RU2848204C1 - Cultural inactivated emulsion vaccine for early protection against sat-1/i genotype foot-and-mouth disease - Google Patents

Abstract

FIELD: veterinary virology; biotechnology.

SUBSTANCE: invention relates to the field of veterinary virology and biotechnology, namely to the development of a culture-inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I. The culture-inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I is manufactured using the oil adjuvant DMIXVAC 76 V.

EFFECT: 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; the vaccine, manufactured from the SAT-1/I/2017 strain in accordance with the proposed invention, has high immunogenic activity and is capable of providing early effective protection of susceptible animals against epizootic strains of the SAT-1/I foot-and-mouth disease virus circulating in African and Middle Eastern countries.

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 inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype.

Foot-and-mouth disease is a highly contagious viral disease characterized by vesicular-erosive lesions of the mucous membranes of the oral and nasal cavities, as well as the skin of the interdigital folds and the periungual bed [1].

There are seven following serotypes of foot-and-mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, with representatives of serotype SAT-1 most often found in Africa and the Middle East, which determines the high relevance of producing vaccines for early protection against foot-and-mouth disease of this serotype.

Each serotype is divided into more than 60 genotypes. Differences between them are determined by analyzing the nucleotide sequence of the highly variable 1D gene, which encodes the amino acid sequence of the _VP1 surface protein [2].

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

The SAT-1 serotype comprises the following 13 topotypes: I (NORTHWEST ZIMBABWE, NWZ), II (SOUTHEAST ZIMBABWE, SEZ), III (WESTERП ZIMBABWE, WZ), IV (EAST AFRICA1, EA-1), V, VI, VII (EAST AFRICA2, EA-2), VIII (EAST AFRICA3, EA-3), IX, X, XI, XII, XIII [1, 3]. Such high genetic and antigen diversity leads to problems in the specific prevention of foot-and-mouth disease using culture-based inactivated foot-and-mouth disease vaccines, and also complicates the strain-specific diagnosis of isolated FMD virus isolates. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis for early protection against foot-and-mouth disease of the SAT-1/I genotype, representatives of which are actively spreading in Africa and the Middle East.

In order to prevent the occurrence of foot-and-mouth disease in farms of the Russian Federation, a set of measures for the control and prevention of foot-and-mouth disease is used, which is aimed at preventing the introduction of the virus into the country, systematic vaccination of animals in the buffer zone, monitoring the immune status of cattle, small cattle and pigs [6, 7, 8, 9, 10, 11].

This infection remains an economically significant problem. Whole-virus vaccines against foot-and-mouth disease are still recognized as the most effective, which is crucial for combating the most contagious disease in the world [8-12].

Conducting an effective vaccination campaign requires the availability of an effective and safe vaccine containing a viral antigen corresponding to the epizootic isolate of a specific genotype as the immunogenic component. The development of such vaccines remains a highly pressing issue. Achieving this goal will enable the effective use of the vaccine in vulnerable regions and at-risk zones to promote immunity against a specific genotype of foot-and-mouth disease virus, ensuring veterinary safety [13-16].

Emergency vaccination is one of several measures that can be used to control foot-and-mouth disease outbreaks. It is a valuable addition to the application of basic animal health control measures, which should include rapid diagnosis, tracking, movement control, and disinfection, as well as the culling of infected and contact animals and their safe disposal. Criteria for the successful implementation of emergency vaccination for early protection include access to a vaccine that: 1) contains a foot-and-mouth disease virus strain with sufficient antigenic relatedness to isolates circulating in the outbreak; 2) belongs to the required type among the developed vaccines; 3) has acceptable safety and efficacy; 4) has adequate availability, including batch size and timely delivery.

Contingency planning should include provision for emergency vaccination and consider the possibility of complex decisions not only regarding when, where and how to administer a vaccine but also the cost-effectiveness of its use [8].

Literary sources indicate that emergency vaccination of animals can be effective in preventing the disease within the first 4-5 days after vaccination. Research data show that the risk of spreading infection decreases as the interval between vaccination and infection with the foot-and-mouth disease virus increases. Furthermore, vaccination can reduce the amount of virus shed compared to unimmunized animals [9, 10].

Outbreaks of foot-and-mouth disease (FMD) genotype SAT-1/I are now sporadic across Africa and the Middle East. Due to the strengthening of trade and economic ties between the Russian Federation and countries in Africa and the Middle East, the risk of introducing isolates of this genotype into the Russian Federation is extremely high, threatening our country's biosecurity with respect to foot-and-mouth disease. Furthermore, to prevent this disease on these continents, it is important to produce a vaccine for early protection against periodic outbreaks of FMD of this genotype.

