RU2812330C1 - Associated vaccine against panleukopenia, calicivirus and feline viral rhinotracheitis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: vaccine contains an active substance and a target additive. As an active substance, the vaccine contains a mixture of attenuated purified antigenic material from Sheba strain of feline panleukopenia virus, Parvoviridae family, Protoparvovirus genus, Carnivore protoparvovirus 1 species, from inactivated and purified antigenic material from Pers strain of feline calicivirus, Caliciviridae family, Vesivirus genus, Feline calicivirus species, from inactivated and purified antigenic material from Fauna strain of feline calicivirus, Caliciviridae family, Vesivirus genus, Feline calicivirus species, from inactivated and purified antigenic material from Lavr strain of feline viral rhinotracheitis, Herpesviridae family, Varicellovirus genus, Alpaherpesvirus 1 species, as targeted additives: 3% aluminum hydroxide gel and stabilizer; taken in an effective ratio.

EFFECT: vaccine provides protective immune activity of each antigen in the body of dogs after administration of the target drug.

7 cl, 3 dwg, 6 tbl, 4 ex

Description

The invention relates to the field of veterinary virology and biotechnology, in particular, the development of an associated vaccine against panleukopenia, calicivirus and viral rhinotracheitis.

To reduce the number of cases of viral diseases among cats, preventive vaccination has proven to be most effective. It is justified from the point of view of economic costs, improvement of the epizootological situation and the immediate health status of animals. Among infectious diseases of cats, panleukopenia, calicivirosis and viral rhinotracheitis are of particular importance. In recent years, a significant number of domestic and imported biological products for cats have been developed and widely used. Based on comprehensively studied strains of the causative agents of these diseases in domestic and foreign practice, associated vaccines were created, which are currently produced by 1 enterprise in the Russian Federation and many enterprises abroad.

According to the Center for Veterinary Medicine for 2020-2022, the epizootic situation for these three viral diseases of cats in the Russian Federation is as follows: the leading place is occupied by calicivirus - it was recorded in 38.17% of cases, viral rhinotracheitis is in second place - 35.23%, in third place is panleukopenia - 26.60%. These diseases are found everywhere in cats of different breeds and ages [2, 3].

To prevent and prevent the spread of panleukopenia, calicivirosis and viral rhinotracheitis viruses among cats, cultured live and inactivated vaccines from various strains are developed and used. When developing vaccines, special attention is paid to the safety of the finished product and the strength of the immune system. To form more intense and long-lasting immunity in vaccinated animals, a wide range of adjuvants are used as nonspecific stimulators of immunogenesis, in particular, aluminum oxide hydrate (AHO) in the production of sorbed vaccines [4, 5].

The use of associated vaccines is the optimal solution not only for cats kept individually, but especially for use in nurseries and shelters, where crowded keeping of animals contributes to the occurrence and spread of infections. In general, the use of associated vaccines, including several immunogens, makes it possible to reduce stressful situations in vaccinated animals, create intense immunity in a short time and reduce labor costs [4].

Feline panleukopenia virus (FPV, feline panleukopenia virus) is a highly contagious disease of viral etiology, characterized by fever, acute hemorrhagic enteritis, leukopenia, dehydration and high mortality (up to 90%) [2]. The causative agent of feline panleukopenia is a non-enveloped virus with single-stranded DNA and an icosahedral capsid, belonging to the family Parvoviridae, genus Protoparvovirus, species Carnivore protoparvovirus 1 [6].

The most favorable for the reproduction of the FPV pathogen are mitotically active cells of the bone marrow, lymphatic tissue and epithelial cells of the crypts of the small intestine [7]. The virus is released with all the secretions and excreta of the animal during the active stage of infection, but the maximum concentration of the pathogen is achieved in feces [2]. Typically, virus shedding occurs about two days after the onset of clinical signs, but FPV can be shed in feces and urine for up to 6 months after recovery [8]. Transmission factors can include contaminated objects, shoes, and clothing, so the risk of infection in domestic unvaccinated cats that do not have free access to the outdoors remains high.

FPV has been known since the beginning of the 20th century and has maintained a certain genetic stability over the subsequent decades [9]. But in the 1970s, the causative agent of canine parvovirus enteritis (CPV-2 - Canine parvovirus-2), which evolved from FPV, acquired about 6 amino acid changes in the capsid, which allowed the virus to also infect dogs [10]. While dogs are susceptible only to the original CPV-2 and all its variants, cats are susceptible to both FPV and CPV-2 variants [7]. This feature demonstrates that, despite the general conservation of DNA-containing viruses, this pathogen is changing, and there is a risk of infection of other animal species [11]. According to the World Small Animal Veterinary Association (WSAVA) vaccination guidelines for dogs and cats, the feline panleukopenia virus vaccine is a core vaccine that all cats should receive regardless of circumstances (they provide protection against diseases that are life-threatening to animals and widespread throughout the world) [12]. Despite the widespread availability and significant choice of vaccines for cats, panleukopenia remains an important disease. In this regard, the question of the need to develop vaccine prophylaxis agents that provide reliable protection against new strains of feline panleukopenia remains relevant.

Currently, the following industrial strains of the feline panleukopenia virus are best known and are used to produce specific prophylactic agents against this disease:

- Strain “RY IV” [13];

- Strain “MW-1” [14];

- Strain “LR 72” [15];

- Strain “Snow Leopard” [16];

- Strain “Meow” [2].

Strains “PLI IV”, “MW-1”, “LR 72”, “Snow Leopard” are included in the composition of imported vaccines against cat diseases, which are created on the basis of strains that are most widespread in the producing countries. The “Meow” strain is used for the production of vaccines by a domestic manufacturer. The trend towards an annual increase in cases of feline panleukopenia may indicate the insufficient effectiveness of foreign and domestic vaccines against strains circulating in the cat population in the Russian Federation. In addition, these vaccines are expensive.

The viral genome is represented by single-stranded DNA without an envelope. The shape of virions is polymorphic, predominantly spherical. The internal part of the virion is a spiral-twisted thread of ribonucleoprotein, which is surrounded by an envelope with radially located processes. The virus is homogeneous in immunobiological terms. The virion size ranges from 115 to 160 nm [2].

The virus retains a certain degree of genetic stability [17], but molecular studies have not been conducted for several decades, so the antigenic properties of currently circulating strains are not well known. In addition, the genetic and immunological relatedness between field FPV strains and vaccine strains has not been assessed, although all known vaccines are produced using long-established FPV strains. Several studies have demonstrated that some outbreaks of panleukopenia in cats are not caused by classical FPV strains, but by antigenic variants of canine CPV-2 [18].

Feline calicivirus (feline calicivirus infection, Feline calicivirus, FCV) is a highly contagious viral disease characterized primarily by damage to the oral mucosa and upper respiratory tract [2]. The virus is widespread and affects domestic cats of all breeds and ages, as well as zoo and wild felines. According to the literature, FCV was found in 18-30% of cases of diseases associated with damage to the upper respiratory tract [19, 20, 21]. Virus carriage is also widespread (up to 75%), especially among stray cats [2]. In addition to the common symptoms of ulcerative stomatitis, rhinitis and conjunctivitis, the disease can manifest itself as lameness, swelling of the head and limbs, pneumonia, necrosis of the tongue and palate, and damage to the gastrointestinal tract [22, 23]. Various sources describe a systemic infection that causes death in up to 60% of sick animals [19]. FCV is characterized by a high degree of variability and a large antigenic diversity of strains, which significantly reduces the effectiveness of existing vaccines.

The causative agent of the disease is Feline calicivirus (FCV), belongs to the family Caliciviridae, genus Vesivirus.

Currently, the following strains of the feline calicivirus virus are known, which are used for the diagnosis of FCV, as well as the production of vaccines against this disease:

- Strain “F-9” [24].

- Strain “Lare” [25].

- Strain “Ashley” [26].

Genetically, FCV strains belong to one diverse genogroup, however, a second genogroup has been described in East Asia. Phylogenetic analysis usually results in a "star" phylogeny. This genetic diversity is accompanied by antigenic diversity, although cross-reactivity is sufficient for all isolates to be considered to belong to the same serotype. Studies of the spatial and temporal distribution of FCV strains have described very high genetic and antigenic complexity among FCV strains, with many different FCV strains circulating in the cat population and no single field strain being dominant [23, 25].

