RU2847814C1 - Vaccine against foot-and-mouth disease genotype a/africa/g-i, cultured 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 A/AFRICA/G-I, 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 to susceptible animals against the epizootic strain of foot-and-mouth disease virus genotype A/AFRICA/G-I. The vaccine against foot-and-mouth disease genotype A/AFRICA/G-I is a cultured monovalent inactivated emulsion vaccine manufactured using the oil adjuvant Montanide ISA-61 VG. Based on the results of a control infection, it was established that the vaccine contains 55.90 PD₅₀ and 0.04 ImD₅₀ for pigs.

EFFECT: high efficacy of the vaccine.

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 A/AFRICA/G-I genotype.

Foot-and-mouth disease causes enormous economic losses to the livestock sectors in endemic countries due to trade restrictions on livestock products, reduced animal productivity, and high mortality [1].

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

There are seven serotypes of foot-and-mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, with serotype A being the most common, including in the African continent, particularly in Uganda.

Most animals infected with foot-and-mouth disease are found in Asia, Africa, the Middle East, and Venezuela in South America. The distribution of foot-and-mouth disease is characterized by seven geographic endemic groups, each containing distinct viral lineages: Group 1 - East Asia, Group 2 - South Asia, Group 3 - West Eurasia, Group 4 - East Africa, Group 5 - West Africa, Group 6 - Southern Africa, and Group 7 - Southern Africa/America.

Foot-and-mouth disease virus serological types are divided into more than 60 genotypes, 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 viral attachment and penetration, immune defense, and serotype specificity.

By modeling the evolution of nucleotide sequences derived from the _VP1 coding region, it is possible to define FMDV serotypes, topotypes (≈15% divergence), genetic lineages (≈10% divergence), and sublineages (≈5% divergence) based on genetic similarity thresholds.

Recovery of an animal after foot-and-mouth disease with one genotype does not provide immune protection against another [3-5].

Since vaccination is used as the primary control tool in endemic areas, by analyzing each pool and the genotypes within it, it is possible to select the most specific vaccines that match the topotypes present in that pool, rather than continuing to rely on the more widely available genetic vaccines.

Foot-and-mouth disease virus (FMD) isolates of serotype A exhibit the greatest genetic diversity. Serotype A includes three characterized genotypes: ASIA, AFRICA, and EURO-SA, as well as one poorly studied group of isolates [2, 5], which poses challenges for the specific prevention of FMD using culture-based inactivated FMD vaccines. Consequently, the development of new specific prophylactic agents against FMD serotype A is necessary [1, 6, 7].

To ensure biological security in the Russian Federation, a range of measures are being implemented to combat and prevent foot-and-mouth disease. These measures are aimed at preventing the introduction of the virus into the country, systematically vaccinating animals in the buffer zone, and monitoring the immune status of cattle and pigs, for which culture-based inactivated vaccines against foot-and-mouth disease are used.

To immunize animals, a vaccine made from a virus homologous to field isolates should be used, which has been confirmed by research by many scientists [6, 7, 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 A poses challenges in the specific prevention of foot-and-mouth disease using culture-based inactivated foot-and-mouth disease vaccines, and also complicates strain-specific diagnostics of isolated foot-and-mouth disease virus isolates. This necessitates the development of new diagnostic tools and specific immunoprophylaxis against foot-and-mouth disease virus serotype A, particularly genotype A/AFRICA/G-I, whose representatives have spread throughout Africa [3].

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

For many years, outbreaks of foot-and-mouth disease serotype A have been periodically recorded in African countries, in particular in the Democratic Republic of the Congo in 2011, in Kenya in 2021, in Burundi in August 2022, and in the Republic of Uganda in 2023. The spread of foot-and-mouth disease genotype A/AFRICA/G-I is dangerous and requires research into the strains of this genotype to develop diagnostic tools and specific prevention measures for foot-and-mouth disease of this genotype.

There are known industrial strains of foot-and-mouth disease virus serotype A, which are used for the production of specific preventive agents for foot-and-mouth disease:

- strain "A ₂₂ /Iraq/64" (genotype A/ASIA/Iraq-64),

- strain "A/Türkiye/06" (genotype A/ASIA/Iran-05),

- strain "A/Zabaikalsky/2013" (genotype A/ASIA/Sea-97),

- strain "A No. 2269/VNIIZh/2015" (genotype A/ASIA/G-VII),

- strain “A No. 2205/G-IV/2018” (genotype A/AFRICA/G-IV),

- strain “A/Tanzania/2013” (genotype A/AFRICA/G-I).

