RU2802192C1 - Inactivated sorbed vaccine against fmd of o/ea-2 genotype from o/kenya/2017 strain culture - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the development of a cultured inactivated adsorbed vaccine against foot-and-mouth disease from O/Kenya/2017 strain of A/О/ЕА-2 new genotype. The vaccine contains an avirulent and purified concentrated antigen of O/Kenya/2017 strain of foot and mouth disease virus of A/О/ЕА-2 genotype, obtained in a suspension continuous cell line from the kidney of a newborn Syrian hamster (BHK-21/SUSP/ARRIAH), representing a suspension containing predominantly 146S immunogenic component of FMDV, adjuvant aluminum hydroxide and saponin in effective ratios. The vaccine is highly immunogenic and can provide effective protection against a homologous infectious agent circulating in African countries.

EFFECT: vaccine is relevant for ensuring biological safety for FMD in relation to the O/EA-2 genotype.

3 cl, 1 dwg, 8 tbl, 14 ex

Description

The invention relates to the field of veterinary virology and biotechnology, namely to the development of a vaccine against FMD genotype O/EA-2 from the strain "O/Kenya/2017" cultural inactivated adsorbed.

Foot-and-mouth disease is the most contagious mammalian disease and has great potential to cause severe economic losses in susceptible artiodactyls. There are seven serotypes of FMD virus, namely, O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, while the virus of serotype O is the most common and studied, which determines the relevance of the manufacture of vaccines against FMD of this serotype.

Each serotype is genetically divided into different topotypes, and sometimes into separate genetic lineages and even sublines. Differences between them are determined by analyzing the nucleotide sequence of the highly variable 1D gene, which encodes the amino acid sequence of the VP ₁ protein [1]. Given that the FMD causative agent is an RNA-containing virus, it is characterized by the formation of a large number of mutations during the replication of the viral genome (advantage in the 5'-section) and the phenomenon of recombination (mainly in the 3'-section). It is for this reason that many genetic lines and sublines are distinguished within seven serotypes; to date, more than 60 of them have been described [2].

The importance of knowing the genetic diversity of FMDV lies in the possibility of a thorough analysis of the epizootic situation in a particular region and a detailed selection of an appropriate vaccine, which should be adapted specifically to the genotype of interest. Recovery after an animal has been ill with any one serotype does not provide immune protection against another serotype and even some other genetic lines [3]. However, FMD viruses can show partial cross-protection within the same serotype.

Establishing genetic links between different isolates and strains is carried out using nucleotide sequencing, which makes it possible to determine the origin of the virus in outbreak conditions. The antigenic characteristics of the virus associated with the emergence of new strains are important for the characterization of outbreaks and the search for optimal vaccine preparations [4, 6].

FMDV serotype O is divided into 11 topotypes: EAST AFRICA 1-4 (East Africa) (EA-1-4), SOUTHEAST ASIA (Southeast Asia) (SEA), EUROPE-SOUTH AMERICA (Europe-South America) (EURO -SA), INDONESIA-1 and 2 (Indonesia-1 and 2) (ISA-1 and -2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA) (Middle East-South Asia) and WEST AFRICA (West Africa) (WA). This high genetic and antigenic diversity leads to problems in the specific prophylaxis of foot-and-mouth disease using culture-inactivated FMD vaccines. As a result, there is a need to create new means of specific immunoprophylaxis against FMDV serotype O [5-11, 15].

This variety of representatives of the O serotype is due to the high degree of variability of RNA-containing viruses and the active movement of this pathogen in East Africa and neighboring countries.

Due to the easy and rapid spread of the FMD pathogen, this disease can acquire the scope of epizootics [12]. In order to prevent the occurrence of the disease in the farms of the Russian Federation, a system of measures for the control and prevention of foot and mouth disease is used, which is aimed at preventing the introduction of the virus into the country, systematic immunization of cattle and small cattle in the buffer zone, as well as monitoring the immune status of vaccinated animals.

For immunization of animals, a vaccine made from a virus homologous to field isolates should be used [6, 7, 8, 9, 10, 11].

The worldwide outbreaks of foot-and-mouth disease demonstrate that this infection continues to be a significant economic problem throughout the world. The debate about the most effective way to respond to FMD outbreaks in disease-free countries continues to focus on the use of vaccines [8, 9, 12, 13]. An effective vaccination campaign requires the availability of an effective, safe vaccine containing, as an immunogenic part, a virus antigen corresponding to an epizootic spreading isolate of a certain genotype. The creation of such vaccines is an urgent task. Achieving this goal will make it possible to effectively use the vaccine in a disadvantaged area and a threatened zone to form immunity against a specific FMDV genotype.

One of several measures that can be taken to control FMD outbreaks is emergency vaccination. Criteria for the success of such immunization include access to a vaccine that has the following characteristics: 1) contains a FMDV strain with sufficient antigenic relationship to isolates circulating in the outbreak; 2) refers to the required species among the developed vaccines; 3) is characterized by acceptable safety and efficacy; 4) has appropriate access, including the volume of consignments and the timeliness of their deliveries.

Contingency planning must include the provision of vaccination and consider the possibility of complex decisions not only about when, where and how to apply the vaccine, but also the economic feasibility of its use [14].

On the territory of East Africa, in particular, in Nigeria, Kenya, Tanzania, Ethiopia, outbreaks of foot-and-mouth disease of the O / EA-2 genotype are sporadic. It should be noted that in recent years, trade and economic ties with the countries of the African continent, in particular, with the states of East Africa, have intensified. There are risks of introduction of isolates of this genotype into the territory of the Russian Federation.

Analyzing the outbreaks that were registered in Africa, it was found that in Nigeria, Kenya, Tanzania, Ethiopia, FMDV isolates that belong to the O serotype were detected, in particular, in 2009, 2010, outbreaks of FMD of the O / EA-1 genotype were noted , in 2010 - O / EA-4, in 2010, 2011, 2017, 2020, 2021 - O / EA-2. At the same time, it should be noted that representatives of the O/EA-2 genotype of the foot-and-mouth disease virus were found in a single quantity, and since 2021 they began to be massively registered in the countries of East Africa and adjacent territories, which is dangerous and requires the study of strains of this genotype to create diagnostic tools. and specific prevention of foot-and-mouth disease of the O/EA-2 genotype.

