{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T21:28:39Z","timestamp":1779312519754,"version":"3.51.4"},"reference-count":57,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Inventprise INC."},{"name":"Gates foundation","award":["OPP11752192017"],"award-info":[{"award-number":["OPP11752192017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: The immunogenicity of polysaccharide conjugate vaccines is critically influenced by the choice of carrier protein, which promotes a T-cell-dependent immune response mechanism leading to strong antibody production. In this study, the cholera toxin B subunit (CTB), a non-toxic pentameric protein, was evaluated as a novel carrier protein for pneumococcal polysaccharide antigens. Methods: Recombinant CTB was produced in Escherichia coli and purified using scalable chromatographic methods. Pneumococcal polysaccharides from serotypes 7F, 22F, and 33F were chemically activated with CDAP and conjugated to CTB. Results: The resulting glycoconjugates were characterized by SEC-MALS, confirming successful conjugation, high molecular weights, consistent polysaccharide-to-protein ratios, and acceptable endotoxin levels. Immunogenicity was assessed in rabbits following immunization with alum-adjuvanted formulations. Conclusions: Robust IgG responses were elicited by all CTB-based conjugates, with antibody levels found to be comparable to those induced by CRM197 conjugates, demonstrating the potential of CTB as a promising alternative for the next generation of conjugate vaccines.<\/jats:p>","DOI":"10.3390\/vaccines13111159","type":"journal-article","created":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T12:57:13Z","timestamp":1763038633000},"page":"1159","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Evaluation of Cholera Toxin B Subunit as a Novel Carrier Protein for Polysaccharide Conjugate Vaccines"],"prefix":"10.3390","volume":"13","author":[{"given":"Chathuranga","family":"Siriwardhana","sequence":"first","affiliation":[{"name":"Bacterial Research and Development, Inventprise, Inc., Redmond, WA 98052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aakriti","family":"Bajracharya","sequence":"additional","affiliation":[{"name":"Bacterial Research and Development, Inventprise, Inc., Redmond, WA 98052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Florence","family":"Seal","sequence":"additional","affiliation":[{"name":"Immunology, Manufacturing Sciences and Technology, Inventprise, Inc., Redmond, WA 98052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anup","family":"Datta","sequence":"additional","affiliation":[{"name":"Bacterial Research and Development, Inventprise, Inc., Redmond, WA 98052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-6642-1128","authenticated-orcid":false,"given":"Subhash","family":"Kapre","sequence":"additional","affiliation":[{"name":"Inventprise, Inc., Redmond, WA 98052, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"12283","DOI":"10.1073\/pnas.1400472111","article-title":"History of vaccination","volume":"111","author":"Plotkin","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","first-page":"593","article-title":"Vaccination and global health","volume":"21","year":"2003","journal-title":"Vaccine"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"140","DOI":"10.2471\/BLT.07.040089","article-title":"Vaccination greatly reduces disease, disability, death and inequity worldwide","volume":"86","author":"Andre","year":"2008","journal-title":"Bull. World Health Organ."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1016\/S1474-4422(06)70409-4","article-title":"Pneumococcal meningitis in adults: New approaches to management and prevention","volume":"5","author":"Weisfelt","year":"2006","journal-title":"Lancet Neurol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1543","DOI":"10.1016\/S0140-6736(09)61114-4","article-title":"Pathogenesis, treatment, and prevention of pneumococcal pneumonia","volume":"374","author":"Opal","year":"2009","journal-title":"Lancet"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"893","DOI":"10.1016\/S0140-6736(09)61204-6","article-title":"Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: Global estimates","volume":"374","author":"Wolfson","year":"2009","journal-title":"Lancet"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/s41479-022-00097-y","article-title":"The remarkable history of pneumococcal vaccination: An ongoing challenge","volume":"14","author":"Musher","year":"2022","journal-title":"Pneumonia"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1962","DOI":"10.1016\/S0140-6736(10)62225-8","article-title":"Serotype replacement in disease after pneumococcal vaccination","volume":"378","author":"Weinberger","year":"2011","journal-title":"Lancet"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"ofae220","DOI":"10.1093\/ofid\/ofae220","article-title":"Immunogenicity of Current and Next-Generation Pneumococcal Conjugate Vaccines in Children: Current Challenges and Upcoming Opportunities","volume":"11","author":"Feemster","year":"2024","journal-title":"Open Forum Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1278346","DOI":"10.3389\/fimmu.2023.1278346","article-title":"Recent progress in pneumococcal protein vaccines","volume":"14","author":"Li","year":"2023","journal-title":"Front. