RU2665849C1 - Inactivated emulsion vaccine against foot-and-mouth disease type o - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. Invention is an inactivated emulsion vaccine against foot and mouth disease type O. Vaccine contains an avirulent and purified antigenic material from strain O No. 2311/Zabaikalsky/2016 of foot and mouth disease virus obtained in a suspension continuous cell line from the kidney of a newborn golden hamster (BHK-21/2-17 (baby hamster kidney)), which is a suspension containing mainly 146S immunogenic components of the foot and mouth disease virus, oil adjuvants Montanide ISA-70 or Montanide ISA-206 in effective ratios.EFFECT: vaccine has a high immunogenicity and is able to provide effective protection against the homologous pathogen of the infection circulating in Central and South-East Asia.10 cl, 10 ex, 1 dwg, 11 tbl

Description

According to the classification of the World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO), foot and mouth disease belongs to the group of “cross-border infections” that are of great economic importance for trade and food security in all countries. The foot and mouth disease virus can spread easily and become epizootic. In the Russian Federation, a system of measures for the control and prevention of foot and mouth disease has been developed, which provides for the prevention of virus entry into the country, systematic vaccination of cattle and small cattle in the buffer zone and monitoring of the immune status of vaccinated animals. For immunization of animals, a vaccine made from a virus homologous to field isolates should be used [1].

In 2014, outbreaks of foot and mouth disease type O were recorded in the Transbaikal and Primorsky Territories of the Russian Federation, as well as in neighboring countries. Nucleotide sequencing of the field isolates of the virus isolated in the Transbaikal Territory allowed them to be assigned to the PanAsia genetic line of the ME-SA type [2]. The virus of the same line circulated in 2014 in Mongolia. An outbreak of foot and mouth disease in Primorsky Krai is caused by an isolate of the virus, which belongs to the genetic line Mya-98 of the SEA topotype and has a high degree of nucleotide affinity (96.87%) with an isolate isolated in 2014 in South Korea [2].

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.

Successful prophylactic immunization requires information on the antigen compliance of vaccine strains and epizootic isolates circulating in the prophylactic vaccination zone. When epizootics occur precisely by determining the nucleotide and amino acid correspondence of the field isolate to the production strain, it is possible to most quickly select the appropriate strain for the production of vaccines used in risk areas [3].

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 prevention of foot and mouth disease, the necessary types of virus are used, strains are selected with a rich set of antigenic variations within the type and with pronounced cross immunogenicity. A strain having a wide range of immunogenicity and satisfying the requirements of the region is selected using its study in the cross-neutralization reaction. As a rule, a virus population is used as a production strain, which, in combination with the system and conditions of industrial cultivation, ensures a guaranteed and high accumulation of 146S and 75S virus components to obtain an immunogenic vaccine.

The production strain has high demands on the stability of the virus during cleaning from tissue components and concentration, as well as on the preservation of antigen during inactivation and during long-term storage [4].

An important feature of the causative agent of foot and mouth disease is the antigenic variation of strains within the same serotype that occurs at different time intervals in different territories and depends on the species composition of the susceptible livestock of animals, its immune status and many other factors. Mostly substitutions in amino acid sequences occur in the polypeptides of the viral proteins that make up the virion capsid. The arising 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 caused disease [1, 5].

Changes in the antigenic spectrum, corresponding to the renewal of the structure of the new field isolate, can vary from insignificant detected by monoclonal antibodies to significant ones detected by polyclonal immunoglobulins. Significant genetic changes in the natural isolate cause a weakening of specific immunity against FMD induced by a heterologous antigen. As a result, there is a need to create new diagnostic tools and specific immunization of foot and mouth disease.

Known strains of the causative agent of foot and mouth disease type O, which for a long time have been used as production in Russia over the past 50 years. Among them, strain O ₁ No. 1618 Chechen-Ingush / 66, isolated in 1966, and strain O ₁ No. 194, isolated in 1957 in the Volgograd Region, are isolated. These strains were used to obtain diagnosticum and anti-vaccine vaccines used in various regions of the country, they are currently supported by the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health”.

In the Gali district of Abkhazia, cattle strain O 1736 / Abkhazia / 2000 of foot and mouth disease virus was isolated from cattle, which was used for the manufacture of diagnostic and vaccine preparations.

On April 13, 2000, isolate О No. 1734 / Primorsky / 2000 of foot-and-mouth disease virus was isolated from pigs on the territory of the Russian Federation in the Stepnoye OPH of the Elite village of the Ussuriysky district of the Primorsky Territory from pigs. A comparative analysis of the nucleotide sequences of strain O No. 1734 / Primorsky / 2000 showed that this virus belongs to the PanAsia type O line. The pan-Asian virus caused a pandemic of foot and mouth disease in 1999-2000 in most countries in Asia, including Japan, Korea, and Vietnam , Mongolia, as well as Armenia and Georgia. The devastating epidemic of foot and mouth disease in the UK in 2001 was also caused by a virus of this genetic line. Strain O No. 1734 / Primorsky / 2000 has the highest homology level (98.74%) with isolates O Vietnam / 99 and O Taiwan / 99. Moreover, by antigenic characteristics, it differs from all previously isolated strains O ₁ No. 194 and O ₁ No. 1618 / Chechen-Ingush / 1966 used for the manufacture of diagnostic tools and specific prophylaxis [7].