Known production strains of FMD virus serotype O, which are used for the production of specific means of prevention of FMD:

- strain "O/Taiwan/1997" (genotype O/CATHAY/CAM 94),

- strain "O/Primorsky/2014" (genotype O/SEA/Mya-98),

- strain "O/Primorsky/2012" (genotype O/ME-SA/PanAsia),

- strain "O/Saudi Arabia/2008" (genotype O/ME-SA/PanAsia2),

- strain "O/Zabaikalsky/2016" (genotype O/ME-SA/Ind-2001).

The FMDV isolate O/KEN/4/2017 was isolated from pathological material from cattle in the Republic of Kenya in 2017. During scientific research and passage on cell culture at the FGBI ARRIAH, it was characterized and named strain "O/Kenya/2017".

According to the results of a comparative analysis of the nucleotide sequences, the isolated strain belongs to the O/EA-2 genotype of the FMD virus, which differs significantly from the production strains of the FMDV serotype O (Fig. 1) [16].

Given the increase in trade and economic relations between Russia and the countries of the African continent, in particular, Nigeria, Tanzania, Kenya and others, there is a danger of occurrence of foot and mouth disease of this genotype in the Russian Federation and the problem of possible emergency prevention of this disease for the formation of immunity in susceptible animals is of particular importance. Thus, it became necessary to develop a new vaccine against foot-and-mouth disease of the O/EA-2 genotype from the O/Kenya/2017 strain, culturally inactivated and adsorbed to ensure the biological safety of the territory of the Russian Federation and neighboring states.

The closest to the proposed invention in terms of essential features is an inactivated vaccine sorbed against FMD serotype O (strain "O / Primorsky / 2014"), containing the active substance in the form of avirulent and purified cultural antigenic material from a homologous pathogen of FMD strain "O / Primorsky / 2014" obtained in a sensitive biological system, and targeted additives in the form of aluminum hydroxide and saponin (adjuvant): the concentration of antigen (inactivated 146S + 75S components of foot-and-mouth disease virus) is not less than 3.0 μg / cm ³ , the content of 10% aluminum hydroxide solution is -40% by volume, the concentration of saponin is 1.5 mg/cm ³ , the additive in the form of a supporting medium - up to 1.0 cm ³ (prototype).

As a sensitive biological system for the reproduction of foot-and-mouth disease virus, a transplantable suspension cell line of the kidney of a newborn Syrian hamster VNK-21/2-17 is used, Eagle's MEM medium without serum is used as a supporting medium, with the addition of dry enzymatic muscle hydrolyzate, dry blood protein hydrolyzate at medium pH 7.40-7.60.

Aminoethylethyleneimine (AEEI) is used to inactivate the virus. Aluminum hydroxide (Al(OH) ₃ ) serves as an adjuvant in the manufacture of an adsorbed vaccine. Purification of the virus-containing suspension from ballast impurities is carried out using polyhexamethylene guanidine hydrochloride (PHMG).

Avirulent and purified inactivated antigenic material from a strain of foot-and-mouth disease virus serotype O is a suspension consisting of 146S + 75S components of the foot-and-mouth disease virus, that is, not only complete immunogenic particles, but also "empty" capsids.

A significant drawback of the prototype vaccine lies in the composition of the antigen, namely, the use of 146S particles in addition to the 75S component. According to some researchers, in particular, Verlinden Y. (2005), "empty" capsids are the result of unsuccessful assembly of the virion, or a temporary receptacle for pentamers and are not the main immunogenic component of vaccine preparations.

A significant drawback of the prototype vaccine is its very low immunogenic activity against strains/field isolates of FMDV genotype O/EA-2 circulating in African countries.

To solve this problem, the present invention was created, which included the development of a vaccine against foot-and-mouth disease of the O / EA-2 genotype from the culture inactivated sorbed strain "O / Kenya / 2017". This vaccine assumes a high content of the 146S immunogenic component in a dose.

The technical result from the use of the present invention is to expand the arsenal of inactivated cultural adsorbed vaccines to protect animals against isolates and strains of FMDV serotype O and, in particular, genotype O/EA-2.

The specified technical result is achieved by creating a vaccine against foot-and-mouth disease of the O/EA-2 genotype from the strain "O/Kenya/2017" cultured inactivated sorbed, characterized by the following set of features reflected below.

The developed vaccine in 1.0 cm ³ of the preparation contains the following main components: 1) the active substance in the form of avirulent and purified antigenic material from the homologous pathogen of the infection strain "O / Kenya / 2017" (genotype O / EA-2) of the foot-and-mouth disease virus, reproduced preferably in the continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; 2) aluminum hydroxide 10% colloidal solution with a content of 45% by volume (4% on a dry matter basis), 3) saponin in the amount of 1.5 mg, 4) supporting medium - up to 1.0 cm ³ .

The strain "O/Kenya/2017", which was obtained by adapting the isolate "O/KEN/4/2017", was deposited in the All-Russian State Collection of Exotic Types of FMD Virus and Other Animal Pathogens (GKSHM) of the Federal State Budgetary Institution "Federal Center for Animal Health" ”(FGBU “ARRIAH”), under registration number: No. 412 - dep / 22-46 - GKShM FGBI “ARRIAH”.

The strain is adapted to primary trypsinized monolayer cells of the porcine kidney (SP) line and transplantable cell lines from the kidney of a newborn Syrian hamster (BHK-21/SUSP/ARRIAH), the kidney of a pig (IB-RS-2) and the kidney of the Siberian mountain ibex (PSGK- thirty).

For the manufacture of a vaccine, a transplantable suspension culture of BHK-21/SUSP/ARRIAH cells is preferably used as a sensitive biological system, and Eagle's medium DMEM is used as a supporting medium without adding serum, but with the addition of a blood protein hydrolyzate in an amount of 5% of the total volume at pH Wednesdays 7.45-7.65. Virus inactivation is carried out using aminoethylethyleneimine (AEEI) with a concentration of 0.03% of the volume of the virus-containing suspension. The inactivated virus is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.014% of the total volume.

The avirulent and purified antigen of the O/Kenya/2017 strain of foot-and-mouth disease virus is a suspension containing a predominantly inactivated 146S immunogenic component of foot-and-mouth disease virus.

The concentration of the component in the product is estimated using a quantitative version of the complement fixation reaction (CFR) [18]. To prepare the vaccine, a viral material containing at least 4.0 μg of inactivated immunogenic 146S particles of foot-and-mouth disease virus is used in 1.0 cm ³ . The required concentration of the immunogenic component in the vaccine preparation is provided by concentrating the antigen by adsorption on the surface of colloidal particles of aluminum hydroxide (Al(OH) ₃ ).

