RU2366458C1 - Combined emulsion inactivated vaccine against rotaviral and coronaviral cattle infections - Google Patents

Abstract

FIELD: medicine; veterinary science.

SUBSTANCE: vaccine contains mixed avirulent purified antigens of strain No.101 "ВНИИ3Ж-ДЕП" of cattle rotavirus and of strain "ВНИИ3Ж-ДЕП" of cattle coronavirus in amount to provide protective immunoactivity in an animal's organism when a desired preparation is introduced, and an oil adjuvant in effective ratio. In immunised animals, the vaccine induces high level of antibodies against cattle rota- and protects the cattle stock of sex-age groups from the diseases as stated above. The invention can be used in veterinary science.

EFFECT: new compounds possess effective biological activity.

14 cl, 7 tbl, 3 ex

Description

The invention relates to the field of veterinary virology and biotechnology and relates to an associated vaccine for the specific prophylaxis of gastrointestinal diseases of cattle (cattle), mainly calves caused by causative agents of rotavirus and coronavirus cattle infections.

In the structure of calf diseases in the early postnatal period, the predominant place is occupied by disorders of the digestive system, which are clinically manifested by diarrhea, which leads to the development of severe dehydration, toxemia, immunodeficiencies, and metabolic disorders.

Etiopathogenetic lesions of the digestive system are noted by significant polymorphism, including a wide range of various factors, including genetic, physiological, sanitary and hygienic and infectious. Massive digestive dysfunctions, clinically manifested by diarrhea, are recorded in 70-100% of newborn calves by the end of the first day after birth. Sick calves refuse colostrum, are not very mobile, dehydration, intoxication quickly sets in, and their tone decreases sharply. The death of newborn calves, as a rule, occurs on the 2nd-5th or 7th-10th days. Typically, 15 to 55% of newborn calves die (1, 2, 3).

The main cause of gastroenteritis in newborn calves is infectious agents, including viruses, bacteria, protozoa and fungi, the virulence of which increases due to various adverse conditions of feeding and feeding. The causative agents of infectious diseases enter the body usually on the first day of calf life (4, 5).

It is generally accepted that viruses are the trigger for the development of intestinal pathology. With diarrhea of newborn calves, mainly rotavirus and coronavirus are secreted. These viruses play a leading role in the etiology of gastrointestinal diseases of newborn calves, especially in large livestock farms (5, 6, 7).

Rotavirus infection of cattle is widespread in all countries of the world and causes great economic damage to livestock production due to the high incidence of calves, mortality due to diarrhea, weight loss of animals and reduced resistance to other infections.

Cattle rotaviruses are characterized by significant antigenic polymorphism and instability of the antigenic structure. The complex structure of the genome, including its fragmentation, and relative resistance to various factors determine the constant possibility of modifying its antigenic structure and the enzootic course of the disease in farms with poor living conditions for animals.

For specific prophylaxis of rotavirus enteritis, live and inactivated vaccines are used, which are used to vaccinate deep-shelled cows. In this case, colostral immunity due to the intake of specific antibodies neutralizing the pathogen in the lumen of the small intestine, as well as immunocompetent cells and other protective factors with colostrum, is of practical importance for the prevention of rotavirus diarrhea in newborn calves

Coronavirus infection in cattle is manifested in the form of coronavirus diarrhea in newborn calves, coronavirus winter dysentery of adult cattle, and coronavirus respiratory infection.

Extremely widespread, the tendency to cause long-term persistence, and relative resistance to various factors determine the enzootic course of the disease in farms with poor living and feeding conditions. The stationarity of the disease is maintained due to the prolonged persistence of the causative agent of coronavirus infection in the body of animals-convalescents.

Important conditions for the successful fight against the above diseases are their timely and correct diagnosis and specific prophylaxis, for which live and inactivated vaccines are used in the form of mono- or associated drugs (8, 9, 10).

The advantage of live vaccines due to manufacturability and ease of use is beyond doubt. However, the immune response in animals when they are introduced is not always stable and in some cases causes complications after vaccination: the vaccine can cause increased reactogenicity, immunosuppression, and sometimes death of up to 2% of animals during vaccination, depending on the conditions of detention and the presence of co-infections, reproduction problems, prolonged persistence and isolation of the vaccine virus into the environment. For this reason, they are not widely used.

For specific prophylaxis of these diseases, inactivated vaccines, both sorbed and emulsion, are used.

A known vaccine against rotavirus infection of cattle, containing as an active substance an avirulent purified antigenic material from strain No. 101 ARRIAH of cattle rotavirus obtained in animal cell culture selected from the group consisting of MDBK calf kidney cell culture and SPEV porcine kidney cell culture, co the degree of accumulation of not less than 10 ⁶ viral particles / cm ³ and the activity in the enzyme immunoassay (ELISA) of not less than 5.0 log ₂ , and as target additives aluminum hydroxide (GOA) and saponin in the ratio, wt.%:

Antigenic material 50.0-80.0 GOA 19.6-49.6 Saponin 0.4 (11).

A known vaccine against coronavirus infection of cattle is adsorbed inactivated, containing as an active substance an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in animal cell culture, taken from the group consisting of MDBK cell culture, Taurus-1 cell culture, from KST cell culture and from Vero cell culture, and as a target additive, the adjuvant-sorbent GOA in an effective ratio (12).

The disadvantages of monovalent inactivated vaccines are that they stimulate the formation of immunity in animals to only one infection, require prophylactic treatments against each infection separately, which affects the labor costs, increase the timing of the formation of immunity in animals, and contribute to the emergence of stressful phenomena and complications.

These disadvantages are partially eliminated by the creation of associated vaccines containing separately or in combination of causative agents of rotavirus and coronavirus infections of cattle of various strains.

In some cases, the composition of known vaccines, especially designed to protect calves, include antigens of pathogens that cause secondary infections.

An associated vaccine for enteric infection of newborn calves is inactivated emulsion, containing as an active substance a mixture of avirulent purified antigenic materials from strain TM-91 (CAPM V-279) of rotavirus, from strain BCV 9 (CAPM V-280) of coronavirus and from strains 3935 / 1 (CAPM 6233), 4804 (CAPM 6234) and 6304 (CAPM 6235) of E. coli cattle bacteria obtained in sensitive biological systems, and as the target additive, an oil adjuvant in an effective ratio (13).

An associated vaccine is known for viral diarrhea — mucosal disease (VD-BS), rota- and coronavirus infections, and cattle Escherichiosis inactivated emulsion containing a mixture of avirulent purified antigenic materials from strain VDV-Oregon-C24 V (CAPM V-293) as an active substance. VD-BS virus, from strain TM-91 (CAPM V-279) of rotavirus, from strain BCV-9 (CAPM V-280) of coronavirus and from strains 3935/1 (CAPM 6233), 4804 (CAPM 6234) and 6304 (CAPM 6235) E.coli cattle bacteria obtained in sensitive biological systems and, as a target additive, an oil adjuvant in effective relationship (14).