The “F-9” strain is part of imported associated vaccines against infectious diseases of cats, data on it is presented in GenBank, however, studies show that since its isolation, the calicivirus virus has undergone significant genetic changes, and vaccines based on the “F-” strain 9" (and its alternative "F-255") cannot provide reliable protection against infection with new strains [19]. In addition, in the Russian Federation, imported drugs made on the basis of this strain have a high cost.

The “Ashley” strain of calicivirus infection was patented in 2016 to study the antiviral activity of drugs against the causative agent of feline calicivirus and is not used for the manufacture of vaccines [26].

Feline viral rhinotracheitis {feline viral rhinotracheitis, FHV-1, FHV, infectious feline rhinotracheitis) is a contagious viral disease characterized by damage to the upper respiratory tract, conjunctivitis and keratitis [2, 3].

The causative agent of feline viral rhinotracheitis is a virus belonging to the family Herpesviridae, genus Varicellovirus, species Alphaherpesvirus 1 (FHV-1 - Feline alphaherpesvirus-1) [27].

FHV-1 was first isolated in the United States in 1957 from a cat with symptoms of upper respiratory tract disease [28].

Viral rhinotracheitis has been reported in many felines, such as European wild cats (Felis silvestris silvestris), sand cats (Felis margarita), leopard cats (Felis bengalensis), cheetahs (Acinonyx jubatus), mountain lions (Puma concolor), ocelots (Leopardus pardalis) and others [16]. Both adult cats of all breeds and kittens are susceptible to infection, but in young animals infected with FHV-1, death is common as a result of complications of pneumonia [29]. Also in the literature, cases of the development of meningoencephalitis as a result of FHV-1 infection are described [3, 30]. After the acute phase, the disease often passes into a latent form with lifelong viral carriage [31]. Researchers in Australia have estimated the seroprevalence of FHV-1 to be 11-17% in wild felines and 37% in domestic cats, however, other estimates estimate that about 90% of cats are considered seropositive for FHV-1, of which 45%) excrete virus throughout life [28, 32].

Currently, the following strains of feline infectious rhinotracheitis virus are known, which are used for the diagnosis of FHV-1, as well as the production of vaccines against this disease:

- Strain “F-2” [13].

- Strain “G 2620A” [14].

- Strain “FVRm” [16].

- Strain “Grand” [33].

All FHV-1 strains belong to the same serogroup, although minor genetic changes have been documented for some strains [2, 28, 34]. Genetically and antigenically, FHV-1 is similar to canine herpesvirus type 1 (CHV-1), as well as seal herpesvirus type 1 (PhHV-1). There are reports of cross-protection between FHV-1 and PhHV-1 [2].

To date, there are many associated vaccines of Russian and foreign production of various valences against panleukopenia, calicivirus and infectious feline rhinotracheitis.

Currently, there is a huge selection of domestic and imported vaccines.

Of the Russian vaccines on the market, the following are offered on the market: Multifel - 4 - a vaccine made from formalin-inactivated industrial strains of panleukopenia viruses, infectious rhinotracheitis, feline calicivirus, Chlamydia psittaci and an adjuvant in the amount of 10% of the vaccine volume [42].

Among the foreign vaccines on the market are:

• Felocell CVR-C, manufacturer Zoetis LLC 601 W (USA) - lyophilized mass and solution for injection. The vaccine is made from attenuated viruses of rhinotracheitis (strain FVRm - 10 ^5.7 EID50), calicivirus infection (strain F-9 - 10 ^6.2 EID ₅₀ ), feline panleukopenia (strain Snow Leopard - 10 ^3.0 EID ₅₀ ), Chlamydia psittacci (Baker strain) with the addition of gentamicin as a preservative and stabilizer (which includes: casein, gelatin, sucrose) [43];

• Purevax RCPCh - contains: an attenuated strain of the feline infectious rhinotracheitis virus FFTV F-2, a combination of two inactivated strains of the feline calicivirus virus FCV 431 and FCV G1, an attenuated strain of the feline panleukopenia virus PLIIV and an attenuated strain of feline chlamydia 905 [44 ];

• Leucofeligen is a two-component vaccine. The first component contains an attenuated panleukopenia virus (strain LR 72 FPV, min. 10 ^3.7 - 10 ⁴ TCID ₅₀ /ml); calicivirus (strain F-9 FCV, min. 10 ^4.6 - 10 ^6.1 TCID ₅₀ /ml); rhinotracheitis virus (strain F2 min. 10 ⁵ - 10 ^6.6 TCID ₅₀ /ml). The second component contains inactivated feline leukemia virus (purified p45 antigen protein > 102 μg/ml), and auxiliary components: aluminum hydroxide, purified soap tree extract and isotonic solution up to 1 ml [45];

• Quadricat (Qadricat) - a vaccine against panleukopenia, respiratory viruses and feline rabies consists of two components that are mixed directly during use:

- lyophilized vaccine "Rabiffa-Feliniffa", which is a mixture of attenuated feline panleukopenia virus with inactivated rabies virus - this is a dry, homogeneous white mass in the form of a tablet;

- liquid vaccine “Corifelin”, which is an oil solution of the glycoprotein fraction of feline herpesvirus with purified feline calicivirus antigen. In appearance it is an oily liquid of milky white color [46];

This vaccine is not currently available.

• Nobivac Tricat Trio - a dry live vaccine obtained from the culture fluid of a continuous FEF cell line infected with attenuated (weakened) strains of viruses. In 1 ml, the vaccine contains 5.2 lg PFU units of rhinotracheitis virus strain G 2620A, 4.6 lg PFU units of feline calicivirus strain F-9 and 4.3 lg TCD ₅₀ units of feline panleukopenia virus strain MW-1, as well as stabilizers: hydrolyzed gelatin, sorbitol, pancreatic casein hydrolyzate and buffered potassium hydrogen phosphate dihydrate solution [47];

• Nobivac Ducat - dry live vaccine against rhinotracheitis and calicivirus in cats. The vaccine is made from the cultural fluid of a continuous FEF cell line infected with feline rhinotracheitis (strain G 2620A) and calicivirus (strain F9) viruses, with the addition of stabilizers (hydrolyzed gelatin, sucrose) and sodium hydrogen phosphate dihydrate [48];

• Felovax (Fel-O-Vax) - the vaccine contains inactivated feline panleukopenia virus, two strains of feline calicivirus, inactivated feline rhinotracheitis virus and Chlamydia psittaci. Contains thimerosal, neomycin, polymyxin B and amphotericin B as preservatives [49];

• Biofel PCHR (Biofel PCHR) - a vaccine for the prevention of panleukopenia, calicivirus, herpesvirus infections and rabies in cats. The vaccine is made from inactivated strains of panleukopenia virus (strain FPV), calicivirus (strain FCV F), herpesvirus (strain FITV-1) and rabies virus (strain Vnukovo-32), with the addition of excipients (thiomersal, oil adjuvant, emulsifiers: sorbitol derivatives , glyceride, glycerol) 10% [50].

• Biofel RSN - a vaccine for the prevention of panleukopenia, calicivirus, herpesvirus infections of cats. The vaccine is made from inactivated strains of panleukopenia virus (strain FPV), calicivirus (strain FCV F), herpesvirus (strain FITV-1) and rabies virus (strain Vnukovo-32), with the addition of excipients (thiomersal, oil adjuvant, emulsifiers: sorbitol derivatives , glyceride, glycerol) 10% [51].

The following vaccines are the closest in composition to the antigens of infectious disease viruses: Felocel and Nobivak Triket Trio.

A significant disadvantage of these vaccines is that they are created on the basis of industrial strains of viruses obtained abroad and not updated for a long time, as well as the use of live viruses, which leads to the release of viral particles by animals for some time. This can lead to infection of animals, asymptomatic virus carriage and, possibly, the resumption of the pathogenic properties of weakened strains of viruses.

All known combined polyvalent associated vaccines contain live attenuated viruses. They are characterized by insufficient anti-epizootic effectiveness, due to the fact that the strains used for the production of polyvalent vaccines have antigenic and immunobiological differences compared to similar characteristics of pathogens circulating in Russia and the CIS countries.

