Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H

J Infect Dis. 2016 May 15;213(10):1606-14. doi: 10.1093/infdis/jiv770. Epub 2016 Mar 1.

Abstract

Background: Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.

Methods: We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay.

Results: Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody.

Conclusions: This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

Keywords: Clostridium botulinum; bioterrorism; botulinum neurotoxin type H; botulinum toxin; botulism; monoclonal antibodies; select agents.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibodies, Neutralizing / immunology
  • Antitoxins / immunology*
  • Botulinum Toxins / chemistry
  • Botulinum Toxins / immunology*
  • Botulism / drug therapy
  • Botulism / immunology*
  • Botulism / prevention & control
  • Clostridium botulinum / immunology*
  • Disease Models, Animal
  • Goats
  • Horses
  • Humans
  • Mice

Substances

  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Antitoxins
  • Botulinum Toxins