Bivalent inactivated vaccine against the disease teschen

 

(57) Abstract:

The invention relates to the field of veterinary Virology. The vaccine consists of two circulating in Russia and the near abroad antigenically different strains - "Transcarpathian" and "Navlya-96". In addition, the vaccine contains tevtropin at a final concentration of 0.06 to 0.08%, which effectively inactivates the virus without affecting its immunogenic properties. To reduce the viscosity of the vaccine in its composition introduced vaccine emulsifier 139 at a final concentration of 0.5-4%. The vaccine is easy to use, stable in storage, allows you to achieve 100% protection of vaccinated pigs throughout the growing cycle.

The invention relates to the field of veterinary Virology, in particular to the means of prevention of disease Teschen.

To prevent the disease Teschen used live and inactivated vaccines. A live vaccine prepared from a culture of strain Perechyn-346" non-pathogenicity for pigs and inducing antibodies to enteroviruses pigs (Romanenko centuries, Forty Century. And., the locust O., Immunogenicity VirusWall against disease Teschen. Veterinary medicine, 1991, 7, 27-28). However, in modern epizootic conditions it is not effective because of immunologist from the tissues of the Central nervous system of infected pigs (A. Klobouk Encephalomyelitis enzootica suum. Active Immunisation. (Czech. ) Zverolek. Rozpavy, 1935, 9; 73, 85, 217), or using viral culture suspension with the addition of aluminium hydroxide or oil adjuvant (Dudnikov L. A. Sergeev, O. C., Alper T. I., Nepoklonov E. A., Sergeev, C. A. Antigenic activity of inactivated preparation of Teschen disease in pigs depending on the adjuvant. Viral disease C. farm animals. Vladimir, 1995. - 152; Bourden P., H. Serres Vaccination centre la maladie de Teschen avec le virus de culture. Anticorps et immunite. - Ann. Inst. Pasteur, 1959, 97, 4, 583-589). The immunogenicity of these vaccines in modern conditions is insufficient, therefore, immunizations based on them are not quite effective, especially in some regions of the former USSR.

Known vaccine against the disease Teschen inactivated culture emulsified (Rybalkin P. C., Gestures Century. And. Ways to increase the stock of immunogenicity, reduced reactogenicity and viscosity of emulsified vaccine against the disease Teschen. Proc. Dokl. Vsesojuzn. scientific conference "Development, testing and state control of veterinary drugs", M - 1981.-71). When production use of cultural strain "Transcarpathian" of the pathogen grown in a single layer culture of primary trypsinization cells is enzio source activity is not below 107,0TCD50/ml inactivate formaldehyde in the 28oC for 8 days and then mixed in equal volumes (1:1) with oil adjuvant (80% oil +20% emulsifier). The final product is an emulsion type water in oil with a kinematic viscosity of 250-400 JV that depends on mineral oil (vaccine, liquid paraffin, perfume) and fat emulsifier (lanolin).

However, the known vaccine formulation, which can serve as a prototype of the proposed invention, has a number of disadvantages.

Strain "Zakarpattya" is selected in the Transcarpathian oblast of the Ukrainian SSR during the epizootic disease Teschen 1969-1972, Russia Teschen disease was first registered in 1994, According to our data, allocated on the territory of the Bryansk region the strain Navlya-96" disease Teschen different antigenic and immunogenic properties of strain "Zakarpattya". Specific serum neutralization reaction with homologous strains were 8-128 times more than with heterologous (Bosun A. I., Gestures Century. And. About the role nutritioni differences pathogen vaccination Teschen disease of pigs. Proceedings of the International conference on the 40th anniversary of the all-Russian scientific research Institute of vet. Kolosov, V. I. Gestures, Y. F., Celantano. Antigenic differences in industrial strains of the virus Teschen disease. Collection of articles of the international scientific-practical conference dedicated to the 75th anniversary of I. A. Bakalova, , Cover, 2000, 235). Immunological vaccine from strain "Transcarpathian" effective in the Bryansk region only when it is used in higher doses (2 to 3-fold) or at 2 - to 3-fold application of (every 1-2 months).

As part of a prototype drug as inactivant used formalin, requiring exposure to the virus not less than 8 days. In addition, the use of formalin requires compulsory neutralization of residual amounts of formaldehyde as immunodepressant the hyposulphite of sodium to stop his actions. So in the recipe prototype vaccine as balistae substance is present and this component.

