HLA-DR-restricted peptides identified in the Nef protein can induce HIV type 1-specific IL-2/IFN-gamma-secreting CD4+ and CD4+ /CD8+ T cells in humans after lipopeptide vaccination.

We screened the Neflaiprotein to identify new HLA-DR-restricted epitopes, because this small protein is expressed early during infection, and specific CD4(+) T cells are critical for effective immunity in HIV-1 infection. We synthesized a set of peptides that covers the sequence of the Nef protein, and performed binding assays using 10 common HLA-DR molecules. We defined four large regions in this protein able to bind very efficiently to eight HLADR molecules. We took advantage of healthy volunteers immunized with an HIV-1 lipopeptide vaccine that contains three of the four HLA DR-restricted regions to investigate their capacities to stimulate T cells. In 11 vaccinated volunteers, typed for their class II molecules, we were able to correlate sequences of the vaccine displaying binding activities to specific HLA-DR molecules and the induction of CD4(+) T cell proliferation. To identify potential HLA-DR epitopes, we synthesized 31 15-mer peptides and showed that 26 bound to one or more HLA-DR molecules. Interestingly, 12 of the 26 15-mer peptides identified are included in the sequence of lipopeptides. We used IFN-gamma ELISPOT and flow cytometer assays to investigate the capacity of these potential CD4(+) T cell epitopes to induce specific T cell responses. We showed that seven of these peptides were able to stimulate HIV-specific T cell responses in five of six tested volunteers. These cells are Nef-specific CD4(+) and CD4(+) CD8(+) T cells secreting IL-2/INF-gamma or IL-2 alone. To conclude, these 26 Nef HLA-DR-restricted peptides could be helpful to better evaluate CD4(+) deficiencies in HIV infection and, for new vaccine designs.

Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+-T-cell epitopes recognized.

We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4(+)-T-cell responses. To better characterize the CD8(+)-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8(+)-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8(+)-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8(+)-T-cell epitopes were induced at a higher number after a fourth injection (P < 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8(+)-T-cell epitope recognition after the boost.