The vaccine against foot-and-mouth disease from the strain "O/Kenya/2017" of the genotype O/EA-2, cultural inactivated sorbed, is obtained by mixing the purified antigen and a 10% suspension of aluminum hydroxide in a ratio of 55% to 45% by volume, respectively. To enhance the immune response, saponin is used at a concentration of 1.5 mg/cm ³ . The resulting vaccine is a suspension containing particles of water-insoluble aluminum hydroxide carrying on their surface inactivated FMDV virions, which provide the formation of immunity.

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

1. Vaccine against FMD genotype O / EA-2 from the strain "O / Kenya / 2017" culture inactivated sorbed.

2. The active substance in the form of an avirulent and purified antigenic material from the FMDV strain "O/Kenya/2017" homologous to the causative agent of the disease of the O/EA-2 genotype in an effective amount.

3. Target additives.

The essential distinguishing features of the proposed vaccine are that it contains an avirulent purified antigen of the O/Kenya/2017 strain of the O/EA-2 genotype of the foot-and-mouth disease virus as an active substance in an effective amount.

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

1. Avirulent and purified antigen of the strain "O/Kenya/2017" of the genotype O/EA-2 of the foot-and-mouth disease virus, obtained preferably in a suspension continuous cell line BHK-21/SUSP/ARRIAH and which is a suspension containing the 146S immunogenic component of the foot-and-mouth disease virus in an effective quantity.

2. Avirulent and purified antigen of the strain "O/Kenya/2017" of the genotype O/EA-2 of the foot-and-mouth disease virus, obtained preferably in a suspension continuous cell culture BHK-21/SUSP/ARRIAH and representing a suspension containing mainly the 146S immunogenic component of the foot-and-mouth disease virus in amount of at least 4.0 μg per 1.0 cm ³ of the finished vaccine preparation.

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

4. Of the targeted additives, the vaccine contains an adjuvant - aluminum hydroxide in combination with saponin.

5. The vaccine contains an adjuvant - aluminum hydroxide 4.0% in terms of dry residue in combination with saponin at a concentration of 1.5 mg per 1.0 cm ³ of the finished product.

Fig. 6. Avirulent and purified antigen of the strain "O/Kenya/2017" of the O/EA-2 genotype of the foot and mouth disease virus, obtained preferably in a continuous suspension cell line BHK-21/SUSP/ARRTAH, an adjuvant - aluminum hydroxide and saponin, an additive in the finished product in the form of a supporting medium in the following quantities:

Avirulent and purified antigen strain "O/Kenya/2017" genotype O/EA-2 foot and mouth disease virus not less than 4.0 µg/cm ³ aluminum hydroxide 4.0% (based on dry matter) Saponin 1.5 mg / cm ³ Supportive environment up to 1.0 cm ³

The proposed vaccine against foot-and-mouth disease from the O/Kenya/2017 strain of the O/EA-2 genotype, cultured inactivated sorbed, has a high immunogenic activity and provides reliable protection against the FMDV of the O/EA-2 genotype circulating in East Africa and neighboring countries.

The achievement of the technical result from the use of the invention is ensured by the fact that the antigen of the strain "O/Kenya/2017" of the genotype O/EA-2 of the foot-and-mouth disease virus, which has a high immunogenic activity, creating effective protection of susceptible animals against isolates, is introduced into the composition of the proposed FMD vaccine as an active substance. FMDV of the presented genotype, which in recent years have caused outbreaks in African countries.

The essence of the invention is reflected in the graphic image:

Fig. 1. - Dendrogram reflecting the phylogenetic relationship of the strain "O/Kenya/2017" of foot and mouth disease virus with epizootic strains of serotype O. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₁ gene.

The essence of the invention is illustrated by the following sequence listings:

SEQ ID NO: 1 represents the nucleotide sequence of the VP ₁ protein gene of the O/Kenya/2017 strain of foot-and-mouth disease virus of the O/EA-2 genotype;

SEQ ID NO: 2 represents the amino acid sequence of the VP ₁ protein gene of the O/Kenya/2017 strain of foot-and-mouth disease virus of the O/EA-2 genotype.

The strain "O/Kenya/2017" of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The O/Kenya/2017 strain of foot-and-mouth disease virus serotype O belongs to the order Picornavirales, the family Picornaviridae, the genus Aphthovirus, the species Foot-and-Mouth Disease virus and has morphological features characteristic of the foot-and-mouth disease pathogen: the shape of the virion is ixahedral, the size is 20-25 nm . A virion consists of a single-stranded, positively charged RNA molecule enclosed in a protein coat. The 146S component consists of an RNA molecule enclosed in a polypeptide complex consisting of 60 copies of the proteins VP ₄ , VP ₂ , VP ₃ , VP ₁ .

Antigenic properties

According to antigenic properties, the FMDV strain “O/Kenya/2017” belongs to serotype 01. The virus is stably neutralized by homologous antiserum and does not show hemagglutinating activity. In sick animals, type-specific antibodies are formed in the blood serum, which are detected in the enzyme immunoassay (ELISA) and microneutralization reaction (RMN).

The primary structure of the ID gene of the VP ₁ protein of the O/Kenya/2017 strain of foot-and-mouth disease virus was determined by nucleotide sequencing. Comparative analysis of nucleotide sequences showed that the FMDV strain "O/Kenya/2017" belongs to the O/EA-2 genotype (Fig. 1).

Antigenic relatedness (r ₁ ) of the strain "O/Kenya/2017" of FMD virus was studied in the RMN, in a cross-study of the strain with specific sera obtained for the following production strains of FMD virus:

- strain "O/Taiwan/1997" (genotype O/CATHAY/CAM 94),

- strain "O/Primorsky/2014" (genotype O/SEA/Mya-98),

- strain "O/Primorsky/2012" (genotype O/ME-SA/PanAsia),

- strain "O/Saudi Arabia/2008" (genotype O/ME-SA/PanAsia2),

- strain "O/Zabaikalsky/2016" (genotype O/ME-SA/Ind-2001).

The titer of reference blood sera of cattle obtained by immunization of animals with monovalent vaccines from industrial strains of foot and mouth disease virus serotype O against 10 ² TCD ₅₀ of homologous and heterologous virus was determined in RMN during cross titration, calculating values using a linear regression equation, and expressed in lg. The value of _r1 was determined as the antilogarithm of the difference between lg serum titers against heterologous and homologous virus [17, 18, 19].

