The chitinase allergens Der p 15 and Der p 18 from Dermatophagoides pteronyssinus

BACKGROUND: House dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae cause allergic disease in humans as well as in dogs. In geographical regions where the two mite species coexist, they both elicit specific immunoglobulin (Ig E) responses in humans whereas dogs preferentially react to D. farinae extracts. In dogs the main IgE binding is directed to the D. farinae chitinase allergens Der f 15 and Der f 18 and not to the groups 1 and 2 allergens as found for humans. Although the IgE response of humans to Der f 18 has been investigated there is no report on Der f 15-specific IgE in humans. OBJECTIVE: This study aimed to characterize the chitinase allergens Der p 15 and Der p 18 of D. pteronyssinus and to find out whether they are important allergens for humans. METHODS: cDNA was cloned by a polymerase chain reaction strategy from D. pteronyssinus libraries using primers based on conserved chitinase sequences. IgE binding to the recombinant polypeptides was measured by immunosorbent assay. Mice were immunized with the polypeptides and cross-reactivity examined. RESULTS: Two variants of Der p 15 were isolated, encoding mature proteins of 58.8 and 61.4 kDa. The amino acid sequences had 90% identity to Der f 15. The cDNA for Der p 18 encoded a mature protein of 49.2 kDa with 88% sequence identity to Der f 18. Der p 15-specific IgE was detected in 70% and Der p 18-specific IgE in 63% of a panel of 27 human allergic sera. CONCLUSIONS: The D. pteronyssinus chitinases Der p 15 and Der p 18 show a high frequency of binding to IgE in allergic human sera. They are therefore potentially important allergens for humans as well as dogs.     

Comparison of Dermatophagoides pteronyssinus allergens from culture medium extract and whole body extract by using the same probe of pooled human serum

Two distinct allergens of Dermatophagoides pteronyssinus were isolated from culture medium extract and whole body extract monitored by the same probe of pooled human serum and compared. IgE-binding activity of allergens from the two sources was detected by microplate-ELISA with the same probe throughout the column chromatography. An allergen obtained from culture medium extract (Ag-CME) had a molecular weight of 27,000 by SDS-polyacrylamide gel electrophoresis (PAGE) and two isoelectric point ranges, pI 4.0-4.7 and 5.9-6.8. Another allergen from whole body extract (Ag-WBE) had a molecular weight of 15,000 by SDS-PAGE and an isoelectric point range, pI 4.8-5.3 Ag-CME and Ag-WBE are probably the same allergens as antigen P1 and DpX, respectively.     

Interaction of human lung surfactant proteins A and D with mite (Dermatophagoides pteronyssinus) allergens

Human lung surfactant proteins A (SP-A) and D (SP-D) are both collagenous C-type lectins which appear to mediate antimicrobial activity by binding to carbohydrates on micro-organisms and to receptors on phagocytic cells. Purified native SP-A and SP-D, isolated from human bronchoalveolar lavage fluid, were found to bind to whole mite extracts (Dermatophagoides pteronyssinus) and the purified allergen Der p I, in a carbohydrate-specific and calcium-dependent manner. Binding was inhibited by ethylenediamine tetra-acetic acid (EDTA) as well as by maltose in the case of SP-D, or mannose in the case of SP-A. A recombinant polypeptide, which trimerized to form the neck region and carbohydrate recognition domains of SP-D, also inhibited the binding of native SP-D to the whole mite extract and Der p I. Both SP-A and SP-D did not bind to deglycosylated whole mite extracts or to recombinant Der p proteins, which lacked carbohydrate residues. These results suggest that the ability of surfactant proteins to bind certain allergens is mediated through their carbohydrate-recognition domains (CRDs) interacting with carbohydrate residues on the allergens. Moreover, SP-A and SP-D were found to inhibit allergen-specific IgE binding to the mite extracts either via steric hindrance or competitive binding. It is therefore possible that SP-A and SP-D may be involved in the modulation of allergen sensitization and/or the development of allergic reactions.     

