Dust mite allergens : Cloning, characterisation and T-cell responses

Abstract: The number of allergens that have been cloned has increased dramatically during the last 10 years, and today, over 400 allergen sequences are available in the databases. In addition, at least 15 dust mite allergens have been characterised. The group 1 and 2 mite allergens are of great importance, since up to 90% of all mite sensitised individuals have IgE antibodies against those proteins. This thesis focuses on cloning and characterisation of allergens from three different dust mite species, Acarus siro, Tyrophagus putrescentiae and Lepidoglyphus destructor. Two low molecular mass allergens from A. siro were identified and characterised. A 15 kDa component was cloned and showed high sequence homology with fatty acid-binding proteins (FABP). The allergen was named Aca s 13, was produced in E. coh as a recombinant protein and was shown to be detected by 23% of the A. siro sensitised individuals. Amino acid sequencing of peptides from the 17 kDa component also revealed sequence homology to FABP's and Aca s 13, although the 17 kDa component was not classified as an isoform of Aca s 13 due to the size difference and the fact that only a partial sequence was revealed. The cDNA encoding a group 2 allergen from T putrescentiae was sequenced and the deduced sequence consists of 126 amino acids with a 15 amino acid leader peptide. The allergen was named Tyr p 2 due to its homology (about 40% sequence identity) to other mite group 2 allergens. Tyr p 2 was expressed in E. coh and shown to be detected by sera from T putrescentiae sensitised individuals. The lgE binding of recombinant Tyr p 2 and the previously cloned Lep d 2 was evaluated in the Pharmacia RAST CAP system and was cornpared with the lgE binding to the corresponding commercial extracts. Among the L. destructor and T putrescentiae sensitised subjects, 73.3% and 60.5% were positive to rLep d 2 and rTyr p 2, respectively, in the CAP assay. Although rLep d 2 and rTyr p 2 are able do identify the majority of the subjects sensitised to L. destructor and T putrescentiae, respectively, it is obvious that additional recombinant allergens are needed to get reliable diagnostic tools based on recombinant allergens. Therefore, a cDNA phage display expression library was constructed and screened against sera from L. destructor allergic patients, using a modified selection method. Three new allergens were identified, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13. The allergens were expressed in E. coh and were recognised by 9%, 62% and 13% of sera from 45 L. destructor sensitised subjects, respectively. An investigation of T-cell responses to the two cloned isoforms of Lep d 2 (01 and 02), a mutant form of Lep d 2.01 (Lep d 2.6Cys) with a highly reduced IgE reactivity, as well as peptides derived from Lep d 2, was performed in order to investigate the cellular responses to Lep d 2. Isoform 01 evoked greater proliferation of PBMC than isoform 02 and the response to rLep d 2.6Cys was intermediate. Although rLep d 2.6Cys evoked a lower T-cell response than rLep d 2.01, it may be a good candidate for immunotherapy because of its low IgE reactivity. Two immunodominant peptides corresponding to amino acid residue 11-25 and 61-75 were identified and are likely to contain the immunodominant T-cell epitopes of Lep d 2. There was a correlation between the proliferation to rLep d 2.01 and IFN- gamma production in the group of non-allergic controls, suggesting that a strong proliferative response is accompanied by a Th 1 response in non-allergic individuals. In conclusion, five new dust mite allergens have been cloned and characterised. These allergens may be useful to improve the diagnosis of dust mite allergy. The T-cell studies indicated that modified recombinant allergens or peptides may be useful to improve current schemes of allergen-specific immunotherapy of dust mite allergy.