Antigenic determinants of asthma-associated allergens for design of immunotherapy

Indoor Biotechnologies, Charlottesville VA

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Abstract

TITLE: Antigenic determinants of asthma-associated allergens for design of immunotherapy PROJECT SUMMARY/ABSTRACT Asthma is the most common chronic disease among children. One in twelve children are affected. Asthma is a major cause of missed school days and disproportionately affects black, Hispanic and minority populations, and rural Americans who lack access to health care. The CDC estimates that the economic costs of asthma are ~$80 billion. The condition is strongly associated with economic disparities, especially low-income populations living in sub-standard housing. Allergic reactions to potent allergens produced by house dust mites, cat, and dog are major risk factors for the development of asthma and for emergency room admission with asthma. House dust mite allergy affects 85% of asthmatic children, while cat and dog allergens account for >500,000 ER visits per year. The molecular determinants on these allergens that are the targets of allergy antibodies are unknown. The goal of this application is to use human IgE monoclonal antibodies derived from allergic patients to define the allergenic structure of dust mite, cat, and dog allergens for potential development of novel forms of immunotherapy. Mite group 1 allergens are cysteine proteases that contribute to lung inflammation in asthma, whereas group 2 allergens are lipopolysaccharide binding proteins. Fel d 1 and Can f 1 are uteroglobins and lipocalins, respectively. Allergen-specific human IgE monoclonal antibodies (mAb) will be produced from children and adults with allergic asthma as they occur in vivo. Allergens will be co-crystallized with recombinant IgE antibody constructs. The key amino acids involved in IgE antibody binding will be identified and modified. The specific aims are: 1) Production of allergen-specific human IgE mAb and analysis of their immunoreactivity; 2) Mapping of actual IgE binding epitopes on dust mite, cat, and dog allergens by X- ray crystallography; and 3) Site-directed mutagenesis of IgE antibody epitopes for expression of hypoallergenic mutants as candidates for immunotherapy. An analysis of the association between allergen-specific IgE antibodies from the human repertoire and the epitopes recognized by these IgE antibodies will be performed. Unique experimental data sets of three-dimensional structures of B-cell epitopes will be generated to expand databases used for developing tools for B cell epitope prediction. Most importantly, this project will define IgE antibody responses to cat, dog, and mite allergens and will provide the structural basis for rational design of hypoallergens and IgG blocking antibodies for therapy. In Aim #3, IgE antibody binding to the epitope mutants will be analyzed by immunoassays and cell mediator release assays, and T cell reactivity will be evaluated. Mutants will be compared, and hypoallergenic forms will be selected for the design of vaccines for immunotherapy of cat, dog, and mite allergy. The capacity of the mutants to induce a protective immune response will also be tested in vivo in a mouse model of passive anaphylaxis. Defining the allergenic structure of the most important allergens associated with asthma will be a major outcome of this study and is anticipated to result in new diagnostic and therapeutic strategies for treatment of allergic disease.