RU2839344C1 - Cultural inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes sat-2/vii/lib-12 and sat-2/xiv - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: proposed is a cultural inactivated emulsion vaccine containing an active substance – a mixture of two strains of foot-and-mouth disease virus: an avirulent and purified antigen material from a homologous to the infectious agent strain "SAT-2 Lib 39/2012" of genotype SAT-2/VII/Lib-12, deposited in the Russian national collection of exotic types of viruses of foot-and-mouth disease and other animal pathogens (SCSM) FGBI "ARRIAH", under registration number: No. 361 – dep/21-22 – GKShM FGBU "VNIIZZh", reproduced in a continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of not less than 15.0 mcg; and an avirulent and purified antigen material from a homologous to the infectious agent strain "SAT-2/XIV/2023" of the SAT-2/XIV genotype, deposited in the Russian national collection of exotic types of viruses of foot-and-mouth disease and other animal pathogens (SCSM) FGBI "ARRIAH", under registration number: No. 489 – dep/23-39 – GKShM FGBU "VNIIZZh", reproduced in a continuous suspension cell line BHK-21/SUSP/ARRIAH, in an amount of not less than 15.0 mcg; and adjuvant – domestic oil adjuvant DMIXVAC 78 V with content of 70% by weight of finished preparation and supporting medium – up to 2.0 cm³ of finished preparation.

EFFECT: invention provides a wider range of cultural inactivated emulsion vaccines for early protection against epizootic strains of foot-and-mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, circulating in African and Asian countries.

3 cl, 1 dwg, 10 tbl, 14 ex

Description

The 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 of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV.

Foot-and-mouth disease is an acute viral disease from the group of zoonoses, characterized by intoxication and vesicular-erosive (vesicular-ulcerative) 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 the representative of serotype SAT-2 being widespread in Africa, the Middle East and since 2023 in the countries of the Asian continent, which determines the high relevance of the production of vaccines to protect against foot-and-mouth disease of this serotype.

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

Recovery from illness in an animal with one genotype does not provide immune protection against another [3-5].

Since vaccination is used as the primary control tool in endemic areas, analysis of each pool and the genotypes within it could 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.

For the SAT-2 serotype, 14 topotypes (I–XIV) are distinguished, within which there is no division into genetic groups [1, 3]. Such high genetic and antigen diversity leads to problems in the specific prevention of foot-and-mouth disease when using culture-based inactivated foot-and-mouth disease vaccines, and also complicates the strain-specific diagnostics of the isolated isolates of the foot-and-mouth disease virus. As a result, there is a need to create new diagnostic tools and specific immunoprophylaxis against the foot-and-mouth disease virus of the SAT-2 serotype and, in particular, the SAT-2/VII/Lib-12 and SAT-2/XIV genotypes, whose representatives are actively spreading in Asia and Africa.

In order to prevent the occurrence of foot-and-mouth disease in farms of the Russian Federation, a set of measures is used to combat and prevent foot-and-mouth disease, 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.

To immunize animals, a vaccine made from a virus homologous to field isolates should be used, which has been confirmed by studies by many scientists [6, 7, 8, 9, 10, 11].

This infection continues to be an economically significant problem. Whole-virion vaccines against foot-and-mouth disease are still recognized as the most effective today, which is extremely important for combating the most contagious disease in the world [8, 9, 12, 13].

To conduct an effective vaccination campaign, it is necessary to have an effective and safe vaccine containing the antigen of the virus as an immunogenic component, corresponding to the epizootic spreading isolate of a certain genotype. The creation of such vaccines is still an extremely urgent task. Achieving this goal allows for the effective use of the vaccine in unfavorable regions and a threatened zone to form immunity against a specific genotype of the foot-and-mouth disease virus in order to ensure veterinary welfare [14, 15, 16].

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

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

Literature sources indicate that emergency vaccination of cattle, sheep and pigs can be effective in preventing the disease during the first 4-5 days after vaccination. Research data show that the risk of spreading the infection decreases as the interval between vaccination and infection with the virus increases. In addition, vaccination can reduce the amount of virus excreted compared to non-immunized animals [9, 10, 17].

In the territory of the African continent, as well as South-East, South, East Asia, outbreaks of foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV are now sporadic. There are extremely high risks of introducing isolates of these genotypes to the territory of the Russian Federation, which threatens the biological security of our country in terms of foot-and-mouth disease. It is important, for the prevention of this disease on the specified continents, to produce a vaccine and vaccination for early protection in conditions of periodically occurring outbreaks of foot-and-mouth disease of the specified genotypes.

Thus, there was a need to develop a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV to ensure biological safety and veterinary well-being of the territory of the Russian Federation, neighboring states and countries endemic for foot-and-mouth disease, which will use this drug for disease prevention.

The FMD virus isolate was isolated from cattle in the State of Libya in 2012 and was delivered to the All-Russian Research Institute of Animal Health from the World Reference Laboratory for Foot-and-Mouth Disease, the Pirbright Institute, Great Britain, for research and diagnostic work. The strain "SAT-2 Lib 39/2012" was obtained by adapting the isolate to reproduction in primary trypsinized cell culture SP, in continuous cell cultures 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 comparative analysis of nucleotide sequences, the isolated strain belongs to the SAT-2/VII/Lib-12 genotype of foot-and-mouth disease virus, which differs significantly from the production strains of foot-and-mouth disease virus serotype SAT-2 (Fig. 1).

The foot-and-mouth disease virus isolate was obtained as a result of laboratory diagnostic studies at the All-Russian Research Institute of Animal Health from pathological material collected from cattle in the Hashemite Kingdom of Jordan in August 2023. The isolate was characterized and named strain "SAT-2/XIV/2023". According to the results of comparative analysis of nucleotide sequences, the isolated strain belongs to the SAT-2/XIV genotype of the foot-and-mouth disease virus, which differs significantly from the production strains of the SAT-2 serotype of the foot-and-mouth disease virus (Fig. 1).

A vaccine against foot-and-mouth disease of the SAT-2/VII genotype from the strain "SAT-2/Eritrea/1998" is known, 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 foot-and-mouth disease strain "SAT-2/Eritrea/1998", obtained in a sensitive biological system, and target additives in the form of an adjuvant of aluminum hydroxide and saponin: the concentration of the antigen in an amount of at least 3.5 μg, the content of the adjuvant aluminum hydroxide is 4.4% in terms of dry matter, saponin in an amount of 1.9 mg, an additive in the form of a supporting medium - up to 2.0 cm ³ .

This vaccine uses the SAT-2/VII genotype foot-and-mouth disease virus strain, but the vaccine is adsorbed.

The closest to the proposed invention in terms of the set of essential features is a vaccine against foot-and-mouth disease of the SAT-2/XIV genotype from the strain "SAT-2/XIV/2023", a culture inactivated emulsion vaccine [18], containing an active substance in the form of avirulent and purified culture antigen material from the strain "SAT-2/XIV/2023", homologous to the causative agent of foot-and-mouth disease, obtained in a sensitive biological system, and target additives in the form of the oil adjuvant DMIXVAC 76 V: antigen concentration in an amount of at least 5.0 μg, the content of the oil adjuvant DMIXVAC 76 V is 70%, an additive in the form of a supporting medium is up to 10 cm3 (prototype) [18].

