RU2562547C1 - Inactivated sorptive foot-and-mouth disease type a vaccine - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science, namely to virology and biotechnology. The vaccine contains avirulent and purified antigen material of the foot-and-mouth disease type A virus strain No. 2171/Kabardino-Balkarian/2013 prepared in the passaged cell culture VNK-21, representing a suspension primarily containing 146S and 75S immunogenic components of the foot-and-mouth disease, the adjuvants aluminium hydroxide with saponin and a maintenance medium in effective ratios.

EFFECT: vaccine possesses the high immunogeneicity and is able to provide the effective protection against the homologous agent of the infection persistent in Transcaucasus, Central Asia, Middle and Near East.

11 cl, 7 ex, 8 tbl, 1 dwg

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of a vaccine inactivated adsorbed against foot and mouth disease type A.

Foot and mouth disease is a particularly dangerous, quarantine disease of artiodactyl animals, characterized by typical and variant variability of the virus, high contagiousness and aerogenic transmission of the pathogen, acute course and epizootic manifestation [1].

The widespread occurrence of foot and mouth disease, as well as the difficulties encountered in the diagnosis and selection of vaccine strains during anti-epizootic measures, are due to the high mutational variability of the genome and the antigenic diversity of foot and mouth disease [2, 3].

Vaccine prophylactic efficacy, determined by epidemiological indicators, is established in groups of vaccinated animals with a threat of the onset and spread of the disease. Vaccines should ensure the creation of mass immunity and prevent the spread of epizootic strains of foot and mouth disease virus. So, for successful prophylactic group vaccination, information is needed on the antigenicity of vaccine strains and epizootic isolates circulating in the preventive vaccination zone. In case of outbreaks of the disease, it is by determining the antigenic correspondence of the epizootic isolate to the production strain that the most suitable strain can be most quickly determined for the production of vaccines used in the threatened area [1].

To ensure sustainable well-being of the country by foot and mouth disease in the Russian Federation, a system of measures is being implemented, the priority of which is the prevention of the introduction of foot-and-mouth disease virus into the territory of the country, and vaccination prevention in areas of high risk. In the Russian Federation and the CIS countries, inactivated adsorbed vaccines are used for immunization of cattle and small cattle against foot and mouth disease, and inactivated emulsion vaccines are used for immunization of pigs [4].

The manufacturing technology of FMD vaccine from an inactivated virus begins with the selection of production strains based on an epizootological analysis of the dynamics of foot and mouth disease in the country and neighboring countries. When creating drugs for specific prophylaxis, appropriate types of foot-and-mouth disease virus are used and strains with a wide antigenic spectrum within the type with pronounced cross immunogenicity are selected. A strain with a wide range of immunogenicity and satisfying the requirements of the region is selected by testing it in a cross-protection reaction or more often in a cross-neutralization reaction. As a rule, a virus population is used as a production strain, which, together with the system and conditions of industrial cultivation, ensures a guaranteed and high accumulation of 146S and 75S virus components and the production of an immunogenic vaccine.

In addition, the production strain is required to maintain the stability of the virus during purification from tissue components and concentration, as well as the preservation of the virus during inactivation and long-term storage [5].

A well-known feature of the causative agent of foot and mouth disease is the antigenic variability of strains within the same serotype, occurring at different time intervals, in different territories, depending on the species composition of the susceptible livestock of animals, its immune status and many other factors.

It is believed that the main cause of antigenic variation is a change in the amino acid sequence in the polypeptides of the viral proteins that make up the virion capsid. In practice, such antigenic changes can be expressed both in insignificant differences between the strains, captured only using subtle methods of molecular analysis, and in the appearance of completely different strains requiring the use of new means of specific prophylaxis of the disease caused by these strains [2, 6].

Type A foot and mouth disease virus strains isolated in the USSR and used as production strains in the manufacture of FMD inactivated vaccines are known. These include: strain A ₇ No. 103, isolated in 1962 in the Kuibyshev region; strain A _t No. 2, isolated in 1965 in the Tajik SSR; strain A No. 717/73, isolated in 1973 in the Stavropol Territory. After the elimination of foot and mouth disease caused by antigenically similar strains of the virus, they were discontinued and are currently supported only in the Collection of Epizootic Strains of Foot and Mouth Disease Virus and Other Animal Pathogens of FSBI ARRIAH [4, 5, 6].

A known vaccine is inactivated adsorbed against type A foot and mouth disease, containing the active substance in the form of avirulent and purified antigenic material from strain A22 No. 550, homologous to the pathogen, obtained in a sensitive biological system, and targeted additives in the form of an adjuvant and support medium in an effective ratio [7].

Strain A ₂₂ No. 550 of foot-and-mouth disease virus was isolated in 1964 in Azerbaijan and used in the Russian Federation as an industrial one in the manufacture of specific prophylaxis and diagnostics that are used throughout Russia and the CIS countries.

Newborn rabbits are used as a sensitive biological system, and a phosphate-buffered solution is used as a supporting medium.

To clean the virus-containing suspension from ballast impurities, chloroform in 2% concentration and subsequent centrifugation are used.

Formaldehyde is used to inactivate the virus, and aluminum hydroxide (GOA) with saponin is used as adjuvant.

Known vaccine is inactivated adsorbed against foot and mouth disease type A, containing the active substance in the form of avirulent and purified antigenic material from strain A ₂₂ Iraq 24/64, obtained in a sensitive biological system, and target additives in the form of adjuvant and support medium in an effective ratio.

