Molecular details of the IgE antibody response to galactose-alpha-1,3-galactose in alpha-gal syndrome - PROJECT SUMMARY The IgE molecule lies at the center of the pathogenesis of allergic diseases. In sensitized individuals, re-exposure to the offending allergen results in IgE engagement, causing Fcε receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. Studies of the human IgE molecule, and its targeted allergens, have been very limited. Nearly all of our knowledge of this process has come from studies using allergic patient serum, which contains a complex mixture of antibodies, with different specificities, directed toward numerous epitopes, and having dissimilar affinities; thus, studies of the molecular interactions of IgE with target allergens using serum are greatly limited. The ideal way to study this process is to use naturally occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many intrinsic technical hurdles, particularly the scarcity of these cells in the circulation, no such antibodies could be made until now. We have established two unrelated methods to obtain sequences of IgE encoding B cells from the peripheral blood of allergic individuals. In this proposal, we develop an extensive first ever panel of naturally occurring alpha-gal specific human IgE mAbs from subjects with alpha-gal syndrome (also known as red meat allergy). We intend to comprehensively define the precise molecular basis for IgE- mediated reactions to red meat, from both the allergen and the antibody perspective. We will begin by expanding our preliminary panel of IgE antibodies, using both human hybridoma technologies and single-cell RNA sequencing, obtained from peripheral blood B cells of highly characterized research subjects with alpha-gal syndrome. As these IgE mAbs represent the B cells which are induced to undergo class-switch recombination, we will analyze their variable gene sequences to allow for a keen understanding of the B cell population(s) underlying this allergic disease, providing great insights needed to predict individuals’ risk of sensitization. By analyzing IgE mAb variable regions we will understand the alpha-gal specific mAb repertoire in terms of clonality and diversity as a function of glycan specificity within and between affected individuals. Next, we will precisely define critical molecular details of human alpha-gal specific IgE binding and function using a variety of in vitro characterizations, including glycan microarrays, competition-binding assays, kinetic measurements by surface plasmon resonance analysis, and cell-based functional assays. X-ray crystallography will be used to determine structures for IgE clones in complex with glycans, thereby molecularly defining the epitopes underpinning red meat allergy. Together, this proposal will provide comprehensive analyses to molecularly define the glycan epitope targets of the human IgE B cell response, and the origins of the B cells themselves, which will provide much needed details underlying the sensitization and development of hypersensitivity to alpha-gal to inform diagnostic and therapeutic development.
