Rationally designed hypoallergenic mutant variants of the house dust mite allergen Der p 21.

BACKGROUND: Allergic diseases figure among the most common immune-mediated diseases worldwide, affecting more than 25% of the world's population. Allergic reactions can be triggered by house dust mite (HDM) allergens, of which the so-called group 21 of allergens is considered as clinically relevant. METHODS: Herein, we used a structural bioinformatics and immunoinformatics approach to design hypoallergenic mutant variants of the Der p 21 allergen of Dermatophagoides pteronyssinus, which were then recombinantly expressed in bacteria and tested for their IgE-reactivities. For this, we scanned the wild-type Der p 21 protein for all possible single amino acid substitutions in key IgE-binding regions that could render destabilization of the major epitope regions. RESULTS: Four main substitutions (D82P, K110G, E77G, and E87S) were selected to build mutant variants of the Der p 21 allergen, which were produced in their recombinant forms; two of these variants showed reduced reactivity with IgE. Molecular dynamic simulations and immune simulations demonstrated the overall effects of these mutations on the structural stability of the Der p 21 allergen and on the profile of immune response induced through immunotherapy. CONCLUSIONS: When produced in their recombinant forms, two of the Der p 21 mutant variants, namely proteins K110G and E87S, showed significantly reduced IgE reactivities against sera from HDM-allergic individuals (n = 20; p < 0.001). GENERAL SIGNIFICANCE: This study successfully translated a rational in silico mutagenesis design into low IgE-binding mutant variants of the allergen rDer p 21. These novel hypoallergens are promising to compose next-generation allergen-immunotherapy formulations in near future.

Effectiveness and safety of oral lactococci-based vaccine encoding triple common allergens to prevent airway allergy in mice.

Allergic airway disease is the most common chronic airway inflammatory disorder in developed countries. House dust mite, cockroach, and mold are the leading allergens in most tropical and subtropical countries, including Taiwan. As allergen avoidance is difficult for patients allergic to these perennial indoor allergens, allergen-specific immunotherapy (ASIT) is the only available allergen-specific and disease-modifying treatment. However, for patients sensitized to multiple allergens, ASIT using each corresponding allergen is cumbersome. In the present study, we developed a recombinant L. lactis vaccine against the three most common indoor aeroallergens and investigated its effectiveness for preventing respiratory allergy and safety in mice. Three recombinant clones of Der p 2 (mite), Per a 2 (roach), and Cla c 14 (mold) were constructed individually in pNZ8149 vector and then electroporated into host strain L.lactis NZ3900. BALB/c mice were fed with the triple vaccine 5 times per week for 4 weeks prior to sensitization. The effectiveness and safety profile were then determined. Oral administration of the triple vaccine significantly alleviated allergen-induced airway hyper-responsiveness in the vaccinated mice. The allergen-specific IgG2a was upregulated. IL-4 and IL-13 mRNA expressions as well as inflammatory cell infiltration in the lungs decreased significantly in the vaccinated groups. No body weight loss or abnormal findings in the liver and kidneys were found in any of the groups of mice. This is the first report to describe a triple-aeroallergen vaccine using a food-grade lactococcal expression system. We developed a convenient oral delivery system and intend to extend this research to develop a vaccination that can be self-administered at home by patients.

IgE Epitopes of the House Dust Mite Allergen Der p 7 Are Mainly Discontinuous and Conformational.

Background: Several studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus 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. Methods: 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. Results: 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. Conclusion and Clinical Relevance: 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.

Inactivation of common airborne antigens by perfluoroalkyl chemicals modulates early life allergic asthma.

Allergic asthma, driven by T helper 2 cell-mediated immune responses to common environmental antigens, remains the most common respiratory disease in children. Perfluorinated chemicals (PFCs) are environmental contaminants of great concern, because of their wide application, persistence in the environment, and bioaccumulation. PFCs associate with immunological disorders including asthma and attenuate immune responses to vaccines. The influence of PFCs on the immunological response to allergens during childhood is unknown. We report here that a major PFC, perfluorooctane sulfonate (PFOS), inactivates house dust mite (HDM) to dampen 5-wk-old, early weaned mice from developing HDM-induced allergic asthma. PFOS further attenuates the asthma protective effect of the microbial product lipopolysaccharide (LPS). We demonstrate that PFOS prevents desensitization of lung epithelia by LPS, thus abolishing the latter's protective effect. A close mechanistic study reveals that PFOS specifically binds the major HDM allergen Der p1 with high affinity as well as the lipid A moiety of LPS, leading to the inactivation of both antigens. Moreover, PFOS at physiological human (nanomolar) concentrations inactivates Der p1 from HDM and LPS in vitro, although higher doses did not cause further inactivation because of possible formation of PFOS aggregates. This PFOS-induced neutralization of LPS has been further validated in primary human cell models and extended to an in vivo bacterial infection mouse model. This study demonstrates that early life exposure of mice to a PFC blunts airway antigen bioactivity to modulate pulmonary inflammatory responses, which may adversely affect early pulmonary health.