Outbreaks of foot-and-mouth disease (FMD) of the SAT-1/I genotype are consistently reported in countries across the African continent and the Middle East, raising the risk of further outbreaks of this genotype in the Russian Federation. This requires urgent prevention of this disease to build immunity in susceptible animals. Therefore, it became necessary to develop a culture-based, inactivated emulsion vaccine for early protection against FMD of the SAT-1/I genotype 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 foot-and-mouth disease virus isolate "SAT-1/KEN/8/2017" was isolated from pathological material collected from cattle in the Subukia settlement in the Nakuru region of the Republic of Kenya in 2017 and submitted to the All-Russian Research Institute of Animal Health for scientific research in 2021. As a result of research and development, the "SAT-1/I/2017" foot-and-mouth disease virus strain was obtained through adaptation to reproduction in a primary trypsinized SP cell culture, in continuous cell cultures of BHK-21, IB-RS-2, PSGK-30, in the body of pigs and cattle under the conditions of the All-Russian Research Institute of Animal Health. According to the results of a comparative analysis of nucleotide sequences, the isolated strain belongs to the SAT-1/I genotype of the foot-and-mouth disease virus, which differs significantly from the production strains of the SAT-1 serotype of the foot-and-mouth disease virus (Fig. 1).

The closest to the proposed invention in terms of the set of essential features is the SAT-1/NWZ genotype foot-and-mouth disease vaccine, a culture-based inactivated sorbed vaccine [17], containing the following main components in 1.0 cm ³ of the preparation: 1) an active substance in the form of an avirulent and purified 146S component from the homologous to the causative agent of the infection strain "SAT-1/Kenya/2017" of the SAT-1/NWZ genotype of the foot-and-mouth disease virus, reproduced preferably in the transplantable suspension cell line BHK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; 2) aluminum hydroxide 10% colloidal solution with a content of 55% by volume (5.5% in terms of dry matter), 3) saponin in an amount of 1.7 mg, 4) a supporting medium - up to 2.0 cm ³ (prototype).

A significant drawback of the prototype vaccine is its insufficient immunogenic activity in the early stages after immunization (on the 4th day) in relation to strains/isolates of the foot-and-mouth disease virus of the SAT-1/I genotype.

To address this problem, the present invention was developed, which included the development of a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease (FMD) genotype SAT-1/I. This vaccine contains a high dose of the 146S immunogenic component of FMD virus genotype SAT-1/I.

The technical result of using the proposed invention consists in expanding the arsenal of culture-based inactivated emulsion vaccines for early protection against foot-and-mouth disease virus of the SAT-1/I genotype.

The specified technical result has been achieved by creating a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype, characterized by the following set of features, reflected below.

The developed vaccine in 2.0 ^cm3 of the preparation contains the following main components: 1) the active substance in the form of avirulent and purified antigen material from the SAT-1/I/2017 strain (genotype SAT-1/I) 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 30.0 μg; 2) the oil adjuvant DMIXVAC 76 V (Dmitrievsky Chemical Plant, Russian Federation), which provides an enhancement of the immune response in animals with a content of 60% by weight,
3) supporting medium – up to 2.0 cm ³ .

The foot-and-mouth disease virus strain "SAT-1/I/2017" 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. 451 - dep / 23-1 - GKShM of the All-Russian Research Institute of Animal Health.

The specified 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 8.0% of the total volume at a pH of 7.55-7.65. Virus inactivation is performed using aminoethylethyleneimine (AEEI) at a concentration of 0.04% 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 0.04% of the total volume.

The avirulent and purified antigen of the SAT-1/I/2017 strain (genotype SAT-1/I) of foot-and-mouth disease virus is a suspension containing the inactivated 146S immunogenic component of each genotype of foot-and-mouth disease virus. The concentration of the component in the product is assessed using a quantitative complement fixation assay (CFA) [18, 19].

To create a vaccine, viral material containing
2.0 ^cm3 of at least 30.0 μg of inactivated immunogenic 146S particles of foot-and-mouth disease virus genotype SAT-1/I. The stated concentration of the immunogenic component in the vaccine preparation is achieved by concentrating the antigen using a Centramate 500S Pall tangential filtration unit.

A culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I The vaccine is produced by mixing purified antigen and the oil adjuvant DMIXVAC 76 V in a ratio of 40% and 60% by weight, respectively. The resulting vaccine is a stable, simple, reverse emulsion of white or pale pink color, containing the inactivated 146S component of the foot-and-mouth disease virus, genotype SAT-1/I, which provides specific immunity.

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. Culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I.

2. An active substance in the form of avirulent and purified antigenic material from the strain "SAT-1/I/2017" genotype SAT-1/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 SAT-1/I/2017 strain of the SAT-1/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 SAT-1/I/2017 strain of the SAT-1/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 "SAT-1/I/2017" of the genotype SAT-1/I 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 of each strain in an amount of at least 30.0 μg in 2.0 ^cm3 of the finished vaccine preparation.

3. Among the target additives, the vaccine contains the oil adjuvant DMIXVAC 76 V.

4. The vaccine contains the oil adjuvant DMIXVAC 76 V in an amount of 60% (by weight) in 2.0 ^cm3 of the finished product.

