RU2810132C1 - Culture inactivated adsorbed vaccine against fmd of sat-1/x genotype from sat-1/nigeria/2015 strain - Google Patents

Abstract

FIELD: veterinary virology and biotechnology.

SUBSTANCE: culturally inactivated sorbed vaccine against foot and mouth disease has been proposed. The vaccine contains an avirulent and purified concentrated antigen of SAT-1/Nigeria/2015 of SAT-1/Х genotype obtained in a suspension continuous cell line from the kidney of a newborn Syrian hamster (BHK-21/SUSP/ARRIAH), representing a suspension containing predominantly 146S immunogenic component of FMDV, adjuvant aluminum hydroxide and saponin in effective ratios.

EFFECT: vaccine is highly immunogenic and can provide effective protection against a homologous infectious agent circulating in African and Middle East countries.

3 cl, 1 dwg, 8 tbl, 14 ex

Description

The invention relates to the field of veterinary virology and biotechnology, namely to the development of a culture inactivated sorbed vaccine against foot and mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015”.

The most contagious disease of mammals is foot and mouth disease. This disease causes serious economic losses, which are associated with the costs of eradicating the disease. There are seven following serotypes of foot and mouth disease virus: O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1, while the virus serotype SAT-1 is common in countries throughout the African continent and the Middle East, which determines the relevance production of vaccines against foot-and-mouth disease of this serotype.

Each serotype is genetically divided into different topotypes, and sometimes into separate genetic lineages and even sublineages. 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 VP ₁ protein [1]. Considering that the causative agent of foot-and-mouth disease is an RNA-containing virus, it is characterized by the formation of a large number of mutations during replication of the viral genome (predominantly in the 5' region) and the phenomenon of recombination (mainly in the 3' region).

The importance of knowing the genetic diversity of the foot-and-mouth disease virus lies in the possibility of a thorough analysis of the epizootic situation in a particular region and a detailed selection of a suitable vaccine, which should be adapted specifically to the genotype of interest. Animals after being infected with any one serotype do not have immune protection against another serotype and even some other genetic lines [3]. However, foot-and-mouth disease viruses can show partial cross-protection within the same serotype [2].

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

Foot and mouth disease virus serotype SAT-1 is characterized by high genetic variability, namely it includes the following 13 topotypes: I (NORTHWEST ZIMBABWE, NWZ), II (SOUTHEAST ZIMBABWE, SEZ), III (WESTERN ZIMBABWE, WZ), IV (EAST AFRICA 1, EA-1), V, VI, VII (EAST AFRICA 2, EA-2), VIII (EAST AFRICA 3, EA-3), IX, X, XI, XII, XIII [5].

This high genetic and antigenic diversity leads to problems in the specific prevention of FMD when using culture-inactivated FMD vaccines. As a result, there is a need to create a means of specific prevention against foot and mouth disease of the SAT-1 serotype, namely SAT-1/X.

Due to the easy and rapid spread of the foot-and-mouth disease pathogen, this disease can acquire the scope of an epizootic [12]. In order to prevent the occurrence of the disease, farms in the Russian Federation apply a system of measures to combat and prevent foot-and-mouth disease, which is aimed at preventing the introduction of the virus into the country, systematic immunization of large and small livestock in the buffer zone, as well as monitoring the immune status of vaccinated animals.

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

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

One of several measures that can be used to control foot-and-mouth disease outbreaks is emergency vaccination. The access criteria include the following characteristics: 1) contains a strain of foot-and-mouth disease virus with sufficient antigenic affinity in relation to isolates circulating in the outbreak; 2) belongs to the required type among the developed vaccines; 3) characterized by acceptable safety and effectiveness; 4) has appropriate access, including the volume of shipments and the timeliness of their deliveries.

In many countries of Northern, Western, Eastern, Central and Southern Africa, and the Middle East, outbreaks of foot-and-mouth disease of the SAT-1/X genotype are sporadic. Thus, trade and economic relations with the countries of the African continent entail the risk of introducing isolates of this genotype into the territory of the Russian Federation and neighboring states [14].

Analyzing the outbreaks that were registered in Africa, it was found that isolates of different topotypes of the SAT-1 serotype were identified, in particular, II (SAT-1 RV 11 37), III (SAT-1 BOT 1 68), IV (SAT-1 UGA BUFF 21 70), V (SAT-1 NIG 11 75), VI (SAT-1 SUD 3 76), VII (SAT-1 UGA 13 74), VIII (SAT-1 UGA 1 97), IX (SAT- 1 ETH 3 2007), X (SAT-1/X), XI (SAT-1 TCH 1/72), XII (SAT-1/ANG/9/74), XIII (SAT 1 MOZP132010 B16). In recent years, starting from 2015, isolates of the foot-and-mouth disease virus of the serotype SAT-1 topotype X have become widespread. In particular, officially outbreaks of foot-and-mouth disease of the SAT-1/X genotype have been recorded in Nigeria and Cameroon. At the same time, there is a high probability of unreported cases of foot-and-mouth disease virus of this genotype [15].

