Diagnosis of IgE-mediated allergy : new approaches using recombinant allergens

Abstract

More than 10% of the population in industrialized countries suffer from lgE-mediated cat allergy. Allergens produced by recombinant techniques offer new possibilites to diagnose and treat allergic patients but will also help to uncover the mechanisms behind the sensitisation. The aim of this thesis was to investigate the clinical usefulness of a recombinant form of the major cat allergen, Fel d 1, from construction of genes and characterisation to diagnosis of cat allergic patients. Furthermore, tools for treatment were developed using the Fel d 1 structure and the major allergen in timothy, Phl p 5.

Although Fel d 1 was cloned more than a decade ago, attempts to produce a tetrameric allergen by recombinant methods with structural features similar to the natural allergen have been only partially successful. A synthetic gene coding for direct fusion of the two chains of Fel d 1 was constructed. Expression resulted in a 30 kDa non-covalently associated homodimer. Biochemical and biological analysis showed that the overall fold and immunological properties were very similar to those of natural Fel d 1. The recombinant (r)Fel d 1 construct was subsequently used to determine the structure by X-ray crystallography at 1.8 A resolution. The fold of Fel d 1 presents a striking resemblance to uteroglobin, a molecule with anti-inflammatory and immunomodulatory properties. An internal pocket and the surface localisation of three previously defined Fel d 1 lgE epitopes is presented.

We evaluated the diagnostic usefulness of lgE and IgG4 antibodies to rFel d 1 in children and adults with doctors' diagnosis of rhinoconjunctivitis (RC) andlor asthma due to cat. All patients showed positive lgE responses to rFel d 1 by ELISA. Sera from children displayed higher lgE levels to rFel d 1 compared to the adult patients. There was a close correlation between lgE responses by CAP to rFel d 1 and the cat dander extract" however the lgE levels to the single rFel d 1 molecule were significantly higher. Among children with asthma, the lgE levels to rFel d 1 were higher in comparison to both the asthmatic adults and children with RC, whereas the IgG4 levels were elevated in adults with allergic RC compared to adults with asthma.

The only curative treatment of allergic disease is allergy vaccination, however the patients face the risk of side effects. Allergens with decreased lgE-binding capacity, but retained Tcell reactivity (hypoallergens) have been tested in clinical trials. We present a new approach of how to generate hypoallergens using structural information and knowledge of B- and T-cell epitopes. The structure of the model allergen Fel d 1 was systematically altered by duplication of T-cell epitopes and disruption of disulphide bonds. Three derivatives displayed a marked reduction in lgE-binding capacity, induced a lower degree of basophil activation but stimulated T-cell proliferation equally well compared to rFel d 1, and are therefore promising hypoallergen candidates.

The occurrence of side effects caused by the aluminium hydroxide adjuvant (Alum) by allergy vaccination is frequently reported. In a mouse model using the recombinant version of the timothy pollen allergen Phl p 5, we demonstrated that carbohydrate-based particles (CBP) exhibit several potential advantages over aluminium-hydroxide as adjuvant for immunotherapy. CBP covalently bound to rPhl p 5b (CBP-p5) induced a strong antibody response, which cross-reacted with group 5 allergens from other grass species and exhibited characteristics of blocking antibodies. Alum-5b induced a preferential allergen-specific Th2 cytokine profile, whereas CBP-p5 induced a mixed Th1/Th2 response. CBP-p5 yielded a stable vaccine formulation with preserved immunogenic features and, in contrast to Alum, induced no granulomatous tissue reactions.

In conclusion, the work presented in this thesis presents the development of a clinically relevant allergen, from idea, via biochemical, biological and structural characterization to diagnosis of cat-allergic patients and also the rational design of allergens and development of a novel adjuvant for therapeutic purposes.