5. Avirulent and purified antigen of the SAT-1/I/2017 strain of the SAT-1/I genotype of the foot-and-mouth disease virus, obtained in the continuous suspension cell line BHK-21/SUSP/ARRIAH, oil adjuvant DMIXVAC 76 V, additive in the finished product with a volume of 2.0 ^cm3 in the form of a maintenance medium in the following quantities:

Avirulent and purified antigen of the strain "SAT-1/I/2017" of the genotype SAT-1/I of the foot-and-mouth disease virus not less than 30.0 mcg Oil adjuvant DMIXVAC 76 V 60% (by weight) Supportive environment up to 2.0 cm ³

The proposed culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I has high immunogenic activity and provides reliable protection against isolates of foot-and-mouth disease virus of the SAT-1/I genotype.

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 emulsion vaccine contains, as an active substance, an antigen of the SAT-1/I/2017 strain of the SAT-1/I 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 in recent years has caused outbreaks in the countries of the Middle East and Africa.

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

Fig. 1 – Dendrogram reflecting the phylogenetic relationship of the SAT-1/I/2017 strain of foot-and-mouth disease virus with epizootic strains of the serological type SAT-1. 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 strain "SAT-1/I/2017" of the foot-and-mouth disease virus of the genotype SAT-1/I;

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

The strain "SAT-1/I/2017" of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The strain "SAT-1/I/2017" of foot-and-mouth disease virus has the following taxonomy: sphere Riboviria , kingdom Orthornavirae , phylum Pisuviricota , class Pisoniviricetes , order Picornavirales , family Picornaviridae , genus Aphthovirus , species Foot-and-mouth disease virus , serotype SAT-1 , genotype SAT-1/I. The strain of the pathogen has the following morphological features characteristic of the causative agent of foot-and-mouth disease: the shape of the virion is icosahedral, the size is 26 nm. The virion consists of a molecule of a single-stranded positively charged RNA molecule and 60 copies of a polypeptide, each of which is represented by proteins VP ₄ , VP ₂ , VP ₃ , VP ₁ .

Antigenic properties

Based on its antigenic properties, the SAT-1/I/2017 foot-and-mouth disease virus strain belongs to the SAT-1 serotype. The virus is reliably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. In animals that have recovered from the disease, type-specific antibodies are produced in the serum, which are 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 "SAT-1/I/2017" of the foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "SAT-1/I/2017" of the foot-and-mouth disease virus belongs to the SAT-1/I genotype (Fig. 1).

The antigenic affinity (r ₁ ) of the SAT-1/I/2017 foot-and-mouth disease virus strain was studied in a virus microneutralization reaction, in a cross-study of the strain with specific sera obtained for the following production strains of foot-and-mouth disease virus:

- strain "SAT-1/Kenya/2017" (genotype SAT-1/NWZ),

- strain “SAT-1/RV/11/37” (genotype SAT-1/SEZ),

- strain "SAT-1/BOT/1/68" (genotype SAT-1/WZ),

- strain “SAT-1/UGA/BUFF/21/70” (genotype SAT-1/EA-1),

- strain "SAT-1/NIG/11/75" (genotype SAT-1/V),

- strain “SAT-1/SUD/3/76” (genotype SAT-1/VI),

- strain “SAT-1/UGA/13/74” (genotype EA-2),

- strain “SAT-1/UGA/1/97” (genotype EA-3),

- strain "SAT-1/ETH/3/2007" (genotype IX),

- strain "SAT-1/NIG/2015" (genotype X),

- strain "SAT-1/TCH 1/72" (genotype XI),

- strain "SAT-1/ANG/9/74" (genotype XII),

- strain “SAT 1/MOZP132010/B16” (genotype XIII).

The titer of reference bovine blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype SAT-1 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, 20, 21].

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 “SAT-1/I/2017” were r ₁ from 0.01 to 0.24, which indicates the absence of obvious antigenic relatedness with the production strains of foot-and-mouth disease virus of serotype SAT-1.

Geno- and chemotaxonomic characteristics

The SAT-1/I/2017 strain of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular mass of 8.080×10 ⁶ D. The nucleic acid is a single-stranded linear molecule with a molecular mass of 2.858×10 ⁶ D. The virion has a protein membrane consisting of four main 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 31.7% RNA and 68.3% 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 8.450×10 ^-18 g. The buoyant density is 1.467 g/cm ³ .

Resistance to external factors

The SAT-1/I/2017 foot-and-mouth disease virus strain is resistant to ether, chloroform, and acetone. It is most stable at a pH of 7.55-7.65. 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 38.3 ^° C).

Additional features and properties

Reactogenicity – the antigen does not have reactogenic properties.

Pathogenicity – the inactivated antigen is not pathogenic for ungulates.

