Immune response of man to various Habu-venom toxoids with different potencies was investigated. A linear relationship was observed between circulating anti-HR1 and the amount of HR1 toxoid used for the basic immunization. A highly purified toxoid was 2.5 times more effective in man than partially purified ones, when compared on the basis of the immunizing unit determined in guinea pigs. Out of 265 vaccinees, 202 (76.2%) produced anti-HR1 exceeding 1 U/ml after two injections of the mixed toxoid and the rate rose to 96% after the booster immunization, when the preparation contained HRl toxoid of not less than 9ImU per dose. The rate fell to 58% and 28.5%, 12 months and 24 months after the last injection, respectively. Although booster immunization was effective to restore the declined antibody production, the level of anti-HR1 after the injection did not exceed that after the basic immunization in the groups primed with partially purified HR1 toxoid. Neither increased number of injection nor changed duration of intervals of successive injections for the basic immunization improved the immune response of man. Booster effect was slightly higher in the groups primed with a highly purified toxoid, but its magnitude was much smaller than that in the case of other kinds of toxoid (e.g., tetanus toxoid) .
Anti-HR2 was invariably higher than 1 U/ml, being more than 5-10 U/ml in many vaccinees. However, the behavior after the booster injection was similar to that of anti-HR1.
Side reaction was not a serious obstacle in continuing immunization. The volunteers amounted to 8, 206, but general reactions were rare, if any. However, allergic reactions of the vaccinees should carefully be checked, since repeated booster immunization may be necessary to maintain immunity.

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Purity of the parent toxin influenced greatly the immunogenicity in guinea pigs of HR1 component of Habu-venom toxoid. The potency of HR1 toxoid in terms of the immunizing unit (ImU) appeared to be related to the antigen and aluminum contents. The immune response of the animals differed depending on the purity of the toxoid when the dose-response curve was examined over a wide range of the antigen dosage. Anti-HR1 titers of the guinea pigs immunized with crude toxoid (adsorbed) reached a plateau at about 10-20 U/ml and further rise was not remarkable even when the amount of the antigen was increased several times, while 200 U/ml (maximum 400 U/ml or higher) of anti-HR1 was produced with a large amount of highly purified HR1 toxoid.
Purified toxoid showed an excellent immunogenicity also in monkeys. The amounts of antigens necessary for the basic immunization to resist the challenge with a certainly lethal dose of crude venom were 18 and 0.5 mg as protein, for a crude and a highly purified toxoid, respectively. The total amount of aluminum was also reduced from 8.1 (crude toxoid) to 0.5 mg (purified toxoid) . Multiple injections for the primary immunization enhanced early production of anti-HR1 in monkeys. However, at later stages of immunization, significant correlation was observed between the amount of HR1 toxoid (in ImU) for the primary immunization and the circulating anti-HR1 titer irrespective of the schedule of immunization. Anti-HR2 higher than 10 U/ml was produced consistently in monkeys with HR2 toxoid of 1.0 ImU for the basic immunization. However, excess HR2 toxoid of more than 10 ImU seemed rather unfavorable for anti-HR2 production.

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