Known associated vaccine against infectious rhinotracheitis (RTI), rota and coronavirus infections and Escherichia cattle inactivated emulsion, containing as an active substance a mixture of avirulent purified antigenic materials from strain IBR-BK-5-PTOB-8 INRV No. 3760 of the IRT virus, from strain TM-91 (CAPM V-279) rotavirus, from strain BCV-9 (CAPM V-280) coronavirus and from strains 3935/1 (CAPM 6234) and 6304 (CAPM 6235) of E. coli cattle bacteria obtained in sensitive biological systems , and as a target additive, an oil adjuvant in effective ratios (15).

Known vaccine associated parainfluenza-3 (PG-3), rotavirus and coronavirus infections and ehsherihioza RNC inactivated emulsion comprising as active ingredient the mixture of purified antigenic material avirulent strain of PI-3 / T _1/11 V-30 virus PG- 3 cattle, from the strain TM-91 (CAPM V-279) of rotavirus, from the strain BCV-9 (CAPM V-280) of coronavirus and from strains 3935/1 (CAPM 6233), 4808 (CAPM 6234) and 6304 (CAPM 6235) E. coli bacteria from cattle obtained in sensitive biological systems and, as a target additive, an oil adjuvant in effective ratios (16).

An associated vaccine against rota-, corona-, herpesvirus and Escherichia diarrhea of newborn calves is known, which is inactivated adsorbed, containing as an active substance a mixture of avirulent purified antigenic materials from Bovine rotavirus strain III-1, from Bovine coronavirus strain CM-1, from strain “TKA-VIEV-V 2” Bovine Herpesvirus and from strains of bacteria E. coli containing adhesins K ₉₉ and A ₂₀ , and as a target additive adjuvant-sorbent GOA in effective ratios (17).

An associated vaccine against rota- and coronavirus infections, Escherichiosis and Clostridiosis of cattle is known, inactivated emulsion containing a mixture of avirulent purified antigenic materials from strains Cody 81-4.10 B223 type G and B 641 of cattle rotavirus, Mebus strain (ATCC No. VR -874) of KPC coronavirus, strains B41, B43, B44 and B141 of E. coli bacteria and strain GL 47 (ATCC PTA-3000) of Clostridium perfringens bacteria obtained in sensitive biological systems, and as the target additive, an oil adjuvant in effective ratios (18 )

There is a known vaccine against IRT, PG-3, viral diarrhea, respiratory syncytial, rota- and coronavirus disease of calves “COMBOVAC” inactivated adsorbed containing as a active substance a mixture of purified virulent antigenic materials from 6 (six) production strains of virus pathogens: IRT, PG-3, VD-BS, respiratory syncytial, rota- and coronavirus disease KPC, obtained in sensitive biological systems, and as a target additive adjuvant-sorbent GOA in effective ratios (19) .

An associated vaccine against cattle viral pneumogastroenteritis and calves is sorbed inactivated, containing as a active substance a mixture of avirulent purified antigenic materials from strains B-25 and TK-A of the IRP KPC virus, from strains BD-N and BD-R of the virus VD-BS KPC, from strains B-19 and PTK-45/86 of the PG-3 KPC virus, from strains KV-90 and K-22 of the coronavirus KPC and from strains PC-89 and PC-71 of the causative agent of respiratory syncytial KPC infection obtained in sensitive biological systems , and as a target additive adjuvant-sorbent GOA in effective ootnosheniyah (20).

There is also a method for the quantitative assessment of virus-containing suspensions using electron microscopy, which arose as a result of many years of experiments on electron microscopic control of viral preparations and comparison of its data with biological test parameters, for example, the virus infectivity titer (21).

The authors of this method established the relationship between the infectivity titer of native FMD virus suspensions and the number of physical virus particles observed under an electron microscope, which served as the basis for determining the total concentration of viral particles per unit volume of the suspension (the degree of accumulation of viral particles in a certain volume of liquid).

To determine the total concentration (N) of viral particles per unit volume of the analyzed suspension on the screen of an electron microscope with an area of S ₂ count the number of simultaneously observed particles n. The well-known instrumental increase in the electron microscope (M) determines the size of the portion of the substrate film S ₁ corresponding to the useful screen area:

.

.

When applying viral preparations to the substrate films, the concentration of viral particles n in this section S ₁ corresponds to the total amount of adsorbed particles and particles deposited on the substrate during evaporation of the residual liquid. Therefore, the number of particles observed simultaneously on the screen is distributed in a microvolume equal to:

v = S ₁ · I, where:

I is the thickness of the residual liquid layer on the substrate (mm).

In the determined volume of the virus-containing suspension, the number of particles is equal to:

,

,

after replacing the values of v and S ₁ :

, где:

where:

V is the volume of the analyzed suspension (1 cm ³ );

N is the arithmetic average of a series of values of the number of particles calculated from the screen of an electron microscope or exposed photographic plates.

The thickness of the residual layer I on the substrate is measured by a pre-cooled micrometer after freezing the liquid on the substrate in liquid nitrogen vapors. It is 0.03-0.04 mm and it is taken as a constant value.

Electron-microscopic determination of the concentration of physical viral particles allows not only to determine the quantitative characteristic of the virus in the analyzed suspensions, but also to assess the morphological state of the studied viral particles.

The specified method turned out to be universal and suitable for determining the quantitative characteristics of various viruses in the studied liquids.

The closest to the present invention in terms of essential features is the associated vaccine against rotational and coronavirus infections KPC inactivated emulsion, containing as a substance a mixture of avirulent purified antigenic materials from strain TM-91 (CAMP V-279) of rotavirus KPC obtained in cell culture kidney calf MDBK with infectious activity from 10 ³ to 10 ⁵ TCD ₅₀ , and from strain BCV 9 (CAMP V-280) coronavirus KPC obtained in a culture of MDBK cells with hemagglutinating activity (HA activity) 1: 256-1: 51 2, and as a target additive, an oil adjuvant in effective ratios (13).

A common significant drawback of the known associated vaccines, including the prototype vaccine, intended for the prevention of rota- and coronavirus infections of KPC of various age and gender groups, is their insufficiently high efficiency due to:

1) inferiority (incompleteness) of the antigenic composition of their constituent ingredients and complex interactions of the antigens from which they are made;

2) the narrowness of the antigenic spectrum of action of the strains used;

3) low immunogenic activity of drugs. The reason for this phenomenon may be the use of formalin for inactivation of viruses, which causes destructive changes in viral proteins.

Therefore, the problem of creating an associated vaccine against rota- and coronavirus infections of KPC, mainly newborn calves, continues to be relevant and is the main direction of research to improve it.

The problem to which the present invention is directed is to obtain an associated inactivated emulsion KPC vaccine against rot and coronavirus infections of KPC that creates effective protection of susceptible animals of various sex and age groups against pathogens of KPC rot and coronavirus infections circulating in the Russian Federation.