The foot-and-mouth disease virus isolate was isolated from cattle in the Republic of Uganda in 2023 and was submitted to the All-Russian Research Institute of Animal Health for scientific research in 2023.

The strain "A / Kiruhara / 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 genotype A/AFRICA/G-I, which differs significantly from the production strains of foot-and-mouth disease virus serotype A, including the strain “A/Tanzania/2013”.

At the All-Russian Research Institute of Animal Health (VNIIZH), 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 "A/Kiruhara/2023" strain (genotype A/AFRICA/G-I). The phylogenetic affiliation of the "A/Kiruhara/2023" foot-and-mouth disease virus strain is shown in Fig. 1.

With the expansion of trade and economic relations between Russia and African countries, where outbreaks of foot-and-mouth disease (FMD) of the A/AFRICA/G-I genotype are periodically reported, the risk of FMD cases of this genotype emerging in the Russian Federation has increased. The issue of emergency prevention of this disease to promote immunity in susceptible animals has become particularly important. Therefore, it has become necessary to develop a culture-based, monovalent, inactivated emulsion vaccine against FMD of the A/AFRICA/G-I genotype to ensure the biological safety and veterinary well-being of 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 A/AFRICA/GI genotype from the strain "A/Tanzania/2013", a cultural inactivated sorbed vaccine [17], containing an active substance in the form of avirulent and purified cultural antigen material from the homologous pathogen of the foot-and-mouth disease strain "A/Tanzania/2013", 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 foot-and-mouth disease virus of the A/AFRICA/G-I genotype, which are currently circulating in East African countries, in particular in Uganda.

To address this problem, the present invention was developed, which included the development of a culture-based, monovalent, inactivated emulsion vaccine against genotype A/AFRICA/G-I foot-and-mouth disease. This vaccine contains a high content of the 146S immunogenic component of the "A/Kiruhara/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 A/AFRICA/G-I genotype, common in East Africa.

The specified technical result has been achieved by creating a vaccine against foot-and-mouth disease of the A/AFRICA/G-I genotype, a monovalent inactivated emulsion culture vaccine, 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 homologous to the causative agent of the infection strain "A / Kiruhara / 2023" (genotype A / AFRICA / GI) of the foot-and-mouth disease virus, reproduced in the transplantable suspension cell line BHK-21 / SUSP / ARRIAH, in an amount of at least 15.0 μg; 2) oil adjuvant Montanide ISA-61 VG, providing an increase in the immune response in animals with a content of 60% by weight, 3) maintenance medium - up to 2.0 ^cm3 .

The strain "A / Kiruhara / 2023" of the 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 FGBU "ARRIAH", under the registration number: No. 524 - dep / 24-04 - GKShM of the FGBU "ARRIAH".

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 0.025% 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 0.035% of the total volume.

The avirulent and purified antigen of the A/Kiruhara/2023 strain (genotype A/AFRICA/G-I) 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 vaccine against foot-and-mouth disease genotype A/AFRICA/G-I, a monovalent inactivated emulsion culture vaccine, is obtained by mixing the purified antigen and the oil adjuvant Montanide ISA-61 VG 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 "A/Kiruhara/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 A/AFRICA/G-I, cultural monovalent inactivated emulsion.

2. An active substance in the form of avirulent and purified antigenic material from the strain "A / Kiruhara / 2023" of the genotype A / AFRICA / G-I 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 A/Kiruhara/2023 strain of the A/AFRICA/G-I 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. An avirulent and purified antigen of the A/Kiruhara/2023 strain of the A/AFRICA/G-I genotype of the foot-and-mouth disease virus, obtained in the suspension-transplantable cell line BHK-21/SUSP/ARRIAH and representing a suspension containing predominantly the 146S immunogenic component of the foot-and-mouth disease virus in an effective amount.

2. Avirulent and purified antigen of the strain "A / Kiruhara / 2023" of the genotype A / AFRICA / GI 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 cm ³ of the finished vaccine preparation.

3. Among the target additives, the vaccine contains the oil adjuvant Montanide ISA-61 VG.

4. 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.