There are known industrial strains of the foot-and-mouth disease virus type SAT-1, which are used or have been used in the world for the production of means for the specific prevention of foot-and-mouth disease:

- strain “SAT-1 T 155 71” (genotype SAT-1/I),

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

- strain “SAT-1 BOT 1 68” (genotype SAT-1/III),

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

- 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 VII),

- strain “SAT-1 UGA 1 97” (genotype VIII),

- strain “SAT-1 ETH 3 2007” (genotype IX),

- strain “SAT-1/X” (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 foot-and-mouth disease virus isolate “SAT-1/NIG/1/2015” was isolated from cattle in the Federal Republic of Nigeria in 2015 and was supplied to the Federal State Budgetary Institution “ARRIAH” from the World Reference Laboratory for Foot-and -Mouth Disease, the Pirbright Institute, UK) for study and research. The strain “SAT-1/Nigeria/2015” was obtained by adaptation to reproduction in the primary trypsinized monolayer pig kidney cell line SP, in continuous cell cultures BNK-21/SUSP/ARRIAH, IB-RS-2, PSGK-30.

According to the results of a comparative analysis of nucleotide sequences, the isolated strain belongs to topotype X, type SAT-1 of the foot-and-mouth disease virus, which differs significantly from the production strains of the foot-and-mouth disease virus type SAT-1 (Fig. 1) [16].

With the increase in trade and economic relations between Russia and the countries of the African continent, in particular Nigeria, Tanzania, Kenya and others, there is a danger of the spread of foot and mouth disease of this genotype in the Russian Federation and, therefore, the problem of possible emergency prevention of this disease arises to build immunity in susceptible animals. Thus, there was a need to develop a culture-inactivated, sorbed vaccine against foot-and-mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015” to ensure the biological safety of the territory of the Russian Federation and neighboring countries.

The closest to the proposed invention in terms of the totality of essential features is an inactivated vaccine sorbed against foot-and-mouth disease serotype SAT-1 (strain “SAT-1 RV 11 37”), containing the active substance in the form of avirulent and purified cultural antigenic material from the strain “SAT-1” homologous to the foot-and-mouth disease pathogen RV 11 37", obtained in a sensitive biological system, and targeted additives in the form of aluminum hydroxide and saponin (adjuvant): antigen concentration (inactivated 146S+75S components of the foot and mouth disease virus) not less than 3.0 μg/cm ³ , content of 10% hydroxide solution aluminum - 40% by volume, additive in the form of a supporting medium - up to 1.0 cm ³ (prototype).

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

Aminoethylethylenimine (AEEI) is used to inactivate the virus. When producing a sorbed vaccine, aluminum hydroxide (Al(OH) ₃ ) is used as an adjuvant. Purification of the virus-containing suspension from ballast impurities is carried out using polyhexamethylene guanidine hydrochloride (PHMG).

Avirulent and purified inactivated antigenic material from the foot-and-mouth disease virus strain serotype SAT-1 is a suspension consisting of 146S+75S components of the foot-and-mouth disease virus, that is, not only complete immunogenic particles, but also “empty” capsids.

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

A significant drawback of the prototype vaccine is its very low immunogenic activity against strains/field isolates of the foot-and-mouth disease virus genotype SAT-1/X circulating in African countries due to the strain “SAT-1 RV 11 37” belonging to the genotype SAT-1/II. In addition, this strain was obtained more than 40 years ago.

To solve this problem, the present invention was created, which included the development of a culturally inactivated sorbed vaccine against foot and mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015”. This vaccine assumes a high content of predominantly 146S immunogenic component in the dose.

The technical result from the use of the proposed invention is to expand the arsenal of inactivated culture-sorbed vaccines for emergency protection of animals against isolates and strains of foot-and-mouth disease virus serotype SAT-1 and, in particular, genotype SAT-1/X.

The specified technical result was achieved by creating a vaccine against foot-and-mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015”, a culture-inactivated sorbed vaccine, characterized by the following set of characteristics reflected below.

The developed vaccine in 1.0 cm ³ of the drug contains the following main components: 1) the active substance in the form of avirulent and purified antigenic material from the strain “SAT-1/Nigeria/2015” (genotype SAT-1/X) of the foot-and-mouth disease virus, homologous to the causative agent of the infection, reproduced preferably in the continuous suspension cell line BNK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; 2) aluminum hydroxide 10% colloidal solution containing 45% by volume (4.5% in terms of dry matter), 3) saponin in the amount of 1.5 mg, 4) supporting medium - up to 1.0 cm ³ .

The strain “SAT-1/Nigeria/2015”, which was obtained by adapting the isolate “SAT-1/NIG/1/2015”, was deposited in the All-Russian State Collection of Exotic Types of Foot-and-Mouth Disease Virus and Other Animal Pathogens (GKSHM) of the Federal State Budgetary Institution “Federal Center for Animal Health" (FGBU "ARRIAH"), under registration number: No. 414 - dep / 22-48 - GKShM FGBU "ARRIAH".

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

To produce a vaccine, a continuous suspension culture of BHK-21/SUSP/ARRIAH cells is preferably used as a sensitive biological system, and Eagle's DMEM medium is used as a supporting medium without adding serum, but with the addition of blood protein hydrolysate in an amount of 5% of the total volume at pH Wednesdays 7.45-7.65. Virus inactivation is carried out using aminoethylethylenimine (AEEI) with a concentration of 0.025% 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 0.012% of the total volume.

The avirulent and purified antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” is a suspension containing predominantly the inactivated 146S immunogenic component of the foot-and-mouth disease virus. The concentration of the component in the product is assessed using a quantitative version of the complement fixation reaction (CRR) [18]. To prepare the vaccine, use viral material containing at least 4.0 μg of inactivated immunogenic 146S particles of foot-and-mouth disease virus per 1.0 cm ³ . The required concentration of the immunogenic component in the vaccine preparation is ensured by concentrating the antigen through sorption on the surface of colloidal particles of aluminum hydroxide (Al(OH) ₃ ).

The vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X, culture-inactivated, sorbed, is prepared by mixing the purified antigen and a 10% suspension of aluminum hydroxide in a ratio of 55% to 45% by volume, respectively. To enhance the immune response, saponin is used at a concentration of 1.5 mg/cm ³ . The resulting vaccine is a suspension containing particles of water-insoluble aluminum hydroxide, carrying on their surface inactivated virions of the foot-and-mouth disease virus, which ensure the formation of immunity.

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

1. Vaccine against foot and mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015”, culture-inactivated, sorbed.

2. The active substance in the form of avirulent and purified antigenic material from the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X homologous to the causative agent of the disease in an effective amount.

3. Targeted supplements.

The significant distinctive features of the proposed vaccine are that as an active substance it contains an avirulent purified antigen of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus in an effective amount.

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

1. Avirulent and purified antigen of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus, obtained preferably in the suspension continuous cell line BHK-21/SUSP/ARRIAH and representing a suspension containing 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/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus, obtained preferably in a suspension continuous culture of BHK-21/SUSP/ARRIAH cells and representing a suspension containing predominantly the 146S immunogenic component of the virus foot and mouth disease in an amount of at least 4.0 mcg per 1.0 cm ³ of the finished vaccine preparation.

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

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

5. The vaccine contains an adjuvant - aluminum hydroxide 4.5% in terms of dry residue in combination with saponin at a concentration of 1.5 mg per 1.0 cm ³ of the finished product.

6. Avirulent and purified antigen of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus, obtained preferably in the continuous suspension cell line BHK-21/SUSP/ARRIAH, adjuvant - aluminum hydroxide and saponin, additive in the finished product the drug in the form of a maintenance medium in the following quantities:

Avirulent and purified antigen strain “SAT-1/Nigeria/2015” genotype Foot and mouth disease virus SAT-1/X not less than 4.0 μg/cm ³ Aluminum hydroxide 4.5% (based on dry matter) Saponin 1.5 mg/cm ³ Supportive environment up to 1.0 cm ³

The proposed vaccine against foot-and-mouth disease from the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X, culturally inactivated, sorbed, has high immunogenic activity and provides reliable protection against isolates of the foot-and-mouth disease virus of the genotype SAT-1/X circulating in East Africa and neighboring countries states.

Achieving a technical result from the use of the invention is ensured by the fact that the antigen of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus, which has high immunogenic activity and creates effective protection for susceptible animals, is introduced into the composition of the proposed foot-and-mouth disease vaccine as an active substance. against foot-and-mouth disease virus isolates of the presented genotype, which in recent years have caused outbreaks of the disease in African countries.

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

Fig. 1. Dendrogram reflecting the phylogenetic relationship of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” with epizootic strains of the serological type SAT-1. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₁ gene.

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

SEQ ID NO:1 represents the nucleotide sequence of the VP ₁ protein gene of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus genotype SAT-1/X;

SEQ ID NO:2 represents the amino acid sequence of the VP ₁ protein gene of the strain “SAT-1/Nigeria/2015” of the foot and mouth disease virus genotype SAT-1/X.

The foot and mouth disease virus strain “SAT-1/Nigeria/2015” is characterized by the following characteristics and properties.

Morphological characteristics

Strain “SAT-1/Nigeria/2015” of foot-and-mouth disease virus serotype SAT-1 belongs to the order Picornavirales, family Picornaviridae, genus Aphthovirus, species Foot-and-Mouth Disease virus and has morphological characteristics characteristic of the causative agent of foot-and-mouth disease: the shape of the virion is ixahedral, the size 24-25 nm. A virion consists of a single-stranded, positively charged RNA molecule enclosed in a protein shell. The 146S component consists of an RNA molecule enclosed in a polypeptide complex consisting of 60 copies of the structural proteins VP ₄ , VP ₂ , VP ₃ , VP ₁ .

Antigenic properties

According to antigenic properties, the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus belongs to the SAT-1/X serotype. The virus is stably neutralized by homologous antiserum and does not exhibit hemagglutinating activity. In recovered animals, type-specific antibodies are formed in the blood serum, which are detected in enzyme-linked immunosorbent assay (ELISA) and microneutralization reaction (RMN).

Using the nucleotide sequencing method, the primary structure of the 1D gene of the VP ₁ protein of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” was determined. Comparative analysis of nucleotide sequences showed that the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus belongs to the SAT-1/X genotype (Fig. 1).

The antigenic affinity (r ₁ ) of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” was studied in a cross-study of the strain with specific sera obtained for the following production strains of the foot-and-mouth disease virus:

- strain “SAT-1 T 155 71” (genotype SAT-1/I),

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

- strain “SAT-1 BOT 1 68” (genotype SAT-1/III),

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

- 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 VII),

- strain “SAT-1 UGA 1 97” (genotype VIII),

- strain “SAT-1 ETH 3 2007” (genotype IX),

- strain “SAT-1/X” (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) in RMN.

The titer of reference cattle blood sera obtained by immunizing animals with monovalent vaccines from production strains of foot-and-mouth disease virus serotype SAT-1 against 10 ² TCD ₅₀ of homologous and heterologous virus was determined in RMN with cross-titration, calculating values using a linear regression equation, and expressed in lg . The r ₁ value was determined as the antilogarithm of the difference in serum lg titers against heterologous and homologous viruses [17, 18, 19].