A significant drawback of the prototype vaccine is its low immunogenic activity against emerging strains/isolates of the foot-and-mouth disease virus of the SAT-2/XIV genotype and the lack of protection against foot-and-mouth disease of the SAT-2/VII/Lib-12 and SAT-2/XIV genotypes in the early stages – on the 4th day after vaccination.

To solve the said problem, the present invention was created, which included the development of a culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of the genotypes SAT-2/VII/Lib-12 and SAT-2/XIV. This vaccine assumes a high content of the 146S immunogenic component of the foot-and-mouth disease virus of the genotypes SAT-2/VII/Lib-12 and SAT-2/XIV in the dose of the preparation.

The technical result of using the proposed invention consists in expanding the arsenal of culture inactivated emulsion vaccines for early protection against foot-and-mouth disease virus of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV.

The specified technical result was achieved by creating a culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, 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 homologous to the causative agent of the infection strains "SAT-2 Lib 39/2012" (genotype SAT-2/VII/Lib-12) and "SAT-2/XIV/2023" (genotype SAT-2/XIV) 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 of each; 2) the oil adjuvant DMIXVAC 78 V ("Dmitrievsky Chemical Plant", Russian Federation), which provides an increase in the immune response in animals with a content of 70% by weight,
3) supporting medium – up to 2.0 ^cm3 .

The foot-and-mouth disease virus strain "SAT-2/XIV/2023" 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. 489 - dep / 23-39 - GKShM of the FGBU "ARRIAH".

The foot-and-mouth disease virus strain "SAT-2 Lib 39/2012" 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. 361 - dep / 21-22 - GKShM of the FGBU "ARRIAH".

The indicated strains are adapted to primary trypsinized monolayer cells of the pig 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 mountain ibex (PSGK-30).

For the production of the vaccine, the transplantable suspension culture of BHK-21/SUSP/ARRIAH cells is preferably used as a sensitive biological system, and Hanks' medium without the addition of serum, but with the addition of blood protein hydrolysate in an amount of 5.0% of the total volume at a pH of the medium of 7.50-7.70, is used as a supporting medium. The virus is inactivated using aminoethylethyleneimine (AEEI) with a concentration of 0.035% of the volume of the virus-containing suspension. The inactivated virus is purified from ballast impurities using polyhexamethylene guanidine (PHMG), which is added to the suspension to a concentration of 0.040% of the total volume.

The avirulent and purified antigen of the SAT-2/XIV/2023 (genotype SAT-2/XIV) and SAT-2 Lib 39/2012 (genotype SAT-2/VII) foot-and-mouth disease virus strains is a suspension containing the inactivated 146S immunogenic component of the foot-and-mouth disease virus of each genotype. The concentration of the component in the product is assessed using the quantitative complement fixation reaction (CFR) [19, 20].

To create a vaccine, viral material containing
2.0 cm ³ not less than 15.0 μg of inactivated immunogenic 146S particles of foot-and-mouth disease virus of each genotype genotype SAT-2/XIV and SAT-2/VII. The declared concentration of the immunogenic component in the vaccine preparation is ensured by concentrating the antigen using a Centramate 500S Pall tangential filtration unit.

Culture inactivated emulsion vaccine for early protection against foot-and-mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV is obtained by mixing the purified antigen and the oil adjuvant DMIXVAC 78 V in a ratio of 30% and 70% 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 of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, which ensures the formation of 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 requested:

1. Culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV.

2. Active substance in the form of avirulent and purified antigen material from strains homologous to the causative agent of the disease
"SAT-2 Lib 39/2012" genotype SAT-2/VII and "SAT-2/XIV/2023" genotype SAT-2/XIV of foot-and-mouth disease virus in effective quantities.

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 strains “SAT-2 Lib 39/2012” of the genotype SAT-2/VII and “SAT-2/XIV/2023” of the genotype SAT-2/XIV 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 implementation or special conditions of its use:

1. An avirulent and purified antigen of the strain "SAT-2 Lib 39/2012" of the genotype SAT-2/VII and the strain "SAT-2/XIV/2023" of the genotype SAT-2/XIV of the foot-and-mouth disease virus, obtained preferably 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. An avirulent and purified antigen of the strain "SAT-2 Lib 39/2012" of the genotype SAT-2/VII and the strain "SAT-2/XIV/2023" of the genotype SAT-2/XIV of the foot-and-mouth disease virus, obtained preferably 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 15.0 μg in 2.0 ^cm3 of the finished vaccine preparation.

3. Of the target additives, the vaccine contains the oil adjuvant DMIXVAC 78V.

4. The vaccine contains the oil adjuvant DMIXVAC 78 V in an amount of 70% (by weight) in 2.0 ^cm3 of the finished product.

5. Avirulent and purified antigen of the strain "SAT-2 Lib 39/2012" genotype SAT-2/VII and the strain "SAT-2/XIV/2023" genotype SAT-2/XIV of the foot-and-mouth disease virus, obtained preferably in the continuous suspension cell line BHK-21/SUSP/ARRIAH, oil adjuvant DMIXVAC 78 V, additive in the finished product with a volume of 2.0 ^cm3 in the form of a supporting medium in the following quantities:

Avirulent and purified antigen of the strain "SAT-2 Lib 39/2012" genotype SAT-2/VII foot and mouth disease virus not less than 15.0 mcg Avirulent and purified antigen of the strain "SAT-2/XIV/2023" genotype SAT-2/XIV foot and mouth disease virus not less than 15.0 mcg Oil adjuvant DMIXVAC 78 V 70% (by weight) Supportive environment up to 2.0 ^cm3

The proposed culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV has high immunogenic activity and provides reliable protection against isolates of foot-and-mouth disease virus of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV.

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 strains "SAT-2 Lib 39/2012" of the genotype SAT-2/VII and "SAT-2/XIV/2023" of the genotype SAT-2/XIV 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 genotypes, which in recent years have caused outbreaks in Asian and African countries.

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

Fig. 1 – Dendrogram reflecting the phylogenetic relationship of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” of foot-and-mouth disease virus with epizootic strains of serological type SAT-2. 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 lists of sequences:

SEQ ID NO:1 represents the nucleotide sequence of the 1D gene of the VP ₁ protein of the strain "SAT-2 Lib 39/2012" of the foot-and-mouth disease virus of the genotype SAT-2/VII/Lib-12;

SEQ ID NO:2 represents the amino acid sequence of the 1D gene of the VP ₁ protein of the strain "SAT-2 Lib 39/2012" of the foot-and-mouth disease virus of the genotype SAT-2/VII/Lib-12.

SEQ ID NO:3 represents the nucleotide sequence of the 1D gene of the VP ₁ protein of the strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus of the genotype SAT-2/XIV;

SEQ ID NO:4 represents the amino acid sequence of the 1D gene of the VP ₁ protein of the strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus of the genotype SAT-2/XIV.

The strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus has the following taxonomy: family Picornaviridae , genus Aphthovirus , species Foot-and-mouth disease virus , serotype SAT-2 , genotype SAT-2/VII/Lib-12. 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 25 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

According to its antigenic properties, the SAT-2 Lib 39/2012 strain of the foot-and-mouth disease virus belongs to the SAT-2 serotype. The virus is stably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. In animals that have recovered from the disease, type-specific antibodies are formed in the blood serum, which are detected in the enzyme-linked immunosorbent assay (ELISA) and the microneutralization reaction (MNR).

The primary structure of the 1D gene of the VP protein was determined using nucleotide sequencing₁strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus belongs to the SAT-2/VII/Lib-12 genotype (Fig. 1).

