Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

• Published in: PloS one Vol. 10; no. 8; p. e0135306
• Main Authors: Fan, Yongfeng, Geren, Isin N, Dong, Jianbo, Lou, Jianlong, Wen, Weihua, Conrad, Fraser, Smith, Theresa J, Smith, Leonard A, Ho, Mengfei, Pires-Alves, Melissa, Wilson, Brenda A, Marks, James D
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14.08.2015; Public Library of Science (PLoS)
• Subjects: Acetylcholine; Alanine; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - metabolism; Antigenic determinants; Antitoxins; Antitoxins - chemistry; Antitoxins - immunology; Antitoxins - metabolism; Biological products; Botulinum toxin type A; Botulinum Toxins, Type A - immunology; Botulism; Catalysis; Catalytic activity; Chains; Cloning; Clostridium botulinum; Epitope Mapping; Epitopes; Female; Flow cytometry; Fluorescence; Food contamination; Genes; Humans; Immunization; Immunoglobulins; Infectious diseases; Intoxication; Libraries; Mice; Molecular chains; Monoclonal antibodies; Neurotoxins; Neurotoxins - immunology; Neurotransmitter release; Paralysis; Protein Structure, Tertiary; Proteins; Scanning mutagenesis; Serogroup; Single-Chain Antibodies - metabolism; SNAP receptors; Toxins; Waterborne diseases; Yeast; Zinc; Zinc endopeptidase; San Francisco California; California; United States > US; Maryland; Illinois
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0135306

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.