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Recent studies have suggested including nonstructural proteins as Tat and Vpr in HIV vaccines. However, little is known about the CD4+ T-cell response that these small proteins induce in humans. We have therefore evaluated these responses by in vitro priming experiments of CD4+ T lymphocytes harvested in healthy donors. In the Tat protein, only one peptide primed CD4+ T cells of eight HLA unrelated healthy donors. T cells induced by this peptide recognized immature DC loaded with the native Tat protein and are restricted by multiple HLA-DR molecules, in agreement with its binding capacity. This peptide was therefore processed in an appropriate manner and was highly immunoprevalent. CD4+ T-cell response to Vpr peptides was more disperse and involved six different peptides depending on the HLA-DR molecules of the donors. Two overlapping peptides were T-cell stimulating in at least half of the donors. T-cell response to Vpr in multiple donors is the result of a combination of several CD4+ T-cell epitopes with good to moderate immunoprevalence. Altogether, our results show that the frequency of responders to HIV Tat or Vpr proteins relies on one or multiple CD4+ T-cell epitopes, respectively.

To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250-1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients.

Mechanisms underlying successful immunotherapy of allergic patients operate at the level of CD4+ helper T cells. T cell epitopes from allergens may thus constitute interesting molecules for immunotherapy, provided they are efficient for all patients and are not recognized by IgE. In an attempt to define such peptides for allergy to bee venom, we have investigated the capacity of peptides encompassing the sequence of the major bee venom allergen to stimulate PBMC from allergic patients and to react specifically with their IgE. The region 77-110 emerged as the most frequently T cell stimulating. We then analyzed the binding modes of the sequence 81-97 for ten different HLA-DR molecules and introduced punctual mutations to enhance the peptide affinity for these molecules. Six different modes have been identified on the sequence 81-97, one mode being common to eight HLA-DR molecules. Four HLA-DR molecules can bind the P85-97 peptide by two different modes with an equivalent affinity. The peptide N89L has a higher affinity for DRB1*0301 and DRB3*0101 and remains as active as the native peptide towards the other HLA-DR molecules.