This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus. Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

No abstract

Among the vast number of identified protein families, allergens emanate from relatively few families which translates to only a small fraction of identified protein families. In allergy diagnostics and immunotherapy, interactions between immunoglobulin E and allergens are crucial because the formation of an allergen-antibody complex is necessary for triggering an allergic reaction. In allergic diseases, there is a phenomenon known as cross-reactivity. Cross-reactivity describes a situation where an individual has produced antibodies against a particular allergenic protein, but said antibodies fail to discriminate between the original sensitizer and other similar proteins that usually belong to the same family. To expound the concept of cross-reactivity, this study examines ten protein families that include allergens selected specifically for the analysis of cross-reactivity. The selected allergen families had at least 13 representative proteins, overall folds that differ significantly between families, and include relevant allergens with various potencies. The selected allergens were analyzed using information on sequence similarities and identities between members of the families as well as reports on clinically relevant cross-reactivities. Based on our analysis, we propose to introduce a new A-RISC index (Allergens’–Relative Identity, Similarity and Cross-reactivity) which describes homology between two allergens belonging to the same protein family and is used to predict the likelihood of cross-reactivity between them. Information on sequence similarities and identities, as well as on the values of the proposed A-RISC index is used to introduce four categories describing a risk of a cross-reactive reaction, namely: high, medium-high, medium-low and low. The proposed approach can facilitate analysis in component-resolved allergy diagnostics, generation of avoidance guidelines for allergic individuals, and help with the design of immunotherapy.

No abstract