RU2847822C1 - Vaccine against foot-and-mouth disease genotype o/ea-2, monovalent, inactivated, emulsion - Google Patents

Abstract

FIELD: veterinary virology; biotechnology.

SUBSTANCE: invention relates to the field of veterinary virology and biotechnology, namely to the development of a vaccine against foot-and-mouth disease genotype O/EA-2, which is a cultured monovalent inactivated emulsion vaccine. The vaccine embodying the proposed invention is intended as a reserve for use in agriculture, namely in veterinary virology and biotechnology. The feasibility of the invention has been confirmed. This vaccine has high immunogenic activity and is capable of providing effective protection for susceptible animals against the epizootic strain of foot-and-mouth disease virus genotype O/EA-2. The vaccine against foot-and-mouth disease genotype O/EA-2 is a cultured monovalent inactivated emulsion vaccine manufactured using the oil adjuvant Montanide ISA-61 VG. Based on the results of a challenge test, it was established that the vaccine contains 55.90 PD₅₀ and 0.04 ImD₅₀ for pigs in the vaccination dose.

EFFECT: high efficacy of the vaccine preparation.

3 cl, 1 dwg, 7 tbl, 14 ex

Description

The invention relates to the field of veterinary virology and biotechnology, namely to the development of a culture-based monovalent inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-2 genotype.

Foot-and-mouth disease (aphthous fever) is an acute viral disease of animals, known since the 16th century. It is caused by an RNA-containing virus of the genus Aphthovirus , which is highly virulent and pathogenic. Foot-and-mouth disease is most commonly found in cattle and pigs; sheep, goats, and wild ungulates are less susceptible [1].

The FMDV genome is a positive-sense single-stranded RNA approximately 8400-8500 nt long, enclosed in an icosahedral capsid consisting of 60 copies of structural proteins, namely VP ₁ , VP ₂ , VP ₃ and VP ₄ .

Currently, scientists know the following seven characterized serotypes of foot-and-mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, with representatives of serotype O being diverse and particularly common in Asia and Africa.

More than 60 genetic lineages of foot-and-mouth disease virus are known, the differences between which are determined by analyzing the nucleotide sequence of the highly variable 1D gene, which encodes the amino acid sequence of the surface and most variable viral protein VP ₁ [2]. The highly variable structural protein VP ₁ contains two immunogenic regions: the GH loop of the highly conserved RGD tripeptide motif and the C-terminus, which are involved in virus attachment and penetration, immune defense, and serotype specificity.

Recovery of an animal from foot-and-mouth disease caused by a single genotype does not provide immune protection against isolates/strains of a different genetic lineage that are genetically and antigenically distinct from the vaccine strain [3]. Antigenic variations arise from amino acid mutations that alter the immune system's recognition of viral proteins. Surface-exposed viral structures are particularly susceptible to immune attack. The emergence of new antigenic forms is determined by a complex interaction of viral and host cell factors [4].

Vaccination is used as the main control tool in endemic areas, and analysis of each pool and the genotypes in it makes it possible to select the most specific vaccines, containing the corresponding topotypes present in this pool [4, 5].

Serotype O is divided into 11 topotypes: EAST AFRICA 1-4 (EA-1-4), SOUTHEAST ASIA (SEA), EUROPE-SOUTH AMERICA (EURO-SA), INDONESIA-1 and 2 (ISA-1 and -2), CATHAY (China), MIDDLE EAST-SOUTH ASIA (ME-SA) and WEST AFRICA (WA) [1, 5] and is widespread throughout the world and well studied. This leads to problems in the specific prevention of foot-and-mouth disease when using culture-based inactivated foot-and-mouth disease vaccines. There is a need to create new means for specific prevention against foot-and-mouth disease serotype O [1, 6, 7].

To ensure biological security in the Russian Federation, a range of measures to combat and prevent foot-and-mouth disease are being implemented on farms across the country. This range of measures aims to prevent the introduction of the virus into the country, systematically vaccinate animals in the buffer zone, and monitor the immune status of cattle and pigs, which are treated with culture-based inactivated foot-and-mouth disease vaccines [6, 7].

To immunize animals, a vaccine made from a virus homologous to field isolates should be used, this has been confirmed by research by many scientists [8, 9, 10, 11].

Inactivated vaccines, made primarily from the 146S component, are still considered the most effective against foot-and-mouth disease [8, 9, 12, 13-16].

The genetic and antigenic diversity of foot-and-mouth disease virus serotype O poses challenges for specific prophylaxis, complicating strain-specific diagnostics of isolated FMD virus isolates. This necessitates the development of new agents for specific immunoprophylaxis against foot-and-mouth disease virus serotype O, particularly the O/EA-2 genotype, whose representatives have spread throughout East Africa [7, 8].

In Africa, outbreaks of foot-and-mouth disease serotype O are sporadic. In recent years, Russia's trade and economic ties with countries in this region have strengthened, particularly with Tanzania, Uganda, Kenya, the Democratic Republic of the Congo, and others. Consequently, the risk of introducing isolates of this genotype into the Russian Federation is high, threatening our country's biological security with respect to foot-and-mouth disease.

Analyzing outbreaks in Africa, isolates of foot-and-mouth disease virus belonging to serotype O were identified in Nigeria, Kenya, Tanzania, and Ethiopia. In particular, outbreaks of foot-and-mouth disease of the O/EA-1 genotype were noted in 2009 and 2010, as well as O/EA-4 in 2010, and O/EA-2 in 2010, 2011, 2017, 2020, and 2021 [7, 8]. It should be noted that representatives of the O/EA-2 genotype of foot-and-mouth disease virus were found in isolated quantities, but since 2021 they have been registered en masse in East African countries and adjacent territories.

