The present report deals with the production and characterization of inactivated Japanese encephalitis virus vaccine grown in porcine kidney cells for human trials. Bottle cultures of RFVL line of Nakayama strain were completely harvested by freeze-thawing followed by sonication. This improved the potency of the vaccine although there was no apparent rise in virus titer. The paradox may in part be explained by the lability to the temperature of the virus. In fact, the halflife of the virus was measured as follows: 15hr at 30°, 11hr at 33°, 7hr at 36° and 5hr at 37°. The resulting virus suspension was further centrifuged at 10, 000×g for 30min and filtered through membrane filter of 0.45μ pore size with little loss of virus. As inactivating reagent, formalin was superior to β-propiolactione since the latter significantly impaired the antigenicity. Optimal concentration of formalin was found to be 1: 2000 to retain the immunizing potency. However it must be kept in mind that over-neutralization by sodium metasulfate of formalin may be detrimental to the antigenicity. Safety tests were performed at appropriate stages of processing; these included sterility test, safety test in a narrow sense, and exclusion of recidual JEV as well as contaminating or adventitious agents in porcine kidney cells. Potency test was carried out in mice in two ways; one was the direct challenge method required by the Japanese government, and the other was to evaluate the antigenic extinction limit method as determined by the 50% plaque reduction. In our experience the latter technique was bettter in that it was more sensitive and reliable. The potency of the tissue culture vaccine was estimated to be 1/2 to 1/4 of that of the mouse brain type of vaccine. The stability of the vaccine was observed by storing it at refrigerator temperature for 3 years, during which period the relative potency of the tissue culture vaccine to current mouse brain vaccine remained unchanged. Finally the vaccine was tested in both man and monkey for antibody response. It was revealed that the tissue culture vaccine was not satisfactory in a primary response but showed definite booster effects. No untoward reactions were noted among vaccinees.
