Immunological Characterization And Neutralizing Ability Of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H

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.aEuro integral 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.aEuro integral Anti-BoNT/A H-CC-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 [K-D] = 7.56 x 10(-8) M) than its BoNT/F affinity (K-D = 9.1 x 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 K-D = 1.48 x 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 A mu g of total antibody.Conclusions.aEuro integral 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.