Neutralization mechanism of human monoclonal antibodies against type B botulinum neurotoxin

Abstract

Botulism is a deadly neuroparalytic condition caused by the botulinum neurotoxin (BoNT) produced by Clostridium botulinum and related species. Toxin-neutralizing antibodies are the most effective treatments for BoNT intoxication. We generated human monoclonal antibodies neutralizing type B botulinum neurotoxin (BoNT/B), designated M2 and M4. The combination of these antibodies exhibited a strong neutralizing effect against BoNT/B toxicity. In this study, we analyzed the mechanisms of action of these antibodies in vitro. M4 binds to the C-terminus of the heavy chain (the receptor-binding domain) and inhibits BoNT/B binding to neuronal PC12 cells. Although M2 recognized the light (L) chain (the metalloprotease domain), it did not inhibit substrate (VAMP2) cleavage in the cleavage assay. M2 increased the surface localization of BoNT/B in PC12 cells at a later time point, suggesting that M2 inhibits the translocation of the L chain from synaptic vesicles to the cytosol. These results indicate that M2 and M4 inhibit the different processes of BoNT/B individually and that multistep inhibition is important for the synergistic effect of the combination of monoclonal antibodies. Our findings may facilitate the development of effective therapeutic antibodies against BoNTs.