RU2323741C2 - Anti- bird flue inactivated emulsion vaccine - Google Patents

Abstract

FIELD: medicine, veterinary.

SUBSTANCE: vaccine contains active substance and food additive. Active substance consists of efficient amount of avirulent antigenic material from "Novosibirsky" bird flue strain Influenzae virus avicum, Orthomyxoviridae family, serotype A, subtype H5N1, collection of FGU "VGNKI" VNIISZH N. 125 - deposition "Novosibirsky".

EFFECT: vaccine provides the efficient bird protection against the epizootic virus and prevents the environmental propagation of infectious agent from immunized bird.

6 cl, 12 tbl, 7 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of a type A influenza vaccine.

Taxonomically influenza viruses are assigned to the family of orthomyxoviruses (Ortho-myxoviridae, ICTV classification - 00.046). It includes serotypes A, B and C.

The first two of them make up one genus (00.046.0.01), serotype C forms the other (00.046.02.001). Belonging to one of them is determined by the antigenic characteristics of nucleoprotein (NP) and matrix protein (M1) located inside the virion.

Influenza A is a contagious viral disease that causes seasonal epizootics and panzootia with various mortality rates, depending on the degree of pathogenicity of the pathogens.

Influenza A virus is widespread in nature and affects many species of mammals and birds, mainly aquatic and near-water ecological complexes. Viruses with all known combinations of surface proteins were isolated from wild birds, while the infection in wild birds proceeds in the form of enteritis, with no visible signs of the disease. This indicates a high degree of adaptation of influenza A viruses to wild birds, which are the natural hosts of influenza viruses. The virus persists in water for a long time (6-8 months), infection occurs by water-fecal route.

Influenza A virus has a high degree of variability, especially for the surface glycoproteins of the virion: hemagglutinin (HA) and neuraminidase (HA). It has been established that host antibodies to HA and HA provide the basis for humoral immunity, while protective antibodies to GA play a leading role. There are 16 known antigenic subtypes of HA (H1-H16) and 9 subtypes of HA (N1-N9). The influenza virus has a segmented genome, consisting of 8 segments, which have a high ability to reassort.

Various combinations of HA and HA can lead to the emergence of highly virulent strains of the virus, causing a high mortality rate.

Currently, the spread of avian influenza (GP) caused by type A virus, subtype H5N1, is observed in Eurasia. It is believed that this subtype is one of the most dangerous in terms of pandemics (1).

The disease, caused by highly pathogenic strains of the pathogen type A (H5N1), causes tremendous economic damage in industrial poultry farming, as evidenced by recent epizootic outbreaks of "chicken flu" in the Siberian, Central and Southern regions of the Russian Federation, as well as in the countries of the near and far abroad.

In countries where outbreaks of the disease are noted, vaccination is used as one of the methods to combat it. The decrease in economic losses due to vaccination is associated with the prevention of outbreaks of the disease, with a limited spread of the virus and with the possible minimization of the probability of its mutation due to a decrease in the number of birds susceptible to it.

Three types of inactivated influenza vaccines are known:

1) virionic (corpuscular, or whole), which is a mixture of antigenic material from a strain of influenza virus homologous to the pathogen of infection with adjuvant in an effective ratio;

2) split or disintegrated preparations (“split vaccines”), which are a mixture of the structural components of the virion (both surface and internal), separated by various detergents, with adjuvants in an effective ratio;

3) subunit, containing a mixture of surface structures of HA virion and HA carriers of protective antigens of the virus with adjuvants in an effective ratio. By the preparation method, almost all of the listed types of vaccines are mixed, since they contain both whole virions and their structural components, including units of supercapsid (2).

Modern genetic engineering methods make it possible to create vaccines based on plasmids embedded in smallpox viruses or infectious bird laryngotracheitis, which unifies the high virus yield and, most importantly, ensures their labeling by HA. An example is the vaccines created in the United States and some other countries based on avirulent chickenpox and chickenpox viruses.

Another type of vaccine is live virus-based vaccines with a built-in HA gene that can reproduce in birds without causing pathology. Merial recombinant vaccine expressing HA H5 is registered in many countries. These vaccines are highly effective and technologically advanced, easy to use, provide fairly reliable protection against homologous viruses, but they are not currently used, as they are biological preparations of a “strategic stock”. Each of these vaccines has its own advantages and disadvantages (3-7).

In the fight against GP, inactivated preparations are most often used, including full-fledged virions mixed with oil adjuvant.

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain H7N1 / Rostock (access sequence number in Genbank = × 52226) of the HPV virus obtained in a constant culture of chicken embryo fibroblast cells, purified from ballast impurities and inactivated with beta -propiolactone in a final concentration of 1: 3000 with a 2-hour exposure at 37 ° C, and the target additive in the form of an oil adjuvant in an effective ratio (8).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Turkey / Ontario / 7732/66 (H5N9) of the GP virus obtained in 10-day-old chicken embryos with infectious activity of 8.9 lg ELD ₅₀ / ml and inactivated with formalin 0.5% concentration for 72 hours at 4 ° C, and the target additive in the form of an oil adjuvant in a ratio of 1: 9 (9-11).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Chicken / Pakistan / 95 (H7N3) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (12 )

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Queretaro / 14588-19 / 95 (H5N2) of the HPV virus obtained in 10-day-old embryos of SPF-chickens with infectious activity of 7.3 lg ELD ₅₀ / 0.1 ml and hemagglutinating activity (HA activity) 1:64, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 ( 13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Turkey / Wisconsin 68 (H5N9) of the HPV virus obtained in 10-day-old embryos of SPF-chickens with infectious activity of 8.5 lg ELD ₅₀ / 0.1 ml and HA activity of 1: 512, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