Der p 2.1 Peptide Abrogates House Dust Mites-Induced Asthma Features in Mice and Humanized Mice by Inhibiting DC-Mediated T Cell Polarization.

Asthma is a chronic airway disease often due to sensitization to aeroallergens, especially house dust mite allergens (HDMs). The Dermatophagoides pteronyssinus group 2 (Der p 2), is one of the most representative HDM allergens and is recognized by more than 90% of HDM-allergic patients. In mouse models, all asthma-related features can be prevented by prophylactic administration of Dermatophagoides pteronyssinus 2-derived peptide (Der p 2.1). However, it is unknown whether it is able to treat well-established asthma in mice and humans. We aimed here to evaluate the efficacy of Der p 2.1 immunotherapy in a mouse, humanized mouse, and asthmatic patients. Asthma related-features were analyzed through airway hyperresponsiveness (AHR), allergen-specific IgE, and lung histology in mice and humanized mice. Immune profile was analyzed using lung and blood from mice and severe asthmatic patients respectively. T cell and dendritic cell (DC) polarization was evaluated using co-culture of bone marrow derived cells (BMDCs) and naïve T cell from naïve mice. Mice and humanized mice both have a reduced AHR, lung tissue alteration, and HDM-specific IgE under Der p 2.1 treatment. Concerning the immune profile, T helper 2 cells (Th2) and T helper 17 cells (Th17) were significantly reduced in both mice and humanized mice lung and in peripheral blood mononuclear cells (PBMCs) from severe asthmatic patients after Der p 2.1 incubation. The downregulation of T cell polarization seems to be linked to an increase of IL-10-secreting DC under Der p 2.1 treatment in both mice and severe asthmatic patients. This study shows that allergen-derived peptide immunotherapy abrogates asthma-related features in mice and humanized mice by reducing Th2 and Th17 cells polarization via IL-10-secreting DC. These results suggest that Der p 2.1 peptide immunotherapy could be a promising approach to treat both Th2 and Th17 immunity in asthma.

Allergen Stability and Immunological Reactivity during Co-dissolution and Incubation of House Dust Mite and Japanese Cedar Pollen SLIT-Tablets.

Japanese allergic subjects are commonly sensitized to both house dust mite (HDM) and Japanese cedar pollen (JCP) and combined treatment with sublingual immunotherapy (SLIT) tablets is desirable. However, mixing extracts of two non-homologous allergens may compromise allergen stability and affect the clinical outcome. Therefore, we investigated the stability of major allergens and total allergenic reactivity of HDM and JCP SLIT-tablets following dissolution in human saliva or artificial gastric juice. Two fast-dissolving freeze-dried SLIT-tablets were completely dissolved and incubated at 37 °C. Major allergen concentrations and total allergenic reactivity were measured. After mixing and co-incubation of HDM and JCP SLIT tablets in human saliva for 10 min at 37°C, there were no statistically significant changes in major allergen concentrations. In addition, no loss of allergenic reactivity of the mixed two SLIT-tablet solutions was seen. In contrast, complete loss of allergenic reactivity and detectable major allergen concentrations occurred when the two SLIT-tablets were dissolved and incubated in artificial gastric juice. These results demonstrate that HDM or JCP major allergens and the total allergenic reactivity of both SLIT-tablets measured here remain intact after dissolution and co-incubation in human saliva, supporting the possibility of a dual HDM and JCP SLIT-tablet administration regimen if clinically indicated. The complete loss of allergenic reactivity after incubation in artificial gastric juice can furthermore be taken to indicate that the immunological activity of the allergen extracts contained in the two SLIT-tablets is likely to be lost or severely compromised upon swallowing.

[Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae].