The value of r, in RMN was interpreted as follows:

at ≥ 0.3 - the studied and production strains of foot-and-mouth disease virus are closely related;

at < 0.3 - the studied sample of the FMDV strain differs from the production strain.

The indicators of antigenic relationship in the study of the strain "O / Kenya / 2017" amounted to r ₁ from 0.04 to 0.08, which indicates the absence of antigenic relationship with industrial strains of FMDV serotype O (Table 1).

Geno- and chemotaxonomic characteristics

The strain "O/Kenya/2017" of the foot and mouth disease virus is an RNA (+) - containing virus with a molecular weight of 7 × 10 ⁶ D. The nucleic acid is represented by a single-stranded linear molecule with a molecular mass of 2.8 × 10 ⁶ D. The virion has a protein shell consisting of four main 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. Viral 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, an RNA-dependent RNA polymerase (3D gene) is isolated, which is involved in RNA replication for the assembly of virions.

Physical properties

The mass of the virion is 8.4×10 ^-18 g. The floating density is 1.45 g/cm ³ .

Resistance to external factors

The O/Kenya/2017 strain of FMDV is resistant to ether, chloroform, and acetone. Most stable at pH 7.42-7.65. Shifts in pH to the acidic and strongly alkaline side lead to virus inactivation. Sensitive to formaldehyde, UV radiation, y-irradiation, high temperatures (above 38.3°C).

Additional features and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period) on transplanted cultures.

Biotechnological characteristics. The O/Kenya/2017 strain of FMDV is reproduced in transplanted cell cultures: kidneys of the Siberian mountain ibex (PSGK-30), kidneys of a pig (IB-RS-2), kidneys of a Syrian hamster (VNK-21).

When testing, 5 successive passages of the strain "O/Kenya/2017" of foot-and-mouth disease virus were carried out in transplanted cell cultures PSGK-30, VNK-21, IB-RS-2. The biological properties were characterized by determining the infectivity of the virus of each passage in the continuous IB-RS-2 cell line and in naturally susceptible animals - cattle (cattle) and pigs.

Based on the data obtained, it can be argued that the FMDV strain “O/Kenya/2017” according to antigenic and immunological spectra is an original, taxonomically new, previously unknown variant of FMDV of the O/EA-2 genotype.

To reduce the epizootic risk of foot and mouth disease caused by the O/EA-2 genotype and to prevent the occurrence of disease foci, timely vaccination is important, which requires the development of a new highly immunogenic and effective vaccine.

A highly immunogenic and effective vaccine against foot-and-mouth disease of the O/EA-2 genotype from the strain "O/Kenya/2017" cultured inactivated adsorbed was obtained.

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

Example 1. Genetic characterization of the FMDV strain "O/Kenya/2017" according to PCR and nucleotide sequencing.

The studies carried out included studying the primary structure of the 1D gene (VP ₁ protein) (639 n.b.) of the FMDV strain “O/Kenya/2017” by nucleotide sequencing using the Sanger method and determining the position of this strain on the phylogenetic tree of FMDV serotype O. Method is based on determining the primary structure of the 1D gene (VP ₁ protein) of the tested isolate/strain, followed by analysis of phylogenetic relationship with other FMDV serotype O isolates.

It was necessary to conduct a comparative analysis of the nucleotide sequences of the 1D gene encoding the VP protein] of the foot-and-mouth disease virus of the O/Kenya/2017 vaccine strain with other isolates and strains. The nucleotide sequence of the VP ₁ protein gene of the O/Kenya/2017 strain of the O/EA-2 genotype of the foot-and-mouth disease virus is presented by SEQ ID NO: EA-2 of FMDV is shown in SEQ ID NO: 2.

A phylogenetic analysis of the O/Kenya/2017 strain was carried out. The phylogenetic tree was derived using the MEGA6 program and the Neighbor-Joining algorithm [20]. The percentage of repetitive branches in which related taxa are grouped together in the bootstrap test (200 repetitions) is shown next to the branches [21, 22]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [23] and expressed in terms of the number of base substitutions per site. This analysis included 33 nucleotide sequences, including the sequence of the ID gene of the strain "O/Kenya/2017" of foot-and-mouth disease virus. All positions containing spaces and missing data have been removed (complete removal option). There were only 639 positions in the final data set. Evolutionary analysis was carried out in MEGA 6 [24].

As a result of the work performed by comparing the complete nucleotide sequences of the 1D gene (VP ₁ protein) of the O/Kenya/2017 strain and other isolates/strains of FMDV serotype O, the position of the studied strain on the phylogenetic tree was determined (Fig. 1).

Based on the results of the entire analysis, we conclude that the studied strain “O/Kenya/2017” belongs to the O/EA-2 genotype, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of the strain "O/Kenya/2017" to a continuous monolayer cell culture of the kidney of the Siberian mountain ibex PSGK-30.

A 10% aphthous suspension of foot-and-mouth disease virus obtained from aft cattle (2nd passage) was used to infect a continuous monolayer culture of Siberian ibex kidney cells PSGK-30. The inoculum concentration of cells was 0.20-0.25 million cells/cm ³ , the virus infection dose was 0.001 TCD50/CL. According to the results of the study, the reproduction of the foot-and-mouth disease virus of the strain "O/Kenya/2017" took place with a specific destruction of the monolayer by 90-100% in 15-16 hours. During 6 consecutive passages, an increase in the values of the titer of infectious activity of the virus from 6.25 ± 0.11 up to 7.75±0.08 lg TCD ₅₀ / cm ³ .

The maximum value of the titer of infectious activity of the virus was reached at the stage of passage 6 (7.75±0.08 lg TCD ₅₀ /cm ³ ). The concentration of 146S particles from passages 1 to 6 changed from 0.35±0.01 to 1.25±0.03 µg/cm ³ . At the same time, the largest amount of the 146S component was noted at the stage of the 6th passage (1.25±0.03 μg/cm ³ ). The degree of reliability (R ² ) of the results of the study in the quantitative version of RT-PCR-RT ranged from 97.87 to 99.75%. Thus, over the course of ⁶ consecutive passages, the adaptation of the strain “O/Kenya/2017” of foot-and-mouth disease virus to the continuous monolayer cell line PSGK-30 was successfully carried out.

Example 3. Study of the conditions for monolayer cultivation of FMDV strain "O/Kenya/2017" on an industrial scale.

In the process of industrial cultivation of the strain "O/Kenya/2017" of foot-and-mouth disease virus in a transplantable monolayer cell culture PSGK-30, the optimal conditions for cultivating the virus were selected by analyzing the different composition of the supporting medium, the temperature factor and the dose of cell infection.