The antigenic affinity (r ₁ ) of the SAT-2 Lib 39/2012 strain of foot-and-mouth disease virus 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-2/ZIM/14/2002”) (genotype SAT-2/I),

- strain (“SAT-2/ZIM/5/81”) (genotype SAT-2/II),

- strain (“SAT-2/BOT/P3/98”) (genotype SAT-2/III),

- strain (“SAT-2/ETH/1/90”) (genotype SAT-2/IV),

- strain (“SAT-2/GHA/2/90”) (genotype SAT-2/V),

- strain (“SAT-2/GAM/8/79”) (genotype SAT-2/VI),

- strain (“SAT-2/SAU/6/2000”) (genotype SAT-2/VII),

- strain (“SAT-2/RWO/1/00”) (genotype SAT-2/VIII),

- strain (“SAT-2/KEN/2/84”) (genotype SAT-2/IX),

- strain (“SAT-2/UGA/19/98”) (genotype SAT-2/X),

- strain (“SAT-2/ANG/4/74”) (genotype SAT-2/XI),

- strain (“SAT-2/UGA/51/75”) (genotype SAT-2/XII),

- strain (“SAT-2/ETH/2/2007”) (genotype SAT-2/XIII),

- strain (“SAT-2/ETH/2/91”) (genotype SAT-2/XIV).

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype SAT-2 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 lg of serum titers against heterologous and homologous virus [7, 8, 9, 20, 21].

The r ₁ value in the RMM 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 relatedness is observed at the r ₁ value in the RMM tending to 1.0.

The antigenic relationship indices in the study of the strain “SAT-2 Lib 39/2012” were r ₁ from 0.01 to 0.25, which indicates the absence of obvious antigenic relationship with production strains of foot-and-mouth disease virus of the SAT-2 serotype.

Geno- and chemotaxonomic characteristics

The strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular weight of 8.085×10 ⁶ D. The nucleic acid is represented by a single-stranded linear molecule with a molecular weight of 2.854×10 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 VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Viral RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural viral polypeptides, including such an important enzyme as RNA-dependent RNA polymerase (3D gene), which is involved in RNA replication for virion assembly.

Physical properties

The mass of the virion is 8.452×10 ^-18 g. The buoyant density is 1.475 g/cm ³ .

Resistance to external factors

The strain "SAT-2 Lib 39/2012" of the foot-and-mouth disease virus is resistant to ether, chloroform, and acetone. It is most stable at pH 7.45-7.70. Shifts in pH to the acidic and strongly alkaline side lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-irradiation, high temperatures (above 38.8°C).

Additional features and properties

Reactogenicity – the antigen does not have reactogenic properties.

Pathogenicity – the antigen is not pathogenic for ungulates.

Virulence – the antigen is not virulent for naturally susceptible animals upon contact, aerosol and parenteral infection.

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

Biotechnological characteristics

The strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus is reproduced in continuous cell cultures: Siberian ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (VNK-21).

During the test, 5 consecutive passages of the SAT-2 Lib 39/2012 strain of foot-and-mouth disease virus were carried out in continuous cell cultures PSGK-30, BNK-21, IB-RS-2. Biological properties were characterized by determining the infectious activity of the virus of each passage in the continuous cell line IB-RS-2 and in naturally susceptible animals - cattle and pigs.

The strain "SAT-2/XIV/2023" of foot-and-mouth disease virus is characterized by the following features and properties.

Morphological features

The strain "SAT-2/XIV/2023" of foot-and-mouth disease virus has the following taxonomy: family Picornaviridae , genus Aphthovirus , species Foot-and-mouth disease virus , serotype SAT-2 , genotype SAT-2/XIV. 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 ixahedral, the size is 23 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

According to its antigenic properties, the SAT-2/XIV/2023 strain of the foot-and-mouth disease virus belongs to the SAT-2 serotype. The virus is stably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. In animals that have recovered from the disease, type-specific antibodies are formed in the blood serum, which are detected in an enzyme-linked immunosorbent assay and a microneutralization reaction (MNR).

The primary structure of the 1D gene of the VP protein was determined using nucleotide sequencing₁strain "SAT-2/XIV/2023" of foot-and-mouth disease virus. Comparative analysis of nucleotide sequences showed that the strain "SAT-2/XIV/2023" of foot-and-mouth disease virus belongs to the genotype SAT-2/XIV (Fig. 1).

The antigenic affinity (r ₁ ) of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus was studied in the 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-2/ZIM/14/2002”) (genotype SAT-2/I),

- strain (“SAT-2/ZIM/5/81”) (genotype SAT-2/II),

- strain (“SAT-2/BOT/P3/98”) (genotype SAT-2/III),

- strain (“SAT-2/ETH/1/90”) (genotype SAT-2/IV),

- strain (“SAT-2/GHA/2/90”) (genotype SAT-2/V),

- strain (“SAT-2/GAM/8/79”) (genotype SAT-2/VI),

- strain (“SAT-2/SAU/6/2000”) (genotype SAT-2/VII),

- strain (“SAT-2/RWO/1/00”) (genotype SAT-2/VIII),

- strain (“SAT-2/KEN/2/84”) (genotype SAT-2/IX),

- strain (“SAT-2/UGA/19/98”) (genotype SAT-2/X),

- strain (“SAT-2/ANG/4/74”) (genotype SAT-2/XI),

- strain (“SAT-2/UGA/51/75”) (genotype SAT-2/XII),

- strain (“SAT-2/ETH/2/2007”) (genotype SAT-2/XIII),

- strain (“SAT-2/ETH/2/91”) (genotype SAT-2/XIV).

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from industrial strains of foot-and-mouth disease virus serotype SAT-2 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 lg of serum titers against heterologous and homologous virus [7, 8, 9, 20, 21].

The r ₁ value in the RMM 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 relatedness is observed at the r ₁ value in the RMM tending to 1.0.

The antigenic relationship indices in the study of the strain "SAT-2/XIV/2023" were r ₁ from 0.01 to 0.27, which indicates the absence of obvious antigenic relationship with production strains of foot-and-mouth disease virus of serotype SAT-2.

Geno- and chemotaxonomic characteristics

The strain "SAT-2/XIV/2023" of foot-and-mouth disease virus is an RNA(+)-containing virus with a molecular weight of 8.079×10 ⁶ D. The nucleic acid is represented by a single-stranded linear molecule with a molecular weight of 2.84×10 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 VP ₁ . The virion contains approximately 31.5% RNA and 68.5% protein. Viral RNA is infectious and is involved in the formation of precursor proteins in infected cells. The precursors, in turn, are cleaved to form more stable structural and non-structural viral polypeptides, including such an important enzyme as RNA-dependent RNA polymerase (3D gene), which is involved in RNA replication for virion assembly.

Physical properties

The mass of the virion is 8.44×10 ^-18 g. The buoyant density is 1.47 g/cm ³ .

Resistance to external factors

The strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus is resistant to ether, chloroform, and acetone. It is most stable at pH 7.44-7.70. Shifts in pH to the acidic and strongly alkaline side lead to inactivation of the virus. It is sensitive to formaldehyde, UV radiation, γ-irradiation, high temperatures (above 38.7°C).

Additional features and properties

Reactogenicity – the antigen does not have reactogenic properties.

Pathogenicity – the antigen is not pathogenic for ungulates.