Antibodies to Dermatophagoides pteronyssinus allergen Dpt 12 contaminating rabbit antisera to human and mouse anti-immunoglobulins

Antibodies to the allergen Dpt 12 from Dermatophagoides pteronyssinus have been demonstrated in commercially obtained rabbit anti-human IgE, IgG and IgA antisera and in a commercially obtained rabbit anti-mouse IgG1 antiserum using either double or triple antibody radioimmunoassays. Where measurable, the titre of anti-Dpt 12 antibodies varied both between different anti-isotypes and between batches of the same anti-isotype as judged by the dilution of antiserum required to bind 50% of the radiolabelled Dpt 12. The titres ranged from 1/23-1/360. In contrast, a specific rabbit anti-Dpt antiserum gave a titre of 1/4500.     

Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross‐reactivity

Background

Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity.

Methods

A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide‐specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross‐reactivity between the allergens was investigated by IgE inhibition studies.

Results

Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N‐ and C‐terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross‐reactivity could be detected.

Conclusion

Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope‐containing area located on similar portions of their structure, but they lack relevant IgE cross‐reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen‐specific immunotherapy of HDM allergy.     

Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae

Background The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes.
Objective The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic detertninants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences.
Methods In order to identity any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs.
Results None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs.
Conclusion The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.     

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E

BACKGROUND: The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.     

Localization of a major allergen, Der p 2, in the gut and faecal pellets of Dermatophagoides pteronyssinus

BACKGROUND: The house dust mite Dermatophagoides ptronyssinus is one of the most significant indoor sensitizing agents of allergy. Allergen localization may indicate the importance of secreted materials, faeces, and nonexcreted mite body components as allergen sources. OBJECTIVE: This study attempted to localize the sites and concentrations of Der p 2 in the cryostat sections of D. pteronyssinus using antirecombinant Der p 2 monoclonal antibody. METHODS: Male and female mites and mite faeces collected separately from both sexes were used. Live mites were embedded and serial cryostat sections for light microscopy were performed. Anti-recombinant Der p 2 monoclonal antibody previously produced by the authors was used. For immunoprobing, mite cryostat sections were incubated in the following antibody-containing solutions: monoclonal antibody against Der p 2 was initially applied to the sections and fluorescent isothiocyanate conjugated antimouse immunoglobulin G was reacted as the secondary antibody. The faecal pellets were treated the same as described above. RESULTS: Immunofluorescent probing of cryostat sections with the monoclonal antibody showed labelling of the gut lining, gut contents and defecated faecal pellets. No other internal organs were identified as positively labelled. CONCLUSION: This study suggested that a major allergen, Der p 2, found in the house dust mite D. pteronyssinus is derived from the digestive tract and concentrated in the faeces.     

Analysis of antigenic determinants shared by two different allergens recognized by human T cells: house dust mite (Dermatophagoides pteronyssinus) and chironomid midge (Chironomus yoshimatsui)

To analyse the cross-reactivity of T-cell-mediated immunity between Dermatophagoides pteronyssinus (Dp) and Chironomus yoshimatsui (Cy), the most common allergens in Japan, we established antigen-specific human T-cell lines and clones. Some but not all of the Cy-induced T-cell lines showed a significant proliferative response not only to Cy, but also to Dp. No T-cell line responded to other unrelated antigens. When we stimulated the Dp-induced T-cell clones with Cy, 3 of the 40 clones (7.5%) showed a significant proliferation, and 2 of the 3 clones produced interleukin-4 and interferon-gamma, indicating their helper function. Cross-reactivity was diminished significantly after the absorption of Dp antigen in an anti-Cy affinity column. The cross-reactive epitopes were thought to be expressed on the Dp molecule of 45-53 kD. The presence of helper T cells reactive to both allergens suggests a possibility that this cross-reactivity might be involved in part in the high incidence of allergy to the 2 major allergens.     