Strain A ₂₂ Iraq 24/64 of the FMD virus was transmitted in the form of aphthous material from the Razi Institute (Iran) in 1967. In the Russian Federation it is used as an industrial one in the manufacture of specific prophylaxis and diagnostics, used throughout Russia and the CIS countries.

To clean the virus-containing suspension from ballast impurities, chloroform in 2% concentration and subsequent centrifugation are used.

To inactivate the virus, aminoethylethyleneimine (AEEI) is used, and among the adjuvants, aluminum hydroxide (GOA) with saponin is used.

The closest to the invention according to the set of essential features is an inactivated adsorbed vaccine against foot and mouth disease type A containing the active substance in the form of avirulent and purified antigenic material from strain A No. 2045 / Kyrgyzstan / 2007, obtained in a sensitive biological system, and targeted additives in the form of adjuvants and maintenance medium in the ratio, mcg [8]:

antigenic material not less than 3.0 GOA 11000.0 ÷ 15000.0 saponin 500.0 ÷ -1500.0 support environment up to 1,000,000.0

The foot-and-mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007 was isolated in the Kyrgyz Republic from a foot-and-mouth disease heifer in 2007, belongs to the genetic line, called A Iran / 2005 [9, 10], and is used as a production strain in the manufacture of specific prevention and diagnosis used throughout Russia and the CIS countries.

A suspension culture of VNK-21 cells is used as a sensitive biological system, and Serum without serum with the addition of enzymatic dry muscle hydrolysates (FSMS), dry blood protein hydrolysates (GBKS) and antibiotics at pH 7.4 ÷ 7 are used as a supporting medium 6.

Polyhexamethylene guanidine (PHMG) is used to purify the virus-containing suspension from ballast impurities, and aminoethylethyleneimine (AEEI) is used to inactivate the virus. To neutralize AEEI, sodium thiosulfate is added to the suspension.

Avirulent and purified antigenic material from strain A No. 2045 / Kyrgyzstan / 2007 is a suspension mainly of 146S and 75S immunogenic components of foot and mouth disease virus. As adjuvants, GOA with saponin is used.

The main disadvantage of known vaccines, including the prototype vaccine, is the lack of immunogenic activity against epizootic isolates of FMD virus type A, currently circulating in the countries of the Caucasus, Central Asia and the Middle East (Turkey, Iran, etc.), due to significant antigenic differences. They do not provide reliable protection for susceptible animals from the epizootic virus of type A foot and mouth disease, which causes disease in these countries in recent years.

The task of creating the present invention was to develop a vaccine for FMD inactivated adsorbed, which creates effective protection of susceptible animals against the epizootic virus of foot and mouth disease type A circulating in the countries of the Caucasus, Central Asia, the Middle East and the Middle East.

The technical result from the use of the invention is to expand the arsenal of vaccines inactivated adsorbed against foot and mouth disease type A.

The specified technical result was achieved by the creation of a vaccine inactivated adsorbed against foot and mouth disease type A, characterized by the following set of features.

The proposed vaccine contains 1 cm ^{3 of the} preparation: the active substance in the form of avirulent and purified antigenic material from a foot and mouth disease virus strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a suspension culture of BHK-21 cells, in an amount of at least 3.0 μg and target additives: GOA, preferably in an amount of 11000.0 ÷ 15000.0 μg, saponin, preferably in an amount of 500.0 ÷ 1500.0 μg and a support medium in an amount of up to 1,000,000.0 μg.

The initial virus for obtaining strain A No. 2171 / Kabardino-Balkarian / 2013 was isolated in July 2013 in the village of Nizhny Kurkuzhin, Baksan district, Kabardino-Balkaria. The strain was obtained by repeated consecutive passages on sensitive hetero- and homologous cell cultures. The strain is adapted to primary trypsinized pork kidney cells and transplantable cell cultures of BHK-21, IBRS-2 and PSGK-30.

For the manufacture of the vaccine, a suspension culture of BHK-21 cells is preferably used as a sensitive biological system, and Earle's serum-free solution with the addition of FGMS, GBKS, and antibiotics at pH 7.4–7.6 is used as a supporting medium.

For inactivation of the virus using AEEI, which is added to the virus-containing suspension to a concentration of 0.025 ÷ 0.05%. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to the suspension [11].

The resulting antigen is purified from ballast impurities using PHMG, which is introduced into the suspension to a concentration of 0.005 ÷ 0.007% [12].

Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarian / 2013 is a suspension containing predominantly 146S and 758 immunogenic components of foot and mouth disease virus.

The quantitative and qualitative content of viral raw materials is determined by turbidimetry [13].

For the preparation of the vaccine, viral material is used that contains at least 0.5 μg of 146S and 75S immunogenic components of foot and mouth disease virus in 1 cm ³ .

The required concentration of 146S and 75S immunogenic components of foot and mouth disease virus in the proposed vaccine, comprising at least 3 μg in 1 cm ^{3 of the} finished product, is obtained by adding the calculated amount of GOA sorbent adjuvant to the avirulent and purified antigenic material. The optimum is the GOA content in 1 cm ^{3 of the} finished product in the range from 11000.0 μg to 15000.0 μg.

An additional 10% aqueous solution of saponin is added to the resulting concentrate to its content in 1 cm ^{3 of the} finished preparation in the range from 500.0 μg to 1500.0 μg.

The resulting vaccine is a light yellow liquid with a loose precipitate of sorbent, which is formed at the bottom of the vial during storage and is easily broken into a homogeneous suspension with shaking.

