Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity

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 (HC) of the BoNT/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT/A subtypes. This study aimed to generate HC domains of all eight BoNT/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT/A1-A8 were recombinantly produced in Escherichia coli. The bont/HCA1 fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT/A2-A8 were derived from pET45b-HCA1 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 HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC 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 HC of BoNT/A1, two new VHHs were found to recognize the HC domains of all BoNT/A subtypes, of which VHH-A3 displayed EC50 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.