MODIFICATION OF BIOLOGICAL PROPERTIES OF PROTEIN TOXINS BY STEPWISE IODINATION

Abstract

By gradual incorporation of stable iodine into toxins and whole venoms it is possible to abolish completely the physiological, lesion and lethal properties of the native components. The properties of iodinated antigens and from antibodies generated by these detoxified derivatives, are presented. The hapten is incorporated into tyrosyl and histidyl residues. The derivatives can be obtained in less than one hour. Within the same batch of protein, there is a determinable stoichiometric ratio hapten/protein to achieve the desired modified properties of the derivative. The iodinating solutions are easy to prepare, can be accurately standardized and have unlimited shelf lives. The cost of the whole procedure is very low. No side-effects, local or systemic were observed, even with prolonged use of the derivatives. The method was applied to toxic components and whole venom of the scorpion Tityus serrulatus, and the hypertensive, bradipneic, oliguric, lesional, lethal and cytotoxic effects were completely abolished. Polyclonal antibodies generated by these iodinated antigens neutralized the virulent effects of native components and reversed the α effects of the whole venom in frog sciatic nerves. They conferred active immunization in mice, rats, guinea pigs, goats, horses and pigeons. Crotoxin and whole venom of Crotalus durrissus terrificus lost the lesional and lethal activity, but conserving the immunogenic capacity. They produced antibodies against the native components, giving also vaccinal protection. While the virulent crotalic antigens had a cytotoxic activity, the iodinated antigens were highly mitogenic with human white cells. Five bothropic venoms were neutralized in the hemorrhagic, tissue lesion and lethal capacity, the derivatives were immunogenic. Repetitive sublethal doses of scorpionic, crotalic and bothropic venoms lead invariably to an amyloid-like deposit in tissues, whereas the iodinated samples were ineffective. Allergenic extracts of Schistosoma mansoni can be transformed into anallergic derivatives that retains antigenic properties. Violently allergenic extracts of Ascaris lumbricoides suum can be completely deactivated with iodination, but conserved immunological competence. Cholera, tetanus and botulinum toxins, as iodinated toxoids, had its lesional and lethal capacity completely avoided. Physiological proteins with strong biological activity can also be rendered innocuous. Iodinated insulin lost its capacity to lower blood glucose levels, but induced high avidity antibodies in guinea-pigs and rabbits. By iodination, kallikrein can be turned unable to contract rat uterus, and to liberate kinins from kinninogen. Modified tonin do not increase the blood pressure in rats. Aqueous extracts of Leptospira canis and L. icterohaemorrhagiae after iodination, were innocuous to hatched eggs, and immunogenic in mice and rabbits. A lectin from Macrotylema axillare, lost the hemaglutination capacity with only 75% of iodine saturation. The derivative was highly immunogenic in rabbits. Heavy iodination can transform self-antigens in non-self, generating antibodies in same species animals. All derivatives obtained were stable, did not show any reversion to toxicity, generated antibodies against the native antigens and gave active protection when injected in animals. The injections were also apparently painless. The time gap between the accident and the administration of antibodies is discussed for systemic and local effects. A new schedule for immunization, only feasible with toxoided venoms is presented. It is based on a clonal expansion induced by a small dose, followed by an exponential saturation dose of the same toxoid. The attainment of higher levels of protecting antibodies against the native antigen in the generated sera, is unmatched by other procedures. Data for practical use of iodination is presented.

Notes

* The senior author was the first to be innoculated, followed by a long series made by physician Dr. M. Chamone, habilitated to perform human tests.