RU2366457C1 - Antirabic vaccine for animals ("унирэв") - Google Patents

Abstract

FIELD: medicine; veterinary science.

SUBSTANCE: vaccine contains virus-containing culture fluid that includes inactivated rabies virus of strain Shchelkovo-51, reproduced in passaged cell culture "ВНК"-21 and aluminium hydroxide (AHO). In addition, the vaccine contains ethanol in the component ratio as follows (wt %): virus-containing culture fluid containing inactivated rabies virus of strain Shchelkovo-51, passaged cell culture "ВНК"-21 - 70-80, ethanol - 9-11, aluminium hydroxide - the rest. Ethanol in specified concentration reliably protects the liquid vaccine from bacterial growth. Besides, ethanol improves antibody-inducing activity of antirabic vaccines for desired animal (dogs, cats).

EFFECT: higher immunity against rabies.

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Description

The invention relates to the field of biotechnology and can be used in the industrial production of highly immunogenic, harmless, easy-to-use liquid vaccines against rabies in animals.

The rabies vaccine for animals (UNIREV), comprising a virus-containing culture fluid containing the inactivated rabies virus strain Schelkovo-51, reproduced in the culture of transplantable cells BHK-21 and aluminum hydroxide (GOA), characterized in that it additionally contains ethanol in the following ratio of components (wt.%):

Virus-containing culture fluid containing inactivated rabies virus strain Schelkovo-51, reproduced in the culture of transplantable cells BHK-21 70-80 Ethanol 9-11 Aluminum hydroxide rest

Ethanol as a part of a liquid rabies vaccine plays the role of a harmless preservative, which almost completely eliminates the possibility of activation and multiplication of microflora in bottles with finished products (Table 1), where it can accidentally get into (for example, by packaging) and not always be detected during production bacteriological and biological controls. At the same time, there is no need to introduce antibiotics, mercury derivatives, and other preservatives that are not individually and environmentally friendly to the liquid rabies vaccines.

In addition, ethanol increases the antibody-inducing activity of the liquid rabies vaccine for target animals (in particular dogs and cats) and, therefore, their degree of protection against rabies virus (Table 2). Perhaps this is due to the effect of ethanol on nerve receptors at the injection site and the influx of more antigen-presenting cells to them in response to the corresponding signal from a more excited and informed central nervous system.

The invention relates to the field of veterinary biotechnology and can be used in the industrial production of highly immunogenic, harmless, easy-to-use liquid rabies vaccines for animals.

Known dry brain vaccine against rabies in animals, including a virus-containing suspension of brain tissue of sheep infected with rabies virus strain "Sheep", characterized in that the inactivation of the virus is carried out with 20-21% ethanol solution at 30-31 ° C for 12-14 days. Then a protective drying medium is added and the resulting mixture is freeze dried (SU 770196 A, A61K 39/205, 1983). Naturally, when dried along with moisture, ethanol is completely removed from the preparation. Thus, ethanol is present in the composition of the vaccine only at the stage of its manufacture.

A disadvantage of the known vaccine is the presence in its composition of dangerous encephalitogenic brain tissue of sheep, causing post-vaccination complications in vaccinated animals. In addition, due to the significant infection of sheep with the causative agent of scrapie, there is a high probability of it getting into the composition of the manufactured vaccine with brain tissue and, as a result, the spread of prion infection among the vaccinated livestock.

Also known is a dry culture vaccine for the prevention of rabies in cattle, containing the Schelkovo-51 strain of rabies virus inactivated by β-propiolactone, reproduced in the culture of transplantable cells of BHK-21, a protective drying environment and an immunity stimulant - saponin (RU 955577 A, AK61 39 39 / 205, (46) 08/15/1994). The vaccine has high immunogenic activity, but contains saponin, which is toxic to many animals (dogs, cats, horses, pigs, etc.). Therefore, it is used only for immunization of small and cattle.

The closest analogue is the currently widely used rabies inactivated liquid culture vaccine Rabikov manufactured according to the method RU 2191600 C1 AK61 39/205 (46) 10.27.2002), including virus-containing culture fluid containing inactivated rabies virus strain Schelkovo-51 ”, Reproduced in the culture of transplantable cells of BHK-21 and aluminum hydroxide (GOA) (Catalog of medicinal media for animals, Bioprom Trading House, M., 2004, p. 135). In contrast to saponin, GOA is non-toxic and the vaccine can be used for all types of animals. However, its immunostimulating activity does not in all cases manifest itself at a level that ensures the appearance in the blood of initially single-vaccinated animal antibodies with an activity of 0.5 IU / ml, recognized by WHO as sufficient to protect animals from rabies (Table 2).

A disadvantage of the analogue is the lack of an approved preservative in the vaccine, which can remove the threat of post-vaccination complications in case of accidental use of the drug with microflora activated during storage.

The objective of the invention is to improve the liquid vaccine against rabies in animals.

The technical result of the invention is to increase the safety and immunogenicity of a liquid rabies vaccine by introducing ethanol into its composition.