Production strain O No. 1734 / Primorsky / 2000 of foot and mouth disease virus is used in the Russian Federation as a production strain in the development of diagnostic and specific prophylaxis agents used throughout Russia and the CIS countries.

In the East Gobi aimak of Mongolia in February 2004, strain O 1964 / Mongolia / 2004 was isolated from cattle, which is used for the production of diagnostic and vaccine preparations [6].

In the countries of Southeast Asia (Bhutan, Mongolia, Nepal, China, etc.) for 2015-2017. According to quarterly WRLFMD data, FMD virus isolates were repeatedly isolated, which, according to the results of molecular biological analysis of the VP ₁ gene, belong to the O / ME-SA / Ind-01d genetic line. It should be noted that the foot and mouth disease virus of the genetic line O Ind-2001 was isolated on the territory of the Russian Federation for the first time.

The closest to the invention according to the set of essential features is an inactivated emulsion vaccine against foot and mouth disease type O containing the active substance in the form of avirulent and purified antigenic material from strain O No. 1734 / Primorsky / 2000, homologous to the pathogen, obtained in a sensitive biological system, and targeted additives in in the form of an oil adjuvant in a ratio, mcg / cm ³ :

The VNK-21 / 2-17 transplantable suspension cell line is used as a sensitive biological system for the reproduction of foot-and-mouth disease virus, Earl solution without serum is added as a supporting medium, with the addition of dry muscle enzyme hydrolyzate (FGMS), dry blood protein hydrolyzate (GBKS) and antibiotics at pH 7.4-7.6.

For inactivation of the virus, aminoethylethyleneimine (AEEI) is used. In the manufacture of emulsion vaccines, Montanide ISA-70 and Montanide ISA-206 are used as adjuvants. Purification of the virus-containing suspension from ballast impurities is carried out using polyhexamethylene guanidine hydrochloride (PHMG).

A virulent and purified antigenic material from strain O No. 1734 / Primorsky / 2000 is a suspension consisting mainly of 146S and 75S immunogenic components of foot and mouth disease virus.

A new foot-and-mouth disease isolate has appeared, a new line from which it follows that existing vaccines against foot-and-mouth disease virus will not create immunity from the type O epizootic isolate circulating in Central and Southeast Asia.

A significant drawback of the known vaccines, including the prototype vaccine, is their insufficient immunogenic activity, which is explained by the low degree of protection of susceptible animals from epizootic isolates of FMD virus type O circulating in Central and Southeast Asia.

To solve this problem, the present invention was created, which included the development of FMD inactivated emulsion vaccine that provides effective protection of susceptible animals against field isolates of FMD virus type O, common in Central and Southeast Asia.

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

The specified technical result was achieved by creating an inactivated emulsion vaccine against foot and mouth disease type O, characterized by the following set of features.

The developed vaccine in 1 cm ^{3 of the} preparation contains the following components: the active substance in the form of an avirulent and purified antigenic material from a foot and mouth disease virus homologous to the pathogen infection strain O No. 2311 / Zabaykalsky / 2016, preferably reproduced in the transplantable suspension cell line VNK-21 / 2-17, in an amount of not less than 3.0 μg and adjuvants: Montanide ISA-70 or Montanide ISA-206 oil adjuvants, preferably in an amount of 600,000.0-700000.0 or 500000.0-575000.0 μg, respectively.

The foot-and-mouth disease isolate, which served as the source for the creation of strain O No. 2311 / Zabaykalsky / 2016, was isolated on November 28, 2016 in the Shirokaya pond near the village of Molodezhny, Priargunsky region, in the Trans-Baikal Territory in the eastern part of the Russian Federation on the border with Mongolia and China (examination No. 2311/2016) . Production strain O No. 2311 / Transbaikal / 2016 of the type O FMD virus was obtained by successive passages in sensitive homo- and heterologous cell cultures.

The strain is adapted to primary trypsinized pork kidney cells and transplantable cell lines from the kidney of a newborn Syrian hamster (VNK-21 / 2-17), pig's kidney (IB-RS-2) and Siberian mountain capricorn kidney (PSGK-30).

For the manufacture of the vaccine, a transplantable suspension culture of VNK-21 / 2-17 cells is preferably used as a sensitive biological system, and Earl solution without serum supplemented with FGMS, GBKS, and antibiotics at pH 7.4-7.6 is used as a supporting medium. . Inactivation of the virus is carried out using AEEI with a concentration of 0.025-0.050% of the volume of the virus-containing suspension. At the end of the AEEI process. neutralized by adding sodium thiosulfate to the suspension [8]. The inactivated antigen is purified from ballast impurities using PHMG, which is added to the suspension to a concentration of 0.005-0.007% of the total volume [9].

