RU2722868C1 - Antirabic inactivated emulsion culture vaccine for prophylactic immunization of domestic carnivores and farm animals - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, specifically to developing an antirabic inactivated emulsion culture vaccine for prophylactic immunization of domestic carnivores and farm animals. Vaccine contains avirulent and purified antigenic material from strain "VNIIZZh" of rabies virus, obtained in transplantable suspension cell line from newborn golden hamster kidney (BHK-21/SUSP/ARRIAH), which is a suspension, containing predominantly immunogenic components of a rabies virus (glycoprotein and nucleoprotein), an oil adjuvant Montanide ISA-61 VG, a preservative merthiolate in effective ratios.EFFECT: vaccine has high immunogenicity and is capable of providing long-term and reliable protection against rabies virus of the genetic line RABV.10 cl, 16 dwg, 12 tbl, 14 ex

Description

The invention relates to the field of biotechnology and can be used in industrial production of an effective, safe and convenient in use anti-rabies inactivated emulsion culture vaccine for the prophylactic immunization of domestic carnivores and farm animals.

Rabies (Rabies) is a priority in a number of viral diseases of humans and animals, is one of the most dangerous zoonoses, causing damage to the central nervous system, encephalomyelitis, paralysis with an inevitable death. This disease is a global problem, which is given special attention by international organizations (WHO, OIE, FAO, GARC) and veterinary services in many countries of the world [1, 2, 3].

The rabies virus belongs to the order Mononegavirales, the family Rhabdoviridae, the genus Lyssavirus, and the species Rabies lyssavirus [4]. Rabies virus virions are bullet-shaped, ≈180 nm long (100-430 nm), ≈75 nm diameter (45-100 nm). On the outer surface of the viral particle there are protrusions in the form of spikes 10 nm long, which are attached to the bilayer lipid membrane [1]. The rabies virus genome is represented by a non-segmented single-stranded negative helical RNA with a length of about 12000 n.a., which encodes 5 main proteins: nucleoprotein (N-protein), phosphoprotein (P-protein), matrix protein (M-protein), glycoprotein (G-protein ), RNA-dependent RNA polymerase (L-protein) [1]. Moreover, the L-gene occupies more than half of the viral genome. Between the G- and L-cistrons there is a pseudogen (ψ-fragment) [1]. The G protein is responsible for the process of binding rabies virus to the receptors of the target cell and subsequent induction of the immune response in the host [5].

The genus Lyssavirus includes the classic rabies virus (phylogenetic group 1, the RABV genetic line), which is widespread among various animal species around the world, as well as 13 related genotypes, but found mainly in bats [6]. The genus Lyssavirus includes 4 phylogenetic groups. The first phylogroup includes the following types of viruses: Rabies virus, Duvenhage virus, European bat lissavirus 1, European bat lissavirus 2, Australian batlissa virus, Aravan virus, Khujand virus, Irkut virus, the second phylogroup - Mokola virus, Logos bat virus, Shimoni bat , to the third philogroup - West Caucasian virus, to the fourth philogroup - Ikoma [1]. Significant nucleotide differences exist between the presented species (the number of nucleotide substitutions is 0.449-0.543 with a standard error of 0.006-0.008) [1]. The vast majority of cases of rabies in humans and animals are caused by the classic rabies virus [1, 7]. Based on research by Y.T. Arai et al. (1997), the effect of the accumulation of nonsynonymous mutations during the evolution of the rabies virus does not lead to significant changes in epitopes within the genetic line, which confirms the severe inhibition of the process of evolution of the N-gene. Apparently, this particular gene is characterized by the greatest conservatism [8].

An important role in rabies epizootology is played by wild and domestic animals of the following families: canids (Canidae), skunks (Mephitidae), coons (Mustelidae), raccoons (Procyonidae), feline (Felidae), mongoose (Herpestidae), bats (Viverridae), bats (Microchiroptera), rodents (Rodentia), which are reservoirs and carriers of the virus [1, 9].

The disease leads to significant costs associated with the death of animals, the elimination of the consequences of outbreaks of the disease, the implementation of preventive and quarantine measures, the regulation of the number of wild carnivores, the capture of stray cats and dogs and laboratory tests for diagnosis [10, 11]. According to experts, the annual economic damage from rabies is more than 8.6 billion US dollars [7].

The rabies virus spreads easily, for this reason this disease can acquire the scope of epizootics [9]. Currently, a difficult epidemiological and epizootic rabies situation is noted in more than 110 countries of the world, with the exception of some island states and on all continents except Antarctica [12]. So, in the Russian Federation there are a large number of natural foci of the disease, which are supported mainly by wild carnivores, which are the main reservoir and source of rabies virus. The causative agent of the disease is transmitted to domestic and agricultural animals, which requires increased attention in the light of their close proximity to humans [13]. At the same time, it is believed that sick dogs make the largest contribution to the process of transmission of rabies virus to humans.

The most important role in the fight against rabies today is played by the prevention of disease in susceptible animals, which consists in their specific immunization, and assessment of their immune status [1, 14]. Given that domestic carnivores and farm animals began to become more and more involved in the epizootic process, their specific prophylaxis becomes especially relevant [15].

For the prevention of rabies in animals, live oral and inactivated culture rabies vaccines are currently used [16]. Used rabies vaccines have protective properties against lissaviruses of phylogenetic group 1 (the classic rabies virus of the genetic line RABV) [1].

In accordance with OIE international requirements, rabies vaccines must be safe, have an immunogenicity index of at least 1.0 ME at the lowest prescribed dose, form reliable anti-rabies immunity in target animal species, and cause the production of virus-neutralizing antibodies (VNA) against rabies virus with a protective titer ≥0.5 IU / cm ³ [2, 14].

For practical use, many different rabies vaccines for animals have been developed and tested (brain, embryonic, cultural and others). The process of improving manufacturing technology allows you to abandon obsolete samples in favor of more advanced and high-quality drugs. Rabies vaccines made on the basis of the culture of rabies virus are safer, areactogenic, relatively cheap at cost and effective [1].

Currently, rabies inactivated culture vaccines from various fixed strains of rabies virus, including Schelkovo-51 and ARRIAH, are used to prevent rabies in domestic carnivores and farm animals in Russia [16, 17, 18].

The strain Schelkovo-51 of the rabies virus was obtained at the Schelkovo Biocombine (RF) as a result of the adaptation of the Pasteur strain of Ovine-VGNKI to the transplanted cell subline from the kidney of a newborn Syrian hamster (VNK-21/13) [1]. This strain of rabies virus belongs to phylogenetic group 1, the genetic line of RABV [19]. The following vaccine preparations are made on the basis of the Shchelkovo-51 strain of rabies virus: the inactivated dry culture anti-rabies vaccine from the Shchelkovo-51 strain for dogs and cats "Rabikan"; rabies vaccine from the Schelkovo-51 strain inactivated liquid culture "Rabikov"; vaccine antirabies for animals "UNIREV"; dry rabies vaccine for cattle and small cattle.

The vaccine "Rabikan" is intended for the preventive and compulsory immunization of domestic carnivores against rabies. The manufacturer of this vaccine is FKP Shchelkovo Biocombine (RF). The preparation is a fixed rabies virus of the Schelkovo-51 strain, lyophilized, inactivated with β-propiolactone, grown in a cell culture of BHK-21. In primary vaccinated animals, immunity begins to form from 5-7 days after a single injection of the vaccine, reaches its maximum by 30-40 days and lasts up to a year. After secondary vaccination, carried out 30-50 days after the first, the protective reaction of the body is enhanced and intense immunity persists for 2 years. The immunogenic activity of the drug is at least 1.0 IU / cm ³ [16, 17]. The disadvantage of the analogue is that with a single vaccination of domestic carnivores, the post-vaccination rabies antibodies almost completely disappear 12 months after immunization and, therefore, re-inoculation is required. Also, the use of this drug for vaccination of farm animals is not provided.

Rabikov vaccine is intended for the prevention and forced immunization of cattle, sheep and goats, reindeers and horses against rabies. The drug is a homogeneous suspension of light gray color. The vaccine is made from a fixed rabies virus of the Schelkovo-51 strain, reproduced in the BHK-21 cell line, inactivated with β-propiolactone and adsorbed on alumina hydrate (Al ₂ O ₃ ⋅nH ₂ O). In initially vaccinated animals, immunity begins to form from 5-7 days after the vaccine is administered, reaches its maximum by 30-40 days, and lasts up to a year. After secondary vaccination, carried out 30-50 days after the first, the protective reaction of the body is enhanced and intense immunity can be maintained for 2 years. The immunogenic activity of the vaccine is at least 1.0 IU / cm ³ [16, 17]. A disadvantage of the analogue is that during short storage at the bottom a loose sediment of light gray color and a transparent supernatant with a yellowish tinge are formed. In addition, the vaccine uses alumina hydrate, the immunostimulating activity of which does not in all cases [20] manifest itself at a level that, after initial and single vaccination, induces anti-rabies antibodies with a titer of ≥0.5 IU / cm ³ , recognized by OIE as sufficient to protect animals from rabies [2].

The antirabies vaccine for animals "UNIREV" is intended for the prevention of cats, dogs and cattle from rabies. The manufacturer of the drug is the All-Russian Scientific Research and Technological Institute of the Biological Industry of the Russian Agricultural Academy (FGBNU "VNITIBP", Russian Federation). This vaccine is inactivated adsorbed. It includes inactivated using UV light and ethanol rabies virus strain Schelkovo-51 "(wt. 70-80%) and as an adjuvant aluminum hydroxide (wt. 9-21%). Additionally, the preparation contains ethanol (wt. 9-11%), which reliably protects the vaccine from bacterial growth and increases antibody-inducing activity for target animals. The vaccine contains at least 2.0 ME of viral material in 1.0 cm ^{3 of the} preparation. The formation of an immune response against rabies virus occurs on days 14-21 after vaccination of animals. When using a vaccine stored for 14 months at a temperature of 6-8 ° C, the number of virus-neutralizing antibodies in the blood serum of dogs (puppies) immunized initially and once, on the 27th day after vaccination is 2.5-3.4 IU / cm ³ , in kittens - 1.7-2.2 IU / cm ³ . The duration of immunity of domestic carnivores is 12 months. However, information on the tension of animal immunity against rabies during the year is not presented [20].

The disadvantages of the analogue is that 12 months after the primary and single immunization with an anti-rabies sorbed vaccine, the number of antibodies against rabies virus is below the protective level (0.5 IU / cm ³ ), and the possibility of using this drug for the prevention of farm animals is not provided.

Dry rabies culture vaccine for cattle and small cattle (manufacturer - FSBI "VNIIZZH", Russian Federation) includes rabies antigen, cultivation medium, protective medium for drying based on peptone, sucrose and gelatin, as well as an immune stimulator saponin. The drying medium additionally contains alumina hydrate in the following ratio of components in the final product (wt.%): Alumina hydrate with inactivated rabies virus adsorbed on it - 6.0-9.0, saponin - 0.75-1.50, peptone - 30.0-33.0, sucrose - 35.0-37.0, gelatin - 9.0-10.0, cultivation medium - the rest. Dry vaccine has a high level of immunogenic activity. The antibody-inducing activity of the rabies vaccine in the experiment on cattle 45 days after immunization is 3.5-5.1 IU / cm ³ , after 350 days - 0.38-0.65 IU / cm ³ . The immunogenicity of this rabies vaccine in the experiment on sheep and goats 35 days after inoculation is 2.2-2.8 IU / cm ³ and 3.1-4.2 IU / cm ³ , respectively, 14 days after re-vaccination in sheep and goats - 65-87 IU / cm ³ and 40-48 IU / cm ³ , respectively [21].