There are known industrial strains of foot-and-mouth disease virus serotype O, which are used in the production of products for the specific prevention of foot-and-mouth disease:

- 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).

- strain "O/Kenya/2017" (genotype O/EA-2).

An isolate of the O/EA-2 genotype foot-and-mouth disease virus was isolated from cattle in the Republic of Uganda in 2023 and was delivered to the All-Russian Research Institute of Animal Health for scientific research.

The strain "O/Kiruhura/EA-2/2023" was obtained as a result of research work by adapting the isolate to reproduction in the primary trypsinized monolayer cell line of the SP pig kidney, in continuous cell cultures of BHK-21/SUSP/ARRIAH, IB-RS-2, PSGK-30, in the body of cattle and pigs under the conditions of the Federal State Budgetary Institution "All-Russian Research Institute of Animal Health".

According to the results of a comparative analysis of nucleotide sequences, the isolated strain of foot-and-mouth disease virus belongs to the O/EA-2 genotype, which differs significantly from production strains of foot-and-mouth disease virus serotype O, including the O/Kenya/2017 strain.

At the All-Russian Research Institute of Animal Health (VNIIZG), this isolate was adapted to various sensitive cell lines, including the monolayer and suspension-transplantable newborn Syrian hamster kidney cell line BHK-21/SUSP/ARRIAH, resulting in the O/Kiruhura/EA-2/2023 strain (genotype O/EA-2). The phylogenetic affiliation of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain is shown in Fig. 1.

Outbreaks of foot-and-mouth disease (FMD) genotype O/EA-2 are periodically reported in African countries. Due to the significant increase in trade and economic relations with Russia, there is a risk of FMD cases of this genotype emerging in the Russian Federation. The issue of emergency prevention of this disease to promote immunity in susceptible animals is of particular importance. Therefore, it became necessary to develop a culture-based, monovalent, inactivated emulsion vaccine against FMD genotype O/EA-2 to ensure biosecurity and veterinary safety in the Russian Federation, neighboring countries, and countries endemic for FMD, which will use this vaccine for disease prevention.

The closest to the proposed invention in terms of the set of essential features is a vaccine against foot-and-mouth disease of the O/EA-2 genotype from the O/Kenya/2017 strain, a cultural inactivated sorbed vaccine [17], containing an active substance in the form of an avirulent and purified cultural antigen material from the O/Kenya/2017 strain homologous to the causative agent of foot-and-mouth disease, obtained in a sensitive biological system, and target additives in the form of an adjuvant of aluminum hydroxide and saponin: the concentration of the 146S immunogenic component is not less than 4.0 μg in 1.0 cm ³ of the finished product, the content of aluminum hydroxide in the amount of 4.5% in terms of dry matter, saponin - 1.5 mg in 1.0 cm ³ of the finished product (prototype).

A significant drawback of the prototype vaccine is its insufficient immunogenic activity against strains/isolates of the O/EA-2 genotype foot-and-mouth disease virus that are currently circulating in East African countries, particularly in Uganda.

To address this problem, the present invention was developed, which included the development of a culture-based, monovalent, inactivated emulsion vaccine against O/EA-2 genotype foot-and-mouth disease. This vaccine contains a high content of the 146S immunogenic component of the O/Kiruhura/EA-2/2023 strain and contains the oil adjuvant Montanide ISA-61 VG.

The technical result of using the proposed invention consists in expanding the arsenal of anti-foot-and-mouth disease inactivated culture monovalent emulsion vaccines for protecting animals against isolates and strains of the foot-and-mouth disease virus of the O/EA-2 genotype, common in East Africa.

The specified technical result has been achieved by creating a culture-based monovalent inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-2 genotype, characterized by the following set of features, reflected below.

The developed vaccine in a dose of 2.0 ^cm3 contains the following main components: 1) the active substance in the form of avirulent and purified antigen material from the O/Kiruhura/EA-2/2023 strain (O/EA-2 genotype) of the foot-and-mouth disease virus homologous to the causative agent of the infection, reproduced in the transplantable suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg; 2) the oil adjuvant Montanide ISA-61 VG, which provides an enhancement of the immune response in animals with a content of 60% by weight, 3) maintenance medium - up to 2.0 ^cm3 .

The O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus is deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and Other Animal Pathogens (GKShM) of the All-Russian Research Institute of Animal Health, under the registration number: No. 523 - dep / 24-03 - GKShM of the All-Russian Research Institute of Animal Health.

The strain is adapted to primary trypsinized monolayer cells of the porcine kidney (SP) line and continuous cell lines from the kidney of a newborn Syrian hamster (BHK-21/SUSP/ARRIAH), pig kidney (IB-RS-2) and the kidney of the Siberian ibex (PSGK-30).

For vaccine production, the BHK-21/SUSP/ARRIAH cell suspension culture is preferably used as the sensitive biological system. Hanks' medium, without serum, is used as the maintenance medium, but with the addition of blood protein hydrolysate at a concentration of 5.0% of the total volume at a pH of 7.50-7.70. Virus inactivation is performed using aminoethylethyleneimine (AEEI) at a concentration of 0.035% of the virus-containing suspension volume. The inactivated virus is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.02% of the total volume.

The avirulent and purified antigen of the O/Kiruhura/EA-2/2023 strain (genotype O/EA-2) of foot-and-mouth disease virus is a suspension containing the inactivated 146S immunogenic component of the foot-and-mouth disease virus. The concentration of the component in the product is determined using a quantitative complement fixation assay (CFA) [18–20].