5. Avirulent and purified antigen of the strain "A / Kiruhara / 2023" of the genotype A / AFRICA / GI of the 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 cm ³ in the form of a maintenance medium in the following quantities:

Avirulent and purified antigen of the strain "A / Kiruhara / 2023" genotype
A/AFRICA/GI 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 A/AFRICA/G-I genotype, a monovalent inactivated emulsion culture vaccine, has high immunogenic activity and provides reliable protection against isolates of the A/AFRICA/G-I 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 strain "A / Kiruhara / 2023" of the genotype A / AFRICA / G-I of the foot-and-mouth disease virus, which has high immunogenic activity, creating effective protection of susceptible animals against isolates of the foot-and-mouth disease virus of the presented genotype, which in recent years have caused outbreaks around the world.

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

Fig. 1 - Dendrogram reflecting the phylogenetic relationship of the A/Kiruhara/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 A/Kiruhara/2023 strain of foot and mouth disease virus genotype A/AFRICA/GI;

SEQ ID NO:2 represents the amino acid sequence of the 1D gene of the VP ₁ protein of the A/Kiruhara/2023 strain of foot and mouth disease virus genotype A/AFRICA/GI.

The A/Kiruhara/2023 strain of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The A/Kiruhara/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 A, genotype A/AFRICA/G-I. The pathogen strain exhibits the following morphological features characteristic of the foot-and-mouth disease pathogen: the virion is icosahedral in shape and 29 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 A/Kiruhara/2023 foot-and-mouth disease virus strain belongs to serotype A. 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 "A/Kiruhara/2023" of the foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "A/Kiruhara/2023" of the foot-and-mouth disease virus belongs to the genotype A/AFRICA/G-I (Fig. 1).

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

- strain "A ₂₂ /Iraq/64" (genotype A/ASIA/Iraq-64),

- strain "A/Türkiye/06" (genotype A/ASIA/Iran-05),

- strain "A/Zabaikalsky/2013" (genotype A/ASIA/Sea-97),

- strain "A No. 2269/VNIIZZH/2015" (genotype A/ASIA/G-VII),

- strain “А№ 2205/G-IV/2018” (genotype A/AFRICA/G-IV),

- strain “A/Tanzania/2013” (genotype A/AFRICA/G-I).

The titer of reference bovine blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype A 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, 8, 9].

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 A/Kiruhara/2023 strain were r ₁ from 0.04 to 0.25, which indicates the absence of obvious antigenic relatedness with industrial strains of foot-and-mouth disease virus serotype A.

Geno- and chemotaxonomic characteristics

The A/Kiruhara/2023 strain of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular mass of 8.09×10 ⁶ D. The nucleic acid is a single-stranded linear molecule with a molecular mass of 2.90×10 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 31.0% RNA and 69.0% 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 8.49×10 ^-18 g. The buoyant density is 1.49 g/cm ³ .

Resistance to external factors

The A/Kiruhara/2023 strain of foot-and-mouth disease virus is resistant to ether, chloroform, and acetone. It is most stable at a pH of 7.45-7.70. 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 38.0°C).

Additional features and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for ungulates.

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 A/Kiruhara/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 test involved five consecutive passages of the A/Kiruhara/2023 foot-and-mouth disease virus strain 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 A/AFRICA/G-I 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 genotype A/AFRICA/G-I, 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 A/Kiruhara/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 A/Kiruhara/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 A 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 A.

A comparative analysis of the nucleotide sequences of the 1D gene encoding the VP ₁ protein of the A/Kiruhara/2023 foot-and-mouth disease virus vaccine strain was required. The nucleotide sequence of the VP ₁ protein gene of the A/Kiruhara/2023 strain, genotype A/AFRICA/GI 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 A/Kiruhara/2023 strain, genotype A/AFRICA/GI foot-and-mouth disease virus is shown in SEQ ID NO: 2.

A phylogenetic analysis was performed for the A/Kiruhara/2023 strain. The phylogenetic tree was inferred using MEGA and the Neighbor-Joining algorithm [21]. The percentage of repetitive 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 "A/Kiruhara/2023" strain and other isolates/strains of foot-and-mouth disease virus serotype A, the position of the studied strain on the phylogenetic tree was determined (Fig. 1). Thus, the studied strain "A/Kiruhara/2023" belongs to the A/AFRICA/GI genotype, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of the strain “A/Kiruhara/2023” of the genotype A/AFRICA/GI to the transplantable monolayer culture of Siberian ibex kidney cells PSGK-30.

