SMNCs secreting IFN-γ, IL-4, or IL-10 upon stimulation were detected by ELISPOT assay. Groups of six mice were immunized intramuscularly three times at 2-week intervals using 10 µg of NM2e protein in normal saline (NS) or 10 µg of NM2e formulated with Al(OH)3, CpG 1826, or Al(OH)3 plus CpG 1826. Mice immunized with NS or Al(OH)3 plus CpG 1826 were treated as negative controls. All mice in each treatment group were sacrificed on day 38. SMNCs were separated from mouse spleen samples, and 5 µg/ml NP147–155, NP55–69, and M2e peptide pool were used as stimulants in the ELISPOT assays. The numbers of SMNCs producing IFN-γ (A), IL-4 (B), or IL-10 (C) after stimulation for 40 h with NP147–155 (left), NP55–69 (middle), or M2e peptides (right) are presented as spot-forming cells (SFCs)/106 SMNCs. Columns show the average SFCs/106 SMNCs, and bars indicate the standard deviation of each group. Lines above two or more groups indicate that they have the same comparative results. *, p≤0.05; **, p≤0.01; ***, p≤0.001 by one-way ANOVA.