Rapid definition of five novel HLA-A*3002-restricted human immunodeficiency virus-specific cytotoxic T-lymphocyte epitopes by Elispot and intracellular cytokine staining assays

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) p lay a major role in control of viral replication. To understand the contrib ution of this antiviral response, an initial step is to fully define the sp ecific epitopes targeted by CTL. These studies focused on CTL responses res tricted by HLA-A*3002, one of the HLA-A molecules most prominent in African populations. To avoid the time-consuming effort and expense involved in cu lturing CTL prior to defining epitopes and restricting alleles, we develope d a method combining Elispot assays with intracellular gamma interferon sta ining of peripheral blood mononuclear cells to first map the optimal epitop es targeted and then define the HLA restriction of novel epitopes. In two A *3002-positive subjects whose CTL responses were characterized in detail, t he strongest response in both cases was to an epitope in p17 Gag, RSLYNTVAT LY (residues 76 to 86). Using this method, CTL epitopes for which there wer e no motif predictions were optimized and the HLA restriction was establish ed within 48 to 72 h of receipt of blood. This simple and convenient approa ch should prove useful especially in the characterization of CTL responses specific to HIV and other viruses, particularly in localities where perform ing cytotoxicity assays would be problematic.