{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T06:01:52Z","timestamp":1785132112109,"version":"3.55.0"},"reference-count":31,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["AMP 25-574"],"award-info":[{"award-number":["AMP 25-574"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["LP 25-2382"],"award-info":[{"award-number":["LP 25-2382"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["LP 24-2368"],"award-info":[{"award-number":["LP 24-2368"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["AMP 23-1115"],"award-info":[{"award-number":["AMP 23-1115"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["AMP 22-1071"],"award-info":[{"award-number":["AMP 22-1071"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["LP 20-2256"],"award-info":[{"award-number":["LP 20-2256"]}]},{"name":"The Lion\u2019s Cancer Research Foundation at Ume\u00e5 University","award":["AMP 19-982"],"award-info":[{"award-number":["AMP 19-982"]}]},{"name":"Ume\u00e5 University Medical Faculty Planning Grants for COVID-19","award":["3453 16032"],"award-info":[{"award-number":["3453 16032"]}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Despite the widespread deployment of current COVID-19 vaccines, significant gaps remain in understanding the complete biological behavior of the SARS-CoV-2 spike (S) protein. Through systematic characterization of mammalian expression systems, this study shows that the full-length S protein exhibits a complex intracellular distribution, predominantly localizing not only to the cell membrane but also to the cytoplasm, nucleus, and extracellular compartments. Comparative analyses revealed distinct subunit-specific trafficking patterns. The S1 subunit showed increased intracellular accumulation and secretion compared to the full-length S protein, although with reduced surface expression. Conversely, the receptor-binding domain (RBD) and S2 domains were mainly associated with cytoskeletal (CS) structures. Notably, the signal sequence-enhanced RBD (SS-RBD) construct engineered in this study demonstrated dramatically enhanced extracellular accumulation, approximately 100-fold higher than the full-length S protein and 10-fold greater than S1, as measured by proximity extension assay (PEA). Signal peptide modification effectively redirected RBD from CS retention to efficient secretion, significantly improving detection sensitivity. PEA outperformed conventional methods such as flow cytometry (FACS), Western blotting (WB), and immunofluorescence, offering sensitivities several orders of magnitude higher. Consequently, these findings provide: (1) a structural framework for rational antigen design by distinguishing essential versus dispensable domains; (2) experimental support for SS-RBD as a promising vaccine candidate due to its high secretion efficiency and preservation of neutralizing epitopes; and (3) a robust platform using PEA for high-sensitivity antigen characterization. This study enhances the fundamental understanding of spike protein biology and offers actionable insights for developing next-generation vaccines targeting SARS-CoV-2 and related coronaviruses.<\/jats:p>","DOI":"10.3389\/fmicb.2026.1856886","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T05:41:08Z","timestamp":1785130868000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Systematic characterization of SARS-CoV-2 spike protein subunit trafficking and secretion reveals enhanced strategies for vaccine design and quantification"],"prefix":"10.3389","volume":"17","author":[{"given":"Qian","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Clinical Microbiology, Ume\u00e5 University","place":["Ume\u00e5, Sweden"]},{"name":"Ume\u00e5 Center for Microbial Research (UCMR), Ume\u00e5 University","place":["Ume\u00e5, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Hammond","sequence":"additional","affiliation":[{"name":"Department of Immunology, Genetics, Pathology, SciLifeLab; Proximity Proteomics Facility","place":["Uppsala, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Medical Bioscience, Ume\u00e5 University","place":["Ume\u00e5, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zongwei","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology, Ume\u00e5 University","place":["Ume\u00e5, Sweden"]},{"name":"Ume\u00e5 Center for Microbial Research (UCMR), Ume\u00e5 University","place":["Ume\u00e5, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongxing","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Immunology, Genetics, Pathology, SciLifeLab; Proximity Proteomics Facility","place":["Uppsala, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mar\u00e9ne","family":"Landstr\u00f6m","sequence":"additional","affiliation":[{"name":"Department of Medical Bioscience, Ume\u00e5 University","place":["Ume\u00e5, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ya-Fang","family":"Mei","sequence":"additional","affiliation":[{"name":"Department of Clinical Microbiology, Ume\u00e5 University","place":["Ume\u00e5, Sweden"]},{"name":"Ume\u00e5 Center for Microbial Research (UCMR), Ume\u00e5 University","place":["Ume\u00e5, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"1014","DOI":"10.1186\/s12879-026-13281-y","article-title":"Vaccine effectiveness across the omicron evolutionary spectrum (BA.2, BA.5, XBB, JN.1, KP.3): a systematic review of studies published 2022\u20132025","volume":"26","author":"Alhumaid","year":"2026","journal-title":"BMC Infect. 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