Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates.

Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be a much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec(-) (unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo(-) (unable to form oligomers), and DNI (Dominant Negative Inhibitory, unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PA<Rec(-)<SSSR<Oligo(-)<DNI. While the differences between Rec(-) or SSSR and PA were small and not statistically significant, DNI and Oligo(-) were significantly more immunogenic than wild-type PA. One year after immunization and compared with PA-immunized mice, DNI-immunized mice maintained significantly higher levels of anti-PA IgG with correspondingly higher titers of toxin-neutralizing activity. In contrast, Oligo(-)-immunized mice had high levels of anti-PA IgG but lower titers of toxin-neutralizing activity, suggesting that Oligo(-) mutation sites may overlap with critical protective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA non-functional but also simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure vaccination.