The technical result from the use of the present invention is to expand the arsenal of highly immunogenic, harmless and areactogenic associated vaccines against rotational and coronavirus infections of KPC emulsion inactivated, capable of protecting the KPC population of various age and gender groups from pathogens of rotational and coronavirus KPC infections circulating in Russia.

The specified technical result was achieved by the creation of an associated vaccine against rota- and coronavirus infections KPC inactivated emulsion, characterized by the following set of features.

According to the invention, the proposed vaccine contains as an active substance a mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH (author's name) rotavirus KPC and strain "ARRIAH" (author's name) coronavirus KPC obtained in animal cell cultures in quantities that provide protective immune activity in the animal’s body with the introduction of the target drug, and as the target additive oil adjuvant in an effective ratio.

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

1. Associated vaccine against rota- and coronavirus infections KPC emulsion inactivated.

2. The active substance.

3. As an active substance, a mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH of rotavirus KPC and strain "ARRIAH" of coronavirus KPC obtained in animal cell cultures in amounts that provide protective immune activity in the animal’s body when the target drug is administered to it.

4. Targeted supplement.

5. As a target additive, an oil adjuvant.

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. Associated vaccine against rota- and coronavirus infections KPC emulsion inactivated.

2. The active substance.

3. Targeted supplement.

Compared with the prototype vaccine, the essential distinguishing features of the proposed vaccine are that it contains a mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH of rotavirus KPC and strain "ARRIAH" of coronavirus KPC obtained in animal cell cultures in amounts that provide protective immune activity in the body of the animal with the introduction of the target drug.

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

1. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC, obtained in a cell culture of animal origin, taken from the group consisting of culture cells of calf kidney cells MOVK, from cell culture of porcine kidney SPEV, from the cell culture of the kidney green monkey Marc-145, from a Vero monkey kidney cell culture and from a CTF calf coronary cell culture, in an effective amount.

2. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in an MDBK cell culture with a degree of accumulation of at least 10 ⁶ virus particles / cm ³ and activity in an enzyme-linked immunosorbent assay (ELISA) of at least 5.0 log ₂ , not less than 15.0 wt.%.

3. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in cell culture SPEV with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of at least 15.0 wt.%.

4. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in a Marc-145 cell culture with a degree of accumulation of at least 10 ⁶ virus particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of at least 15 , 0 wt.%.

5. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in Vero cell culture with an accumulation degree of at least 10 ⁶ viral particles / cm ³ and an ELISA activity of at least 5.0 log ₂ , in an amount of at least 15.0 wt.%.

6. Avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in KST cell culture with a degree of accumulation of at least 10 ⁶ virus particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of at least 15.0 wt.%.

7. Avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in an animal cell culture taken from the group consisting of MDBK cell culture, Taurus-1 calf kidney cell culture, KCT cell culture, and Vero cell culture, in an effective amount.

8. Avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in an MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the hemagglutination reaction (RGA) of at least 7.0 log ₂ , not less than 15.0 wt.%.

9. Avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in Taurus-1 cell culture with a degree of accumulation of at least 10 ⁹ viral particles / cm ³ and activity in the RGA of at least 9.0 log ₂ , in an amount of at least 15 , 0 wt.%.

10. Avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus, obtained in KCT cell culture with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ , in an amount of at least 15 , 0 wt.%.

11. Avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus, obtained in Vero cell culture with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ , in an amount of at least 15 , 0 wt.%.

12. Oil adjuvant in an amount of not less than 70.0 wt.%

13. A mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH of rotavirus KPC and strain "ARRIAH" of coronavirus KPC obtained in animal cell cultures, and an oil adjuvant in the ratio, wt.%:

Avirulent purified antigenic material from strain No. 101 ARRIAH of the rotavirus KPC virus obtained in cell culture of animal origin taken from the group consisting of MDBK cell culture, from SPEV cell culture, from Marc-145 cell culture, from Vero cell culture and from KCT cell culture, with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA not less than 5.0 log ₂ 15.0 Avirulent purified antigenic material from strain "ARRIAH" KPC coronavirus obtained in culture cells of animal origin, taken from the group consisting of cell culture MDBK, from cell culture Taurus-1, from KCT cell culture and from cell culture Vero, in concentration of viral particles, providing protective immune activity in the body animal with the introduction of the target drug 15.0 Oil adjuvant up to 100.0.

The virus strains included in the proposed vaccine are known and are industrial in the manufacture of monovaccines (22, 23).

As a result of the studies, the authors found that there is no competition between the antigens that make up the proposed vaccine.

The authors also discovered the phenomenon of synergism when combining these antigens in the proposed vaccine, which allowed to reduce their quantitative content in the drug while maintaining the same tension and duration of immunity in vaccinated animals, as when using the corresponding monopreparations for immunization.

An additional technical result from the use of the present invention is achieved due to the fact that for the inactivation of virus-containing materials using aminoethylethyleneimine (AEEI), which can significantly reduce labor and energy costs for the manufacture of vaccines and improve the quality of antigenic materials.

An additional technical result from the use of the invention in terms of increasing the antigenic and immunogenic activity of the target drug is also achieved through the use of Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard) in its composition.

The viral isolate, which served as the source for the production strain No. 101 ARRIAH of rotavirus KPC, was isolated from the feces of a newborn calf with impaired gastrointestinal function. Vaccine strain No. 101 ARRIAH of rotavirus KPC is adapted to cell cultures of MDBK, SPEV, Vero, Marc-145 and KCT.

Strain No. 101 ARRIAH of rotavirus KPC was deposited on March 12, 2001 in the All-Russian State Collection of microorganism strains used in veterinary medicine and animal husbandry, Federal State Institution "All-Russian State Center for Quality and Standardization of Medicines for Animals and Feed" (FGU VGNKI) under the registration name : culture strain No. 101 ARRIAH-DEPT of rotavirus KPC.

Strain No. 101 ARRIAH of rotavirus KPC is characterized by the following features and properties (22).

Morphological properties

Strain No. 101 ARRIAH of rotavirus KPC belongs to the family Reoviridae, genus Rotavirus and has morphological characteristics characteristic of the causative agent of rotavirus infection KPC: spherical shape with a diameter of 65-75 nm, having the form of a "wheel". The virion consists of a core, inner and outer capsid. The core with a diameter of 40-45 nm has a hexagonal shape and consists of 3 proteins (VP1, VP2 and VP3) and RNA. The internal capsid (diameter 15–20 nm) has an icosahedral shape and is composed of 260 morphological units, each of which is represented by VP6 protein. The outer capsid is represented by VP7 protein. On the surface of the external capsid are "spines" represented by the VP4 protein dimer.