The present invention includes the following set of essential features, providing a technical result in all cases for which legal protection is requested.

1. The vaccine is inactivated adsorbed against foot and mouth disease type A.

2. The active substance in the form of avirulent and purified antigenic material from a strain of foot and mouth disease virus A No. 2171 / Kabardino-Balkarsky / 2013 in an effective amount.

3. Targeted supplements.

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. vaccine inactivated adsorbed against foot and mouth disease type A;

2. active substance;

3. targeted supplements.

Compared with the prototype vaccine, an essential distinguishing feature of the proposed vaccine is that it contains an avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarian / 2013 type A foot and mouth disease virus 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. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained in a sensitive biological system and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

2. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarian / 2013, obtained preferably in a transplanted cell culture of animal origin and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

3. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A.

4. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarian / 2013, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing predominantly 146S and 75S immunogenic components of FMD virus type A in an amount of at least 3, 0 mcg in 1 cm ^{3 of the} finished product.

5. As a target additive, the vaccine contains an adjuvant-sorbent GOA.

6. GOA, preferably in an amount of 11000.0 ÷ 15000.0 μg in 1 cm ^{3 of the} finished product.

7. As a targeted supplement, the vaccine contains an adjuvant saponin.

8. Saponin, preferably in an amount of 500.0 ÷ 1500.0 in 1 cm ^{3 of the} finished product.

9. As a targeted supplement, the vaccine contains a supportive environment.

10. Supporting medium in an amount up to 1,000,000.0 mcg in 1 cm ^{3 of the} finished product.

11. Avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplanted cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A, GOA, saponin and supporting medium in the ratio, mcg / cm ³ :

antigenic material not less than 3.0 GOA 11000.0 ÷ 15000.0 saponin 500.0 ÷ 1500.0 support environment up to 1,000,000.0

The proposed vaccine has high immunogenic activity and provides reliable protection against FMD virus serotype A circulating in the countries of the Caucasus, Central Asia, the Middle and Middle East.

The achievement of the technical result from the use of the invention is achieved by the fact that the composition of the proposed vaccine introduced as an active substance antigenic material from the strain

A No. 2171 / Kabardino-Balkarsky / 2013 of the foot-and-mouth disease virus of serotype A, which has high biological, antigenic and immunogenic activity in its native form and, after inactivation, provides the FMD vaccine adsorbed inactivated, which effectively protects susceptible animals against the foot-and-mouth disease virus serotype A, which causes outbreaks of the disease in recent years in the countries of Transcaucasia, Central Asia, the Middle and Middle East.

Strain A No. 2171 / Kabardino-Balkarian / 2013, type A foot-and-mouth disease virus was deposited on January 28, 2014 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI “ARRIAH”), under the registration number (link): virus strain foot and mouth disease A No. 2171 / Kabardino-Balkarian / 2013 (production).

The invention is illustrated in a graphical image in which a dendrogram reflecting phylogenetic relationships of a strain of foot and mouth disease virus A No. 2171 / Kabardino-Balkarian / 2013 with epizootic and vaccine strains of foot and mouth disease virus of serological type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP1 gene.

The invention is explained in the list of sequences in which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP1 protein gene of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus;

SEQ ID NO: 2 represents the amino acid sequence of the VP1 protein gene of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus.

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus is characterized by the following features and properties.

Morphological features

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus belongs to the family Picornaviridae, genus Aphtovirus, serotype A and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the icosahedral form of the virion is 23-25 nm. Virion consists of an RNA molecule enclosed in a protein coat. The protein shell consists of 32 capsomeres arranged in cubic symmetry.

Antigenic properties

According to its antigenic properties, strain A No. 2171 / Kabardino-Balkarian / 2013 of the FMD virus belongs to type A. The virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity (GA activity). In sick animals, antibodies are formed in the blood serum that are detected in an enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN). When cattle are immunized with a vaccine from an inactivated virus, it induces the formation of specific antibodies detected in ELISA and RMN.

During hyperimmunization of guinea pigs, the concentrated antigen from inactivated foot and mouth disease virus type A No. 2171 / Kabardino-Balkarsky / 2013 induces the formation of virus-specific antibodies detected in CSC at a dilution of 1:30.

Using the method of nucleotide sequencing, the primary structure of the type 1 FMD virus of the FMD virus No. 2171 / Kabardino-Balkarsky / 2013 was determined and the primary structure of the VP1 protein was derived. A comparative analysis of the nucleotide sequences showed that the type A foot-and-mouth disease virus strain No. 2171 / Kabardino-Balkarsky / 2013 is significantly different from the production strains of type A. The degree of nucleotide differences in the sequences of strain A No. 2171 / Kabardino-Balkarian / 2013 sequences with foot-and-mouth disease virus strains of serological type A was : A ₂₂ No. 550 - 22.65%, A ₂₂ / Iraq / 64 - 21.66%, A / Iran / 96 - 21.43%, A / Armenia / 98 - 21.98%, A / Turkey / Ob -8.23%, A / Iran / 05 - 8.75%.

Thus, phylogenetic analysis showed that strain A No. 2171 / Kabardino-Balkarsky / 2013 belongs to the Iran 2005 genetic line of the Asia topotype of FMD virus of serological type A and is antigenically different from existing production strains of VJ type A.

