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

Authors :
Yongfeng Fan
Jianbo Dong
Jianlong Lou
Weihua Wen
Fraser Conrad
Isin N. Geren
Consuelo Garcia-Rodriguez
Theresa J. Smith
Leonard A. Smith
Mengfei Ho
Melissa Pires-Alves
Brenda A. Wilson
James D. Marks
Source :
Toxins, Vol 7, Iss 9, Pp 3405-3423 (2015)
Publication Year :
2015
Publisher :
MDPI AG, 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.

Details

Language :
English
ISSN :
20726651
Volume :
7
Issue :
9
Database :
Directory of Open Access Journals
Journal :
Toxins
Publication Type :
Academic Journal
Accession number :
edsdoj.6b1b373bea8b45a2adb6651086bec8a0
Document Type :
article
Full Text :
https://doi.org/10.3390/toxins7093405