Abstract

Human botulism can be caused by botulinum neurotoxin (BoNT) serotypes A to G. Here, we present an antibody-based antitoxin composed of four human monoclonal antibodies (mAbs) against BoNT/C, BoNT/D, and their mosaic toxins. This work built on our success in generating protective mAbs to BoNT /A, B and E serotypes. We generated mAbs from human immune single-chain Fv (scFv) yeast-display libraries and isolated scFvs with high affinity for BoNT/C, BoNT/CD, BoNT/DC and BoNT/D serotypes. We identified four mAbs that bound non-overlapping epitopes on multiple serotypes and mosaic BoNTs. Three of the mAbs underwent molecular evolution to increase affinity. A four-mAb combination provided high-affinity binding and BoNT neutralization of both serotypes and their mosaic toxins. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing and neutralizing BoNT/C and BoNT/D serotypes and their mosaic toxins. A derivative of the four-antibody combination (NTM-1634) completed a Phase 1 clinical trial (Snow et al., Antimicrobial Agents and Chemotherapy, 2019) with no drug-related serious adverse events.

Highlights

  • Botulism is an acute life-threatening flaccid paralysis caused by botulinum neurotoxins (BoNTs) produced by Clostridium species [1,2]

  • While overall BoNT/C and BoNT/D differed from each other by 49%, the HN of the two BoNTs and their mosaics differed at most by 32%. This observation suggested that it might be possible to generate cross-reactive monoclonal antibodies (mAbs) binding the HN domain of BoNT/C and BoNT/D and their mosaic toxins

  • Given the increased potency of the four-mAb combination compared to the threemAb combination versus BoNT/C, we evaluated decreasing doses of the affinity-matured antibody combination of 4C2:4C4.4:4C10.5:8DC4.4 against 40,000 LD50 challenges using BoNT/C, BoNT/CD, and BoNT/DC, and 10,000 LD50 using BoNT/D

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Summary

Introduction

Botulism is an acute life-threatening flaccid paralysis caused by botulinum neurotoxins (BoNTs) produced by Clostridium species [1,2]. The majority of naturally occurring human botulism is caused by serotypes A, B, E, and F. BoNT/CD causes botulism in avian species, both in the wild and in domestic flocks [5,6,7]. BoNT/D and mosaic-type BoNT/DC botulism outbreaks are rare and are usually associated with horses [18] and cattle [19,20,21]. Rare human outbreaks of botulism due to types C and D have been reported [24,25,26]; two cases of foodborne botulism and one case of infant botulism were attributed to BoNT/C [27], while several people fell ill from BoNT/D botulism [24] attributed to tainted ham

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