The vaccine is created using viral material containing at least 15.0 μg of inactivated immunogenic 146S particles of the foot-and-mouth disease virus per 2.0 ^cm³ . The stated concentration of the immunogenic component in the vaccine is achieved by concentrating the antigen using a tangential filtration unit.

The O/EA-2 genotype foot-and-mouth disease vaccine, culture-based inactivated emulsion vaccine, is obtained by mixing purified antigen and Montanide ISA-61 VG oil adjuvant in a ratio of 40% and 60% by weight, respectively.

The resulting vaccine is a stable, white or pale pink water-in-oil reverse emulsion containing the inactivated 146S component of the foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023".

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

1. Vaccine against foot-and-mouth disease genotype O/EA-2, cultural monovalent inactivated emulsion.

2. An active substance in the form of avirulent and purified antigenic material from the O/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus homologous to the causative agent of the disease in an effective amount.

3. Targeted supplements.

The essential distinguishing features of the proposed vaccine are that it contains, as an active substance, an avirulent purified antigen of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus in an effective amount.

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

1. Avirulent and purified antigen of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus, obtained in a suspension transplantable cell culture BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly the 146S immunogenic component of the foot-and-mouth disease virus in an amount of at least 15.0 μg in 2.0 ^cm3 of the finished vaccine preparation.

2. Of the target additives, the vaccine contains the oil adjuvant Montanide ISA-61 VG in an amount of 60% (by weight) in 2.0 ^cm3 of the finished product.

3. Avirulent and purified antigen of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of foot-and-mouth disease virus, obtained in the continuous suspension cell line BHK-21/SUSP/ARRIAH, oil adjuvant Montanide ISA-61 VG, additive in a dose of the finished product of 2.0 ^cm3 in the form of a maintenance medium in the following quantities:

Avirulent and purified antigen of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of foot-and-mouth disease virus not less than 15.0 mcg Oil adjuvant Montanide ISA-61 VG 60% (by weight) Supportive environment up to 2.0 cm ³

The proposed vaccine against foot-and-mouth disease of the O/EA-2 genotype, a monovalent inactivated emulsion culture vaccine, has high immunogenic activity and provides reliable protection against isolates of the O/EA-2 genotype foot-and-mouth disease virus.

The achievement of the technical result from the use of the invention is ensured by the fact that the proposed anti-foot-and-mouth disease culture inactivated emulsion vaccine contains, as an active substance, an antigen of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus, which has high immunogenic activity and creates effective protection of susceptible animals against isolates of the foot-and-mouth disease virus of the presented genotype, which have caused outbreaks worldwide in recent years.

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

Fig. 1 – Dendrogram reflecting the phylogenetic relationship of the O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus with epizootic strains of serological type A. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP gene₁.

The essence of the invention is explained by the following sequence lists:

SEQ ID NO:1 represents the nucleotide sequence of the 1D gene of the VP ₁ protein of the O/Kiruhura/EA-2/2023 strain of the foot-and-mouth disease virus of the O/EA-2 genotype;

SEQ ID NO:2 represents the amino acid sequence of the 1D gene of the VP ₁ protein of the O/Kiruhura/EA-2/2023 strain of the foot-and-mouth disease virus of the O/EA-2 genotype.

The O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The O/Kiruhura/EA-2/2023 strain of foot and mouth disease virus has the following taxonomy: sphereRiboviria, kingdomOrthornavirae, type Pisuviricota, Class Pisoniviricetes, detachmentPicornavirales, familyPicornaviridae, genus Aphthovirus, view Foot-and-mouth disease virus, serotype O, genotype O/EA-2. The pathogen strain exhibits the following morphological features characteristic of the foot-and-mouth disease pathogen: the virion is ixahedral in shape and 27 nm in size. The virion consists of a single-stranded, positively charged RNA molecule and 60 copies of a polypeptide, each of which is represented by VP proteins.₄, VP₂, VP₃, VP₁.

Antigenic properties

Antigenically, the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain belongs to serotype O. The virus is reliably neutralized by homologous antiserum and does not exhibit hemagglutinating activity. In animals that have recovered from the disease, specific antibodies are produced in the serum, detectable by enzyme-linked immunosorbent assay (ELISA) and microneutralization assay (MNA).

The primary structure of the 1D gene of the VP protein was determined using nucleotide sequencing.₁strain "O/Kiruhura/EA-2/2023" of foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "O/Kiruhura/EA-2/2023" of foot-and-mouth disease virus belongs to the O/EA-2 genotype (Fig. 1).

The antigenic relatedness (r ₁ ) of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain was studied in a virus microneutralization reaction using a cross-test of this foot-and-mouth disease pathogen strain with specific sera obtained for the following production strains of foot-and-mouth disease 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).

- strain "O/Kenya/2017" (genotype O/EA-2).

The titer of reference bovine blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype O against ¹⁰² _TCID50 of homologous and heterologous virus was determined in RMN by cross-titration, calculating the values using the linear regression equation, and expressed in lg. The _r1 value was determined as the antilogarithm of the difference in lg serum titers against heterologous and homologous virus [7, 9, 10].

The r ₁ value in the RMN was interpreted as follows:

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

at < 0.3 – the studied sample of the foot-and-mouth disease virus strain differs significantly from the production strain;

The maximum relationship is observed when the value of r ₁ in the RMN tends to 1.0.

The antigenic relatedness indices in the study of the strain "O/Kiruhura/EA-2/2023" were r ₁ from 0.01 to 0.24, which indicates the absence of obvious antigenic relatedness with industrial strains of foot-and-mouth disease virus serotype O.