OBJECTIVE: To obtain the leptin receptor overlapping transcript-like 1 encoding gene (LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene. METHODS: The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein. RESULTS: The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus. CONCLUSIONS: The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

Gene synthesizing, expression and immunogenicity characterization of recombinant translation elongation factor 2 from Dermatophagoides farinae.

House dust mite (HDM) hypersensitivity increasingly affects millions of individuals worldwide. Although numerous major allergens produced by HDM species have been characterized, some of the less potent allergens remain to be studied. The present study aimed to obtain the recombinant allergen of Translation Elongation Factor 2 (TEF 2) from the HDM Dermatophagoides farinae by synthesizing, and then expressing the recombinant TEF 2 to identify its immunogenicity. In the present study, the D. farinae TEF 2 (Der f TEF 2) was synthesized, expressed and purified. The molecular characteristics of Der f TEF 2 were analyzed using bioinformatics approaches. The recombinant protein was purified via affinity chromatography, and the allergenicity was assessed using immunoblotting, ELISAs and skin prick tests. The gene for TEF 2 consists of 2,535 bp and encodes an 844­amino acid protein. A positive response to recombinant Der f TEF 2 was detected in 16.2% of 37 patients with HDM allergies using skin prick tests. In addition, the immunoblotting indicated that the protein showed a high ability to bind serum IgE from patients allergic to HDMs, and that the recombinant TEF 2 was highly immunogenic. Bioinformatics analysis predicted 17 peptides as B cell epitopes (amino acids 29­35, 55­64, 92­99, 173­200, 259­272, 311­318, 360­365, 388­395, 422­428, 496­502, 512­518, 567­572, 580­586, 602­617, 785­790, 811­817 and 827­836) and 14 peptides as T cell epitopes (amino acids 1­15, 65­79, 120­134, 144­159, 236­250, 275­289, 404­418, 426­440, 463­477, 510­524, 644­658, 684­698, 716­730 and 816­830). The software DNAStar predicted the secondary structure of TEF 2, and showed that 27 α­helices and five ß­sheets were found in the protein. In conclusion, the present study cloned and expressed the Der f TEF 2 gene, and the recombinant protein exhibited immunogenicity, providing a theoretical bases, and references, for the diagnosis and treatment of allergic disease.

CRM197-Coupled Der p 2 Peptides Suppress Allergic Airway Inflammation in a Der p 2-Induced Asthma Mouse Model.

Allergic diseases affect more than 25% of the global population. Der p 2 is the major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus. Allergen-specific immunotherapy is the only treatment to change the course of allergic diseases. In this study, two synthesized Der p 2 peptides coupled to cross-reacting material 197 (CRM197) showed reduced IgE reactivity and allergenic activity. CRM197-coupled Der p 2 peptides induced rDer p 2-specific IgG1 antibodies in mice, which could inhibit HDM-allergic patients' IgE binding to rDer p 2. The immunity effects of CRM197-coupled Der p 2 peptides were studied in an rDer p 2-induced asthma mouse model. CRM197-coupled Der p 2 peptides can suppress asthmatic airway inflammation in this model. Analysis of IL-4, IL-5, and IFN-γ levels in bronchoalveolar lavage fluid revealed that the suppression was associated with a shift from a Th2 to a Th1 response. Thus, CRM197-bound Der p 2 peptides exhibited less allergenic activity than the rDer p 2 allergen, which preserved immunogenicity and may be candidates for mite allergy vaccines.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Blo t 2: Group 2 allergen from the dust mite Blomia tropicalis.

Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Here we report the immuno-characterization of its group 2 allergen, Blo t 2. Allergen Blo t 2 was amplified from the cDNA of B. tropicalis using degenerate primers, expressed in Escherichia coli as a recombinant protein and purified to homogeneity. The mature protein of Blo t 2 was 126 amino acids long with 52% sequence identity to Der p 2 and apparent molecular mass of 15 kDa. Circular dichroism spectroscopy showed that Blo t 2 is mainly a beta-sheeted protein. We confirmed the presence of three disulfide bonds in recombinant (r) Blo t 2 protein using electrospray mass spectrometry. Thirty-four percent of dust-mite allergic individuals from the Singapore showed specific IgE binding to rBlo t 2 as tested using immuno dot-blots. IgE-cross reactivity assays showed that Blo t 2 had between 20-50% of unique IgE-epitopes compared to Der p 2. IgE binding of native and recombinant forms of Blo t 2 were highly concordant (r2 = 0.77, p < 0.0001) to rBlo t 2. Dose-dependent in vitro histamine was observed when rBlo t 2 was incubated with whole blood of Blo t 2 sensitized individuals, demonstrating that it is a functional allergen. Nine naturally occurring isoforms of Blo t 2 were identified in this study, each having between 1-3 amino acid variations compared to the reference clone. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp.

