Mice were immunized twice with 20 μg of DENV2 NS1 in combination with ML-RR-cGAMP, ML-RR-cGAMP-D or MPLA, each formulated in AddaVax, and immune responses were evaluated 1 week following the second immunization. Anti-NS1 IgG titers were measured by direct ELISA using sera from WTB6 (A) and Ifnar−/− (D) mice. Calculations of area under the curve (AUC) based on serially diluted serum samples are shown. Antigen-specific IFN-γ responses induced by recombinant DENV2 NS1 were measured by ELISpot with splenocytes from WTB6 (B) and Ifnar−/− (E) mice. Frequencies of DENV2 NS1-specific IFN-γ−, TNF-α− and IL-2-producing CD4+ T cells in the spleen of WTB6 (C) and Ifnar−/− (F) mice were measured by ICS. Data are representative of 2 independent experiments (mean ± SEM; n=3–4; * p<0.05, ** p<0.01).