A study of allergens in the mite Lepidoglyphus destructor, using monoclonal antibodies and recombinant techniques

Sammanfattning: Mites are one of the most common causes of allergic reactions in many parts of the world. They can broadly be divided into two categories: house dust mites, primarily found in dwellings, and storage mites, primarily found in farming environments and grain storage facilities. Storage mites have now also been shown to exist in house dust. Lepidoglyphus destructor (L. destructor) is one of the predominant storage mite species in Europe. This thesis focuses on the characterization of allergens in this mite species with emphasis on the major allergen Lep d 2, previously called Lep d 1. Several monoclonal antibodies (Mabs) were produced against L. destructor. One Mab, 117179, identified a new allergen complex with molecular weights of 79/93 kDa and over 60 % IgE reactivity, indicating that it could be of clinical importance. A second Mab, I 112138, reacted to the major allergen, Lep d 1 (now Lep d 2). The two Mabs were applied for histochemical staining of cut mite sections in combination with confocal laser scanning microscopy in order to localize the two allergens. They were found to be distributed differently in the mite body. The 79/93 kDa allergens were located quite distinctly to the cytoskeleton and were not present in faecal particles while Lep d I showed a widespread distribution around the gut and front regions of the mite as well as in the faecal particles. This indicates differences in biological functions, where the 79/93 kDa allergens might be structural components and Lep d 1 could be involved in digestion. To further characterize Lep d 1, cDNA cloning was performed, revealing a 125 amino acid protein with a 16 amino acid leader sequence. Polymorphism was shown to be present in this allergen and two isoforms were cloned, differing in 13 amino acid positions. The cDNA sequences revealed over 50% homology with the group 2 allergens from the Dermatophagoides mite species leading to the redesignation of Lep d I to Lep d 2. The two isoforms of Lep d 2 were then expressed as recombinant allergens in two different expression systems, the prokaryotic E. coli system and the eukaryotic baculovirus system. Both systems were shown to produce recombinant allergens with IgE reactivities comparable to that of native Lep d 2 and no apparent differences were shown in reactivity between the two isoforms. Also the production yields were similar, leading to the conclusion that these systems are equally efficient in producing recombinant Lep d 2. In the E. coli system recombinant mutants of Lep d 2 were produced using site directed mutagenesis. In these mutants we disrupted disulphide bonds in order to study the implications for IgE reactivity. We found that the bond formed between cysteines 72 and 77 in Lep d 2 is most important for IgE reactivity in this allergen, however, in order to significantly reduce immunogenicity a mutant with all the disulphide bonds had to be produced. This mutant (Lep d 2.6Cys) showed an approximate 100 fold reduction in IgE reactivity but had retained T cell reactivity. In conclusion, monoclonal antibodies and recombinant allergens have been shown useful in the characterization of L. destructor allergens. Moreover, recombinant Lep d 2 and the mutant variant are likely to contribute to improved diagnosis and treatment of L. destructor allergy.