Avirulent and purified antigenic material from strain O No. 2311 / Transbaikal / 2016 is a suspension containing predominantly 146S immunogenic components of foot and mouth disease virus.

The content of components in the product is evaluated using a quantitative version of the complement binding reaction (CSC) [10]. To prepare the vaccine, viral material is used that contains at least 0.5 μg of 146S immunogenic components of foot and mouth disease virus in 1 cm ³ . The required concentration of 146S immunogenic components in the vaccine preparation is ensured by concentration of the antigen by flow ultrafiltration.

An O type emulsion-induced vaccine against foot and mouth disease is obtained by dispersing in a colloid mill a concentrate of foot-and-mouth disease antigen and an oil adjuvant in a ratio of 4: 6 or 1: 1 by weight, respectively. To enhance the immune response, an oil adjuvant manufactured by Seppic Montanide ISA-70 and Montanide ISA-206 is used.

The resulting vaccine is a milk-like liquid, insoluble in water.

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

1. Inactivated emulsion vaccine against foot and mouth disease type O.

2. The active substance in the form of avirulent and purified antigenic material from an effective amount of foot and mouth disease virus homologous to the pathogen of strain O No. 2311 / Zabaykalsky / 2016.

3. Targeted supplements.

The salient features of the proposed vaccine are that it contains the avirulent purified antigenic material from strain O No. 2311 / Zabaykalsky / 2016 in an effective amount as the active substance.

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 the strain

About No. 2311 / Transbaikal / 2016, type O foot and mouth disease virus, obtained preferably in suspension transplantable cell culture BHK-21 / 2-17 and representing a suspension containing predominantly 146S immunogenic components of foot and mouth disease virus in an effective amount.

2. Avirulent and purified antigenic material from strain O No. 2311 / Transbaikal / 2016 of type O foot and mouth disease virus, obtained preferably in suspension transplantable cell culture BHK-21 / 2-17 and representing a suspension containing predominantly 146S immunogenic components of foot and mouth disease virus less than 3.0 mcg in 1 cm ^{3 of the} finished product.

3. Of the targeted additives, the vaccine contains an oil adjuvant.

4. Of the targeted supplements, the vaccine contains Montanide ISA-206 Oil Adjuvant.

5. The vaccine contains Montanide ISA-206 oil adjuvant in an amount of 500,000.0-575000.0 μg in 1 cm ^{3 of the} finished product.

6. Of the targeted additives, the vaccine contains an oil adjuvant.

7. Of the targeted supplements, the vaccine contains Montanide ISA-70 Oil Adjuvant.

8. The vaccine contains Montanide ISA-70 oil adjuvant in an amount of 600,000.0-700000.0 μg in 1 cm ^{3 of the} finished product.

9. Avirulent and purified antigenic material from strain O No. 2311 / Transbaikal / 2016 of foot-and-mouth disease virus, obtained preferably in suspension transplantable cell culture BHK-21 / 2-17 and oil adjuvant Montanide ISA-206 in the amount, μg / cm ^{3 of the} finished product:

10. Avirulent and purified antigenic material from strain O No. 2311 / Transbaikal / 2016 of foot-and-mouth disease virus, obtained preferably in suspension transplantable cell culture BHK-21 / 2-17 and oil adjuvant Montanide ISA-70 in the amount, μg / cm ³ preparation:

The proposed vaccine has a high immunogenic activity and provides reliable protection against FMD virus type O circulating in Central and Southeast Asia.

The achievement of the technical result from the use of the invention is ensured by the fact that antigenic material from strain O No. 2311 / Zabaykalsky / 2016, which has high immunogenic activity, creates effective protection of susceptible animals against foot and mouth disease virus serotype O, causing recent years of disease outbreak in Central and Southeast Asia.

The foot-and-mouth disease isolate, which served as the source for the type VW strain O type No. 2311 / Zabaykalsky / 2016, was isolated in December 2016 from cattle and foot-and-mouth disease patients located in the territory of Shyroka Pad, the rural settlement of Molodezhninsky, Priargunsky District, Trans-Baikal Territory (examination No. 2311). Production strain VJA No. 2311 / Zabaykalsky / 2016 was obtained by successive passages on sensitive hetero- and homologous cell cultures and deposited in the Collection of microorganism strains (KShM) of the Federal Center for Animal Health.

Strain VIA O No. 2311 / Zabaykalsky / 2016 was deposited on August 08, 2017 into the Collection of microorganism strains of the Federal State Budgetary Institution “Federal Center for Animal Health” (FSBI VNIIZZH), under the registration number (link): strain VIA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB).

The invention is presented in a dendrogram that reflects the phylogenetic relationship of strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus with epizootic isolates and vaccine strains of foot and mouth disease virus of serological type O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₁ gene.