Known SE inactivated vaccine emulsion containing the active substance in the form of antigenic material from strain A / Turkey / Oregon / 71 (H7N3) virus GP prepared in 10-day old embryonated SPF-chickens with infectious activity 8,7 lg ELD _50/0 1 ml and HA activity of 1: 2048, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Mallard / Ohio / 556/87 (H5N9) of the HPV virus obtained in 10-day-old SPF chicken embryos with infectious activity of 7.9 lg ELD ₅₀ / 0.1 ml and a GA activity of 1:64, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Mexico / 31381-7 / 94 (H5N2) of the HPV virus obtained in 10-day-old SPF-chicken embryos with infectious activity of 7.7 lg ELD ₅₀ / 0.1 ml and HA activity of 1:48, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Mexico / 26654-1374 / 94 (H5N2) of the GP virus obtained in 10-day-old embryos of SPF-chickens with infectious activity of 8.3 lg ELD ₅₀ / 0.1 ml and HA activity of 1: 128, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Turkey / Minnesota / 10734-5 / 95 (H5N2) of the HPV virus obtained in 10-day-old SPF-chicken embryos with infectious activity of 7.9 lg ELD ₅₀ / 0.1 ml and GA activity 1:96, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known inactivated emulsion vaccine against GP containing the active substance in the form of antigenic material from strain A / Chicken / Jalisco / 14589-660 / 94 (H5N2) of the HP virus obtained in 10-day-old SPF-chicken embryos with infectious activity of 7.7 lg ELD ₅₀ / 0.1 ml and HA activity of 1:48, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Veracruz / 28159-398 / 95 (H5N2) of the HPV virus obtained in 10-day-old SPF-chicken embryos with an infectious activity of 8.9 lg ELD ₅₀ / 0.1 ml and HA activity of 1: 256, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Puebla / 28159-474 / 95 (H5N2) of the GP virus obtained in 10-day-old embryos of SPF-chickens with infectious activity of 8.7 lg ELD ₅₀ / 0.1 ml and HA activity of 1: 256, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Chicken / Chiapas / 28159-488 / 95 (H5N2) of the GP virus obtained in 10-day-old embryos of SPF-chickens with infectious activity of 7.3 lg ELD ₅₀ / 0.1 ml and HA activity of 1:48, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (13).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Turkey / Minnesota / 3689-1551 / 81 (H5N2) of the HPV virus obtained in 10-day-old SPF-chicken embryos with an infectious activity of 8.7 lg ELD ₅₀ / 0.1 ml and HA activity of 1: 1024, purified from ballast impurities and inactivated with formalin, and the target additive in the form of an oil adjuvant in an effective ratio (14).

A known vaccine against GP inactivated emulsion containing the active substance in the form of antigenic material from strain A / Duck / Singapore / F119 / 97 (H5N3) of the HPV virus obtained in 10-day-old embryos of SPF-chickens with an infectious activity of 8.1 lg ELD ₅₀ / 0.1 ml and a GA activity of 1: 128, purified from ballast impurities and inactivated with 2-bromoethylamine hydrobromide taken in an amount of 3 mM, and the target additive in the form of an oil adjuvant in a ratio of 1: 4 (14).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Hong Kong / 156/97 (H5N1) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio ( fourteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Hong Kong / 483/97 (H5N1) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio ( fourteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Duck / Potsdam / 1402/86 (H5N2) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (fifteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Duck / Potsdam / 2243/84 (H5N6) of the HP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (fifteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / CK / Italy / 473/99 (H7N1) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (fifteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of purified and avirulent antigenic material from strain A / Duck / Potsdam / 15/80 (H7N7) of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (fifteen).

A known vaccine against GP inactivated emulsion containing the active substance in the form of a purified and avirulent antigenic material from strain A / SK / IAE / 415/99 of the GP virus obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant in an effective ratio (15) .

The closest to the invention according to the set of essential features is an inactivated emulsion vaccine against GP containing the active substance in the form of purified and avirulent antigenic material from strain A / Hong Kong / 483/97 (H5N1) of the GP virus obtained in a sensitive biological system, and the target additive as an oil adjuvant in an effective ratio (14).

The problem to which the present invention is directed, is to expand the arsenal of vaccines against avian influenza inactivated emulsion, possessing high antigenic and immunogenic activity, providing effective protection of poultry against the type A virus (H5N1) and preventing the vaccine from releasing the infectious agent into the environment .

This problem was solved by creating a vaccine against GP inactivated emulsion, characterized by the following set of features.

The proposed vaccine contains the active substance in the form of purified and avirulent antigenic material from the Novosibirsk strain (author's name) type A GP, subtype H5N1, obtained in 10-12-day-old SPF-chicken embryos with infectious activity of at least 9.0 lg ELD ₅₀ / ml and HA activity of at least 1:64, and the target additive in the form of an oil adjuvant in the ratio, wt.%, 30: 70-40: 60.

For the manufacture of the vaccine, 10-12-day-old SPF-chicken embryos are used as a sensitive biological system. Extraembryonic fluid (EEG) is purified from ballast impurities by centrifugation at 1000 rpm for 10-15 minutes. After centrifugation, the viral material is poured into 10-20 liter bottles and subjected to inactivation. Before inactivation, the infectious activity of the virus should be at least 9.0 lg ELD ₅₀ / ml, and GA activity at least 1:64.

For inactivation of the virus, aminoethylethyleneimine (AEEI), formaldehyde or beta-propiolactone are used (16).

AEEI is added to the virus-containing suspension with stirring in the form of a 10% solution to a final concentration of 0.05-0.1%, formaldehyde to a final concentration of 1: 1000, and beta-propiolactone to a final concentration of 1: 2000-1: 4000. Inactivation of the virus is carried out for 24 hours at a temperature of from 20 to 37 ° C. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to a suspension of antigenic material.

After checking for sterility and avirulence, the antigen is subjected to repeated purification from ballast impurities, passing it through an OTR-102K-01 intermediate centrifuge at 15000-17000 rpm with an antigen feed rate of 40-50 l / h and a filter unit.

The purified and avirulent antigenic material must have a GA activity of at least 1:64 and a pH in the range of 7.2–7.4.

An emulsion vaccine is obtained by dispersing a purified and avirulent antigenic material with an oil adjuvant in a ratio of 30: 70-40: 60 by weight.

Of the adjuvants, it is advisable to use an oil adjuvant based on Marcol-52 mineral oil and non-inogenous surfactant emulsifiers (alkyl-modified polyoxyalkylene-siloxane copolymer - APS and lanolin) or commercial oil adjuvant of the brand Montanide ISA-70 manufactured by France company (Seppic (France )

The content of antigenic material and oil adjuvant in the vaccine dose in the ratio, wt.%, 30: 70-40: 60 is optimal, as it provides a tolerant presentation of antigen in the body of an immunized bird.

The resulting vaccine is a homogeneous white emulsion of the type "water in oil". A layer of oil adjuvant is allowed at the top of the vial. With vigorous shaking, the uniformity of the contents is easily restored.

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. Inactivated emulsion vaccine against GP.

2. The active substance in the form of purified and avirulent antigenic material from the Novosibirsk strain of type A virus, subtype H5N1 in an effective amount.

3. Targeted supplement.

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. Inactivated emulsion vaccine against GP.

2. The active substance.

3. Targeted supplement.

Compared with the prototype vaccine, an essential distinguishing feature of the present invention is that it contains purified and avirulent antigenic material from the Novosibirsk strain of type A virus, subtype H5N1 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. Purified and avirulent antigenic material from the Novosibirsk strain of type A HPV virus, subtype H5N1, obtained in 10-12-day-old SPF chicken embryos with infectious activity of at least 9.0 lg ELD ₅₀ / ml and GA activity of at least 1:64.

