Wendy-Anne Smith

Characterization and immunobiology of house dust mite allergens

The examination of house dust mite extracts has indicated that over 30 different proteins can induce IgE antibody in patients allergic to the house dust mite. There are however dominant specificities especially the group 1 and 2 allergens which can account for much of the allergenicity of extracts. Of the 19 denominated allergens, the major IgE binding has been reported for the group 1, 2, 3, 9, 11, 14 and 15 allergens. The high-molecular-weight group 11, 14 and 15 allergens have only recently been described and although high IgE binding has been anticipated from immunoblotting, there is a need for considerable corroboration. Similarly, the study of the group 3 and 9 serine protease allergens has been incomplete. The group 4, 5, 7 and 8 allergens have shown intermediate IgE binding and the group 10 tropomyosins are of interest because of their potential cross-reactivity with allergen from disparate species. Although the progress with the production of recombinant group 1 allergens has been recent, many of the allergens can be produced as high IgE-binding polypeptides. The tertiary structure of the group 2 allergens has been determined from recombinant proteins and they are an excellent model for the investigation of modified allergens. An unexpected property of the group 1, 2 and 3 allergens has been the high degree of polymorphism found by cDNA analysis. It has however been possible to identify sequences to represent the variation in the natural allergens. The group 7 and 14 allergens show secondary modifications which vary in different extracts creating batch variation. While some estimate of the importance of allergens can be obtained from IgE binding, few analyses of T-cell responses have been made and these regulate both the development of, and the protection from sensitization.

House dust-mite allergens

Dermatophagoides pteronyssinus and D. farinae are the major source of HDM allergens, although Euroglyphus mayrtei, in temperate regions, and Blomia tropicalis, in tropical and subtropical areas, can be important. D. microcerus in Europe and D. siboney in the Caribbean have been occasionally studied. Lepidoglyphus destructor is the source of important occupational allergens, which in some locations can contribute to house-dust allergy (1). The results of immunoblotting studies of the allergens in extracts of the mites have differed in detail, but the broad picture shows both complexity and the existence of some dominant specificities. Western blotting using sera selected on the basis of high antimite IgE has revealed 32 different IgE-binding bands with molecular weights from 11 to over 100 K (2, 3). Most sera recognized a unique combination of 6-10 bands. Of the 32 bands, nine were recognized at high frequency. A subsequent study found that most sera reacted to 1-7 bands, although some did have a broader reactivity (4). This pattern is consistent with several similar studies using either D. pteronyssinus or D. farinae extracts (5-8). Although the blotting studies have provided a snapshot, their limitations should be recognized. The concentration of allergens varies from extract to extract, and blotting procedures can have sharp dose-response characteristics. Some allergens, such as Der p 2, have their IgE-binding activity enhanced by denaturation (9), while others. W. R. Thomas, W. Smith

House dust mite allergens in asthma and allergy

IgE antibodies in house dust mite (HDM) allergy follow a predictable pattern. Half are directed against two dominant allergens and the remainder largely against four midpotency allergens. This hierarchical pattern is not changed by the titre of the IgE response or severity of disease. The structures of these allergens are known and they can be produced as authentic recombinant allergens. There is also evidence that the allergenicity is augmented by the biological activity of the key allergens, validating them as targets for vaccination. Collectively, these developments should facilitate the development of new diagnostics, improve immunotherapy and allow vaccination with defined reagents. Highly purified recombinant polypeptides representing the important mite allergens are now available so that informative and reproducible experiments can be performed with mite allergens in place of poorly defined and variable extracts.

Fel d 4, a cat lipocalin allergen

Background Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin‐like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals.