The antigenic affinity of strain VYA A No. 2171 / Kabardino-Balkarsky / 2013 with existing production strains of VYA A was investigated by the serological method in the cross microneutralization reaction (RMN). Strain A No. 2171 / Kabardino-Balkarian / 2013 antigenically differs from production strains A22 No. 550, A22 Iraq / 64, A / Iran / 97, A / Turkey / Ob (A / Iran / 05), A / Kyrgyzstan / 07 (A / Iran / 05).

The results of the studies in the RMN are presented in table 1. Antigenic compliance (r ₁ ) was for A22 No. 550 - 0.013, A ₂₂ Iraq / 64 - 0.02, A / Iran / 97 - 0.03, A / Turkey / 06-0 , 19 and A / Kyrgyzstan / 2007 - 0.03. With a value of r ₁ ≥0.3, the field isolate and the production strain are closely related and the vaccine from the production strain will protect against an epizootic virus, with a value of r ₁ <0.3 the field isolate is different from the production strain and the vaccine from this strain does not protect against an epizootic virus [14, 15].

Biotechnological characteristics

Strain A No. 2171 / Kabardino-Balkarsky / 2013 is reproduced in monolayer cell cultures: a primary trypsinized cell culture of pig kidneys (SP), transplantable cultures of kidney cells of the Siberian mountain ibex (PSGK-30), BHK-21 and IB-RS-2. Within 18 ÷ 24 hours of incubation, the virus harvest in these cultures reaches values from 6.00 to 7.50 Ig TCD ₅₀ / cm ³ . With a high multiplicity of infection (1 ÷ 10 TCD / cell), the virus causes CPD after 5 hours. The virus retains its original characteristics when passaged in cell cultures for 5 passages (observation period).

Gene and chemotaxonomic characteristics

Strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus is an RNA-containing virus with a molecular weight of 7 × 10 ⁹ D.

Nucleic acid is a single-chain linear molecule with a molecular weight of 2.8 × 10 ⁶ D. The virion has a protein shell consisting of four basic proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ . The lipoprotein membrane is absent.

The main antigenic protein is VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Virionic RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural virus polypeptides. Of the 8 non-structural polypeptides that accumulate in infected cells, one (VP _66a ) is an RNA-dependent RNA polymerase involved in RNA replication of new virions.

Physical properties

The virion mass is 8.4 × 10 ⁻¹⁸ g. A sedimentation coefficient of 146S in the sucrose gradient. The floating density of 1.45 g / cm ³ .

Resistance to external factors

Strain A No. 2171 / Kabardino-Balkarsky / 2013 is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.2 ÷ 7.6. PH shifts to both the acidic and alkaline sides lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-radiation, high temperatures.

Additional features and properties

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

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals, newborn mice, guinea pigs.

Virulence - virulence for naturally susceptible animals with contact, aerosol and parenteral infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 10 passages (observation period).

Based on the data obtained, it can be argued that strain A No. 2171 / Kabardino-Balkarsky / 2013 in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMD virus type A, has a higher protective and immunogenic activity.

To reduce its epizootic danger, timely vaccine prophylaxis is important to prevent the emergence of new foci of the disease, for which a highly immunogenic vaccine is needed.

A highly immunogenic inactivated sorbed vaccine against foot and mouth disease type A was obtained from strain A No. 2171 / Kabardino-Balkarsky / 2013.

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

Example 1

Strain A No. 2171 / Kabardino-Balkarian / 2013, type A foot-and-mouth disease virus was isolated from field material received at the Federal State Budget Scientific Institution ARRIAH in the form of epithelium aft from cattle suspected of foot-and-mouth disease during laboratory diagnosis of this disease and its differentiation from other vesicular diseases. When isolating the virus, a complex of biological, virological and biochemical methods was used.

Biological and virological methods included the isolation of the virus on a culture of primary trypsinized SP cells, transplanted PSGK-30, IB-RS-2 cell lines with subsequent adaptation (3 passages). For staging biological samples in primary and transplanted cell cultures, they were grown on appropriate nutrient media, under stationary conditions in bottles with a surface area of 25 cm ² , washed from the growth medium and infected with a 10% suspension of aphthous material (the multiplicity of infection was 1 ÷ 10 TCD ₅₀ per cell ) prepared in a Hanks solution with 0.5% lactalbumin hydrolyzate (GLA) and antibiotics according to a standard formulation. To remove microflora and ballast cell components, the suspension was treated with 10% chloroform. After 30 minutes of contact at 37 ° C, 5 cm ^{3 of the} support medium was added to the vials and incubated at 37 ° C until the appearance of the virus CPD. In the presence of CPD (rounding of cells, increasing their optical density, degeneration and separation of cells from glass), the bottles were subjected to freezing, thawing, purification of the cell suspension with chloroform and centrifugation at 3000 g for 15 min. The resulting virus-containing material was used for subsequent passages and studies in the CSC for the presence of a viral antigen, using commercial type-specific sera stored in the Museum of strains of the Federal State Budget Scientific Institution VNIIZZH. The virus was considered adapted to cell cultures if, within 18–24 hours, (%) 90–100 CPDs appeared in the monolayer.

A virus adapted to IB-RS-2 cell cultures was used to produce antigen for CSC.

The virus adapted to the PSGK-30 cell culture was used to infect the VNK-21 cell suspension in order to obtain antigen for the manufacture of an experimental vaccine series, as well as for hyperimmunization of guinea pigs.

The results of the adaptation of the virus to cell cultures are presented in table 2.

The data shown in table 2 indicate a high adaptive activity of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus to the used cell cultures.

