HLA-DR-Restricted Peptides Identified in The Nef Protein Can Induce HIV Type 1-Specific IL-2/IFN-γ-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-γ 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-γ 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.