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Abstract SUMMARY Hepatitis C virus is a widespread infectious disease in humans with the negative implication of becoming chronic in most persons. Patients infected with HCV are at risk of liver cirrhosis or hepatocellular carcinoma at later stages. In Egypt, there is a high prevalence averaging from 12 to 18% of the entire population. Despite difficulties associated with extreme variability and mutability of HCV, several vaccines that prevent initial infection or viral persistence, or that clear viraemia in individuals with chronic HCV infections, are currently in development. Trials are underway in search of vaccine that may prevent chronic persistent infections in humans. Unfortunately none of these trials are directed towards GT- 4 that is the predominant genotype in Egypt. This study was designed to investigate the possibility that Egyptians patients could get benefit from vaccine candidates directed towards the HCV GT- 1b. This cross sectional study was carried out to compare their HCVspecific CMI responses to HCV GT- 4a and 1b peptide pools. Forty-five subjects, who agreed to participate from the original HCV incidence study among 859 HCW at risk of exposure to-, and infection with- HCV at the Menoufiya University’s NLI, formed the study population. All study subjects (21 chronic, 14 resolved and 10 controls) were tested for HCV viral RNA and Abs then HCV RNA positive subjects were confirmed to have GT- 4 HCV infections. Summary 121 HCV-specific CMI in study groups was quantified using the interferon gamma (IFN-γ) enzyme-linked immunospot (Elispot) assay in response to two sets of 7 HCV genotype 4a and1b overlapping 15mer peptide pools covering most of the viral genome. The phenotype of the responding T cells to the HCV antigens and Treg cells was also characterized by flow cytometry. In addition Interleukin 17 (IL-17) gene expression upon stimulation by 4a and1b pool peptides was quantified by real time PCR. The results revealed a significant difference in ALT activities among the studied groups while there was no significant difference among the aforementioned groups in the demographic and clinical data. A positive HCV-specific IFN- γ was elicited for 1-14 HCV pools in 17 (37%) of subjects tested. The total mean (±SEM) was 199±55 SFC/Million PBMCs for genotype 1b and 216 ±55.8 SFC/Million PBMCs for 4a, respectively. There was no statistical difference in total response between the two genotype antigen pools (p=0.833). Twenty eight subjects out of 45 (62.2%) didn’t respond to any of the 14 tested pools (non- responders). Differences between responders and nonresponders for both genotypes 4a and 1b were statistically significant (p=0.003; p=0.001, respectively). The average positive response to any of the tested pools for each antigen set was 1.65±0.27 pools and 1.65±0.24 pools, respectively (p=0.5). A strong correlation was found in the CMI responses to both GT-1b and GT-4a antigens among Egyptians exposed to HCV infection. These antigens were immunogenic, flow cytometric analysis of the phenotype of cells producing IFN-γ and IL-2 indicated that both CD4+ and CD8+ T-cell subsets are able to produce Summary 120 these cytokines with higher frequency in the CD4+ T cell subset at least by being double the percentage of CD8+ T cells in the blood. A higher frequency of Treg was detected in the chronic group rather than in the resolved and control groups but with no significant difference. Upon stimulation of PBMCs with mixture of GT- 4a pooled Ag peptides, enhanced IL-17 expression (up-regulation) was recorded only in chronic subjects while it was down-regulated in resolved and control. IL-17 was up-regulated after stimulation with mixture of GT- 1b pooled Ag peptides in resolved and control groups whereas it was down-regulated in chronic subjects. In conclusion, cross-strain recognition was indicated to be possible and this will open the path for future considerations of HCV as prophylactic and/or therapeutic vaccine candidate against HCV. The present results revealed specific T cell immune responses, including CD4+ and CD8+ T cell responses in HCV chronic and resolved subjects in response to stimulation with 4a and 1b pool peptides |