NOTES IMMUNOGENICITY IN MONKEYS OF A COMBINED TOXOID FROM THE MAIN TOXIC PRINCIPLES SEPARATED FROM HABU SNAKE VENOM

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1 Japan. J. Med. Sci. Biol., 23, , 1970 NOTES IMMUNOGENICITY IN MONKEYS OF A COMBINED TOXOID FROM THE MAIN TOXIC PRINCIPLES SEPARATED FROM HABU SNAKE VENOM Antivenine has been proved useful as a treatment of envenomation in man from snake bite. It is, however, not very effective unless administered within a few hours after the bite. Active immunization with toxoid (Td) is needed, therefore, to prevent the envenomation. Many workers attempted to toxoid a variety of snake venoms by treating with formalin (Boquet and Vendrely, 1943; Christensen, 1947; Wiener, 1960; Moroz-Perlmutter et al., 1963) or other chemicals (Okonogi and Hattori, 1968; Sawai and Kawamura, 1969), by irradiation (Flowers, 1963) and by photo-oxidation (Kocholaty, 1966). All these treatments succeeded in detoxifying venoms but the resulting toxoids were poorly immunogenic. We have recently attempted to detoxify Habu (Trimeresurus flavoviridis) venom with f ormalin* and found that the immunogenicity was preserved well when the venom was treated with formalin initially at 0.2 % and then at concentrations increased by 0.2 % (Kondo et al., 1970; Sadehiro et al., 1970). The present paper describes the immunogenicity in monkeys of a combined toxoid from the main toxic principles separated from Habu venom. The venom used (Batch No. 68-B) was a pool of dried venom taken from specimens of Trimeresurus flavoviridis collected in Amami Oshima Islands in Of several toxic principles contained in this venom, a major lethal toxin (Toxin I) and two immunologically distinct hemorrhagic principles, HR 1 and HR 2, have been regarded as playing the most important role in snake poisoning in man (Ohsaka and Kondo, 1960; Kondo et al., 1965 a, b). We, therefore, separated HR1 and HR2 fractions from the crude venom by gel filtration on sephadex G-100 (Omori-Satoh et al., 1967; Ohsaka et al., 1970). The HR 2 fraction was further purified by chromatagraphy on Amberlite CG-50 as reported (Takahashi and Ohsaka, 1970; Ohsaka et al., 1970). The HR 1 fraction contained the major lethal toxin (Toxin I) of the original venom. The HR 1 and HR 2 fractions thus obtained were then toxoided with increasing concentrations of formalin as described previously (Kondo et al., 1970; Sadahiro et al., 1970). Formalin had been added to each fraction initially at 0.2 % and then every other day at concentrations increasingly elevated by 0.2 % increments until the fraction was completely detoxified. The temperature had been kept at 37 C throughout, and sodium hydroxide added to maintain a constant ph of 7.0. The final concentration of formalin ranged from 1.2 to 1.6 %. The toxicities were determined at intervals by intravenous injection into mice (Ohsaka * Formalin contains 37% formaldehyde. 413

2 414 NOTE Vol. 23 et al., 1960) and by intracutaneous injection into rabbits (Kondo et al., 1960). The solution became non-toxic in days. The two toxoids (HR 1-Td and HR 2-Td) thus obtained were mixed at a ratio of 1:1 on the basis of protein content to prepare a mixed toxoid (Mixed-Td). The Mixed-Td was dialyzed against M acetate-buffered saline (ph 6.0). To the dialysis residue were added half volume each of 0.2 M AlCl3 and 0.2 M Na3PO4 in this order, resulting in an alminium-phosphate-gel suspension. The suspension adjusted to ph 6.0 contained protein at 1 mg/ml and aluminium at 1.35 mg/ml. The Mixed-Td (adsorbed), designated as Lot No. 12, showed a high immunogenicity for guinea pigs (Kondo et al., 1970) and rabbits (unpublished data). Monkeys were then immunized with Mixed-Td (adsorbed). Twenty cynomolgus monkeys (Macaca irus) weighing Kg were injected subcutaneously with this toxoid in a constant dose of 0.5 ml, 1.0 ml or 2.0 ml three times at about 4 weeks' intervals, and individual serum specimens were titrated for antilethal (Kondo et al., 1965 a; 1970) as well as antihemorrhagic, anti-hr1 and anti-hr2, potencies (Kondo et al., 1965 b; Ohsaka et al., 1966; Kondo et al., 1970). The antihemorrhagic potency of a serum specimen was expressed as a value relative to the potency of a standard antivenine, which was arbitrarily determined as 200 units/ml in respect to both anti-hr1 and anti-hr2; roughly 10 units and 50 units of the antivenine actually neutralized 0.1 mg of the HR1 fraction and of the HR2 fraction, respectively. Figure 1 shows an example of antitoxin production in monkeys which received 0.5 ml of the toxoid. The anti-hr1 and Fig. 1. Immunogenicity in monkeys of a combined toxoid (Mixed-Td). The Mixed-Td (adsorbed) was injected subcutaneously into 6 monkeys in 3 doses of 0.5 ml at 4 weeks' intervals. The arrows indicate the time of injection. Each animal was bled three times, prior to the 1st injection, and 10 days after the 2nd and the 3rd injections. The anti- HR1 and anti-hr2 titers of individual serum specimens were determined. Each circle represents the geometric mean of antihemorrhagic titers obtained. \, anti-hr1 titer; ----, anti-hr2 titer.