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1146\/annurev.iy.13.040195.003255","article-title":"T Cell-Independent Antigens Type 2","volume":"13","author":"Mond","year":"1995","journal-title":"Annu. Rev. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Siegrist, C.A. (2018). Vaccine Immunology. Plotkin\u2019s Vaccines, Elsevier. Available online: https:\/\/linkinghub.elsevier.com\/retrieve\/pii\/B978032335761600002X.","DOI":"10.1016\/B978-0-323-35761-6.00002-X"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2505","DOI":"10.4161\/hv.26109","article-title":"Protein carriers of conjugate vaccines: Characteristics, development and clinical trials","volume":"9","author":"Pichichero","year":"2013","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1602","DOI":"10.1038\/nm.2535","article-title":"A mechanism for glycoconjugate vaccine activation of the adaptive immune system and its implications for vaccine design","volume":"17","author":"Avci","year":"2011","journal-title":"Nat. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2194","DOI":"10.1016\/j.vaccine.2006.11.032","article-title":"Immunogenicity, efficacy, safety and effectiveness of pneumococcal conjugate vaccines (1998\u20132006)","volume":"25","author":"Beutels","year":"2007","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1016\/S1473-3099(08)70152-X","article-title":"Haemophilus influenzae type b conjugate vaccine use and effectiveness","volume":"8","author":"Morris","year":"2008","journal-title":"Lancet Infect. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4222","DOI":"10.1016\/j.vaccine.2018.06.002","article-title":"Meningococcal C conjugate vaccine effectiveness before and during an outbreak of invasive meningococcal disease due to Neisseria meningitidis serogroup C\/cc11, Tuscany, Italy","volume":"36","author":"Pezzotti","year":"2018","journal-title":"Vaccine"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1174","DOI":"10.1086\/651418","article-title":"Cellular CD4 T Cell Responses to the Diphtheria-Derived Carrier Protein of Conjugated Pneumococcal Vaccine and Antibody Response to Pneumococcal Vaccination in HIV-Infected Adults","volume":"50","author":"Rabian","year":"2010","journal-title":"Clin. Infect. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4335","DOI":"10.1016\/j.vaccine.2010.04.072","article-title":"Overview of the development and current use of CRM197 conjugate vaccines for pediatric use","volume":"28","author":"Shinefield","year":"2010","journal-title":"Vaccine"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1385834","DOI":"10.3389\/fmicb.2024.1385834","article-title":"Safety and immunogenicity of Vi-diphtheria toxoid typhoid conjugate vaccine among children below 2 years: A systematic review and meta-analysis","volume":"15","author":"Taha","year":"2024","journal-title":"Front. Microbiol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5103","DOI":"10.1016\/j.vaccine.2022.07.011","article-title":"Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein","volume":"40","author":"Chang","year":"2022","journal-title":"Vaccine"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4881","DOI":"10.1016\/j.vaccine.2011.04.053","article-title":"Comparative effects of carrier proteins on vaccine-induced immune response","volume":"29","author":"Knuf","year":"2011","journal-title":"Vaccine"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1423","DOI":"10.1016\/j.vaccine.2014.01.047","article-title":"Carrier priming or suppression: Understanding carrier priming enhancement of anti-polysaccharide antibody response to conjugate vaccines","volume":"32","author":"Pobre","year":"2014","journal-title":"Vaccine"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2319","DOI":"10.4049\/jimmunol.135.4.2319","article-title":"Carrier-induced epitopic suppression, a major issue for future synthetic vaccines","volume":"135","author":"Schutze","year":"1985","journal-title":"J. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.4161\/hv.28371","article-title":"Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice","volume":"10","author":"Hou","year":"2014","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4621","DOI":"10.4049\/jimmunol.155.10.4621","article-title":"Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4","volume":"155","author":"Marinaro","year":"1995","journal-title":"J. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1016\/0264-410X(93)90039-Z","article-title":"Cholera toxin and cholera B subunit as oral\u2014Mucosal adjuvant and antigen vector systems","volume":"11","author":"Holmgren","year":"1993","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"432","DOI":"10.1046\/j.1365-3083.2003.01248.x","article-title":"The Cholera Toxin B Subunit is a Mucosal Adjuvant for Oral Tolerance Induction in Type 1 Diabetes","volume":"57","author":"Bregenholt","year":"2003","journal-title":"Scand. J. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"S45","DOI":"10.1038\/nm1213","article-title":"Mucosal immunity and vaccines","volume":"11","author":"Holmgren","year":"2005","journal-title":"Nat. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1038\/nri1777","article-title":"Mucosal vaccines: The promise and the challenge","volume":"6","author":"Neutra","year":"2006","journal-title":"Nat. Rev. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1038\/nri3251","article-title":"Recent progress in mucosal vaccine development: Potential and limitations","volume":"12","author":"Lycke","year":"2012","journal-title":"Nat. Rev. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"579","DOI":"10.3390\/vaccines3030579","article-title":"Cholera Toxin Subunit B as Adjuvant\u2013\u2013An Accelerator in Protective Immunity and a Break in Autoimmunity","volume":"3","author":"Stratmann","year":"2015","journal-title":"Vaccines"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5716","DOI":"10.1128\/IAI.69.9.5716-5725.2001","article-title":"Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells","volume":"69","author":"Eriksson","year":"2001","journal-title":"Infect. Immun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1740","DOI":"10.1128\/IAI.71.4.1740-1747.2003","article-title":"Cholera Toxin and Its B Subunit Promote Dendritic Cell Vaccination with Different Influences on Th1 and Th2 Development","volume":"71","author":"Eriksson","year":"2003","journal-title":"Infect. Immun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"8278","DOI":"10.4049\/jimmunol.181.12.8278","article-title":"B Lymphocytes Promote Expansion of Regulatory T Cells in Oral Tolerance: Powerful Induction by Antigen Coupled to Cholera Toxin B Subunit","volume":"181","author":"Sun","year":"2008","journal-title":"J. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1362289","DOI":"10.3389\/fimmu.2024.1362289","article-title":"The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity","volume":"15","author":"Bonifaz","year":"2024","journal-title":"Front. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1038\/s41577-021-00583-2","article-title":"Mucosal vaccines\u2014Fortifying the frontiers","volume":"22","author":"Lavelle","year":"2022","journal-title":"Nat. Rev. Immunol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e13972","DOI":"10.1371\/journal.pone.0013972","article-title":"Intranasal Immunization with Influenza VLPs Incorporating Membrane-Anchored Flagellin Induces Strong Heterosubtypic Protection","volume":"5","author":"Wang","year":"2010","journal-title":"PLoS ONE"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3594","DOI":"10.1128\/iai.62.8.3594-3597.1994","article-title":"Successful immunization against gastric infection with Helicobacter species: Use of a cholera toxin B-subunit-whole-cell vaccine","volume":"62","author":"Lee","year":"1994","journal-title":"Infect. Immun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"100","DOI":"10.1086\/313608","article-title":"Which Pneumococcal Serogroups Cause the Most Invasive Disease: Implications for Conjugate Vaccine Formulation and Use, Part, I","volume":"30","author":"Hausdorff","year":"2000","journal-title":"Clin. Infect. Dis."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2790","DOI":"10.1098\/rstb.2011.0032","article-title":"Contribution of vaccines to our understanding of pneumococcal disease","volume":"12","author":"Klugman","year":"2011","journal-title":"Philos. Trans. R. Soc. B"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1016\/S1473-3099(04)00938-7","article-title":"Streptococcus pneumoniae colonisation: The key to pneumococcal disease","volume":"4","author":"Bogaert","year":"2004","journal-title":"Lancet Infect. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1038\/nrd3012","article-title":"Carbohydrate vaccines: Developing sweet solutions to sticky situations?","volume":"9","author":"Astronomo","year":"2010","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"3823","DOI":"10.1128\/iai.57.12.3823-3827.1989","article-title":"Comparative immunogenicities of Vi polysaccharide-protein conjugates composed of cholera toxin or its B subunit as a carrier bound to high- or lower-molecular-weight Vi","volume":"57","author":"Szu","year":"1989","journal-title":"Infect. Immun."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1093\/femsre\/fuy011","article-title":"Potential targets for next generation antimicrobial glycoconjugate vaccines","volume":"42","author":"Micoli","year":"2018","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1257","DOI":"10.1586\/erv.10.120","article-title":"Carbohydrate-based vaccines: Challenges and opportunities","volume":"9","author":"Huang","year":"2010","journal-title":"Expert Rev. Vaccines"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1016\/j.vaccine.2017.12.045","article-title":"Development of an FHbp-CTB holotoxin-like chimera and the elicitation of bactericidal antibodies against serogroup B Neisseria meningitidis","volume":"36","author":"Price","year":"2018","journal-title":"Vaccine"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1128\/IAI.69.1.297-306.2001","article-title":"Group B Streptococcus Capsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccines Prepared by Different Methods for Intranasal Immunization","volume":"69","author":"Shen","year":"2001","journal-title":"Infect. Immun."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2021","DOI":"10.1128\/iai.63.5.2021-2025.1995","article-title":"Local and systemic antibody responses to dextran-cholera toxin B subunit conjugates","volume":"63","author":"Bergquist","year":"1995","journal-title":"Infect. Immun."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"800","DOI":"10.1111\/j.1699-0463.1998.tb00226.x","article-title":"Antibody responses in serum and lung to intranasal immunization with Haemophilus influenzae type b polysaccharide conjugated to cholera toxin B subunit and tetanus toxoid","volume":"106","author":"Bergquist","year":"1998","journal-title":"APMIS"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/0264-410X(95)00195-7","article-title":"Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate for use in protein\u2014Polysaccharide conjugate vaccines and immunological reagents","volume":"14","author":"Lees","year":"1996","journal-title":"Vaccine"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/0003-2697(72)90042-5","article-title":"Differential determination of glucose and fructose, and glucose- and fructose-yielding substances with anthrone","volume":"50","author":"Halhoul","year":"1972","journal-title":"Anal. Biochem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1016\/S0731-7085(99)00183-1","article-title":"Quantitation of low level unconjugated polysaccharide in tetanus toxoid-conjugate vaccine by HPAEC\/PAD following rapid separation by deoxycholate\/HCl","volume":"21","author":"Lei","year":"2000","journal-title":"J. Pharm. Biomed. Anal."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1093\/protein\/11.7.577","article-title":"The affinity of cholera toxin for Ni2+ ion","volume":"11","author":"Dertzbaugh","year":"1998","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_55","first-page":"113","article-title":"Production and Purifi-cation of Recombinant B Subunit of Vibrio cholerae Toxin in Escherichia coli","volume":"13","author":"Khastar","year":"2022","journal-title":"J. Cell Mol. Res."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"1268","DOI":"10.1016\/j.vaccine.2016.01.034","article-title":"Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination","volume":"34","author":"Tamaki","year":"2016","journal-title":"Vaccine"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1006\/prep.1994.1071","article-title":"Recombinant Cholera Toxin B-Subunit in Escherichia coli: High-Level Secretion, Purification, and Characterization","volume":"5","author":"Slos","year":"1994","journal-title":"Protein Expr. Purif."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/11\/1159\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T12:25:25Z","timestamp":1764937525000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/11\/1159"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,13]]},"references-count":57,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["vaccines13111159"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13111159","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,13]]}}}