Conformational immunoglobulin E epitopes von Bet v 1

Up to 70% of birch pollen allergic patients show adverse reactions to plant foods. In most cases, this cross- reactivity is a consequence of sensitization to the major birch pollen allergen, Bet v 1, and subsequent immunoglobulin E (IgE) cross-reactivity to homologous plant food allergens. Most patients, however, tolerate at least some of the typical birch pollen-related foods, even if their IgE binds to a broad range of Bet v 1-related plant food allergens. Thus, no clear correlation between the spectrum of IgE reactivities and the clinical symptoms in the birch pollen-plant food syndrome could yet be established. Several factors that might contribute to the varying clinical relevance of cross-reactive IgE are discussed. Of those, the epitope specificity and affinity of allergen- specific IgE will be examined in this project. The aim of this project is the definition of conformational IgE epitopes of Bet v 1 and Bet v 1-related allergens. In order to achieve this goal, an innovative directed in vitro evolution approach will be employed. Artificial proteins carrying single IgE epitopes will be generated from non-IgE binding structural homologues of Bet v 1 by several rounds of random mutagenesis and phage display-based selection using Bet v 1-specific IgE purified from patients` sera. A panel of single epitope carriers will then be used for the following experiments: (1) determination of the frequencies of IgE recognition and the IgE affinities in a group of Bet v 1-sensitized, birch pollen allergic patients, (2) assessment of the contributions of single IgE epitopes to the cross-reactivity between Bet v 1 and Bet v 1- related food allergens, (3) establishment of a correlation between the IgE epitope recognition profile or the affinities of IgE binding to single epitopes and the spectrum of clinically manifest reactions to birch pollen related plant food in a large panel of birch pollen allergic patients. The outcome of this project will be relevant both for basic research in allergology and for allergy diagnostics. Immunogenetic studies have shown that the IgE repertoire consists of a limited number of highly affinity-matured clones, Results obtained in this project will complement these data on the protein level by determining the epitope recognition profile for IgE directed to a well-defined model allergen. Second, the establishment of a link between the epitope specificity of Bet v 1-specific IgE and the clinical spectrum of plant food allergy will pave the way for an epitope-based diagnosis of plant food allergy with increased diagnostic specificity.

Birch pollen an important elicitor of respiratory allergy in temperate zones of the northern hemisphere. Up to 70% of birch pollen allergic patients show adverse reactions to certain plant foods due to sensitisation to the major birch pollen allergen, Bet v 1, and subsequent cross-reactivity of Bet v 1-specific IgE antibodies with related plant food allergens. Most patients, however, tolerate at least some of the typical birch pollen-related foods, even if their IgE binds to a broad range of Bet v 1-related allergens. The cause of this limited clinical relevance of cross-reactive allergen-specific IgE is still unknown. The hypothesis underlying this project was that the extent and clinical relevance of IgE cross-reactivity is determined by the composition and the fine-specificity of the allergen-specific antibody repertoire, as different antibodies bind to different binding sites (epitopes) on the allergen with different extents of cross-reactivity. Therefore, we aimed at identifying IgE-binding epitopes on Bet v 1 and measuring their recognition by allergen-specific antibodies. To this end, we employed different experimental approaches: We examined IgE cross-reactivity between Bet v 1 and the distantly-related mung bean allergen Vig r 6. Moreover, we constructed four chimeric proteins in which different parts of the molecular surface of Bet v 1 were grafted onto the surface of the low IgE-binding celeriac allergen Api g 1 and used these proteins to assess the epitope recognition of Bet v 1-specific IgE from birch pollen allergic patients. Finally, we studied the binding of IgE and other types of antibodies (IgG and IgA) specific to Bet v 1 and several Bet v 1-related food allergens in sera of well-characterised birch pollen allergic patients with different patterns of plant food allergies. We showed that the composition of Bet v 1-specific IgE from birch pollen allergic patients shows a high degree of variability with respect to epitope binding and extent of cross-reactivity. Some patients contain highly cross-reactive IgE that binds to distantly-related food allergens such as those from celeriac, soybean or mung bean, while specificity of IgE from other patients is restricted to allergens with high similarity to Bet v 1, such as those from hazelnut, apple and peach. Nevertheless, we could not establish a clear connection between the composition of Bet v 1-specific IgE or other types of antibodies and the pattern of birch pollen associated food allergies. However, we established a strategy for determining the epitope specificity of allergen-specific antibodies based on the construction of chimeric proteins that carry single epitopes. Further improvements of this approach could lead to the development of an epitope-based diagnosis, which has the potential to increase the reliability of IgE-based diagnosis of birch pollen-associated food allergies but which may also be applicable to other types of allergy.