Recombinant cat and mite allergens as tools in the study of diagnosis and treatment of allergy
Sammanfattning: Allergic diseases, such as allergic asthma, allergic rhinitis and atopic eczema, are a major health problem world-wide, affecting up to 30% of the population. A large proportion of the patients are allergic to indoor allergens, such as mite and cat allergens, which are airborne and thus cause symptoms in the airways upon inhalation. Hundreds of single allergens from different sources have been identified, cloned and expressed as highly pure and well-defined recombinant allergens. They can be produced in large amounts and are gradually replacing allergen extracts in allergy diagnosis and allergen-specific immunotherapy (SIT), the only treatment that may affect the natural course of allergic diseases. SIT may be further improved by genetically modifying allergens into hypoallergens, which have a reduced allergenic capacity, but a retained T-cell reactivity. The aims of this thesis were to clone and characterise new allergens from the storage mite Lepidoglyphus destructor, and to apply the major recombinant cat allergen Fel d 1 in allergy diagnosis and in the construction and evaluation of hypoallergens for use in therapy of cat allergy. In paper I, PCR and screening with sera were used to identify and isolate new allergen clones from a phage display cDNA library that was constructed previously from L. destructor. Two new clones, showing homology to tropomyosin and ?-tubulin, were obtained and subsequently expressed in Escherichia coli. The recombinant proteins bound to IgE antibodies in sera of mite-allergic patients and were characterised as putative minor allergens Lep d 10 and L. destructor ?-tubulin. Lep d 10 was found to be cross-reactive with tropomyosins from other mite species and crustaceans. In paper II, the previously well-characterised major recombinant cat allergen rFel d 1 was compared to cat dander extract (CDE) in the diagnosis of cat allergy in children of a large prospective birth cohort. The rFel d 1 was at least as good as CDE in detecting allergen-specific IgE and may be a better marker for early cat sensitisation. The study showed that children with allergen-specific IgE, but without symptoms to cat at 4 years of age all developed allergic symptoms at 8 years of age. In paper III, the three-dimensional structure of rFel d 1 was genetically altered in a rational approach comprising duplication of known T-cell epitopes and disruption of disulphide bonds through introduction of point mutations. Three out of seven Fel d 1 derivatives generated were identified as hypoallergens with a strongly reduced IgE-binding capacity, a reduced allergenicity and a retained T-cell reactivity. The most promising candidate for SIT, rFel d 1 (DTE III) was further evaluated in vivo in paper IV. Therapeutic treatment of cat-allergic mice with the hypoallergen resulted in decreased airway hyperreactivity and induction of allergen-specific IgG with blocking capacity. In contrast to unmodified rFel d 1, the hypoallergen was tolerated at a high treatment dose without any observed side effects. In addition, rFel d 1 (DTE III) induced less skin prick test-reactivity compared to rFel d 1 in cat-allergic patients. In summary, the results of this thesis show the wide usage of recombinant allergens. They are important in the characterisation of single allergens of allergen sources, as shown for the mite L. destructor. Furthermore, they reveal sensitisation to a specific allergen component, e.g. the major cat allergen Fel d 1, which in respect to diagnosis of cat allergy seems to be at least as good as CDE. In addition, recombinant allergens can be used in the development of safer and more efficient tools for SIT, as demonstrated with Fel d 1 (DTE III).
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