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.     

Cytokine responses to Der p 1 and Der p 7: house dust mite allergens with different IgE-binding activities

BACKGROUND: There is very limited information comparing T-cell responses to different house dust mite (HDM) allergens even though T cells are essential in the initiation and regulation of immunoglobulin (Ig) E synthesis and eosinophilia. OBJECTIVE: To compare the level of T-cell proliferation and cytokine production to the group 1 and group 7 HDM allergens which are known to have different IgE-binding capabilities. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) from HDM-allergic and HDM-nonallergic donors were stimulated with the group 1 and group 7 allergens of Dermatophagoides pteronyssinus and the level of proliferation as well as IL-5 and IFNgamma production were measured. RESULTS: The proliferative and IL-5 production to the group 1 and group 7 allergens were equivalent despite the group 7 allergen's lower frequency of IgE-binding. However more IFNgamma was produced to Der p 7 than to Der p 1, particularly for the nonallergic donors. As expected IL-5 production was much higher for PBMC from the allergic donors than for cells from nonallergics; however, there was no difference in the level of T-cell proliferation between the donor groups. CONCLUSION: The relative importance of the individual HDM allergens is normally determined by measuring the frequency of IgE-binding to the allergen in sera from an allergic population. The equivalent increased IL-5 response of PBMC from allergic people to the group 1 and group 7 allergens despite the different IgE-inducing activity indicates that these allergens may be equally capable of contributing to an asthmatic response by inducing eosinophilia.     

Molecular characterization of Der p 10: a diagnostic marker for broad sensitization in house dust mite allergy

Background Tropomyosins represent clinically relevant seafood allergens but the role of mite tropomyosin, Der p 10, in house dust mite (HDM) allergy has not been studied in detail. Objective To express and purify a recombinant Der p 10 with equivalent IgE reactivity as natural Der p 10 and to evaluate its IgE reactivity and allergenic activity in HDM‐allergic patients. Methods rDer p 10 was expressed in Escherichia coli, purified and characterized by mass spectrometry and circular dichroism. It was tested for IgE reactivity in 1322 HDM‐allergic patients. Detailed IgE‐reactivity profiles to six HDM allergens (Der p 1, 2, 5, 7, 10, 21) were established for subgroups of Der p 10‐positive and ‐negative patients. The allergenic activity of rDer p 10 was evaluated in basophil degranulation experiments. Results rDer p 10 is an α‐helical protein sharing IgE epitopes with nDer p 10. It is recognized by 15.2% of HDM‐allergic patients. Der p 10‐negative patients were primarily sensitized to Der p 1 and/or Der p 2, whereas Der p 10‐positive patients reacted to several other HDM allergens besides the major allergens (Der p 1, Der p 2) or showed a rather selective Der p 10 reactivity. The allergenic activity of Der p 10 was generally low but patients could be identified who suffered from clinically relevant HDM allergy due to Der p 10 sensitization. Conclusion and Clinical Relevance Der p 10 may be a diagnostic marker for HDM‐allergic patients with additional sensitization to allergens other than Der p 1 and Der p 2. Such patients may require attention when allergen‐specific immunotherapy is considered. Cite this as: Y. Resch, M. Weghofer, S. Seiberler, F. Horak, S. Scheiblhofer, B. Linhart, I. Swoboda, W. R. Thomas, J. Thalhamer, R. Valenta and S. Vrtala, Clinical & Experimental Allergy, 2011 (41) 1468–1477.     

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.     

Sequence polymorphisms of the Der p 3 house dust mite allergen

Background The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectricfocusing studies with the natural Der p 3 protein have indicated that several isoforms exist. Objective To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. Methods Five cDNA clones of Der p 3 have been isolated from a λ gt 10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco RI digested genomic DNA was performed. Results Southern blot analysis of Eco RI digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones ditfered only in their 3′untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. Conclusions These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group I and group 2 house dust mite allergens.     

Genetic polymorphisms of major house dust mite allergens

BACKGROUND: Polymorphic sequence substitutions in the major mite allergens can markedly affect immunoglobulin E binding and T cell responses, but there are few studies on environmental isolates from Dermatophagoides pteronyssinus and none for D. farinae. OBJECTIVE: To determine the sequence variation of the group 1 and 2 allergens from environmental D. pteronyssinus and D. farinae. METHODS: RNA from each species was isolated from homes in Bangkok and the sequence of Der p 1, Der p 2, Der f 1, and Der f 2 determined from cDNA produced by high fidelity polymerase chain reactions. RESULTS: The enlarged data set revealed preferred amino acid substitutions in residues 19, 81, and 215 of Der p 1 as well as sporadic changes. Der p 2 showed frequent variations with clusters of amino acid substitutions, but the canonical Der p 2.0101 was not found in any of 17 sequences. Der f 2 showed variants with clusters of substitutions similar to Der p 2 but in different amino acid positions and without any structural concordance. Der f 1 in contrast to the other allergens had few amino acid sequence substitutions. CONCLUSIONS: The sequence information on variants provides data important for the optimal design of allergen formulations and useful for the genetic engineering and structure-function analyses of the major allergens.     

