CS233279B1 - A method of preparing a subunit vaccine against tick-borne encephalitis - Google Patents

Abstract

The invention relates to a method of preparing a subunit vaccine for tick-borne encephalitis. The essence of the invention is that the thermosensitive strain of tick-borne encephalitis virus Skalica Cl. gl. 53 is propagated in tissue cultures of smoking embryo cells, the virus is semipurified by differential centrifugation, the sediment is resuspended in saline, digested three times with ether, which is removed, the vaccine is lyophilized. The invention has use in humane and veterinary practice.

Description

233 279

The invention relates to a method of preparing a subunit vaccine against tick-borne encephalitis. E. N. Levkovic and also Ch. Kunz prepared inactivating vaccines against tick-borne encephalitis on smoking embryonic cells followed by formaldehyde inactivation. N.levkovic: Biology of the Tick borne encephalitis complex.Czechoslovak Acad. Sci. 317 (1962); Ch. Kunz: Arzneim. Porsch.Drug. Res. 28, 10, 1806, (1978) J. A disadvantage of inactivated vaccines is their preparation with a full virulent strain of tick-borne encephalitis virus. Mayer and Mitr prepared a live attenuated vaccine with the Langat / Mayer and Mitra: Biological works. Sci. The disadvantage of the attenuated vaccine is that the Langat strain has reverted and cannot be used to prepare a live vaccine.

The above disadvantages are substantially eliminated by the method of preparing a subunit vaccine against tick-borne encephalitis, which is based on the fact that the thermosensitive strain of virus encephalitis -Cl. gl. 53? is propagated on tissue cultures of embryonic cell smokers; the virus is semipurified by differential centrifugation, resuspended in physiological saline, cleaved 3 times with ether, removed, the vaccine clarified and lyophilized. The advantage of the proposed method of preparing the subunit vaccine is that a thermosensitive strain of tick-borne virus is used that is non-pathogenic after subcutaneous administration to white-age mice, which is significant in terms of safety at work.

AND

The vaccine contains virus subunits that induce the formation of antibodies and are free of ballast. This method of preparation is economically unpretentious. The vaccine may be stored after lyophilization. Example 1

Tissue cultures are prepared from 11 day C / E smoking cells to remove eyes and offal. Tissue cultures are cultured in basal Eagle's medium (BEM) admixed with 5% fetal calf serum and antibiotics (penicillin and streptomycin). After culturing for 24 hours, the medium is removed and the tissue cultures are infected with tick-borne encephalitis virus (Scalica strain, deposited in the Czechoslovak Collection of Viral Sciences of the SAS under Cl. Gl. 53) with 7.5 logs. Ο, ΟΙ ml.Adsorption takes place for 90 min at 37 ° C. Then add fresh BEM with 5% inactivated serum and antibiotic. On the first day, the medium is withdrawn and cleared. Virususa purification is performed by differential centrifugation at 25,000 rpm. for min. for 2 h. The supernatant is removed and the sediment is resuspended 100 times less isotonic saline pH 7.2 with 50 µg gentamycin per ml admixture. After clarification, the titer of the virus is determined by the hemagglutination test. The continuous carbon black preparation was digested 3 times with ether, 1 time for 5 minutes, second time with the same portion for 18 hours at 4 ° C, 3 times for 5 minutes. After clarification, the ether was filtered off with suction, and the ether was removed with nitrogen. The vaccine is lyophilized and stored at -20 ° C. Vaccine safety studies are done by intracerebral - 3 "233,279 inoculation of 0.1 ml inoculum of the next group of white mice. During the observation period of 14 days, none of the experimental animals showed any health impairment related to the application of the test preparation. A test for the absence of a live vivirus is done on 5 to 8 g of white mice. The mice are inoculated with 0.03 ml of the Intracerebral vaccine undiluted and the saline group inoculated with a 0.03 ml 1:10 dilution vaccine. After 7 days, the brain will be taken from the half to Salši passage. Antibody formation is done on 15 mice, which are injected intraperitoneally with 0.1 ml vaccine, 7 days after the first dose, the blood is removed from the mouse and the serum is examined in a hemagglutinin-inhibition assay. The second dose of vaccine is given 1 month. Seven days after the first vaccination, serum murine presence of haemagglutinin-inhibiting antibodies is examined. The protective test is performed on adult white mice immunized with a single dose of 0.1 ml intraperitoneal vaccine. After 7 to 8 days a live virus challenge is made. Mice are protected against 100 LD 50 (murine lethal doses) of the virus. Vacuum sterility test is performed according to Sel. 3. Example 2

The procedure is as in Example 1 except that the virus is cultured at 37 ° C for 48 h.

The procedure is as in Example 1, except that the viral particles are separated in a sucrose density gradient (10 to 30% sucrose).

It replaces aa as in Example 1, with the difference that the final preparation of ether extraction and treatment with formalin in a 1: 2000 dilution, maintains e for 3 days at 4 ° C, formalin and is removed by adding 1 to 100 ml of sample. , 7 ml of 5% pyroeurate. After mixing, the sample is filled into a dialysis bath and this is dialysed in 3 liters of physiological solution at 4 ° C for 20 hours. Vakoin and lyophilized after dialysis ·

The invention can be widely used in the medium as a vaccine against tick-borne encephalitis.

Claims (1)

OBJECT OF THE INVENTION 233 279 A method for preparing subunit anti-fecalencephalitis, characterized in that the thermosensitive strain, virus cleavage encephalitis C1. gl. The rock is propagated by embryonic cell smoker cell cultures, the virus is sasemipurified by differential centrifugation, resuspended in saline, digested 3 times with ether, which is removed, the vaccine cleared and lyophilized.