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

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

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

Indicators of antigenic relatedness when studying the strain “SAT-1/Nigeria/2015” amounted to r ₁ 0.01 - 0.32, which indicates the absence of antigenic relatedness with production strains of foot-and-mouth disease virus serotype SAT-1 (Table 1). It should be noted that the relationship between the strains “SAT-1/X” and “SAT-1/Nigeria/2015”, belonging to the SAT-1/X genotype, is only 0.32, based on this, they have a relationship, but it insignificant, the differences are significant. The “SAT-1/X” strain was isolated in 1980 and does not correspond to epizootic isolates isolated from foot-and-mouth disease outbreaks on the African continent in a more recent period. This is the basis for creating a vaccine based on the SAT-1/Nigeria/2015 strain.

Geno- and chemotaxonomic characteristics

Strain “SAT-1/Nigeria/2015” of foot-and-mouth disease virus is an RNA-containing virus with a molecular weight of 7 × 10 ⁶ D. Nucleic acid is represented by a single-chain linear molecule with a molecular weight of 2.8 × 10 ⁶ D. The virion has a protein shell consisting only of four main proteins VP ₁ , VP ₂ , VP ₃ and VP ₄ .

The main antigenic protein is VP ₁ . The virion contains 31.5% RNA and 68.5% protein. Viral RNA is positively charged and 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 nonstructural polypeptides of the virus. Of the 8 non-structural polypeptides that accumulate in infected cells, the protein - RNA-dependent RNA polymerase (encoded by the 3D gene), necessary for viral RNA replication, is of particular importance.

Physical properties

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

Resistance to external factors

Strain “SAT-1/Nigeria/2015” of foot-and-mouth disease virus is resistant to ether, chloroform, freon, and acetone. Most stable at pH 7.35-7.70. Shifts in pH both to the acidic and alkaline sides lead to inactivation of the virus. Sensitive to formaldehyde, UV irradiation, γ-irradiation, high temperatures (above 38.2°C).

Additional characteristics and properties

Reactogenicity - does not have reactogenic properties.

Pathogenicity - pathogenic for artiodactyl animals.

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

Stability - retains the original biological properties when passaging in sensitive biological systems for 5 passages (observation period) on continuous crops.

Biotechnological characteristics

The foot and mouth disease virus strain “SAT-1/Nigeria/2015” is reproduced in continuous cell cultures: Siberian mountain ibex kidneys (PSGK-30), pig kidneys (IB-RS-2), Syrian hamster kidneys (VNK-21).

During the test, 5 consecutive passages of the “SAT-1/Nigeria/2015” 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 (cattle) and pigs.

Based on the data obtained, it can be argued that the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in its antigenic and immunological spectra is the original variant of the foot-and-mouth disease virus of the SAT-1/X genotype.

To reduce the epizootic risk of foot and mouth disease caused by the SAT-1/X genotype and prevent the emergence of new foci of the disease, timely vaccine prevention is important, which requires the development of a highly immunogenic and effective vaccine.

A highly immunogenic and effective vaccine against foot and mouth disease of the SAT-1/X genotype was obtained from the strain “SAT-1/Nigeria/2015”, culture inactivated sorbed.

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

Example 1. Genetic characteristics of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” according to PCR and nucleotide sequencing data.

The studies carried out consisted of studying the primary structure of the 1D gene (VP ₁ protein) (663 nt) of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” using the Sanger nucleotide sequencing method and determining the position of this strain on the phylogenetic tree of foot-and-mouth disease virus serotype O The method is based on determining the primary structure of the 1D gene (VP ₁ protein) of the tested isolate/strain, followed by analysis of phylogenetic relationship with other isolates of foot-and-mouth disease virus serotype SAT-1.

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/Nigeria/2015” with other isolates and strains was carried out. The nucleotide sequence of the VP protein gene of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus is presented by SEQ ID NO:1. The amino acid sequence of the 1D gene encoding the VP ₁ protein of the strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus is reflected in SEQ ID NO:2.

A phylogenetic analysis of the strain “SAT-1/Nigeria/2015” was carried out. The phylogenetic tree was derived using the MEGA6 program and the Neighbor-Joining algorithm [20]. The percentage of replicate branches in which related taxa cluster together in a bootstrap test (200 replicates) is shown next to the branches [21, 22]. Evolutionary distances were calculated using the Maximum Composite Likelihood method [23] and expressed in units of the number of base substitutions per site. This analysis included 14 nucleotide sequences, including the sequence of the 1D gene of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015”. All items containing spaces and missing data were removed (complete deletion option). There were a total of 663 items in the final data set. Evolutionary analysis was carried out in MEGA 6 [24].

As a result of the work carried out by comparing the complete nucleotide sequences of the 1D gene (VP1 protein) of the strain “SAT-1/Nigeria/2015” and other isolates/strains of foot-and-mouth disease virus serotype SAT-1/X, the position of the studied strain on the phylogenetic tree was determined (Fig. 1) .

Based on the results of the entire analysis, we conclude that the studied strain “SAT-1/Nigeria/2015” belongs to the SAT-1/X genotype, which is confirmed by the data of nucleotide and amino acid analysis.

Example 2. Adaptation of the strain “SAT-1/Nigeria/2015” to a continuous monolayer culture of kidney cells of the Siberian mountain ibex PSGK-30.

To infect a continuous monolayer culture of Siberian mountain ibex kidney cells PSGK-30, a 10% aphthous suspension of foot-and-mouth disease virus obtained from bovine aphthae (passage 2) was used. The inoculating cell concentration was 0.20-0.25 million cells/cm ³ , the virus infection dose was 0.001 TCD ₅₀ /cell. According to the results of the study, the reproduction of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” took place with a specific destruction of the monolayer by 90-100% in 18-20 hours. Over the course of 6 consecutive passages, an increase in the titer of infectious activity of the virus was noted from 6.14 ± 0 .12 to 7.62±0.12 lg TCD ₅₀ /cm ³ .

