Abstract
Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HC(N)) and a receptor binding domain (HC(C)). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC(N)/A1 and HC(C)/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >10(6)-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1(W). M-BoNT/A1(W) vaccination protected against challenge by 10(6) LD(50) Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1(W) vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1(W) surviving BoNT challenge had dominant antibody responses to the LCHC(N) domain, but varied antibody responses to HC(C). Sera from mice vaccinated with M-BoNT/A1(W) also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1(W) elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1(W), with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.