Virulence – the inactivated antigen is not 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 SAT-1/I/2017 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 SAT-1/I/2017 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 SAT-1/I genotype virus 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 for early protection.

A safe and effective culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I 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 SAT-1/I/2017 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 SAT-1/I/2017 foot-and-mouth disease virus vaccine strain was analyzed using Sanger nucleotide sequencing and the position of this strain on the phylogenetic tree of foot-and-mouth disease virus serotype SAT-1 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 SAT-1.

It was necessary to conduct a comparative analysis of the nucleotide sequences of the 1D gene encoding the VP ₁ protein of the foot-and-mouth disease virus vaccine strain “SAT-1/I/2017” with other isolates and strains.

The nucleotide sequence of the gene encoding the VP ₁ protein of the SAT-1/I/2017 strain of the SAT-1/I genotype of foot-and-mouth disease virus is shown in SEQ ID NO: 1. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the SAT-1/I/2017 strain of the SAT-1/I genotype of foot-and-mouth disease virus is shown in SEQ ID NO: 2.

A phylogenetic analysis was performed for the foot-and-mouth disease virus vaccine strain SAT-1/I/2017. The phylogenetic tree was inferred using MEGA 6 and the Neighbor-Joining algorithm [21]. The percentage of repeat branches in which related taxa are grouped together in a bootstrap test (1000 replicates) 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. This analysis included 14 nucleotide sequences, including the 1D gene sequence of the foot-and-mouth disease virus vaccine strain SAT-1/I/2017. All positions containing gaps and missing data were removed (complete deletion option). The evolutionary analysis was performed in MEGA 6 [24].

A comparison of the complete nucleotide sequences of the 1D gene (VP ₁ protein) of the SAT-1/I/2017 foot-and-mouth disease virus vaccine strain and other isolates/strains of the SAT-1 serotype allowed us to determine the position of the studied strain on the phylogenetic tree (Fig. 1). Thus, the SAT-1/I/2017 strain belongs to the SAT-1/I genotype, as confirmed by nucleotide and amino acid analysis data.

Example 2. Adaptation of the foot-and-mouth disease virus strain "SAT-1/I/2017" to the transplantable monolayer culture of Siberian ibex kidney cells PSGK-30.

A 25% aphthous suspension of foot-and-mouth disease virus obtained from bovine aphthae (passage 2) was used to inoculate a continuous monolayer culture of PSGK-30 Siberian ibex kidney cells. The cell inoculation concentration was 0.40-0.45 million cells/ ^cm3 , and the virus infection dose was 0.001 _TCID50 /cell.

According to the results of the study, the reproduction of the foot-and-mouth disease virus strain "SAT-1/I/2017" 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 values of the titer of infectious activity of the virus was noted from 5.87±0.10 to 7.20±0.10 lg TCID ₅₀ /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of passage 6 (7.20±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from passages 1 to 6 changed from 0.20±0.02 to 0.60±0.02 μg/ ^cm3 . Moreover, the highest amount of 146S component was observed at the stage of passage 6 (0.60±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 99.27 to 100.00%. Thus, over the course of 6 consecutive passages, the adaptation of the SAT-1/I/2017 foot-and-mouth disease virus strain 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 “SAT-1/I/2017” on an industrial scale.

During the industrial cultivation of the SAT-1/I/2017 foot-and-mouth disease virus strain in the continuous monolayer cell culture PSGK-30, 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 SAT-1/I/2017 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 MEM medium; 2) Leibovitz's medium; 3) Hanks' solution. The cell inoculation concentration was 0.30 million cells/cm ³ , the virus infection dose was 0.1000-0.0001 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 85%. The results of reproduction of the SAT-1/I/2017 foot-and-mouth disease virus strain with support media of different compositions are shown in Table 1.

From the data in Table 1 it is evident that during the reproduction of the SAT-1/I/2017 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 with 8% GBC and 2% bovine blood serum, which allows achieving specific destruction of the cell monolayer by 95-100% in 15-16 hours with the accumulation of the 146S component in the resulting suspension in an amount of 0.60±0.02 μg/cm ³ and an infectious activity titer of 7.20±0.10 lg TCID ₅₀ /cm ^3. When using Leibovitz and Eagle's MEM media, 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 "SAT-1/I/2017" 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 with 8% GBK and 2% bovine blood serum 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.

Cultivation of the foot-and-mouth disease virus strain "SAT-1/I/2017" was carried out until the destruction of the cell monolayer by 85-100% for 15-32 hours (Table 1). Based on the results of the study, it was revealed that for complete reproduction of the foot-and-mouth disease virus strain "SAT-1/I/2017" in a monolayer culture of PSGK-30 cells, the optimal medium temperature is 37.0±0.2 ⁰ C, at which in 15-16 hours the development of CPE reached 95-100% with the accumulation of 146S particles equal to 0.60±0.02 μg/cm ³ and the titer of infectious activity of the virus of 7.20±0.10 lg TCID ₅₀ /cm ³ .