Therefore, the problem of obtaining a highly effective associated vaccine against the main causative agents of viral diseases of cats remains relevant and is the main direction of research to create the desired drug.

In this regard, the objective of the invention is to create a safe and highly immunogenic vaccine against the main infectious diseases of cats, containing antigenically and immunogenically balanced viral antigens that ensure the formation of stable immunity against the virus of panleukopenia, calicivirosis and feline viral rhinotracheitis.

The associated vaccine against panleukopenia, calicivirosis and feline viral rhinotracheitis “Carnifel RSN” has not been registered in the Russian Federation, despite the fact that these diseases are highly contagious for cats, leading to death.

According to the patent and scientific and technical literature, a similar set of features has not been found, in particular, the composition of the components - strains of microorganisms, which makes it possible to judge the inventive level of the claimed technical solution.

The technical result of the invention is to expand the arsenal of associated vaccines against the main causative agents of infectious diseases of cats, to increase the stability, antigenic and immunogenic activity of the vaccine, as well as to enhance the intensity of immunity in vaccinated animals by obtaining vaccine components with a full spectrum of antigens.

The technical result of the invention is achieved by using in the vaccine composition new production strains of the panleukopenia virus, two strains of the calicivirus virus and feline viral rhinotracheitis with high antigenic and immunogenic activity, and the formation of a synergistic composition of a polyvalent associated vaccine by combining the antigens of the viruses of the new production strains.

The technical result of the invention is also achieved by using an adjuvant in the vaccine, which enhances the effect of the pharmaceutical substance (antigens) on the immune system of vaccinated animals, initiating a cascade of immune reactions and ensuring the development of intense immunity.

The specified technical result was achieved by the creation of an associated vaccine against panleukopenia, calicivirus and feline viral rhinotracheitis, characterized by the following set of characteristics.

The essence of the invention is as follows: the proposed associated vaccine against panleukopenia, calicivirus and feline viral rhinotracheitis contains as an active substance a mixture of:

• inactivated purified antigenic material of the Sheba strain of feline panleukopenia;

• inactivated purified antigenic material of the Persian strain of feline calicivirus virus;

• inactivated purified antigenic material of the “Fauna” strain of feline calicivirus virus;

• inactivated purified antigenic material of the “Lavr” strain of the feline infectious rhinotracheitis virus;

taken in effective quantities that provide protective immune activity in the cat’s body after administration of the target drug. As a targeted additive, the proposed vaccine contains aluminum hydroxide adjuvant (produced by ARRIAH).

In the proposed vaccine, the active substance and the target additive are combined in the ratio: vol.%: 90.0÷10.0.

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

1. Vaccine associated against panleukopenia, calicivirosis and viral rhinotracheitis of cats “Carnifel RSN”;

2. Active substance in the form of a mixture of inactivated purified antigenic material from the Sheba strain of the panleukopenia virus, family Parvoviridae, genus Protoparvovirus, species Carnivore protoparvovirus 1, collection of the Federal State Budgetary Institution "ARRIAH" under registration number: No. 455 - dep / 23-5 - GKShM FSBI "ARRIAH"; from inactivated purified antigenic material from the “Pers” strain of feline calicivirosis virus, family Caliciviridae, genus Vesivirus, species Feline calicivirus, collection of the Federal State Budgetary Institution “ARRIAH” under registration number: No. 458 - dep / 23-8 - GKShM FGBU “ARRIAH”; from inactivated purified antigenic material from the “Fauna” strain of the feline calicivirosis virus, family Caliciviridae, genus Vesivirus, species Feline calicivirus, collection of the Federal State Budgetary Institution “ARRIAH” under registration number: No. 471 - dep / 23-21 - GKShM FGBU “ARRIAH”; from inactivated purified antigenic material from the “Lavr” strain of the infectious rhinotracheitis virus of the Herpesviridae family, genus Varicellovirus, species Alphaherpesvirus I, collection of the FSBI “ARRIAH” under registration number: No. 457 - dep / 23-7 - GKShM FSBI “ARRIAH.

3. Targeted supplements.

The significant distinctive features of the proposed vaccine are that as an active substance it contains a mixture of inactivated purified antigenic materials from the “Sheba” strain of the panleukopenia virus, the “Pers” and “Fauna” strains of the calicivirosis virus and the “Lavr” strain of the feline infectious rhinotracheitis virus.

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

1. Inactivated and purified antigenic material from the Sheba strain of feline panleukopenia virus (FPV), obtained in a continuous culture of CrFK cells, in an effective amount.

2. Inactivated and purified antigenic material from the “Sheba” strain of the feline panleukopenia virus (FPV), obtained preferably in a continuous culture of CrFK cells, representing a viral suspension with a hemagglutinating activity of 9.33 ± 0.57 log ₂ HA, in an amount of 30.0 vol . %.

3. Inactivated and purified antigenic material from the Persian strain of feline calicivirus virus (FCV), obtained in a continuous culture of CrFK cells, which is a suspension in an effective amount.

4. Inactivated and purified antigenic material from the Persian strain of feline calicivirus virus (FCV), obtained preferably in a continuous culture of CrFK cells, representing a viral suspension with an infectious activity of 7.75 ± 0.25 lg TCD ₅₀ / cm ³ , in the amount 15.0 rev. %.

5. Inactivated and purified antigenic material from the “Fauna” strain of the feline calicivirus virus (FCV), obtained in a continuous culture of CrFK cells, which is a suspension in an effective amount.

6. Inactivated and purified antigenic material from the “Fauna” strain of the feline calicivirus virus (FCV), obtained preferably in a continuous culture of CrFK cells, representing a viral suspension with an infectious activity of 7.75 ± 0.25 lg TCD ₅₀ / cm ³ , in the amount 15.0 rev. %.

5. Inactivated and purified antigenic material from the “Lavr” strain of the feline infectious rhinotracheitis virus (FHV), obtained in a continuous culture of CrFK cells, in an effective amount.

6. Inactivated and purified antigenic material from the “Lavr” strain of feline infectious rhinotracheitis virus (FHV), obtained preferably in a continuous culture of CrFK cells, representing a viral suspension with an infectious activity of 6.5 ± 0.25 lg TCD ₅₀ / cm ³ , in quantity 30.0 vol. %.

7. Among the targeted additives, the vaccine contains aluminum hydroxide.

8. 3% aluminum hydroxide gel.

9. The vaccine contains 3% aluminum hydroxide gel in an amount of at least 10.0 vol. %.

10. A mixture of inactivated and purified antigenic materials from the “Sheba” strain of the feline panleukopenia virus, from the “Pers” strain of the feline calicivirus virus, from the “Fauna” strain of the feline calicivirus virus, from the “Lavr” strain of the feline infectious rhinotracheitis virus and a target additive: adjuvant aluminum hydroxide in quantity (vol. %):

As a result of the research, the authors identified the most important technological indicators that made it possible to create a drug in which there is no competition between the antigens included in the proposed vaccine, and its immunogenic properties were comparable to those after the use of monopreparations. A high immunological effect in relation to the specific antigens included in the vaccine was achieved by obtaining viral components in the vaccine that are harmless, immunogenic and antigenic activity, and an option was selected for their optimal ratio in the vaccine, ensuring the production of a protective level of antibodies, i.e. e. creation of intense immunity to viral diseases in cats.

The proposed vaccine has high immunogenic activity and provides reliable protection against panleukopenia, calicivirus and feline viral rhinotracheitis viruses circulating in the Russian Federation.

An additional technical result from the use of the proposed invention is achieved due to the fact that to inactivate virus-containing materials, aluminum hydroxide (AHA) produced by ARRIAH is used, which can significantly reduce labor and energy costs for vaccine production and improve the quality of antigenic materials, and is also available on the territory Russia at the moment.

An additional technical result of the proposed invention is achieved due to the fact that the associated vaccine includes new industrial strains of panleukopenia virus, viral rhinotracheitis and two strains of feline calicivirus.

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

Fig. 1 - Dendrogram, reflects the phylogenetic relationship of FPV “Sheba” strains with epizootic and vaccine strains of the feline panleukopenia virus. The dendrogram is based on a comparison of the complete nucleotide sequences of the VP2 gene.

