Studies with Shigella Dysenteriae (Shiga) V. Factors Involved in the Preparation of Toxoid

Discussion and Summary The usual concept of toxoid preparation has involved, first, reducing the toxicity by some means until no longer demonstrable, then, determining the antigenicity of the toxoid preparation. There has been no attempt to measure the antigenicity of the toxin at the start and to find what part of the antigenicity remained after the detoxification process has been completed. By the use of this concept, toxoids have been prepared for well-known bacterial toxins such as botulinum, diphtheria, staphylococcus and tetanus. Potent toxoids have resulted from the action of formaldehyde on the toxins and there has been no need for further study of the detoxification process. The detoxification of Shiga toxin has presented some difficulties not encountered with other bacterial toxins. Treatment of this toxin with formal-dehyde or ultraviolet irradiation has produced toxoids which have stimulated protection in experimental animals when multiple injections were administered. As shown in Table I, protection was not achieved from a single injection of these toxoids unless the exposure to ultraviolet irradiation was less than the minimum needed to destroy the toxicity. By the use of heat at 80°C for eight minutes, as shown in Tables II and IV, it was possible to detoxify Shiga toxin to the same extent as was achieved with either formaldehyde or ultraviolet irradiation and yet protect mice with one injection of the toxoid. When the antigenicity of the toxin was determined before and after heating, as shown in Tables V and VI, it was found that from 86% to 97% of the antigenicity had been destroyed in the heating process. This degree of destruction was not as great as that of the toxicity, because 97% to 99% of the toxicity was destroyed by the heating process. This raises the question whether the same end,—that is, toxoiding, would not have been accomplished by diluting the toxin to a point where the toxicity would not be detectable. The data in Table VI show that the antigenic values of the diluted toxins would not be as great as those of the heated toxins. There has been no effort to correlate Lf of the toxoid with its immunizing power in mice. The data in Tables II and IV show that the immunizing activity is stable over a wide range of pH values from pH 2.2 to pH 9.0 at temperatures up to 88°C, but that heating at 90°C for 8 minutes at pH 8.5 and at 80°C at pH 9.8 for the same period decreased the immunizing activity of the toxin. These data partially confirm the work of Anderson, Brown and Macsween (6) who reported that the flocculating power of the toxin was completely destroyed by heating 30 minutes at 90°C and pH 9.4. The method for determining the antigenicity of Shiga toxoid by the experimental immunization of mice provides a tool for studying, not only methods of detoxification which would conserve most of the antigenicity of the toxin, but also methods of fractionation of toxins and toxoids which would give an index of the immunizing power of each fraction. By this method it was found that toxoid prepared by heating the toxin contained less than 10% of the immunizing power of the unaltered toxin. With other methods of converting toxin to toxoid, e.g. ultraviolet irradiation and formaldehyde, considerably less of the immunizing power was recovered.