{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T18:43:36Z","timestamp":1785437016481,"version":"3.56.0"},"posted":{"date-parts":[[2025,5,30]]},"group-title":"Biochemistry","reference-count":88,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T00:00:00Z","timestamp":1748563200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["R01AI123383"],"award-info":[{"award-number":["R01AI123383"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01AI152766"],"award-info":[{"award-number":["R01AI152766"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R41AI157287"],"award-info":[{"award-number":["R41AI157287"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Nucleic acid vaccines (DNA and mRNA) induce immunity by driving in situ antigen expression in host cells. For non-viral pathogens, however, host expression can impose post-translational modifications absent from the native microbial antigen. Tuberculosis (TB) remains a leading cause of infectious mortality, and nucleic acid vaccines targeting the\n                  <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                  antigen 85 (Ag85) complex did not confer protective efficacy in clinical trials. We hypothesized that host-derived N-glycosylation of Ag85 immunogens expressed in mammalian cells compromises immune recognition. Here, we define structural, biochemical and immunological mechanisms by which host-imposed N-glycosylation remodels a bacterial antigen expressed in mammalian cells. We show that Ag85B expressed in human Expi293 cells is microheterogeneously N-glycosylated at four canonical sequons (N52, N224, N234, N280) with predominantly complex, highly fucosylated, and frequently sialylated glycans. Molecular dynamics simulations indicate that these glycans occupy substantial conformational space and reduce solvent and antibody-accessible surface area, occluding multiple established B-cell and T-cell epitope regions. Consistent with glycan-mediated shielding, mammalian-expressed Ag85B shows markedly reduced binding to an Ag85-complex monoclonal antibody by competitive ELISA and biolayer interferometry, and sialylated glycans enable Siglec-9 binding that is abrogated by sialidase treatment. Together, these findings define the structural and biochemical mechanisms by which host glycosylation can remodel bacterial vaccine antigens, supporting glycosylation-aware immunogen engineering as a design principle for nucleic acid vaccines targeting non-viral pathogens.\n                <\/jats:p>","DOI":"10.1101\/2025.05.29.656815","type":"posted-content","created":{"date-parts":[[2025,5,30]],"date-time":"2025-05-30T14:20:11Z","timestamp":1748614811000},"source":"Crossref","is-referenced-by-count":0,"title":["Molecular mechanisms of immune evasion by host protein glycosylation of a bacterial immunogen used in nucleic acid vaccines"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-5949-019X","authenticated-orcid":false,"given":"M.S.","family":"Cinar","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6661-1694","authenticated-orcid":false,"given":"T.M.","family":"Adams","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Z.","family":"Nawaz","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"E.S.","family":"Demir","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.E.","family":"Demirturk","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.P.","family":"Keelaghan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.M.","family":"Nazaar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"B.R.","family":"Roberts","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Ozdilek","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1902-7532","authenticated-orcid":false,"given":"F.Y.","family":"Avci","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, 30322"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041200110951000_2025.05.29.656815v2.1","first-page":"1182","volume-title":"Vaccines","volume":"60","year":"2013"},{"key":"2026041200110951000_2025.05.29.656815v2.2","doi-asserted-by":"publisher","DOI":"10.1002\/emmm.201403876"},{"key":"2026041200110951000_2025.05.29.656815v2.3","doi-asserted-by":"crossref","unstructured":"Paschall, A. 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