{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T20:46:29Z","timestamp":1780433189127,"version":"3.54.1"},"reference-count":30,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2025,2,5]],"date-time":"2025-02-05T00:00:00Z","timestamp":1738713600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100019600","name":"Hanoi University of Science and Technology","doi-asserted-by":"publisher","award":["T2022-T\u0110-002"],"award-info":[{"award-number":["T2022-T\u0110-002"]}],"id":[{"id":"10.13039\/100019600","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    Background Botulinum neurotoxin type A (BoNT\/A) represents a major threat to global public health because of its most potent toxicity with the longest persistence. Several camelid single-domain antibodies (or VHHs) have been reported to exhibit high neutralizing activity against the receptor binding domain (HC) of the BoNT\/A subtype used to generate them. However, it remains unclear if these VHHs can neutralize effectively HC of other BoNT\/A subtypes. This study aimed to generate HC domains of all eight BoNT\/A subtypes and to screen for VHHs with broad reactivity against these domains. Methods HC domains of BoNT\/A1-A8 were recombinantly produced in\n                    <ns3:italic>Escherichia coli.<\/ns3:italic>\n                    The\n                    <ns3:italic>bont\/HCA1<\/ns3:italic>\n                    fragment was amplified from sludge sample and cloned into pET45b vector by Gibson assembly. Expression vectors for HC domains of BoNT\/A2-A8 were derived from pET45b-HCA1 by site-directed mutagenesis and\/or in-house gene synthesis. Similarly, VHHs were synthesized and cloned into pET22b vector. Recombinant protein were purified by Ni-NTA spin columns and analyzed by SDS-PAGE. ELISA was used to confirm the antigenicity of HC domains and to evaluate the reactivity of VHHs to these domains. Results SDS-PAGE analysis and ELISA results with commercial polyclonal antibody demonstrated the HC domains of all eight BoNT\/A subtypes were correctly produced. ELISA results using a VHH panel indicated that, apart from ciA-C2, a well-characterized VHH specific for HC of BoNT\/A1, two new VHHs were found to recognize the HC domains of all BoNT\/A subtypes, of which VHH-A3 displayed EC50 values for these domains close to those of ciA-C2. Conclusion This study provided a resource to comprehensively identify antitoxins conferring broad protection against BoNT\/A.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.160607.1","type":"journal-article","created":{"date-parts":[[2025,2,5]],"date-time":"2025-02-05T04:20:06Z","timestamp":1738729206000},"page":"163","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Recombinant expression of receptor binding domains of all eight subtypes of botulinum neurotoxin type A for generation of antitoxins with broad reactivity"],"prefix":"10.12688","volume":"14","author":[{"given":"Nga","family":"Quynh Pham","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tam Trang","family":"Mai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tran Bao Anh","family":"Dang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-6759-6312","authenticated-orcid":false,"given":"Ly","family":"Huong Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Quynh","family":"Mai Vu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chien","family":"Trong Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anh","family":"Thi Phuong Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tran Nhat Minh","family":"Dang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Van Anh","family":"Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thinh","family":"Huy Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Van Khanh","family":"Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1853-7397","authenticated-orcid":false,"given":"Hoa","family":"Quang Le","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2025,2,5]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","DOI":"10.3390\/toxins11120686","article-title":"Tables of toxicity of botulinum and tetanus neurotoxins.","volume":"11","author":"O Rossetto","year":"2019","journal-title":"Toxins (Basel)."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"535-549","DOI":"10.1038\/nrmicro3295","article-title":"Botulinum neurotoxins: Genetic, structural and mechanistic insights.","volume":"12","author":"O Rossetto","year":"2014","journal-title":"Nat. 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Microbiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"e0310623","DOI":"10.1128\/mbio.03106-23","article-title":"Botulinum neurotoxin X lacks potency in mice and in human neurons.","volume":"15","author":"B Gregg","year":"2024","journal-title":"MBio."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"e0116381","DOI":"10.1371\/journal.pone.0116381","article-title":"Isolation and functional characterization of the novel clostridium botulinum neurotoxin A8 subtype.","volume":"10","author":"S Kull","year":"2015","journal-title":"PLoS One."},{"key":"ref5","doi-asserted-by":"publisher","DOI":"10.3390\/toxins11090528","article-title":"Antibodies and vaccines against botulinum toxins: Available measures and novel approaches.","volume":"11","author":"C Rasetti-Escargueil","year":"2019","journal-title":"Toxins (Basel)."},{"key":"ref6","doi-asserted-by":"publisher","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":"Y Fan","year":"2015","journal-title":"PLoS One."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"3405-3423","DOI":"10.3390\/toxins7093405","article-title":"Monoclonal antibodies that inhibit the proteolytic activity of botulinum neurotoxin serotype\/B.","volume":"7","author":"Y Fan","year":"2015","journal-title":"Toxins (Basel)."},{"key":"ref8","doi-asserted-by":"publisher","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 (Basel)."},{"key":"ref9","doi-asserted-by":"publisher","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 One."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"e29941","DOI":"10.1371\/journal.pone.0029941","article-title":"A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model.","volume":"7","author":"J Mukherjee","year":"2012","journal-title":"PLoS One."