2. Purified and avirulent antigenic material from the Novosibirsk strain of type A HPV virus, subtype H5N1, obtained in 10-12-day-old SPF chicken embryos with infectious activity of at least 9.0 lg ELD ₅₀ / ml and GA activity of at least 1:64, in an amount of 30.0-40.0 wt.%.

3. As a targeted supplement, the vaccine contains an oil adjuvant.

4. The vaccine contains an oil adjuvant in an amount that provides a tolerant presentation of antigen in the body of an immunized bird.

5. The vaccine contains an oil adjuvant in an amount of 60.0-70.0 wt.%.

6. The vaccine contains purified and avirulent antigenic material from the Novosibirsk strain of type A HPV virus, subtype H5N1 and an oil adjuvant in the ratio, wt.%:

Antigenic material 30.0-40.0 Oil adjuvant Up to 100.0

The results of the tests showed that the proposed vaccine has high antigenic and immunogenic activity and provides reliable protection of poultry against the type A virus, type H5N1 subtype, and also prevents the pathogen from being infected with the vaccinated bird in the environment.

The achievement of the indicated result from the use of the invention is explained by the fact that the antigenic material from the Novosibirsk strain of type A virus, subtype H5N1, which has high infectious, antigenic and immunogenic activity in its native form and retains antigenic and immunogenicity, is introduced into the composition of the proposed vaccine as an active substance. activity after inactivation, thereby providing a vaccine against type A GP, subtype H5N1, which creates effective protection of poultry against a homologous virus and prevents increasing the release of the causative agent of infection into the environment by the vaccinated bird.

The Novosibirsk strain of type A virus, subtype H5N1, was deposited on April 12, 2006, in the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, of the Federal State Institution All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed (FGU “ VGNKI ”) under the registration number (link) - the production strain“ ARRIAH ”No. 125-DEP“ Novosibirsk ”of the type A virus, subtype H5N1.

Compared with the initial isolate, the Novosibirsk strain of the type A HPV virus, the H5N1 subtype, has genetic and phenotypic features. It is cloned in an MDCK culture; when propagated in chicken embryos, it has high virulence and retains antigenic strength after inactivation. The possibility of its use for the preparation of inactivated vaccine preparations has been experimentally confirmed.

The strain "Novosibirsk" of the GP virus is characterized by the following features and properties.

Morphological features

The strain "Novosibirsk" of the GP virus belongs to the family Orthomyxoviridae, serotype A, subtype H5N1 and has morphological characteristics characteristic of influenza virus type A.

Influenza virus virions are membranous, pleomorphic. The diameter of the sphere-like particles is 50-120 nm; filamentous particles about 20 nm in diameter, 200-300 nm in length or more. On the surface of the virion, towering above the lipid membrane by 10-14 nm, there are about 500 spike-like protrusions (spikes) formed by GA homotrimers.

In the intervals between spike clusters in type A viruses, as a rule, about 100 HA molecules are located.

The lipid membrane surrounds loop-like filaments of nucleocapsids with eight fragments of the viral genome, each of which is represented by a linear single-stranded antisense RNA.

The total genome length is about 13,600 nucleotides (nt). The largest fragment (No. 1) contains about 2350 nt; No. 2 - a little less than 2350 nt; No. 3 - as a rule, 2250 nt; No. 4 - 1780 nt; No. 5 - 1575 nt; No. 6 - 1420 nt; No. 7 - 1050 nt; No. 8 - 900 nt.

At both ends of each RNA genomic chain, repeating segments are contained, and repeats of the 5′-terminal region (for type A viruses they look like 5′-AGUAGAAACAAGG) are complementary to inverted repeats located at the 3′-end. In addition, the 3'-terminal sections of some fragments of the genome contain conserved segments typical of viral strains that infect subjects of a particular biological species [ICTV-Intemational Committee on Taxonomy of Viruses (2006). http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/].

Antigenic properties

By its antigenic properties, the Novosibirsk strain of avian influenza virus belongs to type A, subtype H5N1.

The virus is stably neutralized by homologous antiserum.

Inactivated virus induces antibodies detected by rtga, pH and ELISA. So, 12 days after a single immunization of chickens with the proposed vaccine, the titer of antibodies to GA, providing a hemagglutination inhibition reaction, amounted to 5.0 log ₂ ; the titer of antibodies determined by ELISA was 1: 1243, precipitating antibodies were detected by diluting the test sera from 1: 2 to 1: 4.

The determination and comparative analysis of the primary structure of amplified cDNA fragments showed that the test sample contained fragments of the genome of field isolates of avian influenza virus. The primary structure of the site for cutting the HA isolate protein was PQGRERRRKKR * GLF, which indicates that it belongs to the group of highly pathogenic strains of the GP virus.

The obtained sequences were compared with the sequences of the HA gene type H5 and the HA gene type N1 from the Flu. The Influenza Sequence Database ”(http://www.flu.lanl.gov/). It was shown that the HA of the isolated isolate belongs to the H5 subtype, and HA to the N1 subtype of the extensive line of highly pathogenic GP isolates A / Goose / Guandong / 1/96 (H5N1) isolated in Southeast Asia, including Indonesia and China, from 1996 to the present.

Biotechnological characteristics

Strain "Novosibirsk" shows a high biological, antigenic and immunogenic activity both in the native form and after inactivation. The strain is intended for the production of diagnostic sera and antigenic preparations, as well as for the manufacture of an inactivated vaccine against the GP virus.

The Novosibirsk strain of type A virus, subtype H5N1, is reproduced in 10-12 day old chicken embryos at a temperature of 37.0 ± 0.5 ° C and a humidity of 50-70%. An infectious dose ranging from 1 to 4.0 lg ELD ₅₀ causes a specific death of TBE, limited to a time period of 24 to 48 hours of incubation. During the specified period of time, the virus accumulates in titers from 9.0 to 10.2 lg ELD ₅₀ / ml. Such a crop allows using one chicken embryo to obtain material for 50-70 vaccine doses. The Novosibirsk strain of avian influenza virus is stable, has high virulence and is adapted for propagation in chicken embryos.

Chemo- and genotaxonomic characterization

The genome of the Novosibirsk virus strain of the GP virus consists of eight unequal (900-2350 nucleotides) fragments of single-stranded minus RNA, the total length of which is about 14 thousand nucleotides with a molecular weight of 4.5 MD. Virionic RNA is not infectious. 10 proteins were found in virions. Proteins are divided into surface (GA, HA, M2) and internal (PB1, PB2, PA, NP, M1, NS1, NS2).

GA and HA have the most important antigenic sites on which protective immunity is generated, primarily in the form of neutralizing antibodies.

Physical properties

The molecular weight of the virions is 250 MD, the floating density in sucrose is 1.19 g / cm ³ , and the virion sedimentation coefficient is 700-800 S 20w.

