Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm

Conventional analysis of the cytotoxic T lymphocyte (CTL) response to HIV-1 may underestimate the true breadth of CTL epitopes recognized. This undere stimation could be due to several reasons, including (1) the use of laborat ory-adapted stains of HIV or consensus sequences, which would lead to the i dentification of only highly conserved epitopes, (2) the use of EBV-transfo rmed B cells (B-LCLs) and vaccinia virus constructs in standard assays that may obscure low level CTL responses due to high EBV or vaccinia reactivity , and (3) relatively insensitive assays wherein PBMCs instead of profession al APCs are used to stimulate CTL responses. To address these problems, we first identified an immunodominant HLA-B7-restricted CTL epitope, by standa rd cloning methods, in a long-term nonprogressor (LTNP), To determine wheth er the patient had CTLs specific for autologous viral sequences other than the dominant epitope, proviral DNA was cloned and sequenced. A matrix-based epitope algorithm (EpiMatrix) was used to identify the top 2% of peptides from the viral sequences with the highest likelihood of binding to HLA-B7, These 55 peptides were synthesized and tested for HLA-B7 binding in a T2/B7 cell line; 10 peptides were able to stabilize HLA-B7 on the cell surface. By using peptide-pulsed autologous dendritic cells as a more sensitive meth od of CTL stimulation, we found three additional subdominant CTL epitopes.