The disadvantage of the analogue is that 350 days after immunization with this vaccine, the number of antibodies against rabies virus in some animals was below the protective level (0.5 IU / cm ³ ), and the possibility of using this drug for the prevention of rabies in domestic carnivores is not provided . The vaccine has high immunogenic activity for 11-12 months, however, it contains saponin, which can cause toxic effects in small cattle, manifested in impaired sexual function and nervous system disorders [22].

In 1997, by adapting the rabies virus Schelkovo-51 vaccine strain to the BHK-21 / 2-17b suspension cell subline, the rabies virus production strain VNIIZH was obtained.

Known vaccine anti-rabies inactivated liquid culture (FSBI "ARRIAH") [1, 23, 24, 25], which is intended for the prophylactic and emergency immunization of cattle, small cattle, pigs, horses, dogs and cats against rabies. This vaccine is inactivated adsorbed. It contains the active substance in the form of avirulent and purified rabies antigen of the strain "ARRIAH", obtained in a sensitive biological system, and targeted additives in the form of a mineral adjuvant aluminum hydroxide (Al (OH) ₃ ) and a preservative of merthiolate in the ratio, wt. %:

Antigenic material 89.95-90.00 Aluminum hydroxide 9.95-10.00 Merthiolate 0.05

As a sensitive biological system for the reproduction of rabies virus using transplantable suspension cell line from the kidney of a newborn Syrian hamster VNK-21. The support medium is Earl's solution without serum, with the addition of dry muscle enzyme hydrolyzate (FGMS), dry blood protein hydrolyzate (GBKS) and antibiotics (pH 7.7-7.9). For inactivation of the virus, aminoethylethyleneimine (AEEI) is used. Purification of the virus-containing suspension from ballast impurities is carried out by the method of simple sedimentation of particles.

The vaccine causes the formation of an immune response to rabies virus 21 days after a single application, which lasts for 12 months.

The disadvantage of this vaccine is that when using the sorbed vaccine after 12 months, repeated inoculation of animals is required to form a tense immunity against rabies.

Significant disadvantages of the known dry and sorbed rabies vaccines are their insufficient immunogenic activity and the need for cases of additional immunization of animals, which is explained by the absence of a longer action and better preservation of the antigen, as well as the impossibility of introducing a more concentrated antigenic part.

The closest analogue of the claimed vaccine in terms of essential features is a culture inactivated rabies vaccine from the strain "ARRIAH" (vaccine analogue), which is intended for the prophylactic immunization of cattle and small cattle, horses and dogs against rabies [26]. This vaccine contains the active substance in the form of an inactivated rabies virus antigen of the strain "ARRIAH", obtained in a sensitive biological system, and the target additive in the form of an oil adjuvant ISA-70 in the ratio, wt. %:

Antigenic material thirty Oil Adjuvant ISA-70 70

As a sensitive biological system for the reproduction of rabies virus using transplantable suspension cell line from the kidney of a newborn Syrian hamster VNK-21-2-17b. The support medium consists of Earl's solution without serum, with the addition of dry muscle enzyme hydrolyzate (FGMS), dry blood protein hydrolyzate (GBKS) and antibiotics (pH 7.4-7.6). For inactivation of the virus, aminoethylethyleneimine (AEEI) is used in an amount of 0.02% (by volume). In the manufacture of the vaccine, ISA-70 is used as an oil adjuvant. Purification of the virus-containing suspension from ballast impurities is carried out by the method of simple sedimentation of particles.

Immunization with this vaccine in a single dose induces the production of anti-rabies antibodies in cattle in an amount of 5.2; 4.7; 5.1; 5.0 log ₂ or 2.30; 1.62; 2.14; 2.00 IU / cm ³ after 1, 2, 3 and 4 months after vaccination, respectively. The antibody titer against rabies virus in horses was 5.0; 5.3; 5.3; 5.0 log ₂ or 2.00; 2.46; 2.46; 2.00 IU / cm ³ after 1, 2, 3 and 4 months after a single immunization with this drug, respectively. A single inoculation of a sheep with this vaccine causes the production of anti-rabies antibodies in titers of 5.50; 5.53; 5.00; 5.00 log ₂ or 2.83; 2.89; 2.00; 2.00 IU / cm ³ 1, 2, 3 and 4 months after the administration of this vaccine, respectively. In this case, the introduction of the indicated vaccine, which includes the adjuvant ISA-70, activates only humoral immunity. Information about the level of post-vaccination immunity tension after 5 or more months after vaccination with the proposed experimental vaccine is not presented [26].

The immunogenicity index of this vaccine after manufacture is 1.30-1.45 IU / cm ³ , and after 12 months of storage at a temperature of 4-8 ° C - 1.30-1.40 IU / cm ³ [26]. Thus, the culture inactivated rabies vaccine from the strain "ARRIAH" during the year, subject to storage conditions, retains the necessary indicators of immunogenicity.

A significant drawback of the anti-rabies vaccine analogue is the absence of a more prolonged action of the inactivated rabies antigen and the possibility of administering a concentrated antigenic part of the vaccine [27] in an amount of more than 30%, which, in turn, does not allow the formation of a more prolonged intense immunity against rabies infection. In addition, the use of a vaccine analogue does not provide activation of cellular immunity, which plays a significant role in the formation of protection against the causative agent of rabies [1].

To solve this problem, the present invention was created, which included the development of an inactivated emulsion culture anti-rabies vaccine for the prophylactic immunization of domestic carnivores and farm animals.

The technical result from the use of the present invention is to increase the immunogenicity of the rabies emulsion vaccine, which ensures the activation of long-term intense humoral and cellular immunity of domestic carnivores and farm animals due to the introduction of a larger amount of antigenic part (40% by weight) in the vaccine preparation and the use of Montanide ISA oil adjuvant -61 VG [28].

The specified technical result was achieved by the creation of an inactivated emulsion culture anti-rabies vaccine for the prophylactic immunization of domestic carnivores and farm animals, characterized by the following set of features.

The developed vaccine in 1.0 cm ^{3 of the} preparation contains the following components: 1) the active substance in the form of avirulent and purified antigenic material from the strain "ARRIAH" of the rabies virus, preferably reproduced in transplantable suspension cell subline VNK-21 / SUSP / ARRIAH, with quantities of glycoprotein a viral envelope of at least 1.5 μg and a virion nucleoprotein of at least 1.5 μg, which corresponds to an immunogenicity index (JJ) of at least 4.5 ME; 2) Montanide ISA-61 VG oil adjuvant ("SEPPIC"), preferably in an amount of 575000.0-700000.0 μg.

The production Schelkovo-51 strain of rabies virus, which served as the source for the strain ARRIAH, was created by conducting more than 50 consecutive passages in the cell culture of BHK-21/2 in 1997, followed by adaptation in 2016-2018. to suspension cultivation in the cell subline BHK-21 / SUSP / ARRIAH.

The strain "ARRIAH" of the rabies virus was deposited in the collection of microorganism strains of the Federal State Budgetary Institution "Federal Center for Animal Health" (FSBI "ARRIAH"), under the registration number: "ARRIAH" -DEP for fixed rabies virus dated 12/20/2018

The strain "ARRIAH" of the rabies virus is adapted to the primary culture of pig kidney cells (SP) (Abelseth MK, 1964), the primary hamster kidney cell line (Kissling R.E., 1958), a monolayer cell line of mouse neuroblastoma (NA-2), monolayer cell culture from the green monkey kidney (Vero) (Montagnon BJ et al., 1985), a monolayer cell culture from the kidney of a newborn Syrian hamster (VNK-21 / 2-17) (FSBI ARRIAH), suspension cell subline from the kidney of a newborn Syrian hamster (VNK-21 / SUSP / ARRIAH) (FSBI ARRIAH) and others.

For the manufacture of the vaccine, a transplantable suspension culture of BHK-21 / SUSP / ARRIAH is preferably used as a sensitive biological system. As a supporting medium, MEM Needle medium is used without introducing cattle blood serum with the addition of FGMS and a liquid hydrolyzate of blood proteins (pH 7.4–7.6). Inactivation of rabies virus is carried out using AEEI with a concentration of up to 0.025% (by volume). After the end of inactivation, the suspension is cooled to a temperature of 8-10 ° C, merthiolate is added to 0.002% (by weight) and left for 48 hours for the sedimentation process. The required concentration of antigen in the vaccine is provided due to its concentration by flow ultrafiltration [29, 30].

To prepare the vaccine, avirulent and purified antigenic material from the strain “ARRIAH” of rabies virus is used in the form of a suspension with an amount of glycoprotein of at least 3.75 μg / cm ³ and a nucleoprotein of at least 3.75 μg / cm ³ , which corresponds to an immunogenicity index of at least 4 5 ME in 1.0 cm ³ vaccine. These concentrations of immunogenic components provide the content of glycoprotein and nucleoprotein in quantities of each not less than 1.5 ME in 1.0 cm ^{3 of the} drug, given that in the manufacture of the vaccine the antigenic part is 40% (by weight). Determination of the concentration of rabies virus glycoprotein and nucleoprotein is carried out using enzyme-linked immunosorbent assay using polyclonal antibodies specific against these antigens [19]. Evaluation of immunogenic potency is carried out in accordance with the recommendations of Manual of diagnostic tests and vaccines for terrestrial animals [2].

An anti-rabies inactivated emulsion culture vaccine is obtained by dispersing with a homogenizer the rabies virus antigen concentrate of the ARRIAH strain and the Montanide ISA-61 VG oil adjuvant manufactured by SEPPIC in a ratio of 40/60 by weight, respectively. The resulting vaccine is a milk-like liquid that is not soluble in water.

The present invention includes the following set of essential features that provide a technical result in all cases for which legal protection is requested:

1. Anti-rabies inactivated emulsion culture vaccine for the prophylactic immunization of domestic carnivores and farm animals.

2. The active substance in the form of avirulent and purified antigenic material of rabies virus from the strain "ARRIAH" (a classic version of the phylogenetic group 1, the genetic line of RABV) in an effective amount.

3. Targeted supplements.

The salient features of the proposed vaccine are that the active substance is an avirulent purified antigenic material of rabies virus of the ARRIAH strain of an effective amount (antigenic concentrate in an amount of 40% by weight of the drug), and an oil-based immune response is used adjuvant Montanide ISA-61 VG.

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

1. Avirulent and purified antigenic material from the ARRIAH strain of the rabies virus of the RABV genetic line, preferably obtained in the suspension transplantable cell line BHK-21 / SUSP / ARRIAH and which is a suspension containing predominantly immunogenic components of the rabies virus in effective amounts.

2. Avirulent and purified antigenic material from the ARRIAH strain of the rabies virus of the RABV genetic line, preferably obtained in the suspension transplantable cell line BHK-21 / SUSP / ARRIAH and representing a suspension containing the immunogenic components of the rabies virus in the form of at least 3 glycoprotein , 75 μg / cm ³ and a nucleoprotein of at least 3.75 μg / cm ³ (40% by weight of the drug), which corresponds to a vaccine immunogenicity index of at least 4.5 ME in 1.0 cm ^{3 of the} finished drug.

3. Of the targeted additives, the vaccine contains an oil adjuvant.

4. Of the targeted supplements, the vaccine contains the Montanide ISA-61 VG Oil Adjuvant.

5. The vaccine contains Montanide ISA-61 VG oil adjuvant in an amount of 575000.0-700000.0 μg (60% by weight of the preparation) in 1.0 cm ^{3 of the} finished preparation.

6. The vaccine contains the preservative merthiolate in an amount of 0.002% (by weight).

7. Avirulent and purified antigenic material from the ARRIAH strain of the rabies virus of the RABV genetic line, obtained preferably in the suspension transplantable cell line BHK-21 / SUSP / ARRIAH, Montanide ISA-61 VG oil adjuvant and the preservative merthiolate in the following amounts of the finished preparation (1 , 0 cm ³ ):

The proposed vaccine has high immunogenic activity and provides reliable long-term protection against rabies virus of the genetic line RABV, circulating around the world [1].