To infect the continuous monolayer culture of Siberian ibex kidney cells PSGK-30, a 15% 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 "A / Kiruhara / 2023" occurred with a specific destruction of the monolayer by 90-100% in 23 hours. Over the course of 6 consecutive passages, an increase in the titer of infectious activity of the virus was noted from 5.56 ± 0.10 to 7.47 ± 0.10 lg TCID ₅₀ /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of passage 6 (7.47±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from passages 1 to 6 changed from 0.26±0.02 to 1.35±0.02 μg/ ^cm3 . Moreover, the highest amount of the 146S component was noted at the stage of passage 6 (1.35±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 99.03 to 100.00%. Thus, over the course of 6 consecutive passages, the adaptation of the foot and mouth disease virus strain "A / Kiruhara / 2023" to the continuous monolayer cell line PSGK-30 was successfully carried out.

Example 3. Study of conditions for monolayer cultivation of foot-and-mouth disease virus strain "A/Kiruhara/2023" genotype A/AFRICA/GI on an industrial scale.

During the industrial cultivation of the A/Kiruhara/2023 strain of the A/AFRICA/G-I 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 A/Kiruhara/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 medium DMEM; 2) RPMI-1640 medium; 3) Hanks' solution with 5% blood protein hydrolysate. The cell inoculation concentration was 0.1 million cells/ ^cm3 , the virus infection dose was 0.010-0.001 _TCID50 /cell. Virus cultivation was carried out at temperatures of 35±0.2 - 38±0.2°C until the cell monolayer was destroyed by at least 90%. The results of reproduction of the A/Kiruhara/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 A/Kiruhara/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 19 hours with the accumulation of the 146S component in the resulting suspension in an amount of 1.35±0.02 μg/ ^cm3 and an infectious activity titer of 7.47±0.10 lg _TCID50 / ^cm3 . When using RPMI-1640 and Eagle DMEM medium, the virus reproduction rates were lower.

At the next stage of studying the conditions of monolayer cultivation of the foot-and-mouth disease virus strain "A / Kiruhara / 2023" in the 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 °C. The dose of infection of the cell culture with the foot-and-mouth disease virus was 0.010-0.001 TCID ₅₀ /cell. Virus cultivation was carried out until the cell monolayer was destroyed by 90-100% for 19-24 hours (Table 1). The results of the study revealed that for the complete reproduction of the A/Kiruhara/2023 strain of foot-and-mouth disease virus in a monolayer culture of PSGK-30 cells, the optimal temperature is 37.0±0.2°C, at which, within 19 hours, the development of CPE reached 95-100% with the accumulation of 146S particles equal to 1.35±0.02 μg/ ^cm3 and the titer of infectious activity of the virus of 7.47±0.10 lg _TCID50 / ^cm3 .

The effect of the infection dose of the PSGK-30 cell line monolayer with the A/Kiruhara/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°C for 19-28 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 1.35±0.02 μg/cm ³ with a titer of infectious activity of the virus of 7.47±0.10 lg TCID ₅₀ /cm ³ .

Thus, the conditions of monolayer cultivation of the foot-and-mouth disease virus strain "A / Kiruhura / 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 "A / Kiruhura / 2023" in the monolayer PSGK-30 cell line were determined: 1) maintenance medium - Hanks'medium; 2) cultivation temperature - 37.0 ± 0.2 ° C; 3) virus infection dose - 0.01-0.001 TCID ₅₀ / cell.

Example 4. Study of the conditions of suspension cultivation of the strain “A/Kiruhara/2023” of the genotype A/AFRICA/GI of the foot-and-mouth disease virus on an industrial scale.

During industrial cultivation of the strain “A / Kiruhara / 2023” genotype A/AFRICA/G-I 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 strain "A/Kiruhara/2023" was determined. foot-and-mouth disease virus on an industrial scale. For analysis, suspensions with the following cell concentrations were prepared: 3.0; 3.5; 4.0; 4.5 million cells/cm³The virus cultivation temperature was 37.0±0.2°C, the virus infection dose was 0.010-0.001 TCID₅₀/cell. Virus replication was carried out until specific cell death was 95-100%. Results of suspension cultivation of strain "A/Kiruhara/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 "A / Kiruhara / 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 3.0 million cells/cm³amounted to 1.08±0.02 μg/cm³, with a concentration of 3.5 million cells/cm³- 1.74±0.02 μg/cm³, with a concentration of 4.0 million cells/cm³- 2.24±0.02 μg/cm³, and with a concentration of 4.5 million cells/cm³- 2.27±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "A / Kiruhara / 2023" For the foot-and-mouth disease virus, a sufficient concentration of BHK-21/SUSP/ARRIAH cells in suspension is 4.0 million cells/cm³(at a concentration of 4.5 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 4.0 million cells/cm³). The value of the titer of infectious activity of the virus was 8.94±0.10 lg TCD₅₀/cm³.

