{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T17:45:27Z","timestamp":1769622327189,"version":"3.49.0"},"reference-count":57,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,1,26]],"date-time":"2026-01-26T00:00:00Z","timestamp":1769385600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"crossref","award":["RS-2024-00454175"],"award-info":[{"award-number":["RS-2024-00454175"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Intramural Research Program of KIST"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: The emergence of immune-evasive SARS-CoV-2 variants highlights the need for adaptable vaccine strategies. Trimeric receptor-binding domain (tRBD) antigens offer structural and immunological advantages over monomeric RBDs, but DNA vaccine efficacy has been limited by inefficient antigen expression, particularly in non-dividing antigen-presenting cells. Although cytoplasmic transcription\u2013based DNA platforms have been developed to overcome nuclear entry barriers, their utility for antigen structure\u2013function optimization remains underexplored. This study evaluated whether integrating a rationally designed trimeric RBD with a T7-driven cytoplasmic transcription system could enhance immunogenic performance. Methods: A DNA vaccine encoding a tandem trimeric SARS-CoV-2 RBD was delivered using a T7 RNA polymerase-driven cytoplasmic transcription system. In vitro antigen expression was assessed following Lipofectamine 3000-mediated transfection. In vivo, mice were immunized with the SM-102-based Rpol\/tRBD\/LNP formulation, and immunogenicity was assessed by antigen-specific antibody titers, serum neutralizing activity, and T-cell response profiling, together with basic safety\/tolerability evaluations. Results: The T7-driven cytoplasmic transcription system markedly increased antigen mRNA and protein expression compared with conventional plasmid delivery. Rpol\/tRBD vaccination induced higher anti-RBD IgG titers, enhanced neutralizing antibody activity, and robust CD8\u207a T cell responses relative to monomeric RBD and plasmid-based trimeric RBD vaccines. Immune responses were Th1-skewed and accompanied by germinal center activation without excessive inflammatory cytokine induction, body-weight loss, or hepatic and renal toxicity. Conclusions: This study demonstrates that integrating rational trimeric antigen engineering with direct cytoplasmic transcription enables balanced and well-tolerated immune activation in a DNA vaccine context. The T7 autogene-based platform provides a flexible framework for antigen structure\u2013function optimization and supports the development of next-generation DNA vaccines targeting rapidly evolving viral pathogens.<\/jats:p>","DOI":"10.3390\/pharmaceutics18020164","type":"journal-article","created":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T09:05:27Z","timestamp":1769504727000},"page":"164","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Direct Cytoplasmic Transcription and Trimeric RBD Design Synergize to Enhance DNA Vaccine Potency Against SARS-CoV-2"],"prefix":"10.3390","volume":"18","author":[{"given":"Yunju","family":"Nam","sequence":"first","affiliation":[{"name":"Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0156-680X","authenticated-orcid":false,"given":"Sang Chul","family":"Shin","sequence":"additional","affiliation":[{"name":"Technological Convergence Center, Research Resources Division, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3996-7457","authenticated-orcid":false,"given":"Sang Won","family":"Cho","sequence":"additional","affiliation":[{"name":"Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9008-832X","authenticated-orcid":false,"given":"Hyung Jun","family":"Ahn","sequence":"additional","affiliation":[{"name":"Medicinal Materials Research Center, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,26]]},"reference":[{"key":"ref_1","first-page":"162","article-title":"SARS-CoV-2 variant biology: Immune escape, transmission and fitness","volume":"21","author":"Carabelli","year":"2023","journal-title":"Nat. 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