Resistance to external factors

The strain "Novosibirsk" of the GP virus is sensitive to detergents, formaldehyde, beta-propiolactone, derivatives of aziridine, is rapidly inactivated by heating (56 ° C), ultraviolet radiation and at pH below 5.0.

Additional features and properties

Immunogenic activity - immunogen in the composition of an inactivated vaccine.

Reactogenicity - does not possess reactogenic properties. Immunization of chickens with a dose twice the vaccination dose does not cause visible clinical changes in the general condition of the bird, as well as local tissue lesions at the injection site.

Pathogenicity is high (intravenous index of 2.87, which corresponds to highly pathogenic strains of the GP virus).

Virulence - 100% death of intramuscularly infected chickens at a dose of 10 ^6.0 ELD ₅₀ .

Oncogenicity is absent.

Contagiousness is contagious. Non-immune chickens become infected when kept together with infected ones.

Stability of the strain was observed with five consecutive passages on sensitive chickens.

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

Example 1

For the manufacture of an inactivated emulsion vaccine against GP, viral material of the Novosibirsk strain obtained by culturing in chicken embryos is used. First-order inoculum virus (mother seed virus) is stored in a lyophilized state in hermetically sealed vials in a dark place at a temperature not exceeding -40 ° C and not more than 12 months from the date of manufacture.

To obtain a second-order inoculum virus, the contents of three vials are diluted with sterile phosphate-buffered saline (PBS) containing 500 IU / penicillin and 500 μg / cm ³ streptomycin at a pH of 7.2-7.4 so that the virus dose a volume of 0.2 ml, intended for infection of one embryo of SPF-hens, contained at least 2.0 lg ELD ₅₀ virus. After infection, chicken embryos are incubated for 24-48 hours at 37 ± 0.5 ° C and relative humidity of 60-70%. Ovoscopy of infected embryos is carried out up to a 24-hour incubation period once, and then every 5 hours. Embryos die in 24-48 hours. Embryos that die before 24 hours are discarded, and the rest are used as a seed virus. Under sterile conditions, from every 5-7 embryos, an EEG is taken into a separate sterile container that is tightly closed. The contents of each container are checked for bacterial contamination and fungal contamination. After the control, the sterile virus-containing material is poured into one vessel and packed into sterile penicillin vials, closed with sterile rubber stoppers, filled with paraffin and stored at a temperature not exceeding minus 40 ° С.

The contents of one of the vials are used to determine the antigen content, the identity of the viral raw material and the biological activity of the virus.

The seed virus is considered suitable for infection of a production batch of embryos, if the virus is identified, the GA activity is not lower than 1:64, the infectious activity is not lower than 9.0 lg ELD ₅₀ / ml.

The inoculum virus is stored at a temperature not exceeding minus 30 ° C for no more than 4-6 months, preventing even its thawing once.

To obtain a production seed crop, the seed virus is thawed at a temperature of 18-24 ° C under sterile conditions, the bottle is opened and its contents are centrifuged at 2500 rpm for 10 minutes. The supernatant is removed, diluted with a sterile physiological solution of pH 7.0-7.4 at the rate of 1000-10000 ELD ₅₀ / ml, add 500 μg of streptomycin and 500 IU of penicillin per 1 ml of material. Virus-containing material prepared in this way is used to infect chicken embryos. Chicken embryos at the age of 10-12 days are infected in the allantoic cavity with virus-containing material in a volume of 0.2 ml. Infected embryos are placed in a thermostat at a temperature of 37.0 ± 0.5 ° C and a relative humidity of 50-70%. An ovoscopy of infected embryos is carried out daily 2 times a day.

Embryos die in 24-48 hours. At the end of the incubation, the embryos are placed in refrigerated chambers at a temperature of 2-6 ° C for 6-18 hours, so that during the opening they prevent the ingress of red blood cells into the allantoic-embryonic fluid. The cooled embryos are opened. EEG from every 20-30 embryos is collected in sterile bottles with a capacity of 200-500 ml. Before collecting EEG, benzylpenicillin sodium salt 200 IU / ml and streptomycin sulfate 200 μg / ml are added to sterile vials. EEG contact with antibiotics is carried out for 2 hours at a temperature of 24 ± 6 ° C.

The resulting EEG was centrifuged at 1000 rpm for 10-15 minutes to remove red blood cells. After centrifugation, the viral material is poured into 15-20 liter bottles and transferred to inactivation. A sample of 1 ml was taken from each bottle to determine the titer of HA and the infectious activity of the GP virus.

Infectious activity of the virus-containing material used to compile the vaccine series should not be lower than 9.0 lg ELD ₅₀ / ml, and GA activity not lower than 1:64.

Inactivation of the GP virus is carried out with a working solution of AEEI, formaldehyde or beta-propiolactone.

The viral suspension is warmed to room temperature or to a temperature of 37 ° C, depending on the inactivant used. With stirring, a 10% AEEI solution is added to the suspension to a final concentration of 0.05-0.1% or formaldehyde to a final concentration of 1/1000, or beta-propiolactone to a final concentration of 1 / 2000-1 / 4000.

In addition, merthiolate is added to the suspension at the rate of 1/10000 and benzylpenicillin sodium salt or some other antibiotic selected by subtitling in the amount of 500 U / ml.

After adding the inactant, the mixture is stirred for 20-30 minutes. Inactivation of the virus is carried out for 24 hours and at a temperature of 37 ± 0.5 ° C. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to a suspension of antigenic material. After conducting a sterility and avirulence test, the antigenic material is subjected to repeated purification from ballast impurities by passing it through an OTR-102K-01 flowing centrifuge at 15000-17000 rpm with a liquid flow rate of 40-50 l / h and a filter unit.

The purified and avirulent antigenic material must have a GA activity of at least 1:64 and a pH in the range of 7.2–7.4.

An emulsion vaccine is obtained by dispersing a purified and avirulent antigenic material with an oil adjuvant in the ratio, wt.%, 30: 70-40: 60.

Of the adjuvants, it is advisable to use an oil adjuvant based on Marcol-52 mineral oil and emulsifiers of nonionic surfactants (alkyl-modified polyoxyalkylene siloxane copolymer - APS and lanolin) or a commercial oil adjuvant of the Montanide ISA-70 brand manufactured by Seppic (France).

The manufacture of a vaccine is carried out in two stages: obtaining a pre-emulsion and obtaining an emulsion. At the stage of preparation of the pre-emulsion, the required volume of antigenic material is supplied to the reactor with a sterile oil adjuvant with the stirrer switched on (250-300 rpm) at a speed of 100-120 l / h. After feeding the antigen into the oil adjuvant, the mixture is stirred at the same speed for 60-90 minutes, maintaining the temperature of the mixture within 12-16 ° C.