At the next stage of the work, the influence of temperature on the reproduction of the strain "A / Kiruhara / 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±0.2°C. The dose of infection of the cell culture with the virus was 0.010-0.001 TCID₅₀/cell. The virus was cultured to a CPE of 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 "A/Kiruhara/2023" strain For the growth of foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells, the optimal temperature is 37.0±0.2°C, at which specific cell death of 95-100% with a high accumulation of the 146S component (2.24±0.02 μg/cm) is observed within 10 hours.³) and the titer of infectious activity of the virus is 8.94±0.10 lg TCID₅₀/cm³.

In the course of studying the conditions of suspension cultivation of the strain "A / Kiruhara / 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 ± 0.2 ° C until the cytopathic effect was 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. (2.24±0.02 µg/cm³) with a titer of infectious activity of the virus equal to 8.94±0.10 lg TCID₅₀/cm³.

Thus, a study of three parameters of suspension cultivation of the strain "A / Kiruhara / 2023" was carried out. genotype A/AFRICA/G-I 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 "A/Kiruhara/2023" strain in the BHK-21/SUSP/ARRIAH suspension cell culture were identified: 1) cell concentration before infection - 4.0 million cells/cm³; 2) cultivation temperature regime - 37.0±0.2°C; 3) virus infection dose - 0.010-0.001 TCID₅₀/cl.

Example 5. Inactivation of a suspension of foot-and-mouth disease virus strain “A/Kiruhura/2023” genotype A/AFRICA/GI.

At the end of the reproduction cycle of the foot-and-mouth disease virus, without stopping the incubation process (37.0 ± 0.2 ° 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 0.025%. Inactivation of the infectious activity of the foot-and-mouth disease virus strain "A / Kiruhara / 2023" genotype A/AFRICA/G-I was carried out for 12 hours at a temperature of 37.0 ± 0.1 ° 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 the inactivation of FMD virus strain "A/Kiruhara/2023" reproduced in culture vessels to 0.0 lg TCID.₅₀/ml occurred 6 hours after the introduction of 1,2-AEEI at a dose of 0.025%, and to a titer of -10.0 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 2.17±0.02 μg/ ^cm3 .

Example 6. Selection of conditions for purification of a suspension of foot-and-mouth disease virus strain "A/Kiruhara/2023" genotype A/AFRICA/GI.

A suspension of the foot-and-mouth disease virus strain "A/Kiruhara/2023" genotype A/AFRICA/G-I, obtained by reproduction in the BHK-21/SUSP/ARRIAH suspension cell line, was purified. Polyhexamethylene guanidine (PHMG) at concentrations of 0.030, 0.035, and 0.040% was used to obtain purified foot-and-mouth disease virus antigen. Before and after the purification process of the foot-and-mouth disease virus strain "A/Kiruhara/2023" antigen. 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 "A/Kiruhara/2023" strain Using PHMG at a concentration of 0.030%, a decrease in the concentration of ballast protein by 8% and the 146S component by 1.5% was observed. When using PHMG at a concentration of 0.035%, a decrease in the amount of ballast protein by 21% and the 146S component by 5.1% was observed. Using PHMG at a concentration of 0.040%, the content of ballast protein by 31% and immunogenic components by 21%.

Thus, having studied the conditions for purifying the antigen of the strain “A/Kiruhara/2023”, we came to the conclusion that it is optimal to use PHMG with a concentration of 0.035% to obtain a purified product.

Example 7. Selection of conditions for concentrating the suspension of the antigen of the strain "A / Kiruhara / 2023" of the foot-and-mouth disease virus genotype A/AFRICA/GI.

