Fig. 4

RBD-PP can elicit a cellular immune response and memory T cells. Mice were immunized three times with 50 µg doses of different vaccines. Fourteen days after the last immunization, splenocytes were isolated and stimulated with the RBD protein expressed in HEK293 cells. Flow cytometry was used to analyze the ratio of RBD-specific (A) CD4⁺ T (CD3⁺CD4⁺), (B) CD8⁺ T (CD3⁺CD8⁺), (C) Th1 (CD3⁺CD4⁺IFN-γ⁺), and (D) Th2 (CD3⁺CD4⁺IL-4⁺) cells. The proportion of multifunctional CD8⁺ T cells, which are defined as CD8⁺ T cells that release (E) IFN-γ, (F) IL-2, and (G) TNF-α, was detected (n = 3). H Splenocytes of immunized mice were stimulated with RBD protein expressed in HEK293 cells, and the culture supernatant after stimulation was used to analyze the release of cytokines (n = 3). I Representative ELISpot images of spots and bar graphs corresponding to IFN-γ- and (J) IL-4-secreting lymphocytes in RBD-stimulated splenocytes of immunized mice (n = 3). K Flow cytometry analyses of memory T cells in splenocytes from immunized mice. Bar graphs summarizing the flow cytometry results for CD4⁺ central memory T cells (CD3⁺CD4⁺CD44^highCD62L^high) (n = 3), (L) CD4⁺ effector memory T cells (CD3⁺CD4⁺CD44^highCD62L^low) (n = 3), (M) CD8⁺ central memory T cells (CD3⁺CD8⁺CD44^highCD62L^high) (n = 3), and (N) CD8⁺ effector memory T cells (CD3⁺CD8⁺CD44^highCD62L^low) (n = 3) in the spleen. Data are shown as the mean ± SD. Statistical significance was calculated via one-way ANOVA (A–N) to obtain p values: ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05