The maximum titer of infectious activity of the virus was achieved at stage 6 of passage (7.62±0.12 lg TCD ₅₀ /cm ³ ). The concentration of 146S particles from passages 1 to 6 changed from 0.27±0.03 to 1.10±0.03 μg/cm3. In this case, the largest amount of the 146S component was noted at stage 6 of passage (1.10±0.03 μg/cm ³ ). The degree of reliability (R ² ) of the study results in the quantitative version of RT-PCR-RT ranged from 96.97 to 99.85%). Thus, over the course of 6 consecutive passages, the adaptation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” 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/Nigeria/2015” on an industrial scale.

In the process of industrial cultivation of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in a continuous monolayer cell culture PSGK-30, the optimal conditions for cultivating the virus were selected, analyzing the different composition of the supporting medium, the temperature factor and the dose of infection of the cells.

At the first stage of the study, the effect of the composition of the supporting medium on the reproduction of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in a monolayer culture of PSGK-30 cells was assessed on a production scale (at the stage from the 5th to the 6th passages). For comparison, three media were used with the addition of 2% fetal calf serum: 1) Eagle's medium DMEM; 2) RPMI-1640 environment; 3) Hanks solution with 3.0% blood protein hydrolyzate. The inoculating cell concentration was 0.20-0.25 million cells/cm ³ , the dose of virus infection was 0.1000-0.0001 TCD ₅₀ /cell. Cultivation of the virus was carried out at temperatures of 35±0.2 - 38±0.2°C until the cell monolayer was destroyed by 95-100% within 15-30 hours. Results of adaptation of the foot-and-mouth disease virus strain "SAT-1/Nigeria/2015" with supporting media of different compositions are shown in Table 2.

From the data in Table 2 it can be seen that when reproducing the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in a monolayer culture of PSGK-30 cells using supporting media of different compositions, the optimal medium is Eagle DMEM, which allows achieving specific destruction in 15-16 hours cell monolayer by 95-100% with accumulation of the 146S component in the resulting suspension in an amount of 1.10±0.03 μg/cm ³ and an infectious activity titer of 7.62±0.12 lg TCD ₅₀ /cm ³ . Viral reproduction rates were lower when using RPMI-1640 medium and Hanks' solution.

At the next stage of studying the conditions for monolayer cultivation of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in the PSGK-30 cell culture under production conditions, the influence of the temperature factor on the virus reproduction process was assessed. To do this, the virus was cultivated in Eagle's medium DMEM 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 TCD ₅₀ /cell. The virus was cultivated until the cell monolayer was destroyed by 80-100%) for 15-30 hours (Table 2). Based on the results of the study, it was revealed that for complete reproduction of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in a monolayer cell culture PSGK-30, the optimal environmental temperature is 37.0±0.02°C, at which the development of The CPE reached 95-100%) with the accumulation of 146S particles equal to 1.10±0.03 μg/cm ³ and the titer of infectious activity of the virus 7.62±0.12 lg TCD ₅₀ /cm ³ .

We assessed the effect of the dose of infection of a monolayer of PSGK-30 cells with the strain “SAT-1/Nigeria/2015” during cultivation on a production scale. For this, different doses of the virus were used: 0.10-0.01; 0.010-0.001; 0.0010-0.0001 TCD ₅₀ /cl. Eagle's MEM medium with 2% cattle blood serum was used as a supporting medium. Virus reproduction was carried out at a temperature of 37.0±0.2°C for 15-24 hours until specific cell destruction was 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 greatest accumulation of “full” virus particles was achieved at an infection dose of 0.01-0.001 TCD ₅₀ /cell. and amounted to 1.10±0.03 μg/cm ³ with a titer of infectious activity of the virus of 7.62±0.12 lg TCD ₅₀ /cm ³ .

Thus, we studied the conditions for monolayer cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in PSGK-30 cell culture on an industrial scale. As a result of the study, the following optimal parameters for the reproduction of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in the monolayer cell line PSGK-30 were determined: 1) maintenance medium - Eagle’s medium DMEM; 2) cultivation temperature - 37.0±0.02°C; 3) dose of virus infection - 0.01-0.001 TCD ₅₀ / cell.

Example 4. Study of conditions for suspension cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” on an industrial scale.

When industrially cultivating the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in a continuous suspension cell culture BHK-21/SUSP/ARRIAH, the optimal parameters for the successful reproduction of the foot-and-mouth disease pathogen were searched for, using different cell concentrations, temperatures and infection doses.

At the first stage of the work, the effect of the seed concentration of cells of the BHK-21/SUSP/ARRIAH line in suspension on the reproduction of the “SAT-1/Nigeria/2015” strain of foot-and-mouth disease virus on an industrial scale was determined. Suspensions with the following cell concentrations were prepared for analysis: 2.5; 3.0; 3.5; 4.0 million cells/ ^cm3 . The pH value was maintained in the range of 7.45-7.65. The virus cultivation temperature was 37.0±0.02°C, the dose of virus infection was 0.10-0.01 TCD ₅₀ /cell. Virus reproduction was carried out until specific cell death was 95-100%, which occurred within 9-11 hours.