The culture inactivated suspension of the foot-and-mouth disease virus strain "A / Kiruhara / 2023", obtained using the BHK-21 / SUSP / ARRIAH suspension cell line, was concentrated 12 times by volume. To increase the concentration of 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 "A/Kiruhara/2023" The concentration of total viral protein and immunogenic components was determined in the quantitative version of the RSC. The results of the studies are presented in Table 5, which shows that when concentrating the antigen of the "A/Kiruhara/2023" strain A 4-hour concentration of PEG-6000-treated foot-and-mouth disease virus (FMDV) revealed a 10.4-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 10.8 times. Using tangential filtration for concentration, it was possible to increase the amount of immunogenic components of the A/Kiruhara/2023 strain antigen. The foot-and-mouth disease virus suspension was 12 times more abundant. Thus, tangential filtration technology made it possible to obtain the A/Kiruhara/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 A/Kiruhara/2023 strain of foot-and-mouth disease virus 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 "A / Kiruhara / 2023" genotype A / AFRICA / GI, culture-based monovalent inactivated emulsion.

A monovalent inactivated emulsion culture vaccine against foot-and-mouth disease of the A/AFRICA/G-I genotype was prepared from the obtained antigen concentrate of the A/Kiruhara/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 24.72±0.02 μg/cm³, and in a dose of 19.78 mcg.

Example 10. Immunization of animals with a culture-based monovalent inactivated emulsion vaccine against foot-and-mouth disease of the genotype A/AFRICA/GI .

A series of dilutions for pigs with a 5-fold step were prepared from the obtained foot-and-mouth disease vaccine of the A/Kiruhara/2023 strain, genotype A/AFRICA/GI, culture-inactivated emulsion (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 drug 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 vaccine against foot-and-mouth disease of the genotype A/AFRICA/GI, culture-based monovalent inactivated emulsion vaccine (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 39.9°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 monovalent inactivated emulsion culture vaccine against foot-and-mouth disease of the A/AFRICA/GI genotype (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 vaccine against foot-and-mouth disease of genotype A/AFRICA/GI, culture-based monovalent inactivated emulsion (proposed invention) for the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 21st day after the administration of the foot-and-mouth disease vaccine from the strain "A / Kiruhara / 2023" genotype A / AFRICA / GI, cultured monovalent inactivated emulsion (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 9.00 ± 0.31 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 5.00 ± 0.50 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 3.60 ± 0.22 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 vaccine against foot-and-mouth disease of genotype A/AFRICA/GI, culture-based inactivated emulsion vaccine (proposed invention) on naturally susceptible animal species using the challenge infection method.

Fifteen pigs immunized with the vaccine in whole form and in dilutions were infected with the control strain "A/Kiruhara/2023" of the A/AFRICA/GI genotype of the foot-and-mouth disease virus adapted to these animals at a dose of 10 ^4.0 ID ₅₀ /0.20 cm ³ (in two points of 0.10 ± 0.05 cm ³ ). Seven days after infection, all animals were euthanized and underwent a pathological 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 "A / Kiruhara / 2023" of the genotype A / AFRICA / GI, cultured 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 cm ³ ) protects pigs from infection with a 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 vaccine against foot-and-mouth disease of the A/AFRICA/G-I genotype, 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 "A/Kiruhura/2023" genotype A/AFRICA/G-I 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 A/AFRICA/G-I, in particular, currently in East Africa.

Sources of information

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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.

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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.

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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 11.08.2024).

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17. RF Patent No. 2815536, 03/27/2023 Vaccine against foot-and-mouth disease genotype A/AFRICA/G-I from strain “A/Tanzania/2013”, cultural inactivated sorbed // Application No. 2023107358 / Doronin M.I., Mikhalishin D.V., Borisov A.V., Mikhalishin V.V., Voevodina M.E., Nikiforov V.V.

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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.

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Table 1

Results of adaptation of the A/Kiruhara/2023 strain of foot-and-mouth disease virus during cultivation in a continuous monolayer culture of PSGK-30 cells under different conditions

(n=3, M ± m, p<0.01)