Towards defining the full spectrum of important house dust mite allergens

Tsai et al. [1] describe the cDNA cloning and sequencing of the group 11 house dust mite allergen, paramyosin. It is almost certainly the high molecular weight allergen of about 100 kDa noted to bind immunoglobulin (Ig) E frequently in several Western blotting studies [2±5]. Paramyosin and a discrete degradation product can account for bands representing allergens estimated to migrate as both 98 and 92 kDa proteins. It is also glycosylated which could produce further heterogeneity [6]. Data previously published for puri®ed Der f 11 showed it bound IgE in the serum of 82% of miteallergic subjects and gave dot±blot reactivities with the same staining intensity as reactivity to Der p 2 [6]. This and the ongoing studies of the M-177 (Mag 3) allergen [7,8] are important steps in de®ning the high molecular weight IgE binding speci®cities which have largely eluded the recent progress in the characterization of mite allergens. It is timely to discuss not only paramyosin but also some emerging features of the spectrum of allergens produced by Dermatophagoides mites. Some salient properties of the allergens are summarized in Table 1. Early indications suggested that although many proteins produced by Dermatophagoides mites induced IgE antibody, the bulk of the allergenic activity resided in a small number of dominant allergens. This was particularly indicated by the ®nding that frequently over 50% of the IgE binding activity of mite extracts could be attributed to the group 1 and 2 allergens [9,10]. The picture has changed now that other allergens have been de®ned and produced in suf®cient amounts to conduct studies with known and adequate quantities of allergen. The group 3 allergens from D. pteronyssinus and D. farinae bind IgE with a similar frequency and degree to the group 1 and 2 allergens [11,12] and in the studies reported for D. farinae, paramyosin [6], tropomyosin [13] and the allergen designated M-177 [7,8] have been found to bind IgE to a high degree and at a high frequency. Studies with D. pteronyssinus showed that the IgE binding to the Der p 9 collagenolytic allergen was similar to the binding to the group 1 allergens [11] and was not cross-reactive with the other serine proteases Der p 3 and Der p 6. The allergens Der p 5 and Der p 7 are interesting because although they only bind IgE in the serum of half the mite-allergic subjects, the strength of binding by responders is similar and often greater than the binding to the major allergen, Der p 2 [14,15]. Der p 4 and 8 allergens bind IgE at frequencies of about 40% but the strength of binding has not been compared with the binding to other allergens and the group 6 chymotrypsins bind IgE at a frequency of 40% and at a lower level than the major allergens [16]. The allergens of D. pteronyssinus and D. farinae have 80±90% sequence identity so there is little reason to suspect any difference in the allergenicity of the homologous proteins from each species. There has been a discrepancy reported for the IgE binding of the group 10 allergens, where studies with Der p 10 have not shown the high frequency or strength of IgE binding reported for Der f 10 [17]. The study on Der f 10 was conducted in Japan and the study of Der p 10 was conducted in Europe so factors other than the nature of the allergens are likely to be important, particularly since these allergens have 99% amino acid sequence identity. The group 1 and 2 allergens are found in abundant quantities in mite body extracts [18] while the group 3, 5, 7, 10 and the M-177 allergens are only minor or unstable constituents [7,13,19±21]. It is therefore not known how well the extracts represent the important allergens. The reasons for low concentrations in extracts could be several fold. The group 3 allergens are known to be major constituents of mite faeces despite their paucity in body extracts [19] and degradation products have been shown for the group 7 allergens [21] and M-177 allergens [8]. Additionally, the information available would suggest that mite bodies are not the main source of allergens. The data obtained by Tovey et al. for Der p 1 [22] shows that almost all the allergen in the environment is of faecal origin and assuming a steady state concentration, 15% of the Der p 1 in the environment would have been produced fresh each day. In addition to components of bodies and faeces, mite secretions could release allergens into the environment. For example, the group 2 allergens are homologues of proteins produced by the epithelial cells of respiratory and epididymal surfaces [22] and the M-177 allergen is, amongst other tissues, found in the lining of the oesophagus and gut [7]. Likewise, although paramyosin is a muscle protein it has been found as an abundant non-®lamentous protein in lipid bodies of the tegument and oesophageal lining of Schistosomes [23], a phenomenon which may explain why it can be used as a vaccine. The source of mite paramyosin would therefore be of interest and is discussed by Tsai et al. [1]. The concentration in whole mite extracts appears to be substantial as judged by the strong immunostaining with a monoclonal antibody in Western blotting of mite extract compared with the staining of puri®ed protein [6]. The existence of many proteins with a high frequency of

Cloning and sequencing of the Dermatophagoides pteronyssinus group III allergen, Der p III