Geno- and chemotaxonomic characteristics

The O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular mass of 8.08×10 ⁶ D. The nucleic acid is a single-stranded linear molecule with a molecular mass of 2.88×10 ⁶ D. The virion has a protein membrane consisting of four structural proteins VP ₁ , VP ₂ , VP ₃ and VP _4. The lipoprotein membrane is absent.

The main antigenic protein is _VP1 . The virion contains approximately 31.5% RNA and 68.5% protein. Viral RNA is infectious and participates in the formation of precursor proteins in infected cells. These precursors, in turn, are cleaved to form more stable structural and nonstructural viral polypeptides, including the important enzyme RNA-dependent RNA polymerase (3D gene), which is involved in RNA replication for virion assembly.

Physical properties

The mass of the virion is 8.47×10 ^-18 g. The buoyant density is 1.48 g/cm ³ .

Resistance to external factors

The O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain is resistant to ether, chloroform, and acetone. It is most stable at a pH of 7.44-7.69. Shifts in pH toward acidic and strongly alkaline pH inactivate the virus. It is sensitive to formaldehyde, UV radiation, γ-irradiation, and high temperatures (above 38.2 ^° C).

Additional features and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for cloven-hoofed animals.

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

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

Biotechnological characteristics

The O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus is reproduced in continuous cell cultures: Siberian ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), and Syrian hamster kidneys (BHK-21).

The O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain was tested in five consecutive passages in continuous cell cultures of PSGK-30, BHK-21, and IB-RS-2. Biological properties were characterized by determining the infectious activity of the virus at each passage in the continuous cell line IB-RS-2 and in naturally susceptible animals—cattle and pigs.

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

A safe and effective vaccine against foot-and-mouth disease of the O/EA-2 genotype, a monovalent inactivated emulsion culture vaccine, has been obtained.

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

Example 1. Genetic characteristics of the O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus based on PCR and nucleotide sequencing data.

The primary structure of the 1D gene (VP ₁ protein) of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain was analyzed using Sanger nucleotide sequencing and the position of this strain on the phylogenetic tree of foot-and-mouth disease virus serotype O was determined. The method is based on determining the primary structure of the 1D gene (VP ₁ protein) of the test isolate/strain, followed by an analysis of the phylogenetic relationship with other isolates/strains of foot-and-mouth disease virus serotype O.

A comparative analysis of the nucleotide sequences of the 1D gene encoding the VP ₁ protein of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus vaccine strain with other strains was required. The nucleotide sequence of the VP ₁ protein gene of the O/Kiruhura/EA-2/2023 strain, genotype O/EA-2 foot-and-mouth disease virus is presented in SEQ ID NO: 1. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the O/Kiruhura/EA-2/2023 strain, genotype O/EA-2 foot-and-mouth disease virus is shown in SEQ ID NO: 2.

A phylogenetic analysis was performed for the O/Kiruhura/EA-2/2023 strain. The phylogenetic tree was inferred using MEGA and the Neighbor-Joining algorithm [21]. The percentage of repeated branches in which related taxa are grouped together in a bootstrap test is shown next to the branches [22, 23]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [24] and are expressed as units of base substitutions per site. Evolutionary analysis was performed in MEGA 6 [25].

As a result of the study conducted to compare the complete nucleotide sequences of the 1D gene (VP ₁ protein) of the "O/Kiruhura/EA-2/2023" strain and other isolates/strains of foot-and-mouth disease virus serotype O, the position of the studied strain on the phylogenetic tree was determined (Fig. 1). Thus, the studied strain "O/Kiruhura/EA-2/2023" 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/Kiruhura/EA-2/2023" of the O/EA-2 genotype to the continuous monolayer culture of Siberian ibex kidney cells PSGK-30.

To infect the continuous monolayer culture of Siberian ibex kidney cells PSGK-30, a 40% aphthous suspension of foot-and-mouth disease virus obtained from pig aphthae (passage 2) was used. The inoculation cell concentration was 0.20-0.25 million cells/cm ³ , the virus infection dose was 0.001 TCID ₅₀ /cell. According to the study, the reproduction of foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" occurred with a specific destruction of the monolayer by 90-100% within 28 hours. Over the course of 6 consecutive passages, an increase in the titer of infectious activity of the virus was noted from 5.02 ± 0.10 to 7.05 ± 0.10 lg TCID ₅₀ /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of passage 6 (7.05±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from passages 1 to 6 changed from 0.18±0.02 to 0.98±0.02 μg/ ^cm3 . Moreover, the highest amount of the 146S component was noted at the stage of passage 6 (0.98±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 98.59 to 100.00%. Thus, over the course of 6 consecutive passages, the O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus was successfully adapted to the continuous monolayer cell line PSGK-30.

Example 3. Study of conditions for monolayer cultivation of foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" genotype O/EA-2 on an industrial scale.

During the industrial cultivation of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus in a continuous monolayer culture of PSGK-30 cells, the optimal conditions for culturing the virus were selected based on the supporting medium, temperature factor, and the dose of infection of the cells with the studied virus.

At the first stage of the study, the influence of the composition of the maintenance medium on the reproduction of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain in a monolayer culture of PSGK-30 cells at production scale (at the stage from the 5th to the 6th passage) was assessed. For comparison, three media with the addition of 2.0% fetal calf blood serum were used: 1) Eagle's DMEM medium; 2) RPMI-1640 medium; 3) Hanks' solution with 5% blood protein hydrolysate. The cell inoculation concentration was 0.1 million cells/cm ³ , the virus infection dose was 0.010-0.001 TCID ₅₀ /cell. Virus cultivation was carried out at temperatures of 35±0.2 - 38±0.2 ⁰ C until the cell monolayer was destroyed by at least 90%. The results of reproduction of the O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus with support media of different compositions are shown in Table 1.