The floating density of the complete virions of rotavirus KPC in the gradient of cesium chloride is 1.36 g / cm ³ and single-capsid particles 1.38 g / cm ³ . The sedimentation coefficient of full particles is 500-530 S, and of homogeneous particles 380-400 S.

Antigenic properties

The antigen of rotavirus KPC strain No. 101 ARRIAH induces the formation of virus-specific antibodies in the body of immunized guinea pigs and rabbits, detected in the diffusion precipitation reaction (RDP) in dilutions from 1:16 to 1:64. In ELISA, serum reacted positively in dilutions from 1: 128 to 1: 2048, in the complement binding reaction (CSC) from 1:40 to 1: 160.

In blood serum of deep-skinned cows, 10 days after the second vaccination, antibodies to rotavirus in ELISA were detected in titers from 8.2 to 10.8 log ₂ , in colostrum from vaccinated cows, antibody titers to rotavirus in ELISA were 8.8-14.0 log ₂ . Feeding such colostrum provides a high level of colostral antibodies (7.0-8.5 log ₂ ) in the body of calves.

Strain No. 101 ARRIAH of rotavirus KPC has a wide antigenic spectrum and dominance. The average rate of bilateral antigenic affinity (R%) of the used strain in the neutralization reaction (PH) with 12-reference and epizootic strains of rotavirus KPC was 56.3%.

The research results presented in table 1 indicate that strain No. 101 ARRIAH of rotavirus KPC dominates the studied pathogens of rotavirus infection of KPC.

Biotechnological characteristics

Strain No. 101 ARRIAH of rotavirus KPC adapted to cell cultures MDBK, SPEV, KCT. Vero and Marc-145 and reproduced in them for 18-48 hours at 37 ° C with a degree of accumulation of 10 ⁶ to 10 ⁸ viral particles / cm ³ . The results of studies on the adaptation of strain No. 101 ARRIAH to these cell cultures are presented in table 2.

Strain No. 101 ARRIAH retains the original biological properties when passaged in these cultures for 20 passages (observation period).

Resistance to external factors

Strain No. 101 ARRIAH of rotavirus KPC is resistant to chloroform, freon, ether, acidic (pH 4.0) and alkaline (pH 8.0) environments. The causative agent is resistant to trypsin.

The inactivated antigen of rotavirus KPC strain No. 101 ARRIAH is stable during storage at 4 ° C both in the native state and as part of inactivated vaccine preparations.

Additional features and properties

Antigenic activity - has antigenicity in the composition of inactivated vaccine preparations, a drug for hyperimmunization of donors of diagnostic sera and antibody therapeutic drugs.

Reactogenicity - does not possess reactogenic properties.

Stability - retains the original biological (antigenic) properties when passaged in transplanted cell cultures for 20 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

Strain No. 101 ARRIAH of rotavirus KPC is free from contamination by bacteria, mycoplasmas and other viruses.

To obtain antigenic material from strain No. 101 ARRIAH of rotavirus KPC, mainly MDBK and SPEV cell cultures are used as a sensitive biological system.

If necessary, Marc-145, Vero and KCT cell cultures can be used to obtain antigenic material from strain No. 101 ARRIAH of rotavirus KPC.

The resulting virus-containing material is freed from cellular detritus and other ballast impurities by low-speed centrifugation and the viral raw material is monitored. The production series of rotavirus KPC should have a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA not less than 5.0 log ₂ .

For inactivation of rotavirus, AEEI is used, which is introduced into the purified virus-containing suspension to a concentration of 0.15%. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours. At the end of inactivation, AEEI is neutralized by introducing a 1M sodium thiosulfate solution into the antigen suspension at the rate of 10% of the volume of used AEEI.

For the manufacture of the emulsion form of the proposed vaccine, antigenic material is used from strain No. 101 ARRIAH of rotavirus KPC obtained in a sensitive biological system with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of at least 15.0 wt.%.

The viral isolate, which served as the source for the production strain “ARRIAH” of KPC coronavirus, was isolated from the feces of a newborn calf with impaired gastrointestinal function. The production strain “ARRIAH” of KPC coronavirus is adapted to cell cultures of MDBK, Taurus-1, KCT and Vero.

The strain “ARRIAH” of KPC coronavirus was deposited on March 12, 2001 into the All-Russian State Collection of Microorganism Strains Used in Veterinary and Animal Husbandry, of the Federal State Institution “All-Russian State Center for Quality and Standardization of Medicines for Animals and Feed” (FGU “VGNKI”) under the registration name "ARRIAH-DEPT".

The strain “ARRIAH” of KPC coronavirus is characterized by the following features and properties (23).

Morphological features

The strain “ARRIAH” of the KPC coronavirus belongs to the Coronaviridae family, the genus Coronavirus, and has morphological characteristics characteristic of the causative agent of coronavirus infection KPC: a spherical or pleomorphic virion with a diameter of 80-100 nm consists of a nucleocapsid of spiral symmetry and a lipoprotein sheath, the surface of which has buobular protrusions with a length of 18-24 nm, forming the likeness of the solar corona. The floating density of the virions in the sucrose gradient is 1.15-1.18 g / cm ³ .

Three structural proteins were found in the virions: peplomeric glycoprotein E2 (180-200 kD), nucleocapsid phosphoprotein N (50-60 kD) and matrix glycoprotein E1 (23-30 kD).

Antigenic properties

The antigen of KPC coronavirus strain "ARRIAH" induces the formation of virus-specific antibodies in the body of guinea pigs and rabbits, which are detected in the hemagglutination inhibition reaction (RTGA). In the blood serum of vaccinated rabbits, the antibody level ranges from 5.4 ± 0.25 to 7.25 ± 0.47 log ₂ (an average of 6.32 ± 0.39 log ₂ ), and in the blood serum of immunized guinea pigs - from 5 , 4 ± 0.38 to 8.2 ± 0.8 log ₂ (on average 6.1 ± 0.49 log ₂ ).

Antibodies in rtga in titers from 7.9 ± 0.35 to 8.9 ± 0.47 log ₂ are detected in blood serum of samples taken 10-15 days before calving, twice vaccinated cows.

Timely feeding of such colostrum provides a high level of colostral antibodies (up to 8.0 log ₂ ) in the body of newborn calves.

Biotechnological characteristics

Coronavirus KPC strain “ARRIAH” is reproduced in calf kidney cell cultures (MDBK, Taurus-1), calf coronary vessels (KCT) and in a heterologous monkey kidney cell culture (Vero). At 37 ° C during the 3-4 days of incubation, the virus accumulates in an amount of up to 10 ⁸ viral particles / cm ³ (electron microscopic data). The results of studies on the adaptation of coronavirus KPC strain "ARRIAH" to cell cultures of MDBK, Taurus-1, Vero and KCT are presented in table 3.

The strain "ARRIAH" of KPC coronavirus has the ability to reproduce in the MDBK cell culture with the same degree of accumulation at 34 ° C. The reproduction period at this temperature increases by almost a day, but allows you to get a double crop of the virus by changing the supporting environment. "Drain" has a high infectious, antigenic and immunogenic activity.