At the stage of emulsion preparation, the Silverson-450 LS homogenizer is turned on and the mixture is emulsified for 3 hours. The mixer is turned off.

Given the thermolability of the viral antigen, the emulsification process is carried out at a temperature of 12-16 ° C.

After the end of emulsification, a sample of the vaccine is taken from the reactor, maintained for one day and the physical parameters of the emulsion are monitored: type of emulsion, viscosity and stability. A vaccine with a positive quality characteristic is supplied for packaging. Packing and capping of the vaccine in bottles is carried out automatically on a filling machine and a seaming machine located under a laminar flow.

The resulting vaccine is monitored for compliance with the indicators established by the standard of the organization.

Example 2

Tests of the immunogenic activity of the vaccine against GP inactivated emulsion (series No. 1), manufactured as described in example 1, and containing, wt.%:

Purified and avirulent antigenic material from the strain "Novosibirsk" virus type A virus, subtype H5N1, obtained in 10-12-day-old embryos of SPF-hens with infectious activity of 9.1 lg ELD ₅₀ / ml and GA activity of 1: 1024 30,0 Oil adjuvant 70.0

For this, 5 chickens were immunized with the vaccine obtained at the age of 50 days intramuscularly in the region of the pectoral muscle group in a volume of 0.5 ml. 16 days after immunization, the chickens were infected with a homologous strain of the GP virus at a dose of 6.0 lg ELD ₅₀ intramuscularly. Immunized chickens (gr. No. 1) were kept in the same cage with the control group of chickens (gr. No. 2), consisting of uninfected and unvaccinated chickens.

After 60 hours, chickens of the second control group (gr. No. 3), consisting of infected chickens, were put into the cage (positive control).

The results of observation with a 12-hour interval for chickens of the experimental groups with fixation of visible clinical signs of the disease are presented in table 1 (this and all subsequent tables, see the end of the description).

The data in table 1 allow us to draw a number of conclusions:

1. Non-immune chickens infected with the virulent Novosibirsk strain of the HPV virus (gr. No. 3) became ill and died with the manifestation of clinical and pathological signs characteristic of severe GP.

2. Chickens of group No. 2 were infected from chickens of group No. 3 by an alimentary route. Chickens of group No. 2 also fell ill and died with the manifestation of clinical and pathological anatomical signs characteristic of severe GP.

3. Chickens immunized with the vaccine against GP emulsion inactivated from the Novosibirsk strain had 100% protection against severe GP disease.

In blood serum samples taken from chickens from the experimental groups, antigenic activity in the hemagglutination inhibition reaction (RTGA) was determined. The research results presented in table 2 indicate that the antibody titer of 5.8 log ₂ in RTGA in immunized chickens (gr. No. 1) at the time of infection (16 days after immunization) was sufficient to ensure 100% immunity from the disease GP in severe form. Control infection of chickens with a homologous strain did not cause an increase in the level of specific antibodies in the blood of the experimental group of chickens (gr. No. 1).

Example 3

Tests of the antigenic activity of the inactivated vaccine against GP (series No. 1), obtained as described in examples 1 and 2.

The purpose of the research was to study the formation of immunity to HP in chickens of different ages vaccinated with a vaccine in the amount of 0.25 ml, as well as the formation of immunity to GP in birds of different types: ducklings, goslings and turkey poults.

To study the immunity in chickens of different ages, 4 experimental and 4 control groups of chickens free of antibodies to the influenza virus were formed. The experimental and control groups contained 5 chickens of the same age. The chickens of the experimental groups were immunized intramuscularly with an inactivated emulsion vaccine in the region of the right breast in a volume of 0.25 ml.

In samples of blood serum taken from chickens at different times, the activity of antibodies to HA in rtga was determined.

The research results presented in table 3 indicate that the vaccine against GP emulsion inactivated from the Novosibirsk strain has high antigenic activity and can cause the formation of antihemagglutinating antibodies in vaccinated chickens aged 1 to 30 days with the introduction of 0.25 ml of the vaccine.

To study the formation of immunity in birds of different species, 3 experimental and 3 control groups of ducklings, goslings and turkey poults free of antibodies to the influenza virus were formed. The birds of the experimental groups were immunized with the proposed vaccine in a volume of 0.5 ml.

In samples of blood serum taken from birds of different species at different times, the activity of antibodies to GA in rtga was determined.

The research results presented in table 4 indicate that the proposed vaccine has high antigenic activity and can cause the formation of anti-hemagglutinating antibodies in birds of various types when the vaccine is administered in a volume of 0.5 ml.

Example 4

Tests of the antigenic activity of the inactivated vaccine against GP (series No. 1), obtained as described in examples 1 and 2.

The antigenic activity of the vaccine was studied in 45-day old chickens free of antibodies to the GP virus. For this, two groups of chickens were formed with 5 goals each, one of which was a control (unvaccinated). The chickens of the experimental group were immunized once intramuscularly in the region of the right breast in a volume of 0.5 ml inactivated emulsion vaccine from the strain Novosibirsk. The chickens were kept in an isolated box with the creation of negative atmospheric pressure in the cage, self-singing and feeding.

In samples of blood serum taken from chickens at different times, the activity of antibodies to HA in rtga was determined.

The research results presented in table 5 indicate that the proposed vaccine has high antigenic activity and can cause the formation of intense immunity against type A (H5N1) GP.

Example 5

Tests of the antigenic activity of the vaccine against GP inactivated emulsion (series No. 1), obtained as described in examples 1 and 2.

In experiments, we used chickens, 38 days old, free from antibodies to the HPV virus, of the Hayseks brown breed, obtained at the Kineshemskaya poultry farm in Ivanovo Region.

For the experiment, two experimental and one control group of birds were formed. The experimental design is presented in table 6.

To set up rtga, the serum to the H5N9 subtype HPV virus was used as a positive control (Institute of Zooprophylaxis, Venice, Italy), the normal blood serum of chickens (FGU ARRIAH) was used as the negative control, and the inactivated GP virus as antigen subtype of H5N1 strain "Novosibirsk". The number of test sera corresponds to the number of chickens in the experiment. Enzyme-linked immunosorbent assay (ELISA) was performed using the commercial Avian Influenza Virus Antibody Test Kit (Synbiotics Co, France, cep. No. OSUCAIV + 45003, November 16, 2005).

The ELISA was performed according to the methodology attached to the kit. A titer higher than 273 was considered positive.

The results of a study of sera in rtga presented in table 7 indicate that the level of antibodies to the hepatitis B virus of the H5N1 subtype 28 days after immunization was 8.5 log ₂ for birds vaccinated in a volume of 0.5 ml and 8.4 log ₂ for birds, immunized with a vaccine in a volume of 0.25 ml.

Presented in table 8, the results of studies in ELISA of blood serum of birds immunized with the proposed vaccine indicate that the proposed drug has a high antigenic activity.