The table data shows that during the reproduction of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain in a monolayer culture of PSGK-30 cells using support media of different compositions, the optimal medium is Hanks' medium, which allows achieving specific destruction of the cell monolayer by 95-100% in 22 hours with the accumulation of the 146S component in the resulting suspension in an amount of 0.98±0.02 μg/ ^cm3 and an infectious activity titer of 7.05±0.10 lg _TCID50 / ^cm3 . When using RPMI-1640 and Eagle's DMEM medium, the virus reproduction rates were lower.

At the next stage of studying the conditions of monolayer cultivation of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus strain in PSGK-30 cell culture under production conditions, the effect of the temperature factor on the virus reproduction process was assessed. For this purpose, the virus was cultivated in Hanks' medium at the following temperatures: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2 ⁰ C. The dose of infection of the cell culture with the foot-and-mouth disease virus was 0.010-0.001 TCID ₅₀ /cell. The virus was cultivated until the cell monolayer was destroyed by 90-100% for 22-28 hours (Table 1). The results of the study revealed that for the complete reproduction of the O/Kiruhura/EA-2/2023 strain of foot and mouth disease virus in a monolayer culture of PSGK-30 cells, the optimal temperature is 37.0±0.2 ⁰ C, at which, in 22 hours, the development of CPE reached 95-100% with the accumulation of 146S particles equal to 0.98±0.02 μg/cm ³ and the titer of infectious activity of the virus of 7.05±0.10 lg TCID ₅₀ /cm ³ .

The effect of the infection dose of the PSGK-30 cell line monolayer with the O/Kiruhura/EA-2/2023 strain was assessed during cultivation on a production scale. Different virus doses were used for this purpose: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCID ₅₀ /cell. Hanks' medium was used as a maintenance medium. Virus reproduction was carried out at a temperature of 37.0±0.2 ⁰ C for 22-32 h until specific cell destruction by 90-100%. Based on the results of cultivation, the concentration of the 146S component and the titer of infectious activity of the foot-and-mouth disease virus were determined. As follows from the data in Table 1, the highest accumulation of the 146S virus component was achieved at an infection dose of 0.01-0.001 TCID ₅₀ /cell. and amounted to 0.98±0.02 μg/cm ³ with a titer of infectious activity of the virus of 7.05±0.10 lg TCID ₅₀ /cm ³ .

Thus, the conditions of monolayer cultivation of the foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" in the PSGK-30 cell culture were studied on an industrial scale. As a result of the study, the following optimal parameters for the reproduction of the foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" in the monolayer PSGK-30 cell line were determined: 1) maintenance medium - Hanks'medium; 2) cultivation temperature - 37.0±0.2 ⁰ C; 3) virus infection dose - 0.01-0.001 TCID ₅₀ /cell.

Example 4. Study of the conditions of suspension cultivation of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of foot-and-mouth disease virus on an industrial scale.

During industrial cultivation of the strain "O/Kiruhura/EA-2/2023" genotype O/EA-2 of the foot-and-mouth disease virus in a continuous suspension culture of BHK-21/SUSP/ARRIAH cells, a search was conducted for optimal parameters for the successful reproduction of the foot-and-mouth disease pathogen, using different cell concentrations, temperature conditions and the dose of virus infection.

At the first stage of the work, the influence of the seeding concentration of BHK-21/SUSP/ARRIAH cell line in suspension on the reproduction of the O/Kiruhura/EA-2/2023 strain was determined. foot-and-mouth disease virus on an industrial scale. For analysis, suspensions with the following cell concentrations were prepared: 3.5; 4.0; 4.5; 5.0 million cells/cm³The virus cultivation temperature was 37.0±0.2⁰C, the dose of virus infection is 0.010-0.001 TCD₅₀/cell. Virus replication was carried out until specific cell death was 95-100%. Results of suspension cultivation of the strain "O/Kiruhura/EA-2/2023" The results of the analysis of the results of the cell suspensions of the foot-and-mouth disease virus are presented in Table 2.

When cultivating the foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" in the suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations, it was determined that the accumulation of the 146S component in a suspension with a cell concentration of 3.5 million cells/cm³amounted to 0.65±0.02 μg/cm³, with a concentration of 4.0 million cells/cm³– 1.30±0.02 μg/cm³, with a concentration of 4.5 million cells/cm³– 1.54±0.02 μg/cm³and with a concentration of 5.0 million cells/cm³– 1.62±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "O/Kiruhura/EA-2/2023" For the foot-and-mouth disease virus, a sufficient concentration of BHK-21/SUSP/ARRIAH cells in suspension is 4.5 million cells/cm³(at a concentration of 5.0 million cells/cm³the concentration is slightly higher, but the production costs per number of cells are higher, based on this, it is economically feasible to use a cell concentration equal to 4.5 million cells/cm³). The titer of infectious activity of the virus was 8.12±0.10 lg TCD₅₀/cm³.