Virulence – the antigen is not virulent for naturally susceptible animals upon contact, aerosol and parenteral infection.

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

Biotechnological characteristics

The strain "SAT-2/XIV/2023" of the foot-and-mouth disease virus is reproduced in continuous cell cultures: Siberian ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (VNK-21).

During the test, 5 consecutive passages of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus were carried out in continuous cell cultures PSGK-30, BHK-21, IB-RS-2. Biological properties were characterized by determining the infectious activity of the virus of 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 virus genotypes SAT-2/VII/Lib-12 and SAT-2/XIV and to prevent the emergence of new foci 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 genotypes SAT-2/VII/Lib-12 and SAT-2/XIV 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 strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” of foot-and-mouth disease virus according to PCR and nucleotide sequencing data

The primary structure of the 1D gene (VP ₁ protein) of the SAT-2 Lib 39/2012 and SAT-2/XIV/2023 foot-and-mouth disease virus vaccine strains was analyzed using Sanger nucleotide sequencing and the position of this strain on the phylogenetic tree of the SAT-2 foot-and-mouth disease virus serotype 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 (19 representatives) of the SAT-2 foot-and-mouth disease virus serotype.

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 strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” with other isolates and strains.

The nucleotide sequence of the gene of the VP ₁ protein of the strain "SAT-2 Lib 39/2012" of the genotype SAT-2/VII/Lib-12 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 strain "SAT-2 Lib 39/2012" of the genotype SAT-2/VII/Lib-12 foot-and-mouth disease virus is reflected in SEQ ID NO: 2.

The nucleotide sequence of the gene of the VP ₁ protein of the strain "SAT-2/XIV/2023" genotype SAT-2/XIV foot-and-mouth disease virus is presented in SEQ ID NO:3. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the strain "SAT-2/XIV/2023" genotype SAT-2/XIV foot-and-mouth disease virus is reflected in SEQ ID NO:4.

A phylogenetic analysis was performed for the SAT-2 Lib 39/2012 and SAT-2/XIV/2023 foot-and-mouth disease virus vaccine strains. 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 the bootstrap test (increased from the standard 100 to 1000 repeats for high confidence) is shown next to the branches [22, 23]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [24] and are expressed as base substitutions per site. This analysis included 19 nucleotide sequences, including the 1D gene sequence of the SAT-2 Lib 39/2012 and SAT-2/XIV/2023 foot-and-mouth disease virus vaccine strains. All positions containing gaps and missing data were removed (complete removal option). Evolutionary analysis was performed in MEGA 6 [25].

As a result of the work carried out on comparison of complete nucleotide sequences of gene 1D (VP ₁ protein) of vaccine strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" of foot-and-mouth disease virus and other isolates/strains of foot-and-mouth disease virus of serotype SAT-2, the position of the studied strain on the phylogenetic tree was determined (Fig. 1). Thus, strain "SAT-2 Lib 39/2012" belongs to the genotype SAT-2/VII/Lib-12, strain "SAT-2/XIV/2023" – to the genotype SAT-2/XIV, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" to the transplantable monolayer culture of Siberian ibex kidney cells PSGK-30

To infect the continuous monolayer culture of Siberian mountain ibex kidney cells PSGK-30, a 10% aphthous suspension of foot-and-mouth disease virus obtained from cattle aphthae (passage 2) was used. The inoculation concentration of cells was 0.20-0.25 million cells/ ^cm3 , the dose of virus infection was 0.001 _TCID50 /cell.

According to the results of the study, reproduction of the foot-and-mouth disease virus strain "SAT-2 Lib 39/2012" occurred with specific destruction of the monolayer by 90-100% in 18 hours. During 6 consecutive passages, an increase in the values of the titer of infectious activity of the virus was noted from 5.90±0.10 to 7.45±0.10 lg TCID ₅₀ /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of the 6th passage (7.45±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from the 1st to the 6th passage changed from 0.28±0.02 to 0.90±0.02 μg/ ^cm3 . Moreover, the highest amount of the 146S component was noted at the stage of the 6th passage (0.90±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 99.14 to 100.00%. Thus, over the course of 6 consecutive passages, the adaptation of the SAT-2 Lib 39/2012 foot-and-mouth disease virus strain to the transplantable monolayer cell line PSGK-30 was successfully carried out.

According to the results of the study, the reproduction of the foot-and-mouth disease virus strain "SAT-2/ _{XIV/2023" occurred with a specific destruction of the monolayer by 90-100% in 14 hours. During 6 consecutive passages, an increase in the values of the titer of infectious activity of the virus was noted from 6.01±0.10 to 7.75±0.10 lg TCID 50} /cm ³ .

The maximum value of the virus infectious activity titer was achieved at the stage of passage 6 (7.75±0.10 lg _TCID50 / ^cm3 ). The concentration of 146S particles from passages 1 to 6 changed from 0.32±0.02 to 0.96±0.02 μg/ ^cm3 . Moreover, the highest amount of the 146S component was noted at the stage of passage 6 (0.96±0.02 μg/ ^cm3 ). The reliability degree ( ^R2 ) of the study results in the quantitative version of RT-PCR-RV ranged from 99.02 to 100.00%. Thus, over the course of 6 consecutive passages, the adaptation of the SAT-2/XIV/2023 foot-and-mouth disease virus strain to the transplantable monolayer cell line PSGK-30 was successfully carried out.

Example 3. Study of conditions for monolayer cultivation of foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" on an industrial scale

In the process of industrial cultivation of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" foot-and-mouth disease virus in the continuous monolayer culture of PSGK-30 cells, the optimal conditions for culturing the virus were selected based on the supporting medium, temperature factor, and dose of infection of the cells with the studied virus.

At the first stage of the study, the influence of the composition of the supporting medium on the reproduction of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” was assessed. foot-and-mouth disease virus in a monolayer culture of PSGK-30 cells on a production scale (at the stage from the 5th to the 6th passage). For comparison, three media with the addition of 2.0% fetal calf blood serum were used: 1) Eagle's DMEM medium; 2) RPMI-1640 medium; 3) Hanks' solution. The inoculation concentration of cells was 0.20 million cells/cm³, the dose of virus infection is 0.1000-0.0001 TCD₅₀/cl. The virus was cultivated at temperatures of 35±0.2 – 38±0.2°C until the cell monolayer was destroyed by at least 85%. Results of reproduction of strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” foot-and-mouth disease virus with supporting media of different compositions are shown in Tables 1 and 2.

It is evident from the data in Table 1 that during reproduction of the SAT-2 Lib 39/2012 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 13-14 hours with accumulation of the 146S component in the resulting suspension in the amount of 1.08±0.02 μg/ ^cm3 and an infectious activity titer of 7.80±0.10 lg _TCID50 / ^cm3 . When using the RPMI-1640 medium and Eagle DMEM, the virus reproduction rates were lower.

Table 2 shows that when reproducing the SAT-2/XIV/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 13-14 hours with the accumulation of the 146S component in the resulting suspension in an amount of 1.25±0.02 μg/cm ³ and an infectious activity titer of 7.91±0.10 lg TCID ₅₀ /cm ³ . When using RPMI-1640 and Eagle DMEM media, the virus reproduction rates were lower.

At the next stage of studying the conditions of monolayer cultivation of strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” foot-and-mouth disease virus in the PSGK-30 cell culture under production conditions, the effect of temperature on the virus reproduction process was assessed. For this purpose, the virus was cultured in Eagle's DMEM medium with 2% bovine serum 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 TCD₅₀/cl.

