Splenocyte proliferation assay
One week after the last immunization, spleens of 3 mice in each group were collected and disrupted with the plunger of a syringe. After straining through a 100 µm mesh, red blood cells were lysed in erythrocyte lysis buffer (Solarbio, Beijing, China). Splenocytes (2.5×105) were plated together with the purified ZIKV-E antigen (10 μg/ml) in 96-well plates. After incubation for 44 h, 10 µl/well CCK-8 (KeyGEN Biotech, Nanjing, China) was added to the cells to determine the cell proliferation in the next step. After cultivation for another 4 h, the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The proliferation index (PI) was calculated as: (ODstimulated cultures − ODnon-stimulated cultures)/(ODnon-stimulated cultures − ODcontrol cultures).