The achievement of the technical result from the use of the invention is ensured by the fact that the proposed rabies vaccine is made of an avirulent rabies virus antigen of the ARRIAH strain in an effective amount in combination with Montanide ISA-61 VG oil adjuvant and conserved merthiolate. This combination determines the high immunogenic activity of the vaccine, which creates effective early and long-term protection of domestic carnivores and farm animals against rabies caused by the RABV genetic line of viruses and also activates not only humoral, but also cellular immunity against rabies infection in target animals.

The invention is reflected in the graphic images:

FIG. 1 - Dendrogram reflecting the phylogenetic relationship of the strain "ARRIAH" with other strains of rabies virus (when comparing the complete nucleotide sequences of the N gene (1350 n.a.)). Marker ♦ indicates the strain "ARRIAH" of the rabies virus. The abbreviation denotes representatives of various genotypes of the genus Lyssavirus: EBLV-1 - European bat lyssavims 1; EBLV-2 - European bat lyssavims 2; KHUV - Khujand virus; BBLV - Bokeloh bat lyssavims; ARAV - Aravan virus; IRKV - Irkut virus; DUVV - Duvenhage virus; ABLV - Australian bat lyssavims; RABV - Rabies virus (rabies virus, lissavirus of the 1st genotype); IKOV - Ikoma virus; WCBV - West Caucasian bat virus; SHIBV - Shimon bat virus; LBV - Lagos bat virus; MOKV - Mokola virus.

FIG. 2 - Antigenic relationship of the strain "ARRIAH" with the production strains of the rabies virus "RV-97", "SAD B19", "Schelkovo-51" and field isolates RV 257 Omsk, 24/2010 Tuva, 1634/2008 Saratov (European group), 1352/2008 Krasnodar, RV 308 Georgia, 18/2005 Krasnodar (Caucasian group), 33/2005 Bryansk, 552/2010 Kaliningrad (North European group), 211/2010 Vladimir, 110/2009 NNovgorod, 705/2009 Moscow (Central group), 304c Chita, SG 21 Yakutia, 1410/2008 Komi (Arctic group) in the neutralization reaction.

FIG. 3 - The results of the cultivation of the strain “ARRIAH” of rabies virus in primary cultures of pig and hamster kidney cells, in heteroploid cell lines from the kidney of the newborn Syrian hamster VNK-21 / 2-17 and VNK-21 / SUSP / ARRIAH, in mouse neuroblastoma cells ( NA-2) and green monkey kidney (Vero) (n = 3).

FIG. 4 - Comparative analysis of the biological properties of the strain "ARRIAH" with production strains of rabies virus of the genetic line RABV ("RV-97", "Schelkovo-51", "CVS-27") (n = 3).

FIG. 5 - Electrophoregram of DNA fragments of the ARRIAH strain of rabies virus, positive and negative controls with primer systems D. Oligo-R721 (a) (for gene N), G0326-G0327 (b) (for gene G), G0341-G0342 (c ) (for gene N).

FIG. 6 - Graphs of amplification of fragments of complementary DNA of production strains "ARRIAH" (A), "RV-97" (B), "SAD B19" (C), "Schelkovo-51" (D) of rabies virus in PCR-RV with different systems primers (F4924-R5063, CF17-R230, F11-SR220) (for gene N) and probes (Cy5 and FAM).

FIG. 7 - Dynamics of the level of subpopulations of CD4 + and CD8 + lymphocytes in cattle blood at different times after administration of a single dose of rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 8 - Dynamics of the level of CD4 + / CD8 + cells in cattle blood at different times after administration of a single dose of the rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 9 - Dynamics of the level of subpopulations of CD4 + and CD8 + cells in goat blood at different times after administration of a single dose of rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 10 - Dynamics of the level of CD4 + / CD8 + cells in goat blood at different times after administration of a single dose of the rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 11 - Dynamics of the level of subpopulations of CD4 + and CD8 + cells in the blood of cats at different times after administration of a single dose of rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 12 - Dynamics of the level of CD4 + / CD8 + cells in the blood of cats at different times after the administration of one dose of the rabies inactivated emulsion culture vaccine (No. 1), the culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 13 - Dynamics of the level of subpopulations of CD4 + and CD8 + cells in the blood of dogs at different times after administration of a single dose of the rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 14 - Dynamics of the level of CD4 + / CD8 + cells in the blood of dogs at different times after the administration of one dose of the rabies inactivated emulsion culture vaccine (No. 1), the culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 15 - Dynamics of the level of subpopulations of CD4 + and CD8 + cells in the blood of pigs at different times after administration of a single dose of rabies inactivated emulsion culture vaccine (No. 1), culture inactivated rabies vaccine from strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

FIG. 16 - Dynamics of the level of CD4 + / CD8 + cells in the blood of pigs at different times after the administration of one dose of the rabies inactivated emulsion culture vaccine (No. 1), the culture inactivated rabies vaccine from the strain "ARRIAH" (No. 2). Control without vaccination - No. 3.

The invention is set forth in the list of sequences in which:

SEQ ID NO: 1 represents the nucleotide sequence of the N gene (1350 n.a.) of the ARRIAH strain of the rabies virus of phylogenetic group 1, the RABV genetic line;

SEQ ID NO: 2 reflects the amino acid sequence (450 aa) corresponding to the nucleotide sequence of the N gene of the ARRIAH strain of the rabies virus of phylogenetic group 1, the RABV genetic line.

SEQ ID NO: 3 represents a fragment of the pseudogen nucleotide sequence (ψ fragment) (540 n.a.) of the ARRIAH strain of phylogenetic group 1, the RABV genetic line;

SEQ ID NO: 4 reflects the amino acid sequence (180 aa) corresponding to a fragment of the nucleotide sequence of the pseudogen (ψ fragment) of the ARRIAH strain of the phylogenetic group 1 of the RABV genetic line.

The strain "ARRIAH" rabies virus of phylogenetic group 1 of the RABV genetic line is characterized by the following features and properties.

Morphological features

The strain "ARRIAH" of the rabies virus belongs to the order Mononegavirales, the family Rhabdoviridae, the genus Lyssavirus, the species Rabies lyssavirus and has morphological characteristics characteristic of the causative agent of rabies: the shape of the virion is bullet-shaped, length ≈180 nm (100-430 nm), diameter ≈75 nm (100 nm) 45-100 nm). On the outer surface of the viral particle there are protrusions in the form of spikes 10 nm long, which are attached to the bilayer lipid membrane. The virion consists of a non-segmented genome represented by a single molecule of helically-twisted negative RNA, approximately 12,000 n.o. [1].

Antigenic properties

The rabies virus virion of the ARRIAH strain contains 2 main antigens: the viral envelope glycoprotein (G-protein) and the virion's internal nucleoprotein (N-protein) [1].

G-protein induces the formation of neutralizing antibodies and determines the development of humoral immunity in animals against rabies virus.

The strain "ARRIAH" and most other strains and field isolates of rabies virus isolated on different continents, contain a common nucleoprotein antigen, which is detected by the immunofluorescence reaction (RIF), or by the method of fluorescent antibodies (MFA), as well as in the reaction of diffuse precipitation (RDP) ) [nineteen]. The nucleoprotein antigen has the ability to stimulate cross-reactions between rabies virus isolates. N-protein plays an important role in the formation of cellular immunity, in particular, interacts with T-helpers, activates CD4 + and CD8 + cells [31]. A nucleoprotein is able to stimulate the immune system without intracellular processing and splicing, and a potentially protective immune response to highly conserved N-protein epitopes is formed (positions 404-418) [32].

The nucleotide sequencing method was used to determine the primary structure of the N gene of the strain "ARRIAH" rabies virus. A comparative analysis of the nucleotide sequences of the N gene showed that the strain "ARRIAH" of the rabies virus belongs to the RABV genetic line of the first phylogenetic group (Fig. 1).

Antigenic relationship of the strain "ARRIAH" with the production strains "RV-97", "SAD B19", "Schelkovo-51" of the rabies virus, as well as with field isolates from different geographical groups in the Russian Federation: RV 257 Omsk, 24/2010 Tuva, 1634/2008 Saratov (European Group), 1352/2008 Krasnodar, RV 308 Georgia, 18/2005 Krasnodar (Caucasian Group), 33/2005 Bryansk, 552/2010 Kaliningrad (North European Group), 211/2010 Vladimir, 110 / 2009 NNovgorod, 705/2009 Moscow (Central group), 304c Chita, SG 21 Yakutia, 1410/2008 Komi (Arctic group) was studied in the neutralization reaction. The reference strain “CVS-27” adapted to the cell culture (ANSES, Nancy, France) was used as the initial control strain in the neutralization reaction. The reference serum was the serum against the strain "ARRIAH" of the rabies virus with a titer of 2.5 IU / cm ³ (FSBI "ARRIAH"). The study of antigenic affinity (matching) in the neutralization reaction was performed in accordance with the recommendations of the OIE [2]. The value of the coefficient of antigenic affinity (r ₁ ) was calculated by the formula:

When r ₁ > 0.30, the studied strains are closely related, and the vaccine from the production strain will protect against epizootic virus, when r ₁ <0.30 the field isolate is different from the production strain, and the vaccine from this strain will not provide protection against field isolate . The research results are presented in table 1 and in FIG. 2.

From the data of table 1 and FIG. 2 it follows that the coefficient r ₁ for the strain "CVS-27" amounted to 0.41; "RV-97" - 0.65; "SAD B19" - 0.42; "Schelkovo-51" - 0.71; RV 257 Omsk - 0.40; 24/2010 Tuva - 0.45; 1634/2008 Saratov - 0.51; 1352/2008 Krasnodar - 0.48; RV 308 Georgia - 0.58; 18/2005 Krasnodar - 0.51; 33/2005 Bryansk - 0.39; 552/2010 Kaliningrad - 0.46; 211/2010 Vladimir - 0.65; 110/2009 NNovgorod - 0.62; 705/2009 Moscow - 0.53; 304c Chita - 0.61; SG 21 Yakutia - 0.64; 1410/2008 Komi - 0.62. Thus, the presented strain “ARRIAH” of rabies virus shows cross-serological reactions with representatives of the first phylogenetic line of the RABV genetic group and allows protection against rabies virus infection of this group by isolates.

Biotechnological characteristics

The strain "ARRIAH" of rabies virus is reproduced in primary cell cultures: pig kidney (Abelseth MK, 1964), hamster kidney (Kissling R.E., 1958); in heteroploid cell lines: monolayer culture of cells from the kidney of a newborn Syrian hamster (VNK-21 / 2-17) (FSBI "ARRIAH", Vladimir); suspension cell subline from the kidney of a newborn Syrian hamster (VNK-21 / SUSP / ARRIAH) (FSBI "ARRIAH", Vladimir); mouse neuroblastoma cells (NA-2); cells from a green monkey kidney (Vero) (Montagnon BJ et al., 1985) and others. The infection dose was 0.005 KKID ₅₀ / cell. To determine the biological activity, each passage virus was titrated in an inoculated monolayer cell culture of BHK-21 / 2-17b [33]. Within 48-96 hours of incubation, the amount of virus in these cell cultures reaches high values from 5.60 ± 0.25 to 7.75 ± 0.20 lg CCID ₅₀ / cm ³ (table 2). The highest rates of increase in the number of strain "ARRIAH" of rabies virus are achieved by reproduction in the suspension cell line BHK-21 / SUSP / ARRIAH. This strain retains its original characteristics when passaged in these cell cultures for 10 consecutive passages (observation period).

