{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T23:24:12Z","timestamp":1787613852333,"version":"build-2736575974"},"update-to":[{"DOI":"10.1371\/journal.ppat.1010169","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,1,19]],"date-time":"2022-01-19T00:00:00Z","timestamp":1642550400000}}],"reference-count":52,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2022,1,6]],"date-time":"2022-01-06T00:00:00Z","timestamp":1641427200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI139087; R21AI139690; R01AI125704; and R21AI123920"],"award-info":[{"award-number":["R01AI139087; R21AI139690; R01AI125704; and R21AI123920"]}],"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":["R01AI125704"],"award-info":[{"award-number":["R01AI125704"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["P41 GM103403"],"award-info":[{"award-number":["P41 GM103403"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000015","name":"U.S. Department of Energy","doi-asserted-by":"publisher","award":["DE-AC02-06CH11357"],"award-info":[{"award-number":["DE-AC02-06CH11357"]}],"id":[{"id":"10.13039\/100000015","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT\/A and BoNT\/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT\/A and BoNT\/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC\/A or LC\/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT\/A or BoNT\/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC\/A, that could aid future development of inhibitors against BoNT\/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.<\/jats:p>","DOI":"10.1371\/journal.ppat.1010169","type":"journal-article","created":{"date-parts":[[2022,1,6]],"date-time":"2022-01-06T18:34:22Z","timestamp":1641494062000},"page":"e1010169","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":20,"title":["Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies"],"prefix":"10.1371","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7173-6600","authenticated-orcid":true,"given":"Kwok-ho","family":"Lam","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacqueline M.","family":"Tremblay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4046-1704","authenticated-orcid":true,"given":"Kay","family":"Perry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2198-1325","authenticated-orcid":true,"given":"Konstantin","family":"Ichtchenko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9738-1361","authenticated-orcid":true,"given":"Charles B.","family":"Shoemaker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0348-7363","authenticated-orcid":true,"given":"Rongsheng","family":"Jin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,1,6]]},"reference":[{"issue":"9","key":"ppat.1010169.ref001","doi-asserted-by":"crossref","first-page":"5047","DOI":"10.1128\/AAC.02830-14","article-title":"Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A","volume":"58","author":"SU Nayak","year":"2014","journal-title":"Antimicrobial agents and chemotherapy"},{"key":"ppat.1010169.ref002","article-title":"Safety and pharmacokinetics of a four monoclonal antibody combination against botulinum C and D neurotoxins","author":"DM Snow","year":"2019","journal-title":"Antimicrobial agents and chemotherapy"},{"issue":"4","key":"ppat.1010169.ref003","doi-asserted-by":"crossref","DOI":"10.3390\/toxins11040208","article-title":"Monoclonal antibody combinations prevent serotype A and serotype B inhalational botulism in a guinea pig model","volume":"11","author":"MT Tomic","year":"2019","journal-title":"Toxins"},{"issue":"3","key":"ppat.1010169.ref004","doi-asserted-by":"crossref","DOI":"10.3390\/toxins10030105","article-title":"A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype E subtypes","volume":"10","author":"C Garcia-Rodriguez","year":"2018","journal-title":"Toxins"},{"issue":"3","key":"ppat.1010169.ref005","doi-asserted-by":"crossref","first-page":"e0174187","DOI":"10.1371\/journal.pone.0174187","article-title":"A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes","volume":"12","author":"Y Fan","year":"2017","journal-title":"PLoS 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