IFN-γ ELISPOT assay

IFN-γ enzyme-linked immunospot (ELISPOT) assays were performed as previously described [36]. In brief, PBMCs were plated in 96-well plates that were precoated with anti-IFN-γ monoclonal antibody at 100,000 cells per well with peptides at a final concentration of 5 μg/ml for pooled peptides, single peptides, and optimal epitopes (BD^TM ELISPOT, USA). Phytohemagglutinin (PHA) served as a positive control at a concentration of 10 μg/ml, and medium alone was the negative control. Spots were counted using the ImmunoSpot® Analyzer (Cellular Technology Ltd, USA), and the number of specific T-cells was counted by subtracting the mean negative control values. Responses were considered positive if the activity was at least three times greater than the mean number of spot-forming cells (SFCs) in the negative control as well as >50 SFCs per 10⁶ PBMCs.