Example 6

Tests of the antigenic activity of the vaccine against avian influenza inactivated emulsion (series No. 2 and No. 3), manufactured as described in example 1, and containing, wt.%:

Series No. 2

Purified and avirulent antigenic material from the strain "Novosibirsk" virus type A virus subtype H5N1, obtained in 10-12-day-old embryos of SPF-hens with infectious activity of 9.1 lg ELD ₅₀ / ml and GA activity of 1: 241 30,0 Oil adjuvant 70.0

Series No. 3

Purified and avirulent antigenic material from the strain "Novosibirsk" virus type A virus, subtype H5N1, obtained in 10-12-day-old embryos of SPF-hens with infectious activity of 9.0 lg ELD ₅₀ / ml and GA activity of 1: 234 40,0 Oil adjuvant 60.0

In experiments, chickens aged 40 days free of antibodies to the HPV virus were used.

For the experiment, 4 experimental and one control group of chickens were formed. The experimental design is presented in table 9.

To set up rtga, the serum to the H5N9 subtype HPV virus was used as a positive control (Institute of Zooprophylaxis, Venice, Italy), the normal blood serum of chickens (FGU ARRIAH) was used as the negative control, and the inactivated GP virus as antigen subtype of H5N1 strain "Novosibirsk". The number of test sera corresponds to the number of chickens taken in the experiment.

The results of a study of sera in rtga presented in table 10 indicate that the level of antibody titers depended on the time elapsed after immunization: after 23 days, the average titers of antibodies ranged from 7.9 to 8.2 log ₂ , after 12 days from 4.9 to 7 , 0 log ₂ depending on the vaccine vaccine volume.

The level of antibody titers in chickens above 4.0 log ₂ 12 days after immunization indicates a high antigenic activity of the proposed vaccine.

Example 7

Tests of the protective activity of the inactivated vaccine against avian influenza (series No. 1) obtained as described in examples 1 and 2.

The study of the protective activity of the vaccine with respect to the homologous Novosibirsk strain of the HPV virus was carried out by two methods: by placing contact chickens in vaccinated infected chickens in the same cell and by isolating the virus in embryos of SPF hens from cloacal smears of experimental chickens (as a group of vaccinated chickens, as well as groups of chickens, “contactees”). To control the selection of the virus from all groups of chickens with a time interval of 12 hours, cloacal smears were selected. To isolate the virus from each sample, 3 chicken embryos were used.

The presence or absence of the virus in the embryos was confirmed by the study of allantoic fluid in the RGA. Table 11 presents the results of the isolation of the virus from cloacal smears of experimental chickens, selected in the time interval from 0 to 60 hours after infection control, and the results of the RGA. The data presented in table 11 indicate that:

1) experimental chickens (vaccinated (gr. No. 1) and unvaccinated (gr. No. 2) virus with feces were not isolated within 60 hours after infection with the virulent strain "Novosibirsk" of the GP virus;

2) vaccinated chickens were protected from infection with a homologous strain of the GP virus.

Table 12 presents the results of a continuation of the study of the protective activity of the inactivated vaccine against GP. 60 hours after the control infection of vaccinated chickens (gr. No. 1), a control group of non-immune chickens (gr. No. 3) was placed in the same box, which was infected with the strain Novosibirsk virus of the GP virus. To control the isolation of the virus from all groups of chickens, cloacal smears were performed at a time interval of 12 hours. To isolate the virus from each sample, 3 chicken embryos were used.

As can be seen from the results presented in table 12, from two non-immune infected chickens, starting from 24 hours after infection, the virus was isolated, which is confirmed by the specific death of chicken embryos and a positive titer in the RGA. The virus was isolated from all chickens in this group 60 hours after infection. At the same time, the disease in this period of time manifested itself vividly: two chickens were sick with an acute course, and one died. 72 hours after infection, the remaining two chickens died. From chickens “contactees” (gr. No. 2), the virus was isolated from one chicken 36 and 48 hours after infection control. After 84 hours, one chicken showed clinical signs of the disease, after 120 hours, all three chickens from this group died. Vaccinated chickens (gr. No. 1) were not sick and did not secrete the virus.

Thus, the results of this experiment showed a high virulence of the Novosibirsk strain, the ability to be transmitted alimentarily and cause the death of non-immune chickens. At the same time, vaccinated chickens were completely protected from infection with a virulent virus, which was proved by the absence of clinical signs of the disease and the absence of its replication.

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

- vaccine against GP inactivated emulsion, 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 has been confirmed;

- inactivated emulsion vaccine against GP, made on the basis of the Novosibirsk strain in accordance with the invention, has high antigenic and immunogenic activity and is able to provide effective protection of the susceptible bird from the epizootic virus of the H5N1 subtype, as well as to prevent the pathogen from being infected by the immunized bird into the environment .

Information sources

1. Grebennikova T.V., Zaberezhny A.D. and Alipper T.I. "Diagnosis of influenza A using molecular methods." Veterinary life, 2006, No. 2 (50), 4.

2. Peradze TV, Fridman EA, Shild J. Anti-influenza prophylactic drugs. - M .: Medicine, 1986, 100-139.

3. Yamnikova S. S., Smolensky V. I., Vlasov N. A. et al. Prevention and control strategy for avian influenza. - Mater. II International vet. congress on poultry farming. - M.: March 21-23, 2006 - C.38-41.

4. Pat. France No. 2751225; A61K 48/00, 39/225, 39/17, 39/215; C12N 15/85; 01/23/1998

5. Pat. US No. 5916879, 514/44, 06/29/1999

6. WO 03/086453; A61K 39/295, 39/145; 10/23/2003

7. FAO Recommendations on the Prevention, Control and Eradication of Highly Pathogenic Avian Influenza (HPAI) in Asia. - September 2004, 1-59.

8. Ronda E., Garcia-Gancedo A., Alonso M.L. et al. Classical plague vaccine made from chicken embryo cells inactivated with beta-propiolactone. Mater. IX International congress on microbiology. Thes. Dokl., M., 1966, 465.

9. Narayan G., Rouse B.T., Lang G. A virus infection in turkeys. VI Artificial immunization against the Malignant virus strain Turkey / Ontario / 7732/66 / - Gen. J. Comp. Med., 1970, 34, 1, 72-79.

10. Rouse B.T., Lang G. and Narayan G. A new Influenza A virus Infection in Turkeys. VII. Comparative Immunology. - Canad. J. Comp. Med., 1971, 35, 44-51.

11. Kiyoko Iwatsuki-Horimoto et. al. The index influenza A virus subtype H5N1 isolated from a human in 1997 differs in its receptor-binding properties from a virulent avian influenza virus. - J. of Gen. ViroL, 2004, 85, 1001-1005.