House dust mites are widely recognized as major factors involved in the triggering of allergic diseases such as asthma. It is now apparent that the group III allergens of the Dermatophagoides mite species may play a significant role in a number of house dust mite allergic cases. Natural Der p III was isolated by gel filtration of salt precipitated Dermatophagoides pteronyssinus extract and as reported previously ran as a doublet of Mr 28 and 30 K on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Natural Der fIII was isolated by affinity purification with the 5A12 monoclonal antibody. Amino acid sequence data was generated for both these proteins which was used to construct DNA probes to screen a Dermatophagoides pteronyssinus cDNA library by hybridization and resulted in the isolation of a recombinant Der p III cDNA clone, P3WSI. The 1059 bp cDNA fragment included a 786 bp open reading frame which encodes a pre‐pro region of 29 amino acids and a mature protein of 232 amino acids with a calculated Mr 24985. A search of the BLAST protein database has confirmed that the Der p III P3WS1 clone is approximately 50% homologous with other trypsin proteins. We have confirmed with both our natural protein sequence and the P3WS1 amino acid sequence data that the group III allergens are trypsin‐like proteins.

Genetic variation of Der p 2 allergens: effects on T cell responses and immunoglobulin E binding

Background Der p 2 is a highly polymorphic allergen that shows a distinct pattern of sequence divergence. The effect of the variations on T cell and antibody responses has not been compared.

Antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children

Background Infants who develop house dust mite (HDM) allergy and HDM-sensitised children with severe persistent asthma have low antibody responses to the P6 antigen of Haemophilus influenzae. Objective To measure the development of antibody to two ubiquitous bacteria of the respiratory mucosa in a prospective birth cohort at high risk of allergic disease and to assess which responses are associated with asthma and atopy. Methods IgG1 and IgG4 antibody to H influenzae (P4 and P6) and Streptoccocus pneumoniae (PspA and PspC) surface antigens was measured in yearly blood samples of children aged 1–5 years. IgE to the P6 antigen was examined for the 5-year group. The children were stratified based on HDM sensitisation and asthma at 5 years of age. Results HDM-sensitised children had lower IgG1 antibody titres to the bacterial antigens, and early responses (<3 years and before the development of HDM sensitisation and asthma) corrected for multiple antigens were significantly reduced for P4, P6 and PspC (p=0.008, p=0.004 and p=0.028, respectively). Similar associations with asthma were also found (p=0.008, p=0.004 and p=0.032 for P4, P6 and PspC, respectively). The IgG4 antibody titre and prevalence were similar in both HDM-sensitised and non-sensitised groups, but sensitised children had a slower downregulation of the IgG4 response. Children with asthma (27/145 at 5 years) had lower anti-P6 IgE responses (p<0.05). Conclusions HDM-sensitised children have early defective antibody responses to bacteria that are associated with asthma. Surprisingly, antibacterial IgE was associated with a reduced risk for asthma.

Pyroglyphid House Dust Mite Allergens

Mites of the family Pyroglyphidae are the most important source of house dust mite allergens. A small number of allergens, namely those of groups 1, 2, 4, 5 and 7 constitute the known major and mid-potency specificities, with possible important contributions of the groups 11, 14 and 15 requiring further definition. Most of the allergens can be identified by sequence homologies and the structures of the major allergens have been solved. There are however challenges in determining the nature of the group 5 and 7 allergens and in obtaining detailed structures of the significant allergens to be used for genetic engineering.

Molecular analysis of the group 1 and 2 allergens from the house dust mite, Euroglyphus maynei

Background: There is increasing evidence that the house dust mite Euroglyphus maynei may be a significant source of allergic sensitization. The structural information for the E. maynei allergens is largely restricted to a single partial genomic sequence of Eur m 1. Methods: A cDNA library was constructed from a culture of E. maynei. Clones encoding the major group 1 and 2 allergens were isolated by DNA hybridization and sequenced. Results: The sequence of several full length clones of Eur m 1 and Eur m 2 were obtained. The full pre–proenzyme sequence of the cysteine protease Eur m 1 was determined. The translated amino acid sequence of Eur m 1 and Eur m 2 had 84–86% sequence identity with the corresponding allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae mites. This is the same as the degree of sequence identity found between D. pteronyssinus and D. farinae despite Euroglyphus being a member of the Pyroglyphinae subfamily rather than the Dermatophagoidinae subfamily. Conclusion: The sequences of the major Eur m 1 and Eur m 2 allergens are described. Their degree of divergence from the Dermatophagoides spp. is similar to that observed between D. pteronyssinus and D. farinae group 1 and group 2 allergens.