Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening.

BACKGROUND: The incidence of allergic diseases in developed countries has been increasing constantly in the last 2 decades. The only curative treatment available thus far is specific immunotherapy (SIT). The problematic side effects that can occur during SIT with allergen extracts led to the search for safer alternatives, such as recombinant hypoallergenic proteins with reduced allergenic potential and preserved T-cell immunogenicity. OBJECTIVE: We created hypoallergenic variants of the allergens Bet v 1a and Phl p 5b by using in silico mutation and screening and characterized the biochemical and immunologic properties of selected mutant proteins. METHODS: Knowledge-based potentials were used to estimate structural changes of the protein structures under sequence variation. IgE antibodies and their cross-linking capacity were determined by using a basophil release assay, binding of human birch pollen-specific IgE was investigated by means of ELISA, and ELISPOT assays were performed to examine the T-cell immunogenicity. RESULTS: Selected mutated proteins showed significantly reduced IgE-binding and cross-linking ability but retained their T cell-stimulating properties. Immunization with the hypoallergenic mutants induced blocking antibodies against murine and human IgE epitopes. CONCLUSION: In silico calculation and selection of mutations that render a protein hypoallergenic represents a novel and rapid tool to create candidate molecules for SIT with recombinant allergens.

The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine protease allergen-family, which stresses the importance of these proteins with respect to fungal IgE-cross-reactivity.

Calcium-binding proteins and their role in allergic diseases.

Calcium-binding proteins (CBPs) are ubiquitous pollen allergens and important food allergens in fish and amphibians. Calcium-binding allergens containing two EF-hands (polcalcins) have been detected and characterized in pollen from trees, grasses, and weeds. Timothy grass Phl p 7 is the most cross-reactive allergen among polcalcins. Although there is cross-reactivity described within the subfamilies of calcium-binding allergens, there are no strong indications for IgE cross-reactivity between CBPs from plants, fish, and humans. Therefore, Phl p 7 could be used as marker to identify multiple pollen-sensitized patients, whereas cod Gad c 1 or carp Cyp c 1 could be selected for the diagnosis of fish allergy. Hom s 4, a calcium-binding autoantigen, might be an interesting candidate to monitor chronic skin inflammation in atopic and nonatopic individuals. Diagnostic tests containing these molecules could allow the identification of most patients sensitized to calcium-binding allergens/antigens. In general, IgE recognition of calcium-binding allergens is influenced by binding or release of calcium ions. This knowledge could be used to engineer hypoallergenic CBPs for specific immunotherapy.