Resistance to external factors

The strain "ARRIAH" coronavirus KPC shows maximum resistance in the pH range of 6.0-7.2. Acidic (pH 3.0) and alkaline (pH 11.0) media have an inactivating effect on the virus. The causative agent is resistant to trypsin. The virus is destroyed by fat solvents: chloroform, methylene chloride, ether, tween and Triton X-100. The inactivated KPC coronavirus antigen of the ARRIAH strain is stable during storage at 4 ° C both in the native state and as part of inactivated vaccines.

Additional features and properties

Antigenic activity - has antigenicity in the composition of an inactivated vaccine and drug for hyperimmunization of donors of diagnostic and therapeutic sera.

Reactogenicity - does not possess reactogenic properties.

Stability - retains the original biological (antigenic) properties when passaged in MDBK cell culture for 30 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

The strain "ARRIAH" of KPC coronavirus is free from contamination by bacteria, mycoplasmas and other hemagglutinating viruses.

To obtain antigenic material from the ARRIAH strain of KPC coronavirus, a MDBK cell culture is mainly used as a sensitive biological system.

If necessary, Taurus-1, KCT and Vero cell cultures can be used to obtain antigenic material from the ARRIAH strain of KPC coronavirus.

The resulting virus-containing material is purified from ballast impurities by centrifugation or any other known method.

For inactivation of the virus, AEEI is used, which is introduced into the purified virus-containing suspension to a concentration of 0.1%. Inactivation of the virus is carried out at 37 ° C for 24 hours. At the end of inactivation, AEEI is neutralized by introducing a 1M sodium thiosulfate solution into the antigen suspension at the rate of 10% of the volume of used AEEI.

For the manufacture of the proposed vaccine, the avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus, obtained mainly in the MDBK cell culture with a degree of accumulation of at least 10 ⁸ virus particles / cm ³ and activity in the RGA of at least 7.0 log _{2, is} used as an active substance , in an amount of not less than 15.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain KPC coronavirus obtained in Taurus-1 cell culture with an accumulation degree of at least 10 ⁹ viral particles / cm ³ and activity in the RGA of at least 9, 0 log ₂ , in an amount of not less than 15.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus, obtained in KCT cell culture with an accumulation degree of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA of at least 6, 0 log ₂ , in an amount of not less than 15.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain KPC coronavirus obtained in a Vero cell culture with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6, 0 log ₂ , in an amount of not less than 15.0 wt.%.

Thus obtained antigenic material from the strain "ARRIAH" coronavirus KPC used for the manufacture of the emulsion form of the proposed vaccine.

To obtain the associated vaccine, antigenic materials are mixed in equal weight fractions and the mixture is thoroughly emulsified with an oil adjuvant in the homogenizer in the ratio, wt.%: 30.0: 70.0.

From oil adjuvants it is advisable to use Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard).

The content of antigens in the proposed vaccine in the above ratio is their effective amount in the drug, ensuring the achievement of a technical result.

The use of the present invention expands the arsenal of highly immunogenic, harmless and areactogenic associated vaccines against rota- and coronavirus infections of KPC emulsion inactivated, capable of protecting the livestock of KPCs of various age and gender groups from pathogens of rota- and coronavirus KPC infections circulating in Russia.

The essence of the invention is illustrated by examples of its implementation and use.

Example 1

To obtain the associated vaccine against rota- and coronavirus infections of KPC emulsion inactivated, production virus strains are used: strain No. 101 ARRIAH of rotavirus KPC and strain “ARRIAH” of coronavirus KPC.

Inoculum virus production strain No. 101 ARRIAH of rotavirus KPC, stored at minus 20 ° C, thawed and heated to 37 ° C. Trypsin was added to the suspension to a concentration of 15-20 μg / cm ³ and thermostated at 37 ° C for 30 minutes. Before infection, the cell culture of MDBK or SPEV with a high-quality monolayer is washed twice from growth serum with Hanks solution. Trypsin-treated virus is introduced into the culture vessels. The dose of infection should be 0.1-1.0 viral particles / cell. After infection, a supportive culture medium containing trypsin at a concentration of 10-15 μg / cm ^{3 is} introduced into the cell culture. Vessels are incubated at 37 ° C until the appearance of the virus CPP in more than 90% of monolayer cells (24-48 hours). The resulting virus-containing suspension is frozen-thawed. The cooled suspension is cleaned of ballast impurities by low-speed centrifugation (2500-3000 rpm) for 30 minutes. The resulting virus is subjected to control for infectious and antigenic activity. The production series of rotavirus should have an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and an activity in ELISA of at least 4.0 log ₂ .

Inactivation of the virus is carried out with a working solution of AEEI. In order to avoid changing the pH of the viral suspension, the AEEI solution was previously adjusted with a 5M solution of acetic acid to a pH of 7.8-8.2. The final concentration of inactivant in the viral suspension should be at least 0.15%. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours with periodic stirring of the suspension (3-5 minutes every 5-6 hours). At the end of inactivation, to neutralize the AEEI, a 1M sodium thiosulfate solution is added to the antigenic material suspension at the rate of 10% of the volume of used AEEI. Then the antigenic material is cooled to 2-8 ° C and samples are taken to check for residual infectious activity in the MDBK cell culture.

For the manufacture of the proposed vaccine using avirulent purified antigenic material from strain No. 101 ARRIAH rotavirus KPC, obtained mainly in a monolayer culture of MDBK cells or SPEV with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of not less than 15.0 wt.%.

Inoculum virus production strain "ARRIAH" of KPC coronavirus is stored before use at a temperature of -20 ° C. The seed virus should be free of bacterial and fungal microflora, specific, cause agglutination of mouse red blood cells, have an activity in the RGA of not less than 1: 8 and a concentration of viral particles (according to electron microscopy) ranging from 10 ^5.0 to 10 ^6.5 viral particles / cm ³ (21). The inoculum coronavirus KPC is frozen twice, thawed and used to infect a monolayer culture of MDBK cells. Before infection, the MDBK cell culture with a high-quality monolayer is washed twice from the growth serum with a Hanks solution containing trypsin (15-20 μg / cm ³ ). The multiplicity of infection is 0.1-1.0 viral particles / cell. After infection, the culture vessels are incubated at 37 ° C for 1.5-2 hours, periodically shaking. Then, the virus-containing material is drained and a support medium containing trypsin (15-20 μg / cm ³ ) is introduced into it. The vessels are incubated at 34 ° C or 37 ° C until the appearance of the virus CPP in more than 90% of the cells. After this, the culture is twice frozen-thawed. The resulting viral material is purified from ballast impurities by low-speed centrifugation at 2500-3000 rpm for 30 minutes. Inactivation of the virus is carried out using AEEI. For this, a 6% AEI working solution, acidified with glacial acetic acid to a pH of 7.8-8.2, is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be at least 0.1%. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours with periodic stirring of the suspension (3-5 minutes every 5-6 hours). The remainder of the AEEI is neutralized by adding a 1M sodium thiosulfate solution at the rate of 10% of the volume of the used AEEI. The resulting antigenic material is examined for sterility, avirulence, the content of viral particles in suspension using an electron microscope and activity in the RGA. For the manufacture of the proposed vaccine, the avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus, obtained mainly in the MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the RGA of at least 7.0 log ₂ , in the amount of not less than 15.0 wt.%.