Due to the use in the prototype lanolin as an emulsifier target product has a high kinematic viscosity: 400 CPs. This fact significantly complicates the packaging and especially the use of the drug, because in winter conditions it is necessary to warm up (the Vaccine against the disease Teschen inactivated culture the vaccine preparation against disease Teschen, providing immunological protection of vaccinated animals against both serological subtypes of the pathogen that currently circulate in notariale. An additional objective is the improvement of the technological parameters of the target product (the reduction of the duration of the technological process of production and decrease of kinematic viscosity).

This goal is achieved by the fact that the vaccine composition is administered two antigenically distinct strain of the virus Teschen disease - "Transcarpathian" and "Navlya-96" that circulate in contemporary notarile, inactivator tevtropin (TU 9336-02-0000864-98), and emulsifier for vaccines 139 (TU 6-02-011-1213942-99).

The strain Navlya-96" selected in culture transplantable kidney pig (SPAW) of the brain gilt, showing signs of neuroinfections and owned OPH one of the institutions on the territory of the Bryansk region. The virus causes JRS characteristic of enteroviruses pigs of the 1st type, in cultures of kidney cells pigs (primary and transplantable) and Siberian mountain goat (PSGC), transplantable cells of the testes pig (POPS) accumulate in the credits 3,0-8,0 lg TCD50/mlunder cultivation in the stationary and roller cultivation is but is passed from them to the pigs causing them to typical clinical form of the disease Teschen.

The essential feature of the invention is used as a source of viral components raw materials two strains of the virus Teschen disease - "Navlya-96" and "Zakarpattya". Raw materials produced in permissive culture systems, inactivated and mixed in ratios of 0.5:1, 1:1 or 2:1 before linking vaccines. The use of other isolates and strains not improve the quality of the product, because the claimed components of the vaccine cover their antigenic spectrum sufficient for immunization disease Teschen degree. The use of these strains in monovaccines against Teschen disease requires further investigation on the typing of the pathogen circulating in the hearth, therefore less efficient in economic terms.

The second distinguishing feature of the invention is the presence in its composition inactivant of atropina, which is chemically very similar to the urotropine and unlike formalin and other balastik components of a prototype vaccine is not an immunosuppressant and is rapidly excreted from the body of the pig. On the other hand, tevtropin unlike formalin slices viral the NTA of the claimed vaccine. Finally, the application of atropina allows to reduce time of inactivation of raw materials in the production of vaccines from 8 to 2 days and get rid of the introduction to the recipe substances to neutralize free inactivator, as the latter is used at a final concentration of (0,06-0,08)% and almost completely associated with vaccinated raw materials. A smaller number of atropina not inactivate the virus, and more does not improve the quality of the drug.

The third significant distinguishing feature of the invention is used in the composition of the adjuvant emulsifier 139 domestic production (TU 6-02-011-1213942-99). The amount of emulsifier in Freund is 2.0-7.5 percent. Adjuvant mixed with inaktivirovannye vaccinated raw materials in a ratio of from 3:1 to 1:1. Kinematic viscosity derived vaccine at a ratio of 3: 1 is 6-15 JV at 2:1-18-22 JV at 1:1-56-96 SP. Reducing the amount of emulsifier (<2%) in Freund leads to instability of the resulting emulsion, and increased (>7.5%) to increase the viscosity of the vaccine (108-250 SP). The ratio of the adjuvant and antigenaemia raw is best, because the use of adjuvant in higher or lower concentrations in the final product the spine of the vaccine, or makes it impossible to obtain emulsions of the type water-in-oil". Protected parameters, on the one hand, improve the technological capabilities of production of vaccines using automatic systems of the spill, on the other hand, consumer requirements, as the vaccine is easily injected animals without pre-heating, even in winter conditions.

