Phage display enabled the identification of single-domain nanobody antitoxins against botulinum neurotoxin serotype C.

Botulinum neurotoxin serotype C (BoNT/C) exhibits therapeutic efficacy and duration of action comparable to those of BoNT/A. It has been demonstrated to effectively manage movement disorders such as dystonia and blepharospasm, making it a highly promising alternative to BoNT/A. BoNT/C is also one of the primary etiological botulinum isoforms causing animal botulism and may pose a zoonotic risk. So, it is of great need to develop antidote that can treat human iatrogenic, foodborne and animal botulism caused by BoNT/C. Currently, the approved botulism antidotes primarily are polyvalent horse serum antitoxins, which are severely limited due to insufficient supply and adverse reactions. In this study, we developed a high-affinity nanobody A2, a variable domain of a heavy-chain antibody (VHH) against BoNT/C, based on phage display library screening. Our results demonstrated that A2 effectively blocked the binding of BoNT/C to its target cell. Furthermore, A2 exhibited potent neutralizing activity and rescued BoNT/C-intoxicated mice. These findings supported nanobody A2 as a promising antitoxin candidate with therapeutic efficacy against botulism.