing adjuvant timogen in dilution 10 ^-5 (parent drug as 0, 01% solution), which corresponds to its amount of 0.0001 μg in one nasal dose and 0.0000001 wt.% The content of thymogen in the proposed vaccine.

The research results are presented in table 3.

Analysis of the results in table 3 indicates a pronounced antibody-stimulating effect of thymogen when used in a 10 ^-5 dilution of the virus vaccine from La Sota strain.

Similar results were obtained when testing natural resistance and cellular immunity.

Example 5

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 9.7 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,99999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.00001.

In the experiments, 40 chickens at 18 days of age were used, which were divided into 2 groups - control and experimental (20 chickens each). The chickens were vaccinated with the spray method. The control group was immunized with a commercial series of vaccines from the La Sota strain in accordance with the instructions at a dose of 1 cm ³ , and the chickens of the experimental group were immunized with the same dose of a vaccine with thymogen taken in 10 ^-3 dilution (initial preparation in the form of a 0.01% solution ), which corresponds to its amount of 0.01 μg in one nasal dose and 0.00001 wt.% the content of thymogen in the proposed vaccine.

The titer of antihemagglutinins was determined in rtga three times with an interval of 14 days. The degree of protection was also taken into account. RTGA was set according to the generally accepted method. The vaccinated bird was monitored daily. The test results are presented in table 4.

The data presented in table 4 indicate a pronounced antibody-stimulating effect of the thymogen adjuvant in the composition of the virus vaccine from La Sota strain. Antihemagglutinin titers and indicators of the level of protection in the experimental group are higher than in the control.

Example 6

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 9.7 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,999999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.000001.

The studies were carried out as described in example 5. The difference is that the thymogen adjuvant was used in the vaccine in a dilution of 10 ^-4 (the initial preparation in the form of a 0.01% solution), which corresponds to its amount of 0.001 μg in one nasal dose and 0.000001 wt.% The content of thymogen in the proposed vaccine.

The research results are shown in table 5.

The data presented in table 5 indicate a pronounced antibody-stimulating effect of thymogen in the composition of the virus vaccine from La Sota strain. Antihemagglutinin titers and indicators of the level of protection in the experimental group are higher than in the control.

Example 7

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 9.7 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,9999999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.0000001.

The studies were carried out as described in example 5. The difference is that the thymogen adjuvant was used in the vaccine at a dilution of 10 ^-5 (the initial preparation in the form of a 0.01% solution), which corresponds to its amount of 0.0001 μg in one nasal dose and 0.0000001 wt.% the content of thymogen in the proposed vaccine.

The research results are shown in table 6.

The data presented in table 6 indicate a pronounced antibody-stimulating effect of thymogen in the composition of the virus vaccine from La Sota strain. Antihemagglutinin titers and indicators of the level of protection in the experimental group are higher than in the control.

Example 8

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 10.0 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,99999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.00001.

In the experiments used 20 chickens at 18 days of age, which were divided into 2 groups - control and experimental (10 chickens each). Chickens in the control group were immunized with a commercial series of vaccines from La Sota strain in accordance with the instructions for its use, and chickens of the experimental group were immunized with the proposed virus vaccine from La Sota strain in combination with thymogen as an adjuvant, while thymogen was used in 10 ^-3 dilution (the original preparation in the form of a 0.01% solution), which corresponds to its amount of 0.01 μg in one nasal dose and 0.00001 wt.% the content of thymogen in the proposed vaccine. The dose of 1 cm ³ / goal. The method of administration is the spray method. The titer of antihemagglutinins was determined twice with an interval of 14 days. The results were taken into account by the titer of antihemagglutinins. The hemagglutination inhibition reaction (RHCA) was set by the conventional method. The test results are presented in table 7.

The data in table 7 indicate a pronounced antibody-stimulating effect of the thymogen adjuvant in the composition of the virus vaccine from La Sota strain. In the experimental group, in comparison with the control, a longer circulation of antibodies in high titers is provided (within 30 days, the observation period).

Example 9

Tests of a virus vaccine against NB of birds made as described in example 1, and containing, wt.%:

Antigenic material from the strain La Sota virus NB birds, reproduced in 9-10 day old embryos of SPF hens with infectious activity of 10.0 Ig EID ₅₀ / cm ³ and GA activity 1: 512 49,99999 Protective medium containing 20% GLA solution and skim milk in a ratio of 1: 5 50,0 Adjuvant thymogen 0.00001

The studies were carried out as described in example 8. The difference is that the thymogen adjuvant was used in the composition of the proposed vaccine in a dilution of 10 ^-4 (the original drug in the form of a 0.01% solution), which corresponds to its amount of 0.001 μg in one nasal dose and 0.00001 wt.% the content of thymogen in the proposed vaccine.

The research results are shown in table 8.

The data presented in table 8 indicate a pronounced antibody-stimulating effect of the thymogen adjuvant in the composition of the virus vaccine from La Sota strain. In the experimental group, in comparison with the control, a longer circulation of antibodies in high titers is provided (within 30 days, the observation period).

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

- Virus vaccine against NB birds embodying the invention is intended for use in agriculture, namely in veterinary virology and biotechnology;

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

- when using the proposed virus vaccine against NB birds, the technical result achieved by the task of creating the invention is achieved, which consists in expanding the arsenal of virus vaccines against NB birds based on the La Sota strain of a homologous virus that creates intense and prolonged immunity in the vaccinated bird against the epizootic NB virus circulating in Russia birds. Therefore, the present invention meets the condition of patentability "industrial applicability".

