Background:  Early studies demonstrated that CD8⁺ T lymphocytes from HIV-1-infected individuals could suppress HIV-1 replication in autologous CD4⁺ T lymphocytes in vitro and that CD8⁺ responses evoked by NEF gene had the highest inhibition effect on HIV-1 replication. The major problem in the design of a vaccine against HIV is due to the diversity of this virus as well as to the usual techniques to monitor of the breadth of CTL epitopes recognized by CD8⁺ cytotoxic T cells. The main objective of this study is to compare HIV-specific CD8 T-cell immune responses to autologous virus NEF RNA transfected into dendritic cells (DC) and to compare it to reference virus sequences.

Methods:  We recruited 10 chronically aviremic treated HAART HIV-infected patients. We generated autologous NEF mRNA from viral RNA isolated from the patient plasma collected prior to treatment. DC were then electroporated with RNA encoding viral proteins and induced to mature for 24 hours. DC transfected with RNA encoding the NEF HIV-1 protein were then co-incubated with autologous peripheral blood mononuclear cells (PBMC) labeled using CFSE dye for 6 days in a ratio of 1 to 40, respectively, and were then assessed by flow cytometry. For characteristics of CD8⁺ T cell receptor (TCR) repertoire we used DNA heteroduplex mobility assay (HMA) analysis.

Results:  Our results indicated that we can induce HIV-specific CD8⁺ T cell proliferation when we electroporated DC with the autologous NEF RNA. This proliferation occurred in the absence of any detectable CD4⁺ response. We further compared the differences in the proliferative response induced by autologous mRNA to that induced by the reference virus sequences or to consensus peptides. PBMC stimulated with autologous NEF mRNA showed a massive proliferative dose-dependent response in all patients tested (higher amount of RNA electroporated increased HIV-specific CD8⁺ T cell proliferation). In contrast, a small proliferative response was observed using the reference virus sequences or the consensus peptides. Using HMA for TCR repertoire analysis we estimated a significant increase in total CD8⁺ clonal expansion after co-culturing PBMC with DC electroporated with autologous NEF RNA compare to reference virus sequences or consensus peptides.

Conclusions:  We show that the use of autologous NEF mRNA permits the detection of the proliferative capacity of specific CD8⁺ T cells, a significant factor in HIV vaccine design and development.