Example 2

When studying the properties of strain A No. 2171 / Kabardino-Balkarsky / 2013, significant advantages were revealed in comparison with the used production strains.

Tables 3 and 4 show the results of the cultivation of strains A No. 2171 / Kabardino-Balkarian / 2013 and A No. 2045 / Kyrgyzstan / 2007 of type A foot and mouth disease virus, from which it follows:

1) strains A No. 2171 / Kabardino-Balkarian / 2013 and A No. 2045 / Kyrgyzstan / 2007 have different reproduction times, 10.30 ± 0.35 and 12.40 ± 0.45 hours, respectively;

2) the percentage of yield of immunogenic components during the cultivation of foot-and-mouth disease virus No. 2171 / Kabardino-Balkarsky / 2013 is higher than that of strain A No. 2045 / Kyrgyzstan / 2007.

Example 3

An inactivated adsorbed vaccine against foot and mouth disease type A is prepared from a strain of foot and mouth disease virus No. 2171 / Kabardino-Balkarsky / 2013 grown in a suspension culture of BHK-21 cells. As a supporting medium, Earl's solution without serum is used with the addition of FGMS, GBKS and antibiotics at a pH of 7.4 + 7.6. The cell culture is infected with a virus at the rate of 0.005 ÷ 0.2 TCD ₅₀ per cell.

The cultivation of the virus is carried out at a temperature of 36-37 ° C. After 6–7 hours of incubation, living and dead cells are counted with trypan blue staining. If the number of living cells is 15 ÷ 20%, then incubation is continued for another 2 + 3 hours. When the number of dead cells reaches 90 + 95%, the cultivation is stopped and the virus-containing suspension is monitored for sterility and the content of 146S and 75S components. The amount of 146S + 75S components in the suspension should be at least 0.5 μg / cm ³ . Immediately after the end of the virus reproduction cycle, without stopping thermostating, a 15–20% AEEI solution, acidified with glacial acetic acid to pH 8.0–8.5, is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to 0.025 ÷ 0.05%. Inactivation of the infectiousness of the virus is carried out for 12 ÷ 24 hours at 36 ÷ 37 ° C and a pH of 7.2 ÷ 7.6 with stirring after 5 + 6 hours for 3 ÷ 5 minutes. The remainder of the AEEI is neutralized by the addition of sodium thiosulfate.

A 10% solution of PHMG is added to the warm suspension to a concentration of 0.005–0.007% for flocculation of ballast impurities and inactivation of possible contaminants. Flocculated ballast impurities are subjected to sedimentation followed by decantation.

Table 5 shows the results of studies on the effect of inactivation and purification of antigenic material using AEI and PHMG on the immunogenic (146S and 75S) components of foot-and-mouth disease virus of strains A No. 2171 / Kabardino-Balkarsky / 2013 and A No. 2045 / Kyrgyzstan / 2007.

The data presented in table 5 indicate that the immunogenic components of foot and mouth disease virus strain A No. 2171 / Kabardino-Balkarsky / 2013 are not inferior in resistance to inactivation and purification in conditions of production of foot and mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007. Especially important is the fact that during the inactivation and purification of the virus from strain A No. 2171 / Kabardino-Balkarsky / 2013, there were no changes in the number of 146S and 75S components obtained during virus reproduction.

Example 4

The resulting antigen is monitored for avirulence, virus-specific protein content, 146S and 75S virus components, and sterility. The required concentration of 146S and 75S components in 1 cm ^{3 of the} adsorbed vaccine is obtained by concentration of GOA antigen.

The calculated GOA volume of 3% concentration is added to the cooled antigen suspension with the stirrer operating. Stirring is carried out for 30 minutes. After sedimentation of GOA, the calculated volume of the remaining suspension is drained. The final concentration of GOA should be in the range of 1.28 ± 0.22 p <0.001 mg / cm ³ n = 10, and the concentration of 146S and 75S components of the FMD virus is at least 3.0 μg / cm ^{3 of the} finished product. Then, an additional 10% saponin solution is added to the suspension to a final concentration of 0.05 ÷ 0.15%, which corresponds to 500.0 ÷ 1500.0 μg of saponin in 1 cm ^{3 of the} finished vaccine. The resulting vaccine is packaged in glass or plastic bottles and its sterility is monitored in accordance with GOST 28085-89.

Example 5

The virulence and harmlessness of the vaccine is checked on 5 heads of cattle, introducing the vaccine first under the mucous membrane of the tongue at a dose of 2.0 cm ³ and then subcutaneously at a dose of 10.0 cm ³ . Observation of the clinical condition of the animals is carried out for 10 days. Avirulent, harmless and sterile vaccine is tested for immunogenic activity in cattle or guinea pigs.

The resulting vaccine is a light yellow liquid with a loose white precipitate of sorbent, which is formed at the bottom of the vial during storage and is easily broken into a homogeneous suspension with shaking.

The optimal component composition of the resulting sorbed vaccine against foot and mouth disease type A are shown in table 6.

Example 6

The humoral immunity of cattle vaccinated with a sorbed vaccine from a foot-and-mouth disease virus strain A No. 2045 / Kyrgyzstan / 2007 against the heterologous strain of foot-and-mouth disease virus A No. 2171 / Kabardino-Balkarsky / 2013 was studied. The level of humoral immunity was evaluated against vaccine strain A No. 2045 / Kyrgyzstan / 2007 and against strain A No. 2171 / Kabardino-Balkarian / 2013.

