Molecular characterization of crossreactive animal-derived allergens for diagnosis and treatment of Type I allergy

The aim of the proposed project is the molecular characterization of crossreactive animal-derived allergens (e. g., albumin, parvalbumin) that are a frequent cause of perennial Type 1 allergies and/or IgE-mediated food hypersensitivities. According to previous studies on animal derived allergens, we selected albumins from cat and dog as well as parvalbumin from fish because these allergens bind high amounts of specific IgE and mediate IgE crossreactivities in sensitized individuals. T-cell and antibody recognition of these allergens will be determined with molecular biological as well as synthetic peptide technology. The first part of the project will be dedicated to the production of recombinant allergens (albumins and parvalbumins) and defined fragments thereof. Next, the recombinant allergens and allergen fragments will be compared with their natural counterparts regarding their IgE binding capacity and their ability to induce basophil histamine release and T-cell proliferation. B-cell epitopes of the above selected allergens will then be determined by molecular biological techniques (gene fragmentation and subsequent expression). Recombinant animal-derived allergens and their fragments could be used as research tools to explore their interaction with the patients immune system (e.g., antibodies, T-cells, effector cells: mast cells, basophils, eosinophils, antigen presenting cells: dendritic cells) and as defined tools for in vitro as well as for in vivo allergy diagnosis. The molecular characterization of relevant animal-derived allergens might ultimately permit us to generate recombinant and/or synthetic hypoallergenic allergen derivatives for specific immunotherapy.

Molecular characterization of crossreactive animal-derived allergens for diagnosis and treatment of Type I allergies The aim of the project was to characterize relevant crossreactive animal-derived allergens at a molecular level in order i) to study the humoral and cellular immune recognition of these proteins ii) to produce recombinant allergen components which can be used to improve and refine allergy diagnosis and treatment. The project was dedicated to the molecular characterization of cat and dog albumin and fish parvalbumin, which mediate perennial Type I allergies and/or food hypersensitivities. We succeeded in producing recombinant dog albumin, an important crossreactive dog allergen and recombinant carp parvalbumin, the major fish allergen. Furthermore we were able to evaluate recombinant Fel d 1, the major cat allergen, for in vitro diagnosis of cat allergy in a population of cat allergic children and adults. Crossreactivity of recombinant Fel d 1 was also studied. On the basis of the complete cDNAs obtained for cat and dog albumin the major IgE-reactive portions of these allergens were mapped by gene fragmentation technology. IgE-reactive areas were localized at the N-terminus, the C-terminus and in the center of cat and dog albumin. Although the cDNA and deduced amino acid sequences of dog and cat albumin were highly homologous to sequences of albumins from other species (including man), we could define regions with less conserved sequence homology within the IgE-reactive fragments. The latter observation may explain why IgE antibodies of certain patients are able to discriminate between cat and dog albumin. The fact that IgE-recognition of the major fish allergen, parvalbumin, was strongly influenced by the presence of protein-bound calcium gave rise to the idea that site-directed mutagenesis of the calcium-binding sites will deliver hypoallergenic derivatives for specific immunotherapy of fish allergy.