The invention is set forth in the list of sequences in which:

SEQ ID NO: 1 represents the nucleotide sequence of the VP ₁ gene _of strain O No. 2311 / Transbaikal / 2016 of type O foot and mouth disease virus;

SEQ ID NO: 2 reflects the amino acid sequence of the VP ₁ protein _of strain O No. 2311 / Transbaikal / 2016 of type O FMD virus.

Strain O No. 2311 / Transbaikal / 2016 of the FMD virus type O is characterized by the following features and properties.

Morphological features:

Strain VIA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) belongs to the family Picornaviridae, genus Aphtovirus, serotype O and has morphological characteristics characteristic of the causative agent of foot and mouth disease: the virion form is icosahedral, size 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

By its antigenic properties, strain VYA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) belongs to the O serotype. The virus is stably neutralized by homologous antiserum. The virus does not show hemagglutinating activity (GA activity). In cases of sick animals, specific antibodies are formed in the blood serum that are detected in an enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN).

Using the nucleotide sequencing method, the primary structure of the VP1 gene of strain VYa O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) was determined and the primary structure of the VP1 protein was determined. A comparative analysis of the nucleotide sequences showed that the strain VYa O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) belongs to the genetic line Ind-2001 of the type Middle East-South Asia .

Antigenic relationship of strain VYA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) with production strains of foot and mouth disease virus O ₁ Manisa, O No. 1734 / Primorsky / 2000, O PanAsia2, O / Taiwan 3/97, O No. 2222 / Taiwan / 2012, O No. 2102 / Zabaykalsky / 2010, O No. 2147 / Primorsky / 2012 and O No. 2212 / Primorsky / 2014 studied in the Russian Academy of Medical Sciences.

The results of studies in the RMN are presented in table 1. Antigenic compliance (r ₁ ) amounted to 0.41 for O1 Manisa; About PanAsia2 - 0.85; About No. 2102 / Zabaykalsky / 2010 - 0.44; About No. 2147 / Primorsky / 2012 - 0.47; O / Taiwan 3/97 - 0.07; About No. 2222 / Taiwan / 2012 - 0.12; and About No. 2212 / Primorsky / 2014 - 0.33.

When r ₁ > 0.3, the field isolate and the production strain are related; when r ₁ <0.3, the field isolate differs from the production strain.

Biotechnological characteristics

Strain VIA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) is reproduced in the primary monolayer culture of pig kidney cells (SP), transplanted cell cultures: kidneys of the Siberian mountain ibex (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (VNK-21 / 2-17). Within 18-24 hours of incubation, the virus harvest in these cell cultures reaches values from 5.78 to 6.45 lg TCD ₅₀ / cm ³ . With a high multiplicity of infection (1-5 TCD / cell), it causes a CPD after 5 hours. It retains its original characteristics when passaged in cell cultures for 3 passages (observation period).

Geno and chemotaxonomic characteristics

Strain VIA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) of FMD virus type O is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ 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 VIA O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) is resistant to ether, chloroform, freon, acetone and other organic solvents and detergents. Most stable at pH 7.4-7.6. 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

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 its original biological properties when passaged in sensitive biological systems for 3 passages on an IBRS-2 transplanted cell culture (observation period).

During the test, 3 consecutive passages of the strain VYa O No. 2311 / Zabaykalsky / 2016 (production), (control cattle - K-cattle), (control pig - K-CB) in the transplanted IBRS-2 cell culture were performed. To determine the biological activity, each passage virus was titrated in an IBRS-2 transplanted cell culture.

Based on the data obtained, it can be argued that strain O No. 2311 / Zabaykalsky / 2016 in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMD virus type O, has a higher protective and immunogenic activity.

To reduce the epizootic danger of foot and mouth disease type O and to prevent the emergence of new foci of the disease, timely vaccination is important, which requires the development of a highly immunogenic vaccine.

Received highly immunogenic inactivated emulsion vaccine against foot and mouth disease type O from strain O No. 2311 / Transbaikal / 2016.

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

Example 1. The preparation and study of the biological properties of strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus.

Strain O No. 2311 / Zabaykalsky / 2016 of type O foot and mouth disease virus was isolated from field material received at the Federal State Budget Scientific Institution ARRIAH in the form of epithelium of aphthae from pigs suspected of foot and mouth disease during laboratory diagnosis of this disease and its differentiation from other vesicular diseases. In order to isolate the virus, a complex of biological, virological and biochemical methods was used.