3 1970 NOTE 415 Table 1. Circulating antitoxin titers and protection from intramuscular challenging with the crude venom in monkeys immunized with a combined toxoid (Mixed-Td). The monkeys immunized with Mixed-Td were injected intramuscularly into the thigh with a crude venom. The animals were observed for 72 hr for local symptoms * Numbers in parentheses represent death date in days after the challenge. : Strongly positive : Moderately positive +: Slightly positive }: Doubtful -: Negative

4 316 NOTE Vol. 23 anti-hr2 titers increased at similar rates; the average titers (geometric means) reached 8 u/ml and 21 u/ml, respectively, 10 days after the 3 rd injection. In monkeys which received 1.0 ml of the toxoid, the average titers obtained were higher; 10 u/ml and 24 u/ml, respectively, for anti-hr1 and anti-hr2. In those which received 2.0 ml, the corresponding titers were 21 u/ml and 32 u/ml; the anti-lethal titer was below the level of detection. No markedly harmful side-reaction was observed in animals immunized with the toxoid. All the immunized animals were challenged 17 days after the 3 rd injection with 2 to 10 mg of the crude venom. Seventy-two hours later, an incision was made on the thigh under general anesthesia to observe local symptoms. The results were summarized in Table 1. Among five control animals, all the three which received 5 mg or more of the crude venom died in 1-3 days with severe local symptoms; the other two animals which received 2 mg of the venom survived with severe local symptoms. All the immunized animals challenged with the venom of 5 mg or less survived with slight, if any, local symptoms. Out of 6 animals challenged with 10 mg of the venom, three died within 24 hours; the other three survived with slight local symptoms. All the animals which survived were kept for additional one to two weeks without showing any further development of symptoms. These results demonstrate that the monkeys immunized with Mixed-Td (adsorbed) derived from the purified toxic principles can resist well to the challenge with 5 mg of the crude venom as far as the animals have developed sufficient amounts of circulating antibodies against the main toxic principles. The results also indicate that the monkeys immunized with the toxoid can be partially protected from the challenge with the crude venom of 10 mg. It seems justified, therefore, to assume that all the main antigens capable of stimulating production of antibodies necessary for preventing the envenomation are contained in this toxoid. Considering that the body weight of the monkeys used in this experiment is roughly one-twentieth that of human being and that the challenging dose of 10 mg is equal to one half of the amount of venom ejected by a single snake bite (Kondo et al., 1966), the present results may be interpreted as indicating the effectiveness of Mixed-Td (adsorbed) in the prophylaxis of envenomation in man. Since the relative amounts of the toxic principles in Habu venom may vary consideraby from one venom sample to another and since the optimal conditions for toxoiding the individual toxic principles with formalin are not quite similar, it is necessary to separate each toxic principle to be toxoided and, then to mix the resulting toxoids at an appropriate proportion to make a uniform preparation of combined toxoid. The use of highly purified toxic principles is recommended for the preparation of such a combined toxoid effective enough for prophylaxis of snake poisoning in man. With such toxoid, the unfavorable side reaction due to toxoid injection will be minimized. A full account of these experiments will be published elsewhere. ACKNOWLEDGEMENTS This investigation was supported partly by a grant from the Scientific Research Fund of the Ministry of Health and Welfare of Japan and partly by World Health Organization