Thus, the positive effect is achieved in the following ratio, wt.%:

Suspension of inactivated virus "Transcarpathian" - 12,5 - 35,0

Suspension of inactivated virus Navlya-96" - 12,5 - 35,0

Tevtropin - 0,06 - 0,08

Oil synthetic vaccine (type Marcol 52 or MC) - 33,3 - 50,0

Emulsifier 139 (TU 6-02-011-1213942-99) - 0,5 - 4,0

Example 1

2-day cell culture PTP 60 Klin mattresses infect virus Teschen disease strain "Navlya-96" with multiplicity 0,1 TCD50/cell and incubated at 37oC for 72-96 h before the onset of full cytopathic effect of the virus. Then the contents of the mattresses frozen at minus 20oC. After thawing the contents of mattresses poured into a 10 - liter carboys, determine the infectivity titration in cell culture, and then to 10 l virustargetnet it by thorough mixing and incubated at 37oWith 48 hours

Similarly prepare an inactivated virus Teschen disease strain "Zakarpattya".

Mix in a sterile reactor of 10 l inactivated by infectivity vaccinated suspension strains "Navlya-96" and "Transcarpathian" add sterilized by autoclaving for 1 h at 1.5 ATM adjuvant consisting of 38 l oil MC and 2 l of emulsifier 139. Stirred for 24 h at 200 rpm and is drained from the reactor and Packed 1-10 doses.

Serological examination of blood serum samples of pigs immunized once, shows the presence of neutralizing antibodies in titers of 1: 32-1: 256 2 months after vaccination, whereas titers preimmune of blood serum samples do not exceed 1:4.

Example 2

2-day culture of cells in 90 Klin mattresses infect virus Teschen disease strain "Navlya-96" with multiplicity 0,1 TCD50/cell and incubated at 37oC for 72-96 h before the onset of full cytopathic effect of the virus. Then the contents of the mattresses frozen at minus 20oC. After thawing the contents of mattresses poured into a 10-liter carboys, determine the infectivity titration in cell culture, then in vaccinated culture with theWith 48 hours

Similarly prepare an inactivated virus Teschen disease strain "Zakarpattya", using 45 Klin mattresses.

Mix in a sterile reactor 15 liters inactivated by infectivity vaccinated suspension of strain Navlya-96" and 7.5 l - strain "Transcarpathian" add sterile adjuvant consisting of 22 l oil Marcoll 52 and 0.5 l of emulsifier 139. Stirred for 24 h at 200 rpm and is drained from the reactor and Packed 1-10 doses.

The resulting vaccine is an emulsion of white (sometimes pinkish) type water in oil with viscosity 6-96 JV, which does not separate by centrifugation at 3000 x g for 15-20 min, and if kept in the course of the year at 6-10oC. Immunogenicity of the drug and tension immunity meet the requirements set out in existing guidance to vaccine against the disease Teschen.

The resulting laboratory and clinical trial data indicate that the vaccine of the proposed structure allows for the use of 1 time per year to reach 100% protection of vaccinated pigs throughout the growing cycle. The efficiency of the proposed vaccine drug podtverzdeniye on the infectivity of vaccinated suspension, mixed with oil adjuvant, characterized in that it contains two antigenically distinct strain "Transcarpathian" and "Navlya-96", tevtropin and adjuvant consisting of a synthetic oil and emulsifier 139, in the following ratios, wt. %:

Suspension of inactivated virus "Transcarpathian" - 12,5 - 35,0

Suspension of inactivated virus Navlya-96" - 2,5 - 35,0

Tevtropin - 0,06 - 0,08

Oil synthetic vaccine (type Marcol 52 or MC) - 33,3 - 50,0

Emulsifier 139 (TU 6-02-011-1213942-99) - 0,5 - 4,0

 

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FIELD: medicine.

SUBSTANCE: the suggested strain refers to DNA-containing Adenoviridae type being of cubic type of symmetry, has no supercapsid membrane, virions sizes - 70-80 nm, shape - icosahedral. Moreover, it is resistant to the temperature of 56°C for 30 min, not less, and to the action of both ether and chloroform. It is inactivated with formalin, theotropin, dimer ethylenimine. The strain is cultivated in hepatic cell culture of hen's embryos, about 72-96 h after infection it causes characteristic foci of granulation (cells' rounding), the formation of separate rounded cells and foci of cell degeneration. Viral titer is within 105.5 TCD50/ml. The strain enables to reproduce different forms of the disease mentioned for experimental investigations, it is highly useful for manufacturing certain diagnostic kits and vaccines, moreover, being of high antigenic and immunogenic activity.

EFFECT: higher efficiency.

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