Gene and chemotaxonomic characteristics

The strain "ARRIAH" of the rabies virus belongs to the genetic group RABV phylogenetic line 1.

The causative agent is an RNA-containing virus with an average molecular weight of 0.011926 Mb [32]. Nucleic acid is represented by a single-chain negative non-segmented helical molecule with a length of approximately 12000 n.a. The rabies virus virion is bullet-shaped, ≈180 nm long (100-430 nm), ≈75 nm diameter (45-100 nm). On the outer surface of the viral particle there are protrusions in the form of spikes 10 nm long, which are attached to the bilayer lipid membrane [1].

The main antigenic proteins are the viral envelope glycoprotein (G-protein) and the virion's internal nucleoprotein (N-protein). The purified preparations of the strain "ARRIAH" of the rabies virus contain approximately 1% RNA, 72% proteins, 22% lipids and 3% carbohydrates.

The rabies virus RNA of the ARRIAH strain encodes 5 basic proteins: nucleoprotein (N-protein), phosphoprotein (P-protein), matrix protein (M-protein), glycoprotein (G-protein), RNA-dependent RNA polymerase (L- protein) [1]. The presented proteins and the associated RNA form a ribonucleoprotein, which controls transcription processes [34].

Resistance to external factors

The strain "ARRIAH" of the rabies virus is sensitive to ether, chloroform, acetone, methanol, trypsin, phospholipase. Disinfectants, such as 1-5% formaldehyde solution, 1% bleach solution, 1% sodium hypochlorite solution, 3-5% hydrochloric acid solution, 45-70% ethanol solution, β-propiolactone and others, inactivate the rabies virus.

At a temperature of 70 ° C, the strain "ARRIAH" of the rabies virus is instantly inactivated, at a temperature of 60 ° C for 5-10 minutes, at 50 ° C for 60 minutes, at 35 ° C for 20 days, at 23 ° C - within 35 days. Low temperatures preserve this virus. In the frozen state, this strain retains its activity for about 10 years. The virus is well preserved in a lyophilized state and mixed with 50% glycerol.

UV light destroys the strain "ARRIAH" of the rabies virus within 5-10 minutes. The virus is relatively stable at pH values of 5-10. Most stable at pH 7.4-7.6. Rabies virus is highly sensitive to very acidic (pH <3) and very alkaline (pH> 11).

Additional features and properties

Reactogenicity - does not possess reactogenic properties.

Pathogenicity - pathogenic to humans, foxes, wolves, coyotes, jackals, dogs, cats, rats, voles, hamsters, rabbits, sheep, goats, horses, cattle, squirrels, primates and others.

Virulence - is virulent for naturally susceptible animals during contact infection (mainly through a bite, during salivation of damaged surfaces) and aerogenically, as well as during organ and tissue transplantation.

Stability - retains its original biological properties when passaged in sensitive biological systems for 10 passages (observation period) in primary cell lines from a pig’s kidney and hamster’s kidney, as well as in heteroploid cell cultures of BHK-21 / 2-17 (monolayer), BHK- 21 / SUSP / ARRIAH (suspension), NA-2, Vero.

Based on the obtained analysis of morphological, biotechnological, antigenic and additional characteristics, it can be argued that the strain “ARRIAH” of rabies virus is highly stable during reproduction in sensitive cell lines, is characterized by high rates of infection titer, shows cross-serological reactions with various representatives of the first phylogenetic group of the genetic line RABV and allows you to provide protection against infection with isolates of the rabies virus of this genline.

In order to reduce the epizootic danger of the rabies virus of the RABV genetic line and to prevent the emergence of new foci of the disease, timely vaccine prophylaxis is important, which requires the development of a safe, convenient rabies vaccine with high efficiency.

Received anti-rabies inactivated emulsion culture vaccine for the prophylactic immunization of domestic carnivores and farm animals.

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

Example 1. The study of the biological properties of the strain "ARRIAH" rabies virus.

To study the biological properties of the strain "ARRIAH" of the rabies virus used viral material made in the form of a 10% suspension of sheep brain tissue infected with rabies virus.

The cultivation of the strain "ARRIAH" of rabies virus in primary cell cultures: pig kidney (Abelseth M.K., 1964), hamster kidney (Kissling R.E., 1958); in heteroploid cell lines: monolayer culture of cells from the kidney of a newborn Syrian hamster (VNK-21 / 2-17) (FSBI "ARRIAH", Vladimir); suspension cell subline from the kidney of a newborn Syrian hamster (VNK-21 / SUSP / ARRIAH) (FSBI "ARRIAH", Vladimir); mouse neuroblastoma cells (NA-2); cells from a green monkey kidney (Vero) (Montagnon B.J. et al., 1985).

Infectious activity of the strain "ARRIAH" of the rabies virus was determined by titration with a 10-fold step in a monolayer cell culture of BHK-21 / 2-17b. Plates with a cell monolayer and the required amounts of virus were incubated at 37 ° C in an atmosphere of 5% CO ₂ for 48 hours, then the monolayer was fixed with 80% acetone solution, followed by washing the wells and introducing the FITC conjugate. The reaction results are taken into account using a luminescent microscope. Rabies virus antigen in infected cells treated with anti-rabies FITC immunolobulin was detected by emerald or bright green diffuse luminescence of cytoplasm and inclusion granules, both in single affected cells and in cells located in groups (fluorescent foci) [2].

During the reproduction of the strain “ARRIAH” of the rabies virus in the primary culture of pig kidney cells from 1 to 10 passages, an increase in the titer of infectious activity from 5.60 ± 0.25 to 6.50 ± 0.20 lg CCID ₅₀ / cm ^{3 was} observed in the primary cell Hamster kidney line - from 5.60 ± 0.20 to 6.50 ± 0.25 lg CCID ₅₀ / cm ³ , in a monolayer culture of cells from a Syrian hamster kidney (VNK-21 / 2-17) - from 5.85 ± 0.15 to 7.25 ± 0.20 lg CCID ₅₀ / cm ³ , in suspension sublingual cells from a Syrian hamster kidney (BHK-21 / SUSP / ARRIAH) - from 6.00 ± 0.10 to 7.75 ± 0 , 15 lg CCID ₅₀ / cm ³ , in mouse neuroblastoma cells (NA-2) - from 5.70 ± 0.15 to 6.75 ± 0.15 lg CCID ₅₀ / cm ³ , in green monkey kidney cells (Vero) - from 5.60 ± 0.15 to 6.50 ± 0.20 lg CCID ₅₀ / cm ³ (table 2, Fig. 3).

The research results presented in table 2 and in FIG. 3, indicate a high adaptive activity of the strain "ARRIAH" of the rabies virus to these cell cultures. The virus reproduced in the cell suspension culture of BHK-21 / SUSP / ARRIAH was used to produce antigen in order to produce an experimental vaccine series.

Example 2. A comparative analysis of the biological properties of the strain "ARRIAH" with production strains of rabies virus of the genetic line RABV.

When studying the properties of the strain "ARRIAH" of the rabies virus, significant advantages were revealed in comparison with other used industrial strains "RV-97", "Schelkovo-51", "CVS-27" (control). A suspension of BHK-21 / SUSP / ARRIAH subline cells with a concentration of 3.0-3.5 million cells / cm ^{3 was} used as a test system for infection. The dose of infection was 0.005 CCID ₅₀ / cell.

In table 3 and in FIG. 4 shows the results of the cultivation of the presented strains of the rabies virus. Virus reproduction data testify to the advantages of the ARRIAH strain, because after 10 consecutive passages in the sensitive cell subline VNK-21 / SUSP / ARRIAH, higher values of the titer of infectious activity (7.75 ± 0.15 lg CCID ₅₀ / cm ³ ) compared with the strains "RV-97", "Schelkovo-51", "CVS-27" for which the values of the titer of infectivity corresponded to the readings 7.00 ± 0.10, 7.25 ± 0.11, 7.31 ± 0.16 lg CCID ₅₀ / cm ³ . According to the results of a comparative analysis, the accumulation of rabies virus glycoprotein of the strain "ARRIAH" was 2.11 ± 0.11 μg / cm ³ , which is 67.4, 44.5, 41.6% higher compared with the reproduction of strains "RV -97 "," Schelkovo-51 "," CVS-27 ", respectively. The amount of rabies virus nucleoprotein reproduced for the strain “ARRIAH” was 1.90 ± 0.12 μg / cm ³ , which is 140.5; 40.7; 53.2% higher compared with the cultivation of strains "RV-97", "Schelkovo-51", "CVS-27", respectively. Thus, the strain "ARRIAH" of the rabies virus, compared with the production strains presented during reproduction in the sensitive cell subline VNK-21 / SUSP / ARRIAH, is characterized by higher rates of accumulation of immunogenic components for the production of the rabies inactivated emulsion vaccine.

Example 3. Genetic identification of strain "ARRIAH" rabies virus.

At the first stage of the analysis, the affection of the ARRIAH strain of rabies virus was confirmed to belong to the RABV genetic line. RNA isolation of the strain “ARRIAH” of the rabies virus was performed using the RIBO-sorb RNA isolation kit (Federal State Research Institute for Scientific Research, Moscow) in accordance with the manufacturer's instructions. The obtained RNA eluate was used as a matrix for the formulation of the reverse transcription reaction, which was carried out in a C 1000 TM Thermo Cycler thermal cycler (or analog) with the following parameters: 42 ° C for 15 minutes, 99 ° C for 5 minutes. The reaction resulted in DNA complementary to rabies virus RNA, which was used to carry out the polymerase chain reaction (PCR). The reaction was carried out in a Master Cycler GSX1 thermocycler with the following thermal cycling parameters: 1) preliminary denaturation - 95 ° С for 10 minutes; 2) PCR: 35 cycles, each of which includes denaturation, which was carried out at a temperature of 94 ° C for 1 minute, annealing of primers - 55 ° C for 1 minute, elongation - 72 ° C for 1 minute. The reaction was carried out using a system of primers specific for the N gene (D. Oligo-R721, G0341-G0342) and for the G gene (G0326-G0327). The sizes of the fragments were 705, 280, 384 bp, respectively. A positive control was a suspension of the strain "CVS-27" of rabies virus. As a negative control, a BHK-21 / SUSP / ARRIAH cell suspension not infected with rabies virus was used. After obtaining the PCR products, horizontal electrophoresis was carried out in a 2% agarose gel at a voltage of 170 V for 12 minutes, followed by detection in the UV stream of the transilluminator and documentation in the video system “Sight”. The pUC Mix Marker, 8, was used as a marker. The results of the study are presented in FIG. 5, which shows that the strain "ARRIAH" belongs to the rabies virus of the genetic line RABV.

At the next stage of the study, the strain “ARRIAH” was differentiated from other production strains of rabies virus. DNA complementary to the RNA of the strains ARRIAH, RV-97, SAD B19, Schelkovo-51 of the rabies virus was used for real-time polymerase chain reaction (PCR-RV). The reaction was carried out in an iCycler iQ5 thermocycler with the following thermal cycling parameters: 1) preliminary denaturation - 95 ° С for 10 minutes; 2) PCR: 40 cycles, each of which includes denaturation carried out at a temperature of 95 ° C for 10 seconds, annealing the primers and probe, elongation - 60 ° C for 60 seconds.

Considering that rabies virus strains belong to different gene groups that differ in nucleotide sequence, two primer systems (CF 17 - R 230, F 11 - SR 220) specific for rabies virus of different gene groups were used for PCR-PB; the exception of the strain "ARRIAH". For amplification of a fragment of the genome of the strain "ARRIAH" used a specially designed system of primers (F4924-R5063), which allows a high degree of certainty to distinguish the vaccine strain "ARRIAH" from other production strains of rabies virus. The data of the primer system are complementary to the sections of the rabies virus genome at the border of G- and pseudogenes.

