Effect of PEI25k/DOTAP/cholesterol liposomal formulation on the immunogenicity of SARS-CoV-2 spike protein-encoding DNA vaccine

Background The Coronavirus Disease 2019 (COVID-19) pandemic has highlighted the need to upgrade vaccine strategies. DNA vaccines are advantageous because of their stability and ease of large-scale production; however, they often require adjuvants to boost immune response. Liposomal adjuvants have emerged as effective immune response enhancers, offering efficient delivery and compatibility with biological systems. This study evaluated the immune response induced by a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein-encoding DNA vaccine formulated with various liposome adjuvants in mice. Material and methods The interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-a) mRNA expression levels were analyzed by reverse transcription-quantitative polymerase chain reaction. Flow cytometry was conducted to characterize leukocyte populations and assess immune activation status. Five target cell surface markers were analyzed in this study: CD4+ T, CD8+ T, CD11b+, CD14+, and CD45+ leukocytes, along with their corresponding detected cell types. Results Among the tested formulations, the PEI25k : DOTAP : cholesterol (2 : 1 : 1) combination show­ed the most significant immunostimulatory effect. This formulation induced the lowest expression levels of IL-6 (0.17 ± 0.01) and TNF-a (0.25 ± 0.01) mRNAs in the treatment groups; furthermore, flow cytometry revealed robust activation of both adaptive and innate immune subsets, with an increase in CD4+ (44.00 ± 1.00), CD8+ (40.00 ± 1.00), CD11b+ (50.00 ± 2.00), CD14+ (42.00 ± 2.000), and CD45+ (29.00 ± 1.00) T cell populations. These results demonstrate an integrated immune response characterized by strong T cell activation, myeloid engagement, and recruitment of a broad leukocyte population, indicating balanced immunity with reduced inflammation risk. Conclusions The PEI25k : DOTAP : cholesterol (2 : 1 : 1) formulation has high potential as a liposome-based adjuvant for enhancing the immunogenicity of DNA vaccines. The findings of this study support its suitability for further development as a potential adjuvant candidate, particularly for improving DNA vaccine delivery and immunogenicity against SARS-CoV-2.