If necessary, the avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in a KCT cell culture with a degree of accumulation of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ can be used to make the proposed vaccine. , in an amount of not less than 15.0 wt.%.

If necessary, the avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in a Vero cell culture with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ can be used to make the proposed vaccine. , in an amount of not less than 15.0 wt.%.

Thus obtained antigenic materials are used for the manufacture of the proposed associated vaccine. For this, antigenic materials are mixed in equal weight fractions and the mixture is thoroughly emulsified with an oil adjuvant in the homogenizer in the ratio, wt.%: 30.0: 70.0, respectively. From oil adjuvants it is advisable to use Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard).

The proposed vaccine has an optimal component composition, wt.%:

Avirulent purified antigenic material from strain No. 101 ARRIAH rotavirus KPC obtained in culture cells of animal origin, taken from the group consisting of a culture of MDBK cells, from a culture SPEV cells, from Marc-145 cell culture, from Vero cell culture and from KCT cell culture, with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA not less than 5.0 log ₂ 15.0 Avirulent purified antigenic material from strain "ARRIAH" KPC coronavirus obtained in culture cells of animal origin, taken from the group consisting of cell culture MDBK, from cell culture Taurus-1, from KCT cell culture and from cell culture Vero, in the concentration of viral particles, providing protective immune activity in the body of the animal when introduced to it target drug 15.0 Oil adjuvant up to 100.0.

The content of antigenic materials in the proposed vaccine within the above ranges is their effective amount in the preparation, ensuring the achievement of a technical result from the use of the invention.

The resulting vaccine is a homogeneous emulsion of white or white-pink color with a slightly viscous consistency. When storing the vaccine, slight exfoliation of mineral oil is allowed on the upper surface of the emulsion and the aqueous phase at the bottom of the vial with a thickness of not more than 1 cm.

The vaccine is monitored for avirulence and sterility, and then packaged in sterile vials.

Sterility of the vaccine is determined in accordance with GOST 28085-89. The virulence of the drug is determined by the method of three consecutive passages of the inactivated antigen in the transplanted cell culture of MDBK by the absence of CPD.

To test the safety, the vaccine is administered intramuscularly in the thigh area to 10 white mice at a dose of 0.1 cm ³ . The vaccine is considered harmless if white mice remain clinically healthy for 10 days: without any pathological changes. At the injection site, a local tissue reaction is allowed.

The viscosity of the vaccine is determined on a VPZh-2 viscometer and expressed in mm ² / s. The proposed vaccine containing in its composition an oil adjuvant of the brand Montanide ISA-70, forming a type of emulsion "water-oil", has a viscosity of 38.3 ± 0.5 mm ² / s

The vaccine is monitored for emulsion stability by centrifugation at 3000 rpm for 30 minutes, as well as during storage at 2-8 ° C and 37 ° C, followed by a visual assessment of the uniformity of the emulsion. The vaccine with the oil adjuvant of the brand Montanide ISA-70 has a high stability of the emulsion during storage under various temperature conditions and during centrifugation.

To test the reactogenicity of the drug, the proposed vaccine is administered intramuscularly in the thigh area to 3 rabbits and 5 guinea pigs at a dose of 2.0 cm ³ and 1.0 cm ³ respectively. The clinical condition of the animals is monitored for 14 days.

After immunization in rabbits and guinea pigs, a slight local increase in body temperature and the formation of swelling with a diameter of about 1 cm at the injection site were noted. These signs completely disappear by the end of the observation period.

Thus, the proposed vaccine belongs to the type of weakly reactogenic drugs.

The antigenic activity of the proposed vaccine is tested in rabbits. For this, 6 rabbits weighing 2.0-2.5 kg are taken. The vaccine is administered to 4 rabbits subcutaneously in the back region of 1.0 cm ³ . 14 days after the double administration of the vaccine with an interval of 20-25 days in rabbits, 2.0-3.0 cm ^{3 of} blood is taken from the ear vein. The blood is kept at a temperature of 37 ° C, serum is separated and examined for the presence of antibodies to rotavirus KPC in RDP and ELISA, and to coronavirus KPC in rtga. Unvaccinated animals (2 heads) are bled simultaneously with vaccinated animals, serum is separated and used for control.

The vaccine series is considered to have passed the antigenicity control if the titer of virus-specific antibodies to rotavirus in RDP is not lower than 4.0 log ₂ or in ELISA not lower than 7.0 log ₂ , and for KPC coronavirus in rtga not lower than 5.0 log ₂ .

The associated vaccine against rota- and coronavirus infections KPC inactivated emulsion is intended for immunization of pregnant cows and calves in farms that are disadvantaged for these diseases. Specific prophylaxis begins with immunization of the entire KPC population. The vaccine is administered to calves and adult KPC intramuscularly in the middle third of the neck in volume

2.0 cm ³ twice with an interval of 21-30 days, followed by revaccination after 6 months.

To maintain well-being for these diseases, the following vaccination schedule should be followed:

1. Heifers and cows are vaccinated twice with an interval of 21-30 days. Revaccination after 6 months.

2. Deep-skinned cows are vaccinated twice with an interval of 21-30 days for 60-65 days and 40-45 days before calving. Revaccination after 6 months.

Immunity in vaccinated animals is formed 10-15 days after the second injection of the vaccine and lasts 6 months.

Example 2

Tests of the antigenic activity of the associated vaccine against rota- and coronavirus infections KPC inactivated emulsion, made as described in example 1, and containing, wt.%:

Avirulent purified antigenic material from strain No. 101 ARRIAH rotavirus KPC obtained in culture SPEV cells with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in ELISA 5.0 log ₂ 15.0 Avirulent purified antigenic material from strain "ARRIAH" KPC coronavirus obtained in culture MDBK cells with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in the RGA 7.0 log ₂ 15.0 Oil adjuvant up to 100.0.

The antigenic activity of the obtained vaccine was determined according to the following scheme:

- determination of the level of antibodies to rota- and coronavirus infections in the blood serum of immunized rabbits;

- determination of the level of antibodies to rotavirus and KPC coronavirus in the blood serum of vaccinated deep-body cows and in the blood serum of newborn calves obtained from vaccinated cows and in colostrum.

The results of studies of blood serum of rabbits vaccinated with the associated vaccine against rota- and coronavirus infections KPC emulsion inactivated are presented in table 4.

