A six-proline stabilized trimeric spike DNA vaccine induces robust immunity and protects against SARS-CoV-2 delta challenge in K18-hACE2 transgenic mice

Despite the widespread deployment of vaccines, SARS-CoV-2 continues to pose a serious global health challenge due to the emergence of vaccine-resistant variants. DNA vaccines represent a critical platform for pandemic preparedness due to their ability to stimulate both arms of the immune response, their superior temperature stability, and their rapid scalability. In this study, we describe the immunogenicity and protective efficiency of a DNA vaccine encoding a six-proline stabilized trimeric spike protein (pSTDSpike) designed against the SARS-CoV-2 Delta variant. The pSTDSpike encodes Delta Spike protein stabilized in a prefusion trimeric conformation via six proline (6 P) and two alanine substitutions, complemented by a T4 fibritin foldon domain. Robust expression of the STDSpike protein was confirmed in vitro using HEK293T cells. To assess immunogenicity, BALB/c mice were administered three times via intramuscular (IM) injection or intradermal injection coupled with electroporation (ID+EP). Both vaccinations elicited potent humoral immune response, as evidenced by high anti-S1 IgG titers (∼22,000 for IM and ∼83,000 for ID+EP), > 80% inhibition activity, and significantly elevated neutralizing antibody titers (194.01 for IM and 337.79 for ID+EP). Furthermore, pSTDSpike induced a robust Th1-biased cellular response, characterized by significantly elevated IFN-γ secretion and increased cytotoxic CD8 + T-cells in restimulated splenocyte cultures. In a lethal challenge model using K18-hACE2 transgenic mice, pSTDSpike vaccination conferred 77.7% protection, without any detectable viral load, pneumonia or alveolar inflammation in surviving animals. These results suggest that the STDSpike DNA vaccine platform is highly effective and adaptable for addressing emerging variants and future pandemic threats.