Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (
botulism) and is one of the top six biothreat agents.
Botulism is treated with polyclonal
antibodies produced in horses that are associated with a high incidence of systemic reactions. Human
monoclonal antibodies (mAbs) are under development as a safer
therapy. Identifying neutralizing
epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for
vaccine development. Here, we show that the three domains of BoNT serotype A (
BoNT/A) can be displayed on the surface of yeast and used to
epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for
epitope mapping. Using a library of yeast-displayed
BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine
epitopes of three neutralizing
BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin
sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the
epitopes shows how all three mAbs could bind
BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these
antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody
epitopes and the mapping results identify three neutralizing
BoNT/A epitopes.