IgE binding activities and in silico epitope prediction of Der f 32 in Dermatophagoides farinae.

Dermatophagoides farinae is a common indoor allergen source that produces more than 30 allergens, which induces diverse allergic diseases such as allergic rhinitis, allergic asthma and atopic dermatitis. Der f 32 is an inorganic pyrophosphatase and an important allergen from Dermatophagoides farinae. In the present study, Der f 32 was cloned, expressed and purified in order to better understand its structure and immunogenicity. Immunoblotting analysis and ELISA showed 5 of 5 positive reactions to recombinant Der f 32 using serum from house dust mite (HDM)-allergic patients. We constructed homology modeling and predicted epitopes of Der f 32 via bioinformatic tools. The sequence and structural analysis indicated that Der f 32 belonged to the pyrophosphatase family and represented a special structure of external α-helices and internal antiparallel closed ß-sheets. In addition, eight B-cell epitopes and four T-cell epitopes were predicted. B-cell epitopes were 24-31, 111-121, 135-140, 168-172, 200-207, 214-220, 237-243, and 268-274 and T-cell epitopes were 47-55, 78-90, 127-135 and 143-151. The B-cell epitopes were distributed completely on the surface of Der f 32 and were located largely in random coils of secondary structures. Hydrophobic and charged amino acids comprised more than 80% of the residues of B-cell epitopes and may participate in IgE binding. The T-cell epitopes were located primarily in the interior of Der f 32 and, to a certain extent avoided degradation by proteases. The structures of T-cell epitopes were surrounded by B-cell epitopes, and this arrangement may have important biological significance for maintaining the immunogenicity of allergens.

Identification of Der f 23 as a new major allergen of Dermatophagoides farinae.

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 174­amino 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 (Ser56­Thr117) in Der f 23 that was not present in the D. pteronyssinus homolog, which may affect sIgE binding. Der f 23ΔP2 demonstrated binding with HDM allergic sera, whereas the P2 peptide alone did not. The sIgE binding ability of Der f 23 ΔP2 (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.

Novel Method for the Purification of House Dust Mite Allergen Der p 1 and Its Use in Structure-Based Chemical Design of Novel Inhibitors.

House dust mites are globally significant triggers of allergic disease. Notable among their extensive repertoire of allergens are the Group 1 cysteine peptidase allergens which function as digestive enzymes in house dust mites. Compelling evidence suggests that the proteolytic activity of these molecules plays a key role in the development and maintenance of allergic diseases through the activation of innate immune mechanisms which exploit genetic predispositions to allergy. Growing interest in this area creates a requirement for high-quality purified protein, whether natural or recombinantly expressed. It has also identified these allergens as therapeutic targets for a novel approach to allergy treatment through modulation of innate immune responses. The purpose of this chapter is to describe a new method for the purification of Der p 1 and use of the protein produced in a screening assay designed for the discovery of novel inhibitors of Group 1 house dust mite allergens.

Enhanced sensitivity of capture IgE­ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens.

The detection of allergen­specific immunoglobulin (Ig)E is an important method for the diagnosis of IgE­mediated allergic diseases. The sensitivity of the indirect IgE­ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgE­ELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28­Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgE­ELISA. Capture­ELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidase­conjugated streptavidin and 3,3,5,5­tetramethylbenzidine. The relative sensitivities of indirect­ELISA and capture­ELISA for HDM allergen­specific IgE binding were determined; sera from non­allergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgE­specific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDM­allergic sera. Furthermore, in a random sample of HDM­allergic sera (n=71), capture­ELISA (71/71, 100%) was more sensitive than indirect­ELISA (68/71, 95.8%) for the detection of HDM­specific IgEs (P<0.01), indicating that this novel method may be useful for the diagnosis of HDM allergy.

The major allergen Der p 2 is a cholesterol binding protein.

Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p < 0.05) compared to wild-type Der p 2. These results provide the first direct experimental evidence that Der p 2 binds to cholesterol.

The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.

