MOLECULAR CHARACTERIZATION OF MURINE HUMORAL IMMUNE-RESPONSE TO BOTULINUM NEUROTOXIN TYPE-A BINDING DOMAIN AS ASSESSED BY USING PHAGE ANTIBODY LIBRARIES

To produce antibodies capable of neutralizing botulinum neurotoxin typ e A (BoNT/A), the murine humoral immune response to BoNT/A binding dom ain (H-C) was characterized at the molecular level by using phage anti body libraries, Mice were immunized with BoNT/A H-C, the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were co nstructed from the immunoglobulin heavy and light chain variable regio n genes, Phage expressing BoNT/A binding scFv were isolated by selecti on on immobilized BoNT/A and BoNT/A H-C. Twenty-eight unique BoNT/A H- C binding scFv were identified by enzyme-linked immunosorbent assay an d DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epit opes with equilibrium constants (K-d) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 a nd 2 significantly prolonged the time to neuroparalysis, 1.5-and 2.7-f old, respectively, compared to toxin control, scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuro paralysis, which increased to 3.9-fold compared to the control, The re sults suggest that there are two ''productive'' receptor binding sites on H, which lead to toxin internalization and toxicity, Blockade of t hese two epitopes with monoclonal antibodies may provide effective imm unoprophylaxis or therapy against BoNT/A intoxication.