Fig. 2 - Dendrogram, reflects the phylogenetic relationship of the FCV strains “Persian” and “Fauna” with epizootic and vaccine strains of the feline calicivirus virus. The dendrogram is based on a comparison of the complete nucleotide sequences of the gene encoding the VP ₂ protein.

Fig. 3 - Dendrogram, reflects the phylogenetic relationship of FHV “Lavr” strains with epizootic and vaccine strains of feline infectious rhinotracheitis virus. The dendrogram is based on a comparison of the complete nucleotide sequences of the gene encoding the glycoprotein E protein.

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

SEQ ID NO: 1 - Nucleotide sequence of the VP ₂ DNA gene of the feline panleukopenia virus strain "Sheba" (Felinepanleukopenia Virus).

SEQ ID NO:2 - Amino acid sequence of VP ₂ DNA protein of the feline panleukopenia virus strain "Sheba" (Feline panleukopenia Virus).

SEQ ID NO: 3 - Nucleotide sequence of the VP ₂ gene of the Persian strain of feline calicivirus (Feline calicivirus).

SEQ ID NO: 4 - The amino acid sequence of the protein, which is encoded by the VP ₂ gene, of the Persian strain of the feline calicivirus.

SEQ ID NO: 5 - Nucleotide sequence of the VP ₂ gene of the “Fauna” strain of the feline calicivirus virus (Feline calicivirus).

SEQ ID NO: 6 - The amino acid sequence of the protein, which is encoded by the VP ₂ gene, of the “Fauna” strain of the feline calicivirus (Feline calicivirus).

SEQ ID NO: 7 - Nucleotide sequence of the glycoprotein E gene cDNA of the feline infectious rhinotracheitis virus (Feline alphaherpesvirus-1) strain “Lavr”;

SEQ ID NO: 8 - Amino acid sequence of glycoprotein E cDNA of the feline infectious rhinotracheitis virus (Feline alphaherpesvirus-1) strain “Lavr”.

The initial virus for obtaining the “Sheba” strain of the feline panleukopenia virus was obtained from samples of pathological material (small intestine) obtained from a kitten from the animal center of the All-Russian Public Health Center “Valenta” in the city of Vladimir, Vladimir region of the Russian Federation in 2021. It was adapted for reproduction using the limiting dilution method in a continuous culture of cat kidney cells (CC CrFK).

The Sheba strain of the feline panleukopenia virus has been deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Disease Virus and other Animal Pathogens (FMDV) of the FGBU "ARRIAH" under registration number: No. 455 - dep / 23-5 - GKSHM FGBU "ARRIAH".

The Sheba strain of feline panleukopenia virus is characterized by the following signs and properties.

Morphological characteristics

The feline panleukopenia virus has morphological features characteristic of parvoviruses: non-enveloped viruses with single-stranded DNA and an icosahedral capsid.

Antigenic properties

Antigenically, FPV is related to arctic fox parvovirus (BFP), mink enteritis virus (MEV), and canine parvovirus (CPV-2) [2]. Antibodies that are formed in the blood serum of recovered animals are detected in the hemagglutination inhibition reaction (HAI).

Geno- and chemotaxonomic characteristics

FPV is a single-stranded DNA virus with a 5.1 kb genome encoding 2 major genes, nonstructural (NS) and structural protein genes. The NS gene encodes the NS1 and NS2 proteins involved in DNA replication, capsid assembly, and intracellular transport, while the structural gene encodes the VP1 and VP2 proteins of the viral capsid. The viral capsid consists of 60 protein subunits (~10% VP1 and 90% VP2) arranged in icosahedral symmetry.

Genetic identification of the resulting virus and comparative analysis of the DNA sequence were carried out. Reverse transcription and polymerase chain reaction (RT-PCR) were used. Sequencing was performed for identification and phylogenetic analysis.

The BioEdit sequence alignment editor was used to analyze the raw sequences. Alignments containing whole-genome sequences were constructed using the Clustal_W program. The evolutionary history was inferred using a maximum likelihood test based on the 3 parameters of the classic Tamura model. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analysis was carried out in MEGA7. Amino acid sequence alignment was performed using Clustal X.

The dendrogram is based on a comparison of the complete nucleotide sequences of the VP ₂ DNA gene of the feline panleukopenia virus (FPV) (a section of 664 bp was taken). The Sheba strain has a large number of nucleotide differences from other declared strains (the degree of similarity is no more than 39%), and it belongs to genogroup 1 of the Carninove protoparvovirus species (Fig. 1).

Biotechnological characteristics

The Sheba strain of feline panleukopenia virus is reproduced in a continuous culture of cat kidney cells (CrFK), as well as in a primary trypsinized and subculture of kitten kidney cells (KK). A cell culture (CrFK or PC) infected with the panleukopenia virus is incubated in a thermostat at a temperature of (37.5±0.5)°C for 5 days. Virus reproduction in PC and CrFK cell culture is not accompanied by a specific cytopathic effect. Biological properties were characterized by determining the hemagglutinating activity of the virus of each passage using the X-ray method. When cultivating the virus, the hemagglutination activity of the Sheba strain is not less than 9 log ₂ NA. The initial characteristics are preserved when passaging in sensitive biological systems for 5 passages (observation period).

Resistance to external factors

The virus is highly resistant to phenol, ether, chloroform, acids, trypsin, 0.2% sodium deoxycholate solution, pH 3.0. At a temperature of 4-25°C it can be stored for up to 13 months. At a temperature of 60°C it dies in 60 minutes. It is well preserved in 50% glycerol with a pH of 7.2. Tolerates double defrosting and thawing.

Additional characteristics and properties

Reactogenicity - does not have reactogenic properties.

Harmless to rabbits, guinea pigs, white mice with intramuscular and subcutaneous infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period) on a continuous culture of CrFK cells.

To obtain antigenic material, a virus-containing material with a hemagglutinating activity of at least 7 log ₂ HA is introduced into a culture of CrFK cells grown in culture flasks, after draining the growth medium from them. The bottles are placed in a thermostat for one hour at a temperature of (37.0±0.5)°C to bring the culture cells into contact with the virus. After this, the PSS maintenance medium is added with the addition of 2% cattle blood serum. The infected culture is incubated at (37.0±0.5)°C for 5 days, then frozen at minus (45.0±5.0)°C. The sterile infected monolayer was disaggregated from the working surface of the vials by defrosting it at a temperature of (20.0±2)°C and periodically shaking. After defrosting is completed, the remaining monolayer is removed from the working surface by vigorous shaking, then, observing aseptic conditions, the infected suspension is collected in a common container. A sample is taken from the collected material for control. Control of inoculum of the Sheba strain of feline panleukopenia virus is carried out based on the absence of contamination with fungi, bacteria and mycoplasmas. Control of contamination by fungi and bacteria of the inoculum of the Sheba strain of feline panleukopenia virus was carried out according to GOST 28085 [35]. Control of contamination with mycoplasmas was carried out in accordance with the requirements of the Global Fund XIV, volume II, p. 2997-3008 (OFS.1.7.2.0031.15) [36].

Contamination by bacteria, fungi, mycoplasmas and foreign viruses - the Sheba strain is not contaminated with bacteria, fungi, mycoplasmas and foreign viruses.

The hemagglutinating activity of the Sheba FPV strain was monitored according to the “Methodological recommendations for titration of the causative agent of feline panleukopenia” [38]. The essence of the method lies in the ability of FPV to hemagglutinate pig erythrocytes. The virus titer was taken to be its highest dilution causing complete agglutination of erythrocytes. The hemagglutinating activity of the virus before inactivation was 9.33±0.57 log ₂ HA in the RHA.

To inactivate the Sheba FPV strain, aminoethylethylenimine (AEEI) was used, which was added to the purified virus-containing suspension to a concentration of 0.025% with exposure for 24 hours at a temperature of (37.0±0.5)°C. Upon completion of inactivation, the resulting material was placed in a refrigerator at a temperature of (2-8)°C.

Inactivated purified virus-containing material of the Sheba strain FPV with a hemagglutinating activity of at least 9 log ₂ HA was used to produce the Carnifel RSN vaccine.

The isolate of the feline calicivirus virus, which served as the source for obtaining the Persian strain, was isolated from pathological material obtained from a cat admitted for treatment to the veterinary clinic of the VOOO Animal Center “Valenta”, Vladimir, in 2021.

