House dust mites (Dermatophagoides species) commonly occur in households in many parts of the United States and there is now strong evidence that sensitization and exposure to mite allergens is an important cause of asthma.
The aims of this project are to determine the molecular and antigenic structure of the Group II mite allergens, Der p II and Der f II, from D. pteronyssinus and D. farinae, respectively. These allergens are low molecular weight proteins that cause IgE antibody responses in the majority (80-90%) of mite allergic patients. Murine monoclonal antibodies (mAb) and human IgE or IgG antibodies will be used in epitope mapping studies to determine the repertoire of B cell epitopes on the Group II allergens. Site directed mutagenesis, and other mutagenesis techniques, will be used to produce a panel of recombinant Der p II (rDer p II) variants that differ at one or two known amino acid residues. These mutant allergen molecules will be compared with native allergen for binding to mAb or human IgE and IgG antibodies by either direct binding or competitive inhibition immunoassays. The experiments will involve comparisons of antibody specificities using sera from mite allergic patients with perennial rhinitis, asthma or atopic dermatitis. In addition, monoclonal IgE and IgG antibodies will be obtained from Epstein Barr virus transformed human B cells or hybridomas for epitope mapping studies. Expression systems capable of producing large quantities of rDer p II will be developed in E. coli or eukaryotic cells. The three dimensional structure of Der p II will be determined by nuclear magnetic resonance (NMR) methods using isotope labelled rDer p II. The NMR experiments will determine the topology of Der p II and identify surface exposed residues that could form potential antigenic sites and be targeted for mutagenesis. Determination of the three dimensional structure will allow fine mapping of the epitope(s) and analysis of the structural changes that occur in the mutant Der p II molecules. These studies should provide a detailed analysis of the molecular architecture of a major group of dust mite allergens and of the epitopes recognized by human IgE or IgG antibodies. Modifications of antibody binding sites may allow recombinant molecules to be designed for use as immunotherapeutic agents for allergic disease.
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