Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A

Abstract Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX‐MS) with site‐directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the H CN and H CC subdomains of the BoNT/A1 receptor‐binding domain (H C ). The TA12‐binding interface shares common structural features with the ciA‐C2 VHH epitope and lies on the face opposite recognized by ciA‐C2‐ and the CR1/CR2‐neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to H C confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1‐binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.