The isolate of the feline calicivirus virus, which served as the source for obtaining the “Fauna” strain, was isolated from pathological material from samples of clinical material (oral swabs) obtained from a cat admitted for treatment at the “Fauna” veterinary clinic in Vologda, Vologda region of the Russian Federation.

To obtain the Persian strain of feline calicivirus virus, which has optimal biotechnological properties, a continuous culture of cat kidney cells (CrFK) was used. As a result of a series of successive passages on this cell culture, the “Pers” strain of feline calicivirus virus was obtained from a previously isolated isolate, which has stable biotechnological properties and is suitable for use in laboratory research and the production of diagnostic drugs and drugs for the specific prevention of this disease. The strain is adapted to a continuous culture of cat kidney cells (CrFK), which makes it possible to obtain on an industrial scale the viral material of the Persian strain of feline calicivirus virus with infectious activity titers from 7.75 to 8.25 lg TCD ₅₀ /cm ³ .

To obtain the “Fauna” strain of feline calicivirus virus, which has optimal biotechnological properties, a continuous culture of cat kidney cells (CrFK) was used. As a result of a series of successive passages on this cell culture, the “Fauna” strain of feline calicivirus virus was obtained from a previously isolated isolate, which has stable biotechnological properties and is suitable for use in laboratory research and the manufacture of diagnostic drugs and drugs for the specific prevention of this disease. The strain is adapted to a continuous culture of cat kidney cells (CrFK), which allows the production of viral material of the “Fauna” strain of feline calicivirus virus with titers of infectious activity from 7.75 to 8.25 lg TCD ₅₀ /cm ³ on an industrial scale.

The “Persian” and “Fauna” strains of the feline calicivirus virus are characterized by the following characteristics and properties.

Morphological characteristics

The “Pers” and “Fauna” strains of the feline calicivirosis virus belong to the family Caliciviridae, genus Vesivirus, species Feline calicivirus and have morphological characteristics characteristic of representatives of caliciviruses: the diameter of the virions is 37-40 nm, the virions without a supercapsid shell, cubic type of symmetry, consist of 32 capsomeres.

Antigenic properties

Antigenically and genetically, FCV differs from canine calicivirus [2]. All circulating strains represent a single serotype. In cats who have recovered from the disease, antibodies are formed in the blood serum, which are detected in the neutralization reaction (RN). The virus is stably neutralized by homologous antiserum in PH in CrFK cell culture.

Geno- and chemotaxonomic characteristics The genome is a 7.7 kb single-stranded positive polarity RNA with three open reading frames (ORFs). ORF1 encodes nonstructural proteins, ORF ₂ encodes the major capsid protein VP ₁ , and ORF3 encodes the minor capsid protein VP ₂ . ORF2 is divided into six regions (AF) and is commonly used for phylogenetic differentiation of FCV isolates. Genomic regions A, B, D and F are largely conserved, while regions C and E are more variable. The F region of ORF2 (7253–7329 bp) and the start region of ORF3 (7465–7524 bp) are conserved and are therefore convenient for precise identification of FCVs [37]. Recombination is a common mechanism of FCV evolution. Most reported recombination events are in in the FCV genome occur in the VP ₂ gene region, which was taken as the basis for differentiating strains of this virus (a nucleotide region with a length of 318 aa, 106 aa was analyzed) [20].

Biotechnological characteristics

The Persian strain of feline calicivirus virus is reproduced in a continuous culture of cat kidney cells (CrFK). Reproduction of the virus in CrFK cell culture is accompanied by a specific cytopathic effect, leading to rounding of cells and destruction of the monolayer after 24-48 hours of cultivation. Biological properties were characterized by determining the infectious activity of the virus of each passage in a continuous culture of CrFK cells. During cultivation, the “Persian” strain virus accumulates to a titer of at least 7.75±0.25 lg TCD ₅₀ /cm ³ and retains its original characteristics when passaging for at least 5 passages (observation period).

The “Fauna” strain of feline calicivirus virus is reproduced in a continuous culture of cat kidney cells (CrFK). Reproduction of the virus in CrFK cell culture is accompanied by a specific cytopathic effect, leading to rounding of cells and destruction of the monolayer after 24-48 hours of cultivation. Biological properties were characterized by determining the infectious activity of the virus of each passage in a continuous culture of CrFK cells. During cultivation, the “Fauna” strain virus accumulates in a titer of at least 7.5±0.25 lg TCD ₅₀ /cm ³ and retains its original characteristics when passaging for at least 5 passages (observation period).

Resistance to external factors

The virus is quite stable in the external environment. On surfaces at a temperature of (22±2)°C it remains active for 24 hours, and at a temperature of 10°C and below for 30 days. Relatively stable in an acidic environment at pH 4.0. Practically insensitive to ether and chloroform. Sensitive to formaldehyde, glutaraldehyde, chlorine-containing disinfectants. Tolerates double defrosting and thawing.

Additional characteristics and properties

Pathogenicity - pathogenic for naturally susceptible animals.

Virulence - virulent for naturally susceptible animals during intranasal infection.

Harmless to rabbits and white mice with intramuscular and subcutaneous infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period) on a continuous culture of CrFK cells.

To obtain antigenic material, virus-containing material was added to a culture of CrFK cells grown in culture flasks, after draining the growth medium from them, at a dose of 0.001 TCD ₅₀ /cell. The bottles were placed in a thermostat for one hour at a temperature of (37.0±0.5)°C to bring the culture cells into contact with the virus. After this, PSS maintenance medium was added with the addition of 2% cattle blood serum. The infected culture was incubated at (37.0±0.5)°C until the virus CPE appeared. If the monolayer area was affected by at least 80%, the culture flasks were frozen at a temperature of minus (45.0±0.5)°C. The sterile infected monolayer was disaggregated from the working surface of the vials by defrosting it at a temperature of (20±2)°C and periodically shaking. Upon completion of defrosting, the remaining monolayer was removed from the working surface by vigorous shaking, then, observing aseptic conditions, the infected suspension was collected into a common container. A control sample was taken from the collected material. Control of contamination of seed material of the “Persian” and “Fauna” feline calicivirus virus strains by fungi and bacteria was carried out according to GOST 28085 [4]. Control of contamination with mycoplasmas was carried out in accordance with the requirements of the Global Fund XIV, volume II, p. 2997-3008 (OFS.1.7.2.0031.15) [36].

Contamination with bacteria, fungi, mycoplasmas and foreign viruses - the Persian and Fauna strains are not contaminated with bacteria, fungi, mycoplasmas and foreign viruses.

The infectious activity of the “Pers” and “Fauna” FCV strains was monitored according to the “Methodological recommendations for titrating the calicivirus virus using the micromethod” [39]. Control of infectious activity was carried out in a continuous culture of CrFK cells by determining the titer of biological activity of the Persian strain. The essence of titration is based on determining the cytopathogenic effect (CPE) of the virus, which manifested itself in the CrFK cell culture after 48-72 hours. The titer of the virus is considered to be its last dilution, causing a 50% effect, which is calculated using the Kerber formula. The infectious activity of the virus before inactivation was 8.33±0.57 lg TCD ₅₀ /cm ³ . The infectious activity of the virus before inactivation was 8.05±0.46 lg TCD ₅₀ /cm ³ .

To inactivate the “Pers” and “Fauna” FCV strains, aminoethylethylenimine (AEEI) was used, which was added to the purified virus-containing suspension to a concentration of 0.025% with exposure for 24 hours at a temperature of (37.0±0.5)°C. Upon completion of inactivation, the resulting material was placed in a refrigerator at a temperature of (2-8)°C.

To produce the associated vaccine “Carnifel RSN”, inactivated purified antigenic material is used from the “Pers” FCV strain, obtained in a continuous culture of cat kidney cells (CC CrFK), with an activity of 8.33 ± 0.57 lg TCD ₅₀ / cm ³ in an amount of 15 .0 rev. %.

For the production of the associated vaccine “Carnifel RSN”, inactivated purified antigenic material from the “Fauna” FCV strain, obtained in a continuous culture of cat kidney cells (CK CrFK), with an activity of 8.05 ± 0.46 lg TCD ₅ o/cm ³ in the amount of 15.0 rev. %.