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy

Background Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT).
Objective We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment.
Methods Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli , and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice.
Results The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens.
Conclusion QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.     

House dust mite, Dermatophagoides pteronyssinus, and its allergens: effects of washing

The study was undertaken to evaluate the effect washing at various temperatures would have on the house dust mite, Dermatophagoides pteronyssinus, and its allergens. Young and old mite cultures underwent a simulated washing programme. To record the number of live mites prior to and after washing a new counting method was applied. The findings demonstrated that although washing reduced the number of live mites, a washing programme at minimum temperature 58 degrees C was required in order to achieve complete extermination of the house dust mites. A distinct protein loss was observed when washing at higher temperature, thus further reducing the total allergen pool without loss of allergen activity as determined by direct RAST.     

Cloning and sequencing of the group 6 allergen of Dermatophagoides pteronyssinus

Background The allergen Der p 6 from Dermatophagoides pteronyssinus has been described by substrate affinity as mite chymotrypsin. Objective The aim of this paper was to describe a cDNA clone encoding the allergen. Methods cDNA was cloned from a λ library using oligonucleotides published for Der p 6. Results The clone P6.1.1 had the N-terminal amino acid residues as reported for Der p 6, and at position 189 the Ser was conserved in chymotrypsin for substrate specificity as well as the catalytic triad of His57, Asp102 and Ser195 for serine proteases. The estimated Mr was 24.9K and it was 37% identical to the trypsin allergen Der p 3. Conclusion cDNA encoding Der p 6 has been described.     

Comparisons of IgE, IgG, and IgG4 responsiveness to Dermatophagoides farinae in children by immunoblotting

Although asthmatic children are often sensitized to the house-dust mite (HDM) during early childhood, it is not clear what antigenic components are associated with the early immune response of these children. To investigate this problem, we evaluated IgE, IgG, and IgG4 antibody responses to Dermatophagoides farinae (Df) by immunoblotting among three groups of children: group I aged 0–4 years, group II aged 5–9 years, and group III aged 10–14 years. In the group I subjects, positive IgE-binding reactions to 15- and 25-kDa components were found in 76% and 44% of sera, respectively. Those to other components were generally low in frequency. In contrast, positive IgG-binding reactions to 15- and 25-kDa components were found in 44% and 24% of sera, and those to 30- and 110-kDa components in 48% and 88% of sera, respectively. Positive IgG4 reactions to 15- and 25-kDa components were found in 48% and 24%, respectively, and those to other components were very low. Positive IgE-binding reactions to these components gradually became more frequent with increasing age in groups II and III, while IgG and IgG4 reactivities were not markedly different in these age groups. These results suggest that the 15- and 25-kDa proteins of Df are important antigens associated with the initial IgE response to HDM in early childhood, while the 30- and 110-kDa proteins are associated with IgG and 15-kDa components with IgG4.     

Allergenic properties of kiwi-fruit extract: cross-reactivity between kiwi-fruit and birch-pollen allergens

Our investigation aimed to produce and characterize a kiwi extract and to use this extract to investigate a possible cross-reactivity with birch pollen. Kiwi was extracted in two buffers: phosphate-buffered saline (PBS) and borate-buffered saline (BBS). Extraction in BBS produced a double amount of protein, and a more stabile extract. Tandem crossed-immunoelectrophoresis showed that the BBS and PBS extracts had several common, but also a few individual, proteins. The mixture of both extracts was assumed to represent the most complete allergen extract. The allergenic properties of the kiwi extract were investigated by immunoblotting (IB), RAST, and histamine-release (HR) test in 15 birch-pollen-allergic patients (eight of them with clinical kiwi allergy) and one with clinical monoallergy to kiwi. All eight birch-pollen-allergic patients with kiwi allergy and the kiwi-monoallergic patient were positive in kiwi IB binding most frequently to proteins of 10-12 and 20-25 kDa. With our extract, RAST was positive in four kiwi-allergic and one non-kiwi-allergic patient, whereas the HR test was positive in five kiwi-allergic patients and negative in all non-kiwi-allergic patients. RAST and IB inhibition demonstrated cross-reactivity between birch-pollen and kiwi allergens due to a 10-12 kDa protein. In conclusion, a kiwi extract with allergenic properties was produced, and, by the methods used, cross-reactivity was demonstrated between birch-pollen and kiwi allergens.     