The results of suspension cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in suspension with different cell concentrations are presented in Table 3, from which it follows that when cultivating the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in the suspension cell line BHK -21/SUSP/ARRIAH with different cell concentrations, determined that the accumulation of the 146S component in a suspension with a cell concentration of 2.5 million cells/cm ³ was 0.49 ± 0.03 μg/cm ³ , with a concentration of 3.0 million cells/cm ³ - 0.74±0.03 μg/cm ³ , with a concentration of 3.5 million cells/cm ³ - 1.21±0.03 μg/cm ³ , and with a concentration of 4.0 million cells ./cm ³ - 1.42±0.03 μg/cm ³ . As follows from the data obtained, for the reproduction of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus, the optimal concentration of BHK-21/SUSP/ARRIAH cells in the suspension is equal to 4.0 million cells/cm ³ . The titer of infectious activity of the virus was 8.50±0.11 lg TCD ₅₀ /cm ³ . A higher concentration of cells made it possible to obtain the same product yield with a slight increase. From an economic point of view, therefore, for the reproduction of the “SAT-1/Nigeria/2015” strain of foot-and-mouth disease virus, it is optimal to use a cell suspension of the BHK-21/SUSP/ARRIAH line with a concentration of 4.0 million cells/cm ³ .

We studied the influence of temperature on suspension cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in cell culture of the BHK-21/SUSP/ARRIAH 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 the cell culture with the virus was 0.10-0.01 TCD ₅₀ /cell. The pH value of the viral suspension was maintained in the range of 7.45-7.65. Cultivation of the virus was carried out to a CPE of 90-100% for 14-26 hours. Based on the results of the analysis, it was determined that for complete reproduction of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus in a suspension cell culture BHK-21/SUSP/ ARRIAH, the optimal temperature is 37.0±0.02°C, at which 95-100% specific cell death is observed within 14-15 hours with a high accumulation of the 146S component (1.42±0.03 μg/cm ³ ) and the titer of infectious activity of the virus is 8.50±0.11 lg TCD ₅₀ /cm ³ .

The conditions for suspension cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” on an industrial scale were studied using cell culture BHK-21/SUSP/ARRIAH, and the effect of the dose of cell infection was assessed. To do this, cell suspensions were infected with the virus in different doses: 0.10-0.01; 0.01-0.001; 0.001-0.0001 TCD ₅₀ /cl. Virus reproduction was carried out at a temperature of 37.0±0.2°C for 14-26 hours until the cytopathic effect was 90-95%. 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 greatest accumulation of the 146S component was observed at an infection dose of 0.010-0.001 TCD ₅₀ /cell. (1.42±0.03 μg/cm ³ ) with a titer of infectious activity of the virus equal to 8.50±0.11 lg TCD ₅₀ /cm ³ .

Thus, the parameters of suspension cultivation of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in the suspension cell line BHK-21/SUSP/ARRIAH on an industrial scale were studied. The optimal conditions for the reproduction of the strain “SAT-1/Nigeria/2015” in the suspension culture of BNK-21 cells were identified: 1) cell concentration before infection - 4.0 million cells/cm ³ ; 2) cultivation temperature - 37.0±0.02°C; 3) dose of virus infection - 0.010-0.001 TCD ₅₀ / cell.

Example 5. Inactivation of a suspension of foot and mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X.

At the end of the reproduction cycle of the foot-and-mouth disease virus, without stopping the thermostatting process (37.0±0.02°C), an acidified solution of aminoethylethylenimine (AEEI) with a pH of 8.2-8.3 was added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be equal to 0.025%. Inactivation of the infectious activity of the foot and mouth disease virus strain "SAT-1/Nigeria/2015" genotype SAT-1/X was carried out for 12 hours at a temperature of 37.0±0.1°C and pH 7.45-7.65 with stirring.

To determine the time of complete inactivation after the addition of 1,2-AEEI, samples were taken every hour. The obtained samples were tested for the presence of infectious activity of the foot-and-mouth disease virus in the primary culture of pig kidney cells SP. The results are presented in Table 4, from which it can be seen that complete inactivation of the foot and mouth disease virus strain “SAT-1/Nigeria/2015”, reproduced in cultivators, occurred 8 hours after the addition of 1,2-AEEI.

The prepared inactivated viral suspensions were examined in the RSC to assess the content of virus components in them. The concentration of the 146S component was 1.37±0.03 μg/cm ³ (64% of the total viral protein concentration), and the 146S+75S component was 1.80±0.0 μg/cm ³ (88% of the total viral protein concentration ).

Example 6. Selection of conditions for purification of a suspension of foot and mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X.

A suspension of foot and mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X, obtained during reproduction in the suspension cell line BHK-21/SUSP/ARRIAH, was subjected to inactivation and purification. To obtain purified foot-and-mouth disease virus antigen, polyhexamethylene guanidine (PHMG) was used with concentrations of 0.010, 0.012 and 0.014%. Before and after the process of purifying the antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X in the quantitative version of RSC, the concentration of the total viral protein and immunogenic components was determined. The results of the analysis are reflected in Table 5, from which it follows that as a result of purification of the antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” genotype SAT-1/X using PHMG at a concentration of 0.010%, a decrease in the concentration of ballast protein was noted by 6%, immunogenic components - by 0.5%. When using PHMG at a concentration of 0.012%, a decrease in the amount of ballast protein by 43% and the 146S component by 2% was observed. Using PHMG at a concentration of 0.014%, the content of ballast protein decreased by 52%, and immunogenic components by 10%. Thus, by studying the conditions for purifying the antigen of the strain “SAT-1/Nigeria/2015” of the SAT-1/X genotype, we came to the conclusion that to obtain a purified product it is optimal to use PHMG with a concentration of 0.012%.

Example 7. Selection of conditions for concentrating an antigen suspension of the strain “SAT-1/Nigeria/2015” genotype SAT-1/X of the foot-and-mouth disease virus.

