Nam, Yunju Shin, Sang Chul Cho, Sang Won Ahn, Hyung Jun 2026-01-30T09:00:05Z 2026-01-30T09:00:05Z 2026-01-29 2026-02 https://pubs.kist.re.kr/handle/201004/154081 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–based DNA platforms have been developed to overcome nuclear entry barriers, their utility for antigen structure–function 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⁺ 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–function optimization and supports the development of next-generation DNA vaccines targeting rapidly evolving viral pathogens. English Multidisciplinary Digital Publishing Institute (MDPI) Direct Cytoplasmic Transcription and Trimeric RBD Design Synergize to Enhance DNA Vaccine Potency Against SARS-CoV-2 Article 10.3390/pharmaceutics18020164 1 Pharmaceutics, v.18, no.2 Pharmaceutics 18 2 N scie 001700130400001 Pharmacology & Pharmacy Pharmacology & Pharmacy Article LIPID NANOPARTICLES PLASMID DNA EXPRESSION CANDIDATE COVID-19 SPIKE MICE MESSENGER-RNA VACCINES T7 autogene system receptor-binding domain (RBD) SARS-CoV-2 trimeric RBD DNA vaccine cytoplasmic transcription neutralizing antibody