Background: CD8⁺ cytotoxic T-lymphocyte (CTL) responses are likely to play a critical role in effecting the course of HIV-1 infection. To better understand this role, it is important to identify CTL recognition sites on autologous virus. However, generation and testing of peptides specific to autologous virus is prohibitive. Thus, means are needed to identify likely CTL epitopes not present in standard peptide panels. We hypothesize that diverse elements in the viral genome should reflect sites under diversifying selection, potentially resulting from escape from CTL. Furthermore, analysis of viral genome sequences from acute infection may yield insight into the selective forces shaping the viral population at this critical stage of the host-pathogen interaction.

Methods: Whole viral genome sequences were obtained from the first month of infection and targeted gene fragments from gag-p24, pol-RT, vpr, tat, env-gp41 and nef over the first 2.5 yrs of infection of a patient from the Seattle Primary Infection Cohort. PAUP* was used to study gene diversity, and Dnasp and PAML used to identify sites under diversifying pressure. CTL responses were evaluated by ELISPOT using overlapping peptides generated from the HIV-1-HXB2 sequence.

Results: Thirteen (13) sites along the viral genome were identified as probable sites of diversifying pressure, whereas 7 CTL epitopes were identified by ELISPOT. Four (4) of the 7 CTL epitopes were predicted on the basis of diversifying evolution, including the only 2 confirmed as escape mutants. The remaining 3 CTL epitopes, as well as flanking sequences, were invariant over the examined time, suggesting a continuing suppressive response. Nine (9) additional sites were predicted to be undergoing diversifying selection. Peptides covering 2 of them tested negative for CTL response. The other 7 sites were in peptides not found in the panel HIV-HXB2 peptides. Recognition of these peptides is under study. Among all the genome regions examined, vpr diversity increased earliest and fastest, and, CTL response to the corresponding ancestral epitope was the first detected in this host.

Conclusions: Our study demonstrates that genetic signatures of diversifying selection have predictive value in identifying CTL escape. These studies can also inform generation of autologous peptides for testing and thereby, perhaps facilitate identification of autologous CTL recognition sites on a wider scale than currently feasible.