BACKGROUND: Protein crystallographic studies suggest that the house dust mite (HDM) allergen Der p 5 potentially interacts with hydrophobic ligands. Der p 5, in association with its ligand(s), might therefore trigger innate immune signalling pathways in the airway epithelium and influence the initiation of the HDM-allergic response. OBJECTIVE: We investigated the lipid binding propensities of recombinant (r)Der p 5 and characterized the signalling pathways triggered by the allergen in airway epithelial cells. METHODS: rDer p 5 was produced in Pichia pastoris and characterized by mass spectrometry, multi-angle light scattering and circular dichroism. Its interactions with hydrophobic ligands were investigated in fluorescence-based lipid binding assays and in-silico docking simulations. Innate immune signalling pathways triggered by rDer p 5 were investigated in airway epithelial cell activation assays in vitro. RESULTS: Biophysical analysis showed that rDer p 5 was monomeric and adopted a similar α-helix-rich fold at both physiological and acidic pH. Spectrofluorimetry experiments showed that rDer p 5 is able to selectively bind lipid ligands, but only under mild acidic pH conditions. Computer-based docking simulations identified potential binding sites for these ligands. This allergen, with putatively associated lipid(s), triggered the production of IL-8 in respiratory epithelial cells through a TLR2-, NF-kB- and MAPK-dependent signalling pathway. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the fact that Der p 5 represents a HDM allergen of intermediate prevalence, our findings regarding its lipid binding and activation of TLR2 indicate that it could participate in the initiation of the HDM-allergic state.

Mapping Mimotopes for House Dust Mite Allergen Der f 7 Using a Specific Monoclonal Antibody.

BACKGROUND: The dust mite Dermatophagoides farinae is a common worldwide cause of indoor allergies induced by its proteins, including the mid-tier allergen Der f 7. OBJECTIVE: To identify conformational epitopes in Der f 7 using mimotope mapping and computational modelling. METHODS: Here, we used standard hybridoma technology to generate 3 new monoclonal antibodies against Der f 7 and performed mimotope mapping by probing a random peptide phage display library. Computational tools, including Minox and the DiscoTope-2.0 Server were used to assess the structure and potential position of antigenic residues within Der f 7. RESULTS: Thirteen mimotopes sharing the common sequence --XX[LST]P[-E][LI]MLPLR[-S]- were identified. Further, computationally-predicted conformational epitopes were found at residues 1-7, 10, 27, 76-81, 92, and 130-133 of Der f 7, and the key amino acids for these epitopes were deduced to be 2P, 3I, 10E, 27E, 78E, 79E, 81I, 130S, and 132E based on the common mimotope sequence. CONCLUSION: We identified Der f 7 peptide mimotopes that may model binding sites for blocking antibodies. These may guide the development of immunotherapy for individuals with hypersensitivity to Der f 7.

Synergistic effect of Dermatophagoides pteronyssinus allergen and Escherichia coli lipopolysaccharide on human blood cells.

PURPOSE: House dust mites Dermatophagoides pteronyssinus are the main source of major inhalatory allergens inducing inflammatory response. Mite extract contain both allergenic proteins and lipopolysaccharides (LPS). The main allergenic protein, Der p 2, is a functional homolog of sMD-2, a protein providing blood cell response on LPS. Der p 2 may restore the response to LPS in absence of MD-2, but its interaction with LPS in whole blood is unknown. We studied the effect of Der p 2 on LPS-mediated activation of human whole blood cells. METHODS: Interaction of Der p 2 and LPS was studied on eight healthy donors. The whole blood was incubated with extract of house dust mite Dermatophagoides pteronyssinus (DP-e), recombinant antigenic protein Der p 2 variant 5 (rDep 2), Escherichia coli lipopolysaccharide and their combination. Supernatants were collected for ELISA analysis of protein content. Activation degree was determined by change in concentration of TNF-α, IL-8, IL-1Ra cytokines and sMD-2 protein. RESULTS: extract of mite Dermatophagoides pteronyssinus (DP-e) possessed weak inherent activity and did not cause significant increase of cytokine production. Simultaneous activation of blood cells by LPS and DP-e led to considerable increase of pro-inflammatory cytokine production. We have shown the intrinsic inducing activity of Der p 2 allergen on sMD-2 protein and TNF-α cytokine expression. CONCLUSIONS: Der p 2 allergen enhances the response of human whole blood cells to external LPS by inducing additional expression of LPS-transporting protein sMD-2. The obtained data show an important role of LPS contamination of allegrens in the progress of allergic inflammatory response.