At the next stage of the work, the influence of temperature on the reproduction of the strain "O/Kiruhura/EA-2/2023" was investigated. foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells on an industrial scale. For this purpose, virus reproduction was carried out under the following temperature conditions: 35.0±0.2; 36.0±0.2; 37.0±0.2; 38.0±0.2⁰C. The dose of infection of cell culture with the virus was 0.010-0.001 TCID₅₀/cell. Virus cultivation was carried out until the CPE was at least 85% (priority was given to complete specific cell destruction). Based on the analysis results, it was determined that for full reproduction of the strain "O/Kiruhura/EA-2/2023" for the foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells, the optimal temperature is 37.0±0.2⁰C, at which within 13 hours specific cell death is observed by 95-100% with a high accumulation of the 146S component (1.54±0.02 μg/cm³) and the titer of infectious activity of the virus is 8.12±0.10 lg TCID₅₀/cm³.

In the course of studying the conditions of suspension cultivation of the strain "O/Kiruhura/EA-2/2023" The influence of the infection dose on the cells was assessed in the industrial scale production of foot-and-mouth disease virus using the BHK-21/SUSP/ARRIAH cell culture. For this purpose, cell suspensions were infected with the virus at different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD₅₀/cell. Virus reproduction was carried out at a temperature of 37.0±0.2⁰From a cytopathic effect of at least 90%. Based on the results of cultivation, the concentration of 146S particles and the titer of infectious activity of the foot-and-mouth disease virus were determined. As can be seen from the data in Table 2, the highest accumulation of the 146S component was observed at an infection dose of 0.010-0.001 TCID.₅₀/cl. (1.54±0.02 µg/cm³) with a titer of infectious activity of the virus equal to 8.12±0.10 lg TCID₅₀/cm³.

Thus, a study of three parameters of suspension cultivation of the strain "O/Kiruhura/EA-2/2023" was carried out. genotype O/EA-2 of the foot-and-mouth disease virus in the BHK-21/SUSP/ARRIAH suspension cell line on an industrial scale. Optimal conditions for the reproduction of the O/Kiruhura/EA-2/2023 strain in the BHK-21/SUSP/ARRIAH suspension cell culture were identified: 1) cell concentration before infection – 4.5 million cells/cm³; 2) cultivation temperature regime – 37.0±0.2⁰C; 3) the dose of virus infection is 0.010-0.001 TCD₅₀/cl.

Example 5. Inactivation of a suspension of foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" genotype O/EA-2.

At the end of the reproduction cycle of the foot-and-mouth disease virus, without stopping the thermostatting process (37.0 ± 0.2 ⁰ C), an acidified solution of aminoethylethyleneimine (AEEI) with a pH of 8.2-8.5 was added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to 0.035%. Inactivation of the infectious activity of the foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" genotype O/EA-2 was carried out for 12 hours at a temperature of 37.0 ± 0.1 ⁰ C with periodic stirring.

To determine the time of complete inactivation after the addition of 1,2-AEEI, samples were collected every hour. The resulting samples were tested for the presence of FMD virus infectivity in a primary culture of SP pig kidney cells. The results are presented in Table 3, which demonstrates that the inactivation of the O/Kiruhura/EA-2/2023 FMD virus strain, reproduced in culture vessels, was as low as 0.0 lg TCID.₅₀/ml occurred 6 hours after the introduction of 1,2-AEEI at a dose of 0.035%, and to a titer of -10.50 lg TCD₅₀/ml – after 12 hours of incubation (Table 3).

The prepared inactivated viral suspensions were analyzed in the RSC to assess their viral component content. The concentration of the 146S component was 1.51±0.02 μg/ ^cm3 .

Example 6. Selection of conditions for purification of a suspension of foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" genotype O/EA-2.

A suspension of the O/Kiruhura/EA-2/2023 foot-and-mouth disease virus (FMDV) strain, genotype O/EA-2, obtained by propagation in the BHK-21/SUSP/ARRIAH suspension cell line, was purified. Polyhexamethylene guanidine (PHMG) at concentrations of 0.015, 0.020, and 0.025% was used to obtain purified FMDV antigen. Before and after the O/Kiruhura/EA-2/2023 FMDV antigen purification process. In the quantitative version of the RSC, the concentration of total viral protein and immunogenic components was determined. The results of the analysis are presented in Table 4, which shows that as a result of the purification of the foot-and-mouth disease virus antigen of the O/Kiruhura/EA-2/2023 strain Using PHMG at a concentration of 0.015%, a decrease in the concentration of ballast protein by 6% and the 146S component by 0.7% was observed. When using PHMG at a concentration of 0.020%, a decrease in the amount of ballast protein by 15% and the 146S component by 1.3% was observed. Using PHMG at a concentration of 0.025%, the content of ballast protein by 29% and immunogenic components by 17%.

Thus, having studied the conditions for purifying the antigen of the strain “O/Kiruhura/EA-2/2023”, we came to the conclusion that in order to obtain a purified product of this antigen material, it is optimal to use PHMG with a concentration of 0.020%.

Example 7. Selection of conditions for concentrating the antigen suspension of the O/Kiruhura/EA-2/2023 strain of foot-and-mouth disease virus genotype O/EA-2.

The culture inactivated suspension of the O/Kiruhura/EA-2/2023 strain obtained using the BHK-21/SUSP/ARRIAH suspension cell line was concentrated 14 times by volume. To increase the concentration of the immunogenic components of the foot-and-mouth disease virus antigen, the following techniques 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) at a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 12 hours; 3) concentration using a tangential filtration unit for 3.5 hours at an operating pressure on the filter of 2.2-2.3 atm.

