RU2054294C1 - Method of preparing inactivated vaccine against viral hemorrhagic disease in rabbits - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves the use of 25-30% suspension of rabbit liver dead after viral hemorrhagic disease prepared on the buffered solution, pH 7.2, Virus is extracted twice for 20-48 hr at 6 ± 2 C, at periodic stirring and filtration through the sterile cotton-gauze filter. Inactivation of viral raw is carried out at 60 C for 5 hr. Material is stabilized by addition of 5% lactose and 1% gelatin at the ratio 1:1 and stabilizing agent followed by lyophilization. EFFECT: improved method of vaccine preparing. 4 tbl

Description

 The invention relates to biotechnology, in particular to a method for the manufacture of an inactivated vaccine against viral hemorrhagic rabbit disease (HBVC), and can be used in scientific and production laboratories involved in the design of biological products, as well as in enterprises of the biological industry.

 A known method of manufacturing a lyophilized tissue inactivated vaccine against viral hemorrhagic disease of rabbits (Temporary instruction for the manufacture and control of a vaccine against viral hemorrhagic disease of rabbits of tissue inactivated lyophilized, approved by the director of VNIIVViM 02.01.91).

 A known method of manufacturing an inactivated vaccine against viral hemorrhagic rabbit disease. Also filed application N 5043431 from 05.26.92, authors Shevchenko A.A. and others.

As a viral raw material use a 20% suspension of the liver of rabbits that died after infection with a virulent virus of hemorrhagic disease of rabbits in buffered saline (pH 7.2). The resulting suspension was kept at ²⁰ ° C for 3 hours, freed of cellular debris by centrifugation at 3000 rev / min for 20 minutes (total amount of protein is not more than 5 mg / ml). The disadvantage of the reactor is drained and inactivate diendometilen-1,8,3,6-1,3,6,8-th tetraazatritsiklodekan- rate of 1 g of substance in 1000 ml viral feedstock for 48 hr at 27 ^{° C} with occasional stirring. The stock was then clarified by the addition of chloroform for 1 hour 5 hours feedstock is heated 10 h at 20 ° ^C. To maintain native properties as a stabilizing protective medium using a stabilizer which comprises:.. TU 6-09-2293-75 lactose 50.0 g, dry enzymatic peptone GOST 13805-76 50.0 g, monosubstituted potassium phosphate GOST 4198-75 1.0 g, disubstituted potassium phosphate GOST 2493-65 5.2 g, per 1 liter of distilled ox pH 7.2 GOST 6709- 72.

Then, 1 part of the stabilizer is mixed with 2 parts of inactivated material, thoroughly mixed and packaged with a dispenser (AG-5) into 2.0 ml sterile vials or ampoules and lyophilized under vacuum of 80 μm. To this frozen material at minus 45 ^{° C} for 10 hours, kept at a temperature of from -45 ^to 0 ° C for 22 hours. Then the temperature is brought ^to 20 ° C for 3 hours and finally dried for 3 hours. The whole lyophilization cycle is 28 h. Corking of bottles or sealing of ampoules is carried out under vacuum.

 This inactivated tissue vaccine against HBV does not cause post-vaccination complications, immunity is formed on the 3rd day with intramuscular injection (0.5 ml) lasting 12 months. The vaccine is stable during long-term storage.

 However, in the manufacture of this vaccine, 2-43 times more rabbits are required, a chemical inactivator (1,8,3,6-diendomethylene-1,3,6,8-tetraazatricyclodecane) is used for inactivation, which cannot be neutralized, inactivation is carried out for a long time (2-3 days), with clarification, chloroform is used, which is not harmless to workers. The vaccine is used only intramuscularly in a volume of 0.5 ml.

 The proposed method of manufacturing a vaccine eliminates the above disadvantages.

 The objective of the invention is the manufacture of highly active inactivated vaccine against HBV, stable during storage.