The effect of the infection dose on the cell line monolayer was assessed.
PSGK-30 was cultured separately with the "SAT-1/I/2017" strain at production scale. Different virus doses were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 _TCID50 /cell. Hanks' medium with 8% GBC and 2% bovine serum was used as the maintenance medium.

Reproduction of foot-and-mouth disease virus strain "SAT-1/I/2017" was carried out at a temperature of 37.0±0.2 ⁰ C for 15-26 h until specific cell destruction by 85-100%. 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 component of the virus was achieved at an infection dose of 0.01-0.001 TCID ₅₀ /cell and amounted to 0.60±0.02 μg/cm ³ with an infectious activity titer of the virus of 7.20±0.10 lg TCID ₅₀ /cm ³ .

Thus, the conditions of monolayer cultivation of the foot-and-mouth disease virus strain "SAT-1/I/2017" in the PSGK-30 cell culture were studied on an industrial scale. As a result of the study, the following optimal parameters for their reproduction in the PSGK-30 monolayer cell line were determined: 1) maintenance medium - Hanks' medium with 8% GBA and 2% bovine blood serum; 2) cultivation temperature - 37.0±0.2 ⁰ C; 3) virus infection dose - 0.01-0.001 TCID ₅₀ /cell.

Example 4. Study of conditions for suspension cultivation of the SAT-1/I/2017 strain of foot-and-mouth disease virus on an industrial scale.

During industrial cultivation of the SAT-1/I/2017 strain of 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 these strains 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 reproduction was carried out until specific cell death was 95-100%.

Results of suspension cultivation of strain "SAT-1/I/2017" The results of the analysis of the results of the cell suspensions of the foot-and-mouth disease virus are presented in Table 2.

As follows from Table 2, when cultivating the foot-and-mouth disease virus strain “SAT-1/I/2017” 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 0.81±0.02 μg/cm³, with a concentration of 4.0 million cells/cm³– 1.00±0.02 μg/cm³, with a concentration of 4.5 million cells/cm³– 1.24±0.02 μg/cm³, and with a concentration of 5.0 million cells/cm³– 1.31±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "SAT-1/I/2017" For the foot-and-mouth disease virus, the optimal concentration of BHK-21/SUSP/ARRIAH cells in suspension is 4.5 million cells/cm³(at a concentration of 5.0 million cells/cm³The content of the 146S component and the titer of infectious activity of the virus are slightly higher, but the production costs in terms of the number of cells are higher). The titer of infectious activity of the virus in this case was 8.32±0.10 lg TCD₅₀/cm³.

At the next stage of the work, the influence of the temperature factor on the reproduction of the SAT-1/I/2017 foot-and-mouth disease virus strain in a suspension culture of BHK-21/SUSP/ARRIAH cells on an industrial scale was investigated. For this purpose, the 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 ⁰ C. The dose of infection of the cell culture with the virus was 0.010-0.001 TCID ₅₀ /cell. The virus cultivation was carried out until the CPE was at least 90% (priority was given to complete specific destruction of cells).

Based on the analysis results, it was determined that for the full reproduction of the strain “SAT-1/I/2017” 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 15 hours specific cell death is observed by 95-100% with a high accumulation of the 146S component (1.24±0.02 μg/cm³) and the titer of infectious activity of the virus is 8.32±0.10 lg TCID₅₀/cm³.

In the course of studying the conditions of suspension cultivation of the SAT-1/I/2017 foot-and-mouth disease virus strain on an industrial scale using the BHK-21/SUSP/ARRIAH cell culture, the effect of the cell infection dose was assessed. 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 TCID ₅₀ /cell. Virus reproduction was carried out at a temperature of 37.0±0.2 ⁰ 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 of the foot-and-mouth disease virus strain “SAT-1/I/2017” was observed at an infection dose of 0.010-0.001 TCID ₅₀ /cell (1.24±0.02 μg/cm ³ ) with a titer of infectious activity of the virus equal to 8.32±0.10 lg TCID ₅₀ /cm ³ .

Thus, a study of three parameters of suspension cultivation of the SAT-1/I/2017 foot-and-mouth disease virus strain in the BHK-21/SUSP/ARRIAH suspension cell line on an industrial scale was conducted.

The optimal conditions for the reproduction of the SAT-1/I/2017 foot-and-mouth disease virus strain in the suspension culture of BHK-21/SUSP/ARRIAH cells were identified: 1) cell concentration before infection – 4.5 million cells/cm ³ ; 2) cultivation temperature – 37.0±0.2 ⁰ C; 3) virus infection dose – 0.010-0.001 TCID ₅₀ /cell.

Example 5. Inactivation of a suspension of foot-and-mouth disease virus strain
"SAT-1/I/2017" foot-and-mouth disease virus.

