Fig. 7
Effect of the EV-A71 + EV-D68 bivalent vaccine with or without PS-G as adjuvant 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) and EV-D68 (2.5 μg/mouse), and formalin-inactivated EV-A71 (2.5 μg/mouse) and EV-D68 (2.5 μg/mouse) combined with PS-G (10 μg/mouse) as adjuvant thrice at 3-week intervals. Spleen was isolated at 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 the levels of IFN-γ, IL-17, and IL-4 after 2 d in response to heat-inactivated EV-D68 or EV-A71. e, f Information on IFN-γ and IL-17 production by CD4+ and CD8+ T-cells in the spleen were obtained from all mice. Splenocytes were collected from mice immunized with PBS, formalin-inactivated EV-A71 + EV-D68, or PS-G-adjuvanted EV-A71 + EV-D68 and cultured in RPMI medium containing 10 μg/mL of heat-inactivated EV-A71 or EV-D68. Cells were cultured for 5 d, and the levels of IFN-γ and IL-17 produced by CD4+ and CD8+ T-cells were measured by flow cytometry. All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001