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The growing prevalence of multidrug-resistant (MDR) ExPEC strains, which rapidly erode antibiotic efficacy, underscores vaccine development as a critical priority. Bioconjugate vaccines have emerged as a promising approach to mitigate ExPEC-associated infections. Methods and Results: In this study, we utilized protein glycan coupling technology (PGCT) based on oligosaccharyltransferase (OST) PglL to engineer a tetravalent bioconjugate vaccine targeting four predominant ExPEC serotypes (O1, O2, O6, and O25). We conducted a series of experiments to demonstrate the efficacy of the conjugate vaccine in eliciting humoral immune responses and inducing the production of specific antibodies against Escherichia coli O1, O2, O6, or O25 serotypes. Conclusions: This work establishes the first application of the O-linked PGCT system for engineering bioconjugate vaccines against ExPEC infections.<\/jats:p>","DOI":"10.3390\/vaccines13040362","type":"journal-article","created":{"date-parts":[[2025,3,28]],"date-time":"2025-03-28T06:06:35Z","timestamp":1743141995000},"page":"362","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["A Bioconjugate Vaccine Against Extra-Intestinal Pathogenic Escherichia coli (ExPEC)"],"prefix":"10.3390","volume":"13","author":[{"given":"Linhui","family":"Hao","sequence":"first","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4687-2755","authenticated-orcid":false,"given":"Wenhua","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Guo","sequence":"additional","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiankai","family":"Liu","sequence":"additional","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Wu","sequence":"additional","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9975-8978","authenticated-orcid":false,"given":"Li","family":"Zhu","sequence":"additional","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chao","family":"Pan","sequence":"additional","affiliation":[{"name":"Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8476-4320","authenticated-orcid":false,"given":"Hengliang","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing 100071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1093\/infdis\/jiv429","article-title":"Extraintestinal Pathogenic Escherichia coli, a Common Human Pathogen: Challenges for Vaccine Development and Progress in the Field","volume":"213","author":"Poolman","year":"2016","journal-title":"J. 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