Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross-reactivity.
ABSTRACT: 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.
Project description: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.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.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.rDer p 10 is an ?-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.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.
Project description:<h4>Background</h4>Several studies indicate that Der p 7 is an important and clinically relevant allergen of <i>Dermatophagoides pteronyssinus</i> which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7.<h4>Methods</h4>Recombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1-P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes.<h4>Results</h4>rDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7.<h4>Conclusion and clinical relevance</h4>IgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.
Project description:<h4>Background</h4>In the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects.<h4>Methods</h4>IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty-three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively.<h4>Results</h4>House dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM-allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen-specific IgG levels were found in HDM allergics. PBMC from HDM-allergics produced higher levels of IL-5 whereas non-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines, and IL-10.<h4>Conclusion</h4>IgG antibodies in HDM-allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen-specific IgG antibodies do not protect against IgE-mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.
Project description:BACKGROUND:The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated. OBJECTIVE:To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level. METHODS:Der p 18 was expressed in E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources (i.e. mites, crustacea, mollusca and insects). IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments. RESULTS:Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients. CONCLUSION:Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.
Project description:PURPOSE:Measurement of IgE specific to purified house dust mite (HDM) allergens may improve allergy diagnosis. This study aimed to investigate the sensitization profiles of Korean HDM allergic subjects suffering from respiratory allergy and atopic dermatitis (AD) to Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20. METHODS:Recombinant HDM allergens were produced in Pichia pastoris (Der f 1) or Escherichia coli (5 allergens). IgE reactivity to the individual recombinant allergens and total extract of mite was assessed by ELISA. RESULTS:Der f 1 was recognized by 79.1%, Der f 2 by 79.1%, Der f 6 by 9.3%, Der f 8 by 6.2%, Der f 10 by 6.2%, and Der f 20 by 6.6% of the patients' sera tested, while the prevalence of IgE reactivity to total mite extract was 94.7%. Combination of Der f 1 and Der f 2 had a sensitivity of 87.6%. Specific IgE to Der f 2 alone was detected from 89.4% of HDM-sensitized respiratory allergy subjects and 92.3% to the combination of the 2 major allergens Der f 1 and Der f 2. However, sera from fewer patients with AD, namely 72.4% and 71.0%, recognized Der f 1 and Der f 2, respectively. The combination of 2 major allergens allowed diagnosis of 84.5% of the AD patients. No correlation between sensitization to specific allergens and HDM allergy entity was found. CONCLUSIONS:Der f 2 was the most frequently sensitized allergen among the HDM-sensitized respiratory and AD patients in Korea, and the combination of the group 1 and 2 major allergens increased the diagnostic sensitivity. Minor allergens did not significantly improve diagnostic sensitivity. However, further studies are needed to analyze the relationship between sensitization to other HDM allergens and the disease entity of the HDM allergy.
Project description:Allergens from house dust mites (HDM) are a common cause of asthma. Der p and Der f from Dermatophagoides sp. are strong immunogens in humans. Allergen extracts are used to study T helper (Th2) cell responses to HDM, which are implicated in the development and regulation of allergic disease.To define an epitope mixture that recapitulates, and might substitute for, HDM extract in terms of detecting and characterizing Th2 cell responses.Peripheral blood mononuclear cells (PBMC) from 52 HDM allergic and 10 non-allergic individuals were stimulated with HDM extracts and assayed with a set of 178 peptides spanning mite allergens group Der p 1, 2, 23 and Der f group 1 and 2 allergens. A pool of the most dominant T cell epitopes identified in the present study and from published literature was assembled and tested for ex vivo T cell responses. Correlation with HDM-specific IgE titres was examined.Patterns of T cell reactivity to Der p and Der f - derived peptides revealed a large number of epitopes. Clear patterns of immunodominance were apparent, with HDM allergen group 1 and 2 dominant over group 23. Furthermore, within a given antigen, 6-11 epitopes accounted for the vast majority of responses. Based on these results and published data, a comprehensive dust mite pool (DMP) of epitopes was designed and found to allow detection of ex vivo T cell responses. DMP ex vivo reactivity correlated with HDM-specific IgE titres and was similar to that detected with commonly used HDM extracts. Ex vivo DMP stimulation was associated with a predominant Th2 response in allergic donors, and minor reactivity of T cells producing IFNÎ³, IL17 and IL10.A detailed map of Der p and Der f antigens defined a pool of epitopes that can be used to detect ex vivo HDM responses.
Project description:Background:The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s). Methods:Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed. Results:We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell ?-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes. Conclusions:We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.
Project description:The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.
Project description:House dust mites (HDM) are common allergen sources worldwide. At present, 32 of the 37 internationally recognized HDM allergen groups have been identified in Dermatophagoides farinae. The present study study describes the identification of the first known D. farinae Group 23 allergen (Der f 23). Recombinant Der f 23 protein (rDer f 23) was cloned, expressed and purified. The open reading frame of rDer f 23 was 525?base pairs and encoded a 174?amino acid protein (GenBank accession no., KU166910.1). ELISAs indicated that 72/129 HDM allergic serum samples (55.8%) had specific immunoglobulin E (sIgE) binding activity to rDer f 23. Additionally, 3/10 patients with HDM allergies (30%) exhibited positive skin prick test reactions to rDer f 23. IgE western blot analysis data suggested that only 4/11 HDM allergic sera had a positive sIgE binding result. Sequence homology analysis revealed an extra P2 region (Ser56?Thr117) in Der f 23 that was not present in the D. pteronyssinus homolog, which may affect sIgE binding. Der f 23?P2 demonstrated binding with HDM allergic sera, whereas the P2 peptide alone did not. The sIgE binding ability of Der f 23 ?P2 (Der f 23 with a truncated P2 region) was more marked compared with that of Der f 23 in an IgE ELISA. These data indicate that P2 region in Der f 23 attenuates IgE binding ability. In conclusion, the results of the present study indicate that Der f 23 is a major HDM allergen with predominantly conformational sIgE binding epitopes. The allergenic identification of Der f 23 and its inclusion in World Health Organization/International Union of Immunological Societies database contributes to the theoretical basis underlying the diagnosis and treatment of HDM allergic diseases.
Project description:Arginine kinase (AK) was first identified as an allergen in the Indian-meal moth and subsequently shown to occur as allergen in various invertebrates and shellfish. The cDNA coding for AK from the house dust mite (HDM) species <i>Dermatophagoides pteronyssinus</i>, Der p 20, has been isolated, but no recombinant Der p 20 (rDer p 20) allergen has been produced and characterized so far. We report the expression of Der p 20 as recombinant protein in <i>Escherichia coli</i>. rDer p 20 was purified and shown to be a monomeric, folded protein by size exclusion chromatography and circular dichroism spectroscopy, respectively. Using AK-specific antibodies, Der p 20 was found to occur mainly in HDM bodies, but not in fecal particles. Thirty percent of clinically well-characterized HDM allergic patients (n = 98) whose immunoglobulin E (IgE) reactivity profiles had been determined with an extensive panel of purified HDM allergens (Der f 1, 2; Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 and 37) showed IgE reactivity to Der p 20. IgE reactivity to Der p 20 was more frequently associated with lung symptoms. AKs were detected in several invertebrates with specific antibodies and Der p 20 showed IgE cross-reactivity with AK from shrimp (<i>Litopenaeus vannamei</i>). Thus, Der p 20 is a cross-reactive HDM allergen and may serve as a diagnostic marker for HDM-induced lung symptoms such as asthma.