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Toxins 2015, 7(9), 3405-3423; doi:10.3390/toxins7093405

Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

1
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA
2
Molecular and Translational Sciences Division, United States Army Medical Institute of Infectious Diseases, Fort Detrick, MD 21702, USA
3
Medical Countermeasures Technology, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA
4
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
Current address: Department of Molecular Biology and Genetics, Faculty of Science, Istanbul Medeniyet University, Unalan Mahallesi, Unalan Sokak, D100 Karayolu, Uskudar-Istanbul 34700, Turkey;
*
Author to whom correspondence should be addressed.
Academic Editor: Michel R. Popoff
Received: 6 July 2015 / Revised: 10 August 2015 / Accepted: 18 August 2015 / Published: 26 August 2015
(This article belongs to the Section Bacterial Toxins)
View Full-Text   |   Download PDF [1700 KB, uploaded 26 August 2015]   |  

Abstract

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. View Full-Text
Keywords: botulinum antitoxin; inhibitory antibodies; Botulinum neurotoxin serotype B; alpha-exosite botulinum antitoxin; inhibitory antibodies; Botulinum neurotoxin serotype B; alpha-exosite
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Fan, Y.; Dong, J.; Lou, J.; Wen, W.; Conrad, F.; Geren, I.N.; Garcia-Rodriguez, C.; Smith, T.J.; Smith, L.A.; Ho, M.; Pires-Alves, M.; Wilson, B.A.; Marks, J.D. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins 2015, 7, 3405-3423.

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