The isolate of the feline infectious rhinotracheitis virus, which served as the source for obtaining the “Lavr” strain, was isolated from pathological material obtained from a cat admitted for treatment to the veterinary clinic of the VOOO Animal Center “Valenta”, Vladimir, in 2021.

The “Lavr” FHV strain has been deposited in the All-Russian State Collection of Exotic Types of Foot and Mouth Disease Virus and other Animal Pathogens (FMDV) of the FGBU “ARRIAH”, under registration number: No. 457 - dep / 23-7 - GKSHM FGBU “ARRIAH.

The “Lavr” FHV strain is characterized by the following characteristics and properties.

Morphological characteristics

The “Laurel” strain of feline infectious rhinotracheitis virus belongs to the family Herpesviridae, genus Varicellovirus, species Alpaherpesvirus 1 and has morphological characteristics characteristic of representatives of herpes viruses: the size of virions is from 120 to 180 nm, they consist of a core containing double-stranded DNA, an icosahedral capsid, a layer a shell surrounding the capsid and a lipid bilayer from which the glycoprotein spikes protrude.

Antigenic properties

All circulating strains represent a single serotype. In cats who have recovered from the disease, antibodies are formed in the blood serum, which are detected in the neutralization reaction (RN). The virus is stably neutralized by homologous antiserum in PH in CrFK cell culture. Inoculation of the feline infectious rhinotracheitis virus into the body of an animal is accompanied by the formation of virus-neutralizing antibodies in the blood in a titer from 6 log ₂ SN ₅₀ to 8.00 log ₂ SN ₅₀ in rabbits.

Geno- and chemotaxonomic characteristics

The viral genome is approximately 135,800 bp in size, which consists of unique long (UL) and unique short (US) sequences flanked by inverted repeat regions known as terminal and inverted long repeat (TRL, IRL) and inverted and terminal short repeat. (IRL, TRS), respectively. Recombination is a common mechanism of FHV evolution. Most of the reported recombination events in the FHV genome occur at different sites. For strain differentiation, it is customary in the world scientific community to use the DNA section of the feline infectious rhinotracheitis virus, which encodes glycoprotein E (483 bp and 161 aa).

Biotechnological characteristics.

The Laurel strain of infectious feline rhinotracheitis virus is reproduced in a continuous culture of cat kidney cells (CrFK). Reproduction of the virus in CrFK cell culture is accompanied by a specific cytopathic effect, leading to rounding of cells and destruction of the monolayer after 48-72 hours of cultivation. Biological properties were characterized by determining the infectious activity of the virus of each passage in a continuous culture of CrFK cells. During cultivation, the “Lavr” strain virus accumulates in a titer of at least 6.5±0.25 lg TCD ₅₀ /cm ³ and retains its original characteristics when passaging for at least 5 passages (observation period).

Resistance to external factors

The virus is quite stable in the external environment. Sensitive to strongly acidic and slightly alkaline pH values, at a temperature of 56°C it is inactivated in 3-4 minutes, at a temperature of 37°C - in 36 hours, slowly inactivated in formalin, resistant to ethyl alcohol. Standard disinfectants (hypochlorite, quaternary ammonium compounds) destroy the virus shell. Tolerates double defrosting and thawing.

Additional characteristics and properties

Pathogenicity - pathogenic for naturally susceptible animals.

Virulence - virulent for naturally susceptible animals during intranasal infection.

Harmless to rabbits, guinea pigs and white mice with intramuscular and subcutaneous infection.

Stability - retains the original biological properties when passaged in sensitive biological systems for 5 passages (observation period) on a continuous culture of CrFK cells.

To obtain antigenic material, virus-containing material was added to a culture of CrFK cells grown in culture flasks, after draining the growth medium from them, at a dose of 0.001 TCD ₅₀ /cell. The bottles were placed in a thermostat for one hour at a temperature of (37.0±0.5)°C to bring the culture cells into contact with the virus. After this, PSS maintenance medium was added with the addition of 2% cattle blood serum. The infected culture was incubated at (37.0±0.5)°C until the virus CPE appeared. If the monolayer area was affected by at least 80%, the culture flasks were frozen at a temperature of minus (45.0±0.5)°C. The sterile infected monolayer was disaggregated from the working surface of the vials by defrosting it at a temperature of (20±2)°C and periodically shaking. Upon completion of defrosting, the remaining monolayer was removed from the working surface by vigorous shaking, then, observing aseptic conditions, the infected suspension was collected in a common container. A control sample was taken from the collected material. Control of inoculum of the “Lavr” strain of feline infectious rhinotracheitis virus was carried out based on the absence of contamination with fungi, bacteria and mycoplasmas. Control of contamination by fungi and bacteria of the inoculum of the “Lavr” strain of feline infectious rhinotracheitis virus was carried out according to GOST 28085 [35]. Control of contamination with mycoplasmas was carried out in accordance with the requirements of the Global Fund XIV, volume II, p. 2997-3008 (OFS.1.7.2.0031.15) [36].

Contamination with bacteria, fungi, mycoplasmas and foreign viruses - the “Lavr” strain is not contaminated with bacteria, fungi, mycoplasmas and foreign viruses.

Control of the infectious activity of the “Lavr” FHV strain was carried out according to the “Methodological recommendations for titrating the feline infectious rhinotracheitis virus using the micromethod” [40]. Control of infectious activity was carried out in a continuous culture of CrFK cells by determining the titer of biological activity of the “Lavr” strain. The essence of titration is based on determining the cytopathogenic effect (CPE) of the virus, which manifested itself in the CrFK cell culture after 96-120 hours. The titer of the virus is considered to be its last dilution, causing a 50% effect, which is calculated using the Kerber formula. The infectious activity of the virus before inactivation was 6.5±0.25 lg TCD ₅₀ /cm ³ .

To inactivate the “Lavr” FHV strain, aminoethylethylenimine (AEEI) was used, which was added to a purified virus-containing suspension to a concentration of 0.025% with exposure for 24 hours at a temperature of (37.0±0.5)°C. Upon completion of inactivation, the resulting material is placed in a refrigerator at a temperature of (2-8)°C.

To produce the associated vaccine “Carnifel RSN”, inactivated purified antigenic material is used from the “Lavr” FHV strain, reproduced in a continuous culture of cat kidney cells (CC CrFK), with an infectious activity of 6.75 ± 0.25 lg TCD ₅₀ / cm ³ , in quantity 30.0 vol. %.

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

Example 1. Preparation of the proposed associated vaccine “Carnifel RSN”

To prepare the associated vaccine against panleukopenia, calicivirosis and viral rhinotracheitis of cats “Carnifel RSN”, the following strains of viruses were used: strain “Sheba”, strain “Persian”, strain “Fauna”, strain “Lavr”.

The technological process consists of the following technological stages:

• obtaining viral suspensions of the Sheba, Persian, Fauna and Laurel strains;

• determination of the activity of each suspension;

• inactivation of virus-containing liquids of the Sheba, Persian, Fauna and Laurel strains separately;

• combining the resulting inactivated virus suspensions;

• adding an adjuvant to a mixture of inactivated viruses;

• packaging of the finished product.

The seed material used to make the vaccine was obtained in continuous cell cultures.

To produce the Carnifel RSN vaccine associated against panleukopenia, calicivirus and feline viral rhinotracheitis, a cultured attenuated virus of the Sheba strain was used; cultural virus of the Persian strain; cultural virus strain “Fauna”; cultural virus strain "Lavr", grown in culture flasks.

The proposed vaccine "Carnifel RSN" has an optimal component composition, vol. %:

1. Antigenic material from the Sheba strain FPV, reproduced in a continuous culture of cat kidney cells KK CrFK, with a hemagglutinating activity of 9.33±0.57 log ₂ HA, in an amount of 30.0 vol. %.

2. Antigenic material from the Persian FCV strain, reproduced in a continuous culture of cat kidney cells KK CrFK, with an infectious activity of 8.33±0.57 lg TCD ₅₀ /cm ³ , in an amount of 15.0 vol. %.

3. Antigenic material from the “Fauna” FCV strain, reproduced in a continuous culture of cat kidney cells KK CrFK, with an infectious activity of 8.05 ± 0.46 lg TCD ₅ o/cm ³ , in an amount of 15.0 vol. %.