To detect the accumulation of amplified fragments of complementary rabies virus DNA, 2 TaqMan probes were used: ISO specific for many production strains and field isolates of rabies virus (the exception is the VNIIZh vaccine strain); VAC, which is highly specific for the strain "ARRIAH" of the rabies virus. Considering that the probes have fluorescence dyes with different wavelengths, the presence of the cyanine fluorescent dye Cy5 signal was used to judge the detection of the genome of the ARRIAH virus strain, the FAM dye (carboxyfluorescein) - the presence of many field isolates and vaccine virus strains, with the exception of the ARRIAH strain ". The results of the study are presented in Fig.6, from which it is seen that when using the system of primers F4924-R5063, an accumulation of the fluorescence signal was observed, which indicates the presence of DNA of the strain "ARRIAH" rabies virus. Moreover, testing of this material with primer systems CF 17 - R 230, F 11 - SR 220 no signal detection was noted (Fig. 6A). Analysis of production strains "ARRIAH",

"RV-97", "SAD B19", "Schelkovo-51" showed that when using the systems of primers CF 17 - R 230, F 11 - SR 220 identified strains "RV-97", "SAD B19", "Schelkovo- 51 "rabies virus and were not detected using the F4924-R5063 oligonucleotide system (Fig. 6). At the same time, the primer system F4924-R5063 allowed to prove that the strain "ARRIAH" refers to the rabies virus of the genetic line RABV, but identified strains "RV-97", "SAD B19", "Schelkovo-51". The results obtained indicate that the strain "ARRIAH" differs from other production strains of rabies virus in the sequence of pseudogen nucleotides (ψ-fragment).

Example 4. Obtaining an inactivated antigen, studying the effect of inactivation using AEEI and purification of antigenic material on the number of immunogenic components of the strain "ARRIAH" rabies virus.

An anti-rabies inactivated emulsion culture vaccine was obtained on the basis of the strain "ARRIAH" of rabies virus grown in suspension transplantable cell line BHK-21 / SUSP / ARRIAH. As a support medium, MEMA Needle medium was used without cattle serum supplemented with FGMS, a liquid hydrolyzate of blood proteins (pH 7.4-7.6). The cell culture was infected with this strain of rabies virus at the rate of 0.005 KKID ₅₀ / cell. After every 12 hours of cultivation, samples were taken to determine the pH and adjust the pH values within the specified limits.

At the end of the reproduction, the strain “ARRIAH” of the rabies virus was added to the suspension a solution of thiolate to a final concentration of 0.002% (by weight) and an AEEI solution to a concentration of 0.025% (by weight). Inactivation of rabies virus was carried out at a temperature of 36-37 ° C for 24 hours with periodic stirring. After the inactivation process, the suspension of inactivated virus was cooled to a temperature of 4-6 ° C. Purification of the viral suspension from cell detritus was carried out by simple sedimentation of particles.

The results of studies on the effect of inactivation and purification of antigenic material using AEEI on the number of immunogenic components of the strain "ARRIAH" of rabies virus for 10 samples are shown in table 4. The concentration of rabies virus glycoprotein and nucleoprotein was determined using enzyme-linked immunosorbent assay using polyclonal antibodies specific against these antigens [19]. From the data of table 4 it is seen that before inactivation, the average concentration of glycoprotein and nucleoprotein of the strain "ARRIAH" of rabies virus amounted to 2.20 ± 0.06 and 2.02 ± 0.03 μg / cm ³ , respectively. After the inactivation process, the average values for glycoprotein and nucleoprotein decreased by 8.02-10.83 and 7.61-10.44%, and were equal to 2.01 ± 0.06 and 1.84 ± 0.03 μg / cm ³ , respectively. Thus, the immunogenic components of the strain "ARRIAH" of the rabies virus are resistant to inactivation processes using 0.025% AEEI and purification by simple sedimentation.

Example 5. Concentration of the antigen of the strain "ARRIAH" rabies virus to obtain a vaccine.

To achieve the required concentration of the immunogenic components of the strain “ARRIAH” of rabies virus in 1.0 cm ^{3 of the} vaccine (glycoprotein - not less than 1.5 μg, nucleoprotein - not less than 1.5 μg), it is necessary to concentrate antigens to the amounts of each not less than 3.75 μg in 1.0 cm ^{3 of the} viral suspension, because at the dispersion stage the ratio of antigen and adjuvant is 40/60. The required concentration of the immunogenic components of the strain "ARRIAH" of the rabies virus in the vaccine dose of the emulsion vaccine was obtained by concentrating the antigen by flow ultrafiltration using ultrafilters at a pressure of 1.5 atm. The resulting antigen concentrate was stored at a temperature of 4-6 ° C until use. The resulting vaccine is packaged in glass or plastic bottles and sterilized products are monitored in accordance with GOST 28085-89.

Example 6. The process of dispersing the antigen and adjuvant to obtain rabies inactivated emulsion culture vaccine from strain "ARRIAH".

An anti-rabies inactivated emulsion culture vaccine was obtained by dispersing with a homogenizer the rabies virus antigen concentrate of the ARRIAH strain and the Montanide ISA-61 VG oil adjuvant in a ratio of 40/60 by weight, respectively.

The result was an emulsion vaccine, which was a milk-like liquid, insoluble in water. The vaccine had a fluid and easy-to-use consistency, resolved at the injection site, did not cause the formation of abscesses, a general reaction in the form of a temperature increase. The kinematic viscosity of the obtained preparation was 3.5–4.0 cSt, which is an average degree of viscosity and ranges from 3.0 to 7.0 cSt [35].

The optimal component composition of the resulting vaccine is presented on page 15.

Example 7. Evaluation of the immunogenicity index of the rabies inactivated emulsion culture vaccine from the strain "ARRIAH".

Evaluation of immunogenic potency was carried out in accordance with the recommendations of Manual of diagnostic tests and vaccines for terrestrial animals [2]. The developed rabies vaccine (using the Montanide ISA-61 VG adjuvant) and the analogue vaccine (using the ISA-70 adjuvant) were tested. As a control, we used the reference rabies vaccine from the CVS-27 strain with an immunogenicity index (JJ _ref ) of 2.0 IU / cm ³ (manufacturer - FSBI ARRIAH). For analysis of each drug, five mice weighing 18-20 g were used. Each mouse was vaccinated intramuscularly using 1/5 of the lowest dose volume, which was 0.1 cm ³ . 14 days after injection, blood samples were taken. Sera were tested for the presence of antibodies to rabies virus using the FAVN test [2]. The results of the evaluation of the immunogenicity index of the tested vaccines (JJ _isp ) are shown in table 5.

At least 3.0 μg of the immunogenic components of rabies virus (glycoprotein at least 1.5 μg and nucleoprotein at least 1.5 μg) was contained in 1.0 cm ^{3 of the} finished preparation, which corresponded to a vaccine immunogenicity index of at least 4.5 IU / cm ³ . From the data shown in table 5, it follows that the rabies inactivated emulsion culture vaccine from the strain "ARRIAH" reproduced in the suspension cell line BHK-21 / SUSP / ARRIAH and oil adjuvant Montanide ISA-61 VG, was characterized by an immunogenicity index of 5.76 ± 0.10 ME in 1.0 cm ^{3 of the} drug, which is higher compared with the inactivated liquid rabies vaccine from the strain "ARRIAH", reproduced in the cell culture of BHK-21 / 2-17b, and ISA-70 at 4.02 ME in 1.0 cm ^{3 of the} drug. In other words, the developed vaccine is characterized by high values of the immunogenicity index and met the requirements stated above for this drug (JJ not less than 4.50 ME in 1.0 cm ^{3 of the} vaccine).

Example 8. Assessment of the reactogenicity and harmlessness of the rabies inactivated emulsion culture vaccine.

Assessment of the reactogenicity and harmlessness of the rabies inactivated emulsion culture vaccine was performed on cattle, small cattle, pigs, dogs, cats and rabbits.

The vaccine was administered intramuscularly to all animal species at the dose recommended for each animal species. Grafting dose 2.0 cm³ designed for cattle, 1.0 cm³ - for sheep and goats, adult dogs of large and medium breeds, 0.5 cm³ - for cats, puppies from 2 months of age of all breeds, adult dogs of small breeds (lapdogs, dachshunds, spitz, etc.). The animals were clinically observed. Body temperature was measured within 7 days after injection, then 10 and 14 days after vaccination. The criterion for evaluating clinical observation was the development or absence of a general and local reaction to the administration of the test drug. The results of the study are presented in table 6, from which it follows that all the tested animals were clinically healthy, a general and local reaction to the introduction of the drug was not detected. The increase in body temperature in animals was insignificant (0.3-0.7 ° C) in 1-2 days after the vaccine. During this observation time, the temperature difference (ΔT = T_max-T_{on day 0 after vaccination}) in cattle was 0.3-0.6 ° C, in cattle - 0.3-0.4 ° C, in pigs - 0.3-0.7 ° C, in dogs - 0.3-0.6 ° C, in cats - 0.3-0.7 ° C, in rabbits - 0.3-0.5 ° C. Starting from 3 days, the temperature of the animals was normal. Local reaction (edema, soreness, lameness) in target animal species was not observed. Thus, according to the results of studies, the rabies inactivated emulsion culture vaccine is a harmless and aretogenic drug for target animal species.

Example 9. Evaluation of humoral and cellular immunity in cattle when using rabies inactivated emulsion culture vaccine in comparison with the vaccine analogue. The study of the protective properties of the developed vaccine on the example of cows.

The humoral immunity of cattle grafted with the rabies inactivated emulsion culture vaccine was evaluated in comparison with the analogue vaccine. As the closest prototype, a culture-inactivated rabies vaccine from the ARRIAH strain was used, which is intended for prophylactic vaccination of cattle, small cattle, horses and dogs against rabies [26]. This vaccine contains the active substance in the form of the inactivated rabies antigen of the strain "ARRIAH", obtained in a cell culture of BHK-21 / 2-17 (30% by weight), and an oil adjuvant ISA-70 (70% by weight).

Tests of the two vaccines presented were carried out on ten heads of cattle of black-motley breed at the age of 1 year with an average weight of 200 kg. Animals were inoculated with vaccines at a dose of 2.0 cm ³ intramuscularly once. To assess the intensity of post-vaccination anti-rabies immunity in animals, in accordance with the recommendations of the OIE, the neutralization reaction was used in a monolayer culture of BHK-21 cells (reaction modification - FAVN (Fluorescent antibody virus neutralization)), expressing the values of the titer of rabies antibodies in ME / cm ³ [2]. For analysis, a positive serum control standard diluted to immunogenicity of 0.5 IU / cm ³ and a negative serum control standard with immunogenicity <0.1 IU / cm ^{3 were used} [2].

In accordance with international requirements, the level of VNA against rabies virus ≥0.50 IU / cm ³ is protective [2]. In the neutralization reaction, blood serum from cattle was evaluated before and on day 21, as well as 2, 3, 6, 12, 15, 18 months after vaccination.

Before immunization, no rabies antibodies were detected in the blood of animals. The level of anti-rabies virus-neutralizing antibodies in cattle after administration of one recommended dose for this animal species (2.0 cm ³ ) of the anti-rabies inactivated emulsion culture vaccine on day 21 was 25.90 ± 0.42 IU / cm ³ , after 2 months - 42.50 ± 0.65 IU / cm ³ , after 3 months - 38.51 ± 0.68 IU / cm ³ , after 6 months - 24.80 ± 0.46 IU / cm ³ , after 12 months - 13.42 ± 0 , 84 IU / cm ³ , after 15 months - 4.83 ± 0.42 IU / cm ³ , after 18 months - 0.41 ± 0.11 IU / cm ³ (table 7). In other words, the introduction of a single dose (2.0 cm ³ ) of the rabies inactivated emulsion culture vaccine ensures the accumulation of a high level of antibodies (up to 42.50 ± 0.65 IU / cm ³ ) in the blood of cows and preserves the protective level of antibodies against rabies virus (≥ 0.50 IU / cm ³ ) up to 15 months.