At the first stage of the study, the influence of the composition of the supporting medium on the reproduction of the strain "O/Kenya/2017" of foot-and-mouth disease virus in a monolayer cell culture of PSGK-30 was evaluated on a production scale (at the stage from the 5th to the 6th passages). Three media supplemented with 2% fetal calf serum were used for comparison: 1) Eagle's DMEM medium; 2) RPMI-1640 medium; 3) Hank's solution with 3.0% blood protein hydrolysate. The inoculum concentration of cells was 0.20-0.25 million cells/cm ³ , the dose of virus infection was 0.1000-0.0001 TCD ₅₀ /CL. The cultivation of the virus was carried out at temperatures of 35±0.2 - 38±0.2°C until the destruction of the cell monolayer by 95-100% within 12-28 hours. composition are shown in table 2.

From the data of Table 2, it can be seen that during the reproduction of the strain "O/Kenya/2017" of the foot-and-mouth disease virus in a monolayer cell culture of PSGK-30 using supporting media of different composition, the Eagle's DMEM medium is optimal, which allows achieving specific destruction of the cell monolayer in 12-13 hours by 95-100% with the accumulation in the resulting suspension of the 146S component in the amount of 1.25±0.03 μg/cm ³ and the infectious activity titer of 7.75±0.08 lg TCD ₅₀ /CM ³ . When using RPMI-1640 medium and Hank's solution, the rates of virus reproduction were lower.

At the next stage of studying the conditions of monolayer cultivation of the FMDV strain "O/Kenya/2017" in PSGK-30 cell culture under production conditions, the influence of the temperature factor on the reproduction process of the virus was evaluated. For this, the cultivation of the virus was carried out in Eagle's DMEM medium with 2% cattle serum at the following temperatures: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2°С. The dose of infection of the cell culture with foot-and-mouth disease virus was 0.010-0.001 TCD ₅₀ /cell. The cultivation of the virus was carried out until the destruction of the cell monolayer by 80-100% for 12-28 hours (table 2). According to the results of the study, it was found that for the complete reproduction of the strain "O/Kenya/2017" of foot-and-mouth disease virus in a monolayer cell culture of PSGK-30, the optimal temperature is 37.0 ± 0.02 ° C, at which, in 12-13 hours, the development of CPP reached 95-100% with the accumulation of 146S particles equal to 1.25±0.03 μg/cm ³ and the titer of infectious activity of the virus 7.75±0.08 lg TCD ₅₀ /cm ³ .

The influence of the dose of infection of a monolayer of PSGK-30 cells with the O/Kenya/2017 strain during cultivation on a production scale was evaluated. For this, different doses of the virus were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCD50/CL. Eagle's MEM medium with 2% bovine serum was used as a maintenance medium. The reproduction of the virus was carried out at a temperature of 37.0±0.2°C for 12-28 hours until the specific destruction of cells by 90-100%. Based on the results of cultivation, the concentration of the 146S component and the titer of FMDV infectious activity were determined. As follows from the data in Table 2, the greatest accumulation of "complete" virus particles was achieved at an infection dose of 0.1-0.01 TCD ₅₀ /cell. and amounted to 1.25±0.03 μg/cm ³ with a titer of infectious activity of the virus 7.75±0.08 lg TCD ₅₀ /cm ³ .

Thus, we studied the conditions of monolayer cultivation of the strain "O/Kenya/2017" of foot-and-mouth disease virus in PSGK-30 cell culture on an industrial scale. As a result of the study, the following optimal reproduction parameters of the strain "O/Kenya/2017" of foot and mouth disease virus in the monolayer cell line PSGK-30 were determined: 1) supporting medium - Eagle's DMEM medium; 2) temperature regime of cultivation -37.0±0.02°C; 3) the dose of virus infection - 0.1-0.01 TCD ₅₀ /cl.

Example 4. Study of the conditions for suspension cultivation of the strain "O/Kenya/2017" of FMDV on an industrial scale.

During the industrial cultivation of the strain "O/Kenya/2017" of foot-and-mouth disease virus in a transplantable suspension cell culture BHK-21/SUSP/ARRIAH, we searched for the optimal parameters for successful reproduction of the foot-and-mouth disease pathogen using different cell concentrations, temperature and dose of infection.

At the first stage of the work, the influence of the seeding concentration of BHK-21/SUSP/ARRIAH cells in suspension on the reproduction of the O/Kenya/2017 strain of FMDV on an industrial scale was determined. For analysis, suspensions were prepared with the following cell concentrations: 2.5; 3.0; 3.5; 4.0 million cells/cm ³ . The pH was maintained in the range of 7.45-7.65. The virus cultivation temperature was 37.0±0.02°C, the virus infection dose was 0.10-0.01 TCD ₅₀ /cell. Virus reproduction was carried out until specific cell death by 95-100%, which was carried out within 9-11 hours. The results of suspension cultivation of the FMDV strain "O/Kenya/2017" in suspension with different cell concentrations are presented in Table 3.

As follows from Table 3, when cultivating FMDV strain "O/Kenya/2017" in the suspension cell line VNK-21 /SUSP/ARRIAH with different cell concentrations, it was determined that the accumulation of the 146S component in suspension with a cell concentration of 2.5 million cells ./cm ³ was 0.74±0.03 μg/cm ³ , with a concentration of 3.0 million cells/cm ³ - 0.99±0.03 μg/cm ³ , with a concentration of 3.5 million cells/ cm ³ - 1.56 ± 0.02 μg / cm ³ , and with a concentration of 4.0 million cells / cm ³ - 1.97 ± 0.03 μg / cm ³ . As follows from the data obtained, for the reproduction of the strain "O/Kenya/2017" of FMDV, the concentration of BHK-21 /SUSP/ARRIAH cells in suspension is optimal, equal to 4.0 million cells/cm ³ . The value of the titer of infectious activity of the virus in this case amounted to 9.50±0.11 lg TCD ₅₀ /cm ³ . A high concentration of cells made it possible to obtain the same product yield with a slight increase. From an economic point of view, therefore, for the reproduction of the strain "O/Kenya/2017" of foot and mouth disease virus, it is optimal to use a suspension of cells of the BHK-21 /SUSP/ARRIAH line with a concentration of 4.0 million cells/cm ³ .