The cultivation of the foot-and-mouth disease virus strain "SAT-2 Lib 39/2012" was carried out until the destruction of the cell monolayer by 85-100% for 13-25 hours (Table 1). According to the results of the study, it was revealed that for complete reproduction of the foot-and-mouth disease virus strain "SAT-2 Lib 39/2012" in a monolayer culture of PSGK-30 cells, the optimal temperature of the medium is 37.0±0.2°C, at which in 13-14 hours the development of the CPE reached 95-100% with the accumulation of 146S particles equal to 1.08±0.02 μg/cm ³ and the titer of infectious activity of the virus of 7.80±0.10 lg TCID ₅₀ /cm ³ .

The SAT-2/XIV/2023 strain of foot-and-mouth disease virus was cultured until the cell monolayer was 85-100% destroyed over 13-24 hours (Table 2). The results of the study revealed that for complete reproduction of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus in a monolayer culture of PSGK-30 cells, the optimal temperature was 37.0±0.2°C, at which the CPE development reached 95-100% in 13-14 hours with an accumulation of 146S particles equal to 1.25±0.02 μg/cm ³ and an infectious activity titer of the virus of 7.91±0.10 lg TCID ₅₀ /cm ³ .

The effect of the infection dose of the PSGK-30 cell line monolayer with the strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" separately during cultivation on a production scale was assessed. For this, different doses of the virus were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCID ₅₀ /cell. Eagle's DMEM medium with 2% bovine blood serum was used as a maintenance medium.

Reproduction of the foot-and-mouth disease virus strain "SAT-2/Lib 39/2012" was carried out at a temperature of 37.0±0.2°C for 13-23 hours 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 1.08±0.02 μg/cm ³ with an infectious activity titer of the virus of 7.80±0.10 lg TCID ₅₀ /cm ³ .

Reproduction of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" was carried out at a temperature of 37.0±0.2°C for 13-19 hours 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 2, 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 1.25±0.02 μg/cm ³ with an infectious activity titer of the virus of 7.91±0.10 lg TCID ₅₀ /cm ³ .

Thus, the conditions of monolayer cultivation of the SAT-2 Lib 39/2012 and SAT-2/XIV/2023 foot-and-mouth disease virus strains 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; 2) cultivation temperature – 37.0±0.2°C; 3) virus infection dose – 0.01-0.001 _TCID50 /cell.

Example 4. Study of conditions for suspension cultivation of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” of foot-and-mouth disease virus on an industrial scale

During industrial cultivation of the SAT-2 Lib 39/2012 and SAT-2/XIV/2023 foot-and-mouth disease virus strains in a continuous suspension culture of BHK-21/SUSP/ARRIAH cells, a search was conducted for optimal parameters for successful reproduction of the foot-and-mouth disease pathogen using different cell concentrations, temperature conditions, and virus infection doses.

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: 2.5; 3.0; 3.5; 4.0 million cells/cm³The virus cultivation temperature was 37.0±0.2°C, the virus infection dose was 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-2 Lib 39/2012" The results of the analysis of the results of the study of the foot-and-mouth disease virus in suspension with different cell concentrations are presented in Table 3.

As follows from Table 3, when cultivating the foot-and-mouth disease virus strain “SAT-2 Lib 39/2012” 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 2.5 million cells/cm³amounted to 0.96±0.02 μg/cm³, with a concentration of 3.0 million cells/cm³– 1.23±0.02 μg/cm³, with a concentration of 3.5 million cells/cm³– 1.56±0.02 μg/cm³, and with a concentration of 4.0 million cells/cm³– 1.60±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "SAT-2 Lib 39/2012" For the foot-and-mouth disease virus, a sufficient concentration of BHK-21/SUSP/ARRIAH cells in suspension is 3.5 million cells/cm³(at a concentration of 4.0 million cells/cm³the concentration is slightly higher, but the production costs per cell number are higher, based on this, it is economically feasible to use a cell concentration equal to 3.5 million cells/cm³). The titer of infectious activity of the virus was 8.15±0.10 lg TCD₅₀/cm³.

Results of suspension cultivation of strain "SAT-2/XIV/2023" The results of the analysis of the results of the cell suspensions of the foot-and-mouth disease virus are presented in Table 4.

As follows from Table 4, when cultivating the foot-and-mouth disease virus strain “SAT-2/XIV/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 2.5 million cells/cm³amounted to 1.14±0.02 μg/cm³, with a concentration of 3.0 million cells/cm³– 1.40±0.02 μg/cm³, with a concentration of 3.5 million cells/cm³– 2.23±0.02 μg/cm³, and with a concentration of 4.0 million cells/cm³– 2.32±0.02 μg/cm³. As follows from the data obtained for the reproduction of the strain "SAT-2/XIV/2023" For the foot-and-mouth disease virus, a sufficient concentration of BHK-21/SUSP/ARRIAH cells in suspension is 3.5 million cells/cm³(at a concentration of 4.0 million cells/cm³the concentration is slightly higher, but the production costs per cell number are higher, based on this, it is economically feasible to use a cell concentration equal to 3.5 million cells/cm³). The titer of infectious activity of the virus was 8.45±0.10 lg TCD₅₀/cm³.

At the next stage of the work, the influence of the temperature factor on the reproduction of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” was studied. foot-and-mouth disease virus in suspension culture of BHK-21/SUSP/ARRIAH cell line 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 cell culture with the virus was 0.010-0.001 TCD₅₀/cell. The virus was cultivated until the CPE was at least 90% (priority was given to complete specific destruction of cells).

Based on the results of the analysis, it was determined that for the complete reproduction of the strain “SAT-2 Lib 39/2012” foot-and-mouth disease virus in a suspension culture of BHK-21/SUSP/ARRIAH cells, the optimal temperature of the medium is 37.0±0.2^°C, at which within 12-13 hours specific cell death is observed by 95-100% with a high accumulation of the 146S component (1.56±0.02 μg/cm³) and a titer of infectious activity of the virus of 8.15±0.10 lg TCD₅₀/cm³.

Based on the analysis results, it was determined that for the full reproduction of the strain “SAT-2/XIV/2023” 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% is observed within 12-13 hours with a high accumulation of the 146S component (2.23±0.02 μg/cm³) and a titer of infectious activity of the virus of 8.45±0.10 lg TCD₅₀/cm³.

In the course of work on studying the conditions of suspension cultivation of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" of the foot-and-mouth disease virus 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 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 3, the highest accumulation of the 146S component of the foot-and-mouth disease virus strain “SAT-2 Lib 39/2012” was observed at an infection dose of 0.010-0.001 TCID ₅₀ /cell (1.56±0.02 μg/cm ³ ) with a titer of infectious activity of the virus equal to 8.15±0.10 lg TCID ₅₀ /cm ³ .

The highest accumulation of the 146S component of the foot-and-mouth disease virus strain "SAT-2/XIV/2023" was observed at an infection dose of 0.010-0.001 _TCID50 /cell (2.23±0.02 μg/ ^cm3 ) with a titer of infectious activity of the virus equal to 8.45±0.10 lg _TCID50 / ^cm3 (Table 4).

Thus, a study of three parameters of suspension cultivation of strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” was carried out. foot-and-mouth disease virus in the suspension cell line BHK-21/SUSP/ARRIAH on an industrial scale.

