Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM197, a mutant diphtheria toxin

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BC G (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analy zed the potential of CRM197, a mutated nontoxic derivative of diphtheria to xin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM197 in rBCG was achieved using Escherichia coli-mycobacte rium shuttle vectors under the control of pBlaF*, an upregulated beta -lact amase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG -CRM197 elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activi ty. On the other hand, a sub-immunizing dose of the conventional diphtheria -tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM197 was able to prime the induction of a humoral response within sh orter periods. Interestingly, the antibodies produced showed neutralizing a ctivity only when the vaccines had been given as a mixture in combination w ith rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant e ffect of rBCG-FC on the immune response induced by rBCG-CRM197. Isotype ana lysis of the anti-diphtheria toroid antibodies induced by the combined vacc ines, but not rBCG-CRM197 alone, showed an immunoglobulin G1-dominant profi le, as did the conventional vaccine. Our results show that rBCG expressing CRM197 can elicit a neutralizing humoral response and encourage further stu dies on the development of a DPT vaccine with rBCG.