At the next stage of the work, the influence of the temperature factor on the suspension cultivation of the strain "O/Kenya/2017" of the foot-and-mouth disease virus in the cell culture of the BHK-21 /SUSP/ARRIAH cell line was studied on an industrial scale. To do this, the reproduction of the virus was carried out under the following temperature conditions: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2°С. The dose of cell culture infection with the virus was 0.10-0.01 TCD ₅₀ /cell. The pH of the viral suspension was maintained in the range of 7.45-7.65. The cultivation of the virus was carried out until the CPP, equal to 90-100%, for 9-21 hours. According to the results of the analysis, it was determined that for the complete reproduction of the strain "O/Kenya/2017" of foot-and-mouth disease virus in the suspension culture of BHK-21/SUSP/ARRIAH cells, the optimal is the medium temperature of 37.0±0.02°C, at which within 9-11 hours there is a specific cell death by 95-100% with a high accumulation of the 146S component (1.97±0.03 μg/cm ³ ) and a titer infectious activity of the virus 9.50±0.11 lg TCD ₅₀ /cm ³ .

In the course of work on the study of the conditions of suspension cultivation of the strain "O/Kenya/2017" of foot-and-mouth disease virus on an industrial scale using the BHK-21 /SUSP/ARRIAH cell culture, the effect of the dose of cell infection was evaluated. To do this, cell suspensions were infected with the virus at different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD ₅₀ / class. The reproduction of the virus was carried out at a temperature of 37.0±0.2°C for 9-21 hours until the cytopathic effect was 90-95%. Based on the results of cultivation, the concentration of 146S particles and the titer of FMDV infectious activity were determined. As can be seen from the data in Table 3, the greatest accumulation of the 146S component was noted at an infection dose of 0.10-0.01 TCD ₅₀ /cell. (1.97±0.03 μg/cm ³ ) with a titer of infectious activity of the virus equal to 9.50±0.11 lg TCD ₅₀ /cm ³ .

Thus, we studied the parameters of suspension cultivation of the strain "O/Kenya/2017" of FMDV in the suspension cell line VNK-21/SUSP/ARRIAH on an industrial scale. The optimal conditions for the reproduction of the strain "O/Kenya/2017" in the suspension cell culture VNK-21/SUSP/ARRIAH were revealed: 1) the concentration of cells before infection - 4.0 million cells/ ^cm3 ; 2) temperature regime of cultivation - 37.0±0.02°C; 3) the dose of virus infection is 0.10-0.01 TCD ₅₀ /cell.

Example 5. Inactivation of a suspension of FMDV strain "O/Kenya/2017".

At the end of the reproduction cycle of FMDV, without stopping the process of thermostating (37.0±0.02°C), an acidified solution of aminoethylethyleneimine (AEEI) with a pH of 8.2-8.3 was added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be 0.03%. The inactivation of the infectious activity of the FMDV strain "O/Kenya/2017" was carried out for 12 hours at a temperature of 37.0±0.1°C and pH 7.45-7.65 with stirring.

To determine the time of complete inactivation after the addition of 1,2-AEEI, samples were taken every hour. The obtained samples were tested for the presence of FMD virus infectivity in the primary culture of porcine kidney cells SP. The results are presented in Table 4, from the data of which it can be seen that the complete inactivation of the foot-and-mouth disease virus of the O/Kenya/2017 strain, reproduced in cultivators, occurred 7 hours after the introduction of 1,2-AEEI.

Ready-made virus inactivated suspensions were examined in the RSK to assess the content of virus components in them. The concentration of the 146S component was 1.94±0.03 µg/cm ³ , and the 146S+75S component was 2.43±0.05 µg/cm ³ .

Example 6. Selection of conditions for purification of a suspension of foot and mouth disease virus of the strain "O / Kenya / 2017" genotype O / EA-2.

Suspension of foot and mouth disease virus strain "O/Kenya/2017" genotype O/EA-2, obtained during reproduction in the suspension cell line BHK-21/SUSP/ARRIAH, was subjected to inactivation and purification. To obtain a purified FMDV antigen, polyhexamethyleneguanidine (PHMG) was used at concentrations of 0.010, 0.014, and 0.018%. Before and after the process of purification of the FMDV antigen of the O/Kenya/2017 strain, the concentration of the total viral protein and immunogenic components was determined in the quantitative variant of RSK. The results of the analysis are shown in Table 5, from which it follows that as a result of the purification of the FMDV antigen of the O/Kenya/2017 strain using PHMG at a concentration of 0.010%, a decrease in the concentration of ballast protein by 12%, immunogenic components - by 2% was noted. When using PHMG at a concentration of 0.014%, a decrease in the amount of ballast protein by 37%, 146S component - by 3.5% was observed. Using PHMG at a concentration of 0.018%, the content of ballast protein decreased by 54%, and immunogenic components - by 21%. Thus, studying the conditions for purification of the antigen of the strain "O/Kenya/2017", we came to the conclusion that it is optimal to use PHMG with a concentration of 0.014% to obtain a purified product.

Example 7. Selection of conditions for concentrating the suspension of the antigen of the strain "O/Kenya/2017" of the FMDV genotype O/EA-2.

The cultured inactivated suspension of the O/Kenya/2017 strain, obtained using the BHK-21/SUSP/ARRIAH suspension cell line, was concentrated 7 times by volume. To increase the concentration of the immunogenic components of the FMDV antigen, the following methods were used: 1) adding polyethylene glycol (PEG-6000) at a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 4 hours; 2) adding polyethylene glycol (PEG-6000) with a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 12 hours; 3) concentration with aluminum hydroxide (10% colloidal solution in the amount of 45%, virus antigen - 55% of the total volume).

Before and after the process of concentrating the suspension of the FMD virus antigen of the O/Kenya/2017 strain, the concentration of total viral protein and immunogenic components was determined in the quantitative variant of RSC. The results of the studies are presented in Table 6, which shows that when the antigen of the strain "O/Kenya/2017" of the FMD virus was concentrated with PEG-6000 for 4 hours, an increase in the content of the components was noted compared to the initial values (before concentration) of 4, 9 times. Using the same polymer, but increasing the exposure time to 12 hours, the concentration of virus components was increased by 5.4 times. Using the method of sorption using aluminum hydroxide, it was possible to increase the amount of immunogenic components of the antigen of the strain "O/Kenya/2017" of foot-and-mouth disease virus in suspension by 6.8 times (losses from the expected are insignificant - 2.9%). Thus, by examining the conditions for concentrating the antigen of the O/Kenya/2017 strain, we came to the conclusion that the best way to increase the concentration of FMDV components is the sorption method using a colloidal solution of aluminum hydroxide.

