Characterization of a hybrid protein designed with segments of allergens from Blomia tropicalis and Dermatophagoides pteronyssinus.

Sensitization to allergens of the house dust mites Dermatophagoides pteronyssinnus and Blomia tropicalis is an important risk factor for asthma and allergic diseases. Allergen-specific immunotherapy is currently based on natural allergen extracts, however, in the last years recombinant allergens with different modifications have shown promising immunological properties that may be advantageously applied for developing novel allergy vaccines. A hybrid molecule (MAVAC-BD-2) containing epitopes of B. tropicalis (Blo t 5, Blo t 8 and Blo t 10) and D. pteronyssinus (Der p 1, Der p 2, Der p 7 and Der p 8) allergens was constructed, expressed in Escherichia coli and purified by affinity chromatography. Its folding was analyzed by circular dichroism. Antibody reactivities were evaluated by ELISA and non-denaturing dot blot assays using a battery of sera from mite allergic patients and non-allergic subjects. ELISA inhibition and dot blot assays with monoclonal antibodies were used to detect B-cell epitopes. Human basophil activation and induction of IgG-blocking antibodies in mice immunized with the hybrid protein were also evaluated. MAVAC-BD-2, expressed as a 22.8 kDa protein, showed a lower frequency and strength of IgE reactivity compared to Blo t 5, Der p 1, Der p 2 and the extracts of B. tropicalis and D. pteronyssinus. MAVAC-BD-2 inhibited 26% of IgE reactivity to Der p 2 and Blo t 5, reacted with anti-Der p 1 and anti-Der p 2 monoclonal antibodies and did not induce relevant basophil activation. MAVAC-BD-2 immunized mice produced specific antibodies that reacted against mite extracts and the purified allergens, as well as IgG antibodies that blocked the human IgE reactivity to mite extracts. MAVAC-BD-2 has hypoallergenic characteristics and in mice induces IgG antibodies that block the human IgE reactivity to mite extracts.