Fig. 3
Effect of the EV-A71 + EV-D68 bivalent vaccine on the number of antibody-secreting cells (ASCs), T-cell proliferation, and cytokine release in splenocytes. Mice were intranasally immunized with PBS, formalin-inactivated EV-A71 (2.5 μg/mouse) alone, formalin-inactivated EV-D68 (2.5 μg/mouse) alone, and combined formalin-inactivated EV-A71 (2.5 μg/mouse) with EV-D68 (2.5 μg/mouse) thrice at 3-week intervals. Spleen was isolated 2 weeks after the third immunization, and then the numbers of a EV-D68-specific IgG and IgA ASCs and b EV-A71-specific IgG and IgA ASCs were measured using ELISPOT. c, d Splenocytes from immunized mice were harvested and cultured in medium containing 10 μg/mL heat-inactivated EV-D68 or EV-A71. After culture for 5 d, proliferation was measured as [3H]-thymidine incorporation in splenocytes. Culture supernatants of splenocytes were analysed for levels of IFN-γ, IL-17, and IL-4 after 2 d in response to heat-inactivated EV-D68 or EV-A71. Thymidine uptake was determined by harvesting cells and using a scintillation counter to measure the level of incorporation (as counts per minute; cpm). All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001