IFN-c secreting T cells were enumerated by ELISpot. Briefly, MultiScreen HTS IP 96 plates (Cat MSIPS4510, Millipore, Ireland) were pre-wet with 15 ml/well of 35% ethanol and washed twice with PBS (pH 7.4). For mouse ELIspot assays, wells were coated with 75 ml/well of sterile PBS (pH 7.4) containing 10 mg/ ml anti-mouse IFN-c (Cat 551216, Clone R4-6A2, BD Pharmingen, CA) with or without 1:500 anti-His mAb (Cat H1029, Sigma-Aldrich, St. Louis, MO) overnight at room temperature. For human ELIspot assays, wells were coated with 75 ml/well of carbonate buffer (pH 9.6) containing 10 mg/ml anti-human IFN-c (Cat 3420-3, Clone 1-D1K, MABTECH, Sweden) with or without 1:500 anti-His mAb (Cat H1029, Sigma-Aldrich, St. Louis, MO) overnight at 4uC. Plates were washed twice with 200 ml/well DMEM (splenocytes) or RPMI (PBMC) and blocked with 200 ml/ well of medium containing 10% FCS for 3 hrs at 37uC. After blocking, the wells coated with anti-His mAb were incubated with PyCSP, FluM, FluHA, CMVpp65, EBNA3A or empty pIVEX HisHA IVTT products diluted 1:1000 in DMEM or RPMI containing 2% FCS in a volume of 50 ml/well, overnight at 4uC (to absorb the His-tagged IVTT products to the anti-His mAb coated wells). Plates were washed 3 times with DMEM or RPMI, and the cells added to the IFN-c and His coated wells in triplicates. Alternatively, in the absence of anti-His pre-coating, IVTT products unpurified, associated to Polybeads or ProteinG beads, or purified using NI-NTA resin or MagneHis Ni-particles (see above), were added to the IFN-c coated wells together with the cells (100,000 PBMCs or 500,000 splenocytes), in triplicate. After 36 hrs incubation, plates were flicked to remove the cells and washed 6 times with PBS-Tween 0.05% pH 7.4 (PBS-T). Then 75 ml/well of biotinylated anti-mouse IFN-c (Cat 554410, Clone XMG1.2, BD Pharmingen) at 2 mg/ml in PBS or 75 ml of biotinylated anti-human IFN-c (Cat 3420-6, Clone 7-B6-1, MABTECH, Sweden) diluted 1:1000 in PBS was added to each well. Plates were incubated for 3 hrs at room temperature, washed 3 times with PBS-T, and 75 ml/well of Streptavidin-HRP for splenocytes cultures or Streptavidin-AP (Bio-Rad, Hercules, CA) for PBMC cultures diluted 1:1000 in PBS was added. After 1 hour incubation at room temperature, plates were washed 3 times with PBS-T followed by 3 times with PBS alone, and developed using the AEC substrate set (BD Pharmingen) for mouse splenocytes or SigmaFast BCIP/NBT (Sigma-Aldrich) for humans PBMC cultures, according to manufacturer's instructions. After 10 mins, the plates were rinsed extensively with dH2O to stop the enzymatic reaction, dried and stored in the dark. The number of IFN-c secreting cells was determined using the automated ELISpot reader (AID iSpot Reader, Autoimmun Diagnostika GmbH, Strassberg, Germany).