Enhancing toxin-based vaccines against botulism.

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) and a receptor binding domain (HC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHC/A1 and HC/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-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. M-BoNT/A1 vaccination protected against challenge by 10 LD Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1 vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1 surviving BoNT challenge had dominant antibody responses to the LCHC domain, but varied antibody responses to HC. Sera from mice vaccinated with M-BoNT/A1 also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1 elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1, 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.