The test was conducted on 10 heads of cattle. Sorbed vaccine was administered subcutaneously at a dose of 2.0 cm ³ . The research results are presented in table 7.

From the data shown in table 7, it can be seen that the adsorbed vaccine stimulated the production of virus-neutralizing antibodies against strain A No. 2045 / Kyrgyzstan / 2007 in cattle in an amount of 4.80 ± 0.24 to 5.40 ± 0.06 log ₂ . Against the heterologous strain A # 2171 / Kabardino-Balkarsky / 2013, the titer of neutralizing antibodies was 4.3–7.5 times less and ranged from 2.50 ± 0.65 to 2.70 ± 0.73 log ₂ . The difference in the titers of neutralizing antibodies is significant and significant.

It follows that the vaccine made from production strain A No. 2045 / Kyrgyzstan / 2007 does not create humoral immunity to protect animals from infection with the heterologous FMD virus A No. 2171 / Kabardino-Balkarsky / 2013. According to the recommendations of the OIE, the titer of neutralizing antibodies should be at least 5.00 log ₂ [14].

Example 7

Tests of the adsorbate vaccine against foot and mouth disease type A, made as described in examples 3 and 4, and containing, mcg:

avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarian / 2013 type A foot and mouth disease 3.0 GOA 13000.0 saponin 750.0 support environment up to 1,000,000.0

The virulence and safety of the obtained vaccine was tested on 5 heads of cattle. The drug was administered to each animal under the mucous membrane of the tongue at a dose of 2.0 cm ³ and then subcutaneously at a dose of 10.0 cm ³ . Observation of the clinical condition of the animals was carried out for 10 days. Throughout the entire observation period, deviations in the clinical condition of the animals were not detected, which means that the vaccine administered is avirulent and harmless.

At the end of the control for avirulence and safety, the vaccine was tested for immunogenic activity of the cattle vaccine. The test was conducted on 15 heads of cattle. The results are presented in table 8. ImD _{50 of the} drug was 0.11 cm ³ , i.e. the vaccine dose contained 18 PD ₅₀ .

According to the recommendations of the OIE, the vaccine against foot and mouth disease should contain at least 6 PD ₅₀ in the vaccine volume for cattle [14].

Therefore, the manufactured inactivated sorbed vaccine from a foot and mouth disease virus strain A No. 2171 / Kabardino-Balkarian / 2013 has a high immunogenic activity.

Thus, the above information indicates that the vaccine is sorbed inactivated against FMD type A, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology; confirmed the possibility of carrying out the invention; the vaccine made from strain A No. 2171 / Kabardino-Balkarsky / 2013 in accordance with the invention has high immunogenic activity and is able to provide effective protection of susceptible animals against type A foot and mouth disease virus circulating in the countries of the Caucasus, Central Asia, the Middle East and the Middle East .

Information sources

1. Dudnikov A.I. FMD immunity and practical achievements in the field of FMD / A.I. Dudnikov, V.V. Borisov, S.A. Dudnikov // Probl. Zooengineerii that vet. Medicine: ST. sciences. pra - Kharkiv, 2007. - VIP. 15 (40). - Part 2 - T.1. - S.116-120.

2. Domingo, E. Evolution of FMD virus / E. Domingo, C. Escarmis, E. Baranovski // Virus Res. - 2003. - Vol. 91, No. 1. - P. 47-63.

3. Uzyumov V.L. Ultrastructure and physicochemical properties of foot and mouth disease virus / V.L. Uzumov: Frunze: Kyrgyzstan, 1970 .-- 246 p.

4. Viral diseases of animals / V.N. Syurin, A.Ya. Samuilenko, B.V. Soloviev [et al.]. - M.: VNIITIBP, 1998 .-- S.532-548.

5. Rerer X. Foot and mouth disease: Per. with him. / G.A. Surkova; under the editorship of and with the foreword. P.V. Malyarets. - M .: Kolos, 1971. - 432 p.

6. Foot and mouth disease: monograph / A.M. Burdov, A.I. Dudnikov, P.V. Malyarets [et al.]. - M .: Agropromizdat, 1990 .-- 320 p.

7. Temporary instructions for the manufacture and control of FMD concentrated aluminum hydroxide formol vaccine from lapinized virus A22. Approved by the GUV of the USSR on March 25, 1971

8. Application 2012134084 Russian Federation, IPC A61K 39/135, Inactivated type A foot and mouth disease vaccine / D.A. Lozovoi, V.V. Mikhalishin, D.V. Mikhalishin, V.A. Starikov [et al.]; FSBI "ARRIAH". - Declared. 08/09/2012; publ. 02/20/2014

9. The study of the biological properties of the FMD virus line A Iran / 05 production strains of type A ₂₂ / M.V. Zhiltsova, S.R. Kremenchug, A.I. Egorova, V.V. Borisov // Russian Veterinary Journal. - 2008. - September. - S.15-16.

