IFNγ enzyme‐linked immunosorbent spot (ELISPOT) were performed using DENV‐exposed donors’ or acute patients’ thawed PBMCs that were immediately added to ELISPOT plates. Briefly, 96‐well plates (Multiscreen HTS; Millipore, Burlington, MA, USA) were coated overnight at 4°C with 2.5 μg/mL of capture mouse anti‐human IFNγ antibody (clone 1‐DK1; Mabtech, Nacka Strand, Sweden). The plates were washed with phosphate‐buffered saline (PBS) and blocked with RPMI 1640 (Gibco, Invitrogen Co) supplemented with 10% heat‐inactivated FBS for 1 h at room temperature. Blocking solution was removed, and 2 × 105 PBMCs were plated per well in the presence or absence of NS1 protein from all four DENV serotypes at a concentration of 0.1 μg/mL. After 20–24 h of incubation plates were washed and 1 μg/mL of biotinylated anti‐human IFNγ (clone 7‐B6‐1; Mabtech) was added for 2 h at room temperature. After washing, 100 μL of streptavidin‐alkaline phosphatase (Mabtech) was added and the plates were incubated in the dark for 1 h at room temperature. Then, plates were washed, and 50 μL of alkaline‐phosphatase substrate 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate/nitro blue tetrazolium chloride (BCIP‐NBT); KPL (Gaithersburg, MD, USA) was added. After 10–15 min, colorimetric reaction was stopped with running tap water. Spots were counted using an automated ELISPOT reader (ImmunoSpot® S6UV Ultra, Cleveland, OH, USA). The number of IFNγ‐producing cells was expressed as spot‐forming cells (SFC) relative to 106 PBMCs. Values were calculated by subtracting the number of spots detected in unstimulated control wells. Values were considered positive if they were equal or greater than 10 spots and at least two times above the mean of unstimulated control wells. As a positive control, cells were stimulated with phytohemagglutinin (PHA at 5 μg/mL).