{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,1]],"date-time":"2026-04-01T06:28:51Z","timestamp":1775024931977,"version":"3.50.1"},"reference-count":34,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2017,9,3]],"date-time":"2017-09-03T00:00:00Z","timestamp":1504396800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>There are few available medical countermeasures against botulism and the discontinuation of the pentavalent botulinum toxoid vaccine by the Centers for Disease Control and Prevention in 2011 has resulted in the need for a safe and effective prophylactic alternative. Advances in genetic engineering have resulted in subsequent vaccine efforts being primarily focused on the production of highly purified recombinant protein antigens representing one or more domains of the botulinum neurotoxin. Recombinant subunit vaccines based on the carboxy one-third of the toxin (Hc) developed in our lab against serotypes A-F have been shown to be safe and effective. However, in response to the identification of an ever increasing number of BoNT subtypes with significant amino acid heterogeneity, we have developed catalytically inactive BoNT holoproteins (ciBoNT HPs) in an attempt to elicit greater protective immunity to address these toxin variants. Here we report the production of ciBoNT\/B1 HP, ciBoNT\/C1 HP, ciBoNT\/E1 HP and ciBoNT\/F1 HP and compare the immunological and protective abilities of ciBoNT HPs and BoNT\/A Hc, BoNT\/B Hc, BoNT\/C Hc, BoNT\/E Hc and BoNT\/F Hc vaccines when challenged with homologous and heterologous toxins. Our results suggest the ciBoNT HP vaccines exhibit superior potency after single vaccinations but multiple vaccinations with BoNT\/Hc antigens resulted in increased survival rates at the toxin challenge levels used.<\/jats:p>","DOI":"10.3390\/toxins9090269","type":"journal-article","created":{"date-parts":[[2017,9,4]],"date-time":"2017-09-04T11:11:52Z","timestamp":1504523512000},"page":"269","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":44,"title":["Recombinant Botulinum Neurotoxin Hc Subunit (BoNT Hc) and Catalytically Inactive Clostridium botulinum Holoproteins (ciBoNT HPs) as Vaccine Candidates for the Prevention of Botulism"],"prefix":"10.3390","volume":"9","author":[{"given":"Robert P.","family":"Webb","sequence":"first","affiliation":[{"name":"US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Theresa J.","family":"Smith","sequence":"additional","affiliation":[{"name":"Ke\u2019aki Technologies LLC, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leonard A.","family":"Smith","sequence":"additional","affiliation":[{"name":"Office of the Chief Scientist, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick M.","family":"Wright","sequence":"additional","affiliation":[{"name":"Clinical Research Management, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21707, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rebecca L.","family":"Guernieri","sequence":"additional","affiliation":[{"name":"Ke\u2019aki Technologies LLC, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer L.","family":"Brown","sequence":"additional","affiliation":[{"name":"Ke\u2019aki Technologies LLC, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janet C.","family":"Skerry","sequence":"additional","affiliation":[{"name":"Ke\u2019aki Technologies LLC, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2017,9,3]]},"reference":[{"key":"ref_1","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_2","doi-asserted-by":"crossref","first-page":"1917","DOI":"10.1016\/j.vaccine.2012.01.035","article-title":"Preclinical safety assessment of recombinant botulinum vaccine A\/B (rBV A\/B)","volume":"30","author":"Shearer","year":"2012","journal-title":"Vaccine"},{"key":"ref_3","unstructured":"Flynn, D. (2012, March 19). Canned ripe california olives spread botulism in 1919. Available online: http:\/\/www.foodsafetynews.com\/2012\/03\/canned-ripe-california-olives-spread-botulism-in-1919\/#.WarKm2c4Gac."},{"key":"ref_4","first-page":"003","article-title":"Botulinum neurotoxins as biothreat agents","volume":"S2","author":"Smith","year":"2012","journal-title":"J. Bioterror. Biodef."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"309","DOI":"10.4049\/jimmunol.55.4.309","article-title":"Studies on botulinum toxoids, types A and B iii. Immunization of man","volume":"55","author":"Reames","year":"1947","journal-title":"J. Immunol."},{"key":"ref_6","unstructured":"Lewis, K.H., and Cassel, K. (1966). Botulism: Proceedings of a symposium, Public Health Service."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"D33","DOI":"10.1016\/j.vaccine.2009.08.059","article-title":"Botulism and vaccines for its prevention","volume":"27","author":"Smith","year":"2009","journal-title":"Vaccine"},{"key":"ref_8","unstructured":"CDC (2011). Notice of CDC\u2019s discontinuation of investigational entavalent (abcde) botulinum toxoid vaccine for workers at risk for potential exposure to botulinum toxins. MMWR, 60, 1454\u20131455."},{"key":"ref_9","unstructured":"Ellis, R.W., and Brodeur, B.R. (2012). New Bacterial Vaccines, Springer Science & Business Media."},{"key":"ref_10","unstructured":"Plotkin, S.A., Offit, P.A., Orenstein, W.A., and Edwards, K.M. (2017). Plotkin\u2019s vaccines, Elsevier. [7th ed.]."