10. Pat. 2451745 Russian Federation, IPC A61K 39/135, Strain A 2045 / Kyrgyzstan / 2007, foot and mouth disease virus Aphtae epizooticae type A for monitoring the antigenic and immunogenic activity of FMD vaccines and for the manufacture of biological products for the diagnosis and specific prevention of foot and mouth disease type A / E.V. Belik, V.V. Borisov, A.V. Shcherbakov, S.R. Kremenchug [and others]; FSBI "ARRIAH". - Declared. 07/26/2010; publ. 05/27/2012

11. Pat. 594771 Russian Federation, IPC A61K 39/12, Tool for inactivation of viruses in the manufacture of antiviral drugs / N.A. Ulupov, A.I. Dudnikov, P.A. Gembitsky, D.S. Beetle; ARRIAH. - Declared. 05/07/1973; publ. 07/07/93

12. Pat. 2054039 Russian Federation, IPC A61K 39/135, Method for cleaning and sterilizing a culture of foot and mouth disease virus / T.N. Lezova, N.A. Ulupov, V.V. Borisov, V.V. Mikhalishin, A.I. Dudnikov, P.A. Gembitsky; ARRIAH. - Declared. 02/07/1992; publ. 02/10/1996

13. Auth. testimonial. 784335 USSR, C12Q 1/02, Method for determining the quality of viral raw materials / A.F. Bondarenko; ARRIAH. - Declared. 07/04/1979; publ. 03/20/2000

14. OIE. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. - 7th ed. - Paris. 2012 .-- Vol. 1, Chapter 2.1.5. - P.166-169.

15. Selection of foot-and-mouth disease vaccine strains a review / D.J. Paton, J.F. Valacher, J. Bergman [et al.] // Rev. Sci. Tech. OIE. - 2005. - Vol.24, No. 3. - P.981-983.

Table 1 Antigenic correspondence (r ₁ ) in the RMN of strain A No. 2171 / Kabardino-Balkarian / 2013 of type A foot and mouth disease virus with production of type A foot and mouth disease virus strains Strain Exponent r ₁ A ₂₂ No. 550 0.013 A ₂₂ / Iraq / 64 0.02 A / Iran / 97 0,03 A / Turkey / 06 0.19 A No. 2045 / Kyrgyzstan / 2007 0,03 r ₁ ≥0.3 - the field isolate is antigenically related to the production strain, the vaccine from the production strain can be used if a more related strain is not found and provided that the animals are immunized more than 1 time;
r _i <0.3 - the field isolate differs from the production strain, the vaccine from which is not acceptable for protection against infection by the field isolate

table 2 Biological properties of the virus of epizootic strain A No. 2171 / Kabardino-Balkarian / 2013 FMD virus type A Virus reproduction system The time of manifestation of biological activity (hours) Number of adaptation passages Adapted material characteristics Activity in RSK The titer of infectious activity (Ig TCD ₅₀ / cm ³ ) PSGK-30 twenty four 1: 4 6.45 IB-RS-2 24 3 1: 2 6.0 VNK-21 twenty 5 1: 2 7.5

Table 3 The cultivation of a strain of foot and mouth disease virus A No. 2171 / Kabardino-Balkarian / 2013 in a suspension of BHK-21 cells n = 10 No. p / p The concentration of cells, million / cm ³ Dose of infection, TCD ₅₀ / cl Duration of reproduction, hours The titer of the infectivity of the virus, Ig TCD ₅₀ / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ % yield of 146S + 75S components one 3.8 0.005 eleven 8.00 1,69 1.47 87.0 2 4.2 0.02 eleven 7.75 1.84 1.71 92.9 3 3.9 0.02 11.5 8.00 1,64 1.3 79.3 four 4.2 0.02 9.5 7.25 1.26 1.22 96.8 5 3.8 0,025 eleven 7.75 1.46 1.19 81.5 6 4.1 0,03 8 7.25 1.42 1.22 85.9 7 4.2 0,03 11.5 8.00 1,63 1.34 82,2 8 4.1 0.004 9.5 7.00 1.24 1.15 92.7 9 3.9 0,03 10 7.50 1.26 1.16 92.0 10 3.6 0.05 10 7.50 1.32 1.10 83.3 M ± m 3.98 ± 0.07 p <0.001 0.023 ± 0.004 p <0.001 10.30 ± 0.35 p <0.001 7.60 ± 0.11 p <0.001 1.48 ± 0.07 p <0.001 1.29 ± 0.06 p <0.001 87.4 ± 1.87 p <0.001

Table 4 The cultivation of a strain of foot and mouth disease virus A No. 2045 / Kyrgyzstan / 2007 in a suspension of BHK-21 cells n = 10 No. p / p The concentration of cells, million / cm ³ Dose of infection, TCD ₅₀ / cl Duration of reproduction, hours The titer of the infectivity of the virus, Ig TCD ₅₀ / cm ³ VSB, m kg / cm ³ 146S + 75S components, mcg / cm ³ % yield of 146S + 75S components one 3,7 0.06 13 7.50 2.10 1.72 81.9 2 4.0 0.06 11.5 7.50 2.72 2.47 90.8 3 3,7 0.06 12 7.75 3.24 2.48 76.5 four 3,7 0.04 eleven 8.00 2.59 1.99 76.8 5 3,7 0.04 12 8.25 3.30 2.73 82.7 6 3,5 0.02 12.5 7.00 2.18 2.05 94 7 3.6 0.05 13.5 6.75 2.08 1.73 83,2 8 3.4 0.05 13.5 6.50 2.00 1.90 95 9 3,7 0.002 10 7.75 2.02 1.89 93.6 10 4.0 0.002 fifteen 7.25 1.87 1,60 85.6 M ± m 3.70 ± 0.06 p <0.001 0.038 ± 0.007 p <0.001 12.40 ± 0.45 p <0.001 7.42 ± 0.17 p <0.001 2.41 ± 0.17 p <0.001 2.06 ± 0.12 p <0.001 86.0 ± 2.20 p <0.001

