837 Frequent Detection of ex vivo CD8+ Responses against Hepatitis C Virus in HIV Co-infected Individuals on HAART A. Y. Kim*1, G. M. Lauer1, K. Ouchi1, C. L. Day1, R. T. Chung1, C. Corcoran1, M. A. Boczanowski1, A. Wurcel1, P. Klenerman2, Bruce D Walker1 1Partners AIDS Res Ctr, Charlestown, MA and 2Oxford Univ, UK
Background: Co-infection with HIV accelerates the course of HCV, now a leading cause of morbidity and mortality in this population. HCV-specific CD8+ lymphocytes may be involved in the pathogenesis of accelerated hepatic fibrosis as well as immune reconstitution syndromes following initiation of highly-active antiretroviral therapy (HAART). Prior studies have detected these responses only in HIV+ persons not on HAART. However, these studies may have been limited due to reliance on only previously described epitopes.
Methods: We have developed a peptide-based ELISpot that screens peripheral blood mononuclear cells (PBMC) ex vivo for interferon-gamma production (IFN-gamma) in response to the whole expressed HCV polyprotein. Fifteen (15) to 20-mer peptides overlapping by 10 amino acids were placed in a matrix of pools containing 20 peptides each. PBMC were added and IFN-gamma secretion measured in SFC/million PBMC after an overnight incubation. Confirmation was performed via single-peptide ELISpot as well as generation of T-lymphocyte cell lines. Ex vivo intracellular cytokine staining (ICS) and tetramer staining was performed on expanded cell lines. This methodology was applied for cross-sectional study of 17 HIV+ HCV+ persons, each of whom had chronic HCV viremia. Thirteen (13) were taking HAART for a minimum of 1 year.
Results: Application of the above techniques in co-infected individuals detected immune responses against HCV in 7/17 (41.2%) of patients (pts) studied. Five (5) of 13 (38.4%) pts on HAART exhibited responses. We found as many as 7 ex vivo CD8+ responses within the same individual (range 0-7 responses). The magnitude of ex vivo responses varied between 40-750 SFC/million PBMC per response. Expansion of cells found additional responses beneath the level of detection of the ELISpot, revealing a hierarchy of responses. Multiple novel HCV-specific responses were detected and mapped.
Conclusions: HCV-specific CD8+ cells secreting IFN-gamma can be detected at high frequencies in HIV+ individuals using novel and comprehensive techniques. These responses are present in pts on long-term HAART and despite chronic HCV viremia. Direct comparison of breadth, magnitude, and phenotype of HCV- and HIV-specific immune responses is now possible and will shed insight into the reciprocal effects of these 2 viruses. These cross-sectional data provide the basis for longitudinal study to determine correlates of HCV-specific immune reconstitution while on HAART.
