Unstimulated or empty vaccinia stimulated cells were used as a negative control. PMA/ION stimulated cells were used a positive control. After 48 hrs of incubation, the cells were removed by washing and a biotinylated antibody against IFN-γ (10 μg/ml in PBS) was added. In the subsequent, the streptavidin conjugated with enzyme ALP was added. Finally, a precipitation substrate (BCIP) for ALP was added and the plates were incubated until spots emerged at the site of the responding cells. The spots were examined and counted in an image analyzer system. The mean number of specific spot-forming cells (SFCs) was calculated by subtracting the mean number of spots from unstimulated cells or empty vaccinia stimulated cells Bafilomycin A1 from

the mean number of spots in cells stimulated with core, E1 and E2 or core peptides or recombinant HCV poly vaccinia. Lymphocytes proliferation assay The CD4+ T cell proliferation was assessed after labeling the lymphocytes derived from the spleen

using CFSE dye (Invitrogen Molecular Probes). Labeling cells with CFSE Ten mM of CFSE stock solution was prepared by adding 90 μl Dimethyl Sulfoxide (DMSO) to 500 μg lyophilized Smoothened Agonist purchase powder of CFSE dye. The stock solution was diluted in sterile PBS/0.1% BSA to get the desired working concentration of 10 μM. Purified lymphocytes were resuspended to a concentration of 50 million cells per ml in PBS/0.1% BSA before the addition of CFSE dye. An equal volume of 10 μM of CFSE dye was added to the cell suspension in a tube 6 times more than the volume of the cell suspension and mixed well by vortexing. The labeled lymphocytes (-)-p-Bromotetramisole Oxalate were incubated for 15 min at 37°C. The staining was quenched by adding 5 volumes ice-cold complete RPMI media followed by a 5 min incubation on ice. The cells were washed three times in complete RPMI media and re-suspended in complete RPMI (2 million cells per ml for the proliferation assay and 40 million cells in 75 μl PBS for injecting to mice). To verify the CFSE-labeled cells, samples of the cell suspensions were run on a flow cytometer and were also

analyzed by fluorescent microscopy. The proliferation was assessed after stimulation of the cells with core, E1 and E2 proteins (10 μg/ml) or core peptides (10 μg/ml). PMA (10 ng/ml) and ionomycine (1 μg/ml) were added to the cells as a positive control. After adding the stimulant, the cells were incubated at 37° in 5% CO2 for 4 days. The stimulated cells were then harvested by centrifugation at 1600 rpm for 5 min. The prodedures for statining and manipulation of CFSE labeled cells should be done in the dark. Surface stain each stimulated cell with CD3 TC and CD4 PE for 3 colour flow cytometry The cells were incubated 15 min in the dark at room temperature. After washing with PBS/0.1 azide/5% FCS, the cells were immediately analyzed on FacScan or were fixed by adding an equal volume of 2% paraformaldehyde and stored overnight at 4°C before the analysis. Cells stained with CFSE have very bright fluorescence.