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 suspensions. The final concentration of AEEI in the virus-containing suspension should be equal to 0.04%. Inactivation of the foot-and-mouth disease virus strain "SAT-1/I/2017" 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 addition
Samples were collected after 1, 2, 4, 6, and 12 hours of 1,2-AEEI. The resulting samples were tested for the presence of foot-and-mouth disease virus infectivity in a primary culture of SP pig kidney cells. The results are presented in Table 3, which demonstrates that complete inactivation of the SAT-1/I/2017 strain of foot-and-mouth disease virus, individually reproduced in culture vessels, occurred 6 hours after the addition of 1,2-AEEI.

The prepared inactivated viral suspensions were analyzed at the Russian State Research Center to assess their viral component content. For the "SAT-1/I/2017" strain, the 146S component concentration was 1.19±0.02 μg/ ^cm3 .

Example 6. Selection of conditions for purification of suspensions of foot-and-mouth disease virus strain “SAT-1/I/2017”.

Foot-and-mouth disease virus (FMDV) suspensions of the SAT-1/I/2017 strain, obtained by propagation in the BHK-21/SUSP/ARRIAH suspension cell line, were purified. Polyhexamethylene guanidine (PHMG) was used at concentrations of 0.035, 0.040, and 0.045% to obtain purified FMDV antigen. Before and after the FMDV antigen purification process for the above strains In the quantitative version of the RSC, the concentration of total viral protein and immunogenic components was determined.

The results of the analysis are reflected in Table 4, from which it follows that as a result of purification of the foot-and-mouth disease virus antigen of the SAT-1/I/2017 strain using PHMG at a concentration of 0.035%, a decrease in the concentration of ballast protein by 5.9% and immunogenic components by 0.8% was observed. When using PHMG at a concentration of 0.040%, a decrease in the amount of ballast protein by 12% and the 146S component by 2.5% was observed. Using PHMG at a concentration of 0.045%, the content of ballast protein decreased by 29% and immunogenic components by 18%.

Thus, by investigating the conditions for purification of the strain antigen
"SAT-1/I/2017" concluded that for obtaining a purified product it is optimal to use PHMG with a concentration of 0.040%.

Example 7. Selection of conditions for concentrating a suspension of foot-and-mouth disease virus antigen strain "SAT-1/I/2017".

Cultural inactivated suspension of the strain
« SAT-1/I/2017 » , obtained using the BHK-21/SUSP/ARRIAH suspension cell line, as described in Example 5, was concentrated 35 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) concentrating using a Centramate 500S Pall tangential filtration unit for 4 hours at an operating pressure on the filter of 2.2-2.3 atm.

Before and after concentrating the foot-and-mouth disease virus antigen suspensions, the concentrations of total viral protein and immunogenic components were determined using the quantitative method of the complete protein concentration (CPC). The results are presented in Table 5.

From the data in Table 5 it is evident that when concentrating the antigen of the strain “SAT-1/I/2017” A 4-hour concentration of PEG-6000-treated foot-and-mouth disease virus revealed a 28-fold increase in component content compared to baseline values (before concentration). Using the same polymer but increasing the exposure time to 12 hours, the concentration of virus components was increased 30-fold. Using a tangential filtration concentration method, it was possible to increase the amount of immunogenic components of the SAT-1/I/2017 strain antigen. foot-and-mouth disease virus in suspension by 35 times.

Thus, the tangential filtration technology made it possible to obtain the antigen of the foot-and-mouth disease virus strain "SAT-1/I/2017" with a high concentration of the 146S component for the production of a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype.

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

To produce a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I, the oil adjuvant DMIXVAC 76 V was selected as the adjuvant at 60% by weight. The antigen comprised 40% by weight.

Example 9. Composition of a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I.

From the obtained concentrate of the foot-and-mouth disease virus antigen of the strain "SAT-1/I/2017", prepared according to Example 7, a culture-based inactivated emulsion vaccine was produced for early protection against foot-and-mouth disease of the genotype SAT-1/I using the oil adjuvant DMIXVAC 76 V. The amount of 146S immunogenic component in a dose of 2.0 cm³vaccine component 146S foot-and-mouth disease virus strain "SAT-1/I/2017" was 32.47 μg/cm³, respectively.

Example 10. Immunization of animals with a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I .

A series of dilutions for pigs with a 5-fold step were prepared from the obtained culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I (the proposed invention) . Fifteen pigs were divided into three groups of five 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 virus controls.

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 a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype (proposed invention) .

To determine the avirulence of the culture-based inactivated emulsion vaccine for early protection against genotype SAT-1/I foot-and-mouth disease, a sample of the vaccine preparation was administered intradermally to pigs at a dose of 0.1 ^cm3 . Landrace 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 40.5 ^° C and remain at this level for 1-2 days, which is within the normal range after administration of the vaccine.

According to the research results, a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I (proposed invention)was found to be harmless, all animals remained clinically healthy during the observation period, and pathological analysis did not reveal tissue necrosis at the site of vaccine administration.

