Figure 3.

Extended immunization inhibited the production of RBD-specific memory B cells

(A) The ratio of CD19⁻ CD138⁺ plasma cells of lymphocytes were detected by flow cytometry in the blood on the seventh day after the last immunization.

(B) The percentages of CD19⁺ CD27⁺ memory B cells (gated on CD19⁺ B cells) from the splenocytes were detected by flow cytometry on the seventh day after the last immunization.

(C) R848 (2 μg/mL) combined with 100 U/mL mouse IL-2 were stimulated to induce memory B cells to differentiation into plasma cells, on day 7 after the last immunization. ELISPOT results were showed as the numbers of RBD-specific IgG spots per 5 × 10⁵ splenocytes of each mouse subtracted the numbers from the corresponding DMSO groups. The stimulation with an equal volume of media was performed as the negative control. Data were representative of two independent experiments.

(D) RBD-specific IgG antibodies in the supernatant of 5 × 10⁵ splenocytes per mL were detected by ELISA. IL-4 (E) and IL-5 (F) in the serum were detected by ELISA on the seventh day after the last immunization. Data were presented as mean ± SEM Representative data of two independent experiments were shown. ∗p< 0.05, ∗∗p< 0.01, ∗∗∗p< 0.001, ∗∗∗∗p< 0.0001. ns represented non-significant.