Example 8 Adjuvant selection and antigen-adjuvant ratio.

Aluminum hydroxide in combination with saponin was chosen as an adjuvant for the manufacture of an FMD monovalent adsorbed vaccine from the antigen of the O/Kenya/2017 strain. In the manufacture of monovalent adsorbed vaccine against FMDV used 4.0% aluminum hydroxide in terms of dry matter and saponin in the amount of 1.5 mg/cm ³ .

Example 9. The layout of the FMD vaccine from the strain "O / Kenya / 2017" of the genotype O / EA-2, cultured inactivated sorbed.

From the obtained concentrate of the antigen of the FMD virus of the strain "O/Kenya/2017", a culture-inactivated sorbed vaccine against foot-and-mouth disease of the O/EA-2 genotype was prepared using aluminum hydroxide and saponin as adjuvants. The amount of 146S immunogenic component in 1.0 cm ³ of the prepared antigen was 12.72±0.09 μg/cm ³ (table 6).

Example 10. Immunization of animals with a vaccine against foot-and-mouth disease from the strain "O/Kenya/2017" of the genotype O/EA-2 culture inactivated sorbed.

From the obtained vaccine against foot and mouth disease from the strain "O/Kenya/2017" of the genotype O/EA-2, cultural inactivated sorbed, a series of dilutions for cattle was prepared with a 4-fold step. 15 heads of cattle were divided into 3 groups of 5 heads each. The immunizing dose was 2.0 cm ³ . The first group of cattle (No. 1-5) was immunized with the vaccine without dilution, the second group of cattle (No. 6-10) was vaccinated with the vaccine diluted 1/4, the third group of cattle (No. 11-15) was vaccinated with the vaccine diluted 1/16 . The drug was administered intramuscularly in the middle third of the neck. As a virus control, 2 heads were left without vaccination.

Example 11. Examination of the blood sera of vaccinated animals for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus.

Blood sera from cattle obtained before immunization of animals, 14 days after immunization, were examined for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus using a blocking enzyme-linked immunosorbent assay (ELISA) in accordance with the recommendations of the OIE (OIE) [3]. The obtained sera were tested using the Prio CHECK®FMDV NSP ELISA test system for in vitro detection of antibodies against Foot and Mouth Disease Virus in serum of cattle, sheep and pigs (Prionics Lelystad B.V., Netherlands). The results of the studies are shown in Table 7, which shows that the blood sera of animals did not contain antibodies to non-structural proteins of the foot-and-mouth disease virus (PI values for bovine blood sera < 50%).

Example 12. Evaluation of the avirulence and safety of the vaccine against foot and mouth disease from the strain "O/Kenya/2017" of the genotype O/EA-2 cultured inactivated adsorbed.

To determine the avirulence of the vaccine in cattle, a selected sample of the vaccine preparation was injected intradermally at 0.1 cm ³ . The study used cattle weighing 250-300 kg. Within 10 days of observation, the animals remained clinically healthy, and pathoanatomical examination did not reveal changes characteristic of foot and mouth disease. This indicated the absence of virulent properties of the developed vaccine.

Control of the safety of the product on cattle was carried out by intramuscular injection of the vaccine at a dose of 6.0 cm ³ (triple dose of 2.0 cm ³ ). The observation period was also 10 days. It should be noted that after immunization, the animal's body temperature can rise to 41.2°C and be maintained at this level for 1-2 days, which does not go beyond the norm after the introduction of the vaccine preparation.

According to the results of the studies, the culture-inactivated sorbed vaccine against foot and mouth disease from the O/Kenya/2017 strain of the O/EA-2 genotype was found to be harmless, all animals remained clinically healthy during the observation period, and no tissue necrosis at the site of vaccine administration was detected during the pathoanatomical analysis.

Example 13. Study of the immunogenic properties of the FMD vaccine from the O/Kenya/2017 strain of the O/EA-2 genotype, culturally inactivated, sorbed by the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 21st day after the introduction of the vaccine against foot and mouth disease from the strain "O/Kenya/2017" of the genotype O/EA-2, the cultured inactivated sorbed blood was taken from cattle and the obtained blood sera were studied in the microneutralization reaction [3]. It was found that in cattle immunized with the vaccine in a whole dose (5 heads), the average values of antibody titer were 7.90±0.14 log2 SN ₅₀ , with a dilution of 14 (5 heads) - 4.55±0.21 log2 SN ₅₀ , with a dilution of 1/16 (5 goals) - 3.05 ± 0.45 log ₂ SN ₅₀ . (table 8). The data obtained from the microneutralization reaction indicate that after the administration of the whole vaccine, the formation of humoral immunity with protective titers of strain-specific antibodies (5.5 or more log ₂ SN ₅₀ ) is ensured, which meets the requirements of international standards [3].

Example 14 Challenge of naturally susceptible animals. Study of the protective ability of the vaccine against foot-and-mouth disease from the O/Kenya/2017 strain of the O/EA-2 genotype, cultured inactivated adsorbed on naturally susceptible animal species.

Cattle, in the amount of 15 heads, immunized with the vaccine in whole form and in dilutions, were infected with the control strain "O / Kenya / 2017" of the O / EA-2 genotype of the foot-and-mouth disease virus, adapted to these animals in the mucous membrane of the tongue at a dose of 10 ^{4, 0} ID ₅₀ /0.20 cm ³ (in two points of 0.10±0.05 cm ³ ). 7 days after infection, all animals were euthanized and postmortem examination was performed. Animals were considered protected from foot-and-mouth disease, in which there were no lesions on the limbs. Primary aphthae were not taken into account.

The results of the control infection are presented in Table 8, from which it follows that the FMD vaccine from the strain "O / Kenya / 2017" of the O / EA-2 genotype is cultured inactivated sorbed in whole form and in a dilution of 1/4, administered in a single dose (2 ,0 cm ³ ) protects cattle from infection with a homologous strain of all animals (5 out of 5 heads).

The drug, inoculated at a dilution of 1/16, protected 4 out of 5 animals. According to the results of the control infection, it was found that the inoculation volume of this vaccine contains 24.25 PD ₅₀ and 0.08 IMD ₅₀ for cattle.