Before and after the process of concentrating the antigen suspension of foot-and-mouth disease virus strain "O/Kiruhura/EA-2/2023" The concentration of total viral protein and immunogenic components was determined in the quantitative version of the CSC. The results of the studies are presented in Table 5, which shows that when concentrating the antigen of the strain "O/Kiruhura/EA-2/2023" A 4-hour concentration of PEG-6000-treated foot-and-mouth disease virus revealed a 12-fold increase in component content compared to baseline (before concentration). Using the same polymer but increasing the exposure time to 12 hours, the viral component concentration was increased by 12.5 times. Using a tangential filtration concentration method, it was possible to increase the amount of immunogenic components of the O/Kiruhura/EA-2/2023 strain antigen. The foot-and-mouth disease virus in suspension was 13.9 times more abundant. Thus, tangential filtration technology made it possible to obtain the O/Kiruhura/EA-2/2023 strain antigen with a high concentration of structural viral proteins.

Example 8. Selection of adjuvant and antigen-adjuvant ratio.

For the production of a monovalent emulsion vaccine against foot-and-mouth disease from the antigen of the strain "O/Kiruhura/EA-2/2023" The oil adjuvant Montanide ISA-61 VG was selected as an adjuvant in an amount of 60% by weight.

Example 9. Composition of a vaccine against foot-and-mouth disease from the strain "O/Kiruhura/EA-2/2023" of the genotype O/EA-2, cultural monovalent inactivated emulsion.

A monovalent inactivated emulsion culture vaccine against foot-and-mouth disease of the O/EA-2 genotype (the proposed invention) was prepared from the obtained antigen concentrate of the O/Kiruhura/EA-2/2023 strain of the foot-and-mouth disease virus using the Montanide ISA-61 VG oil adjuvant. The amount of 146S immunogenic component in 1.0 cm³antigen amounted to 20.99±0.02 μg/cm³, and in a dose (2.0 cm³) – 16.79 mcg.

Example 10. Immunization of animals with a culture-based inactivated emulsion vaccine against foot-and-mouth disease of the O/EA-2 genotype .

A series of dilutions for pigs with a 5-fold step were prepared from the obtained O/Kiruhura/EA-2/2023 strain, genotype O/EA-2, culture-inactivated emulsion vaccine (the proposed invention) . 15 pigs were divided into 3 groups of 5 pigs each. The immunizing dose was 2.0 ^cm3 . The first group of animals (Nos. 1-5) was immunized with the vaccine without dilution, the second group of pigs (Nos. 6-10) were vaccinated with the vaccine diluted 1/5, and the third group of animals (Nos. 11-15) – with the vaccine diluted 1/25. The preparation was administered intramuscularly in the middle third of the neck. Two unvaccinated pigs were left as a virus control.

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

Blood sera from pigs obtained before immunization and 14 days after immunization were tested 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 OIE recommendations [3]. Testing of the obtained sera was carried out using the ELISA test system "Prio CHECK ^® FMDV NSP ELISA for in vitro detection of antibodies against Foot and Mouth Disease Virus in serum of cattle, sheep and pigs" (Prionics Lelystad BV, the Netherlands). The obtained research results are presented in Table 6, which shows that the animal blood sera did not contain antibodies to non-structural proteins of the foot and mouth disease virus (PI values for pig blood sera < 50%).

Example 12. Evaluation of the avirulence and harmlessness of the O/EA-2 genotype culture-based inactivated emulsion vaccine against foot-and-mouth disease (proposed invention) .

To determine the vaccine's avirulence in pigs, a sample of the vaccine preparation was administered intradermally at a dose of 0.1 ^cm3 . Pigs weighing 30-40 kg were used in the study. Over a 10-day observation period, the animals remained clinically healthy, and pathological examination revealed no changes characteristic of foot-and-mouth disease. This indicated the lack of virulence properties of the developed vaccine.

The safety of the product was tested in animals by intramuscular administration of the vaccine at a dose of 6.0 ^cm3 (a triple dose of 2.0 ^cm3 ). The observation period also lasted 10 days. It should be noted that after immunization, the animal's body temperature may rise to 40.3 ^° C and remain at this level for 1-2 days, which is within the normal range after administration of the vaccine.

Based on the results of the studies, the O/EA-2 genotype monovalent inactivated emulsion culture vaccine against foot-and-mouth disease (the proposed invention) was found to be harmless; all animals remained clinically healthy during the observation period; no tissue necrosis was detected at the site of vaccine administration during the pathological analysis.

Example 13. Study of the immunogenic properties of the O/EA-2 genotype culture-based inactivated emulsion vaccine against foot-and-mouth disease (proposed invention) for the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 21st day after the administration of the O/Kiruhura/EA-2/2023 strain, genotype O/EA-2, culture-inactivated emulsion vaccine against foot-and-mouth disease (the proposed invention) , blood was collected from the pigs and the obtained blood sera were tested in the microneutralization reaction [3]. It was found that in pigs immunized with the vaccine in a whole dose (5 heads), the average antibody titer values were 8.36±0.21 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.60±0.14 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 3.92±0.12 log ₂ SN ₅₀ (Table 7). The obtained data from the microneutralization reaction indicate that after the administration of the vaccine in its whole form, 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. Study of the protective capacity of the O/EA-2 genotype culture-inactivated emulsion vaccine against foot-and-mouth disease (proposed invention) by the method of challenge infection of naturally susceptible animals.

Fifteen pigs immunized with the whole and diluted vaccines were challenged with the O/Kiruhura/EA-2/2023 control strain of the O/EA-2 genotype of foot-and-mouth disease virus adapted to these animals at a dose of ^104.0 _ID50 /0.20 ^cm3 (in two spots of 0.10±0.05 ^cm3 ). Seven days after challenge, all animals were euthanized and underwent a postmortem examination. Animals with no lesions on their extremities were considered protected from foot-and-mouth disease. Primary aphthae were not taken into account.