Detection of immunoglobulin antibodies in the sera of patients using purified latex allergens

BACKGROUND: Latex allergy is largely an occupational allergy due to sensitization to natural rubber latex allergens present in a number of health care and household products. Although several purified allergens are currently available for study, information on the usefulness of these purified, native or recombinant allergens in the demonstration of specific immunoglobulin (Ig) E in the sera of patients is lacking. OBJECTIVE: To evaluate the purified latex allergens and to demonstrate specific IgE antibody in the sera of health care workers and spina bifida patients with clinical latex allergy. METHODS: Two radioallergosorbent and an enzyme-linked immunosorbent assay (ELISA) using latex proteins Hev b 1, 2, 3, 4, 6 and 7 along with two glove extracts and Malaysian nonammoniated latex (MNA) were evaluated to demonstrate IgE in the sera of health care workers and spina bifida with latex allergy and controls with no history of latex allergy. RESULTS: ELISA using the purified latex allergens demonstrated specific IgE in 32-65% health care workers and 54-100% of spina bifida patients with latex allergy. The corresponding figures for RAST were 13-48 and 23-85 for RAST-1 and 19-61 and 36-57 for RAST-2. These results were comparable with the results obtained with glove extracts and crude rubber latex proteins. CONCLUSIONS: When used simultaneously, latex proteins Hev b 2 and Hev b 7 reacted significantly with specific serum IgE in 80% of health care workers and 92% of spina bifida patients with latex allergy by ELISA technique, while this combination gave lower positivity when the RASTs were used. By the addition of Hev b 3, specific IgE was detected in all spina bifida patients with latex allergy. Both RASTs failed to show specific IgE in the control subjects, while the ELISA showed significant latex-specific IgE in 22% of controls.     

Production of native and modified recombinant Der p 1 molecules in tobacco plants

Background As a complex molecule requiring post‐translational processing, it has been difficult to produce the Der p 1 major allergen from the Dermatophagoides pteronyssinus house dust mite in a recombinant form. Objective Here, we tested whether transgenic tobacco plants are suitable to express Der p 1, either as a wild‐type molecule or as variants lacking N‐glycosylation sites (Gly^?) and/or cysteine protease activity (Enz^?). Methods Using Agrobacterium tumefaciens‐based transformation, pro Der p 1 molecules bearing mutations within either the N‐glycosylation sites (N34Q, N150Q) and/or the cysteine protease‐active site (C132V) were expressed in tobacco plants. After purification by ion exchange chromatography, allergens were characterized using immunoblotting, circular dichroism (CD), as well as basophil and T lymphocyte stimulation assays. Results Four forms of recombinant Der p 1 (i.e. wild‐type Gly⁺/Enz⁺, as well as Gly^?/Enz⁺, Gly⁺/Enz^? or Gly^?/Enz^? variants) were successfully expressed in tobacco leaves as pro Der p 1 molecules. Spontaneous cleavage of the pro‐peptide was observed in tobacco leaf extracts for all forms of recombinant Der p 1 (r Der p 1). CD confirmed that all r Der p 1 molecules, with the exception of the Gly^?/Enz^? variant, exhibited secondary structures comparable to the natural protein. A cysteine protease activity was associated only with the Gly⁺/Enz⁺ form. All these molecules exhibit a profile similar to natural Der p 1 with respect to IgE immunoreactivity, basophil activation and T cell recognition. Conclusion A tobacco plant expression system allows the production of various forms of mature Der p 1, which could be used for diagnostic or immunotherapeutic purposes.     

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.