Parameter Cultivation conditions Reproduction time, h CPD, % Concentration of 146S particles according to data
RT-PCR-RV, μg/cm ³ Infectious activity titer,
lg TCD ₅₀ /cm ³ Composition of the supporting environment Medium Needle DMEM+ 2% S _KPC 23 95-100 0.84±0.02 7.02±0.10 RPMI-1640 medium + 2% S _KPC 23 95-100 0.86±0.02 7.01±0.10 Hanks' solution with 5% HBC +
2% S _KPC 19 95-100 1.35±0.02 7.47±0.10 Temperature, °C 35.0±0.2 24 90-95 0.42±0.02 5.88±0.10 36.0±0.2 22 90-95 0.75±0.03 6.75±0.10 37.0±0.2 19 95-100 1.35±0.02 7.47±0.10 38.0±0.2 20 90-95 1.02±0.02 7.19±0.10 Dose of infection 0.1-0.01 24 90-95 0.45±0.02 5.89±0.10 0.01-0.001 19 95-100 1.35±0.02 7.47±0.10 0.001-0.0001 28 90-95 0.22±0.02 5.11±0.10

Note: S _KPC - bovine serum,

GBC - blood protein hydrolysate,

DMEM is the name of Eagle's medium,

RPMI-1640 is the name of the culture medium,

CPE - cytopathic effect.

Table 2

Results of reproduction of the A/Kiruhara/2023 strain of foot-and-mouth disease virus in the continuous suspension cell line BHK-21/SUSP/ARRIAH with different cell concentrations

(n=3, M ± m, p<0.01)

Parameter Cultivation conditions Reproduction time, h CPD, % Concentration of 146S particles according to data
RT-PCR-RV, μg/cm ³ Infectious activity titer,
lg TCD ₅₀ /cm ³ Cell concentration before infection,
million cells/ ^cm3 3.0 9 95-100 1.08±0.02 7.31±0.10 3.5 10 95-100 1.74±0.02 7.56±0.10 4.0 10 95-100 2.24±0.02 8.94±0.10 4.5 11 95-100 2.27±0.02 9.00±0.10 Temperature, °C 35.0±0.2 18 85-90 0.60±0.02 6.98±0.10 36.0±0.2 16 90-95 0.92±0.02 7.14±0.10 37.0±0.2 10 95-100 2.24±0.02 8.94±0.10 38.0±0.2 15 95-100 1.41±0.02 7.33±0.10 Dose of infection 0.1-0.01 15 95-100 1.47±0.02 7.35±0.10 0.01-0.001 10 95-100 2.24±0.02 8.94±0.10 0.001-0.0001 13 90-95 0.60±0.02 6.98±0.10

Note: RT-PCR-RV - reverse transcription and polymerase chain reaction,

TCD - tissue cytopathic dose,

CPE - cytopathic effect.

Table 3

Study of the kinetics of inactivation of a suspension of foot-and-mouth disease virus strain "A/Kiruhara/2023" genotype A/AFRICA/G-I

(n=3, M ± m, p<0.05)

Sampling time from the moment of inactivation, h Titer of infectious activity of foot-and-mouth disease virus, lg TCID ₅₀ /cm ³ 0 8.94±0.10 1 7.43±0.10 2 5.82±0.10 3 4.35±0.10 4 2.73±0.10 5 1.43±0.10 6 0 12 -10.00

Table 4

The content of components of the strain "A / Kiruhara / 2023" genotype
A/AFRICA/GI foot-and-mouth disease virus in suspensions before and after purification

(n=3, M ± m, p<0.005)

Before cleaning Cleaning conditions After cleaning Ballast protein concentration, mg/ ^cm3 Concentration of 146S component, μg/cm ³
Ballast protein concentration, mg/cm ³ Concentration of 146S component, μg/cm ³ 9.85±0.02 2.17±0.02 PGMG,
0.030% 9.06±0.02 2.14±0.02 PHMG, 0.035% 7.78±0.02 2.06±0.02 PGMG,
0.040% 6.80±0.02 1.71±0.02

Note: The concentration of ballast protein was determined using the Kalkar formula:

C=1.45 × A ₂₈₀ -0.74 × A ₂₆₀ ,

where A ₂₈₀ is the optical density value at a wavelength of 280 nm,

A ₂₆₀ is the optical density value at a wavelength of 260 nm,

PHMG - polyhexamethylene guanidine (flocculant).

Table 5

The content of antigen components of the strain "A / Kiruhara / 2023" genotype A / AFRICA / G-I of the foot and mouth disease virus in suspensions before and after concentration

(n=3, M ± m, p<0.01)

Concentration of 146S component before concentration, μg/cm ³ Concentration methods
(12 times) Concentration of 146S component after concentration, μg/cm ³ 2.06±0.02 PEG-6000,
4 hours 21.42±0.02 PEG-6000,
12 hours 22.25±0.02 Tangential filtration,
3.5 hours 24.72±0.02

Note: PEG - polyethylene glycol,

OVB - total viral protein.

