Molecular approaches for characterization, diagnosis and therapy of allergy to pets

Abstract: Allergy to furred animals is a common affliction, where up to 20% of the population in affluent countries are sensitized to one or more pet allergens. Several causative allergens from an array of species have been identified, but there are still gaps in our knowledge. This complicates diagnosis and treatment of allergy to pets. Conventional methods rely on naturally derived allergen extracts, with poor characterization and lack of broadly accepted standards. Due to the nature of the extracts, some allergens could be missing or present in clinically non-significant concentrations. The aim of the thesis is to improve diagnostics by characterizing extracts and evaluating component based microarrays to conventional methods. Further, to identify new allergens as well as describe previously only partially characterized allergens, their distribution and properties. Paper I compares two multiplex microarray assays in allergy diagnostics to the standard methods ImmunoCAP and skin prick test with doctor’s diagnosis in a cohort of children with persistent asthma (n=71). The results showed that 75% of all children were sensitized to at least one allergen and that nearly half was multisensitized to three or more allergens. The accuracy of the methods was comparable, they all had a pair-wise concordance of 90% and above. However, the microarray assays contributed with positive IgE responses to new allergens not described in the diagnosis in almost half the patients (47%). The added value of microarray analysis in multisensitized patients is not to belittle. In paper II, an analysis of the content and composition of dog dander extracts for skin prick test is conducted. Results from five commercial manufacturers display a large variation, not only between the different suppliers, but also between batches albeit to a lesser extent. Anatomical location of the source material also differs, hair, dander and epithelia was analyzed. Two of the allergens were detected only at low amounts and patient sera sensitized to these failed to induce an allergic response to the extracts in a basophil activation test. Moreover, a population of dogs was investigated in regard to their allergen profile, also resulting in a substantial divergence between individuals. The heterogeneity of the extracts calls for better content characterization and standardization. Substandard quality could render flawed diagnosis and jeopardize patient safety. Paper III characterizes sensitization to horse allergens, where several allergens are only described partially, or analysis of IgE binding performed in a small cohort. We present sensitization data for four horse allergens including a novel protein, designated Equ c 7, with a prevalence of 38%. Equ c 7 is a homologue to the cat allergen Fel d 1, suggesting possible cross-reactivity. Further, we describe two forms of full-length Equ c 2 and the potential roles of isoforms. Here we contribute to the panel of horse allergens. Allergy to horse is less investigated than to other pets and likely more allergens await discovery. Aerodynamic properties of allergens are investigated in paper IV, where three dog allergens were sampled, however, only two could be detected. Particles associated with airborne allergen differed in size. Can f 1 was only detected on particles larger than 2.8 μm, diversely from Can f 4 which was found on particles of all sampled sizes >8.3-<0.34 μm. Smaller particles reach the small airways and are causative of inflammation and airway responsiveness. Different sampling methods also yielded different proportions of the allergens, indicating that they possess different airborne properties. To summarize, furred animals produce a wide array of allergens and there is a large individual variation in expression levels/profiles. This is mirrored in the extracts used for diagnosing allergy, where poor quality counteracts correct diagnosis. Characterization of novel allergens and production of recombinant equivalents could improve the accuracy of diagnostics and therapy.