Thus, the above information indicates that the vaccine against FMD genotype O / EA-2 from the strain "O / Kenya / 2017" cultural inactivated sorbed, embodying the proposed invention, is intended as an experimental reserve vaccine for use in agriculture, namely in veterinary virology and biotechnology; confirmed the possibility of implementing the presented; the vaccine made from the strain "O/Kenya/2017" genotype O/EA-2 in accordance with the invention, has a high immunogenic activity and is able to provide effective protection of susceptible animals against an epizootic strain of foot-and-mouth disease virus serotype O, genotype O/EA-2, circulating in Africa.

Sources of information taken into account when compiling the description of the invention to the application for a patent of the Russian Federation for the invention "Vaccine against FMD genotype O / EA-2 from the strain

"O/Kenya/2017" culture inactivated sorbed":

1. Foot and mouth disease. Prevention measures. URL: http://89.rospotrebnadzor.ru/directions/epid_nadzor/146902 (Date of access: 08.10.2022).

2. Danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Date of access: 10/18/2022).

3. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed. - Paris, 2015-Vol.1, Chapter 2.1.5. - P. 166-169.

4. Burdov A. N., Dudnikov A. I., Malyarets P. V. et al. -M.: Agropromizdat, 1990. - 320 p.

5. Ponomarev A. P., Uzyumov V. L., Gruzdev K. N. Foot-and-mouth disease virus: structure, biological and physico-chemical properties. - Vladimir: Folio, 2006. - 250 p.

6. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M/ Garland, R.P. Kitching [et. al.] // Comparative Immunology, Microbiology and Infectious Diseases. - October 2002. - V. 25. - P. 345-364.

7. Brown F. A brief history of FMD and its casual agent. FMD: Control Strateies: Proc. Jnt. Symp.2-5. June 2002, Lyons, France. - Paris, 2003. - p.13-21.

8. Vaccination against foot-and-mouth disease virus confers complete clinical protection in 7 days and partial protection in 4 days: Use in emergency outbreak response / T.G. William, J. M. Pachecoa, T. Doel [et.al.] // Vaccine. - December 2005. - V. 23. - P. 5775-5782.

9. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M/ Garland, R.P. Kitching [et. al.] // Comparative Immunology, Microbiology and Infectious Diseases. - October 2002. - V. 25. - P. 345-364.

10 Barnett P.V. A review of emergency foot-and-mouth disease (FMD) vaccines / Vaccine. - February 2002. - V. 20. - P. 1505-1514.

11. Official website of The FAO World Reference Laboratory for Foot-and-Mouth Disease - URL: http://www.wrlfmd.org/ref labs/find ref lab reports.htm (Accessed 10/13/2022).

12. Salt I.S. Emergency vaccination of pigs against foot-and-mouth disease: protection against disease and reduction in contact transmission / Vaccine. - April 1998. -V. 16.-P. 746-754.

13. Rakhmanov A.M. Modern epizootic situation in the world on foot and mouth disease and measures of its control // Veterinary Medicine M1zhvgd. topics. Sciences. Kharyuv, 2013.-p.37-38.

14. Longjam N., Tauo T. Antigenic variation of Foot and Mouth Disease Virus // Vet. World. - 2011. - Vol.4(10). - P. 475-479.

15. Melnik R.H., Khaustova H.V., Melnik H.V., Samuylenko A.Ya., Grin S.A., Svyatenko M.S., Litenkova I.Yu. Antigenic variability of foot-and-mouth disease virus serotype A and justification for the need to obtain new relevant industrial strains / Veterinary medicine and feeding. - 2019. - No. 3. - P. 29-31.

16. Paton D.J., Sumption K.J., Charleston B. Options for control of foot-and-mouth disease: knowledge, capability and policy/ Phil. Trans. R. Soc. In (2009) 364. - P. 2657-2667.

17 Barnett P.V. A review of emergency foot-and-mouth disease (FMD) vaccines / Vaccine. - February 2002. - V. 20. - P. 1505-1514.

18. Guidelines for determining the concentration of 146S, 75S, 12S components of the vaccine strains of the cultured foot-and-mouth disease virus in the complement fixation test (CFR) / FGBU "ARRIAH". - Approved. 09/21/17.

19. Guidelines for determining the antigenic correspondence between epizootic isolates and industrial strains of foot-and-mouth disease virus in the cross-reaction of microneutralization: approved. Rosselkhoznadzor 13.09.2017 / FGBU "ARRIAH". - Vladimir: 2017. - 24 p.

20. Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406-42.

21. Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783-791.

22. Tamura K., Nei M., and Kumar S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA) 101:11030-11035.

23. Kumar S., Stecher G., Li M., Knyaz C, and Tamura K. (2018). MEGA 6: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35:1547-1549.

24. Barnett P. Aspects of emergency vaccination against foot-and-mouth disease / P. Barnett, J.M/ Garland, R.P. Kitching [et.al.] // Comparative Immunology, Microbiology and Infectious Diseases. - 2002. - V. 25. - P. 345-364.

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Claims (4)

1. The vaccine against FMD cultural inactivated sorbed, containing the active substance and adjuvant, characterized in that as the active substance it contains avirulent and purified antigenic material homologous to the infectious agent strain "O/Kenya/2017" genotype O/EA-2, reproduced in transplantable suspension cell line of the kidney of a newborn Syrian hamster BHK-21 / SUSP / ARRIAH, in an amount of at least 4.0 μg; as an adjuvant aluminum hydroxide 10% colloidal solution with a content of 45% by volume of 4.0% in terms of dry matter in complex with saponin in the amount of 1.5 mg and supporting medium - up to 1.0 cm ³ . 2. The vaccine according to claim 1, characterized in that it contains an avirulent and purified antigenic material from an O/Kenya/2017 strain of O/EA-2 genotype homologous to the infectious agent, obtained in a sensitive biological system and representing a suspension containing predominantly 146S immunogenic component of FMDV genotype O/EA-2. 3. The vaccine according to any one of paragraphs. 1, 2, characterized in that it contains avirulent and purified antigenic material from the O/Kenya/2017 strain homologous to the infectious agent of the O/EA-2 genotype, obtained preferably in a continuous suspension cell line of the kidney of a newborn Syrian hamster VNK-21/SUSP /ARRIAH and is a suspension containing mainly the 146S immunogenic component of the FMDV genotype O/EA-2, an adjuvant - aluminum hydroxide and an additive in the finished product in the following amounts: Avirulent and purified antigen strain "O/Kenya/2017" genotype O/EA-2 foot and mouth disease virus not less than 4.0 µg/cm ³ aluminum hydroxide 4.0% (based on dry matter) Saponin 1.5 mg / cm ³ Supportive environment up to 1.0 cm ³