ABSTRACT Objectives: House dust mite (HDM) allergy represents one of the most important allergies worldwide and about 50% of all allergic patients are sensitized to HDMs. However, immunotherapy is less efficient for HDM allergy than for other allergies (e.g.,pollen allergy), probably because of the bad quality of the used HDM extracts which do not contain all important allergens. Immunotherapy with individual allergens would improve the safety and efficacy of the treatment but requires the identification of the clinically relevant allergens and the production of hypoallergenic derivatives thereof. In this thesis we aimed to characterize two high molecular weight HDM allergens, Der p 11 and Der p 14, for their importance to be incorporated into a HDM vaccine and to produce a derivative of the major HDM allergen, Der p 23, with reduced allergenic activity for the treatment of HDM allergic patients. Material and Methods: Synthetic genes coding for Der p 11 and Der p 14 were expressed in Escherichia coli and purified to homogeneity. The secondary structure of both proteins was determined by circular dichroism analysis. IgE reactivity of rDer p 11 and rDer p 14 was tested with sera from HDM allergic patients from European and African populations in RAST-based dot-blot assays. Rabbits were immunized with the two allergens to study the localization of the allergens in mites using immunogold electron microscopy. A synthetic gene coding for non-allergenic Der p 23 derived peptides with reduced capacity to induce T cell proliferation and a hepatitis B virus-derived PreS domain for T cell help was expressed in E. coli and purified to homogeneity (PreS-2XP4P5). The IgE reactivity and allergenic activity of PreS-2XP4P5 was compared with rDer p 23 in dot blot assays and basophil activation tests using blood from HDM allergic individuals. Rabbits were immunized subcutaneously with Der p 23 or PreS-2XP4P5 to investigate whether the molecules induce specific IgG antibodies which are able to inhibit patients' IgE binding to Der p 23. Results: rDer p 11 is an alpha helical protein with high homology to paramyosins from dust mites, tropical mites, skin mites, ticks and other invertebrates. rDer p 14 has a mixed alpha helical and beta sheet conformation and shows high homology to C terminal fragments of group 14 allergens from different species of mites. In contrast to most other mite allergens which are mainly located in mite feces, both proteins are located predominantly in mite bodies. The IgE-binding frequency and allergenic activity of both proteins was low in European populations.
However, both proteins showed higher IgE-binding frequencies (30 %) in African populations. When we analysed sera from patients suffering from atopic dermatitis (AD), we found that Der p 11 as well as Der p 14 are important allergens for AD patients.
The recombinant derivative of Der p 23 (PreS-2XP4P5) showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests compared to Der p 23. Upon immunization of rabbits, PreS-2XP4P5 induced IgG antibodies that inhibited the binding of patients' IgE to Der p 23 comparable to IgG antibodies induced with Der p 23. Conclusions:
We found rDer p 11 and rDer p 14 as useful serological markers for the identification of a subgroup of HDM allergic patients suffering from HDM-associated AD, whereas for HDM allergic patients suffering only from respiratory allergy the importance of the two allergens is rather low.
Thus, Der p 11 and Der p 14 need not to be included into a vaccine for the treatment of HDM allergic patients. The hypoallergenic derivative of the major HDM allergen, Der p 23 (PreS-2XP4P5) represents a promising candidate for the treatment of HDM allergic patients, due to its reduced IgE-reactivity, reduced allergenic activity and capacity to induce blocking IgG antibodies.