Mice were immunized with Ub-S DNA vaccine, unmodified S DNA (S DNA) control, full-length S protein (S Protein) vaccine control, or PBS control in the presence of either imiquimod adjuvant or Alum plus MPL adjuvants, and boosted with the same immunogens plus the respective adjuvants twice at 3-week intervals and once at 6 months. PBS without adjuvant was included as a background control. Splenocytes collected 10 days after the last dose from mice immunized with each immunogen plus imiquimod adjuvant (A–D) or Alum plus MPL adjuvants (E–H) were analyzed for SARS-CoV-2 S–specific CD8⁺ (A and B or E and F) and CD4⁺ (C and D or G and H) T cells by flow cytometry. Splenocytes were stimulated with pooled peptides (final concentration 5 μg/mL) from SARS-CoV-2 S protein predicted to contain B6 mouse CTL and Th cell epitopes, and IFN-γ– or TNF-α–producing CD45⁺CD8⁺ and CD45⁺CD4⁺ T cells were stained for respective cell surface and intracellular cytokine markers. Sera collected 10 days after the third dose from mice immunized with each immunogen plus imiquimod adjuvant (I–M) or Alum plus MPL adjuvants (N–R) were detected for SARS-CoV-2 S–, NTD–, RBD–, S1–, or S2–specific IgG antibodies by ELISA. The ELISA plates were respectively coated with SARS-CoV-2 full-length S protein, as well as NTD, RBD, S1, or S2 fragment (1 μg/mL), and IgG antibody (Ab) titers were calculated as the endpoint dilution that remained positively detectable. Data are expressed as mean ± SEM of individual samples (for T cell detection) or quadruplicate wells from pooled sera of mice in each group (n = 4–5). *P < 0.05, **P < 0.01, ***P < 0.001 by ordinary 1-way ANOVA with Tukey’s multiple-comparison test. Experiments were repeated twice, and similar results were obtained.