The level of anti-rabies virus-neutralizing antibodies in cattle after administration of the recommended one dose (2.0 cm ³ ) of the inactivated rabies analogue of rabies vaccine of the ARRIAH strain for 21 days was 2.30 ± 0.43 IU / cm ³ , after 2 months - 1.62 ± 0.56 IU / cm ³ , after 3 months - 2.14 ± 0.33 IU / cm ³ , after 6 months - 1.95 ± 0.39 IU / cm ³ , after 12 months - 0.67 ± 0.19 IU / cm ³ , after 15 months - 0.15 ± 0.09 IU / cm ³ , after 18 months - 0.07 ± 0.02 IU / cm ³ . In other words, the introduction of a single dose (2.0 cm ³ ) of the culture of inactivated rabies vaccine from the strain "ARRIAH" provides the accumulation of the protective level of antibodies against rabies virus up to 12 months. Moreover, the analogue vaccine induces the production of anti-rabies antibodies 18.48 times lower than the developed vaccine.

At the next stage of the study, we evaluated cellular immunity in cattle after administering a single dose (2.0 cm ³ ) of the developed vaccine and the analogue vaccine.

Immunization of animals was carried out as described above in example 9. Blood from cattle was taken 1, 2, 3 weeks after vaccination. Lymphocytes were isolated from cattle blood. For this, the blood was treated with a 2% solution of ethylene diamine tetraacetate (EDTA) and centrifuged in a standard density gradient Ficoll-Paque PLUS. 3 cm ³ of Ficoll-Paque was layered with 2.0 cm ^{3 of} blood diluted with a 1: 1 solution of MEM Eagle medium and centrifuged for 30 minutes at 400 g. The isolated lymphocyte fraction was washed with 1/15 M phosphate buffered saline (PBS) and the resulting pellet was resuspended in 200 μl PBS. Cell counting was performed on a Countess Automated Cell Counter. The concentration of lymphocytes was adjusted with an FBI solution to 10 ⁶ -10 ⁷ cells / cm ³ .

An analysis of the subpopulation of CD4 + and CD8 + lymphocytes and determination of their relative amount in the blood of cattle was carried out by the cytofluorimetric method. The dynamics of the level of subpopulations of CD4 + and CD8 + cells in cattle blood at different times after the administration of a single dose (2.0 cm ³ ) of the rabies inactivated emulsion culture vaccine, culture inactivated rabies vaccine from the strain “ARRIAH” in comparison with the control without vaccination is presented in FIG. . 7. From the data of FIG. 7A and 7B, it follows that the relative number of CD4 + and CD8 + cells in the animal body after administration of the developed vaccine increased and amounted to 12-14 and 6-10%, respectively. The content of CD4 + and CD8 + lymphocytes after administration of the analogue vaccine was at the level of the values of control animals without vaccination and amounted to 3-5%.

Analyzing the ratio of CD4 + / CD8 + cells after the introduction of 2.0 cm ^{3 of the} developed vaccine, one can note a tendency to its decrease in cattle from 2.0 to 1.2, in other words, an increase in the relative number of CD8 + cells was observed (Fig. 8). Thus, the introduction of the rabies inactivated emulsion culture vaccine into cattle using Montanide ISA-61 VG oil adjuvant caused a change in the T-cell phenotype, which indicated activation of cellular immunity. After immunization of cattle with an inactivated liquid rabies analogue against rabies from the ARRIAH strain using the ISA-70 adjuvant, no induction of cellular immunity was noted.

To determine the protective properties of the developed rabies inactivated emulsion culture vaccine, we carried out a control infection with the strain CVS-27 of the rabies virus at a dose of 10 ⁴ LD ₅₀ / 0.20 cm ³ ten heads of cattle on 21 days, as well as 6, 12 and 15 months after immunization and five heads of unvaccinated animals. The animals of the experimental groups remained alive and clinically healthy; in the control group, the death of all heads of cattle was noted. Thus, the developed rabies inactivated emulsion culture vaccine is effective for immunization of cattle.

Example 10. Evaluation of humoral and cellular immunity in MP using an anti-rabies inactivated emulsion culture vaccine in comparison with an analogue vaccine. The study of the protective properties of the developed vaccine on the example of goats.

Evaluation of humoral immunity in goats vaccinated with the rabies inactivated emulsion culture vaccine was carried out in comparison with the inactivated liquid rabies vaccine analogue from the strain “ARRIAH”.

Tests of these vaccines were carried out on ten heads of goats of the Zaanensky breed at the age of 1 year. Animals were vaccinated with a vaccine in a dose of 1.0 cm ³ intramuscularly once. Before immunization and after 21 days, as well as 2, 3, 6, 12, 15, 18 months after vaccination, goats were taken from the goats and the obtained sera were examined for the presence of rabies antibodies in the neutralization reaction (FAVN modification) [2].

Before immunization, no rabies antibodies were detected in the blood of animals. The level of anti-rabies virus-neutralizing antibodies in goats after administration of one recommended dose (1.0 cm ³ ) of the anti-rabies inactivated emulsion culture vaccine for this animal species was 52.70 ± 0.99 IU / cm ³ after 2 months, 84.65 ± 0.85 IU / cm ³ , after 3 months - 79.65 ± 0.74 IU / cm ³ , after 6 months - 45.16 ± 0.89 IU / cm ³ , after 12 months - 25.42 ± 0 , 74 IU / cm ³ , after 15 months - 7.56 ± 0.40 IU / cm ³ , after 18 months - 0.49 ± 0.06 IU / cm ³ (table 8). Thus, the introduction of a single dose (1.0 cm ³ ) of the rabies inactivated emulsion culture vaccine ensures the accumulation of a high level of antibodies (up to 84.65 ± 0.85 IU / cm ³ ) in the blood of goats and preserves the protective level of antibodies against rabies virus (≥ 0.50 IU / cm ³ ) up to 15-18 months.

The titer of anti-rabies virus-neutralizing antibodies in goats after administration of the recommended single dose (1.0 cm ³ ) of the culture-inactivated rabies vaccine analogue from the ARRIAH strain for 21 days was 2.98 ± 0.51 IU / cm ³ , after 2 months - 3.15 ± 0.64 IU / cm ³ , after 3 months - 3.09 ± 0.41 IU / cm ³ , after 6 months - 2.43 ± 0.53 IU / cm ³ , after 12 months - 0.85 ± 0.22 IU / cm ³ , after 15 months - 0.38 ± 0.11 IU / cm ³ , after 18 months - 0.11 ± 0.03 IU / cm ³ . In other words, the use of a single dose (1.0 cm ³ ) of the inactivated rabies vaccine from the ARRIAH strain ensures the accumulation of the protective level of antibodies against rabies virus for up to 12 months. Moreover, this analogue vaccine induces the production of anti-rabies antibodies 26.87 times lower compared to the developed vaccine.

The next stage of the study was devoted to the assessment of cellular immunity in goats after the introduction of a single dose (1.0 cm ³ ) of the presented vaccine and vaccine analogue.

Immunization of animals, blood sampling, isolation and analysis of lymphocytes was carried out as described above in example 9. Dynamics of the level of subpopulations of CD4 + and CD8 + lymphocytes in the blood of goats 1, 2 and 3 weeks after the administration of a single dose (1.0 cm ³ ) of rabies inactivated emulsion culture vaccine, culture inactivated rabies vaccine from the strain "ARRIAH" in comparison with the control without vaccination is presented in Fig. 9. From the data of FIG. 9A and 9B shows that the relative number of CD4 + and CD8 + cells in the animal body after administration of the developed vaccine increased and amounted to 19-24 and 10-13%, respectively. The content of CD4 + and CD8 + lymphocytes after administration of the analogue vaccine was at the level of the values of control animals without vaccination and amounted to 3-6%.

Analysis of the ratio of CD4 + / CD8 + cells after administration of the developed vaccine showed that there was a slight tendency to decrease in goats from 1.90 to 1.83 (Fig. 10). The maximum relative number of CD4 + and CD8 + lymphocytes was observed 2 weeks after vaccination. In general, it was determined that administration of the rabies inactivated emulsion culture vaccine to goats using the Montanide ISA-61 VG oil adjuvant activated cellular immunity, which manifested itself in a change in the T cell phenotype. After immunization of the goats with a culture of inactivated rabies vaccine from the ARRIAH strain using the ISA-70 adjuvant, activation of cellular immunity was not observed.

The protective properties of the developed rabies inactivated emulsion culture vaccine were evaluated as shown in Example 9. After the challenge, all 10 goats vaccinated with a single dose (1.0 cm ³ ) of the vaccine remained alive and clinically healthy. In the control group, the death of all goats was noted. Thus, the developed rabies inactivated emulsion culture vaccine is effective for immunizing goats.

Example 11. Evaluation of humoral and cellular immunity in cats using the rabies inactivated emulsion culture vaccine in comparison with the vaccine analogue. The study of the protective properties of the developed vaccine on the example of cats.

The determination of the tension of humoral immunity in cats vaccinated with the rabies inactivated emulsion culture vaccine was carried out in comparison with the inactivated liquid rabies vaccine analogue of the strain "ARRIAH".

Testing of these vaccines was carried out on ten heads of mongrel cats aged 2 to 6 months weighing 0.5 kg. All animals were injected with the vaccine intramuscularly in a volume of 0.5 cm ³ once. Before vaccination and after 21 days, as well as 2, 3, 6, 12, 15, 18 months after immunization, cats were bled and the serum obtained was examined for the presence of antibodies against rabies virus in the neutralization reaction (FAVN modification) [2].

Before immunization, rabies antibodies were not detected in the blood of animals. The number of antibodies against rabies virus in cats after the introduction of one recommended dose (0.5 cm ³ ) of the rabies inactivated emulsion culture vaccine on day 21 was 82.65 ± 1.03 IU / cm ³ , after 2 months - 92.14 ± 1.21 IU / cm ³ , after 3 months - 85.12 ± 0.85 IU / cm ³ , after 6 months - 49.16 ± 0.98 IU / cm ³ , after 12 months - 28.31 ± 0, 82 IU / cm ³ , after 15 months - 8.12 ± 0.52 IU / cm ³ , after 18 months - 0.76 ± 0.14 IU / cm ³ (table 9). Thus, the introduction of a single dose (0.5 cm ³ ) of the rabies inactivated emulsion culture vaccine ensures the accumulation of a high level of antibodies (up to 92.14 ± 1.21 IU / cm ³ ) in the blood of cats and preserves the protective level of antibodies against rabies virus (≥ 0.50 IU / cm ³ ) up to 15-18 months.

The level of anti-rabies virus-neutralizing antibodies in the blood of cats after administration of the recommended single dose (0.5 cm ³ ) of the inactivated rabies vaccine analogue from the ARRIAH strain for 21 days was 3.15 ± 0.68 IU / cm ³ , after 2 months - 3.35 ± 0.74 IU / cm ³ , after 3 months - 3.14 ± 0.50 IU / cm ³ , after 6 months - 2.75 ± 0.62 IU / cm ³ , after 12 months - 0.91 ± 0.31 IU / cm ³ , after 15 months - 0.44 ± 0.13 IU / cm ³ , after 18 months - 0.21 ± 0.05 IU / cm ³ . In other words, the introduction of a single dose (0.5 cm ³ ) of the inactivated rabies analogue vaccine from the ARRIAH strain ensures the accumulation of the protective level of antibodies in the blood of cats against rabies virus for up to 12 months. At the same time, this analogue vaccine induces the production of rabies antibodies 27.50 times lower compared to the developed vaccine.

