# Direct Cytoplasmic Transcription and Trimeric RBD Design Synergize to Enhance DNA Vaccine Potency Against SARS-CoV-2

## TL;DR

This study shows that combining a trimeric RBD antigen with cytoplasmic transcription improves DNA vaccine effectiveness against SARS-CoV-2.

## Contribution

The integration of trimeric RBD design with cytoplasmic transcription is novel for enhancing DNA vaccine performance.

## Key findings

- T7-driven cytoplasmic transcription significantly boosts antigen expression compared to conventional plasmid delivery.
- Rpol/tRBD vaccines induce higher antibody titers, neutralizing activity, and CD8⁺ T cell responses than monomeric or plasmid-based trimeric RBD vaccines.
- The vaccine elicits Th1-skewed immune responses with minimal toxicity and germinal center activation.

## Abstract

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.