Example 13. Study of the immunogenic properties of the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I (proposed invention) for the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 4th and 7th days after the administration of the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotype SAT-1/I (the proposed invention), blood was collected from the pigs and the obtained blood serum was tested in a microneutralization reaction [3].

It was found that in pigs immunized with the vaccine in a whole dose (5 heads), the average values of antibody titer against the strain “SAT-1/I/2017” on the 4th day after immunization were 5.70±0.27 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.15±0.14 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 3.20±0.11 log ₂ SN ₅₀ (Table 7). The average values of antibody titers against the SAT-1/I strain on the 7th day after immunization were 7.15±0.42 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) – 5.30±0.27 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) – 4.15±0.29 log ₂ SN _50. The obtained data from the microneutralization reaction indicate that after 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 already on the 4th day after vaccination with the proposed invention, which meets the requirements of international standards [3].

Example 14. Study of the protective capacity of the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype (proposed invention) on naturally susceptible animal species.

Study of the protective capacity of the vaccine (the proposed invention) on naturally susceptible animal species using the challenge method. Pigs immunized in a quantity of 15 heads with the vaccine in whole form and in dilutions as described in Example 10 were infected with the control strain "SAT-1/I/2017" 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 a pathological examination was performed. Animals with no lesions on the 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 culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype (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 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 55.9 PD ₅₀ and 0.036 IMD ₅₀ of the foot-and-mouth disease virus antigen for pigs.

Thus, the above information indicates that the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype, embodying the proposed invention, is intended as a reserve vaccine for use in agriculture, namely in veterinary virology and biotechnology; the possibility of implementing the presented is confirmed; the vaccine made from the SAT-1/I/2017 strain, in accordance with the proposed invention, has high immunogenic activity and is capable of providing early effective protection of susceptible animals against epizootic strains of the foot-and-mouth disease virus of the SAT-1/I genotype circulating in Africa and the Middle East. In addition, the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype is made using the oil adjuvant DMIXVAC 76 V.

Sources of information taken into account when preparing the description of the invention for the application for the issuance of a Russian patent for the invention "Culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I genotype":

1. Foot and mouth disease. Prevention measures. URL: http://89.rospotrebnadzor.ru
/directions/epid_nadzor/146902 (Accessed: 14.10.2024).

2. The danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Accessed: 18.09.2024).

3. OIE. Manual of diagnostic tests and vaccines for terrestrial animals - 24th Ed. - Paris, 2023 – 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.
M.: 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. Brown F. A brief history of FMD and its casual agent. FMD: Control Strategies: Proc. Jnt. Symp. 2-5. June 2002, Lyons, France. – Paris, 2003. – p. 13-21.

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

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

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

11. Official website of the FAO World Reference Laboratory for Foot-and-Mouth Disease – URL: http://www.wrlfmd.org/ref_labs/fmd_ref_lab_
reports.htm (Accessed 14.10.2024).

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. Patent for invention No. 2,809,219 “Cultural inactivated sorbed vaccine against foot-and-mouth disease of the SAT-1/NWZ genotype” / invention priority: May 11, 2023, application No. 2023112436.

18. 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.

19. 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.

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

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

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

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

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

Table 1

Results of adaptation of the SAT-1/I/2017 foot-and-mouth disease virus strain when cultivated in a continuous monolayer cell culture
PSGK-30 in different conditions

(n=3, M±m, p<0.01)

Parameter Cultivation conditions
vaniya 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 MEM+ 2% S _cattle 17-18 95-100 0.36±0.02 6.39±0.10 Leibowitz medium + 2% S _cattle 17-18 95-100 0.41±0.02 6.52±0.10 Hanks' solution with 8% HBC +
2% S _Cattle 15-16 95-100 0.60±0.02 7.20±0.10 Temperature, ⁰ C 35.0±0.2 30-32 85-90 0.15±0.02 4.89±0.10 36.0±0.2 28-29 85-90 0.35±0.03 6.42±0.10 37.0±0.2 15-16 95-100 0.60±0.02 7.20±0.10 38.0±0.2 19-20 95-100 0.42±0.02 6.53±0.10 Infection dose 0.1-0.01 17-18 90-95 0.44±0.02 6.58±0.10 0.01-0.001 15-16 95-100 0.60±0.02 7.20±0.10 0.001-0.0001 25-26 85-90 0.38±0.02 6.44±0.10

Note: S _KRS – bovine blood serum,

GBC – blood protein hydrolysate,

MEM is the name of the Eagle environment,

CPE – cytopathic effect.