Biological and virological methods included the isolation of the virus in a culture of primary trypsinized SP cells, transplanted PSGK-30, IB-RS-2 cell lines, followed by adaptation for 3 consecutive passages. For staging biological samples in primary and transplantable 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 / 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 incubation at a temperature of 37 ° C for 0.5 hours, 5 cm ^{3 of the} supporting medium was added to the vials and incubated at 37 ° C until a virus CPD appeared, characterized by rounding of cells, degeneration and separation of cells from glass, and an increase in the optical density of the suspension. After the development of the CPD, the bottles were subjected to freezing-thawing, purification of the cell suspension with chloroform and centrifugation at 3000 rpm (1200 g) for 0.25 hours. The obtained virus-containing material was used for subsequent passages and studies in the CSC for the detection of viral antigen, while used commercial type-specific sera stored in the Museum of strains of FSBI "ARRIAH". The causative agent of foot and mouth disease was considered adapted to cell cultures if within 18-24 hours the cell monolayer was destroyed by 90-100% under the influence of the virus.

The virus adapted to the IB-RS-2 cell culture was used to produce antigen for CSC.

The virus adapted to the PSGK-30 cell culture was used to infect the VNK-21 / 2-17 cell suspension in order to obtain antigen for the manufacture of an experimental vaccine series, as well as for hyperimmunization of guinea pigs.

The research results are presented in table 2, the data of which testify to the high adaptive activity of strain O No. 2311 / Transbaikal / 2016 of the FMD virus type O to the used cell cultures.

Example 2. A comparative analysis of the biological properties of strain O No. 2311 / Zabaykalsky / 2016 with production of a strain of foot and mouth disease virus type O.

When studying the properties of strain O No. 2311 / Zabaykalsky / 2016 of the foot and mouth disease virus, significant advantages were revealed in comparison with other used industrial strains.

Tables 3 and 4 show the results of the cultivation of strains O No. 2311 / Zabaykalsky / 2016 and O No. 1734 / Primorsky / 2000 of type O FMD virus, from which it follows:

1) strains O No. 2311 / Zabaykalsky / 2016 and O No. 1734 / Primorsky / 2000 have different reproduction times - 10.67 ± 0.36 and 12.33 ± 0.25 hours, respectively;

2) the accumulation of immunogenic components during the cultivation of foot and mouth disease virus No. 2311 / Zabaykalsky / 2016 is higher than that of strain O No. 1734 / Primorsky / 2000 - 1.53 ± 0.07 and 1.11 ± 0.08 μg / cm ³ , respectively .

Example 3. Obtaining an inactivated antigen and studying the effect of inactivation and purification of antigenic material using AEEI and PHMG on the immunogenic (146S) components of foot and mouth disease virus of strains No. 2311 / Zabaykalsky / 2016 and No. 1734 / Primorsky / 2000.

An inactivated emulsion vaccine against foot and mouth disease type O is prepared from strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus grown in suspension transplantable cell line BHK-21 / 2-17. As a supporting medium, an Earle 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 0.005-0.200 TCD ₅₀ / cell.

The cultivation of the virus is carried out at a temperature of 35-37 ° C. After 6-7 hours of incubation, the concentration of living and dead cells was measured by trypan blue staining. If the number of living cells is 15-20%, then exposure to the virus continues for another 2-4 hours. When a cytopathic effect is achieved, about 90-95% of the reproduction of foot and mouth disease virus is stopped, and the suspension obtained is monitored for sterility and the content of 146S components. The concentration of 146S components of foot and mouth disease virus in suspension should be at least 0.5 μg / cm ³ . Immediately after the end of the virus reproduction cycle, without stopping the thermostating process, 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 0.025-0.050%. Inactivation of the infectiousness of the virus is carried out for 12-24 hours at a temperature of 36-37 ° C and a pH of 7.2-7.6 with stirring after 5-6 hours for 3-5 minutes. The aminoethylethyleneimine residue is neutralized by the addition of sodium thiosulfate. For flocculation of ballast impurities and inactivation of possible contaminants, a 10% solution of PHMG is added to a warm suspension to a concentration of 0.005-0.007%. Flocculated ballast impurities are subjected to sedimentation followed by decantation.

The results of studies on the effect of inactivation and purification of antigenic material using AEI and PHMG on the immunogenic (146S) components of strains O No. 2311 / Zabaykalsky / 2014 and O No. 1734 / Primorsky / 2000 of FMD virus are shown in table 5.

The data in table 5 indicate that the immunogenic components of strain O No. 2311 / Zabaykalsky / 2016 of the FMD virus are not inferior in resistance to inactivation and purification under production conditions of strain O No. 1734 / Primorsky / 2000. Especially important is the fact that the use of strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus in comparison with the vaccine strain O No. 1734 / Primorsky / 2000 does not lead to a decrease in the concentration of the 146S component.

Example 4. Concentration of antigen of foot and mouth disease virus strain No. 2311 / Transbaikal / 2016 to obtain a vaccine.

The required concentration of 146S components in the graft dose of the emulsion vaccine from strain O No. 2311 / Zabaykalsky / 2016 is obtained by concentration of the antigen by flow ultrafiltration using BTU 0.5 / 2-A1 ultrafilters at a pressure of 1.5 atm. The resulting antigen concentrate is stored at a temperature of 4-6 ° C until use. The resulting vaccine is packaged in glass or plastic bottles and sterilized products are monitored in accordance with GOST 28085-89.

