{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,19]],"date-time":"2026-02-19T18:42:53Z","timestamp":1771526573811,"version":"3.50.1"},"reference-count":41,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2020,2,7]],"date-time":"2020-02-07T00:00:00Z","timestamp":1581033600000},"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>The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of \u03b2-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.<\/jats:p>","DOI":"10.3390\/vaccines8010073","type":"journal-article","created":{"date-parts":[[2020,2,7]],"date-time":"2020-02-07T11:50:28Z","timestamp":1581076228000},"page":"73","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine"],"prefix":"10.3390","volume":"8","author":[{"given":"Annie","family":"Aubry","sequence":"first","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Zou","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Evguenii","family":"Vinogradov","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dean","family":"Williams","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wangxue","family":"Chen","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Greg","family":"Harris","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongyan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Melissa J.","family":"Schur","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michel","family":"Gilbert","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6654-7346","authenticated-orcid":false,"given":"Gillian R.","family":"Douce","sequence":"additional","affiliation":[{"name":"Institute of Infection, Immunity, Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, Scotland, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan M.","family":"Logan","sequence":"additional","affiliation":[{"name":"Vaccine Program, Human Health Therapeutics Research Centre, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,2,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1038\/nrmicro2164","article-title":"Clostridium difficile infection: new developments in epidiemiology and pathogenesis","volume":"7","author":"Rupnik","year":"2009","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1111\/1469-0691.12064","article-title":"Consequences of Clostridium difficile infection: understanding the healthcare burden","volume":"18","author":"Bouza","year":"2012","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2704","DOI":"10.1128\/IAI.00147-12","article-title":"The Clostridium difficile spo0A gene is a persistence and transmission factor","volume":"80","author":"Deakin","year":"2012","journal-title":"Infect. Immun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1038\/nrgastro.2010.190","article-title":"Clostridium difficile infection: epidiemiology, risk factors and management","volume":"8","author":"Ananthakrishnan","year":"2011","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1099\/mic.0.26681-0","article-title":"Identification of proteins in the exosporium of Bacillus anthracis","volume":"150","author":"Redmond","year":"2004","journal-title":"Microbiology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1046\/j.1365-2958.2000.03000.x","article-title":"A collagen-like glycoprotein is a structural component of the Bacillus anthracis exosporium","volume":"45","author":"Sylvestre","year":"2002","journal-title":"Mol. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1016\/j.tim.2014.04.003","article-title":"Clostridium difficile spore biology: sporulation, germination and spore structural proteins","volume":"22","author":"Shen","year":"2014","journal-title":"Trends Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5377","DOI":"10.1128\/JB.00597-09","article-title":"Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores","volume":"191","author":"Lawley","year":"2009","journal-title":"J. Bacteriol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1492","DOI":"10.1128\/JB.02104-12","article-title":"Functional characterization of Clostridium difficile spore coat proteins","volume":"195","author":"Permpoonpattana","year":"2013","journal-title":"J. Bacteriol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4507","DOI":"10.1021\/pr4005629","article-title":"In pursuit of protein targets: proteomic characterisation of bacterial spore outer layers","volume":"12","author":"Abhyankar","year":"2013","journal-title":"J. Proteomic. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6461","DOI":"10.1128\/JB.05182-11","article-title":"Surface layers of Clostridium difficile endospores","volume":"193","author":"Permpoonpattana","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1111\/mmi.13361","article-title":"A novel regulator controls Clostridium difficile sporulation, motility and toxin production","volume":"100","author":"Edwards","year":"2016","journal-title":"Mol. Micro."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e01991-18","DOI":"10.1128\/mBio.01991-18","article-title":"RstA is a major regulator of Clostridioides difficile toxin production and motility","volume":"10","author":"Edwards","year":"2019","journal-title":"Mbio."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2627","DOI":"10.1128\/JB.01469-14","article-title":"Identification and characterization of glycoproteins on the spore surface of Clostridium difficile","volume":"196","author":"Strong","year":"2014","journal-title":"J. Bacteriol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2202","DOI":"10.1128\/AEM.03410-15","article-title":"Ultrastructural variability of the exosporium layer of Clostridium difficile spores","volume":"82","year":"2016","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1056\/NEJMoa1602615","article-title":"Bezlotoxumab for prevention of recurrent Clostridium difficile infection","volume":"376","author":"Wilcox","year":"2017","journal-title":"New Eng. J. Med."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Simeon, R., Jiang, M., Chamoun-Emanuelli, A.M., Yu, H., Zhang, Y., Meng, R., Peng, Z., Jakana, J., Zhang, J., and Feng, H. (2019). Selection and characterisation of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. PLoS Biol., 17.","DOI":"10.1371\/journal.pbio.3000514"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Hussack, G., Ryan, S., van Faassen, H., Rossotti, M., MacKenzie, C.R., and Tanha, J. (2018). Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains. PLoS Biol., 13.","DOI":"10.1371\/journal.pone.0208978"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1452","DOI":"10.1093\/infdis\/jix488","article-title":"The spore coat protein cotE facilitates host colonization by Clostridium difficile","volume":"216","author":"Hong","year":"2017","journal-title":"J. Infect Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1016\/j.mimet.2007.05.021","article-title":"The ClosTron: a universal gene knock-out system for the genus Clostridium","volume":"70","author":"Heap","year":"2007","journal-title":"J. Microbiol. Methods."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/978-1-60327-365-7_11","article-title":"ClosTron-targeted mutagenesis","volume":"646","author":"Heap","year":"2010","journal-title":"Methods Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1093\/glycob\/cwm090","article-title":"Variants of the beta 1,3-galactosyltransferase CgtB from the bacterium Campylobacter jejuni have distinct acceptor specificities","volume":"17","author":"Bernatchez","year":"2007","journal-title":"Glycobiology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1984","DOI":"10.1053\/j.gastro.2008.09.002","article-title":"A mouse model of Clostridium difficile-associated disease","volume":"135","author":"Chen","year":"2008","journal-title":"Gastroenterology"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1006\/jmbi.2000.4315","article-title":"Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes","volume":"305","author":"Krogh","year":"2001","journal-title":"J. Mol. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"764","DOI":"10.1038\/nsmb.1443","article-title":"Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation","volume":"15","author":"Macauley","year":"2008","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10983","DOI":"10.1074\/jbc.275.15.10983","article-title":"Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity","volume":"275","author":"Lubas","year":"2000","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"24608","DOI":"10.1074\/jbc.M300036200","article-title":"Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity","volume":"278","author":"Iyer","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1007\/978-1-61737-967-3_16","article-title":"Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner","volume":"705","author":"Sun","year":"2011","journal-title":"Methods. Mol. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1080\/21645515.2017.1395538","article-title":"Safety and immunogenicity of Clostridium difficile toxoid vaccine in Japanese adults","volume":"14","author":"Matsuoka","year":"2018","journal-title":"Hum. Vaccin. Immunother."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2170","DOI":"10.1016\/j.vaccine.2016.03.028","article-title":"Defining the optimal formulation and schedule of a candidate toxoid vaccine against Clostridium difficile infection: A randomized phase 2 clinical trial","volume":"34","author":"Saleh","year":"2016","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2585","DOI":"10.1016\/j.vaccine.2016.03.098","article-title":"Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers","volume":"34","author":"Ayad","year":"2016","journal-title":"Vaccine"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1136","DOI":"10.1093\/infdis\/148.6.1136","article-title":"Considerations for formulating the second-generation pneumococcal capsular polysaccharide vaccine with emphasis on the cross-reactive types within groups","volume":"148","author":"Robbins","year":"1983","journal-title":"J. Infect. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1542\/peds.60.5.730","article-title":"Haemophilus influenzae type b capsular polysaccharide vaccine in children: A double\u2014blind field trial of 100,000 vaccinees 3 months to 5 years of age in Finland","volume":"60","author":"Peltola","year":"1977","journal-title":"Pediatrics."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/S0140-6736(00)03594-7","article-title":"Efficacy of meningococcal serogroup C conjugate vaccine in teenagers and toddlers in England","volume":"357","author":"Ramsay","year":"2001","journal-title":"Lancet."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1007\/s10719-013-9489-3","article-title":"Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection","volume":"30","author":"Cox","year":"2013","journal-title":"Glycoconj. J."},{"key":"ref_36","first-page":"1","article-title":"Characterization of the adherence of Clostridium difficile spores: the integrity of the outermost layer affects adherence properties of spores of the epidemic strain R20291 to components of the intestinal mucosa","volume":"6","author":"Gil","year":"2016","journal-title":"Front Cell Infect Microbial."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"30945","DOI":"10.1074\/jbc.M401613200","article-title":"Novel oligosaccharide side chains of the collagen-like region of BclA, the major glycoprotein of the Bacillus anthracis exosporium","volume":"279","author":"Daubenspeck","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jprot.2015.03.035","article-title":"Protein composition of the outermost exosporium-like layer","volume":"123","author":"Milano","year":"2015","journal-title":"J. Proteomics."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.anaerobe.2015.12.001","article-title":"Immunogenicity and protective efficacy of Clostridium difficile spore proteins","volume":"37","author":"Ghose","year":"2016","journal-title":"Anaerobe"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2720","DOI":"10.1021\/acschembio.9b00642","article-title":"Synthetic oligosaccharide-based vaccines protect mice from Clostridioides difficile infections","volume":"14","author":"Broecker","year":"2019","journal-title":"ACS Chem. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1007\/978-1-4939-3387-7_21","article-title":"The design of a Clostridium difficile carbohydrate-based vaccine","volume":"1403","author":"Monteiro","year":"2016","journal-title":"Methods. Mol. Biol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/8\/1\/73\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T08:55:32Z","timestamp":1760172932000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/8\/1\/73"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,2,7]]},"references-count":41,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["vaccines8010073"],"URL":"https:\/\/doi.org\/10.3390\/vaccines8010073","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,2,7]]}}}