Antigenic sites on the H N domain of botulinum neurotoxin A stimulate protective antibody responses against active toxin

Abstract

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (H C) and light chain (L C) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (H N) region as a vaccine candidate. Mice were immunized with recombinant H N 519-845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of H N 519-845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-H N antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that H N 519-593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A H N domain.