title: A novel mechanism for antibody-based anthrax toxin neutralization: inhibition of prepore-to-pore conversion.
abstract: 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. We have previously reported the isolation and chimerization of cAb29, 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 PA(63), 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 2α(1) loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.
authors.affiliation: Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
authors.forename: Adva
authors.initials: A
authors.lastname: Mechaly
authors.forename: Haim
authors.initials: H
authors.lastname: Levy
authors.forename: Eyal
authors.initials: E
authors.lastname: Epstein
authors.forename: Ronit
authors.initials: R
authors.lastname: Rosenfeld
authors.forename: Hadar
authors.initials: H
authors.lastname: Marcus
authors.forename: Einat
authors.initials: E
authors.lastname: Ben-Arie
authors.forename: Avigdor
authors.initials: A
authors.lastname: Shafferman
authors.forename: Arie
authors.initials: A
authors.lastname: Ordentlich
authors.forename: Ohad
authors.initials: O
authors.lastname: Mazor
mesh_terms: D000818:Animals; D000881:Anthrax / Q000188:drug therapy / Q000276:immunology; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000942:Antigens, Bacterial / Q000276:immunology; D001408:Bacillus anthracis / Q000276:immunology; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D055503:Protein Multimerization / Q000187:drug effects / Q000276:immunology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D000939:Epitopes; C030325:anthrax toxin