Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display.
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.
Levy
R
R
Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, San Francisco General Hospital, CA 94110, USA.
Forsyth
C M
CM
LaPorte
S L
SL
Geren
I N
IN
Smith
L A
LA
Marks
J D
JD
D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000276:immunology*; D003014:Clostridium botulinum / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D008958:Models, Molecular; D016296:Mutagenesis; D009500:Neutralization Tests; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D019151:Peptide Library; D019274:Botulinum Toxins, Type A