Example 5. The process of dispersing the antigen and adjuvant to obtain an inactivated emulsion vaccine against foot and mouth disease from strain O No. 2311 / Zabaykalsky / 2016.

An inactivated emulsion vaccine against foot and mouth disease type O is obtained by dispersing antigen concentrate and an oil adjuvant in colloid mills. As adjuvants, products of the Montanide ISA-70 or Montanide ISA-206 brands manufactured by Seppic (France, ISO 9001 standard) are used.

In the manufacture of an emulsion vaccine based on Montanide ISA-206, the adjuvant and antigen are heated to a temperature of 25-30 ° C and dispersed in colloidal mills in a ratio of 1: 1.

An emulsion vaccine based on Montanide ISA-70 is made by dispersing the antigen and adjuvant in a ratio of 4: 6 at a temperature of 18-25 ° C.

The result is an inactivated emulsion vaccine against foot and mouth disease from the production strain O No. 2311 / Zabaykalsky / 2016, which is a milk-like liquid insoluble in water. The vaccine has a liquid consistency, easily dissolves at the injection site, does not cause the formation of abscesses, a general reaction in the form of an increase in temperature. It has a pronounced immunogenic activity for pigs at a vaccination dose of 2 cm ³ 21 days after administration. At least 3 μg of the 146S component of the foot and mouth disease virus should be contained in 1 cm ³ . The optimal component composition of the obtained vaccine are presented in tables 6 and 7.

Example 6. Evaluation of the avirulence and safety of inactivated emulsion vaccine against foot and mouth disease from strain O No. 2311 / Transbaikal / 2016.

To determine the virulence of the vaccine in pigs, a sample of the vaccine preparation was injected intracutaneously at 0.1 cm ³ at two points of the corolla on each limb. The study used pigs weighing 35-40 kg. During the 10 days of observation, the animals remained clinically healthy, and the pathological study did not reveal any changes characteristic of foot and mouth disease. This indicated the absence of virulent properties of the developed vaccine.

The safety of the product in pigs was controlled by intramuscular injection of the vaccine in the region of the upper third of the neck at a dose of 6 cm ³ . The observation period was also 10 days. It should be noted that after inoculation, the body temperature of the animal can increase to 41 ° C and stay at this level for 1-3 days.

According to the results of the studies, the vaccine was found to be harmless, all animals remained clinically healthy during the observation period, and no pathological anatomical analysis of tissue necrosis was detected at the injection site.

Example 7. Evaluation of humoral immunity in pigs when using FMD vaccine from strain O No. 1734 / Primorsky / 2000 against a homologous strain and heterologous strain O No. 2311 / Trans-Baikal / 2016.

The humoral immunity of pigs vaccinated with inactivated FMD vaccine from strain O No. 1734 / Primorsky / 2000 against the heterologous strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease was evaluated. The level of humoral immunity was evaluated against production strains O No. 1734 / Primorsky / 2000 and O No. 2311 / Zabaykalsky / 2016.

Tested emulsion vaccine against foot and mouth disease type O, made as described in examples 4, 5, 6 and containing, mcg in 2 cm ³ :

avirulent and purified strain antigenic material About No. 1734 / Seaside / 2000, type O FMD virus 6.9 oil adjuvant ISA-70 1,200,000.0.

The test was carried out on 10 heads of pigs. The emulsion vaccine was administered intramuscularly at a dose of 2.0 cm ³ . The research results are presented in table 8, the data of which shows that the emulsion vaccine stimulated the production of virus-neutralizing antibodies (VNA) in pigs against homologous strain O No. 1734 / Primorsky / 2000 in the amount of 5.19 ± 0.21 log ₂ , against heterologous strain O No. 2311 / Zabaykalsky / 2016 - 2.46 ± 0.28 log ₂ . The difference in the titers of neutralizing antibodies is significant and significant. It follows that the vaccine made from the production strain O No. 1734 / Primorsky / 2000 does not create humoral immunity to protect animals from infection with the heterologous foot and mouth disease virus No. 2311 / Zabaykalsky / 2016. According to the recommendations of the OIE (OIE), the titer of neutralizing antibodies should be at least 5.00 log ₂ [1].

Example 8. Evaluation of the antigenic and protective activity of an inactivated emulsion vaccine from strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus.

The antigenic and protective activity of the inactivated emulsion FMD vaccine from strain O No. 2311 / Zabaykalsky / 2016, against a homologous strain of foot and mouth disease virus was evaluated.

Tested emulsion vaccine against foot and mouth disease type O, made as described in examples 4, 5, 6 and containing, mcg in 2 cm ³ :

avirulent and purified strain antigenic material About No. 2311 / Transbaikal / 2016, type O FMD virus 6.2 oil adjuvant ISA-70 1,400,000.0.