Optimal conditions for reproduction of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" have been identified foot-and-mouth disease virus in suspension culture of BHK-21/SUSP/ARRIAH cells: 1) cell concentration before infection – 3.5 million cells/cm³; 2) cultivation temperature regime – 37.0±0.2°С; 3) virus infection dose – 0.010-0.001 TCD₅₀/cl.

Example 5. Inactivation of the foot-and-mouth disease virus suspension of the strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" of the 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°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 0.035%. Inactivation of the foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" 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 taken every hour. The obtained samples were tested for the presence of infectious activity of the foot-and-mouth disease virus in the primary culture of SP pig kidney cells. The results are presented in Table 5, from which it is evident that complete inactivation of the foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" separately, reproduced in cultivators, occurred 5 hours after the addition of 1,2-AEEI.

The prepared viral inactivated suspensions were studied in the RSC to assess the content of viral components in them. For the strain "SAT-2 Lib 39/2012", the concentration of the 146S component was 1.54±0.02 μg/cm ³ , for the strain "SAT-2/XIV/2023" - 2.20±0.02 μg/cm ³ .

Example 6. Selection of conditions for purification of suspensions of foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023"

The suspensions of the foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" obtained by reproduction in the suspension cell line BHK-21/SUSP/ARRIAH were purified. Polyhexamethylene guanidine (PHMG) with concentrations of 0.035, 0.040 and 0.045% was used to obtain purified foot-and-mouth disease virus antigen. Before and after the process of purification of the foot-and-mouth disease virus antigen of the specified 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 6, from which it follows that as a result of purification of the foot-and-mouth disease virus antigen of the SAT-2 Lib 39/2012 strain using PHMG at a concentration of 0.035%, a decrease in the concentration of ballast protein by 4% and immunogenic components by 0.3% was noted. When using PHMG at a concentration of 0.040%, a decrease in the amount of ballast protein by 15% and the 146S component by 1.2% was observed. Using PHMG at a concentration of 0.045%, the content of ballast protein decreased by 25% and immunogenic components by 7%.

As a result of purification of the foot-and-mouth disease virus antigen of the SAT-2/XIV/2023 strain using PHMG at a concentration of 0.035%, a decrease in the concentration of ballast protein by 5% and immunogenic components by 0.4% was observed. When using PHMG at a concentration of 0.040%, a decrease in the amount of ballast protein by 17% and the 146S component by 1.3% was observed. Using PHMG at a concentration of 0.045%, the content of ballast protein decreased by 27% and immunogenic components by 8%.

Thus, having studied the conditions for purifying the antigen of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023”, we came to the conclusion that it is optimal to use PHMG with a concentration of 0.040% to obtain a purified product.

Example 7. Selection of conditions for concentrating the suspension of foot-and-mouth disease virus antigen of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023"

The culture inactivated suspension of the strain " SAT-2 Lib 39/2012 " , obtained using the suspension cell line BHK-21/SUSP/ARRIAH, was concentrated by 40 times by volume, and the strain "SAT-2/XIV/2023" - by 28 times. The following techniques were used to increase the concentration of immunogenic components of the foot-and-mouth disease virus antigen: 1) addition of 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) addition of 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 Centramate 500S Pall tangential filtration unit for 3 hours at a working pressure on the filter of 2.2-2.3 atm.

Before and after the process of concentrating the FMD virus antigen suspensions, the concentration of total viral protein and immunogenic components in the quantitative version of the RSC was determined. The results of the studies are presented in Table 7.

From the data in Table 7 it is evident that when concentrating the antigen of the strain “SAT-2 Lib 39/2012” of foot-and-mouth disease virus using PEG-6000 for 4 hours, an increase in the content of components compared to the initial values (before concentration) was noted by 33 times. Using the same polymer, but increasing the exposure time to 12 hours, the concentration of virus components was increased by 35 times. Using the concentration method using tangential filtration, it was possible to increase the amount of immunogenic components of the antigen of the strain "SAT-2 / XIV / 2023" foot-and-mouth disease virus in suspension by 39.6 times.

When concentrating the antigen of the strain "SAT-2/XIV/2023" of foot-and-mouth disease virus using PEG-6000 for 4 hours, an increase in the content of components compared to the initial values (before concentration) was noted by 22 times. Using the same polymer, but increasing the exposure time to 12 hours, the concentration of virus components was increased by 25.5 times. Using the concentration method using tangential filtration, it was possible to increase the amount of immunogenic components of the antigen of the strain "SAT-2 / XIV / 2023" foot-and-mouth disease virus in suspension by 27.8 times.

Thus, the tangential filtration technology made it possible to obtain the foot-and-mouth disease virus antigen of the strains “SAT-2 Lib 39/2012” and “SAT-2/XIV/2023” with a high concentration of the 146S component to obtain a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV. The resulting concentrates were combined in a 1:1 ratio.

Example 8. Selection of the antigen-adjuvant ratio

To produce the vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, the oil adjuvant DMIXVAC 78 V was selected as an adjuvant in an amount of 70% by weight. The antigen was 30% by weight (15% for each antigen).

Example 9. Formulation of a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV

From the obtained concentrate of foot-and-mouth disease virus antigen of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023", a culture inactivated emulsion vaccine was produced for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV using the oil adjuvant DMIXVAC 78 V. The amount of 146S immunogenic component in a dose of 2.0 cm³146S component vaccines foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" amounted to 18.06 and 18.10 μg/cm³ respectively.

Example 10. Immunization of animals with a culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV

A series of dilutions for pigs with a 5-fold step were prepared from the obtained culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (the proposed invention) . 15 pigs were divided into 3 groups of 5 pigs each. The immunizing dose was 2.0 cm ³ . 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, the third group of animals (Nos. 11-15) - with the vaccine diluted 1/25. The preparation was administered intramuscularly into the middle third of the neck. Two heads without vaccination 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 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 version of the enzyme-linked immunosorbent assay (ELISA) in accordance with OIE recommendations [3]. The obtained sera were tested 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 reflected in Table 8 for each declared strain of foot-and-mouth disease virus separately, from which it is evident that the blood sera of animals 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 genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (proposed invention)

To determine the avirulence of the culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of the genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, a selected sample of the vaccine preparation was administered intradermally to pigs at 0.1 cm ³ . The study used Landrace pigs weighing 30-40 kg. During 10 days of observation, the animals remained clinically healthy, and pathological examination did not reveal changes characteristic of foot-and-mouth disease. This indicated the absence of virulent properties of the developed vaccine.

The safety of the product was monitored on animals by intramuscular administration of the vaccine at a dose of 6.0 cm ³ (a triple dose of 2.0 cm ³ ). The observation period was also 10 days. It should be noted that after immunization, the animal's body temperature can rise to 40.3°C and remain at this level for 1-2 days, which does not go beyond the norm after the administration of the vaccine.

According to the results of the research, the vaccine is a culture inactivated emulsion vaccine for early protection against foot and mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (proposed invention)was recognized as 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 inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (proposed invention) for the ability to induce virus-neutralizing antibodies in naturally susceptible animals

On the 4th and 7th days after the introduction of the culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (proposed invention) , blood was collected from 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 the antibody titer against the strain "SAT-2/VII/Lib-12" on the 4th day after immunization were 5.40±0.14 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.05±0.11 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 2.90±0.29 log ₂ SN ₅₀ (Table 9). The average values of antibody titers against the SAT-2/VII/Lib-12 strain on the 7th day after immunization were 7.75±0.18 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) – 5.05±0.21 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) – 4.40±0.22 log ₂ SN ₅₀ .

