J. subjects Kenpaullone with acute HCV infection (n= 30) who either spontaneously cleared the virus (n= 8) or progressed to chronic infection (n= 22) recruited through the Montreal Acute Hep C cohort. 22Cryopreserved peripheral blood mononuclear cells (PBMCs) collected before infection, during the acute phase (13 months), and following resolution or establishment of chronic infection (> 6 months) were used. ELISpot assays were performed as previously described23using antigenic peptides, 1519 amino acids in length with 1112 residues overlap, divided in 4 pools of 2546 peptides corresponding to HCV core, NS3 (10271339), NS3 (13401658) and ARFP proteins (BEI Resources Repository, Manassas, VA). Consistent with previous reports, 1821NS3-specific T cells were detected at high frequency in the majority of study subjects, and were not significantly different between spontaneous resolvers and chronics (Fig. 1and data not shown). In contrast, ARFP-specific T cell responses were not observed in any of the study subjects, irrespective of the outcome of acute HCV infection and in spite of the fact that all patients exhibited detectable levels of ARFP-specific antibodies in serum (data not shown). These results strongly suggest that ARFP-specific cell-mediated immune responses that involve IFN- production either develop late or are of low magnitude during acute HCV infection, and that they play no major role in spontaneous viral clearance. It was recently reported that ARFP expression can be suppressed by core protein, 24and that ARFP binds the proteasome 3 subunit and is degraded by an ubiquitin-independent pathway. 25These results confirm that ARFP is short-lived and are consistent with the low frequency of ARFP-specific T cells observed in our study group. Overall, data presented herein indicate that resolution of acute HCV infection is not associated with the presence of significant ARFP-specific cell-mediated immune responses. Interestingly, this also suggests that enhancement of these responses through immunization against ARFP could potentially lead to heightened rates of spontaneous viral clearance. == Fig. 1 . == HCV-specific cell-mediated immune responses in peripheral blood mononuclear cells from subjects acutely infected with HCV. IFN- production was quantified in individuals acutely infected with HCV followed by spontaneous viral clearance (gray symbols) or persistent infection (black symbols) using ELISpot followingin vitrostimulation Kenpaullone with pooled overlapping peptide panels representing HCV core protein (26 peptides), NS3 pool 1 (residues 10271339) (45 peptides), NS3 pool 2 (residues 13401658) (46 peptides), and ARFP (25 peptides). 2 105cryopreserved PBMCs were stimulated in duplicates with HCV peptide pools at a final concentration of 3 g/ml of each peptide for 36 h as previously described. 23Specific spot CAV1 forming units (SFU) were calculated as (mean number of spots in test wells-mean number of spots in media control wells) and normalized to SFU/106PBMCs. A response was scored positive if greater than 50 SFU/106PBMCs. Peptides were based on the sequence of HCV-1a (H77) or HCV-3a (K3a/650), depending on the infecting viral genotype. Statistical analysis was performed using the KruskallWallis test with Dunns post test (GraphPad Prism 4, GraphPad Software, San Diego, CA; *p < 0. 05; **p < 0. 01). == Acknowledgments == Supported by grants from the Canadian Institutes for Health Research (CIHR) (MOP-74524) to NHS, CIHR-Health Canada Research Initiative on Hepatitis C (EOP-41537) and UNIVALOR SEC to HS and the FRSQ-AIDS and Infectious Disease Network (SIDA-MI). J. Bruneau holds a senior clinical research award from FRSQ. N. H. Shoukry holds a joint New Investigator Award from the Canadian Foundation for Infectious Diseases and CIHR. == Footnotes == Conflicts of interest None. == References Kenpaullone ==.