Three groups of pigs of 5 goals were injected with 2.0 cm ^{3 of the} whole preparation, as well as in dilutions of 1: 5 and 1:25, respectively. 2 pig heads, taken as a control, were not vaccinated. 21 days after immunization, all animals were injected intradermolingually at two points with 10,000 ID _{50 of the} control virus of foot and mouth disease strain O No. 2311 / Zabaykalsky / 2016, obtained as described above. The results of the study are shown in table 9, from which it follows that the pigs vaccinated with the whole preparation, on the 21st day after inoculation, had a titer of neutralizing antibodies (VNA) equal to 6.35 ± 0.52 log ₂ , pigs immunized with the vaccine in dilutions 1: 5 and 1:25 - 4.85 ± 0.55 log ₂ and 2.35 ± 0.80 log ₂ , respectively. Clinical signs characteristic of foot and mouth disease did not develop in animals immunized with a whole vaccine and at a dilution of 1: 5. In 4 out of 5 pigs inoculated with a vaccine preparation at a 1:25 dilution, the development of a generalized form of the disease was observed. Two control animals without vaccination fell ill with foot and mouth disease at a BHA titer of ≤0.5 log ₂ .

Inactivated emulsion FMD vaccine from strain O No. 2311 / Zabaykalsky / 2016 contained 15 PD ₅₀ (protective doses with a probability of 50%), which meets the requirements of the OIE (OIE).

Example 9. Evaluation of the immunogenic and protective activity of an inactivated emulsion vaccine from strain O No. 2311 / Transbaikal / 2016 of foot and mouth disease virus.

A study was made of the immunogenic and protective activity of an inactivated emulsion FMD vaccine from strain O No. 2311 / Zabaykalsky / 2016, against a homologous strain of foot and mouth disease virus.

Tested emulsion vaccine against foot and mouth disease type O, made as described in examples 4, 5, 6 and containing, mcg in 2 cm ³ :

avirulent and purified strain antigenic material About No. 2311 / Transbaikal / 2016, type O FMD virus 6.2 oil adjuvant ISA-70 1,000,000.0

The results of the study are presented in table 10, which shows that the animals vaccinated with the whole preparation, on the 21st day after immunization, had a BHA titer of 6.80 ± 0.33 log ₂ , cattle heads inoculated with the vaccine in 1: 4 and 1 dilutions : 16 - 5.05 ± 0.11 log ₂ and 3.20 ± 0.31 log ₂ , respectively. The BHA titer in the control animals was ≤0.5 log ₂ . The development of clinical signs of foot and mouth disease in cattle immunized with a whole vaccine and at a dilution of 1: 4 was not observed. In 1 out of 5 goals immunized with a vaccine preparation at a 1:16 dilution, the development of a generalized form of the disease was noted. Generalization of the process also developed in two control animals with a BHA titer of ≤0.5 log ₂ .

Inactivated emulsion FMD vaccine from strain O No. 2311 / Zabaykalsky / 2016 contained 22 PD ₅₀ (protective doses with a probability of 50%), which meets the requirements of the OIE (OIE).

According to the recommendations of the OIE (OIE), the vaccine against foot and mouth disease should contain at least 6 PD ₅₀ in the vaccine volume [1]. Therefore, the manufactured inactivated emulsion vaccine from strain O No. 2311 / Transbaikal / 2016 of the foot and mouth disease virus exhibits high immunogenic activity.

Example 10. Evaluation of antigenic activity when using FMD vaccine from strain O No. 1734 / Primorsky / 2000 against heterologous strain O No. 2311 / Trans-Baikal / 2016

avirulent and purified strain antigenic material About No. 1734 / Seaside / 2000, type O FMD virus 6.1 oil adjuvant ISA-206 1,100,000.0.

The results of the study are presented in table 11, which shows that the animals vaccinated with the vaccine, on the 21st day after immunization, had a BHA titer against the homologous strain O No. 1734 / Primorsky / 2000 5.50 ± 0.43 log ₂ , and against the heterologous strain O No. 2311 / Transbaikal / 2016 3.10 ± 0.23 log ₂ .

Antibody titers against a heterologous strain are 5 times lower than against a homologous virus.

One of the five heads noted the development of a generalized form of the disease.

Generalization of the process also developed in two control animals with a BHA titer of ≤0.5 log ₂ .

Thus, the above information indicates that the inactivated emulsion vaccine against foot and mouth disease type O, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology; confirmed the possibility of implementing the presented; the vaccine made from strain O No. 2311 / Zabaykalsky / 2016 in accordance with the invention has high immunogenic activity and is able to provide effective protection of susceptible animals against the epizootic strain of FMD virus type O circulating in Central and Southeast Asia.

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Inactivated emulsion vaccine against foot and mouth disease type O":

1. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed.-, Paris, 2016 - Vol. 1, Chapter 2.1.5. - P. 166-169.

2. The official website of Pervbright - URL: http://www.wrlfmd.org/fmd_genotyping/fmd_genotyping.htm (accessed 08.08.2017).