It was found that in pigs immunized with the vaccine in a whole dose (5 heads), the average values of the antibody titer against the strain "SAT-2/XIV/2023" on the 4th day after immunization were 5.75±0.25 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 4.30±0.33 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 3.30±0.33 log ₂ SN ₅₀ (Table 10). The average values of antibody titers against the SAT-2/XIV/2023 strain on the 7th day after immunization were 8.00±0.25 log ₂ SN ₅₀ , with a dilution of 1/5 (5 heads) - 5.40±0.29 log ₂ SN ₅₀ , with a dilution of 1/25 (5 heads) - 4.55±0.37 log ₂ SN ₅₀ .

The obtained data from the microneutralization reaction indicate that after the administration of the vaccine in its entirety, 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 culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (proposed invention), by the method of control infection of naturally susceptible animals

Pigs immunized in the amount of 15 heads with the vaccine in whole form and in dilutions were infected with the control strain "SAT-2 Lib 39/2012", the other 15 heads were infected with the foot-and-mouth disease virus of the strain "SAT-2/XIV/2023" 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 limbs were considered protected from foot-and-mouth disease. Primary aphthae were not taken into account.

The results of the control infection are presented in Tables 9 and 10, from which it follows that the culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV (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 for each of the strains of foot-and-mouth disease virus antigen included in the preparation contains 55.9 PD ₅₀ and 0.036 IMD ₅₀ for pigs.

Thus, the above information indicates that the culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of the SAT-2/VII/Lib-12 and SAT-2/XIV genotypes, 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 strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023", 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 foot-and-mouth disease virus of the SAT-2/VII/Lib-12 and SAT-2/XIV genotypes circulating in Africa and Asia. In addition, the culture-based inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV is manufactured using the domestically produced oil adjuvant DMIXVAC 78 V (Dmitrievsky Chemical Plant, Russian Federation).

Sources of information taken into account when drafting the description of the invention for the application for the issuance of a Russian Federation patent for the invention "Culture inactivated emulsion vaccine for early protection against foot-and-mouth disease of genotypes SAT-2/VII/Lib-12 and SAT-2/XIV"

1. Foot-and-mouth disease. Preventive measures. URL: http://89.rospotrebnadzor.ru/directions/epid_nadzor/146902 (Accessed: 01.06.2024).

2. The danger of foot-and-mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Accessed: 05.06.2024).

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. – Moscow: Agropromizdat, 1990. – 320 p.

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

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

7. Brown F. A brief history of FMD and its causal 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 09.05.2024).

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 on 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 rationale for the need to obtain new relevant production strains / Veterinary science and feeding. - 2019. - No. 3. - P. 29-31.

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17. Patent for invention No. 2804875 “Vaccine against foot-and-mouth disease of genotype SAT-2/VII from strain “SAT-2/Eritrea/1998”, culture-based inactivated sorbed vaccine” / invention priority: May 11, 2023, application No. 2023112437.

18. Patent for invention No. 2824660 “Foot-and-mouth disease vaccine of genotype SAT-2/XIV from strain “SAT-2/XIV/2023”, culture-based inactivated emulsion vaccine” / invention priority: April 3, 2024, application No. 2024108919.

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

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

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

Table 1

Results of adaptation of the strain "SAT-2 Lib 39/2012" 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 RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCD ₅₀ /cm ³ Composition of the supporting environment Medium Needle DMEM+ 2% S _cattle 24-25 95-100 0.42±0.02 6.15±0.10 RPMI-1640 + 2% S _Cattle Medium 23-24 95-100 0.65±0.02 6.54±0.10 Hanks solution with 5% GBC + 2% S _cattle 13-14 95-100 1.08±0.02 7.80±0.10 Temperature, °C 35.0±0.2 24-25 85-90 0.60±0.02 6.48±0.10 36.0±0.2 19-20 85-90 0.45±0.03 6.17±0.10 37.0±0.2 13-14 95-100 1.08±0.02 7.80±0.10 38.0±0.2 18-19 95-100 0.70±0.02 6.63±0.10 Dose of infection 0.1-0.01 15-16 90-95 0.70±0.02 6.61±0.10 0.01-0.001 13-14 95-100 1.08±0.02 7.80±0.10 0.001-0.0001 22-23 85-90 0.50±0.02 6.11±0.10

Note: S _KRS – bovine serum,

GBP – blood protein hydrolysate,

DMEM is the name of the Eagle medium,

RPMI-1640 is the name of the culture medium,

CPE – cytopathic effect.

Table 2

Results of adaptation of the SAT-2/XIV/2023 strain of foot-and-mouth disease virus when cultivated 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 RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCD ₅₀ /cm ³ Composition of the supporting environment Medium Needle DMEM+ 2% S _cattle 24-25 95-100 0.50±0.02 6.30±0.10 RPMI-1640 + 2% S _Cattle Medium 22-23 95-100 0.71±0.02 6.50±0.10 Hanks solution with 5% GBC + 2% S _cattle 13-14 95-100 1.25±0.02 7.91±0.10 Temperature, °C 35.0±0.2 23-24 85-90 0.60±0.02 6.42±0.10 36.0±0.2 20-21 90-95 0.75±0.03 6.59±0.10 37.0±0.2 13-14 95-100 1.25±0.02 7.91±0.10 38.0±0.2 17-18 95-100 0.85±0.02 6.78±0.10 Dose of infection 0.1-0.01 14-15 90-95 0.85±0.02 6.61±0.10 0.01-0.001 13-14 95-100 1.25±0.02 7.91±0.10 0.001-0.0001 18-19 90-95 0.79±0.02 6.55±0.10

Note: S _KRS – bovine serum,

GBP – blood protein hydrolysate,

DMEM is the name of the Eagle medium,

RPMI-1640 is the name of the culture medium,

CPE – cytopathic effect.

Table 3

Results of reproduction of the strain "SAT-2 Lib 39/2012" of foot-and-mouth disease virus in the transplantable 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 RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCD ₅₀ /cm ³ Cell concentration before infection, million cells/ ^cm3 2.5 11-12 95-100 0.96±0.02 7.45±0.10 3.0 12-13 95-100 1.23±0.02 7.96±0.10 3.5 12-13 95-100 1.56±0.02 8.15±0.10 4.0 14 95-100 1.60±0.02 8.20±0.10 Temperature, °C 35.0±0.2 21-22 90-95 0.42±0.02 6.01±0.10 36.0±0.2 16-18 90-95 1.20±0.02 7.92±0.10 37.0±0.2 12-13 95-100 1.56±0.02 8.15±0.10 38.0±0.2 15-16 95-100 1.10±0.02 7.72±0.10 Dose of infection 0.1-0.01 15 95-100 0.80±0.02 6.70±0.10 0.01-0.001 12-13 95-100 1.56±0.02 8.15±0.10 0.001-0.0001 20-21 90-95 0.95±0.02 7.45±0.10

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

TCD – tissue cytopathic dose,

CPE – cytopathic effect.

