Allergy to pets : molecular approaches for improved diagnostics and vaccination

Abstract

Allergic diseases are a major health problem, affecting up to 30% of the population in affluent countries. Domestic pets such as cats, dogs and horses are among the most common agents of IgE-mediated allergies. Conventional diagnostics and allergen-specific immunotherapy (SIT) still rely on crude allergen extracts, even though several commercial extracts have demonstrated poor quality with regard to the included allergen content. Particularly in dog allergy, SIT may show a low clinical effectiveness upon treatment with allergenic extracts. As an alternative, recombinant DNA technologies allow for the construction of recombinant (r) allergens for improved allergy diagnostics and induction of a long-lasting protection by SIT. However, this therapy is still limited by unwanted side-effects such as IgE-mediated acute reactions, or late phase reactions (LPR). To combat these limitations, allergens have been modified for safer use in SIT, i.e. hypoallergens, or several allergenic molecules have been combined in one construct for improved diagnostics and treatment of allergic patients.

The aim of this thesis was to demonstrate how development in molecular allergology may increase the understanding of the allergenicity and cross-reactivity of dog allergens. We also set out to demonstrate how this knowledge can be used to engineer safer and more efficacious allergenic constructs for improved diagnostics and therapy.

In paper I, we described the crystal structure and the structural characteristics of the dog lipocalin allergen Can f 2. The recombinant protein was shown to mimick the IgE-binding characteristics of the natural allergen. Furthermore, we demonstrated an IgE- mediated cross-reactivity between Can f 2 and the cat lipocalin allergen Fel d 4, and suggested a potential conserved epitope as a cross-reactive site. In paper II, the primary structures for Fel d 4 and the major horse allergen Equ c 1 were used to identify a novel dog lipocalin allergen, Can f 6. The recombinant allergen elicited a positive IgE-reaction in ~40% of dog-sensitized subjects, and most individuals also had a positive reaction to the homologous allergens Fel d 4 and Equ c 1. The biological activity of rCan f 6 and the homologous allergens was demonstrated by basophil activation test (BAT). Finally, we identified IgE-mediated cross-reactivity between these three allergens, which may account for cross-species sensitization between dog, cat and horse.

In paper III, the method phage display was for the first time used to construct hypoallergenic mutants of the major cat allergen, Fel d 1. The four vaccine candidates were produced as folded proteins in E. coli and showed a lower IgE-binding than the wild type protein in ELISA. By T cell proliferation and BAT assays, we demonstrated that two of the four candidates had a significantly lower T cell activation capacity. Moreover all mutants had a lower allergenic activity than rFel d 1, which could indicate a safer profile for SIT of allergic patients. The effectiveness of the mutants was demonstrated in a mouse model for cat allergy, by the induction of blocking IgG antibodies. In paper IV, a different vaccine concept was described, where four dog lipocalin allergens were assembled in one molecule. The corresponding fusion protein comprised the biochemical and immunological properties of the original allergens, investigated by CD-spectra and ELISA. Importantly, the linked construct bound more IgE than the individual allergens, demonstrating the usefulness for diagnostics. Finally, the linked molecule induced comparable IgG levels to all included allergens compared with an equimolar mix, while showing a lower T cell reactivity which could implicate a lower risk of LPR.

To summarize, this thesis demonstrates how molecular approaches may be used to improve our understanding of allergenicity, to characterize new allergens and define their relationship with other allergens. This detailed knowledge was used to formulate modified allergen constructs for improved diagnosis and vaccination of allergic patients.