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

Area of Science:

• Vaccinology
• Molecular Biology
• Immunology

Background:

• Emerging SARS-CoV-2 variants necessitate adaptable vaccine strategies.
• Trimeric RBD antigens offer advantages over monomeric forms, but DNA vaccine efficacy is limited by poor antigen expression.
• Cytoplasmic transcription DNA platforms can overcome nuclear entry barriers, but their use for antigen optimization is underexplored.

Purpose of the Study:

• To evaluate if integrating a trimeric RBD with a T7-driven cytoplasmic transcription system enhances DNA vaccine immunogenic performance.
• To assess the utility of this platform for antigen structure-function optimization.

Main Methods:

• A DNA vaccine encoding a tandem trimeric SARS-CoV-2 RBD was delivered via a T7 RNA polymerase-driven cytoplasmic transcription system.
• In vitro antigen expression was assessed after transfection.
• In vivo immunogenicity and safety were evaluated in mice immunized with the Rpol/tRBD/LNP formulation.

Main Results:

• The T7-driven system significantly increased antigen mRNA and protein expression compared to conventional plasmid delivery.
• Rpol/tRBD vaccination elicited higher anti-RBD IgG titers, enhanced neutralizing antibody activity, and robust CD8+ T cell responses.
• Immune responses were Th1-skewed, with germinal center activation and no significant toxicity.

Conclusions:

• Integrating trimeric antigen engineering with cytoplasmic transcription enables balanced, well-tolerated immune activation in DNA vaccines.
• The T7 autogene-based platform facilitates antigen structure-function optimization for next-generation vaccines against evolving viruses.