Table 1
Test results of antigenic properties of virus vaccine against Newcastle disease of birds with thymogen adjuvant Group of chickens Antibody titers in rtga 21 days after vaccination, in log ₂ Group 1 (chickens immunized with thymogen-free vaccine) 5, 5, 6, 4, 5, 6, 6, 5, 4, 4, 6, 6, 5, 6, 5 Group 2 (chickens immunized with thymogen vaccine) 6, 6, 7, 8, 7, 6, 7, 7, 5, 6, 7, 7, 6, 7, 6 Control (unvaccinated chickens) 2, 1, 2, 2, 2, 1, 2, 2, 1, 2, 1, 2, 1, 2, 1

Table 4
The dynamics of antibody formation and the degree of protection in young chickens during immunization with a vaccine from La Sota strain in combination with thymogen adjuvant Age of the bird, (days) Groups control experienced 32

46

60

Note: the numerator is the titer of antihemagglutinins (log ₂ ); denominator - degree of protection (%).

Table 5
The dynamics of antibody formation and the degree of protection in young chickens during immunization with a vaccine from La Sota strain in combination with thymogen adjuvant Age of the bird, (days) Groups control experienced 32

46

60

Note: the numerator is the titer of antihemagglutinins (log ₂ ); denominator - degree of protection (%).

Table 6
The dynamics of antibody formation and the degree of protection in young chickens during immunization with a vaccine from La Sota strain in combination with thymogen adjuvant Age of the bird, (days) Groups control experienced 32

46

60

Note: the numerator is the titer of antihemagglutinins (log ₂ ); denominator - degree of protection (%).

Sources of information

1. Syurin V.N. and other viral diseases of animals. - M.: VNITIBP, 1998 .-- S.214-233.

2. Auth. testimonial. USSR No. 132771; 30 h, 6; 1960 year

3. Auth. testimonial. USSR No. 177042; 30 h, 6; 12/01/65 g

4. Instructions for the manufacture and control of dry vaccine against pseudo-plague of birds from strain B ₁ . Approved by the GUV Ministry of Agriculture of the USSR on 10.09.73

5. Instructions for the manufacture and control of dry vaccine against pseudo-plague of birds from the strain of La Sota. Approved by the GUV Ministry of Agriculture of the USSR on 10.09.73

6. Auth. testimonial. USSR No. 826584, A 61 K 39/17, C 12 N 7/00, 11/7/82.

7. Auth. testimonial. USSR No. 1124585, A 61 K 39/17, 1984

8. Auth. testimonial. USSR No. 1212045, A 61 K 39/12, C 12 N 7/00, 1990

9. Autosvid. USSR No. 1785098, A 61 K 39/17, 08/09/95

10. RF patent No. 2035917, A 61 K 39/17, 05.27.95.

11. RF patent №2122429, A 61 K 39/17, 11/27/98.

12. Sergeev V.A. Viral vaccines. Kiev: Harvest, 1993 .-- S.158-172.

13. US patent No. 4235876, A 61 K 39/12, C 12 K 7/00, 11.25.80 g.

14. French patent No. 2577139, A 61 K 39/00, 39/17, 39/102, 39/10, 39/07, 08/14/86.

15. US patent No. 4824668, A 61 K 35/75, 04/25/89.

16. US Patent No. 5118502, A 61 K 39/12, C 12 N 7/00, 02/02/92.

17. US patent No. 5149530, A 61 K 39/12, C 12 N 7/00, 09/22/92.

18. US patent No. 5152981, A 61 K 39/12, 39/00, 10/06/92.

19. US patent No. 5250298, A 61 K 39/12, 10/05/93

20. RF patent No. 2111756, A 61 K 35/55, 38/05, 05/27/98.

21. Guidance on the use of a thymogen solution of 0.01% for veterinary medicine. Approved by the GUV Ministry of Agriculture of the USSR on September 13, 1991

22. US patent No. 5733556, A 61 K 39/17, 03/31/98 (prototype).

Claims (7)

1. The vaccine virus against Newcastle disease of birds, containing antigenic material from the La Sota strain of the Newcastle disease virus, reproduced in a sensitive biological system, a protective environment and adjuvant, characterized in that it contains thymogen in an effective amount as adjuvant. 2. The vaccine virus according to claim 1, characterized in that it contains antigenic material from the La Sota strain of the Newcastle disease virus reproduced in 9-10 day old SPF chicken embryos with an infectious activity of at least 9.7 lg EID ₅₀ / cm ³ and hemagglutinating activity of at least 1: 512. 3. The vaccine virus according to claim 2, characterized in that it contains antigenic material from the La Sota strain of the Newcastle disease virus in an amount of 49.9999999-49.99999 wt.%. 4. The vaccine virus according to claim 1, characterized in that it contains a protective medium containing a 20% solution of lactalbumin hydrolysis and skim milk in a ratio of 1: 5. 5. The vaccine virus according to claim 4, characterized in that it contains a protective environment in an amount of 50.0 wt.%. 6. The vaccine virus according to claim 1, characterized in that it contains thymogen in an amount of 0.0000001-0.00001 wt.%. 7. The vaccine virus according to any one of claims 1 to 6, characterized in that it contains antigenic material from the La Sota strain of the Newcastle disease virus, a protective environment and thymogen adjuvant in the ratio, wt.%: Antigenic material 49,9999999-49,99999 Protective environment 50,0 Thymogen 0.0000001-0.00001