12. WO 03/086453; A61K 39/295, A61K 39/145; 10/23/2003

13. Swayne D.E., Beck J.R., Garcia M. et. al. Influents of virus strain and antigen mass on efficacy of H5 avian influenza inactivated vaccines. - Avian Pathol., 1999, 28, 245-255.

14. Swayne D.E., Beck J.R., Perdue M.L. et. al. Efficacy of vaccines in Chickens against Highly Pathogenic Hong Kong H5N1 Avian Influenza. - Avian Dis., 2001, 45, N2, 355-365.

15. Website: www.intervet.com.

16. Pat. RF №594771, А61К 39/12, 07/07/1993

Table 1 Clinical state of chickens after infection with the H5N1 "Novosibirsk" strain Watch hours 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Gr. No. 1 3 * 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 Gr. Number 2 3 3 3 3 3 3 3 3 3 3 3 B ** B 1st 3M 3-M ^{^} 2-M Gr. Number 3 - - - - 3 3 B B B 2-B 3M 3M 3M 3M 3M 3M 1M Note: З * - healthy; B ** - patients; M ^ are dead.

table 2 The titers of specific antibodies in chickens determined using rtga after control infection with the strain of the Novosibirsk virus of GP Group number After infection, days. 16 23 28 No. 1 * No. 2 ** Number 3*** No. 1 Number 2 Number 3 No. 1 Number 2 Number 3 Gr.№1 one 1:64 1:64 1: 128 1: 128 1: 128 N / a 1: 128 1: 128 1: 128 2 1: 8 1:16 1:64 1:32 1: 256 N / a 1:64 1:32 1: 256 3 1:16 1:32 1:32 1:32 1:64 N / A 1:32 1:32 1:64 four 1:32 1:64 1:64 1:64 1: 128 N / a 1:64 1:64 1: 128 5 1:64 1: 256 1: 128 1: 128 1: 256 N / a 1: 128 1: 128 1: 256 Avg caption 4.8 5.8 6.2 6.0 7.2 6.2 6.0 7.2 Gr.№2 one - - N / a N / a N / a N / a N / a N / a N / a 2 - - N / a N / a N / a N / a N / a N / a N / a 3 - - N / a N / a N / a N / a N / a N / a N / a * No. 1 - antigen of the HPV virus subtype H5N2 produced by the Institute of Zooprophylaxis (Venice, Italy);
** No. 2 - antigen of the HPV subtype H5N3 virus produced by Pokrovsky Biological Product Plant OJSC (Pokrov, Russia);
*** No. 3 - antigen of the HPV virus subtype H5N1 produced by the Federal State Institution “Federal Center for Animal Health” (Federal State Institution VNIIZZH, Vladimir, Russia);
N / a - not investigated.

Table 3 The study of the formation of immunity in chickens of different ages to GP vaccinated with inactivated vaccine from the strain Novosibirsk in the amount of 0.25 ml Groups of chickens of different ages, days Group average titers of antihemagglutinating antibodies (log ₂ ) by time, days. fourteen 21 28 one Control 0.3 0.2 0.1 Experienced 3,5 4,5 4.8 10 Control 0.2 0.6 0.7 Experienced 3.9 4.6 5.1 twenty Control 0.4 0.8 0.5 Experienced 4.1 5,0 5.7 thirty Control 0.7 0.2 1,0 Experienced 4.3 5.2 5.8 Table 4 The study of the formation of immunity in birds of different species to GP, immunized with an inactivated vaccine from the strain Novosibirsk in a volume of 5 ml Groups of chickens of different ages, days Group average titers of antihemagglutinating antibodies (log ₂ ) by time, days. fourteen 21 28 Ducks Control 0.3 0.2 0.1 Experienced 4,5 5.8 6.9 Geese Control 0.2 0.6 0.7 Experienced 4.6 6.2 7.0 Turkeys Control 0.4 0.8 0.5 Experienced 4.3 5.8 7.0

Table 5 Antigenic activity of inactivated vaccine against GP from the strain "Novosibirsk" Groups of chickens Group average titers of antihemagglutinating antibodies (log ₂ ) by time, days. 0 fourteen 21 28 Experienced 1,2 4,5 5.1 5.3 Control 0.5 0.4 0.7 Table 6 Scheme of setting up an experiment to test the antigenic activity of an inactivated vaccine against GP from the Novosibirsk strain Group No. The term blood tests after immunization, days. The volume of vaccine vaccine, ml The number of vaccinated goals No. 1 28 0.5 10 Number 2 28 0.25 10 The control 28 - 5

Table 7 The results of determining the antigenic activity of an inactivated vaccine against GP from the strain "Novosibirsk" Group number Anti-HA antibody titers (dilution ratio, log ₂ dilution ratios) in triplicate one 2 3 Control group Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. No. 1 one 1: 1024 (10) 1: 512 (9) 1: 512 (9) 2 1: 256 (8) 1: 128 (7) 1: 256 (8) 3 1: 256 (8) 1: 128 (7) 1: 256 (8) four 1: 512 (9) 1: 512 (9) 1: 256 (8) 5 1: 256 (8) 1: 128 (7) 1: 256 (8) 6 1: 512 (9) 1: 128 (7) 1: 256 (8) 7 1: 512 (9) 1: 256 (8) 1: 256 (8) 8 1: 1024 (10) 1: 512 (9) 1: 512 (9) 9 1: 512 (9) 1: 128 (7) 1: 256 (8) 10 1: 512 (9) 1: 256 (8) 1: 256 (8) Geometric mean caption 8.9 7.8 8.2 8.3 log ₂ Number 2 one 1: 1024 (10) 1: 512 (9) 1: 512 (9) 2 1: 512 (9) 1: 512 (9) 1: 512 (9) 3 1: 128 (7) 1:64 (6) 1:64 (6) four 1: 1024 (10) 1: 512 (9) 1: 1024 (10) 5 1: 512 (9) 1: 256 (8) 1: 256 (8) 6 1: 1024 (10) 1: 512 (9) 1: 512 (9) 7 1: 512 (9) 1: 512 (9) 1: 512 (9) 8 1: 128 (7) 1:64 (6) 1:64 (6) 9 1: 1024 (10) 1: 512 (9) 1: 1024 (10) 10 1: 256 (8) 1: 256 (8) 1: 128 (7) 1: 1024 (10) 1: 512 (9) 1: 512 (9) 1: 128 (7) 1: 128 (7) 1: 128 (7) 1: 256 (8) 1: 128 (7) 1: 128 (7) Geometric mean caption 8.8 8.1 8.2 8.4 log ₂ Table 8 The results of determining the antigenic activity of an inactivated vaccine against GP from the strain "Novosibirsk" by ELISA Group number Average titer one 3578 2 1018 Control 47