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

4. Rerer X. Foot and mouth disease / Translation from it. G.A. Surkova. Ed. and with the foreword. Cand. vet. sciences P.V. Painter. - M .: Kolos, 1971. - 432 p.

5. Burdov A.N., Dudnikov A.I., Malyarets P.V. and other foot and mouth disease / Ed. A.N. Burdova. - M .: Agropromizdat, 1990 .-- S. 231-250.

6. Immunobiological properties of the epizootic strain of foot and mouth disease virus type O No. 1964 / Mongolia / 2004 / T.A. Fomina, V.K. Spirin, A.I. Egorova [et al.] // Proceedings of the Federal Center for Animal Health. - Vladimir, 2005. - T. 3. - S. 94-104.

7. Immunobiological properties of the epizootic strain of foot and mouth disease virus type O No. 1734 Primorsky / 2000 / V.K. Spirin, A.I. Egorova, S.R. Kremenchug [et al.] // Tr. Federal Center for Women's Health. - Vladimir, 2007.- V. 5. - S. 52-58.

8. RF patent No. 2054039, C12N 7/02, A61K 39/135, 02/10/1996

9. RF patent No. 594771, A61K 39/12, 07/07/1993

10. Bondarenko A.F. Method for determining the content of virus-specific protein and component composition of the virus of foot and mouth disease using quantitative CSC: approved. Director of VNIIII. - Vladimir, 1987.

11. Archetti, J. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologus immune serum / J. Archetti, F.L. Horsfall // J. Exp. Med. - 1950. - Vol. 92, No. 5. - P. 441-462.

Note: RMN - microneutralization reaction

* BFS - virus-specific protein

* BFS - virus-specific protein

Note: * BSC - virus-specific protein

Claims (12)

1. Inactivated emulsion vaccine against foot and mouth disease type O, containing the active substance and adjuvants, characterized in that as the active substance it contains avirulent and purified antigenic material from the virus strain Aphtae epizooticae, fam. Picornaviridae, genus Aphtovirus, type O, deposited in the Collection of microorganism strains of the Federal Center for Animal Health (FSBI ARRIAH) under registration number (author's name): strain VIA O No. 2311 / Zabaykalsky / 2016 (production), (control Cattle - K-cattle), (control pig - K-CB), in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from strain O No. 2311 / Zabaykalsky / 2016 homologous to the pathogen, obtained in a sensitive biological system and consisting of a suspension containing mainly 146S immunogenic components of foot and mouth disease virus type ABOUT. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from strain O No. 2311 / Zabaykalsky / 2016 homologous to the pathogen, preferably obtained in a transplanted cell culture of BHK-21 / 2-17 and which is a suspension, containing predominantly 146S immunogenic components of the FMD virus type O. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from strain O No. 2311 / Zabaykalsky / 2016 homologous to the pathogen, preferably obtained in a transplantable cell culture of BHK-21 / 2-17 and which is a suspension, containing predominantly 146S immunogenic components of FMD virus type O 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 an adjuvant it contains an oil adjuvant of Montanide ISA-70. 6. The vaccine according to claim 5, characterized in that it contains an oil adjuvant of Montanide ISA-70, preferably in an amount of 600,000.0-700000.0 μg in 1 cm ^{3 of the} finished product. 7. The vaccine according to any one of paragraphs. 1-6 characterized in that it contains avirulent and purified antigenic material homologous to the pathogen of strain O strain No. 2311 / Zabaykalsky / 2016, obtained preferably in the transplanted cell culture of BHK-21 / 2-17 and which is a suspension containing mainly 146S immunogenic components type O foot and mouth disease virus, Montanide ISA-70 oil adjuvant in the ratio, μg / 1 cm ^{3 of the} finished product: Avirulent and purified antigenic strain material About No. 2311 / Transbaikal / 2016, type O FMD virus not less than 3.0 Oil Adjuvant Montanide ISA-70 600000.0-700000.0 8. The vaccine according to claim 1, characterized in that as an adjuvant it contains an oil adjuvant of Montanide ISA-206. 9. The vaccine according to claim 8, characterized in that it contains an oil adjuvant of Montanide ISA-206, preferably in an amount of 500,000.0-575000.0 μg in 1 cm ^{3 of the} finished product. 10. The vaccine according to any one of paragraphs. 1-4 and 8, 9, characterized in that it contains avirulent and purified antigenic material homologous to the pathogen of strain O No. 2311 / Zabaykalsky / 2016, obtained preferably in a transplantable cell culture of BHK-21 / 2-17 and which is a suspension, containing predominantly 146S immunogenic components of FMD virus type O, oil adjuvant Montanide ISA-206 in the ratio, μg 1 cm ^{3 of the} finished product: Avirulent and purified antigenic material from strain O No. 2311 / Transbaikal / 16 not less than 3.0 type O foot and mouth disease Oil Adjuvant Montanide ISA-206 500000.0-575000.0

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