HLA-DR restricted peptide candidates for bee venom immunotherapy.

T cell epitopes containing peptides have been recently proposed as an alternative to conventional immunotherapy of allergic diseases because they are expected to be better tolerated than allergen extracts. A principal limitation to their clinical use is that they present an important diversity, which primarily results from the polymorphism of HLA class II molecules. In Caucasian populations, however, seven alleles of the most expressed molecules (namely DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*1101, DRB1*1301, and DRB1*1501) predominate. Peptides from allergens that would efficiently bind to them should be potential candidates for specific immunotherapy. In this paper, we have determined the peptides present in the major bee venom allergen by investigating the capacity of synthetic peptides that encompass its whole sequence to bind to each allele. Several efficient binders have been identified and are either allele-specific or common to several HLA-DR molecules. Interestingly enough, the 81-97 sequence is universal in the sense that it binds to all studied molecules. This sequence is surrounded by several active regions, which make the 76-106 sequence particularly rich of binding determinants and a good candidate for specific immunotherapy. Statistical analyses of the binding data also provide an overview of the preponderant HLA-DR alleles specificity.

On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.

Pseudopeptide ligands for MHC II-restricted T cells.

The potential therapeutic use of peptides to activate or anergize specific T cells is seriously limited by their susceptibility to proteolytic degradation. Classically, peptides are stabilized by incorporation of non-natural modifications including main chain modifications. In the case of MHC II-restricted peptides, the peptide backbone actively participates to the interaction with the MHC molecule and hence may preclude the peptidomimetic approach. We thus investigated whether a single amide bond modification influenced the peptide capacity to bind to a MHC II molecule and to stimulate specific T cells. Twenty pseudopeptide analogs of the I-Ed binder 24-36 peptide, whose sequence was derived from a snake neurotoxin, were obtained by replacing each amide bond of the peptide central part, by either a reduced psi[CH2-NH] or N-methylated psi[CO-NMe] peptide bond. In agreement with the major interacting role played by the peptide backbone, several peptides displayed a low, if any, capacity to bind to the MHC II molecule and did not lead to T cell stimulation. However, one-third of the peptides were almost as active as the 24-36 peptide in I-Ed binding assays and one-fifth in T cell stimulation assays. Among them, two pseudopeptides displayed native-like activity. Good binders were not necessarily good at stimulating T cells, demonstrating that main chain modification also affected T cell recognition. We thus showed that a peptidomimetic approach could create a new type of MHC II ligand to control T cell responses.