Molecular characterisation of major allergens from mite (Lep d 2) and cat (Fel d 1)

The prevalence of allergic disease has increased significantly during the last decades and today more than 30% of the Swedish population is affected. By using recombinant allergens, diagnostics and treatment can be improved and the mechanisms of allergic reactions may be further explored. The objective of this thesis was to characterise allergens from two of the most important sources of allergens world-wide, cats and mites, at the molecular level.

(Study I) Traditionally, mites grown in culture for years have been the source of allergens for use in research, diagnostics and therapy. However, the actual source of mite sensitisation is wild mites. The aim of this study was to investigate the occurrence of polymorphism in the gene encoding the allergen Lep d 2 in the dust mite Lepidoglyphus destructor derived from different sources. We could identify two Lep d 2 genes in both cultured mites and wild mites isolated from a hay sample. In addition, two new variant forms of Lep d 2 with single amino acid exchanges were found at different frequencies in wild and cultured mites. Neither in ELISA inhibition studies nor in T-cell experiments could any major differences between the variants be detected.

(Studies II, III) To increase the understanding of the mechanisms underlying cat allergy and to obtain knowledge for new treatment strategies, we have solved the crystal structure of the most potent cat allergen, Fel d 1. The structure of Fel d 1 was determined 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. This resemblance provides possible explanations for the allergenicity of Fel d 1. An internal pocket was found within Fel d 1, and residual electron density within the cavity indicates the presence of a ligand. The shape of the cavity as well as the properties of the residues lining it indicate an amphipathic ligand. Finally, the surface localisation of three previously defined Fel d 1 IgE epitopes is presented.

(Study IV) Today, the only curative treatment for allergic disease is immunotherapy. Although this treatment often results in reduced allergic symptoms, a major disadvantage is the risk of side effects. To reduce these risks, allergens with decreased IgE-binding capacity, but retained T-cell reactivity (hypoallergens) can be used. In this study, 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. In addition, disruption of disulphide bonds was performed. A panel of seven Fel d 1 derivatives was generated, and three of the derivatives displayed a marked reduction in IgEbinding capacity. In addition, they induced a lower degree of basophil activation and similar or stronger T-cell proliferation than Fel d 1.

In conclusion, the work presented in this thesis exemplifies how detailed molecular analyses of allergens may provide knowledge about allergen function and offer tools for improved diagnosis and treatment of allergic disease. Specifically, the Lep d 2 sequence diversity identified in cultured and wild L. destructor mites seemed not to have any significant impact on IgE-binding or T-cell response. The determination of the crystal structure of Fel d 1 has provided clues about the pathogenesis of cat allergy as well as tools for improved diagnostics and therapy. Moreover, the new strategy to generate hypoallergens may be applied to any allergen to improve immunotherapy.

List of scientific papers

I. Kaiser L, Gafvelin G, Johansson E, van Hage-Hamsten M, Rasool O (2003). Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites. Eur J Biochem. 270(4): 646-53.
https://pubmed.ncbi.nlm.nih.gov/12581204

II. Kaiser L, Gronlund H, Sandalova T, Ljunggren HG, Schneider G, van Hage-Hamsten M, Achour A (2003). Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1. Acta Crystallogr D Biol Crystallogr. 59(Pt 6): 1103-5.
https://pubmed.ncbi.nlm.nih.gov/12777788

III. Kaiser L, Gronlund H, Sandalova T, Ljunggren HG, van Hage-Hamsten M, Achour A, Schneider G (2003). The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family. J Biol Chem. 278(39): 37730-5. Epub 2003 Jul 08
https://pubmed.ncbi.nlm.nih.gov/12851385

IV. Saarne T, Kaiser L, Gronlund H, Rasool O, Gafvelin G, van Hage-Harmsten M (2004). Rational design of hypoallergens applied to the major cat allergen Fel d 1. [Submitted]