Molecular Biotechnology for Allergy Diagnosis and Therapy (GENALL)

The objective of the proposed multilateral project is to utilize recombinant DNA technology for molecular allergy diagnosis and the development of novel approaches of allergen-specific immunotherapy. The project focuses on perennial indoor and seasonal outdoor inhalant allergies that are currently predominant in India, but may become clinically more important in Europe in the future. In line with this, the aim of the project is to 1. identify and characterize clinically relevant inhalant allergens and gain insight in potential cross-reactive allergens relevant in both, Europe and India. 2. apply biotechnological approaches for vaccine development to improve safety and efficacy of allergen- specific immunotherapy for inhalant allergies. Our laboratory will focus on the identification, recombinant production, and characterization of Prosopsis pollen allergens; on the recombinant production of the major weed pollen allergens Art v 1, Amb a 4, and Par h 1; on the recombinant production and characterization of cockroach allergens; and on the physicochemical characterization of Art v 1-derived vaccines. Serum IgE reactivity and cross-reactivity profiles of allergic patients from India and Europe will be investigated using panels of recombinant Prosopsis and weed pollen allergens. The molecular analysis of new allergens from clinically relevant pollen abundant in India and their cross-reactive properties will contribute to an improved risk assessment of such plants for the European population. In addition, the IgE reactivity and cross-reactivity of cockroach allergens, one of the most prevalent sources of perennial indoor allergens in India, will also be investigated using sera from European patients. The knowledge about clinical relevance and cross-reactive properties of individual allergenic molecules is the prerequisite for novel innovative treatment options using genetically engineered allergen vaccines. In this respect, the project will also develop allergy vaccines based on T cell epitopes and other hypoallergenic derivatives, which will be combined with immune modulating adjuvants. In summary, the present project will contribute to harmonize methods for allergen characterization and allergy diagnosis, and to provide general standards to determine the quality and safety of recombinant allergen and vaccine preparations in Europe and India.

The prevalence of IgE-mediated allergies is increasing worldwide particularly in newly industrializing countries causing high socio-economic costs and strongly affecting the quality of life. This pandemic appearance of allergies is supposed to be due to changing lifestyle and by spreading of allergens due to global climate change. The focus of this multilateral allergy research project was the application of biotechnological approaches for allergen characterization, molecule-based diagnostics and vaccine development. In specific, we investigated clinically relevant inhalant perennial indoor (cockroach) and seasonal outdoor (mugwort, feverfew and mesquite pollen) allergen sources from India and Europe. For this, ten allergen molecules of American cockroach were biotechnologically produced and tested for IgE reactivity using sera from 117 allergic patients. We observed divergent sensitization profiles in patients from different countries and a higher sensitivity to the allergen molecules compared to the commonly used extract. Allergic reactions to pollen of the invasive weed feverfew and mesquite are frequently observed in highly exposed subjects. Though those allergen sources prevalent in parts of Asia and America are of clinical relevance, comprehensive molecule-based investigations were missing. Using a mass spectrometry- based approach, a panel of novel IgE-binding molecules was identified in both plant sources. Among those, a defensin-like molecule from feverfew was identified and biotechnologically produced making this novel allergen now available for molecule-based diagnostics. This allergen was structurally and immunologically compared to homologous molecules from mugwort and ragweed. Interestingly, weed pollen-allergic patients from Austria and Canada demonstrated similar recognition pattern while Korean patients were mostly sensitized to Art v 1, the defensin-polyproline-linked allergen from mugwort. Knowledge of the allergologic and immunologic patterns in different cohorts worldwide provides better understanding for the design of vaccines. In this respect, the Art v 1 allergen and a hypoallergenic variant were fused to the bacterial component flagellin for potential application in allergen-specific immunotherapy. Specific IgG antibody production in mice provided evidence that incorporation of hypoallergens into flagellin-allergen fusion proteins is suitable to further improve vaccine candidates. Results of this project enabled to better understand the complex sensitization pattern towards the investigated inhalant allergen sources and provided the basis for enhanced diagnostics and vaccines in allergen-specific immunotherapy.