},{"key":"ref_11","first-page":"59","article-title":"Analysis of the immune response to papain digestion products of tetanus toxin","volume":"92","author":"Helting","year":"1984","journal-title":"Acta Pathol. Microbiol. Immunol. Scand. C"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2541","DOI":"10.1128\/iai.55.11.2541-2545.1987","article-title":"Immunization of mice against tetanus with fragments of tetanus toxin synthesized in escherichia coli","volume":"55","author":"Fairweather","year":"1987","journal-title":"Infect. Immun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1627","DOI":"10.1128\/iai.59.5.1627-1632.1991","article-title":"Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system","volume":"59","author":"Charles","year":"1991","journal-title":"Infect. Immun."},{"key":"ref_14","first-page":"1","article-title":"Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes","volume":"364","author":"Hill","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5450","DOI":"10.1128\/IAI.73.9.5450-5457.2005","article-title":"Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization","volume":"73","author":"Smith","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1314","DOI":"10.1128\/IAI.01025-07","article-title":"Subunit vaccine against the seven serotypes of botulism","volume":"76","author":"Baldwin","year":"2008","journal-title":"Infect. Immun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3043","DOI":"10.1128\/IAI.01893-06","article-title":"Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route","volume":"75","author":"Ravichandran","year":"2007","journal-title":"Infect. Immun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1128\/CVI.00496-09","article-title":"Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system","volume":"17","author":"Zichel","year":"2010","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_19","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_20","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.pep.2009.12.013","article-title":"Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery","volume":"71","author":"Band","year":"2010","journal-title":"Protein Expr. Purif."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Vazquez-Cintron, E.J., Vakulenko, M., Band, P.A., Stanker, L.H., Johnson, E.A., and Ichtchenko, K. (2014). Atoxic derivative of botulinum neurotoxin A as a prototype molecular vehicle for targeted delivery to the neuronal cytoplasm. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0085517"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"42923","DOI":"10.1038\/srep42923","article-title":"Engineering botulinum neurotoxin C1 as a molecular vehicle for intra-neuronal drug delivery","volume":"7","author":"Beske","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4586","DOI":"10.1128\/iai.65.11.4586-4591.1997","article-title":"Induction of an immune response by oral administration of recombinant botulinum toxin","volume":"65","author":"Kiyatkin","year":"1997","journal-title":"Infect. Immun."},{"key":"ref_24","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_25","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_26","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1007\/s11095-015-1816-x","article-title":"In vivo toxicity and immunological characterization of detoxified recombinant botulinum neurotoxin type A","volume":"33","author":"Ravichandran","year":"2016","journal-title":"Pharm. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1016\/j.pep.2005.08.015","article-title":"Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris gs115","volume":"45","author":"Dux","year":"2006","journal-title":"Protein Expr. Purif."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3997","DOI":"10.1074\/jbc.M707917200","article-title":"Molecular architecture of botulinum neurotoxin E revealed by single particle electron microscopy","volume":"283","author":"Fischer","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.jmb.2008.12.027","article-title":"Domain organization in clostridium botulinum neurotoxin type E is unique: its implication in faster translocation","volume":"386","author":"Kumaran","year":"2009","journal-title":"J. Mol. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"16993","DOI":"10.1074\/jbc.M710442200","article-title":"Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics","volume":"283","author":"Wang","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4273","DOI":"10.1016\/j.vaccine.2007.02.081","article-title":"Protection with recombinant clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins","volume":"25","author":"Webb","year":"2007","journal-title":"Vaccine"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4817","DOI":"10.1128\/IAI.66.10.4817-4822.1998","article-title":"Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate","volume":"66","author":"Byrne","year":"1998","journal-title":"Infect. Immun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1006\/prep.1998.0910","article-title":"Production and purification of the heavy-chain fragment C of botulinum neurotoxin, serotype B, expressed in the methylotrophic yeast Pichia pastoris","volume":"13","author":"Potter","year":"1998","journal-title":"Protein Expr. Purif."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1006\/prep.2000.1200","article-title":"Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris","volume":"18","author":"Byrne","year":"2000","journal-title":"Protein Expr. 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