IgE-binding Reactivity Research Articles

Changing the IgE Binding Capacity of Tropomyosin in Shrimp through Structural Modification Induced by Cold Plasma and Glycation Treatment

Tropomyosin (TM) is the major allergen of shrimp (Penaeus chinensis). Previous studies showed that separate cold plasma or glycation have their drawback in reducing allergenicity of TM, including effectiveness and reliability. In the current study, a new processing combining cold plasma (CP) and glycation was proposed and its effect on changing IgE binding capacity of TM from shrimp was investigated. Obtained results showed the IgE binding capacity of TM was reduced by up to 40% after CP (dielectric barrier discharge, 60 kV, 1.0 A) combined with glycation treatment (4 h, 80 °C), compared with the less than 5% reduction after single CP or glycation treatment. Notably, in contrast to the general way of CP prompting glycation, this study devised a new mode of glycation with ribose after CP pretreatment. The structural changes of TM were explored to explain the decreased IgE binding reactivity. The results of multi-spectroscopies showed that the secondary and tertiary structures of TM were further destroyed after combined treatment, including the transformation of 50% α-helix to β-sheet and random coils, the modification and exposure of aromatic amino acids, and the increase of surface hydrophobicity. The morphology analysis using atomic force microscope revealed that the combined processing made the distribution of TM particles tend to disperse circularly, while it would aggregate after either processing treatment alone. These findings confirmed the unfolding and reaggregation of TM during combined processing treatment, which may result in the remarkable reduction of IgE binding ability. Therefore, the processing of CP pretreatment combined with glycation has the potential to reduce or even eliminate the allergenicity of seafood.

Dermatophagoides pteronyssinus allergen Der p 22: Cloning, expression, IgE‐binding in asthmatic children, and immunogenicity

Dust mite extract contains multiple components that, while useful in clinical allergy diagnosis and treatment, can cause serious side effects. Defining components of dust mite extract is important their contributions to allergic disease. This study aimed to characterize a novel dust mite allergen, Der p 22. We amplified the cDNA encoding Der p 22 from total RNA of the mite Dermatophagoides pteronyssinus, and inserted it into an expression construct for transformation to competent cells. Purified recombinant (r) Der p 22 was tested for IgE-binding reactivity in sera obtained from children with allergic asthma by the Affiliated Wuxi Children's Hospital of Nanjing Medical University (Jiangsu, China). rDer p 22 also was used to challenge BALB/c mice to assess effects on T helper cells and cytokine levels and applied to cultured lung epithelial cells to evaluate apoptosis and cytokine secretion. rDer p 22 bound to IgE in 93.75% of sera from pediatric allergic asthma patients. Mice challenged with rDer p 22 had altered Th1/Th2 ratios in spleen and lymph, and lower levels of cytokines IFN-γ but higher levels of IL-4 and IL-10 in alveolar lavage fluid compared with controls (p < .05). Cultured lung epithelial cells had greater apoptosis rates and exhibited higher levels of IL-6, IL-8, and GM-CSF when treated with rDer p 22 compared with control treatment (p < .05). Recombinant Der p 22 exhibited high IgE-binding rates in allergic children, indicating the activity of the recombinant protein and suggesting this novel allergen may be appropriate for inclusion in an allergy diagnostic workup. This finding is supported by in vitro and mouse in vivo studies showing rDer p 22 induced strong allergenic reactivity and apoptosis.

Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprint

The relatively easy access to fish worldwide, alongside the increase of aquaculture production contributes to increased fish consumption which result in higher prevalence of respective allergies. Allergies to fish constitute a significant concern worldwide. β-parvalbumin is the main elicitor for IgE-mediated reactions. Creatine, involved in the muscle energy metabolism, and ethylenediamine tetraacetic acid (EDTA), a calcium chelator, are potential molecules to modulate parvalbumin. The purpose of this study was to test creatine (2, 5 and 8%) and EDTA (1.5, 3 and 4.5%) supplementation in fish diets to modulate β-parvalbumin expression and structure and its allergenicity in farmed European seabass (Dicentrarchus labrax) while assessing its effects on the end-product quality. Fish welfare and muscle quality parameters were evaluated by plasma metabolites, rigor mortis, muscle pH and sensory and texture analysis. Proteomics was used to assess alterations in muscle proteome profile and metabolic fingerprinting by Fourier transform infrared spectroscopy was used to assess the liver metabolic profile. In addition, IgE-reactivity to parvalbumin was analysed using fish allergic patient sera. Metabolic fingerprinting of liver tissue revealed no major alterations in infrared spectra with creatine supplementation, while with EDTA, only absorption bands characteristic of lipids were altered. Comparative proteomics showed up regulation of (tropo) myosin and phosphoglycerate mutase 2 with Creatine supplementation. In the case of EDTA proteomics showed up regulation of proteins involved in cellular and ion homeostasis. Allergenicity seems not to be modulated with creatine or EDTA supplementation as no decreased expression levels were found and IgE-binding reactivity showed no quantitative differences.

