Towards new forms of safe immunotherapy for insect allergy

Allergens from honeybee and wasp venom belong to the most frequent causes of life-threatening systemic allergic reactions. Allergen-specific immunotherapy (SIT) is currently the only possibility for the prevention of severe anaphylactic reactions in bee and wasp allergic patients. However, venom SIT can cause severe and systemic side effects. Aim of this project is the characterization of allergenic epitopes on bee- and wasp allergens and the development of new vaccines for bee and/or wasp venom allergy. A prerequisite for the development of such vaccines is the identification and characterization of the anaphylactic IgE epitopes of bee- and wasp venom allergens for their subsequent conversion into safe vaccines. The molecular structures of several frequently recognized bee and wasp allergens have been elucidated and these molecules have been expressed as recombinant proteins or glycoproteins. However their allergenic activity has not been studied in detail. In particular, it is not known whether sequential and/or conformational protein epitopes and/or carbohydrate epitopes are responsible for the anaphylactic activity of the individual allergens. Available data indicate, that the allergenic activity of major bee and wasp allergens may reside in a folded protein backbone and not exclusively in the carbohydrate moieties. This assumption will be investigated in detail by eukaryotic expression of venom allergens using codon-optimized genes with or without glycosylation sites and subsequent assessment of the structural fold and the allergenic activity of the individual molecules using in vitro cellular assays (e.g., basophil activation tests). For the conversion into hypoallergenic molecules, two technologies will be tested. One technology (recombinant hypoallergens) will explore the possibilities for the reduction of allergenic activity by fragmentation, mutation and reassembly of allergen sequences. The other technology is based on the selection of hypoallergenic allergen peptides from surface-exposed areas which will be expressed as recombinant fusion proteins with carrier proteins. The allergen derivatives obtained with these technologies will be tested for IgE and T cell reactivity. Furthermore rabbits will be immunized to test, if the derivatives can induce allergen-specific IgG antibodies. The antibodies will then be tested for their ability to inhibit allergic patients` IgE-binding to the natural or recombinant allergens and for their ability to inhibit bee-or wasp venom induced basophil and T cell activation. The ultimate goal is the in vitro characterization of possible candidate vaccines which may then be tested for safety in first clinical trials.

Characterization of anaphylactic epitopes of bee and wasp venom allergens and development of hypoallergenic variants for immunotherapy Allergens from honeybee and wasp venom belong to the most frequent causes of life-threatening systemic allergic reactions in adults and are the second most common cause of anaphylaxis in children. Venom immunotherapy (VIT) is currently the only possibility for the prevention of severe anaphylactic reactions in bee and wasp allergic patients. VIT is based on the administration of disease-eliciting allergens. Due to the allergenic, irritating and toxic venom components, VIT can be accompanied by severe side effects. A prerequisite for the development of hypoallergenic vaccines with reduced allergenic activity is the identification and characterization of the anaphylactic IgE-epitopes of bee- and wasp allergens. With molecular biology technologies, purified recombinant bee and wasp allergens can be generated. The majority of venom allergens are glycoproteins containing carbohydrate chains, which can cause IgE-cross-reactivity between unrelated allergen sources like venom, pollen and plant derived food. We expressed, purified and characterized a comprehensive panel of bee and wasp allergens. The natural glycosylated bee venom allergens, Api m 1 and Api m 10 were expressed in insect cells without glycosylation and with the wasp venom allergens Ves v 1 and Ves v 5, they were suitable for the identification of the culprit insect in bee and/or wasp venom allergic patients. We assessed Api m 1 and Ves v 5-specific antibody-reactivity and showed, that severe sting reactions can be observed in patients showing very low Api m 1 and/or Ves v 5-specific IgE levels. These results are of clinical relevance, because they indicate that even subjects with very low IgE levels against these marker allergens can experience severe reactions. Accordingly one may consider informing patients with a positive IgE test results to the non-glycosylated marker allergens about the potential risk that they may experience a severe sting reaction. Non-glycosylated Api m 1 is equally folded, stable and enzymatically active as its glycosylated natural form Api m 1. It contains anaphylactic epitopes that are independent of the carbohydrate moieties. Api m 1- and Ves v 5-derived peptides showed no IgE-reactivity with sera of venom allergic patients and serve as basis for the generation of hypoallergenic derivatives. Within this project a carbohydrate marker was generated, with the aim to overcome unwanted IgE-cross-reactivity upon unrelated allergen sources in serological-based allergy tests. N-glycosylation sites were engineered into a per se non-allergenic protein. The expressed and purified glycoprotein binds IgE exclusively on the carbohydrate chains. We identified house dust mites as carbohydrate source showing cross-reactivity with plant as well as venom carbohydrate moieties. Using the carbohydrate marker in inhibition-assays, we were able to dissect carbohydrate-reactive IgE from peptide-specific IgE thereby improving in vitro allergy testing.