title: Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H.
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.
authors.affiliation: Department of Anesthesia, University of California San Francisco, San Francisco General Hospital.
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Infant Botulism Treatment and Prevention Program, Division of Communicable Disease Control, Center for Infectious Diseases, California Department of Public Health, Richmond.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Barash
authors.affiliation: Department of Anesthesia, University of California San Francisco, San Francisco General Hospital.
authors.forename: Jianlong
authors.initials: J
authors.lastname: Lou
authors.affiliation: Department of Anesthesia, University of California San Francisco, San Francisco General Hospital.
authors.forename: Fraser
authors.initials: F
authors.lastname: Conrad
authors.affiliation: Department of Anesthesia, University of California San Francisco, San Francisco General Hospital.
authors.forename: James D
authors.initials: JD
authors.lastname: Marks
authors.affiliation: Infant Botulism Treatment and Prevention Program, Division of Communicable Disease Control, Center for Infectious Diseases, California Department of Public Health, Richmond.
authors.forename: Stephen S
authors.initials: SS
authors.lastname: Arnon
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000276:immunology; D000992:Antitoxins / Q000276:immunology*; D001905:Botulinum Toxins / Q000737:chemistry / Q000276:immunology*; D001906:Botulism / Q000188:drug therapy / Q000276:immunology* / Q000517:prevention & control; D003014:Clostridium botulinum / Q000276:immunology*; D004195:Disease Models, Animal; D006041:Goats; D006736:Horses; D006801:Humans; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000992:Antitoxins; D001905:Botulinum Toxins
keywords: Clostridium botulinum; bioterrorism; botulinum neurotoxin type H; botulinum toxin; botulism; monoclonal antibodies; select agents