5 1970 NOTE 417 Grant No. R/00126 for which grants the authors wish to express thanks. We wish to express our gratitude to the Division of Public Health, Kagoshima Prefecture, Japan, for their generous gifts of Habu venom. REFERENCES BOQUET, P. AND VENDRELY, R. (1943): Influence du ph sur la transformation du venin de Cobra en anavenin par 1'aldehyde formique; preparation d'un anavenin solide. Compt. Rend. Soc. Biol., 137, CHRISTENSEN, P. A. (1947): Formol detoxification of Cape cobra (Naja flava) venom. S. African J. Med. Sci., 12, FLOWERS, H. H. (1963): The effects of x-irradiation on the biological activity of Cottonmouth moccasin (Agkistrodon piscivorus) venom. Toxicon, 1, KOCHOLATY, W. F. (1966): Detoxification of Crotalus atrox venom by photooxidation in the presence of methylene blue. Toxicon, 3, KONDO, H., KONDO, S., IKEZAWA, H., MURATA, R. AND OHSAKA, A. (1960): Studies on the quantitative method for the determination of hemorrhagic activity of Habu snake venom. Japan. J. Med. Sci. Biol., 13, KONDO, H., KONDO, S., SADAHIRO, S., YAMAUCHI, K., OHSAKA, A. AND MURATA, R. (1965a): Standardization of antivenine. I. A method for determination of antilethal potency of Habu antivenine. Japan. J. Med. Sci. Biol., 18, KONDO, H., KONDO, S., SADAHIRO, S., YAMAUCHI, K., OHSAKA, A. AND MURATA, R. (1965b): Standardization of antivenine. II. A method for determination of antihemorrhagic potency of Habu antivenine in the presence of two hemorrhagic principles and their antibodies. Japan. J. Med. Sci. Biol., 18, KONDO, H., KONDO, S., SADAHIRO, S., YAMAUCHI, K., OHSAKA, A., MURATA, R., HOKAMA, Z., YAMAKAWA, M. AND NAKACHI, N. (1966): Estimation of the amount of venom ejected by a single snake bite. Abstracts of the Proceedings of the 11th Pacific Science Congress, Vol. 8, Science Council of Japan, Tokyo, 1966, p. 30. (Poisonous or Noxious Animals). KONDO, H., KONDO, S., SADAHIRO, S., YAMAUCHI, K., OHSAKA, A. AND MURATA, R. (1970): Preparation and immunogenicity of Habu (Trimeresurus flavoviridis) toxoid. Proceedings of the 2nd International Symposium on Animal and Plant Toxins, Tel-Aviv, February 22-28, 1970, in press. MOROZ-PERLMUTTER, C., GOLDBLUM, N., DE VRIES, A. AND GITTER, S. (1963): Detoxification of snake venoms and venom fractions by formaldehyde. Proc. Soc. Exptl. Biol. Med., 112, OHSAKA, A. AND KONDO, H. (1960): Biochemistry of snake venoms. Recent Advan. Med. Sci. Biol., 1, OHSAKA, A., IKEZAWA, H., KONDO, H., KONDO, S. AND UCHIDA, N. (1960): Haemorrhagic activities of Habu snake venom, and their relations to lethal toxicity, proteolytic activities and other pathological activities. Brit. J. Exptl. Pathol., 41, OHSAKA, A., KONDO, H., KONDO, S., KUROKAWA, M. AND MURATA, R. (1966): Problems in determination of antihemorrhagic potency of Habu (Trimeresurus flavoviridis) antivenine in the presence of multiple hemorrhagic principles and their antibodies. Mem. Inst. Butantan Simp. Internac. 33, OHSAKA, A., TAKAHASHI, T., OMORI-SATOH, T. AND MURATA, R. (1970): Purification and characterization of the hemorrhagic principles in the venom of Trimeresurus flavoviridis. Proceedings of the 2nd International Symposium on Animal and Plant Toxins, Tel-Aviv, February 22-28, 1970, in press. OKONOGI, T. AND HATTORI, Z. (1968): Attenuation of Habu-snake (Trimeresurus flavoviridis) venom treatment with alcohol and its effect as immunizing antigen. (text in

6 418 NOTE Vol. 23 Japanese), Nihon Saikingaku zasshi (Japan. J. Bacteriol.), 23, OMORI-SATOH, T., OHSAKA, A., KONDO, S. AND KONDO, H. (1967): A simple and rapid method for separating two hemorrhagic principles in the venom of Trimeresurus flavoviridis. Toxicon, 5, SADAHIRO, S., KONDO, S., YAMAUCHI, K., KONDO, H. AND MURATA, R. (1970): Studies on immunogenicity of toxoid from Habu (Trimeresurus lavoviridis) venom. Japan. J. Med. Sci. Biol., SAWAI, Y., KAWAMURA, Y. (1969): Study on the toxoids against the venoms of certain Asian snakes. Toxicon, 7, TAKAHASHI, T. AND OHSAKA, A. (1970): Purification and characterization of a proteinase in the venom of Trimeresurus flavoviridis. Biochem. Biophys. Acta, 198, WIENER, S. (1960): Active immunization of man against the venom of the Australian tiger snake (Notechis scutatus). Amer. J. Trop. Med. Hyg., 9, The 2nd Department of Bacteriology* and Department of Veterinary Science** National Institute of Health, Shinagawa-ku, Tokyo 141, Japan HISASHI KONDO* õ SEIJI SADAHIRO* SATORU KONDO* KIYOSUMI YAMAUCHI* SHIGEO HONJO** FUMIAKI CHO** AKIRA OHSAKA* RYOSUKE MURATA* Received: November 17th, 1970 õ Present address: Chiba Serum Institute, Konodai, Ichikawa, Chiba, 272, Japan.