Cultural inactivated suspension of the strain

“SAT-1/Nigeria/2015”, obtained using the suspension cell line BHK-21 /SUSP/ARRIAH, was concentrated 8 times by volume. To increase the concentration of immunogenic components of the foot-and-mouth disease virus antigen, the following methods were used: 1) adding polyethylene glycol (PEG-6000) with a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 4 hours; 2) adding polyethylene glycol (PEG-6000) with a concentration of 50% to the antigen in a ratio of 1:4, with an exposure of 12 hours; 3) concentration with aluminum hydroxide (10%) colloidal solution in the amount of 45%, virus antigen - 55% of the total volume).

Before and after the process of concentrating the suspension of the antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015”, the concentration of the total viral protein and immunogenic components in the quantitative version of the RSC was determined. The research results are presented in Table 6, from which it can be seen that when concentrating the antigen of the strain “SAT-1/Nigeria/2015” of the foot-and-mouth disease virus using PEG-6000 for 4 hours, an increase in the content of components was noted compared to the initial values (before concentration) in 5.5 times. Using the same polymer, but increasing the exposure time to 12 hours, we increased the concentration of virus components by 6.3 times. Using the sorption method using aluminum hydroxide, it was possible to increase the amount of immunogenic components of the antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015” in suspension by 7.9 times. Thus, by studying the conditions for concentrating the antigen of the “SAT-1/Nigeria/2015” strain, we came to the conclusion that the optimal way to increase the concentration of foot-and-mouth disease virus components is the sorption method using a colloidal solution of aluminum hydroxide.

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

To produce an anti-foot-and-mouth disease monovalent sorbed vaccine from the antigen of the “SAT-1/Nigeria/2015” strain, aluminum hydroxide in combination with saponin was chosen as an adjuvant. In the preparation of a monovalent sorbed vaccine against foot-and-mouth disease virus, 4.5% aluminum hydroxide was used in terms of dry matter and saponin in an amount of 1.5 mg/cm ³ .

Example 9. Composition of a vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culturally inactivated sorbed.

From the resulting concentrate of the antigen of the foot-and-mouth disease virus strain “SAT-1/Nigeria/2015”, a culturally inactivated sorbed vaccine against foot-and-mouth disease genotype SAT-1/X was prepared using aluminum hydroxide and saponin as adjuvants. The amount of 146S immunogenic component in 1.0 cm ³ of the finished antigen was 10.35 ± 0.09 μg/cm ³ (Table 6).

Example 10. Immunization of animals with a vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culturally inactivated sorbed (the proposed invention).

From the resulting vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culture inactivated sorbed (the proposed invention), a series of dilutions for cattle were prepared in 4-fold increments. 15 heads of cattle were divided into 3 groups of 5 heads each and 2 heads were left without vaccination to control the virus. The immunizing dose was 2.0 cm ³ . The first group of cattle (Nos. 1-5) were immunized with the vaccine without dilution, the second group of cattle (Nos. 6-10) were vaccinated with the vaccine diluted 1/4, the third group of cattle (Nos. 11-15) - with the vaccine diluted 1/16 . The drug was administered intramuscularly into the middle third of the neck.

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

Blood sera from cattle obtained before immunization of animals, 14 days after immunization, were examined for the presence of antibodies to non-structural proteins of the foot-and-mouth disease virus using a blocking enzyme-linked immunosorbent assay (ELISA) in accordance with 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 research results obtained are reflected in Table 7, from which it can be seen that the animal blood sera did not contain antibodies to non-structural proteins of the foot-and-mouth disease virus (PI values for cattle blood sera < 50%).

Example 12. Assessment of the avirulence and safety of the vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culturally inactivated sorbed.

To determine the avirulence of the vaccine for cattle, a selected sample of the vaccine preparation (the proposed invention) was injected intradermally at 0.1 cm ³ . The study used cattle weighing 270-300 kg. During 10 days of observation, the animals remained clinically healthy, and the pathological examination did not reveal any changes characteristic of foot-and-mouth disease. This indicated the absence of virulent properties of the developed vaccine (the proposed invention).

Control of the safety of the product on cattle was carried out by intramuscular administration of the vaccine (the present invention) at a dose of 6.0 cm ³ (triple dose of 2.0 cm ³ ). The observation period was also 10 days. It should be noted that after immunization, the animal’s body temperature can rise to 41.4°C and remain at this level for 1-2 days, which does not go beyond the normal range after administration of the vaccine preparation.

According to the research results, the vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, cultural inactivated sorbed (the proposed invention) was found to be harmless, all animals during the observation period remained clinically healthy, with pathological analysis of tissue necrosis in situ no vaccine administration was detected.

Example 13. Study of the immunogenic properties of the vaccine (the proposed invention) based on the ability to induce virus-neutralizing antibodies in naturally susceptible animals.

On the 21st day after the administration of the vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culturally inactivated sorbed, blood was taken from cattle and the resulting blood sera were studied in a microneutralization reaction (RMN) [3]. It was revealed that in cattle immunized with the vaccine in a whole dose (5 heads), the average antibody titer values were 7.45±0.11 log ₂ SN ₅₀ , with a dilution of 1/4 (5 heads) -4.45±0.11 log ₂ SN ₅₀ , with breeding 1/16 (5 heads) - 3.15±0.22 log ₂ SN ₅₀ . (Table 8). The data obtained from the microneutralization reaction indicate that after 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 ability of the vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, culture inactivated, sorbed on naturally susceptible animal species by the method of control infection of naturally susceptible animals.

