Nontoxic, multi-domain botulinum neurotoxin-LCHC_N as vaccines against botulism

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are proteins consisting of an N-terminal catalytic Light Chain (LC) and a C-terminal Heavy Chain (HC), which comprises a LC-translocation domain (HC_N) and a receptor binding domain (HC_C). There are seven BoNT serotypes (A-G) and multiple subtypes. Currently, there is no approved vaccine against botulism available for human use. Several studies have investigated BoNT domains, multi-domains, or mutated holotoxins as candidate BoNT vaccines. However, residual toxicity has been observed in mutated full-length BoNTs and multi-domain (LCHC_N) vaccines. In this study, six-individual point substitutions (6M) were introduced into the substrate binding and cleavage sites, zinc binding motif, and LC translocation region of LCHC_N/A1 and LCHC_N/B1, which possessed no detectable toxicity in mice at 200 μg, being >40-million-fold less toxic than full-length BoNT. IP immunization of mice with 6MLCHC_N/A1 or 6MLCHC_N/B1 elicited a strong IgG response that protected against high dose challenge with BoNT/A1 or BoNT/B1, respectively. 4MBoNT/B1 and 7MBoNT/B1 were also engineered and found to elicit strong IgG responses that protected against high dose challenge with BoNT/B1. Thus, multi-domain inactivation may yield potent and safe vaccines against other BoNT-serotypes and BoNT-like proteins. The strategy of systematic inactivation of multiple functional domains by targeted single amino acid substitutions to decrease toxicity, combined with investigations of the most immunogenic and protective domains, provides a robust platform for protein toxin vaccines for humans and animals.