EPITOPE MAPPING OF THE HOUSE-DUST-MITE ALLERGEN DER-P-2 BY MEANS OF SITE-DIRECTED MUTAGENESIS

Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cere visiae, was used as a tool to determine IgE- and monoclonal antibody ( mAb)-binding sites on this allergen. For this purpose, mutant molecule s were produced by application of site-directed mutagenesis. The amino -acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues i n three predicted antigenic sites (residues 45-48, 67+69, and 88-90) w ere replaced by alanine residues. IgE- and mAb reactivity to these mut ants was compared to that to ''wild type'' Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb alpha DpX. Mutations in the areas adjacent to this loop (i.e., 67+69 and 88- 90) had similar effects on this mAb (10- to 20-fold decreases in react ivity were observed), supporting the suggestion that these areas are i nvolved in this antigenic structure. The area of residues 45-48 was sh own to be involved in an epitope for mAb 2B12. The reactivity of mAb 7 A1 was influenced by substitutions of residues 45-48 as well as 88-90. Deletion of the residues spanning cys21-cys27 resulted in decreased r eactivity to three mAbs (10E11, alpha DpX, and 7A1). From these observ ations, it may be concluded that binding of different mAbs is influenc ed by the same mutations and that the binding of single mAbs is influe nced by two or more mutations scattered over the allergen molecule. Th ese findings can point in two directions: minor aminoacid changes resu lt in disruption of the overall conformation of the allergen, or dista nt sites are close together in the three-dimensional structure of the allergen. Decreased IgE reactivity was observed with all mutant molecu les, varying between patients. The observed effects ranged from 5- to 1000-fold. Deletion of the amino-acid residues spanning cys21-cys27 an d cys73-cys78 had the strongest effect on IgE reactivity, where decrea ses up to 1000-fold were observed. Such mutants might be useful tools to improve the safety of allergen-specific immunotherapy.