Cattle, immunized in the amount of 15 heads, with the vaccine (the proposed invention) in whole form and in dilutions, were infected with the control strain “SAT-1/Nigeria/2015” of the genotype SAT-1/X of the foot-and-mouth disease virus, adapted to these animals in the mucous membrane tongue in a dose of 10 ^4.0 ID ₅₀ /0.20 cm ³ (at two points of 0.10 ± 0.05 cm ³ ). 7 days after infection, all animals were euthanized and a postmortem examination was performed. Animals that had no lesions on their 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 Table 8, from which it follows that the vaccine against foot and mouth disease from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X is culturally inactivated, sorbed in whole form and in a 1/4 dilution, administered in a single dose (2.0 cm ³ ) protects cattle from infection with a homologous strain of all animals (5 out of 5 heads).

The drug (the proposed invention), inoculated at a dilution of 1/16, protected 4 out of 5 animals. Based on the results of the control infection, it was found that the vaccination volume of this vaccine contains 24.25 PD ₅₀ and 0.08 ImD ₅₀ for cattle.

Thus, the above information indicates that the vaccine against foot and mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015” is a cultural inactivated sorbed, embodying the proposed invention, intended as a reserve for use in agriculture, namely in veterinary virology and biotechnology; the possibility of implementing the presented has been confirmed; a vaccine made from the “SAT-1/Nigeria/2015” strain of the SAT-1/X genotype in accordance with the invention has high immunogenic activity and is capable of providing effective protection to susceptible animals against the epizootic strain of foot-and-mouth disease virus serotype SAT-1, genotype SAT- 1/X, circulating in Africa and the Middle East.

Sources of information taken into account when drawing up the description of the invention for the application for a RF patent for the invention “Vaccine against foot-and-mouth disease genotype SAT-1/X from the strain “SAT-1/Nigeria/2015”, culture inactivated sorbed”:

1. Foot and mouth disease. Prevention measures. URL: http://89.rospotrebnadzor.ru /directions/epid_nadzor/146902 (Date of access: 09/13/2022).

2. The danger of foot and mouth disease. URL: https://vet.astrobl.ru/press-release/opasnost-bolezni-yashchura (Access date: 09/24/2022).

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

4. Burdov A.N., Dudnikov A.I., Malyarets P.V. and others. 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: Folio, 2006. - 250 p.

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

7. Brown F. A brief history of FMD and its casual agent. FMD: Control 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.wr1fmd.org/ref_labs/fmd_ref_lab reports.htm (Date accessed 09/16/2022).

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

13. Rakhmanov A.M. Current epizootic situation in the world regarding foot and mouth disease and measures for its control // Veterinary medicine of the interspecies. topics Sci. Kharkiv, 2013. - pp. 37-38.

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

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

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

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

18. Methodological recommendations for determining the concentration of 146S, 75S, 12S components of vaccine strains of cultural foot-and-mouth disease virus in the complement fixation reaction (FRR) / Federal State Budgetary Institution "ARRIAH". - Approved 09.21.17.

19. Guidelines for determining antigenic correspondence between epizootic isolates and industrial strains of foot-and-mouth disease virus in a cross-microneutralization reaction: approved. Rosselkhoznadzor 09/13/2017 / FSBI "ARRIAH". - Vladimir: 2017. - 24 p.

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

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

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

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

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

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tgacgtgtacgtgcgcatgaagcgggccgagttgtactgccctcggcccctactcacaacctacgcccac

accggcgataggtacaaggtgaaactggtggcgccagaaaaacagatggcaaac</INSDSeq_sequen

ce>

</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="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>FMDV</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>TTSAGEGAEVVTVDVSAHGGNARPTRRQHTDVAFLLDRFTKIGKTTDNK

LVLDLLKTKEKALVGALLRSATYYFCDLEVAAVGTNKWVGWVPNGSPVVNEVDDNPVVFSHNGVTRFALP

YTAPHQVLATVYKGSCKYKPTTEAQPAAIRGDFATLAARLSSETHIPTSFNFGMILTESEVDVYVRMKRA

ELYCPRPLLTTYAHTGDRYKVKLVAPEKQMAN</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (3)

1. Cultural inactivated sorbed vaccine against foot-and-mouth disease, containing an active substance and an adjuvant, characterized in that as an active substance it contains avirulent and purified antigenic material from the strain “SAT-1/Nigeria/2015” of genotype SAT-1/X, homologous to the causative agent of the infection , deposited in the All-Russian State Collection of exotic types of foot-and-mouth disease virus and other animal pathogens of the Federal State Budgetary Institution "Federal Center for Animal Health", under registration number: No. 414 - dep / 22-48 - GKShM FSBI "ARRIAH", reproduced in a continuous suspension cell newborn Syrian hamster kidney line BHK-21/SUSP/ARRIAH, in an amount of at least 4.0 μg; as an adjuvant, aluminum hydroxide 10% colloidal solution containing 45% by volume, 4.0% in terms of dry matter in combination with saponin in an amount of 1.5 mg and a supporting medium up to 1.0 cm ³ . 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the strain “SAT-1/Nigeria/2015” genotype SAT-1/X, obtained in the sensitive biological system BHK-21/SUSP/ARRIAH and which is a viral suspension containing predominantly the 146S immunogenic component of the foot-and-mouth disease virus genotype SAT-1/X. 3. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the SAT-1/Nigeria/2015 strain homologous to the infectious agent, genotype SAT-1/X, and an adjuvant - aluminum hydroxide in combination with saponin in an effective ratio: 55% and 45% respectively.

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