At the next stage of work, we studied cellular immunity in cats after administration of a single dose (0.5 cm ³ ) of the presented vaccine and the analogue vaccine.

Vaccination of animals, blood sampling, isolation and analysis of lymphocytes was performed as described above in Example 9. Dynamics of the level of subpopulations of CD4 + and CD8 + cells in the blood of cats 1, 2, and 3 weeks after the administration of one dose (0.5 cm ³ ) the rabies inactivated emulsion culture vaccine, the inactivated rabies analogue vaccine of the ARRIAH strain, in comparison with the control without vaccination, is shown in FIG. 11. From the data of FIG. 11A and 11B, it can be seen that the relative content of CD4 + and CD8 + lymphocytes in the animal body after administration of the developed vaccine increased and amounted to 21-28 and 12-19%, respectively. The number of CD4 + and CD8 + lymphocytes after administration of the analogue vaccine was at the level of the values of control animals without vaccination and amounted to 4-6%.

Analysis of the ratio of CD4 + / CD8 + cells after the introduction of the developed vaccine showed that from 2 weeks after vaccination there was a tendency to decrease this indicator in cats from 1.75 to 1.42 (Fig. 12). The maximum relative number of CD4 + and CD8 + lymphocytes was recorded 1-2 weeks after inoculation of the developed vaccine. The relative number of CD8 + lymphocytes increased from 1 to 3 weeks of testing. It was revealed that the introduction of the rabies inactivated emulsion culture vaccine into cats using Montanide ISA-61 VG oil adjuvant activated cellular immunity, causing quantitative changes in the T-cell phenotype. After immunization of cats with a culture inactivated rabies analogue vaccine from the ARRIAH strain using ISA-70 adjuvant, activation of cellular immunity was not observed.

The protective properties of the developed rabies inactivated emulsion culture vaccine were evaluated as shown in Example 9. After the challenge, all 10 cats vaccinated with a single dose (0.5 cm ³ ) of the vaccine remained alive and clinically healthy. In the control group, the death of all animals was noted. Thus, the developed rabies inactivated emulsion culture vaccine is effective for immunizing cats.

Example 12. Evaluation of humoral and cellular immunity in dogs using an anti-rabies inactivated emulsion culture vaccine in comparison with a similar vaccine. The study of the protective properties of the developed vaccine on the example of dogs.

The study of the tension of humoral immunity in dogs vaccinated with the rabies inactivated emulsion culture vaccine was carried out in comparison with the inactivated liquid rabies analogue of rabies from the strain "ARRIAH".

Testing of these vaccines was carried out on ten heads of mongrel dogs aged 1-3 years with a body weight of 10-25 kg. All animals were injected intramuscularly in a volume of 1.0 cm ³ once. Before immunization and after 21 days, as well as 2, 3, 6, 12, 15, 18 months after immunization, blood was taken from dogs and the obtained sera were examined for the presence of rabies antibodies in the neutralization reaction (FAVN modification) [2].

Before vaccination in the blood of dogs, antibodies against rabies virus were not detected. The number of virus-neutralizing antibodies in dogs after administration of one recommended dose for the given animal species (1.0 cm ³ ) of the rabies inactivated emulsion culture vaccine on day 21 was 24.12 ± 0.56 IU / cm ³ , after 2 months - 29.65 ± 0.65 IU / cm ³ , after 3 months - 25.63 ± 0.74 IU / cm ³ , after 6 months - 18.16 ± 0.51 IU / cm ³ , after 12 months - 6.45 ± 0, 13 IU / cm ³ , after 15 months - 0.95 ± 0.12 IU / cm ³ , after 18 months - 0.32 ± 0.12 IU / cm ³ (table 10). Thus, the introduction of a single dose (1.0 cm ³ ) of the rabies inactivated emulsion culture vaccine ensures the accumulation of a high level of antibodies (up to 29.65 ± 0.65 IU / cm ³ ) in the blood of dogs and preserves the protective level of antibodies against rabies virus (≥ 0.50 IU / cm ³ ) up to 15 months.

The titer of anti-rabies virus-neutralizing antibodies in the blood of dogs after administration of one recommended single dose (1.0 cm ³ ) of the culture inactivated rabies analogue of rabies from the ARRIAH strain on day 21 was 1.84 ± 0.21 IU / cm ³ , after 2 months - 2.17 ± 0.34 IU / cm ³ , after 3 months - 2.75 ± 0.42 IU / cm ³ , after 6 months - 2.01 ± 0.46 IU / cm ³ , after 12 months - 0.79 ± 0.42 IU / cm ³ , after 15 months - 0.36 ± 0.10 IU / cm ³ , after 18 months - 0.09 ± 0.02 IU / cm ³ . Thus, the introduction of a single dose of a culture inactivated rabies analogue vaccine from the ARRIAH strain ensures the accumulation of a protective level of antibodies in the blood of dogs against rabies virus for up to 12 months. Moreover, this analogue vaccine induces the production of anti-rabies antibodies 10.78 times lower compared to the developed vaccine.

At the next stage of testing, we evaluated the cellular immunity in dogs after administration of a single dose (1.0 cm ³ ) of the presented vaccine and vaccine analogue.

Immunization of animals, blood sampling, isolation and analysis of lymphocytes was performed as described above in Example 9. Dynamics of the level of subpopulations of CD4 + and CD8 + cells in the blood of dogs 1, 2, and 3 weeks after the administration of one dose (1.0 cm ³ ) the rabies inactivated emulsion culture vaccine, the culture inactivated rabies analogue vaccine of the ARRIAH strain, in comparison with the control without vaccination, is shown in FIG. 13. From the data of FIG. 13 shows that the relative content of lymphocytes CD4 + and CD8 + in the blood of dogs after administration of the proposed vaccine increased and amounted to 13-19 and 8-13%, respectively. The number of CD4 + and CD8 + cells after administration of the analogue vaccine was at the level of the values of the control animals without vaccination and was 3-6%.

The level of CD4 + / CD8 + cells after the introduction of the developed vaccine showed that from 1 week after vaccination a decrease in this indicator in dogs was observed from 1.63 to 1.31 and, as a result, an increase in the relative number of CD8 + lymphocytes from 8 to 13 (Fig. 14 ) The maximum relative number of CD4 + and CD8 + lymphocytes was observed 1 week after vaccination. It was revealed that the introduction of an rabies inactivated emulsion culture vaccine into dogs using Montanide ISA-61 VG oil adjuvant activated cellular immunity, which was expressed in quantitative changes in the T-cell phenotype. After immunization of dogs with a culture inactivated rabies analogue vaccine from the ARRIAH strain using the ISA-70 adjuvant, activation of cellular immunity was not observed.

The protective properties of the proposed anti-rabies inactivated emulsion culture vaccine were evaluated as shown in Example 9. After the challenge, all 10 dogs vaccinated with a single dose (1.0 cm ³ ) of the vaccine remained alive and clinically healthy. In the control group, the death of all animals was noted. Thus, the developed rabies inactivated emulsion culture vaccine is effective for immunizing dogs.

Example 12. Evaluation of humoral and cellular immunity in pigs when using rabies inactivated emulsion culture vaccines in comparison with the vaccine analogue. The study of the protective properties of the developed vaccine as an example of pigs.

The study of the tension of humoral immunity in pigs inoculated with an anti-rabies inactivated emulsion culture vaccine was carried out in comparison with an inactivated liquid rabies analogue of rabies from the ARRIAH strain.

Testing of these vaccines was performed on ten heads at the age of 2 months weighing about 30 kg. All animals were injected with the vaccine intramuscularly once in a volume of 2.0 cm ³ . Before immunization and after 21 days, as well as 2, 3, 6, 12, 15, 18 months after immunization, blood was taken from dogs and the obtained sera were examined for the presence of rabies antibodies in the neutralization reaction (FAVN modification) [2].

Before vaccination in the blood of pigs, antibodies against rabies virus were not detected. The number of virus-neutralizing antibodies in pigs after the introduction of one recommended dose for this animal species (2.0 cm ³ ) of the rabies inactivated emulsion culture vaccine on day 21 was 4.50 ± 0.31 IU / cm ³ , after 2 months - 5.12 ± 0.37 IU / cm ³ , after 3 months - 5.08 ± 0.44 IU / cm ³ , after 6 months - 3.98 ± 0.45 IU / cm ³ , after 12 months - 1.85 ± 0, 29 IU / cm ³ , after 15 months - 0.63 ± 0.10 IU / cm ³ , after 18 months - 0.08 ± 0.01 IU / cm ³ (table 11). Thus, the introduction of a single dose (2.0 cm ³ ) of the rabies inactivated emulsion culture vaccine ensures the accumulation of a sufficiently high level of antibodies (up to 5.12 ± 0.37 IU / cm ³ ) in the blood of dogs and preserves the protective level of antibodies against rabies virus ( ≥ 0.50 IU / cm ³ ) up to 15 months.

The titer of rabies anti-rabies virus-neutralizing antibodies in the blood of pigs after administration of one recommended single dose (2.0 cm ³ ) of the culture inactivated rabies analogue of rabies from the ARRIAH strain on day 21 was 1.45 ± 0.28 IU / cm ³ , after 2 months - 1.86 ± 0.29 IU / cm ³ , after 3 months - 1.95 ± 0.31 IU / cm ³ , after 6 months - 1.25 ± 0.27 IU / cm ³ , after 12 months - 0.79 ± 0.19 IU / cm ³ , after 15 months - 0.14 ± 0.07 IU / cm ³ , after 18 months - 0.03 ± 0.01 IU / cm ³ . Thus, the introduction of a single dose (2.0 cm ³ ) of the inactivated rabies vaccine from the ARRIAH strain (using the ISA-70 adjuvant) ensures the accumulation of the protective level of antibodies in the blood of dogs against rabies virus for up to 12 months. Moreover, this analogue vaccine induces the production of rabies antibodies 2.63 times lower compared to the developed vaccine.

At the next stage of testing, we assessed cellular immunity in pigs after administration of a single dose (2.0 cm ³ ) of the presented vaccine and the analogue vaccine.

Immunization of animals, blood sampling, isolation and analysis of lymphocytes was carried out as described above in Example 9. Dynamics of the level of subpopulations of CD4 + and CD8 + cells in pig blood 1, 2, and 3 weeks after the administration of one dose (2.0 cm ³ ) the rabies inactivated emulsion culture vaccine, the culture inactivated rabies analogue vaccine of the ARRIAH strain, in comparison with the control without vaccination, is shown in FIG. 15. From the data of FIG. 15 shows that the relative content of lymphocytes CD4 + and CD8 + in the blood of pigs after administration of the proposed vaccine increased and amounted to 7-15 and 6-10%, respectively. The number of CD4 + and CD8 + cells after administration of the analogue vaccine was at the level of the values of the control animals without vaccination and was 3-4%.

The level of CD4 + / CD8 + lymphocytes after administration of the developed vaccine after 1, 2, 3 weeks in pigs was 1.17, 1.67, 1.50, respectively (Fig. 16). This fact indicated that the CD4 + cell content increased from 1 to 2 weeks and remained at 15% for 3 weeks. The relative number of CD8 + lymphocytes from 1 to 3 weeks of assessment increased from 4 to 10% (Fig. 15). It was revealed that the introduction of the rabies inactivated emulsion culture vaccine into pigs using the Montanide ISA-61 VG oil adjuvant activated cell immunity, which was expressed in quantitative changes in the T-cell phenotype. After immunization of pigs with a culture-inactivated rabies analogue vaccine from the ARRIAH strain using the ISA-70 adjuvant, activation of cellular immunity was not observed.