Table 9 Scheme of setting up an experiment to test the antigenic activity of an inactivated vaccine against GP from the Novosibirsk strain №№ The term blood tests after immunization, days. Series of vaccine used Grafted vaccine volume, ml The number of vaccinated goals one 23 Number 2 0.5 10 2 12 Number 3 0.5 10 3 12 Number 3 0.25 10 four 12 Number 3 1,0 5 The control 12 - - 5

Table 10 The results of determining the antigenic activity of an inactivated vaccine against GP from the strain "Novosibirsk" Serum Sample number 1 repetition 2 repetition Series - 2, dose of 0.5 ml one 1: 256 (8) 1: 256 (8) 2 1: 512 (9) 1: 512 (9) 3 1:64 (6) 1:64 (6) four 1: 256 (8) 1: 256 (8) 5 1: 512 (9) 1: 512 (9) 6 1: 512 (9) 1: 512 (9) 7 1: 256 (8) 1: 128 (7) 8 1: 256 (8) 1: 128 (7) 9 1: 128 (7) 1: 128 (7) 10 1: 1024 (10) 1: 512 (9) Avg caption 8.2 7.9 Series - 3, dose 0.5 ml one 1:16 (4) 1:32 (5) 2 1:32 (5) 1:32 (5) 3 1: 8 (3) 1:16 (4) four 1:32 (5) 1:32 (5) 5 1:64 (6) 1:64 (6) 6 1: 128 (7) 1: 128 (7) 7 1:16 (4) 1:32 (5) 8 1:16 (4) 1:32 (5) 9 1:64 (6) 1:64 (6) 10 1:64 (6) 1:64 (6) Avg caption 5.2 5.5 Series - 3, dose 0.25 ml one 1:16 (4) 1:16 (4) 2 1:16 (4) 1:16 (4) 3 1:16 (4) 1:16 (4) four 1:64 (6) 1:64 (6) 5 1:32 (5) 1:32 (5) 6 1:16 (4) 1:16 (4) 7 1:32 (5) 1:32 (5) 8 1:64 (6) 1:64 (6) 9 1:64 (6) 1:64 (6) 10 1:32 (5) 1:32 (5) Avg caption 4.9 4.9 Control group one - - 2 - - 3 - - four - - 5 - - Harmless, dose of 1 ml one 1:64 (6) 1:64 (6) 2 1: 128 (7) 1: 128 (7) 3 1: 128 (7) 1: 128 (7) four 1: 128 (7) 1: 128 (7) 5 1: 256 (8) 1: 128 (7) Avg caption 7.0 6.8

Table 11 The results of the study of the isolation of the virus from cloacal smears of chickens after the control of infection with the strain "Novosibirsk" virus GP (in the time interval from the moment of infection to 60 hours after infection) Group number No. of chicken Hours / day after infection Hours of incubation (h.) / Death number, pcs. / RGA 24 48 72 96 120 one one 12 1 / from 1 / from - - - 2 - ¹ 1 / from - - - 3 1 / from - - - - four 1 / from - - - - 5 - 1 / from - - - 2 one - - - - - 2 1 / from - 1 / from - - 3 - - - - - one one 24 - - - - - 2 1 / from - - - - 3 - 1 / from - - - four - - - - - 5 1 / from - - - - 2 one - - - - - 2 - 1 / from - - - 3 1 / from - - - - one one 36 - - - - - 2 1 / from 1 / from - - - 3 - - - - - four 1 / from - - - - 5 - - - - - 2 one 1 / from - 1 / from - - 2 - - - - - 3 - 1 / from - - - one one 48 - 1 / from - - - 2 1 / from - - - - 3 - 1 / from - - - four - - - - - 5 1 / from - - - - 2 one 1 / from - - - - 2 - - - - - 3 1 / from 1 / from - - - one one 60 1 / from - - - - 2 - 1 / from - - - 3 1 / from - - - - four - - - - - 5 - - - - - 2 one - 1 / from - - - 2 1 / from - - - - 3 - - - - - Note: - ¹ - The death of chicken embryos was not recorded; from. - A negative result in the RSA.

Table 12 The results of the study of the selection of the virus from cloacal smears of chickens after the control of infection with the strain "Novosibirsk" virus GP (in the time interval from 72 to 120 hours after infection) Group number No. of chicken Hours / day after infection of groups Hours of incubation (h.) / Death number, pcs. / RGA 24 48 72 96 120 one one 72 2 3 four 5 2 one 72 2 3 3 one 12 2 3 one one 84 2 3 four 5 2 one 84 2 3 3 one 24 - ¹ 1/1: 512 1/1: 1024 - - 2 1 / from 1/1: 4096 1/1: 4096 - - 3 1 / from - - - one one 96 2 3 four 5 2 one 96 - - 1/1: 128 - - 2 - - - - - 3 1 / from 1/1: 4096 - - - 3 one 36 - - - - - 2 - 1/1: 256 - 1/1: 4096 - 3 - 1 / from - - - one one 108 2 3 four 5 one 108 - 1 / from 1/1: 2048 2 - - - - - 3 1 / from - - - - 3 one 48 - 1/1: 4096 1 / from - - 2 - - 1 / from - - 3 1 / from - - -

one one 120 2 3 four 5 - 2 one 120 - - - - - 2 - - - - - 3 - - - - - 3 one 60 - - 3/1: 256-1024 - - 2 - 1 / from 2/1: 256-1024 - - 3 1/1: 1024 2/1: 128-1024 - - Note: - ¹ - The death of chicken embryos was not recorded; from. - A negative result in the RSA.

Claims (6)

1. Inactivated emulsion vaccine against avian influenza, containing the active substance and the target additive, characterized in that it contains purified and avirulent antigenic material from the Novosibirsk strain of avian influenza virus Influenzae virus avicum, ceM.Orthomyxoviridae, serotype A, subtype H5N1, collection of FGU "VGNKI" ARRIAH No. 125-DEP "Novosibirsk", in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains purified and avirulent antigenic material from the Novosibirsk strain of avian influenza virus obtained in 10-12-day-old SPF-chicken embryos with infectious activity of at least 9.0 lg ELD ₅₀ / ml and hemagglutinating activity of at least 1:64. 3. The vaccine according to claim 2, characterized in that it contains purified and avirulent antigenic material from the Novosibirsk strain of avian influenza virus in an amount of 30.0-40.0 wt.%. 4. The vaccine according to claim 1, characterized in that it contains an oil adjuvant as a target additive. 5. The vaccine according to claim 4, characterized in that it contains an oil adjuvant in an amount of 60.0-70.0 wt.%. 6. The vaccine according to any one of claims 1 to 5, characterized in that it contains purified and avirulent antigenic material from the Novosibirsk strain of avian influenza virus and an oil adjuvant in the ratio, wt.%: Antigenic material 30.0-40.0 Oil adjuvant up to 100.0