Cat allergy : diagnosis and characterization of important allergens

Allergy affects about 25% of the population. Allergic diseases are manifested in many ways, from mild rhinitis to life-threatening anaphylaxis. Globally, more than 15% of children and adults are allergic to pets.

The focus of this thesis is important cat allergens. First we identified novel cat allergens, cat immunoglobulin (Ig)A and cat IgM. Analyses revealed that the IgE-reactivity in human sera was directed to carbohydrates of the immunoglobulins. We also detected IgEreactivity to IgM in 7 of 9 animal species tested, but not to human immunoglobulins. Since our immunoassays were not optimized for testing allergens with carbohydrate epitopes, appropriate controls were performed. These controls revealed that the reaction was not mediated by IgE but by IgM in patient serum by cross-linking animal immunoglobulins and alkaline phosphatase. This IgM fits into the heterophile antibody classification and is directed to carbohydrates on animal immunoglobulins and on calf intestine alkaline phosphatase.

Sera from cat-sensitized patients were further analysed for IgE antibodies to purified cat IgA using the ImmunoCAP System. Thirty-eight percent of the cat-sensitized sera were positive. Indirect enzyme linked immunosorbent assay (ELISA) confirmed the IgE-reactivity to cat IgA and also to cat IgM, but not to deglycosylated cat IgA. Competitive inhibition ELISA was performed and strong inhibition of patient IgE to cat IgA was observed in all sera after pre-incubation with cat IgA and cat IgM. Thus a new group of cross-reactive allergens with carbohydrate epitopes has been identified. This is the first report of mammalian carbohydrate IgE epitopes. These epitopes are also targeted by IgM and may therefore interfere in immunoassays.

Recombinant variants of the major cat allergen Fel d 1 have also been studied in this thesis. We confirmed that recombinant (r)Fel d 1 (1+2), rFel d 1 (2+1) and natural Fel d 1 have equivalent biological and immunological properties. This confirmation was based on similar basophil stimulation, T-cell proliferation, direct and competitive inhibition ELISAs. We also present the crystal structure of the rFel d 1 (1+2) tetramer at 1.6 Å resolution. The crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. The external calcium-binding site corresponds to a putative calcium-binding site previously suggested for uteroglobin. The second calcium-binding site lies within the dimerization interface, inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand.

We evaluated IgE- and IgG4-binding to rFel d 1 among cat allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria. A high correlation of IgE responses to rFel d 1 and cat dander extract in ImmunoCAP among the patients was demonstrated. Using ELISA, 98% of patients and none of the controls had IgE to rFel d 1 and there was a 3-fold increased risk of asthma for those children with the highest IgE levels. IgE responses to rFel d 1 among children with asthma were higher compared to children with RC and adults with asthma. Furthermore, children with asthma displayed higher IgG4 levels than did asthmatic adults. We demonstrated that a single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the currently used extract-based test. We suggest that elevated IgE antibody levels to Fel d 1 is a risk factor for asthma in cat allergic children.

List of scientific papers

I. Adédoyin J, Johansson SG, Grönlund H, van Hage M (2006). Interference in immunoassays by human IgM with specificity for the carbohydrate moiety of animal proteins. J Immunol Methods. 310(1-2): 117-25.
https://pubmed.ncbi.nlm.nih.gov/16507308

II. Adédoyin J, Grönlund H, Oman H, Johansson SG, van Hage M (2007). Cat IgA, representative of new carbohydrate cross-reactive allergens. J Allergy Clin Immunol. 119(3): 640-5.
https://pubmed.ncbi.nlm.nih.gov/17336613

III. Kaiser L, Velickovic TC, Badia-Martinez D, Adedoyin J, Thunberg S, Hallén D, Berndt K, Grönlund H, Gafvelin G, van Hage M, Achour A (2007). Structural characterization of the tetrameric form of the major cat allergen Fel d 1. J Mol Biol. 370(4): 714-27.
https://pubmed.ncbi.nlm.nih.gov/17543334

IV. Grönlund H, Adédoyin J, Reininger R, Varga E, Zach M, Fredriksson M, Kronqvist M, Szepfalusi Z, Spitzauer S, Grönneberg R, Valenta R, Hedlin G, van Hage M (2008). Higher IgE antibody levels to recombinant Fel d 1 in cat allergic children with asthma compared to rhinoconjunctivitis. Clin Exp Allergy. [Accepted]
https://pubmed.ncbi.nlm.nih.gov/18477016