The results of the challenge infection are presented in Table 7, from which it follows that the vaccine against foot-and-mouth disease from the strain "O/Kiruhura/EA-2/2023" of the genotype O/EA-2, culture-based monovalent inactivated emulsion (the proposed invention) in whole form, as well as in dilutions of 1/5 and 1/25, administered in a single dose (2.0 ^cm3 ) protects pigs from infection with the homologous strain of all animals (5 out of 5 heads).

Based on the results of the control infection, it was established that the inoculation volume of this vaccine contains 55.90 PD ₅₀ and 0.04 IMD ₅₀ for pigs.

Thus, the above information indicates that the O/EA-2 genotype foot-and-mouth disease vaccine, a monovalent inactivated emulsion culture vaccine embodying the proposed invention, is intended for use in agriculture, namely in veterinary virology and biotechnology; the possibility of implementing the presented is confirmed; the vaccine is made from the strain "O/Kiruhura/EA-2/2023" genotype O/EA-2 in accordance with the proposed invention, has high immunogenic activity and is capable of providing effective protection to susceptible animals against the epizootic strain of foot-and-mouth disease virus of genotype O/EA-2, in particular, currently in East Africa.

Sources of information

1. Foot-and-mouth disease. Preventive measures. URL: http://89.rospotrebnadzor.ru/directions/epid_nadzor/146902 (Accessed: 28.09.2024).

2. The danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Accessed: 23.09.2024).

3. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed. - Paris, 2022 – Vol. 1, Chapter 2.1.5. – P. 166-169.

4. Burdov A. N., Dudnikov A. I., Malyarets P. V., et al. Foot-and-mouth disease. – Moscow: Agropromizdat, 1990. – 320 p.

5. Ponomarev A. P., Uzyumov V. L., Gruzdev K. N. Foot-and-mouth disease virus: structure, biological and physicochemical properties. - Vladimir: Foliant, 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. Mugezi I, Kimaanga M, Namwabira A, Chevanne E, Nekouei O, Mclaws M, Motta P, Dulu T, Sumption K. Risk of foot and mouth disease spread through cattle movements in Uganda. Rev Sci Tech. 2020 Jan;39(3):847-861. English. doi: 10.20506/rst.39.3.3182. PMID: 35275131.

8. González-Gordon L, Porphyre T, Muwonge A, Nantima N, Ademun R, Ochwo S, Mwiine NF, Boden L, Muhanguzi D, Bronsvoort BMC. Identifying target areas for risk-based surveillance and control of transboundary animal diseases: a seasonal analysis of slaughter and live-trade cattle movements in Uganda. Sci Rep. 2023 Oct 30;13(1):18619. doi:10.1038/s41598-023-44518-4. PMID: 37903814; PMCID: PMC10616094.

9. Muleme M, Barigye R, Khaitsa ML, Berry E, Wamono AW, Ayebazibwe C. Effectiveness of vaccines and vaccination programs for the control of foot-and-mouth disease in Uganda, 2001-2010. Trop Anim Health Prod. 2013 Jan;45(1):35-43. doi:10.1007/s11250-012-0254-6. Epub 2012 Sep 7. PMID: 22956440.

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/fmd_ref_lab_reports.htm (Accessed 09/28/2024).

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. – R. 746-754.

13. Rakhmanov A.M. Current epizootic situation in the world regarding foot-and-mouth disease and measures for its control // Veterinary medicine, 2013. – P. 37-38.

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

15. Melnik R.N., Khaustova N.V., Melnik N.V., Samoylenko 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 production strains / Veterinary science 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. B (2009) 364. – pp. 2657–2667.

17. Russian Federation Patent No. 2 802 192, March 21, 2023. Culture-based inactivated sorbed vaccine against foot-and-mouth disease of the O/EA-2 genotype from the O/Kenya/2017 strain // Application No. 2023106752 / Doronin M.I., Mikhalishin D.V., Borisov A.V., Lugovskaya N.N., Nikiforov V.V., Fomina S.N.

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

19. Methodological recommendations for determining the concentration of 146S, 75S, 12S components of vaccine strains of culture foot-and-mouth disease virus in the complement fixation reaction (CFR) / FGBU "ARRIAH". - Approved. 21.09.17.

20. Guidelines for determining the antigenic match between epizootic isolates and production strains of foot-and-mouth disease virus in a cross-microneutralization reaction: approved by Rosselkhoznadzor on September 13, 2017 / FGBU "ARRIAH". - Vladimir: 2017. - 24 p.

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

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

23. 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.

24. 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.

25. 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 (3)

1. A monovalent inactivated emulsion culture vaccine against foot-and-mouth disease of the O/EA-2 genotype, containing an active substance and an adjuvant, characterized in that it contains, as an active substance, avirulent and purified antigen material from the causative agent of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and Other Animal Pathogens (GKShM) of the FGBU VRIAH, under the registration number: No. 523 - dep / 24-03 - GKShM of the FGBU VRIAH, reproduced in the continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg in a vaccine dose of 2.0 ^cm3 ; as an adjuvant it contains the oil adjuvant Montanide ISA-61 VG with a content of 60% by weight and a supporting medium up to 2.0 cm ³ . 2. The vaccine according to claim 1, characterized in that it contains predominantly the 146S immunogenic component of the foot-and-mouth disease virus of genotype O/EA-2. 3. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigen material from the causative agent of the infection of the O/Kiruhura/EA-2/2023 strain of the O/EA-2 genotype of the foot-and-mouth disease virus and the oil adjuvant Montanide ISA-61 VG in an effective ratio of 40% and 60% by weight, respectively.