4. Antigenic material from the “Lavr” FHV strain, reproduced in a continuous culture of dog kidney cells KK CrFK, with an infectious activity of 6.75±0.25 lg TCD ₅₀ /cm ³ , in an amount of 30.0 vol. %.

5. Adjuvant 3% aluminum hydroxide gel in an amount of at least 10.0 vol. %.

The content of antigenic materials in the proposed vaccine "Carnifel RSN" in the above concentrations is their effective amount in the drug, ensuring the achievement of the technical result from the use of the invention.

The resulting vaccine “Carnifel RSN” is a pink liquid with sediment. When shaken, the sediment easily breaks into a homogeneous suspension (Table 1).

The contents of the vessels with inactivated purified antigenic material from the Sheba strain FPV, from the Persian strain FCV, from the Fauna strain FCV, from the Laurus strain FHV were poured into separate containers and cooled at a temperature of (2-8) ° C, freeing from cellular debris using low-speed centrifugation. The suspension freed from detritus should look like a translucent liquid in shades of pink.

Then samples were taken from the containers to control viral raw materials.

The Sheba FPV strain series must have a hemagglutinating activity of at least 9.0 log ₂ HA;

A series of the Persian FCV strain must have an infectious activity of at least 7.0 lg TCD ₅₀ /cm ³ .

A series of the “Fauna” FCV strain must have an infectious activity of at least 7.0 lg TCD ₅₀ /cm ³ .

A series of the “Lavr” FHV strain must have an infectious activity of at least 6.5 lg TCD ₅₀ /cm ³ .

To inactivate the viral suspension of strains, aminoethylethylenimine (AEEI) was used, which was added to the purified virus-containing suspension to a concentration of 0.025% with exposure for 24 hours at a temperature of (37.0±0.5)°C.

Upon completion of inactivation, the antigenic material of each strain was cooled to a temperature of (2-8) ° C and samples were taken to determine pH, sterility and completeness of inactivation.

To neutralize AEEI, sodium thiosulfate was added to the viral suspension of antigenic material at the rate of 10% of the volume of AEEI used.

The resulting antigenic material from the strains “Sheba”, “Fauna” and “Persian”, “Lavr” was used to produce a sorbed form of the vaccine against panleukopenia, calicivirosis and viral rhinotracheitis of cats “Carnifel RSN”. To do this, the inactivated antigenic material was thoroughly mixed with 3% aluminum hydroxide gel in a quantitative ratio of 30.0: 15.0: 15.0: 30.0: 10.0, respectively.

The vaccine was controlled for stability, sterility and antigenic activity.

The sterility of the vaccine is determined in accordance with GOST 28085-89 “Biological preparations. Methods of biological control of sterility". The essence of the method is to determine the absence of growth of bacterial and fungal microflora in cultures of vaccine samples on nutrient media.

The vaccine was monitored for completeness of inactivation (avirulence), harmlessness and was packaged in sterile vials.

The completeness of drug inactivation was determined by a well-known method, similar to the determination of harmlessness.

The vaccine must be sterile, avirulent and harmless.

The safety and reactogenicity of the vaccine were studied in a group of ferrets, cats and mice that were administered a double vaccination volume of the vaccine. The observation period for the animals was 10 days. In the experimental animals, depression, loss of appetite, or refusal to feed were not observed. Only one mouse developed a slight swelling with a diameter of up to 0.2 cm at the injection site on day 3, which disappeared on day 5.

According to the results of the harmlessness test, the animals remained alive throughout the entire observation period. Observation of the animals for 10 days did not reveal any deviations in the general condition of the animal’s body, which indicates the harmlessness and reactogenicity of the Carnifel RSN vaccine (the proposed invention).

The associated vaccine "Carnifel RSN" (the proposed invention) is sterile, harmless and areactogenic (Table 1-2).

Example 2. Antigenic activity and stability of the associated vaccine “Carnifel RSN”.

The antigenic activity of the vaccine was assessed based on the results of vaccination of kittens with samples obtained at certain storage intervals of the drug (3 months, 6 months, 9 months and 12 months). For this purpose, outbred kittens aged 2-3 months were used in the amount of 5 animals for each vaccine storage period set for a given period of time.

Every 3 months, a newly created group of kittens was injected with the vaccine intramuscularly into the thigh area in a single dose of 1.0 ^cm3 , which was stored for 3 months, 6 months, 9 months. and 12 months Blood samples were taken from all animals before immunization and 21 days after immunization to detect antibodies to the FPV virus in the RTGA and antibodies to the FCV and FHV viruses in the RMN (Table 3). The vaccine, administered once intramuscularly, should cause in animals at least a 2-fold increase in antibodies in the RTGA to the panleukopenia virus, the average titer of antibodies in the RMN to the calicivirus virus should be at least 1:32 (5.0 log ₂ ) and infectious rhinotracheitis should be not lower than 1:16 (3.0 log ₂ ) in relation to antibody titers before vaccination. There should be no increase in antibody titer in the control group.

The stability of the vaccine was studied over 12 months of storage at temperatures from 2°C to 8°C with inspection and research intervals every 3 months (Table 1). All selected vaccine samples were examined every 3 months for sterility in accordance with GOST 28085-89 [35] and for the absence of contamination with mycoplasmas in accordance with the requirements of the Global Fund XIV volume 2, General Pharmacopoeia Monograph 1.7.2.0031.15 [36].

As presented in Table 1, at all test time intervals when storing the vaccine at temperatures from 2°C to 8°C, no changes were found in the quality indicators of the drug: sterility; completeness of inactivation; harmlessness and reactogenicity; antigenic activity.

All animals vaccinated with vaccine samples, both at the time of storage and with samples after 3, 6, 9 and 12 months of storage at temperatures from 2°C to 8°C, were immune after the specified period. The level of specific antibodies to the FPV virus in the blood sera of kittens of the experimental groups averaged for the group 9.00-9.60 log ₂ HI in the RTGA, to the FCV virus 6.40-7.25 log ₂ in the RMN, to the FHV virus 5, 55-6.25 log ₂ in RMN, which indicates the stability of the vaccine for 12 months (Table 3).

The associated vaccine "Carnifel RSN" (the proposed invention) is harmless, areactogenic and stable (within 12 months - observation period).

Example 3. Use of the proposed associated vaccine “Carnifel RSN” for immunization of cats.

Before use, a vial of vaccine containing 1 vaccination dose must be gently shaken until homogeneity is restored.

The vaccine was administered to kittens twice with an interval of 21 days intramuscularly in the area of the middle third of the thigh or subcutaneously in the area of the shoulder blades in a dose of 1.0 cm ³ in compliance with the rules of asepsis.

The vaccine causes the formation of an immune response in cats to the viruses of panleukopenia, calicivirosis and infectious rhinotracheitis 14 days after a double dose, the duration of immunity against the viruses of panleukopenia, calicivirosis and infectious rhinotracheitis is at least 12 months.

Example 4. Efficacy of vaccination with the proposed vaccine The effectiveness of the vaccine associated with panleukopenia, calicivirosis and feline viral rhinotracheitis "Carnifel RSN", manufactured as described in example 1, is presented in tables 4-6 for assessing immunogenic activity.

The immunogenic activity of the resulting drug was tested on 15 kittens 2-3 months of age, not vaccinated against panleukopenia (FPV), calicivirus (FCV) and viral rhinotracheitis (FHV), and 3 kittens were left unvaccinated as a control.

All 15 kittens were divided into 3 groups of 5 animals each. Each group tested different schemes for using the invention.

Kittens of group No. 1 were vaccinated with the vaccine (the proposed invention) subcutaneously twice with an interval of 21 days in a volume of 1.0 cm ³ .

Kittens of group No. 2 were vaccinated twice with an interval of 21 days. At 12 months of age, the kittens were revaccinated. Each time the vaccine (the proposed invention) was administered subcutaneously in a volume of 1.0 cm ³ .

For kittens of group No. 3, the vaccine was administered subcutaneously once in a volume of 1.0 cm ³ .

Schemes for collecting blood samples from animals for serological studies and the results of studies of antibody titers for each component of the vaccine are presented in Tables 4-6. Animal blood sera were examined in the RTGA for the presence of antibodies to the FPV virus, in the RMN for the presence of antibodies to the FCV, FHV viruses. The magnitude of the antibody titer was considered an assessment of the strength of post-vaccination immunity against these viruses. RMN and RTGA were performed according to generally accepted methods.

