{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T19:14:40Z","timestamp":1776971680677,"version":"3.51.4"},"reference-count":41,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2015,8,26]],"date-time":"2015-08-26T00:00:00Z","timestamp":1440547200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["U01AI056493"],"award-info":[{"award-number":["U01AI056493"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R33AI101504"],"award-info":[{"award-number":["R33AI101504"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["U01AI075502"],"award-info":[{"award-number":["U01AI075502"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["U54AI0571530"],"award-info":[{"award-number":["U54AI0571530"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R21AI101504"],"award-info":[{"award-number":["R21AI101504"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000774","name":"Defense Threat Reduction Agency","doi-asserted-by":"publisher","award":["HDTRA1-07-C-0030"],"award-info":[{"award-number":["HDTRA1-07-C-0030"]}],"id":[{"id":"10.13039\/100000774","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/toxins7093405","type":"journal-article","created":{"date-parts":[[2015,8,26]],"date-time":"2015-08-26T10:23:31Z","timestamp":1440584611000},"page":"3405-3423","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":26,"title":["Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype\/B"],"prefix":"10.3390","volume":"7","author":[{"given":"Yongfeng","family":"Fan","sequence":"first","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianbo","family":"Dong","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianlong","family":"Lou","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weihua","family":"Wen","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fraser","family":"Conrad","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Isin","family":"Geren","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Consuelo","family":"Garcia-Rodriguez","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Theresa","family":"Smith","sequence":"additional","affiliation":[{"name":"Molecular and Translational Sciences Division, United States Army Medical Institute of Infectious Diseases, Fort Detrick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leonard","family":"Smith","sequence":"additional","affiliation":[{"name":"Medical Countermeasures Technology, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mengfei","family":"Ho","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Melissa","family":"Pires-Alves","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brenda","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James","family":"Marks","sequence":"additional","affiliation":[{"name":"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"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2015,8,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1128\/mr.46.1.86-94.1982","article-title":"Bacterial toxins: A table of lethal amounts","volume":"46","author":"Gill","year":"1982","journal-title":"Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Botulinum toxin as a biological weapon: medical and public health management","volume":"285","author":"Arnon","year":"2001","journal-title":"JAMA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"898","DOI":"10.1038\/2338","article-title":"Crystal structure of botulinum neurotoxin type A and implications for toxicity","volume":"5","author":"Lacy","year":"1998","journal-title":"Nat. Struct. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1096","DOI":"10.1038\/nature05411","article-title":"Structural basis of cell surface receptor recognition by botulinum neurotoxin B","volume":"444","author":"Chai","year":"2006","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/307457a0","article-title":"Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization","volume":"307","author":"Dolly","year":"1984","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1126\/science.1123654","article-title":"SV2 is the protein receptor for botulinum neurotoxin A","volume":"312","author":"Dong","year":"2006","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/0968-0004(86)90282-3","article-title":"How do tetanus and botulinum toxins bind to neuronal membranes?","volume":"11","author":"Montecucco","year":"1986","journal-title":"Trends Biochem. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1046\/j.1365-2958.2003.03872.x","article-title":"The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction","volume":"51","author":"Rummel","year":"2004","journal-title":"Mol. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/S0022-3565(25)31908-7","article-title":"Kinetic studies on the interaction between botulinum toxin type A and the chloinergic neuromuscular junction","volume":"212","author":"Simpson","year":"1980","journal-title":"J. Pharmacol. Expt. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/0014-5793(87)80562-8","article-title":"The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers","volume":"226","author":"Blaustein","year":"1987","journal-title":"FEBS Lett."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"10447","DOI":"10.1073\/pnas.0700046104","article-title":"Single molecule detection of intermediates during botulinum neurotoxin translocation across membranes","volume":"104","author":"Fischer","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"832","DOI":"10.1038\/359832a0","article-title":"Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin","volume":"359","author":"Schiavo","year":"1992","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3941","DOI":"10.1021\/bi3000098","article-title":"Insights into the different catalytic activities of Clostridium neurotoxins","volume":"51","author":"Chen","year":"2012","journal-title":"Biochemistry"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1038\/nature03123","article-title":"Substrate recognition strategy for botulinum neurotoxin serotype A","volume":"432","author":"Breidenbach","year":"2004","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1056\/NEJMoa051926","article-title":"Human botulism immune globulin for the treatment of infant botulism","volume":"354","author":"Arnon","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_16","unstructured":"Centers for Disease Control and Prevention (2010). Investigational heptavalent botulinum antitoxin (HBAT) to replace licensed botulinum antitoxin AB and investigational botulinum antitoxin E. MMWR Morb. Mortal Wkly. Rep., 59, 299."