Ethylenimine-inactivated rabies vaccine of tissue culture origin

The replication of seven rabies virus strains (CVS, HEP, PV, ERA, WIRAB, CPZ and BOLIVAR) in BHK cells and the inactivation dynamics of these strains by beta-propiolactone, acetylethylenimine, and ethylenimine were studied to find the most immunogenic strain and the most economic and stable inactivating agent for the production of an inactivated tissue culture rabies vaccine for animal use. The seven strains reached the peak of virus production 3 to 5 days after inoculation of the cell culture; PV yielded the highest virus titer (10(9) plaque-forming units/ml). The infectivity of virus suspensions containing 10(7) to 10(8) plaque-forming units/0.1 ml was inactivated by beta-propiolactone in 0.5 h, acetylethylenimine in 3.0 h, and ethylenimine in 1.0 h. Most of the vaccine lots prepared with the different strains and inactivating agents passed a modified National Institutes of Health potency test. The vaccines prepared with the PV strain had consistently higher antigenic values (equal or better than four) than the other six strains. This difference was highly significant (F6,12=59.8), whereas there were no statistically significant differences among the antigenic values of the vaccine lots prepared with the three inactivating agents. Batches of lyophilized and liquid vaccine stored at 4 C maintained potency for over 1 year. Ten dogs vaccinated with a vaccine prepared with the PV strain and inactivated with ethylenimine developed a good antibody response and resisted challenge 60 days after vaccination, while seven of eight nonvaccinated controls died of rabies. This information indicates that an inactivated, stable, economic, and easy-to-prepare rabies vaccine can be produced in BHK cells by using the PV strain and ethylenimine as an inactivating agent.