title: Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.
abstract: To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection 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 and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, 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 neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.
authors.affiliation: Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, 94110, USA.
authors.forename: P
authors.initials: P
authors.lastname: Amersdorfer
authors.forename: C
authors.initials: C
authors.lastname: Wong
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: T
authors.initials: T
authors.lastname: Smith
authors.forename: S
authors.initials: S
authors.lastname: Deshpande
authors.forename: R
authors.initials: R
authors.lastname: Sheridan
authors.forename: R
authors.initials: R
authors.lastname: Finnern
authors.forename: J D
authors.initials: JD
authors.lastname: Marks
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000276:immunology* / Q000378:metabolism; D003090:Coliphages / Q000235:genetics; D018604:Epitope Mapping; D007128:Immunoglobulin Fragments / Q000276:immunology; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library
chemical_list: D000907:Antibodies, Bacterial; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D019274:Botulinum Toxins, Type A