To cite this article: Twardosz-Kropfmü ller A, Singh MB, Niederberger V, Horak F, Kraft D, Spitzauer S, Valenta R, Swoboda I. Association of allergic patients' phenotypes with IgE reactivity to recombinant pollen marker allergens. Allergy 2010; 65: 296-303.Diagnosis of Type I allergy is based on the documentation of a positive case history, in vivo provocation tests (e.g., skin prick test, nasal, oral and bronchial challenge) and the in vitro detection and measurement of allergen-specific IgE antibodies in patient's serum (1). Natural allergen extracts used for allergy diagnosis and immunotherapy have a number of disadvantages: they contain many different, also nonallergenic proteins and various ill-defined substances (e.g., carbohydrates, enzymes, and fats). The standardization of natural allergen extracts is difficult and the allergen contents and composition of extracts vary from manufacturer to manufacturer and from batch to batch (2-6). Extracts can lack certain allergenic proteins which might have got degraded during the production process and storage and they can be contaminated with proteins from other allergen sources which can deliver false diagnostic results (7). Moreover, allergy tests performed with natural extracts determine only the allergen source a patient reacts with but do not give information about the nature and the number of the disease-eliciting molecules the patient is sensitized to (8).The identification and characterization of allergenic molecules by cDNA cloning allows the large scale production AbstractBackground: During the last decade allergen molecules from several allergen sources have been produced by recombinant DNA technology. The aim of this study was to investigate whether IgE reactivity to recombinant pollen allergens with broad and narrow cross-reactivity is associated with clinical phenotypes of allergic sensitization. Methods: Serum IgE reactivity to a panel of six recombinant birch and grass pollen allergens was measured by ELISA in pollen sensitized patients from Central Europe to define groups of patients with exclusive IgE reactivity to rBet v 1, with exclusive reactivity to major grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5) and with IgE reactivity to cross-reactive pollen allergens (rBet v 2, rPhl p 7). Patients' clinical phenotypes were recorded. IgE responses to tree, grass and weed pollen as well as plant food extracts were evaluated in vitro by CAP-FEIA and clinical sensitivities were confirmed in vivo by skin prick testing. Results: IgE reactivity to the recombinant major birch pollen allergen, rBet v 1, was associated with sensitization to pollen from birch, taxonomically related trees and to certain plant-derived food. Reactivity to the recombinant timothy grass pollen allergens, rPhl p 1, rPhl p 2, rPhl p 5, indicated sensitization to pollen from grasses. Patients reacting with the highly cross-reactive allergen rPhl p 7 were polysensitized to pollen from unrelated trees, grasses and weeds and rBet v 2-positive patients were polysensitized to pollen...

The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy.Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calciumbinding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE.Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.