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"11346","DOI":"10.1073\/pnas.172229899","article-title":"Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody","volume":"99","author":"Nowakowski","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1038\/nbt1269","article-title":"Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin","volume":"25","author":"Levy","year":"2007","journal-title":"Nat. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1093\/protein\/gzq001","article-title":"Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating","volume":"23","author":"Lou","year":"2010","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.ab.2012.08.005","article-title":"Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein","volume":"430","author":"Meng","year":"2012","journal-title":"Anal. Biochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.ab.2011.09.030","article-title":"Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein","volume":"421","author":"Meng","year":"2012","journal-title":"Anal. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2602","DOI":"10.1073\/pnas.0611213104","article-title":"An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists","volume":"104","author":"Eubanks","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"978","DOI":"10.3390\/toxins2050978","article-title":"The zinc-dependent protease activity of the botulinum neurotoxins","volume":"2","author":"Lebeda","year":"2010","journal-title":"Toxins"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e10129","DOI":"10.1371\/journal.pone.0010129","article-title":"Small molecules showing significant protection of mice against botulinum neurotoxin serotype A","volume":"5","author":"Pang","year":"2010","journal-title":"PLoS ONE"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2044","DOI":"10.2174\/1568026614666141022093350","article-title":"Recent advances in botulinum neurotoxin inhibitor development","volume":"14","author":"Kiris","year":"2014","journal-title":"Curr. Top Med. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2081","DOI":"10.2174\/1568026614666141022094517","article-title":"Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism","volume":"14","author":"Wilson","year":"2014","journal-title":"Curr. Top Med. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2555","DOI":"10.1111\/j.1742-4658.2012.08638.x","article-title":"Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin","volume":"279","author":"Edupuganti","year":"2012","journal-title":"FEBS J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1106","DOI":"10.1016\/j.jmb.2010.01.070","article-title":"A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region","volume":"397","author":"Dong","year":"2010","journal-title":"J. Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e0135306","DOI":"10.1371\/journal.pone.0135306","article-title":"Monoclonal antibodies targeting the alpha-exosite of botulinum neurotoxin serotype\/A inhibit catalytic activity","volume":"10","author":"Fan","year":"2015","journal-title":"PLoS ONE"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.jmb.2006.09.084","article-title":"Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display","volume":"365","author":"Levy","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"693","DOI":"10.1038\/78005","article-title":"Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B","volume":"7","author":"Swaminathan","year":"2000","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e113","DOI":"10.1371\/journal.ppat.0030113","article-title":"Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain","volume":"3","author":"Brunger","year":"2007","journal-title":"PLoS Pathog."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3071","DOI":"10.1073\/pnas.0812952106","article-title":"Two-state selection of conformation-specific antibodies","volume":"106","author":"Gao","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1021\/bi035844k","article-title":"Role of metals in the biological activity of Clostridium botulinum neurotoxins","volume":"43","author":"Eswaramoorthy","year":"2004","journal-title":"Biochemistry"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"15365","DOI":"10.1021\/bi00255a017","article-title":"Differences in the protease activities of tetanus and botulinum B toxins revealed by the cleavage of vesicle-associated membrane protein and various sized fragments","volume":"33","author":"Foran","year":"1994","journal-title":"Biochemistry"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"739","DOI":"10.1093\/protein\/15.9.739","article-title":"Free-energy contributions to complex formation between botulinum neurotoxin type B and synaptobrevin fragment","volume":"15","author":"Olson","year":"2002","journal-title":"Protein Eng."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/0022-2836(91)90498-U","article-title":"By-passing immunization. Human antibodies from V-gene libraries displayed on phage","volume":"222","author":"Marks","year":"1991","journal-title":"J. Mol. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1006\/jmbi.1996.0004","article-title":"Isolation of high affinity monomeric human anti-c-erbB-2 single chain Fv using affinity driven selection","volume":"255","author":"Schier","year":"1996","journal-title":"J. Mol. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1016\/j.toxicon.2008.12.016","article-title":"Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity","volume":"53","author":"Ho","year":"2009","journal-title":"Toxicon"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1002\/pro.2017","article-title":"Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A","volume":"21","author":"Ho","year":"2012","journal-title":"Protein Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.jmb.2005.06.003","article-title":"Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A","volume":"351","author":"Razai","year":"2005","journal-title":"J. Mol. Biol."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/7\/9\/3405\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T20:47:24Z","timestamp":1760215644000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/7\/9\/3405"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2015,8,26]]},"references-count":41,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2015,9]]}},"alternative-id":["toxins7093405"],"URL":"https:\/\/doi.org\/10.3390\/toxins7093405","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2015,8,26]]}}}