Table 2

Results of reproduction of the SAT-1/I/2017 foot-and-mouth disease virus strain 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 3.5 14-15 95-100 0.81±0.02 7.47±0.10 4.0 14-15 95-100 1.00±0.02 7.82±0.10 4.5 15 95-100 1.24±0.02 8.32±0.10 5.0 16 95-100 1.31±0.02 8.42±0.10 Temperature, ⁰ C 35.0±0.2 24-25 90-95 0.51±0.02 7.02±0.10 36.0±0.2 17-18 90-95 0.87±0.02 7.51±0.10 37.0±0.2 15 95-100 1.24±0.02 8.32±0.10 38.0±0.2 17-18 95-100 0.91±0.02 7.55±0.10 Infection dose 0.1-0.01 14 95-100 0.62±0.02 7.24±0.10 0.01-0.001 15 95-100 1.24±0.02 8.32±0.10 0.001-0.0001 22-23 90-95 0.77±0.02 7.43±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 suspensions of foot-and-mouth disease virus strain

SAT-1/I/2017

(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 strain "SAT-1/I/2017", lg TCD ₅₀ /cm ³ 0 8.32±0.10 1 6.14±0.10 2 4.25±0.10 4 1.65±0.10 6 0 12 -11.00

Table 4

Content of components of foot-and-mouth disease virus strain "SAT-1/I/2017" in suspensions before and after purification

(n=3, M±m, p<0.005)

Strain name Before cleaning Concentration of PHMG, % After cleaning concentration of ballast protein, mg/cm ³ concentration of 146S component, μg/cm ³ concentration of ballast protein, mg/cm ³ concentration of 146S component, μg/cm ³ SAT-1/I/2017 8.67 1.19 0.035 8.16 1.18 0.040 7.63 1.16 0.045 6.16 0.98

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 components of the foot-and-mouth disease virus antigen of the strain "SAT-1/I/2017" in suspensions before and after concentration

(n=3, M±m, p<0.01)

Strain name Concentration of 146S component before concentration, μg/cm ³ Concentration methods Concentration of 146S component after concentration, μg/cm ³ SAT-1/I/2017 1.16 PEG-6000,
4 hours 32.48 PEG-6000,
12 hours 34.82 Tangential filtration,
4 hours 40.59

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 foot-and-mouth disease virus antigen strain "SAT-1/I/2017"

No. of living person 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 0.472 -//- SPK 52,610 -//- PC 0.072 PC 92,771 OK 0.996 OK 0,000 1 -//- 0.956 0.925 -//- 4,016 7,129 2 0.953 0.927 4,317 6,928

Note: SPC – weak positive control,

PC – positive control,

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

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<INSDSeq_sequence>accacctcggcgggtgagggcgcagaacccgtgaccaccgatgcatcgcaacacggtgg

Tgggcgccgcgccacacgcaggcaacacactgacgtgtctttccttcttgaccggttcacactggtcggaaagactg

Ccaacaacaagctgacactggatctgctccagaccaaggagaaggcgctagtgggcgcaatcctgcgcgctgctacg

Tactacttctcggacctggaggtggcatgtgttggaacgaacaagtgggttggctggacaccgaatggtgcgcctga

Actcagtgaggtcggcgacaacccagtcgtcttctctcacaacgggaccacccgctttgctctaccatacactgccc

Cgcacagatgtcttgccactgcctacaatggcgactgcaagtacaagccagatagtgaggcaccacggacgcacatt

Cgtggagaacctcgcgacgctcgctgagcgcatcgctagcgagacacacatcccaaccactttcaactacggcaggat

Ctacacggaggcggaagtcgacgtgtacgtgcggatgaagcgagcggagctctactgcccccgcccggttctgactc

actatgaccaccaaggtaggaatcgctacaaagtggccctgacaaagcctgccaagcaactgtgc</INSDSeq_se

quence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>221</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..221</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>FMDV</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TTSAGEGAEPVTTDASQHGGGRRATRRQHTDVSFLLDRFTLVGKTANNKLTLDLLQTKE

KALVGAILRAATYYFSDLEVACVGTNKWVGWTPNGAPELSEVGDNPVVFSHNGTTRFALPYTAPHRCLATAYNGDCK

YKPDSEAPRTHIRGDLATLAERIASETHIPTTFNYGRIYTEAEVDVYVRMKRAELYCPRPVLTHYDHQGRNRYKVAL

TKPAKQLC</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (3)

1. A culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-1/I 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 infection, strain "SAT-1/I/2017" of the foot-and-mouth disease virus of the SAT-1/I 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. 451 - dep/23-1 - GKShM of the FGBU "ARRIAH", reproduced in the continuous suspension cell line BNK-21/SUSP/ARRIAH, in an amount of at least 30.0 μg; as an adjuvant it contains the domestic oil adjuvant DMIXVAC 76 V 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 the 146S immunogenic component of the foot-and-mouth disease virus of the SAT-1/I genotype. 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 SAT-1/I/2017 strain of the foot-and-mouth disease virus of the SAT-1/I genotype and the oil adjuvant DMIXVAC 76 V in an effective ratio of 40% and 60% by weight, respectively.