Diploid somatic cell encephalitis B vaccine and method for preparing purified encephalitis B vaccine

Abstract

The invention relates to a preparation method for diploid cell Japanese encephalitis vaccine and purified encephalitis vaccine. The method uses a serum-free medium to culture human diploid cell Japanese encephalitis purified vaccine, thus greatly reducing anaphylactic reaction and being beneficial to purification.

Description

Technical field:

[0001] The invention relates to a preparation method of a purified Japanese encephalitis vaccine, in particular to a preparation method of a purified Japanese encephalitis vaccine obtained by inoculating Japanese encephalitis virus seeds on human diploid cells. Background technique:

[0002] At present, there are three kinds of Japanese encephalitis vaccines produced in large quantities in the world. The first one is a purified Japanese encephalitis vaccine prepared by grinding, emulsifying, concentrating, and ultracentrifuging the brain tissue of mice infected with JE virus, represented by Japan; It is the inactivated JE vaccine cultured from primary hamster kidney cells and the live JE vaccine from primary hamster kidney produced in China. The cell substrates of these two vaccines are derived from ordinary animals, and there is no guarantee that they will not carry foreign factors; The third is the freeze-dried JE purified and inactivated vaccine produced ...

Examples

Embodiment 1

[0065] Diploid cells come from the Chinese Center for Disease Control and Prevention, use 38-50 generations, the cell nutrient solution is serum-free medium, add 25IU / ml gentamycin or 25IU / ml kanamycin, and adjust the pH to 7.2, use The 3L spinner bottle was cultured at 37°C to form a dense monolayer, and then amplified according to the seeding ratio of 1:2. Somatic JE virus, using JE murine encephalovirus species P 3 The second-generation working virus strain (P 3-2 ), virus seed concentration MOI0.05, cell maintenance medium is 199 culture medium containing 0.4-0.5% (W / W) human albumin, pH 7.6, cultivated at 35°C, after 24 hours, replace with fresh cell maintenance medium to continue Cultivate for 2 days, start to harvest the virus, harvest once every 3-4 days, and collect 5 times in total. Each batch of virus liquid is sampled for virus titration and sterility test, and the virus titer of the harvested virus liquid is required to reach 10. 7 LD50 / ml or above; add formalde...

Embodiment 2

[0069] Diploid cells come from the Chinese Center for Disease Control and Prevention, use 38-50 generations, the cell nutrient solution is serum-free medium, add 25IU / ml gentamycin and 25IU / ml kanamycin, and adjust the pH to 7.2, use After cultured in a 3L spinner bottle at 37°C to form a dense monolayer, amplify according to the seeding ratio of 1:2. When the expansion reaches the production batch, after about 4 days, when the cells grow into a dense monolayer, the cell nutrient solution is discarded, and the pH7. 2's Earl's solution washes the cell surface, inoculates the diploid cell JE virus, and uses the second-generation working virus (P 3-2 ), virus seed concentration MOI0.05, cell maintenance medium is 199 culture medium containing 0.4-0.5% (W / W) human albumin, pH 7.6, cultivated at 35°C, after 24 hours, replace with fresh cell maintenance medium to continue Cultivate for 2 days, start to harvest the virus, harvest once every 3-4 days, and collect 5 times in total. Eac...

Embodiment 3

[0073] The JE inactivated vaccine prepared by the method of claim 1 is tested (including giving injections to 12 children aged 4-6 years, and those who have not been vaccinated against JE vaccine are inoculated with 2 needles of JE inactivated vaccine, with an interval of 7-10 days between the two needles. .) proved that the positive conversion rate of neutralizing antibodies was 91.7%, and no side effects occurred.

Technical field:

[0001] The invention relates to a preparation method of a purified Japanese encephalitis vaccine, in particular to a preparation method of a purified Japanese encephalitis vaccine obtained by inoculating Japanese encephalitis virus seeds on human diploid cells. Background technique:

[0002] At present, there are three kinds of Japanese encephalitis vaccines produced in large quantities in the world. The first one is a purified Japanese encephalitis vaccine prepared by grinding, emulsifying, concentrating, and ultracentrifuging the brain tissue of mice infected with JE virus, represented by Japan; It is the inactivated JE vaccine cultured from primary hamster kidney cells and the live JE vaccine from primary hamster kidney produced in China. The cell substrates of these two vaccines are derived from ordinary animals, and there is no guarantee that they will not carry foreign factors; The third is the freeze-dried JE purified and inactivated vaccine produced ...

Examples

Embodiment 1

[0065] Diploid cells come from the Chinese Center for Disease Control and Prevention, use 38-50 generations, the cell nutrient solution is serum-free medium, add 25IU / ml gentamycin or 25IU / ml kanamycin, and adjust the pH to 7.2, use The 3L spinner bottle was cultured at 37°C to form a dense monolayer, and then amplified according to the seeding ratio of 1:2. Somatic JE virus, using JE murine encephalovirus species P 3 The second-generation working virus strain (P 3-2 ), virus seed concentration MOI0.05, cell maintenance medium is 199 culture medium containing 0.4-0.5% (W / W) human albumin, pH 7.6, cultivated at 35°C, after 24 hours, replace with fresh cell maintenance medium to continue Cultivate for 2 days, start to harvest the virus, harvest once every 3-4 days, and collect 5 times in total. Each batch of virus liquid is sampled for virus titration and sterility test, and the virus titer of the harvested virus liquid is required to reach 10. 7 LD50 / ml or above; add formalde...

Embodiment 2

[0069] Diploid cells come from the Chinese Center for Disease Control and Prevention, use 38-50 generations, the cell nutrient solution is serum-free medium, add 25IU / ml gentamycin and 25IU / ml kanamycin, and adjust the pH to 7.2, use After cultured in a 3L spinner bottle at 37°C to form a dense monolayer, amplify according to the seeding ratio of 1:2. When the expansion reaches the production batch, after about 4 days, when the cells grow into a dense monolayer, the cell nutrient solution is discarded, and the pH7. 2's Earl's solution washes the cell surface, inoculates the diploid cell JE virus, and uses the second-generation working virus (P 3-2 ), virus seed concentration MOI0.05, cell maintenance medium is 199 culture medium containing 0.4-0.5% (W / W) human albumin, pH 7.6, cultivated at 35°C, after 24 hours, replace with fresh cell maintenance medium to continue Cultivate for 2 days, start to harvest the virus, harvest once every 3-4 days, and collect 5 times in total. Eac...

Embodiment 3

[0073] The JE inactivated vaccine prepared by the method of claim 1 is tested (including giving injections to 12 children aged 4-6 years, and those who have not been vaccinated against JE vaccine are inoculated with 2 needles of JE inactivated vaccine, with an interval of 7-10 days between the two needles. .) proved that the positive conversion rate of neutralizing antibodies was 91.7%, and no side effects occurred.