Molecular cloning, expression, purification and in silico epitope prediction of cobalamin-independent methionine synthase (Mor a 2), as a novel allergen from Morus alba pollen

IntroductionMorus alba (white mulberry) pollen is an aero-allergen source that can trigger allergic diseases. Cobalamin-independent methionine synthase (MetE) in M. alba pollen has been proved to be one of the major allergens for some patients living in Istanbul (Turkey). The aim of the present study was the recombinant production and identification of MetE (Mor a 2), a novel allergen from M. alba pollen. The IgE binding reactivity of rMor a 2 produced for the first time was evaluated and some structural features were investigated by in silico methods to better understand its immunogenicity.Material and methodsThe gene encoding Mor a 2 was cloned in fission yeast, Schizosaccharomyces pombe ura4-D18h- strain, using pSLF1073 vector. This is the first report of the production of recombinant pollen allergen in S. pombe. After the purification, immunoreactivity of rMor a 2 was confirmed by immunoblotting using sera of patient allergic to M. alba pollen. Besides, B-cell epitopes of rMor a 2 were predicted using various bioinformatic tools, namely Bioinformatics Predicted Antigenic Peptides, BepiPred 2.0 and Immune Epitope Database whereas T-cell epitopes were estimated using NetMHCIIpan-3.2 and NetMHCII 2.3 servers.ResultsThe immunoblotting analysis yielded 11 of 11 positive reactions to rMor a 2. In silico predictions exerted seven B-cell epitopes (22-33, 384-394, 407-423, 547-553, 571-577, 671-678, 736-741) and seven T-cell epitopes (54-62, 161-170, 197-205, 347-358, 622-630, 657-665, 756-764).ConclusionsThese findings may help the use of rMor a 2 in the diagnosis and treatment of allergic diseases associated with M. alba and/or MetE.

Mechanisms of plastein formation influence the IgE-binding activity of egg white protein hydrolysates after simulated static digestion

Egg is the second most common food allergen among infants and young children. This work investigated the influence of plastein reaction on immunoglobulin E (IgE)-binding activities of egg white protein hydrolysates after simulated gastrointestinal (GIT) digestion. Compared to hydrolysate precursors, the IgE-binding activity of Pepsin-Plastein significantly decreased from 35 ± 7% to 8 ± 2% (P < 0.05), and Papain-Plastein from 70 ± 5% to 59 ± 4%. Further GIT hydrolysis of Pepsin-Plastein maintained the reduced IgE-binding activity (7 ± 3%) whereas Papain-Plastein digestion restored the IgE-binding reactivity to 66 ± 7%. This discrepancy is related to the different mechanisms of plastein formation. Covalent modifications (decreased free amino nitrogen and sulfhydryl contents) provided biostability for Pepsin-Plastein, whereas hydrophobic interactions (increased surface hydrophobicity) mainly contributed to Papain-Plastein formation. The latter can be destroyed during GIT digestion leading to re-exposure of hidden IgE-binding epitopes. Taken together, plastein reaction is a promising strategy for inducing structural modifications that reduce the immune reactivity of allergenic proteins.

Structure and IgE-binding properties of α-casein treated by high hydrostatic pressure, UV-C, and far-IR radiations

α-Casein was treated by high hydrostatic pressure (HHP), UV-C, or far-IR (FIR). These treatments increased roughness, α-helicity, and β-turn, but decreased β-sheet and IgE-binding reactivity. One 5-min cycle at 600-MPa pressure caused maximum α-helicity, β-turn, and surface hydrophobicity (Ho), but minimum stimulated intestinal fluid from α-casein. UV-C (15min) produced the maximum kurtosis, free sulfhydryl content (FSC), and stimulated intestinal fluid, minimum Ho, R, and simulated gastric fluid. FIR (15min) caused the minimum α-helicity and FSC, but maximum R and β-sheet. The NMR peaks of the main allergenic characteristics affected were 15–17, 23–26, 40, 53, 59 and 85–88, respectively. Generally, all treatments decreased the allergenicity of α-casein by modifying its morphology, ultrastructure, characteristic domains, and peptides. Based on the stimulated digestion tests, UV-C (15min) was more efficient for lowering α-casein allergenicity, thus decreasing the allergenicity of milk.

Effects of ozone in Plantago lanceolata and Salix atrocinerea pollen

Pollen can be modified by the increasing environmental pollution which can lead to a rising trend in pollen-induced respiratory allergies. The aim of this work was to study the fertility, protein profile and allergenicity of Plantago lanceolata and Salix atrocinerea pollen after exposure to ozone. The pollen collected directly from the anthers of both species was exposed to O3 in an environmental chamber, for 6 h at different concentrations. After exposing the pollen, viability tests and in vitro germination were performed. Total protein content was determined by the Bradford method, the polypeptides were separated by SDS-PAGE and the antigenic properties were analyzed by immunological techniques using patients’ sera. The concentrations of O3 tested affected the pollen fertility and the total protein content, but the protein profiles did not show any differences. Furthermore, the immunological tests revealed an increase in IgE binding reactivity when pollen was exposed to the pollutant.

