{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:57:27Z","timestamp":1760057847732,"version":"build-2065373602"},"reference-count":30,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,2,26]],"date-time":"2025-02-26T00:00:00Z","timestamp":1740528000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Vaccination may represent a suitable strategy for preventing botulism. The recombinant expression of toxin functional domains can induce effective immune responses against botulism. This study aims to develop a safe and effective recombinant detoxified full-length BoNTA vaccine. In this study, we engineered and mutated the toxin activity-related sites on the basis of the full-length BoNTA protein and constructed three detoxified full-length toxin mutants. They were recombinant expressed and purified in Escherichia coli, and the BoNTA\/M4 mutant was determined to have the highest safety, with a murine lethal dose of 50% (MLD50). The M4 protein was used as the antigen for three immunizations, and the serum titers, neutralizing activity, and BoNTA protective effects of immunized mice were evaluated. The results show that, in comparison to the receptor-binding domain BoNTA\/Hc protein, the full-length detoxified mutant M4 protein exhibited superior immunogenicity and could induce higher levels of specific antibodies, and the resulting immune serum could effectively protect mice against higher doses of BoNTA challenge. This study laid the foundation for research on a novel recombinant detoxified full-length botulinum toxin vaccine.<\/jats:p>","DOI":"10.3390\/vaccines13030243","type":"journal-article","created":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T03:37:57Z","timestamp":1740627477000},"page":"243","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Recombinant Detoxified Holotoxin as a Potent Candidate Vaccine Against Botulism"],"prefix":"10.3390","volume":"13","author":[{"given":"Zhixin","family":"Meng","sequence":"first","affiliation":[{"name":"Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Department of Microbial and Biochemical Pharmaceutics, College of Life Science, Hebei University, 180 Wusi East Road Street, Baoding 071002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chunlin","family":"Cheng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Department of Microbial and Biochemical Pharmaceutics, College of Life Science, Hebei University, 180 Wusi East Road Street, Baoding 071002, China"},{"name":"Academy of Military Medical Sciences, Beijing 100071, China"},{"name":"Lanzhou University of Information Science and Technology, Lanzhou 730300, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guoqing","family":"Xiong","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiazhen","family":"Cui","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuzhong","family":"Feng","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhili","family":"Chen","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuanyuan","family":"Lu","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xuan","family":"Huang","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Yang","sequence":"additional","affiliation":[{"name":"Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Xin","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Department of Microbial and Biochemical Pharmaceutics, College of Life Science, Hebei University, 180 Wusi East Road Street, Baoding 071002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin","family":"Ge","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Department of Microbial and Biochemical Pharmaceutics, College of Life Science, Hebei University, 180 Wusi East Road Street, Baoding 071002, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.15585\/mmwr.rr7002a1","article-title":"Clinical Guidelines for Diagnosis and Treatment of Botulism","volume":"70","author":"Rao","year":"2021","journal-title":"MMWR Recomm. Rep."},{"key":"ref_2","first-page":"33","article-title":"Botulism\u2014A rare but still present, life-threatening disease","volume":"68","year":"2019","journal-title":"Epidemiol. Mikrobiol. Imunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1128\/JB.01180-06","article-title":"Genetic diversity among botulinum neurotoxin-producing clostridial strains","volume":"189","author":"Hill","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2175","DOI":"10.3201\/eid2910.230433","article-title":"Emergence of Novel Type C Botulism Strain in Household Outbreak, Japan","volume":"29","author":"Maeda","year":"2023","journal-title":"Emerg. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1152\/physrev.2000.80.2.717","article-title":"Neurotoxins affecting neuroexocytosis","volume":"80","author":"Giampietro","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Winner, B.M., Bodt, S.M.L., and Mcnutt, P.M. (2020). Special Delivery: Potential Mechanisms of Botulinum Neurotoxin Uptake and Trafficking within Motor Nerve Terminals. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21228715"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1038\/nsb879","article-title":"Translocation of botulinum neurotoxin light chain protease through the heavy chain channel","volume":"10","author":"Koriazova","year":"2003","journal-title":"Nat. Struct. Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"9621","DOI":"10.1074\/jbc.M611211200","article-title":"Mechanism of Substrate Recognition by Botulinum Neurotoxin Serotype A","volume":"282","author":"Chen","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1137","DOI":"10.1002\/1097-4598(200007)23:7<1137::AID-MUS21>3.0.CO;2-7","article-title":"Botulism","volume":"23","author":"Maselli","year":"2000","journal-title":"Muscle Nerve"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Sundeen, G., and Barbieri, J.T. (2017). Vaccines against Botulism. Toxins, 9.","DOI":"10.3390\/toxins9090268"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1586\/erv.13.37","article-title":"What next for botulism vaccine development?","volume":"12","author":"Webb","year":"2013","journal-title":"Expert. Rev. Vaccines"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1016\/S0300-9084(00)01173-1","article-title":"Development of vaccines for prevention of botulism","volume":"82","author":"Byrne","year":"2000","journal-title":"Biochimie"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1016\/0002-9343(80)90469-6","article-title":"Hypersensitivity reactions associated with botulinal antitoxin","volume":"69","author":"Black","year":"1980","journal-title":"Am. J. Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/j.toxicon.2020.02.001","article-title":"Potency and stability of a trivalent, catalytically inactive vaccine against botulinum neurotoxin serotypes C, E and F (triCEF)","volume":"176","author":"Webb","year":"2020","journal-title":"Toxicon"},{"key":"ref_15","unstructured":"Centers for Disease Control and Prevention (CDC) (2011). Notice of CDC\u2019s discontinuation of investigational pentavalent (ABCDE) botulinum toxoid vaccine for workers at risk for occupational exposure to botulinum toxins. MMWR Morb. Mortal. Wkly. Rep., 60, 1454\u20131455."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4490","DOI":"10.1016\/j.vaccine.2009.05.030","article-title":"Production of catalytically inactive BoNT\/A1 holoprotein and comparison with BoNT\/A1 subunit vaccines against toxin subtypes A1, A2, and A3","volume":"27","author":"Webb","year":"2009","journal-title":"Vaccine"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1781","DOI":"10.1128\/IAI.68.4.1781-1786.2000","article-title":"Role of Toxin Functional Domains in Anthrax Pathogenesis","volume":"68","author":"Brossier","year":"2000","journal-title":"Infect. Immun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2795","DOI":"10.1128\/IAI.01252-08","article-title":"Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes","volume":"77","author":"Shone","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Webb, R., Smith, T.J., Smith, L.A., Wright, P.M., Guernieri, R.L., Brown, L.J., and Skerry, C. (2017). Recombinant botulinum neurotoxin Hc subunit (BoNT Hc) and catalytically inactive Clostridium botulinum holoproteins (ciBoNT HPs) as vaccine candidates for the prevention of botulism. Toxins, 9.","DOI":"10.3390\/toxins9090269"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"827","DOI":"10.1016\/j.vaccine.2017.12.064","article-title":"Enhancing toxin-based vaccines against botulism","volume":"36","author":"Przedpelski","year":"2018","journal-title":"Vaccine"},{"key":"ref_21","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_22","doi-asserted-by":"crossref","first-page":"23479","DOI":"10.1016\/S0021-9258(18)35863-0","article-title":"Botulinum neurotoxins are zinc proteins","volume":"267","author":"Schiavo","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"6637","DOI":"10.1021\/bi036278w","article-title":"Structural Analysis of Botulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212\u2192Gln Reveals the Pivotal Role of the Glu212 Carboxylate in the Catalytic Pathway","volume":"43","author":"Agarwal","year":"2004","journal-title":"Biochemistry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2399","DOI":"10.1021\/bi992321x","article-title":"Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain","volume":"39","author":"Li","year":"2000","journal-title":"Biochemistry"},{"key":"ref_25","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. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1717","DOI":"10.1021\/bi0157969","article-title":"Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization","volume":"41","author":"Binz","year":"2002","journal-title":"Biochemistry"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1151","DOI":"10.1016\/S0041-0101(00)00252-X","article-title":"Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity","volume":"39","author":"Rossetto","year":"2001","journal-title":"Toxicon"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3755","DOI":"10.1080\/21645515.2018.1547613","article-title":"Development and evaluation of candidate subunit vaccine against botulinum neurotoxin serotype B","volume":"15","author":"Shi","year":"2019","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1128\/IAI.00843-07","article-title":"Recombinant holotoxoid vaccine against botulism","volume":"76","author":"Pier","year":"2008","journal-title":"Infect. Immun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1615\/CritRevImmunol.v27.i4.20","article-title":"Botulinum neurotoxin vaccines: Past, present and future","volume":"27","author":"Smith","year":"2007","journal-title":"Crit. Rev. Immunol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/3\/243\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:43:34Z","timestamp":1760028214000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/3\/243"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,26]]},"references-count":30,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["vaccines13030243"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13030243","relation":{},"ISSN":["2076-393X"],"issn-type":[{"type":"electronic","value":"2076-393X"}],"subject":[],"published":{"date-parts":[[2025,2,26]]}}}