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"7438","DOI":"10.1038\/s41598-017-07457-5","article-title":"A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding.","volume":"7","author":"G Yao","year":"2017","journal-title":"Sci. Rep."},{"key":"ref12","doi-asserted-by":"publisher","DOI":"10.3390\/toxins11080464","article-title":"Camelid VHHs fused to human fc fragments provide long term protection against botulinum neurotoxin a in mice.","volume":"11","author":"S Godakova","year":"2019","journal-title":"Toxins (Basel)."},{"key":"ref13","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159514.v1","article-title":"Construction of pET45b-HCA1 plasmid.","author":"N Pham","year":"2025"},{"key":"ref14","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159544.v1","article-title":"Construction of pET45b-HCA2 plasmid.","author":"N Pham","year":"2025"},{"key":"ref15","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159553.v1","article-title":"Construction of pET45b-HCA3 plasmid.","author":"N Pham","year":"2025"},{"key":"ref16","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159559.v1","article-title":"Construction of pET45b-HCA4 plasmid.","author":"N Pham","year":"2025"},{"key":"ref17","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159574.v1","article-title":"Construction of pET45b-HCA5 plasmid.","author":"N Pham","year":"2025"},{"key":"ref18","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159586.v1","article-title":"Construction of pET45b-HCA6 plasmid.","author":"N Pham","year":"2025"},{"key":"ref19","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159592.v1","article-title":"Construction of pET45b-HCA7 plasmid.","author":"N Pham","year":"2025"},{"key":"ref20","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159598.v1","article-title":"Construction of pET45b-HCA8 plasmid.","author":"N Pham","year":"2025"},{"key":"ref21","doi-asserted-by":"publisher","first-page":"e8818","DOI":"10.1371\/journal.pone.0008818","article-title":"Llama single domain antibodies specific for the 7 botulinum neurotoxin serotypes as heptaplex immunoreagents.","volume":"5","author":"J Conway","year":"2010","journal-title":"PLoS One."},{"key":"ref22","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159622.v1","article-title":"Construction of pET22b-VHH plasmids.","author":"N Pham","year":"2025"},{"key":"ref23","doi-asserted-by":"publisher","first-page":"3205-3216","DOI":"10.1007\/s00253-023-12515-2","article-title":"Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs.","volume":"107","author":"D Shi","year":"2023","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"1314-1318","DOI":"10.1128\/IAI.01025-07","article-title":"Subunit vaccine against the seven serotypes of botulism.","volume":"76","author":"M Baldwin","year":"2008","journal-title":"Infect. Immun."},{"key":"ref25","doi-asserted-by":"publisher","DOI":"10.1126\/SCITRANSLMED.AAZ4197","article-title":"Delivery of single-domain antibodies into neurons using a chimeric toxin\u2013based platform is therapeutic in mouse models of botulism.","volume":"13","author":"S Miyashita","year":"2021","journal-title":"Sci. Transl. Med."},{"key":"ref26","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159754.v1","article-title":"Reactivity of HCA1-HCA8 to the Rabbit Anti-Botulinum Toxin A and B IgG.","author":"N Pham","year":"2025"},{"key":"ref27","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28159982.v1","article-title":"Reactivity of HCA1-HCA8 to VHHs.","author":"N Pham","year":"2025"},{"key":"ref28","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28160042.v1","article-title":"Determination of EC50 values of ciA-C2 and VHH-A3 against HCA1-HCA8.","author":"N Pham","year":"2025"},{"key":"ref29","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28160051.v1","article-title":"The overall amino acid sequence identity among BoNT\/A subtypes using A1 as the benchmark.","author":"N Pham","year":"2025"},{"key":"ref30","doi-asserted-by":"publisher","DOI":"10.6084\/m9.figshare.28171994.v1","author":"N Pham","year":"2025 cited 2025Jan10","journal-title":"figshare."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/14-163\/v1\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/14-163\/v1\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/14-163\/v1\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,3,7]],"date-time":"2025-03-07T01:15:20Z","timestamp":1741310120000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/14-163\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,5]]},"references-count":30,"URL":"https:\/\/doi.org\/10.12688\/f1000research.160607.1","relation":{"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.176528.r366733","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.176528.r366732","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.176528.r366729","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.176528.r366729","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.176528.r366732","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.176528.r366733","asserted-by":"object"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,5]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/14-163\/v1#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.176528.r366732, Ilias B Esmagambetov, Gamaleya Research Center for Epidemiology and Microbiology, Moscow, 28 Feb 2025, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/14-163\/v1#referee-response-366732","order":0,"name":"referee-response-366732","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.176528.r366733, Jianlong Lou, University of California San Francisco, San Francisco, California, 03 Mar 2025, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/14-163\/v1#referee-response-366733","order":1,"name":"referee-response-366733","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.176528.r366729, Charles B Shoemaker, Tufts University, Medford, Massachusetts, 04 Mar 2025, version 1, indexed","URL":"https:\/\/f1000research.com\/articles\/14-163\/v1#referee-response-366729","order":2,"name":"referee-response-366729","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Research reported in this publication was supported by Hanoi University of Science and Technology [Grant number: T2022-T\u0110-002]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":3,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}