Background: Botulinum neurotoxin type A (BoNT/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (H C) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively H C of other BoNT/A subtypes. This study aimed to generate H C domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains.
Methods: H C domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/H CA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for H C domains of BoNT/A2-A8 were derived from pET45b-H CA1 by site-directed mutagenesis and/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of H C domains and to evaluate the reactivity of VHHs to these domains.
Results: SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the H C domains of all eight BoNT/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for H C of BoNT/A1, two new VHHs were found to recognize the H C domains of all BoNT/A subtypes, of which VHH-A3 displayed EC 50 values for these domains close to those of ciA-C2.
Conclusion: This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT/A.
Keywords: HC; VHH; antitoxin; botulinum neurotoxins; botulism; nanobody; neutralization; receptor-binding domain; recombinant proteins.
Copyright: © 2025 Quynh Pham N et al.