Hydrogen exchange nuclear magnetic resonance spectroscopy mapping of antibody epitopes on the house dust mite allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10-100-fold less skin te st reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigeni c surface of Der p 2, we report here the identification of three epitopes u sing hydrogen protection nuclear magnetic resonance spectroscopy. The epito pes are defined by monoclonal antibodies that are able to inhibit IgE antib ody binding to the allergen. Each monoclonal antibody affected the amide ex change rate of 2-3 continuous residues in different regions of Der p 2. Bas ed on these data, a number of other residues were predicted to belong to ea ch epitope, and this prediction was tested for monoclonal antibody 7A1 by g enerating alanine point mutants. The results indicate that only a small num ber of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will en able us to target limited positions for mutagenesis and to expand our studi es of hypoallergenic variants for immunotherapy.