Table 5 Comparative data on the resistance of foot and mouth disease virus of strain A No. 2045 / Kyrgyzstan / 2007 and strain A No. 2171 / Kabardino-Balkarsky / 2013 to inactivation by aminoethylethyleneimine and sterilizing purification with polyhexamethylene guanidine No. p / p A No. 2045 / Kyrgyzstan / 2007 A No. 2171 / Kabardino-Balkarian / 2013 cultivation termination after inactivation and cleaning cultivation termination after inactivation and cleaning VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ VSB, mcg / cm ³ 146S + 75S components, mcg / cm ³ one 2.02 1.89 2.14 2.00 1,69 1.47 2.08 1.81 2 3.24 2.48 2.95 2.44 1.84 1.71 1.78 1,67 3 2.59 1.99 2.69 2.16 1.26 1.22 1,53 1.48 four 1.87 1,60 2.17 1.98 1.46 1.19 1.88 1,53 5 3.30 2.73 2.72 2.42 1.42 1.22 1.77 1,52 6 2.72 2.47 2.91 2.65 1,63 1.34 1.92 1,58 M ± m 2.6210.24 p <0.001 2.19 ± 0.18 p <0.001 2.60 ± 0.15 p <0.001 2.28 ± 0.11 p <0.001 1.55 ± 0.09 p <0.001 1.36 ± 0.08 p <0.001 1.83 ± 0.08 p <0.001 1.60 ± 0.05 p <0.001

Table 6 Optimal formulation of FMD inactivated adsorbate vaccine from foot and mouth disease virus type A, strain A No. 2171 / Kabardino-Balkarian / 2013 (μg in 1 cm ^{3 of the} finished vaccine) Vaccine components Minimum Average Maximum antigenic material 3.0 > 3.0 > 3.0 GOA 11000.0 13000.0 15000.0 saponin 500,0 750.0 1,500.0 support environment up to 1,000,000.0 up to 1,000,000.0 up to 1,000,000.0

Table 7 A study of humoral immunity in cattle grafted with an inactivated sorbed vaccine from the FMD culture virus A No. 2045 / Kyrgyzstan / 2007 against heterologous strain A No. 2171 / Kabardino-Balkarsky / 2013 No. p / p Vaccine characterization Vaccination rate The number of neutralizing antibodies at 21 days after vaccination, log ₂ A No. 2045 / Kyrgyzstan / 2007 A No. 2171 / Kabardino-Balkarian / 2013 one Absorbed PD-2 cm ³ 1468 + 758-7.4 μgА№2045 / Kyrgyzstan / 2007 one 4.80 ± 0.24 p <0.001 2.70 ± 0.73 p <0.05 2 Sorbed PD - 2 cm ³ 1468 + 758 - 7.05 μgА№2045 / Kyrgyzstan / 2007 one 5.40 ± 0.06 p <0.001 2.50 ± 0.65 p <0.05

Table 8 Control of the immunogenic and protective activity of an inactivated sorbed vaccine against foot and mouth disease type A from strain A No. 2171 / Kabardino-Balkarsky / 2013 Animal number Breeding Generalization availability ImD ₅₀ , cm ³ PD ₅₀ one Whole - 0.11 eighteen 2 - 3 - four - 5 - 6 1: 4 - 7 - 8 - 9 - 10 - eleven 1:16 - 12 - 13 + fourteen - fifteen + Control 1 + Control2 +

Claims (11)

1. The vaccine is inactivated adsorbed against foot and mouth disease type A, containing the active substance and target additives, characterized in that as the active substance it contains avirulent and purified antigenic material from the virus strain Aphtae epizooticae, fam. Picornaviridae, genus Aphtovirus, serotype A, collection of FSBI "ARRIAH" A No. 2171 / Kabardino-Balkarian / 2013, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained in a sensitive biological system and consisting of a suspension containing mainly 146S and 75S immunogenic components of foot and mouth disease virus type A. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A. 4. The vaccine according to p. 3, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplantable cell culture of BHK-21 and which is a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A in an amount of at least 3.0 μg in 1 cm ^{3 of the} finished product. 5. The vaccine according to claim 1, characterized in that as the target additive it contains an adjuvant-sorbent aluminum hydroxide (GOA). 6. The vaccine according to claim 5, characterized in that it contains GOA, preferably in an amount of 11000.0 ÷ 15000.0 μg in 1 cm ^{3 of the} finished product. 7. The vaccine according to claim 1, characterized in that it contains saponin adjuvant as a target additive. 8. The vaccine according to claim 7, characterized in that it contains an adjuvant saponin, preferably in an amount of 500.0 ÷ 1500.0 μg in 1 cm ^{3 of the} finished product. 9. The vaccine according to claim 1, characterized in that it contains a supportive medium as a target additive. 10. The vaccine according to claim 9, characterized in that it contains a support medium in an amount up to 1,000,000.0 μg in 1 cm ^{3 of the} finished product. 11. The vaccine according to any one of paragraphs. 1-10, characterized in that it contains avirulent and purified antigenic material from strain A No. 2171 / Kabardino-Balkarsky / 2013, obtained preferably in a transplantable cell culture of BHK-21 and representing a suspension containing mainly 146S and 75S immunogenic components of FMD virus type A, GOA, saponin and support medium in the ratio, μg / cm ³ :
antigenic material not less than 3.0 GOA 11000.0 ÷ 15000.0 saponin 500.0 ÷ 1500.0 support environment up to 1,000,000.0

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