According to the data given in table 4, the test vaccine induced the formation of virus-specific antibodies to rotavirus in the body of vaccinated rabbits in RDP in titers of 5.87 ± 0.55 log ₂ and in ELISA in titers of 8.90 ± 0.17 log ₂ , and coronavirus in rtga in titers of 6.79 ± 0.30 log ₂ .

To study the effect of the vaccinated dose of the vaccine on the immune response, 2 groups of rabbits were formed, 9 animals each. Rabbits of the first group were injected with samples of the test vaccine at a dose of 0.5 cm ³ and rabbits of the second group at a dose of 1.0 cm ³ . Before vaccination, 21 days and 35 days after vaccination, blood samples were taken from the animals, the obtained sera were examined in ELISA and RTGA to determine titers of specific antibodies to rotavirus and KPC coronavirus, respectively, which were expressed as log ₂ . The research results are presented in table 5.

The data obtained indicate that the test vaccine at a dose of 0.5 cm ³ and 1.0 cm ³ on the 35th day after vaccination induces the formation of antibodies to rotavirus in titers of 8.88 ± 0.27 log ₂ and 10.08 ± 0, 35 log ₂ , and for coronavirus in the titers of 6.30 ± 0.27 log ₂ and 7.50 ± 0.45 log _2, respectively, which also confirms the existence of the dose-effect relationship.

The proposed vaccine was tested on deep-skinned cows. The results of these studies are presented in table 6.

The data presented in table 6 indicate that the test vaccine induces the formation of antibodies to rotavirus and coronavirus in high titers in the body of deep-shelled cows.

Example 3

Tests of the effectiveness of the associated vaccine against rota- and coronavirus infections KPC inactivated emulsion, made as described in example 1, and containing, wt.%:

Avirulent purified antigenic material from strain No. 101 ARRIAH rotavirus KPC obtained in culture SPEV cells with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in ELISA 5.0 log ₂ 15.0 Avirulent purified antigenic material from strain "ARRIAH" KPC coronavirus obtained in culture MDBK cells with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in the RGA 7.0 log ₂ 15.0 Oil adjuvant up to 100.0.

In 11 livestock farms, the prophylaxis of viral diarrhea caused by rotavirus and KPC coronavirus was tested. Immunization of deep-skinned cows was carried out with the proposed vaccine 40-45 and 20-25 days before calving. The vaccination results are presented in table 7.

The data in table 7 indicate that in 78% of newborn calves obtained from unvaccinated (control) cows, signs of diarrhea were recorded. Within 20 days, 25.1% of the calves died in the control group. In 11% of newborn calves obtained from vaccinated cows, digestive disorders were noted, of which 3.9% of calves fell.

Information sources

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2. Miskevich S.V. Development of a complement binding reaction for the diagnosis of calf rotavirus diarrhea: Author. dis. for the competition degrees of cand. Vet.science. - Kiev, 1985 .-- 22 p.

3. Ogbu Ogbonaya, Onufriev VP, Skibitsky VG et al. Experiments of inactivation of cattle rotavirus // Tez. doc. Republic scientific production. conf. - Kharkov, 1990 .-- p.262.

4. Onufriev V.P., Skibitsky V.G., Miskevich S.V. et al. The construction of multivalent combined specific biological products for the prevention of gastrointestinal diseases of newborn calves // Prevention and control of diseases of young farm animals: Abstracts. doc. Rep. scientific production conf. - Minsk, 1990 .-- p.14.

5. Skibitsky V.G. Cattle rotavirus infection (rotavirus enteritis). - Kiev. Ukr. IHTEI, 1999 .-- 208 p.

6. Gaffarov Kh.Z., Ivanov A.V., Nepoklonov E.A., Ravilov A.Z. Mono- and mixed diarrhea of newborn calves. - Kazan: FEN, 2002 .-- 590 p.

7. Miskevich S.V. and Skibitsky V.G. Prophylactic efficacy of an associated vaccine against mixed rota- and coronavirus infection in cattle // Vet. honey. Ukraine. - 2000. - No. 10. - p. 18.

8. Infectious diseases of animals: a Handbook. Ed. D.F. Ozidze. - M .: Agropromizdat, 1987, 288 p.

9. Syurin V.N., Samuilenko A.Ya., Soloviev B.V., Fomina N.V. Viral diseases of animals. - M., VNIITIBP, 1998, 928 p.

10. Infectious pathology of animals: In 2 t. / Ed. A.Ya. Samuilenko, B.V. Soloviev, E.A. Nepoklonova, E.S. Voronin. - M.: "Academic Book", 2006. - T.1. - 911 p.

11. Pat. RF №2302258; A61K 39/15, A61P 31/12; 07/10/2007

12. Pat. RF №2301079; A61K 39/215, A61P 31/12; 06/20/2007

13. Pat. Czechoslovakia No. 238179; A61K 39/295, 05/01/1987 (prototype)

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Table 1
The results of the study of antigenic affinity (R%) of rotavirus strains in the neutralization reaction №№ The name of the strains of rotavirus The average (R%) antigenic affinity of the studied strains one Tiverval 31.6 2 Lincoln 44.5 3 SA-11 12.5 four No. 101 56.3 5 3 21.3 6 LA 31.6 7 AT 26.7 8 FROM 22.7 9 IN 28.8 10 TO 26.0 eleven P 30.7 12 D 32.8 13 B 26.9

table 2
Comparison of the productivity of various cell cultures when infected with strain No. 101 ARRIAH of rotavirus KPC Cell culture Reproduction time (hours) The concentration of viral particles (particles / cm ³ ) MDBK 18-20 10 ^7.5-8.0 SPEV 18-20 10 ^8.0 Marc-145 24 10 ^6.5 Vero 48 10 ^6.0 Kct 48 10 ^6.0

Table 3
Sensitivity of transplanted cell cultures to KPC coronavirus strain ARRIAH Cell culture The accumulation of viral particles in cm ³ (electron microscopy) The titer in the hemagglutination reaction (RGA) in log ₂ MDBK 10 ^7-9 7.0-10.0 Taurus-1 10 ⁹ 9.0-10.0 Vero 10 ^7.5 6.0-7.0 Kct 10 ^7.5 7.0

Table 4
Antigenic activity of the associated vaccine against rota- and coronavirus infections KPC emulsion inactivated n = 3 №№ Average antibody titers (log ₂ ) to rotavirus in to coronavirus in rtga RDP IFA one 6.02 ± 0.51 8.22 ± 0.15 6.40 ± 0.28 2 5.80 ± 0.76 8.74 ± 0.12 7.40 ± 0.36 3 4.87 ± 0.72 7.56 ± 0.20 6.60 ± 0.21 four 5.90 ± 0.51 8.62 ± 0.11 7.20 ± 0.31 5 6.03 ± 0.74 8.20 ± 0.20 6.28 ± 0.34 6 5.80 ± 0.76 9.12 ± 0.20 7.30 ± 0.33 7 6.20 ± 0.20 10.06 ± 0.12 7.00 ± 0.48 8 5.42 ± 0.41 9.10 ± 0.35 6.60 ± 0.19 9 6.30 ± 0.70 10.10 ± 0.01 6.70 ± 0.26 10 6.35 ± 0.20 9.35 ± 0.24 6.50 ± 0.27 Average value 5.87 ± 0.55 8.90 ± 0.17 6.79 ± 0.30

