The HIV-1 regulatory proteins Tat and Rev are frequently targeted by cytotoxic T lymphocytes derived from HIV-1-infected individuals

Abstract

The HIV-1 regulatory proteins Rev and Tat are expressed early in the virus life cycle and thus may be important targets for the immune control of HIV-1-infection and for effective vaccines. However, the extent to which these proteins are targeted in natural HIV-1 infection as well as precise epitopes targeted by human cytotoxic T lymphocytes (CTL) remain to be defined. In the present study, 57 HIV-1-infected individuals were screened for responses against Tat and Rev by using overlapping peptides spanning the entire Tat and Rev proteins. CD8+ T cell responses against Tat and Rev were found in up to 19 and 37% of HIV-1-infected individuals, respectively, indicating that these regulatory proteins are important targets for HIV-1-specific CTL. Despite the small size of these proteins, multiple CTL epitopes were identified in each. These data indicate that Tat and Rev are frequently targeted by CTL in natural HIV-1 infection and may be important targets for HIV vaccines.

Figure 1

Number of study subjects (dark columns) with specific CD8 responses against the individual overlapping Tat peptides (1–15) as measured by ELISPOT. The clear horizontal bar indicates the HIV-1 Tat protein domains corresponding to the overlapping peptides. A, acidic amino terminal; Cy-rich, cysteine-rich region; C, core domain; Q-rich, glutamine-rich domain; a total of 57 subjects were evaluated.

Figure 2

Number of study subjects (dark columns) with specific CD8 responses against the individual overlapping Rev peptides (1–21) as measured by ELISPOT. The clear horizontal bar indicates the regions of the HIV-1 Rev protein corresponding to the overlapping peptides. M, multimerization regions; nls, nuclear localization signal; rre, rev responsive element binding; nes, nuclear export signal. A total of 57 subjects were evaluated.

Figure 3

Functional characteristics exemplified for a Tat-1-specific CD8+ T cell line. (a) Evaluation of ability to produce IFN-γ by flow-based intracellular cytokine staining. (b) Cytotoxic activity for the T cell line as measured by standard ⁵¹Cr-release assay at effector-to-target ratios 20:1 and 10:1. Targets included peptide-pulsed autologous B-lymphoblastoid cell line (LCL) and autologous B-LCL infected with rVV expressing Tat rVVTat. Control conditions were established by using unpulsed or rVVLac infected autologous B-cell lines (20).

Figure 4

Characterization of three CTL epitopes within HIV-1 Tat and Rev. The responses were further investigated in study subjects 6007 (HLA A*3002/-, B53/*5801, Cw4/7) (a–d) and AC04 (HLA A2/11, B18/44, Cw5/Cw12)(e and f). HLA restriction was performed by using peptides presented by autologous and partially HLA-mismatched L-BCL in a ⁵¹Cr-release assay (a, c, and e). Fine mapping of the optimal epitope was done by using serial dilutions of truncated peptides in cytotoxicity assays (b, d, and f). Effector-to-target ratio for all assays was 20:1.