Table 6

Results of a study of pig blood serum for antibodies to non-structural proteins of the foot-and-mouth disease virus before and after vaccination with a preparation (proposed invention) manufactured using the antigen of the foot-and-mouth disease virus strain "A/Kiruhara/2023" genotype A/AFRICA/GI

Animal No. Optical density value of the sample Percentage of inhibition (PI), % sample type reference values before vaccination 14 days after vaccination sample type reference values before vaccination 14 days after vaccination SPK 0.470 -//- SPK 53.42 -//- PC 0.050 PC 95.05 OK 1,009 OK 0.00 1 -//- 0.940 0.890 -//- 6.84 11.79 2 0.949 0.881 5.85 12.79

Note: SPC - weak positive control,

PC - positive control,

OK - negative control, serum is negative at PI less than 50%.

Table 7

Study of humoral immunity and immunogenic activity of the monovalent inactivated emulsion culture vaccine against foot-and-mouth disease genotype A/AFRICA/G-I in pigs

(n=5, p<0.01, M ± m)

Name of the strain for challenge infection Dilution of the administered vaccine No. of animals Antibody titer in RMN, log ₂ SN ₅₀ Results of challenge infection IMD ₅₀ / PD ₅₀ A / Kiruhara / 2023 without dilution 1 8.75 ─ 0.04/
55.90 2 9:00 ─ 3 9:00 ─ 4 9.50 ─ 5 8.75 ─ M±m 9.00±0.31 0/5 1/5 6 4.50 ─ 7 5.50 ─ 8 5.00 ─ 9 4.50 ─ 10 5.50 ─ M±m 5.00±0.50 0/5 1/25 11 3.50 ─ 12 3.50 ─ 13 4.00 ─ 14 3.50 ─ 15 3.50 ─ M±m 3.60±0.22 0/5 control 1 ─ + control 2 ─ +

Note: "-" absence of generalization of the foot-and-mouth disease process,

RMN - microneutralization reaction,

ImD ₅₀ - immunogenic dose,

PD ₅₀ is a protective dose.

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softwareName="WIPO Sequence" softwareVersion="2.1.2"

productionDate="2024-09-28">

<ApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

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<FilingDate>2024-09-28</FilingDate>

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<ApplicantName languageCode="ru">Federal State Budgetary Institution "Federal Security Center"

Animal Health" (Federal State Budgetary Institution "All-Russian Research Institute of Animal Health")</ApplicantName>

<ApplicantNameLatin> Federal State-Financed Institution Federal

Center for Animal Health (FGBI ARRIAH)</ApplicantNameLatin>

<InventorName languageCode="ru">Doronin Maxim

Igorevich</InventorName>

<InventorNameLatin>Doronin Maksim Igorevich </InventorNameLatin>

<InventionTitle languageCode="en">Foot-and-mouth disease vaccine genotype

A/AFRICA/G-I culture monovalent inactivated

emulsion

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<INSDSeq_length>639</INSDSeq_length>

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<INSDQualifier id="q1">

<INSDQualifier_name>organism</INSDQualifier_name>

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<INSDSeq_sequence>accaccgcgacgggagagtcagcggaccctgtcaccaccactgttgaga

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gaaattaggagtttccagccccacacatgtcattgacctcatgcaaactcaccaacacggcctggtcggc

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<---

Claims (3)

1. A monovalent inactivated emulsion culture vaccine against foot-and-mouth disease of the A/AFRICA/GI genotype, containing an active substance and an adjuvant, characterized in that as an active substance it contains avirulent and purified antigen material from the causative agent of the infection, strain "A/Kiruhara/2023" of the foot-and-mouth disease virus of the A/AFRICA/GI genotype, deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Viruses and Other Animal Pathogens (GKShM) of the FGBU "ARRIAH", under the registration number: No. 524 - dep / 24-04 - GKShM of the FGBU "ARRIAH", 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 - oil adjuvant Montanide ISA-61 VG with a content of 60% by weight, 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 A/AFRICA/G-I. 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 strain "A / Kiruhara / 2023" of the foot-and-mouth disease virus of the genotype A / AFRICA / G-I and the oil adjuvant Montanide ISA-61 VG in an effective ratio of 40% and 60% by weight, respectively.