哮喘患儿家庭内尘螨过敏原分布特征及其临床意义

目的了解北京地区尘螨过敏性哮喘患儿家庭环境内尘螨过敏原含量分布特征,初步探讨尘螨过敏原暴露水平的临床意义。方法选取54例尘螨过敏性哮喘患儿,其中男性37例,女性17例;年龄3～16岁,平均年龄8岁2个月。采集患儿家庭中床垫、枕头、卧室地板、客厅地板及沙发的灰尘,采用酶联免疫吸附分析（ELISA）测定以上灰尘样本中户尘螨1组过敏原（Der p1）和粉尘螨1组过敏原（Der f1）的含量;应用荧光ELISA测定患儿血清尘螨特异性IgE浓度;评估患儿哮喘临床控制情况,应用化学发光法测定患儿呼出气一氧化氮浓度（FeNO）。结果采集灰尘样本255份,以中位数（最小值～最大值）表示尘螨过敏原含量,床垫、枕头和沙发灰尘样本中Derf1和Derp1的含量显著高于卧室地板和客厅地板灰尘样本中尘螨过敏原含量。Derf1平均含量为0.13μg/g,显著高于Derp1平均含量0.02μg/g（P〈0.05）。Derp1和Derf1联合暴露的最高含量平均为2.18（0.07～54.59）μg/g。Der p1和Der f1联合最高暴露含量≥10.00μg/g、2.00～10.00μg/g、0.05～2.00μg/g的例数分别为4例（7.4%）、24例（44.4%）、26例（48.1%）。其中未控制组患儿家庭内尘螨过敏原最高暴露水平为27.41（0.23～54.59）μg/g,均显著高于部分控制组和控制组哮喘患儿尘螨过敏原最高暴露水平1.66（0.07～26.27）μg/g、2.90（0.37～33.75）μg/g（P〈0.05）。不同sIgE浓度分级组间尘螨过敏原最高暴露水平的差异、不同FeNO浓度范围组间尘螨过敏原最高暴露水平差异均无统计学意义。结论北京地区尘螨过敏性哮喘患儿家庭尘螨以Der f1为主,床垫、枕头及沙发灰尘样本是Der p1和Der f1的主要来源;哮喘未控制者的尘螨过敏原最高暴露水平明显增高。     

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non-allergic subjects

BACKGROUND: Haemophilus influenzae are ubiquitous colonizers of the nasopharynx, Little is known about the T cell cytokine responses to the antigens of these bacteria and whether or not the responses may interact with responses to aeroallergen. OBJECTIVE: To measure the T cell cytokine responses to conserved outer membrane protein antigens of Haemophilus influenzae and to house dust mite allergen of subjects allergic to the house dust mite and of subjects without allergic sensitization. METHODS: T cell responses were measured by in vitro proliferation and cytokine release from peripheral blood monocytes (PBMC). The allergen used was Der p 1 and outer membrane proteins were recombinant polypeptides representing the OMP6 and D15 antigens. RESULTS: The PBMC of most subjects had proliferative responses to OMP6 and D15, which were highly correlated. The pattern of cytokine release was Th1 biased with high levels of IFN-gamma and usually little IL-5 or IL-13 although PBMC from a few subjects did release IL-5 independent of allergic status. IL-10 release was readily detected. There was no difference in the anti-OMP cytokine response of PBMC from subjects without any known allergy and the responses of PBMC from subjects who were highly allergic to house dust mite. The responses to the Der p 1 allergen showed the expected Th2 cytokine release. CONCLUSION: The outer membrane protein antigens of the ubiquitous colonizing bacteria Haemophilus influenzae induce Th1 cytokine responses which are similar for PBMC from non-allergic individuals and subjects with a high degree of allergy to the perennial house dust mite allergen and strong Th2 responses to Der p 1.     