SUMMARY OF THE INVENTION UNIREV animal rabies vaccine, including virus-containing culture fluid containing inactivated rabies virus strain Schelkovo-51, reproduced in the culture of transplantable cells BHK-21 and aluminum hydroxide (GOA), characterized in that it additionally contains ethanol in the following ratio of components (wt.%):

Virus-containing culture fluid containing inactivated rabies virus strain Schelkovo-51, reproduced in the culture of transplantable cells BHK-21 70-80 Ethanol 9-11 Aluminum hydroxide rest

Ethanol in the indicated concentrations reliably protects the liquid vaccine from bacterial growth (Table 1). In addition, ethanol increases the antibody-inducing activity of the rabies vaccine for the target animals and, therefore, their protection against rabies disease (Table 2).

The vaccine (UNIREV), manufactured by the present method is universal, safer to use and has greater immunogenicity. Its use will more effectively protect animals from rabies.

The invention is illustrated by the following examples.

Example 1. The cultivation of rabies virus infected BHK-21 cells is carried out by roller or pseudosuspension methods at a temperature of 37 ° C. As a growth and support medium, Eagle and 199 nutrient media are used with the addition of native cattle serum. The collection of virus-containing culture fluid is carried out after 3, 4, 5 and 6 days of cultivation. The replacement of the nutrient medium is done after each collection of the virus (virus-containing fluid).

To 8 l of virus-containing culture fluid containing rabies virus strain Schelkovo-51, cooled to 4 ° C, is added with constant stirring 2.0 ml of β-propiolactone. Then, after 2 hours, 1.17 L of 96% rectified ethyl alcohol and 2 L of 6% aluminum hydroxide gel (GOA) are added to it. The result is the claimed inactivated ethanol vaccine (UNIREV) containing 71.63 wt.% Virus-containing culture fluid, 10.47 wt.% 96% ethanol (10% pure ethanol) and the remaining GOA (17.9 wt.% )

Example 2. The rabies virus, prepared as in example 1, is inactivated by UV light with a constant flow of virus-containing culture fluid over the surface of a lamp like BUV-30 from UV glass. The thickness of the layer of the irradiated material is not more than 0.5 ml, the irradiation time is 10 seconds.

To 10 l of the culture fluid containing UV light inactivated rabies virus strain Schelkovo-51, add 1.3 l of 96% ethyl alcohol rectified and 2.6 l of 6% GOA. The result is the claimed inactivated ethanol vaccine (UNIREV) containing 71.94 wt.% Virus-containing culture fluid, 9.35 wt.% 96% ethanol (9% pure ethanol) and the remaining GOA (18.7 wt.% )

Example 3. The rabies virus, prepared as in example 1, is inactivated with ethanol. To do this, to 10 l of the culture fluid is added with constant stirring 1.6 l of 96% ethyl alcohol rectified. A mixture is obtained containing 13.79% 96% ethanol. The mixture is incubated for 20 hours at 36-37 ° C to inactivate the infectiousness of rabies virus. Then, 2.35 L of 6% GOA is added to the inactivated virus (virus-containing culture fluid). The result is the inventive inactivated ethanol vaccine containing 71.68 wt.% Virus-containing culture fluid, 11.47% 96% ethanol (11% pure ethanol) and the remaining GOA (16.85 wt.%).

For the manufacture of the analogue, the same virus (previously selected from the same container) is used as for the manufacture of the claimed vaccine. To 10 l of the culture fluid containing the Schelkovo-51 rabies virus, reproduced in the culture of transplantable BHK-21 cells, cooled to 4 ° C, 2.5 ml of β-propiolactone are added with constant stirring, then after 2 hours 2.3 l GOA. The result is a well-known drug containing 18.7 wt.% GOA.

The prepared preparations do not contain live rabies virus and have almost the same immunogenic potency (2.53; 2.45; 2.50 and 2.47 IU / ml, respectively) when tested on mice by the WHO recommended NIH method (national health institutes) USA). However, for the target animals most dangerous for humans (dogs and cats), the proposed vaccine has a much greater antibody-inducing activity than the analogue (Table 2). Therefore, it will more reliably protect them and, as a consequence of this, a person from rabies.

During storage, the proposed vaccine is more protected from random contaminants compared with the analogue (table 1).

Table 1 The result of microbial growth in liquid rabies vaccines after their special and identical contamination Vaccine Storage Temperature 6-10 ° C 15-25 ° C 36-37 ° C The shelf life of the drug, months fourteen 6 2 Proposed vaccine with 9% ethanol The number of bottles in the experiment 500 300 200 No. of vials with microbial seedlings 0 one 3 with 10% ethanol The number of bottles in the experiment 300 300 200 No. of vials with microbial seedlings 0 0 0 with 11% ethanol The number of bottles in the experiment 400 400 200 No. of vials with microbial seedlings 0 0 0 Famous vaccine ethanol free The number of bottles in the experiment 500 200 200 No. of vials with microbial seedlings 32 172 200

Claims (1)

An anti-rabies vaccine for animals, including a virus-containing culture fluid containing an inactivated rabies virus strain Schelkovo-51, reproduced in a culture of transplantable cells BHK-21, and aluminum hydroxide (GOA), characterized in that it additionally contains ethanol in the following ratio of components , wt.%:
Virus-containing culture fluid containing inactivated rabies virus Schelkovo-51 strain reproduced in culture transplantable cells BHK-21 70-80 Ethanol 9-11 Aluminum hydroxide Rest