Studies on Botulinum Toxoids, Types A and B
Authors: Reames H. R. , Kadull P. J. , Housewright R. D. , Wilson J. B. 
Published: January 2023

Summary and Conclusions
Fluid and alum-precipitated botulinum toxoids were used to immunize man against botulinum toxin. There were no cases of botulism among individuals immunized with these preparations. A few individuals who were immunized received botulinum antitoxin immediately after a known gross exposure.
An antitoxic level of 0.02 unit per ml of serum was selected as a presumptive protective level and was the objective in the immunological program of persons exposed.
A schedule of 4 doses of fluid toxoid at 2 week intervals elicited antitoxic titers of the same magnitude as those titers attained by individuals on a schedule of 3 doses at 3 to 4 week intervals. On these schedules protective antitoxic titers were reached slowly, and not until after the last dose of toxoid was administered did more than half of the persons tested have a protective level of antitoxin. Attempts to increase the speed of the response by administering 4 doses of toxoid at weekly intervals or by administering a larger initial dose were unsuccessful. Within 5 months after starting immunization on a schedule of 4 biweekly injections of fluid toxoid over 90 per cent of the individuals had protective levels, and about 30 per cent had titers exceeding 0.1 of a unit per ml. This schedule was accepted for routine immunization, and because there appeared to be a gradual decline of the antitoxic titer after it reached a maximum at 5 months, booster injections were given every 6 months. These booster injections caused a rapid increase in the level of antitoxin present in the serum.
Using alum-precipitated type A toxoid it was found that 3 injections at 3 to 4 week intervals was the most effective of the immunization schedules which were tested. The most important factor seemed to be the longer interval between the first and third injections.
The importance of the time of injections was illustrated also in the experiments with a combined type A and type B alum-precipitated toxoid. A schedule consisting of 2 doses 8 weeks apart was more effective than one that called for 2 doses 3 to 4 weeks apart. Over three-fourths of the sera tested 2½ months after starting immunization, which had type B antitoxic titers classified as greater than 0.02 unit per ml, consisted of sera with levels exceeding 0.1 of a unit per ml.
All of the data indicate that the best results were obtained by allowing a period of about 2 months to elapse between the first injection of toxoid and one of the subsequent injections.
A small number of persons did not attain a measurable level of antitoxin in the serum.
A study of the local and constitutional reactions caused by the combined alum-precipitated toxoid showed that moderate or marked reactions were few in number.
Acknowledgement is made to Dr. Gail M. Dack, of the University of Chicago and Dr. N. Paul Hudson, of the Ohio State University for their continued interest and valuable advice throughout the course of the work.
Acknowledgement is due Joanne R. Smith, Lt. (jg)H(S) and Pearl D. Fisher, Ens. H(S) USNR for their technical assistance.