The decay of house dust mite allergens, Der p I and Der p II, under natural conditions

Background: Fluctuations in the level of mite allergens in domestic house dust are the result of changes in the balance between synthesis, removal and decay. Purely physical forces as well as enzymatic degradation, mediated by house dust inhabiting microbes, may contribute to the decay of allergens in domestic dust. Knowledge about the speed of decay is essential for an understanding of the dynamics of allergen levels. Objective: The present study is a quantitative assessment of the speed of decay at nine combinations of temperature (15°C, 20°C and 25°C) and relative humidity (33%, 55% and 75%). Methods: Samples of mite infested material of an old rug were stored at these temperature/relative humidity-combinations for 6, 12 or 18 months, after the mites were killed by cither a freezing treatment or an acaricide (lindane). The microbes living in the rug presumably survive these treatments. Concentrations of Der p I and Der p II + Der f II. in extracts of the rug material, were measured by a radio immunoassay. Results: No significant changes in the levels of Der p I and Der p II +Der f II, could be detected even after 1¹/₂ year at a high temperature and humidity. Conclusion: These findings incidate that mite allergens can be extremely stable under normal domestic circumstances.     

Biochemical and immunological characterization of a recombinant precursor form of the house dust mite allergen Der p 1 produced by Drosophila cells

BACKGROUND: The major house dust mite allergen Der p 1 elicits strong IgE antibody responses in patients suffering from mite allergy. OBJECTIVE: This study reports the expression and characterization of a recombinant precursor form of Der p 1 secreted as ProDer p 1 from insect cells. METHODS: The cDNA coding for ProDer p 1 was cloned downstream to the gp67 signal peptide, starting from commercial cDNA encoding Der p 1 and PCR-amplified ProDer p 1 genomic fragment. ProDer p 1, expressed in Drosophila cells and purified from culture medium, was compared to Der p 1 isolated from mite culture, in terms of glycosylation, enzymatic activity as well as IgG- and IgE-binding capacity. RESULTS: Sequence analysis of the genomic clone of ProDer p 1 revealed that, besides two introns in the mature Der p 1 coding sequence, two introns were also present in the propeptide coding sequence. ProDer p 1 was purifed to homogeneity by a combination of ion-exchange, hydroxyapatite and gel filtration chromatographies. The precursor form of Der p 1 could be processed in vitro into mature Der p 1 under acidic and reducing conditions. Carbohydrate analysis clearly indicated that ProDer p 1 expressed from insect cells was glycosylated and that glycan structures were located only in the prosequence. ProDer p 1 displayed a similar immunoreactivity towards IgE, monoclonal and polyclonal IgG antibodies compared to natural Der p 1. Specific activity measurements using synthetic substrates clearly indicated that, contrary to natural Der p 1, ProDer p 1 was totally enzymatically inactive. CONCLUSIONS: The expression of an enzymatically inactive and highly antigenic ProDer p 1 zymogen molecule could be a suitable strategy for the development of in vitro diagnosis test as well as for specific immunotherapy.     

Clinical improvement and immunological changes in atopic dermatitis patients undergoing subcutaneous immunotherapy with a house dust mite allergoid: a pilot study

BACKGROUND: House dust mites (HDMs) represent significant indoor allergen sources for patients with atopic dermatitis (AD). Subcutaneous allergen-specific immunotherapy (SCIT) has been shown to be successful in patients with allergic rhinitis and mild asthma and might represent an attractive therapeutic option for the long-term treatment of HDM sensitizations in AD patients. However, only a few studies have been conducted on the effectiveness of HDM SCIT in AD, resulting in controversial clinical results. Data on immunological changes induced by SCIT in AD patients are rare. OBJECTIVES: We performed an open pilot study to assess clinical changes and objective laboratory parameters and evaluate the benefit of HDM SCIT in 25 AD patients with IgE-mediated sensitization against HDM. METHODS: The severity of AD was evaluated by the severity scoring of atopic dermatitis system (SCORAD). Specific IgE and IgG4 against HDM and serum levels of TARC/CCL17, MDC/CCL22, IL-16, IL-4, IFN-gamma, IL-10 and TGF-beta1 were measured during SCIT. RESULTS: Subjective and objective SCORAD improved significantly within only 4 weeks of treatment. The level of the tolerogenic cytokine IL-10 increased, whereas CCL17 and IL-16 decreased in the sera of the patients during SCIT. Allergen specific IgE decreased, while IgG4 increased during SCIT. CONCLUSION: In this open-label pilot study, SCIT with an HDM extract in patients with AD led to a significant improvement of AD mirrored by a reduction of SCORAD as well as serological and immunological changes, which might serve as valuable parameters to estimate the therapeutic effect of SCIT.