Table 5
Dependence of the level of the immune response of rabbits on the size of the vaccine dose of the associated vaccine against rota- and coronavirus infections KPC emulsion inactivated Grafted dose, cm ³ Number of rabbits The average level of antibodies after vaccination, log ₂ to rotavirus, ELISA to coronavirus, rtga 21 day 35 day 21 day 35 days 0.5 9 6.48 ± 0.24 8.88 ± 0.27 3.90 ± 0.22 6.30 ± 0.27 1,0 9 7.68 ± 0.29 10.08 ± 0.35 5.10 ± 0.33 7.50 ± 0.45

Table 6
The dynamics of the formation of antibodies to rotavirus and KPC coronavirus in deep-skinned cows immunized with the associated vaccine against rota- and coronavirus infections KPC emulsion inactivated №№ Serum Characterization The average level of antibodies to rotavirus KPC in ELISA, log ₂ The average level of antibodies to coronavirus KPC in rtga, log ₂ one Serum of cows prior to vaccination 5.71 ± 0.76 4.80 ± 0.74 2 Serum of cows 10-15 days before calving 8.40 ± 0.78 8.36 ± 0.66 3 Colostrum - the first day after calving 12.10 ± 0.68 9.80 ± 0.66 four Blood serum of 3-4 day old calves obtained from vaccinated cows 7.86 ± 0.42 7.70 ± 0.53

Table 7
Test results of the associated vaccine against rota- and coronavirus infections KPC emulsion inactivated in deep-shelled cows No. p / p Unvaccinated cows (goal) Sick calves (%) Calves fell (%) Vaccinated Cows (Goals) Sick calves (%) Calves fell (%) Protected calves (%) one twenty 70 25.0 52 13 3.8 96.2 2 22 77 31.8 36 eighteen 2.7 97.3 3 56 88 23,2 393 eleven 2.5 97.5 four 24 79 25.0 28 fourteen 3,5 96.5 5 57 72 22.8 124 fifteen 3.2 96.8 6 62 81 27.4 268 13 6.3 93.7 7 102 79 29.4 143 8 2,8 97.2 8 66 67 24.2 98 13 2.0 98.0 9 45 89 28.9 173 9 5.2 94.8 10 56 88 21,4 215 8 6.1 93.9 eleven 34 68 17.6 156 10 5.1 94.9 Total 544 78 25.1 1686 eleven 3.9 97.1

Claims (14)

1. Associated vaccine against rotavirus and coronavirus infections of cattle (KPC) inactivated emulsion containing the active substance and the target additive, characterized in that it contains a mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH of rotavirus KPC, family. Reoviridae, genus Rotavirus, collection of FGU VGNKI No. 101 ARRIAH-DEPT, and from the strain ARRIAH of the coronavirus KPC, fam. Coronaviridae, a genus of Coronavirus, a collection of FSI “VGNKI” “ARRIAH-DEPT” obtained in cell cultures of animal origin in amounts that provide protective immune activity in the animal’s body when the target drug is administered to it. 2. The associated vaccine according to claim 1, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in animal cell culture, taken from the group consisting of MDBK calf kidney cell culture, from pig cell culture SPEV kidneys, from a monkey monkey kidney cell culture Marc-145, from a Vero monkey kidney cell culture and from a CTF calf coronary cell culture, in an effective amount. 3. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in an MDBK cell culture with an accumulation degree of at least 10 ⁶ virus particles / cm ³ and activity in an enzyme-linked immunosorbent assay ( ELISA) not less than 5.0 log ₂ in an amount of not less than 15.0 wt.%. 4. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in a culture of SPEV cells with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of not less than 15.0 wt.%. 5. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in a Marc-145 cell culture with an accumulation degree of at least 10 ⁶ viral particles / cm ³ and ELISA activity not less than 5.0 log ₂ , in an amount of not less than 15.0 wt.%. 6. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in Vero cell culture with an accumulation degree of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of not less than 15.0 wt.%. 7. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from strain No. 101 ARRIAH of rotavirus KPC obtained in a KTC cell culture with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA of at least 5.0 log ₂ , in an amount of not less than 15.0 wt.%. 8. The associated vaccine according to claim 1, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in animal cell culture, taken from the group consisting of MDBK cell culture, Taurus calf kidney cell culture -1, from a culture of CCT cells and from a culture of Vero cells, in an effective amount. 9. The associated vaccine of claim 8, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in an MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the hemagglutination reaction ( RGA) not less than 7.0 log ₂ , in an amount of not less than 15.0 wt.%. 10. The associated vaccine according to claim 8, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in Taurus-1 cell culture with an accumulation degree of at least 10 ⁹ viral particles / cm ³ and activity in the RGA not less than 9.0 log ₂ , in an amount of not less than 15.0 wt.%. 11. The associated vaccine of claim 8, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in KCT cell culture with an accumulation degree of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA not less than 6.0 log ₂ , in an amount of not less than 15.0 wt.%. 12. The associated vaccine of claim 8, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of KPC coronavirus obtained in Vero cell culture with an accumulation degree of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA not less than 6.0 log ₂ , in an amount of not less than 15.0 wt.%. 13. The associated vaccine according to claim 1, characterized in that as the target additive it contains an oil adjuvant in an amount of not less than 70.0 wt.%. 14. The associated vaccine according to any one of claims 1 to 13, characterized in that it contains a mixture of avirulent purified antigenic materials from strain No. 101 ARRIAH of rotavirus KPC and strain "ARRIAH" of coronavirus KPC obtained in animal cell cultures and an oil adjuvant in the ratio, wt.%:
Avirulent purified antigenic material from strain No. 101 ARRIAH of the rotavirus KPC virus obtained in cell culture of animal origin taken from the group consisting of MDBK cell culture, from SPEV cell culture, from Marc-145 cell culture, from Vero cell culture and from KTC cell culture, with a degree of accumulation of at least 10 ⁶ viral particles / cm ³ and activity in ELISA not less than 5.0 log ₂ 15.0 Avirulent purified antigenic material from strain "ARRIAH" KPC coronavirus obtained in culture cells of animal origin, taken from the group consisting of cell culture MDBK, from cell culture Taurus-1, from KCT cell culture and from cell culture Vero, in the concentration of viral particles, providing protective immune activity in the animal’s body when introduced to it target drug 15.0 Oil adjuvant Up to 100.0