The protective properties of the proposed anti-rabies inactivated emulsion culture vaccine were evaluated as shown in Example 9. After the challenge, all 10 pigs vaccinated with a single dose (2.0 cm ³ ) of the vaccine remained alive and clinically healthy. In the control group, the death of all animals was noted. Thus, the developed rabies inactivated emulsion culture vaccine is effective for immunizing pigs.

Example 14. The determination of the index of immunogenicity and stability of the rabies inactivated emulsion culture vaccine during storage for 30 months in comparison with the vaccine analogue.

The shelf life of the immunogenic potency of the rabies inactivated emulsion culture vaccine and the inactivated liquid rabies vaccine analogue of the ARRIAH strain was determined in trials with 5 experimental series of these vaccines stored for 30 months after manufacture at a temperature of 2.0-8.0 ° C . Vaccine testing was performed every 6 months after manufacture. The determination of the immunogenicity index of the preparations was carried out as described in example 7 [2]. The research results of the developed vaccine and the analogue vaccine are presented in table 12, which shows that after 12, 18, 24, 30 months of storage under the indicated conditions, the immunogenicity index of the developed vaccine decreased by 1.19; 2.00; 2.61; 3.44%, and the analogue vaccine - by 2.94; 4.80; 6.06; 7.36%, respectively. In other words, the proposed vaccine made it possible to maintain a high immunogenicity index throughout the observation.

It should be noted that all series of the studied vaccines for 24 months maintained the stability of the emulsion. The separation of the aqueous phase was noted only after 30 months of storage at a temperature of 2.0-8.0 ° C.

The results obtained led to the conclusion that the anti-rabies inactivated emulsion culture vaccine was guaranteed to retain its physical and biological properties for 24 months, provided that it was stored at a temperature of 2.0-8.0 ° C.

Thus, the above information indicates that the rabies inactivated emulsion culture vaccine embodying the invention is intended for use in agriculture, namely in veterinary virology and biotechnology; confirmed the possibility of implementing the presented; the vaccine made from the strain "ARRIAH", Montanide ISA-61 VG oil adjuvant and merthiolate in accordance with the invention, has high immunogenic activity, is able to activate humoral and cellular immunity in domestic carnivores and farm animals, providing them with early and long-term protection against the virus rabies genetic line RABV.

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601 ccgaatttca gatttttggc cgggacctac gacatgtttt tctcccggat tgagcatcta 660

661 tattcagcaa tcagagtggg cacggttgtt actgcttatg aagactgttc aggtctggta 720

721 tcatttacag ggttcataaa gcagatcaat ctcaccgcaa gagaagcaat actatacttc 780

781 ttccacaaga actttgagga agagataaga agaatgtttg agccagggca ggagacagct 840

841 gttcctcact cctatttcat ccacttccgt tcactaggct tgagcgggaa gtctccttat 900

901 tcatcaaatg ctgttggtca tgtgttcaat ctcattcact ttgttggatg ctatatgggt 960

961 caagtcagat ccctaaacgc aacagttatt gccgcatgtg ctcctcatga gatgtctgtt 1020

1021 ttagggggct atctgggaga agagttcttc gggaaaggaa cattcgagag aagattcttc 1080

1081 agagatgaga aagaacttca agaatacgag gcagctgaac tgacaaagac tgacgtggct 1140

1141 ttggcagatg atggaactgt ccactcggac gatgaggact acttttccgg agagaccaga 1200

1201 agtccagaag ccgtttatac tcgaatcatg atgaatggag gtcgactaaa gagattgcat 1260

1261 atacggagat atgtttcagt cagttcaaat catcaagctc gtcctaactc atttgccgag 1320

1321 tttctaaaca agacgtattc gaacgactcc 1350

<210> 2

<211> 450

<212> PRT

<213> rabies virus

<400> 2

1 MetAspAlaAspLysIleValPheLysValAsnAsnGlnValValSerLeuLysProGlu 20

21 IleIleValAspGlnTyrGluTyrLysTyrProAlaIleLysAspLeuLysLysProCys 40

41 IleThrLeuGlyLysAlaProAspLeuAsnLysAlaTyrLysSerIleLeuSerGlyMet 60

61 AsnAlaAlaLysLeuAspProAspAspValCysSerTyrLeuAlaAlaAlaMetGlnPhe 80

81 PheGluGlyThrCysProGluAspTrpThrSerTyrGlyIleLeuIleAlaArgLysGly 100

101 AsnLysIleThrProAspSerLeuValGluIleLysArgThrAspValGluGlyAsnTrp 120

121 AlaLeuThrGlyGlyMetGluLeuThrArgAspProThrValSerGluHisAlaSerLeu 140

141 ValGlyLeuLeuLeuSerLeuTyrArgLeuSerLysIleSerGlyGlnAsnThrGlyAsn 160

161 TyrLysThrAsnIleAlaAspArgIleGluGlnIlePheGluThrAlaProPheValLys 180

181 IleValGluHisHisThrLeuMetThrThrHisLysMetCysAlaAsnTrpSerThrIle 200

201 ProAsnPheArgPheLeuAlaGlyThrTyrAspMetPhePheSerArgIleGluHisLeu 220

221 TyrSerAlaIleArgValGlyThrValValThrAlaTyrGluAspCysSerGlyLeuVal 240

241 SerPheThrGlyPheIleLysGlnIleAsnLeuThrAlaArgGluAlaIleLeuTyrPhe 260

261 PheHisLysAsnPheGluGluGluIleArgArgMetPheGluProGlyGlnGluThrAla 280

281 ValProHisSerTyrPheIleHisPheArgSerLeuGlyLeuSerGlyLysSerProTyr 300

301 SerSerAsnAlaValGlyHisValPheAsnLeuIleHisPheValGlyCysTyrMetGly 320

321 GlnValArgSerLeuAsnAlaThrValIleAlaAlaCysAlaProHisGluMetSerVal 340

341 LeuGlyGlyTyrLeuGlyGluGluPhePheGlyLysGlyThrPheGluArgArgPhePhe 360

361 ArgAspGluLysGluLeuGlnGluTyrGluAlaAlaGluLeuThrLysThrAspValAla 380

381 LeuAlaAspAspGlyThrValHisSerAspAspGluAspTyrPheSerGlyGluThrArg 400

401 SerProGluAlaValTyrThrArgIleMetMetAsnGlyGlyArgLeuLysArgLeuHis 420

421 IleArgArgTyrValSerValSerSerAsnHisGlnAlaArgProAsnSerPheAlaGlu 440

441 PheLeuAsnLysThrTyrSerAsnAspSer 450

<210> 3

<211> 540

<212> DNA

<213> rabies virus

<400> 1

1 ccgaagatca cctccccttt aagtttgctt tattgggggg aatctccggg ttcaagagtc 60

61 ctccttgaac tccatgcgac agagtatatt caagagtcat gtgactctca ttattcctct 120

121 cagttgatca aatcaagtca tgtagattct cataacgcgg gagatcttct agcagtttca 180

181 gtgcccaact gtgctttcat tctccaggaa ctaatactaa gggttgtgga cggaccaaga 240

241 ggtatctccccg atgatcccgt gcttgggcac gggtataggt catattacgt cccacgatat 300

301 cggactccac atgaaccgat tgagaaaggc aatctgcctc ccatgaagga cgcaagtaat 360

361 agctcacaat cgtcttgcat ctcagcgaag tatgtatatt tatatagagc tgggtcctct 420

421 aagcttttca gtcaagaaaa aaactgtaga tcaaaagagc aactggcaac acttctcatc 480

481 ctaagaccta catcaagatg ctatatccag gagaggtcta tgatgacccc attgatccaa 540

<210> 4

<211> 180

<212> PRT

<213> rabies virus

<400> 2

1 ProLysIleThrSerProLeuSerLeuLeuTyrTrpGlyGluSerProGlySerArgVal 20

21 LeuLeuGluLeuHisAlaThrGluTyrIleGlnGluSerCysAspSerHisTyrSerSer 40

41 GlnLeuIleLysSerSerHisValAspSerHisAsnAlaGlyAspLeuLeuAlaValSer 60

61 ValProAsnCysAlaPheIleLeuGlnGluLeuIleLeuArgValValAspGlyProArg 80

81 GlyIleSerAspAspProValLeuGlyHisGlyTyrArgSerTyrTyrValProArgTyr 100

101 ArgThrProHisGluProIleGluLysGlyAsnLeuProProMetLysAspAlaSerAsn 120

121 SerSerGlnSerSerCysIleSerAlaLysTyrValTyrLeuTyrArgAlaGlySerSer 140

141 LysLeuPheSerGlnGluLysAsnCysArgSerLysGluGlnLeuAlaThrLeuLeuIle 160

161 LeuArgProThrSerArgCysTyrIleGlnGluArgSerMetMetThrProLeuIleGln 180

<---

Claims (11)

1. Inactivated emulsion culture vaccine for prophylactic immunization of domestic carnivores and farm animals, containing an active substance, adjuvant, preservative, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" of the rabies virus, family Rhabdoviridae, Lyssavirus genus, Rabies lyssavirus species, deposited in the Collection of microorganism strains of the Federal Center for Animal Health (FSBI ARRIAH), under the registration number ARRIAH-DEPT for fixed rabies virus dated December 20, 2018, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the rabies virus strain ARRIAH homologous to the pathogen, obtained in a sensitive biological system and consisting of a suspension containing mainly immunogenic components (glycoprotein and nucleoprotein) of the virus rabies genetic line RABV. 3. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from the rabies virus ARRIAH strain homologous to the pathogen, preferably obtained in the transplantable suspension cell sublayer VNK-21 / SUSP / ARRIAH and which is a suspension containing mainly immunogenic components (glycoprotein and nucleoprotein) of rabies virus of the genetic line RABV. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from the rabies virus ARRIAH strain homologous to the pathogen, preferably obtained in a transplantable suspension cell subline from the kidney of a newborn Syrian hamster BHK-21 / SUSP / ARRIAH and which is a suspension containing mainly immunogenic components of the rabies virus of the RABV genetic line: glycoprotein in an amount of at least 1.5 μg and nucleoprotein in an amount of at least 1.5 μg in 1.0 cm ^{3 of the} finished product, which corresponds to a vaccine immunogenicity index of at least 4 5 IU / cm. 5. The vaccine according to claim 1, characterized in that, as a modulator of the immune response, it contains an oil adjuvant of Montanide ISA-61 VG. 6. The vaccine according to claim 5, characterized in that it contains an oil adjuvant of Montanide ISA-61 VG, preferably in an amount of 575000.0-700000.0 μg in 1.0 cm ^{3 of the} finished product. 7. The vaccine according to claim 5, characterized in that it contains Montanide ISA-61 VG oil adjuvant, preferably in an amount of 575000.0-700000.0 mcg in 1.0 cm ^{3 of the} finished product and activates humoral and cellular immunity in domestic carnivores and agricultural animals. 8. The vaccine according to claim 1, characterized in that it contains merthiolate as a preservative. 9. The vaccine according to claim 8, characterized in that as a preservative contains merthiolate in an amount of 0.002% (by weight). 10. The vaccine according to any one of paragraphs. 1-9, characterized in that it contains avirulent and purified antigenic material from a rabies virus strain ARRIAH homologous to the pathogen, preferably obtained in a transplantable suspension cell subline from the kidney of a newborn Syrian hamster VNK-21 / SUSP / ARRIAH and which is a suspension, containing predominantly immunogenic components of the rabies virus of the RABV genetic line, Montanide ISA-61 VG oil adjuvant, a thiolate preservative in the ratio, μg in 1.0 cm ^{3 of the} finished preparation:

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