The essence of the proposed method lies in the fact that a 25-30% suspension of the liver of rabbits killed by HBV is used in a buffered solution (pH 7.2). Twofold extraction was carried virus for 20-48 hours at 6 ± 2 ^{° C} with occasional stirring, and filtering through a sterile cotton-gauze filter. Inactivation of viral raw materials is carried out at a temperature of 60 ^about C for 5-6 hours

 Material stabilization is carried out by introducing 5% lactose and 1% gelatin in a 1: 1 ratio of material and stabilizer.

 Lyophilized according to the previously proposed regimen.

For the manufacture of a vaccine against viral hemorrhagic disease of rabbits weighing 2.5 kg and above, they are infected with GBK virus (registration number 1652 in the collection of VGNKI) with an infectious activity of 10 ^3.0 LD 50 / ml. After 24-72 rabbits die, they take the liver from them, freeze them, pass them through a meat grinder, and forcemeat grind in a homogenizer. The resulting material was adjusted with physiological saline pH 7.2 to 30% suspension extracted with virus for 20 hours at 6 ^{° C,} the supernatant was decanted, and to the residue was added saline 1: 2, and re-extracted for 20 hours with occasional stirring.

 Cell and tissue detritus are freed by filtration through a sterile cotton-gauze filter. The results are presented in table 1.

From the table. 1 shows that the use of a 30% suspension of the liver, double extraction of the virus, the release of cellular detritus by filtration through a cotton-gauze filter can increase the yield of viral raw materials by 2 times in the manufacture of the vaccine according to the proposed method compared to the prototype. The filtered material is poured into a container and inactivated in a water bath at ⁶⁰ ° C for 5 hours with occasional stirring. The results are presented in table.2.

From Table 2 it is seen that the best temperature for the virus inactivation is ⁶⁰ ° C, while the activity decreases only one order of magnitude. Such inactivation treatment at ⁶⁰ ° C for 5 hours allows completely secure and maintain pathogenicity immunogenic properties.

 To preserve the original properties, a mixture of 5% lactose and 1% gelatin is used as a stabilizing protective medium. Then, the inactivated material is mixed with a stabilizing medium in a ratio of 1: 1, mixed thoroughly, and using a dispenser (AG-5), 2.0 ml are packaged in sterile ampoules, frozen and lyophilized according to the accepted regimen. The results are presented in table.3.

From the table. Figure 3 shows that the use of 5% lactose and 1% gelatin mixed with the material in a 1: 1 ratio as the stabilizing protective medium allows preserving the initial properties of the vaccine for 3-6 months of storage. The use of the proposed method by various methods of administration (intramuscularly, subcutaneously, intradermally) at doses of 0.20-0.50 ml does not cause post-vaccination complications in animals, immunity is formed for 3 days, and protection after control of infection with a virulent virus is 90-100%
The results of the comparative characteristics of vaccines against HBV are presented in table. 4.

Thus, the proposed twofold extraction of virus release from the cell detritus can increase raw material yield is 2 times, the inactivation of the virus at ⁶⁰ ° C for 5 hours, and can suppress virulence retain immunogenic properties.

 The use of 5% lactose and 1% gelatin as a protective medium preserves the biological properties of the lyophilized preparation and storage stability.

 The proposed method is simple to implement, affordable and is industrially applicable.

Claims (1)

METHOD FOR PRODUCING INACTIVATED VACCINE AGAINST VIRUS HEMORRHAGIC RABBIT DISEASE, including the preparation of a liver suspension of rabbits that died after infection with a virulent virus of hemorrhagic disease, its inactivation, lyophilization, characterized in that the suspension is prepared before it is inactivated - 25-30% twice before 48 h at 6 ^o C, inactivation is carried out at 60 ^o C for 5 h, after which the suspension is stabilized with an equal amount of a mixture of 5% lactose and 1% gelatin on a buffered physiological solution solution pH 7.2.

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