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First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.

There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+ CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-gamma secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.

The p53 protein is accumulated in tumor cells of many human cancers and can elicit in vivo humoral and proliferative responses. Rare reports about p53-mediated tumor recognition by CTLs have remained questioned. We therefore studied a panel of breast tumor and melanoma cell lines that we assayed for the presence of accumulated p53 and surface HLA-A2 and for the presentation of p53 epitopes. From PBMC of a healthy donor, we have generated a CTL line, D5/L9V, directed against HLA-A2-restricted peptide 264-272 from wild-type p53. It efficiently lysed breast adenocarcinomas MCF-7, MCF7/RA1, and MDA-MB-231, and melanoma M8, which all accumulate the p53 protein. Using competition assays, we made sure that tumor lysis by D5/L9V was due to recognition of endogenously produced p53 peptide 264-272 associated with the HLA-A2.1 molecule on the surface of these tumor cells. Cells with undetectable levels of wild-type p53, such as lymphoblastoid cells and melanoma M74, were not recognized by D5/L9V. Neither were breast tumor cell line MCF7/ADR nor melanoma line M44 because of HLA loss. This study therefore shows that it is possible to obtain in vitro CTL lines that specifically recognize a p53 epitope spontaneously presented by a variety of HLA-A2+ transformed cell lines provided they display abnormal patterns of p53 expression. This work points out that breast tumors and melanomas share a p53 epitope, and raises hopes for future immunotherapeutic approaches.

To detect HLA-binding peptides in 10 HIV-1 proteins (Rev, Tat, Vif, Vpr, Vpu, Gag p18, Gag p24, Gag p15, Env gp120 and Env gp41), the peptide binding assay (PBA) has been performed using three HLA class I molecules. Correlations have been searched between the PBA results and the peptide competitor activity in a functional test using HLA-A2-restricted CTL and target cells. A correlation between the data found in the PBA and well-defined CTL epitopes could be attempted only for the three Gag proteins. For these proteins, our results are in agreement with the known existence of epitopes reacting with human CD8+ CTL, with some exceptions. Together with the results reported with a panel of Nef peptides, these experiments showed that at least 18/20 of the already reported CTL epitopes from HIV-1 Gag, Nef, and Env proteins could be detected by the PBA, most (17/18) corresponding to strong reactivities. Perhaps more important, the regions of HIV-1 Gag p24 or Nef proteins that contain multiple associated CTL epitopes, with different HLA restrictions, were clearly identified by the reactivities in the PBA of several overlapping peptides and the major practical interest of the PBA might be the detection of such polyepitopic regions. Prediction are proposed in this report for 10 proteins, including several proteins for which CTL epitopes remain presently unknown.

The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.

The lymphoid cell surface receptor for Moloney leukemia virus envelope glycoprotein gp71 was isolated by using anti-gp71 antibodies to immunoprecipitate the receptor-gp71 complex from detergent extract of radiolabeled murine thymus cells. Upon sodium dodecyl sulfate gel electrophoresis, a single molecule with an apparent m.w. of 190,000 was identified as the putative gp71-receptor. Analysis in nonreducing conditions indicated that the receptor may be composed of at least 2 subunits of 190,000 daltons.

Antisera have been prepared in rabbits and in mice against different peptides corresponding to four hydrophilic and variable regions of HLA-B7 heavy chain (65-82, 99-118, 138-157, and 164-187). Specific antipeptide sera have been obtained with all synthetic peptides; for three of them which were more than 20 amino acids long, highly potent sera were elicited by injection of the free peptide. Three overlapping peptides included in region 138-157 have been used, and two different antigenic sites were detected in this region. HLA molecules solubilized in nonionic detergent were precipitated by antipeptide sera directed against regions 65-82, 138-157, and 164-187, but not by antipeptide serum directed against the less hydrophilic region 99-118. Analysis by two-dimensional electrophoresis of the isolated molecules confirmed the anti-HLA specificity of the antipeptide 65-82 and 138-157 sera. Variable numbers of HLA-related spots were found according to the antisera used. Antipeptide 138-157 serum precipitated numerous HLA molecules and therefore probably reacted with monomorphic determinants whereas antipeptide 65-82 appeared specific for a more limited number of HLA antigens. Such reagents directed against well-defined regions of the HLA class I heavy chain are of considerable interest, notably for the mapping of antigenic epitopes on the molecule and for the study of relationships between structure and function.

The characteristics of Moloney leukemia virus (M-MuLV) gp71 binding on lymphoid cells have been studied by a very sensitive assay. Analysis of the results indicated the presence of 1 class of high affinity binding sites (Ka = 1.2 X 10(9) M-1) on BALB/c thymus cells. The total number of binding sites was estimated around 1.3 X 10(4) per thymus cell. No significative differences were observed for the binding of Moloney leukemia virus gp71 on thymus cells from different inbred strains of mice, suggesting that the genetic susceptibility to Moloney leukemia virus infection does not depend on the presence of gp71 receptor on the target cells. On the other hand, it seems that M-MuLV gp71 binds preferentially on T cells, which are the final target of Moloney leukemia virus-induced transformation. Very little binding was found on Moloney, Rauscher, or x-ray-induced lymphoma cells.