Evaluation of IFN-γ producing cells using ELISPOT assay
An ELISPOT assay was used to quantify IFN-γ production by PBMC isolated from the study subjects in response to nine HCV genotype 4a isolate ED43 peptide antigen pools composed of 15 (15mer) and overlapping by 10 amino acids. These were 600 peptides combined in nine pools corresponding to all the HCV proteins. These synthetic peptides were custom synthesized by Mimotopes (Australia). Approximately 15ml of whole blood were collected into EDTA vacutainer tubes (Becton Dickinson Biosciences, NJ, USA). PBMC were isolated by Ficoll-Hypaque density gradient centrifugation and viability was determined by trypan blue exclusion method. Briefly, 2x105 PBMC (200µl/well) were incubated in triplicate cultures in the ELISpot plates (Whatman Unifilter, USA) coated with anti-human IFNγ antibody and incubated for ~16 hours with or without recombinant HCV antigens at 3μg/ml of each single peptide in complete RMPI-1640 medium. Negative and positive controls included medium containing DMSO alone and 0.1μg/ml of SEB or other polyclonal stimuli, respectively. At the end of the incubation period, the assay was developed until the appearance of spots, and then the wells were rinsed with tap water to stop the reaction. The number of spots per well was counted using an automated ELISpot reader (Cellular Technology Ltd., Cleveland, USA) as described [18]. The average number of spot forming cells (SFC) in control wells were subtracted from the average number of peptide-stimulated wells to correct for background cytokine production and are expressed as SFC/million PBMC. A positive HCV antigen-specific response was considered if the SFC in the presence of antigen were at least three-fold the number of SFC in the medium control and if there was >55 SFC/million PBMC, as we previously reported [19].