Splenocyte proliferation after ex vivo stimulation
On the 35th day (one week after the last immunization), the effect of the immunogen on splenocyte proliferation responses was evaluated. After ZIKV-E protein stimulation ex vivo, splenocytes from immunized mice proliferated more efficiently than those of the control mice (Fig. 5A). Moreover, the ZI-△-PA-GEM group showed the greatest increase in proliferation.
Next, the ability of splenocytes to produce IFN-γ and interleukin-4 (IL-4) in response to specific antigens in vitro was evaluated. To calculate the frequency of cells secreting the cytokine, an enzyme-linked immunosorbent spot (ELISpot) assay was performed. Significantly stronger IFN-γ and IL-4 responses were detected in the immune groups (Fig. 5B). Futhermore, through MSD, we tested the levels of multiple cytokines secreted into supernatants by stimulated splenocytes. The levels of the cytokines Th1 (IFN-γ) and Th2 (IL-4, IL-6 and IL-10) were significantly higher in the ZI-△-PA-GEM group than in the control group (Fig. 5C). The tumor necrosis factor (TNF)-α levels were also increased slightly, but the difference was not significant. In the signal peptide replacement groups (ZI-JE-△-PA-GEM, ZI-GP-△-PA-GEM), the levels of the above cytokines were also increased to varying degrees. Moreover, the trend of IFN-γ and IL-4 cytokine levels in the splenocyte culture supernatants was consistent with the ELISpot results. These data suggested that ZI-△-PA-GEM could induce immune cell proliferation and that both Th1 and Th2 responses were elicited.