Fig. 3

Antigen-specific immunity induced by LNP-M-delivered DNA- or mRNA-encoded spike. A Anti-spike antibodies in sera. Mice were immunized intramuscularly with the vaccines three times at 2-week intervals. Two weeks after the final immunization, the levels of serum IgG subtype antibodies were detected using ELISA. B Cytokine secretion by activated splenocytes. Splenocytes from immunized mice were cultured with IL-2 (100 U/ml) and a spike peptide pool (10 μg/ml) for 72 h before ELISA analysis of IFN-γ, TNF-α, IL-10, and IL-4. C and D T cell proliferation. The percentages of BrdU⁺ cells were assessed in gated splenic CD4⁺ or CD8⁺ T cells stimulated with a spike peptide pool. E and F ELISPOT analysis of splenic IFN-γ-secreting T cells. G CTL activity of splenic T cells. H-M Functional analysis of splenocytes. Cells were stimulated with the spike peptide pool (10 μg/ml) protein for 67 h before being treated with 500 ng/ml ionomycin, 50 ng/ml PMA, and 5 μg/ml Brefeldin A for an additional 5 h. FACS analyses were conducted using intracellular cytokine staining to assess the percentages of IFN-γ⁺, IL-2⁺, and TNF-α⁺ CD4⁺ or CD8.⁺ T cells. The experiments were performed with 5 mice per group. Data were represented as means ± SD. Statistical significance was set at ***p < 0.001, ****p < 0.0001