Table 4

Results of reproduction of the strain "SAT-2/XIV/2023" of foot-and-mouth disease virus in the transplantable 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 RT-PCR-RV data, μg/cm ³ Infectious activity titer, lg TCD ₅₀ /cm ³ Cell concentration before infection, million cells/ ^cm3 2.5 11-12 95-100 1.14±0.02 7.62±0.10 3.0 12-13 95-100 1.40±0.02 8.10±0.10 3.5 12-13 95-100 2.23±0.02 8.45±0.10 4.0 15 95-100 2.32±0.02 8.57±0.10 Temperature, °C 35.0±0.2 23-24 90-95 0.50±0.02 6.01±0.10 36.0±0.2 15-16 90-95 1.40±0.02 8.08±0.10 37.0±0.2 12-13 95-100 2.23±0.02 8.45±0.10 38.0±0.2 15-16 95-100 1.45±0.02 8.14±0.10 Dose of infection 0.1-0.01 13-14 95-100 1.49±0.02 8.19±0.10 0.01-0.001 12-13 95-100 2.23±0.02 8.45±0.10 0.001-0.0001 17-18 90-95 1.15±0.02 7.61±0.10

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

TCD – tissue cytopathic dose,

CPE – cytopathic effect.

Table 5

Study of the kinetics of inactivation of foot-and-mouth disease virus suspensions of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023"

(n=3, M±m, p<0.05)

Sampling time from inactivation, h Titer of infectious activity of foot-and-mouth disease virus, lg TCD ₅₀ /cm ³ strain "SAT-2 Lib 39/2012" strain "SAT-2/XIV/2023" 0 8.15±0.10 8.50±0.10 1 5.80±0.10 6.00±0.10 2 3.76±0.10 4.00±0.10 4 1.50±0.10 1.75±0.10 5 0 0 12 -12.00 -12.00

Table 6

Content of foot-and-mouth disease virus components of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" 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-2 Lib 39/2012 10.25 1.54 0.035 9.84 1.53 0,040 8.71 1.52 0.045 7.69 1.43 SAT-2/XIV/2023 9.87 2.20 0.035 9.38 2.19 0,040 8.19 2.17 0.045 7.90 2.02

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 7

Content of antigen components of foot-and-mouth disease virus strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023" 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-2 Lib 39/2012 1.52 PEG-6000,
4 h 50.16 PEG-6000,
12 h 53.20 Tangential filtration,
3 h 60.19 SAT-2/XIV/2023 2.17 PEG-6000,
4 h 47.74 PEG-6000,
12 h 55.34 Tangential filtration,
3 h 60.33

Note: PEG – polyethylene glycol,

OVB – total viral protein.

Table 8

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 of strains "SAT-2 Lib 39/2012" and "SAT-2/XIV/2023"

No. of living person Optical density value of the sample Percentage of inhibition (PI), % sample type reference values before vaccination on the 14th day after vaccination sample type reference values before vaccination on the 14th day after vaccination SPK 0.404 -//- SPK 59.56 -//- PC 0.052 PC 94.80 OK 0.999 OK 0,000 1 -//- 0.952 0.912 -//- 4,705 8,709 2 0.953 0.916 4,605 8,308

Note: SPC – weak positive control,

PC – positive control,

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

Table 9

Study of humoral immunity and immunogenic activity of the culture inactivated emulsion vaccine for early protection against foot and mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV in pigs

(analysis for strain "SAT-2 Lib 39/2012")

(n=5, p<0.01, M±m)

Name of the challenge strain Dilution of the administered vaccine No. of animals Antibody titer in RMN, log ₂ SN ₅₀ Results of the challenge test conducted on day 7 ImD ₅₀ /PD ₅₀ 4 days after vaccination 7 days after vaccination SAT-2 Lib 39/2012 without dilution 1 5.50 8.00 ─ 0.036/
55.9 2 5.50 7.50 ─ 3 5.25 7.75 ─ 4 5.25 7.75 ─ 5 5.50 7.75 ─ M±m 5.40±0.14 7.75±0.18 0/5 1/5 6 4.00 5.25 ─ 7 4.00 5.25 ─ 8 4.25 4.75 ─ 9 4.00 5.00 ─ 10 4.00 5.00 ─ M±m 4.05±0.11 5.05±0.21 0/5 1/25 11 3.00 4.50 ─ 12 3.00 4.50 ─ 13 3.25 4.50 ─ 14 2.50 4.50 ─ 15 2.75 4.00 ─ M±m 2.90±0.29 4.40±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.

Table 10

Study of humoral immunity and immunogenic activity of the culture inactivated emulsion vaccine for early protection against foot and mouth disease genotypes SAT-2/VII/Lib-12 and SAT-2/XIV in pigs

(analysis for strain "SAT-2/XIV/2023")

(n=5, p<0.01, M±m)

Name of the challenge strain Dilution of the administered vaccine No. of animals Antibody titer in RMN, log ₂ SN ₅₀ Results of the challenge test conducted on day 7 ImD ₅₀ /PD ₅₀ 4 days after vaccination 7 days after vaccination SAT-2/XIV/2023 without dilution 1 5.50 8.00 ─ 0.036/
55.9 2 5.50 8.25 ─ 3 6.00 7.75 ─ 4 6.00 7.75 ─ 5 5.75 8.25 ─ M±m 5.75±0.25 8.00±0.25 0/5 1/5 6 4.00 5.75 ─ 7 4.25 5.25 ─ 8 4.75 5.50 ─ 9 4.00 5.00 ─ 10 4.50 5.50 ─ M±m 4.30±0.33 5.40±0.29 0/5 1/25 11 3.50 5.00 ─ 12 3.75 4.50 ─ 13 3.25 4.50 ─ 14 3.00 4.75 ─ 15 3.00 4.00 ─ M±m 3.30±0.33 4.55±0.37 05 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.

Claims (3)

1. A culture-based inactivated emulsion vaccine against foot-and-mouth disease containing an active substance and an adjuvant, which contains a mixture of two strains of foot-and-mouth disease virus as an active substance: avirulent and purified antigen material from the strain "SAT-2 Lib 39/2012" homologous to the causative agent of the infection, genotype SAT-2/VII/Lib-12, 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. 361 - dep / 21-22 - GKShM of the FGBU "ARRIAH", reproduced in the transplantable suspension cell line BNK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg; and avirulent and purified antigen material from the strain "SAT-2/XIV/2023" homologous to the infectious agent, genotype SAT-2/XIV, 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 registration number: No. 489 - dep / 23-39 - ГКШМ FGBU "ARRIAH", reproduced in the transplantable suspension cell line BNK-21/SUSP/ARRIAH, in an amount of at least 15.0 μg; as an adjuvant it contains the domestic oil adjuvant DMIXVAC 78 V with a content of 70% by weight of the finished product and a maintenance medium - up to 2.0 ^cm3 of the finished product. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigen material from the strain "SAT-2 Lib 39/2012" of the genotype SAT-2/VII/Lib-12, homologous to the infectious agent, and the strain "SAT-2/XIV/2023" of the genotype SAT-2/XIV, obtained in a sensitive biological system and representing a suspension containing the 146S immunogenic component of the foot-and-mouth disease virus of the genotypes SAT-2/VII/Lib-12 and SAT-2/XIV, in a ratio of 1:1. 3. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigen material from the strain “SAT-2 Lib 39/2012” of the genotype SAT-2/VII/Lib-12, homologous to the infectious agent, and the strain “SAT-2/XIV/2023” of the genotype SAT-2/XIV, and the oil adjuvant DMIXVAC 78 V in a ratio of 30% to 70% by weight.