It has long been recognized that the allergy to mites of the genus Dermatophagoides is associated with diseases such as asthma, rhinitis, and atopic dermatitis (1, 2). In this regard, the species D. pteronyssinus and D. farinae predominate and many studies have been performed to identify the allergens they produce. Studies on D. pteronyssinus have, for example, demonstrated that at least six major allergens are of clinical importance (3, 4). One of the major mite allergens, designated Derp l, has been purified and shown to react with anti-mite IgE antibodies in up to 80% of allergic sera (3-5). This allergen, reportedly a glycoprotein of mol wt 27 x 1Os , appears to be an excretory product associated with fecal particles (6).Currently, the diagnosis and immunotherapy of the house dust mite allergy is based on the use of crude mite preparations (7). Developments in this field have been hampered by the small or variable quantities of mite allergens present in the extracts . To help overcome these limitations, and to facilitate further studies, the molecular cloning of mite allergens is being investigated in our laboratory . To date, a cDNA clone coding for the allergen Der p 1 has been isolated and shown to contain a 0 .8-kb cDNA insert (8) . We now report the sequence analyses of the Der p 1 cDNA clone and the similarity between its inferred amino acid sequence and the group of cysteine proteases.

A cDNA clone coding for Der f I, a major allergen from the house dust mite Dermatophagoides farinae has been isolated and sequenced. It codes for a putative 18-residue signal peptide, an 80-residue proenzyme region, and a 223-residue mature protein with a derived molecular weight of 25,191. The deduced amino-acid sequence shows significant homology to other cysteine proteases in the proregion as well as in the mature protein. Sequence alignment of the mature Der f I protein with the homologous allergen Der p I from the related mite D. pteronyssinus revealed a high degree of homology (81%) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues comprising the active site of these enzymes and the cysteine residues were conserved. A potential N-glycosylation site was present at an equivalent position in both mite allergens. It is anticipated that the availability of recombinant Der f I will facilitate epitope mapping studies and studies of T-cell function in mite allergy by providing high levels of pure allergen.

A cDNA clone coding for the major house dust mite allergen Der p 1 was isolated from a lambda gt 11 library. Its sequence correlates with known amino acid sequences of Der p 1 and it produces a fusion protein which reacts with rabbit anti-Der p 1 antiserum.

Lambda gt11 clones expressing the major house dust mite allergen, Der p II, have been reported to react with IgE in the serum of a high proportion of allergic patients. The clones described, however, only produced small quantities of protein which was not fused to the β-galactosidase of the vector. A construct of the Der p II is described which produces a fusion of Der p II, minus its leader sequence, with the glutathione-S transferase in the pGEX vector. This could be readily isolated and was shown to react with IgE in 22 of 24 patient sera showing reactivity to native Der p II, the sera not reacting having low reactivity to native protein. Absorption analysis showed that the recombinant material removed most of the IgE reactivity of patients to native Der p II. The construct described, therefore, should be valuable for quantitative studies of a pure mite allergen.

An Escherichia coli clone producing a high-molecular-weight surface antigen of Haemophilus influenzae type b (Hib) was isolated from a library of Hib DNA fragments cloned as lysogens in a lambda replacement vector. The antigen is found in sarcosyl-insoluble outer membrane protein preparations and was produced by all 36 H. influenzae isolates tested. Absorption studies indicated that the antigen is a surface determinant on all isolates tested. Antibodies to the antigen (D15) were found in eight of nine convalescent-phase sera from children with invasive Hib infection. Affinity-purified antibodies prepared against the cloned antigen gave protection against the development of bacteremia in a rat pup model.

Background: A 349–residue recombinant polypeptide of Dermatophagoides farinae, Mag 3, has been shown to represent part of a larger 177–kD (M–177) allergen with very high IgE–binding activity. Methods: Cloning and sequencing of cDNA from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei was used to characterise the polypeptide containing the Mag 3 sequence. Results: cDNA clones containing the complete sequence of the E. maynei homologue of the M–177 allergen were isolated and analysed. The translation contained not only an amino acid sequence with 90% identity to the 349–residue Mag–3 fragment but also a further sequence with 90% identity to another IgE–binding recombinant D. farinae polypeptide designated Mag 1. The complete sequence encoded a mature polypeptide of 1,650 residues and a molecular mass of 189.5 kD. cDNA clones from D. pteronyssinus also encoded sequences equivalent to the Mag 1 and 3 polypeptides. The M–177 sequence showed strong similarity to the lipid transport apolipophorins found in insect lipophorins. Conclusions: cDNA sequence data show that the D. pteronyssinus and E. maynei homologues of the M–177 high–molecular–weight D. farinae allergen contain sequences equivalent to both the Mag 1 and Mag 3 recombinant IgE–binding fragments. The N–terminal sequence of the full–length 1,650 amino–acid allergen showed strong similarity to the insect apolipophorins which are poorly soluble in aqueous extracts and exist in the lipid transport particles in haemolymph. It is proposed that presentation in lipid particles could be a factor which enhances the immunogenicity of this group of allergens.

Background: The IgE-binding peptides Mag 1 and Mag 3 and the high-molecular-weight protein M-177 have been identified as parts of the apolipophorin-like group 14 house dust mite allergen. By analogy with the homologous insect proteins, apolipophorins are hydrophobic proteins found in lipid bodies and lipid transport particles. This explains why they degrade and are poorly represented in extracts. Methods: We have examined the T cell stimulation induced by a 341-residue recombinant Der p 14 peptide equivalent to the Mag 1 polypeptide examined by others. Results: Peripheral blood mononuclear cells from both allergic and non-allergic donors responded strongly in in vitro proliferation assays to the Der p 14 peptide to induce markedly more ³H-thymidine incorporation than Der p 2 and the release of Th2 cytokines. Conclusions: The apolipophorin-like group 14 allergens, despite their predicted hydrophobicity and lipid-binding activity, can induce high IgE responses and T cell stimulation. They appear to be important mite allergens unsuited to representation by aqueous extracts of mites.

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