Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.
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.
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Yongfeng
Y
Fan
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Isin N
IN
Geren
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Jianbo
J
Dong
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Jianlong
J
Lou
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Weihua
W
Wen
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
Fraser
F
Conrad
Molecular and Translational Sciences Division, United States Army Medical Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
Theresa J
TJ
Smith
Medical Countermeasures Technology, United States Army Medical Research and Material Command, United States Army Medical Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
Leonard A
LA
Smith
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Mengfei
M
Ho
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Melissa
M
Pires-Alves
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
Brenda A
BA
Wilson
Department of Anesthesia, University of California, San Francisco, San Francisco, California, United States of America.
James D
JD
Marks
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000992:Antitoxins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000276:immunology*; D002384:Catalysis; D018604:Epitope Mapping; D005260:Female; D006801:Humans; D051379:Mice; D009498:Neurotoxins / Q000276:immunology*; D017434:Protein Structure, Tertiary; D065288:Serogroup; D057127:Single-Chain Antibodies / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000992:Antitoxins; D009498:Neurotoxins; D057127:Single-Chain Antibodies; D019274:Botulinum Toxins, Type A