Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. cAb29 is an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA63, oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 21 loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, it was found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, cAb29 is a potent PA-neutralizing antibody that exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.