After vaccination with the vaccine against panleukopenia, calicivirosis and viral rhinotracheitis “Carnifel RSN”, the animals’ immune system actively reacted to the antigens included in the drug. An intense immune response to FPV, FCV, FHV was observed in animals of all groups throughout the entire observation period. The first administration of the vaccine will protect cats from panleukopenia, calicivirus and rhinotracheitis viruses, based on the data presented in Table 4-6. However, in kittens of group No. 3 after a single vaccination after 5 months there was a decrease in the level of antibodies to FCV viruses and the average for the group was 4.90-6.40 log ₂ in RMN; after 8 months there was a decrease in the level of antibodies to the FHV virus, the average for the group was 3.80 log ₂ in RMN; after 11 months, a decrease in the level of antibodies to the FPV virus was observed, the average for the group was 6.45 log ₂ HI in the RTGA (Table 6).

In kittens of group No. 1 and group No. 2 after double vaccination, the geometric mean titer of antibodies to FPV in the RTGA was 9.60 ± 0.54 log ₂ and 9.05 ± 0.59 log ₂ HI, respectively, the geometric mean titer of antibodies to FCV viruses in RMN in group No. 1 was 7.05 ± 0.48 log ₂ and 8.20 ± 0.32 log ₂ , in group No. 2 it was 7.25 ± 0.39 log ₂ and 8.25 ± 0.25 log ₂ , the geometric mean titer of antibodies to the FHV virus in the RMN was 6.20±0.74 log ₂ and 6.20±0.28 log ₂ , respectively. Throughout the entire observation period, the level of specific antibodies to the FPV, FCV, FHV viruses in the blood serum of kittens of groups No. 1 and No. 2 remained at a high level (Table 4-5).

After revaccination at the age of 12 months of animals of group No. 2 with the Carnifel RSN vaccine, an increase in antibodies to FPV, FCV, FHV was established. The data in Table 5 indicates that antibody titers exceeded the required minimum titers for all antigenic components of the vaccine. Antibody titers to all antigenic components in the control group remained approximately at the same level throughout the entire observation period.

The data indicate that the associated vaccine (the proposed invention) induces the formation of high levels of antibodies to the Sheba strain of feline panleukopenia virus in vaccinated kittens. The maximum level of antibody titers observed 21 days after revaccination was 9.45±0.44 log ₂ HI.

Data indicate that the associated vaccine (the proposed invention) induces the formation of high levels of antibodies to the feline calicivirus strain “Persian” in vaccinated kittens. The maximum level of antibody titers, which was observed 21 days after revaccination, was 8.35 ± 0.22 log ₂ .

Data indicate that the associated vaccine (the proposed invention) induces the formation of high levels of antibodies to the feline calicivirus strain “Fauna” in vaccinated kittens. The maximum level of antibody titers observed 21 days after revaccination was 7.40 ± 0.41 log ₂ .

Data indicate that the associated vaccine (the proposed invention) induces in vaccinated kittens the formation of high levels of antibodies to the feline infectious rhinotracheitis virus of the Laurel strain. The maximum level of antibody titers observed 21 days after revaccination was 7.15±0.48 _log2 .

Based on the studies conducted, it was concluded that the associated vaccine against panleukopenia, calicivirosis and viral rhinotracheitis of cats "Carnifel RSN" (the proposed invention) forms in immunized animals a persistent immune response against panleukopenia, calicivirosis and viral rhinotracheitis of cats lasting at least 12 months at administration of the drug twice with an interval of 21 days and revaccination of animals at 12 months of age.

Based on the results, we can conclude that the associated vaccine against panleukopenia, calicivirosis and feline viral rhinotracheitis “Carnifel RSN” (the proposed invention) does not prevent the formation of an immune response to the causative agents of panleukopenia, calicivirosis and feline viral rhinotracheitis. After administration of the vaccine against panleukopenia, calicivirosis and viral rhinotracheitis of cats "Carnifel RSN", intense immunity is developed that can provide reliable protection against these pathogens. On this basis, we believe that the components of the vaccine against panleukopenia, calicivirus and feline viral rhinotracheitis are compatible with each other.

Thus, the associated vaccine against panleukopenia, calicivirosis and feline viral rhinotracheitis “Carnifel RSN” (the proposed invention) has high immunogenic and antigenic activity against all 4 antigenic components and is harmless for cats.

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--->

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Claims (8)

1. A vaccine associated against panleukopenia, calicivirosis and feline viral rhinotracheitis, containing an active substance and a target additive, characterized in that as an active substance it contains a mixture of inactivated purified antigenic material: from the “Sheba” strain of feline panleukopenia, fam. Parvoviridae, genus Protoparvovirus, species Carnivore protoparvovirus 1, collection of the FGBU "ARRIAH" under registration number: No. 455 - dep / 23-5 - GKSHM FGBU "ARRIAH"; from the “Pers” strain of feline calicivirus, family Caliciviridae, genus Vesivirus, species Feline calicivirus, collection of the FGBU “ARRIAH” under registration number: No. 458 - dep / 23-8 GKShM FGBU “ARRIAH”; from the “Fauna” strain of feline calicivirus, family Caliciviridae, genus Vesivirus, species Feline calicivirus, collection of the Federal State Budgetary Institution “ARRIAH” under registration number: No. 471 - dep / 23-21 GKShM FGBU “ARRIAH”; from the “Lavr” strain of infectious feline rhinotracheitis to the family Herpesviridae, genus Varicellovirus, species Alpaherpesvirus 1, collection of the Federal State Budgetary Institution “ARRIAH” under registration number: No. 457 - dep / 23-7; and as a targeted additive: 3% aluminum hydroxide gel, taken in a volume ratio of 30.0: 15.0: 15.0: 30.0: 10.0 and in quantities that ensure the protective immunogenic activity of each antigen in the body of cats after administration of the target drug. 2. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the Sheba strain of feline panleukopenia (FPV) Carnivore protoparvovirus 1, obtained in a continuous culture of CrFK cells, representing a viral suspension in an amount of 30.0 vol . %. 3. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the “Pers” strain of feline calicivirus (FCV) Feline calicivirus, obtained in a continuous culture of CrFK cells, which is a suspension in an amount of 15.0 vol. %. 4. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the “Fauna” strain of feline calicivirus (FCV) Feline calicivirus, obtained in a continuous culture of CrFK cells, which is a suspension in the amount of 15.0 vol. %. 5. The vaccine according to claim 1, characterized in that it contains inactivated and purified antigenic material from the “Laurel” strain of infectious rhinotracheitis (FHV) Alphaherpesvirus 1, obtained in a continuous culture of CrFK cells, which is a suspension in the amount of 30.0 vol. %. 6. The vaccine according to claim 1, characterized in that as an adjuvant it contains 3% aluminum hydroxide gel in an amount of at least 10.0 vol. %. 7. The vaccine according to any one of paragraphs. 1-5, characterized in that it contains a mixture of inactivated and purified antigenic materials from the Sheba strain of feline panleukopenia (FPV), obtained in a continuous culture of CrFK cells, with an infectious activity of 9.00±0.57 log ₂ HA; from the “Pers” strain of feline calicivirus virus (FCV), obtained in a continuous culture of CrFK cells, with an infectious activity of 8.33±0.57 lg lg TCD ₅₀ /cm ³ ; from the “Fauna” strain of feline calicivirus virus (FCV), obtained in a continuous culture of CrFK cells, with an infectious activity of 8.05±0.46 lg TCD ₅₀ /cm ³ ; from the “Lavr” strain of infectious rhinotracheitis virus (FHV), obtained in a continuous culture of CrFK cells, with an infectious activity of 6.5 ± 0.25 lg TCD ₅₀ / cm ³ ; and targeted additives: adjuvant in quantity, vol. %: antigenic material from the Sheba strain of feline panleukopenia virus 30.0 antigenic material from the Persian strain of feline calicivirus virus 15.0 antigenic material from the “Fauna” strain of feline calicivorosis virus 15.0 antigenic material from the “Lavr” strain of feline infectious rhinotracheitis virus 30.0 adjuvant 3% aluminum hydroxide gel 10.0