Immunochemical and molecular characterization of allergenic profilin (Koc s 2) from Kochia scoparia pollen

Kochia scoparia pollen has been demonstrated as an important cause of pollinosis in tropical and sub-tropical regions of the world. The aim of this study was to characterize the IgE-binding protein of the Kochia pollen extract and production of recombinant form of allergenic profilin of this weed. To predict its allergenic cross-reactivity with profilins of common allergenic plants, nucleotide sequence homology of Kochia profilin was evaluated. Specific ELISA and immunoblotting assay were applied to determine the IgE-binding reactivity of 28 sera collected from patients who were sensitized to Kochia pollen. In cloning procedure, the Kochia profilin-coding sequence was inserted into PTZ57R/T vector and expressed using pET-21b(+) vector. IgE-binding competence of purified recombinant Kochia profilin (rKoc s 2) was analyzed by in vitro assays. There were several protein bands in Kochia pollen extract with molecular weights approximately ranging from 14.5 to 85 kDa. Nucleotide sequencing revealed an open reading frame of 399 bp encoding for 133 amino acid residues which belonged to the profilin family; 15 patients (15/28, 53.1 %) had significant specific IgE levels for the rKoc s 2. Immunodetection and inhibition assays indicated that the purified rKoc s 2 might be the same as that in the crude extract. Koc s 2, the first allergen from the Kochia pollen was identified as a member of the family of profilins. High degree of homology was found among amino acid sequences of Kochia profilin and several profilin molecules from unrelated plant families.

Immunization with Hypoallergens of shrimp allergen tropomyosin inhibits shrimp tropomyosin specific IgE reactivity.

Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their in vitro reactivity towards IgE from shrimp allergy patients and Met e 1-sensitized mice, as well as considerable decrease in induction of mast cell degranulation as demonstrated in passive cutaneous anaphylaxis assay. Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.

Expression, cloning, and IgE-binding of the full-length dust mite allergen Der f 8.

Dermatophagoides farinae, a domestic mite species, produces some of the most potent allergens that contribute to allergy in China and worldwide. We sought to clone and express the group 8 allergen of D. farinae (Der f 8) to investigate its IgE-binding reactivity. The full-length cDNA encoding Der f 8 was generated by using RT-PCR and 5' RACE, cloned into pCold-TF expression vector, confirmed by nucleotide sequencing, sub-cloned into pET-28b (+), and transfected into E. coli BL21 cells for expression. After purification by nickel affinity chromatography and identified by SDS-PAGE, the recombinant Der f 8 bound with sera from 40.9% (9/22) of mite-allergic patients according to ELISA testing. Analysis of the recombinant DNA sequence revealed a 231 amino acid open reading frame encoding a protein with a derived molecular mass of 26.4kDa and an isoelectric point of 6.84. The deduced amino acid sequence has nine phosphorylation sites, displaying strong homology with glutathione S-transferase, and its secondary structure comprises alpha helix (45.5%), extended strand (11.3%), and random coils (43.3%). BLAST through the National Center for Biotechnology Information database and alignment identified similarity with group 8 allergens or glutathione S-transferases of Dermatophagoides pteronyssinus, Suidasia medanensis, Lepidoglyphus destructor, Glycyphagus domesticus, and Aleuroglyphus ovatus (64, 65, 53, 53, and 50%, respectively). The first recombinant Der f 8 protein produced in full length successfully bound with patient IgE, demonstrating the importance of Der f 8 in mite allergy.

A recombinant lipid transfer protein from Cannabis sativa: IgE-binding properties in patients with symptoms to Cannabis

Methods To identify the mature peptide sequence of the lipid transfer protein (LTP) and to study the IgE-binding reactivity of a recombinant Can s 3 (rCan s 3), a cDNA was synthesized from total RNA of Cannabis sativa L. ssp. sativa cv. Kompolti leaves obtained from the botanical garden of the University Bonn. The LTP gene was amplified with a primer mix deduced from published amino acid sequences. The LTP variant was identified in five independent clones by sequencing in the pDrive vector system followed by the expression of the mature LTP in the pMAL-vector. After isolation of a soluble recombinant maltose-binding protein (MBP)-Can s 3 fusion protein, aliquots were biotinylated and coupled to Streptavidin-ImmunoCAPs.

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

Background: Peanut flour is a high-protein, low-oil, powdered material prepared from roasted peanut seed. In addition to being a well-established food ingredient, peanut flour is also the active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a processing strategy to generate hydrolysates from peanut flour with reduced allergenicity. Methods: Soluble fractions of 10% (w/v) light roasted peanut flour dispersions were hydrolyzed with the following proteases: Alcalase (pH 8.0, 60°C), pepsin (pH 2.0, 37°C) or Flavourzyme (pH 7.0, 50°C) for 60 min. Western blotting, inhibition ELISA and basophil activation tests were used to examine IgE reactivity. Results: Western blotting experiments revealed the hydrolysates retained IgE binding reactivity and these IgE-reactive peptides were primarily Ara h 2 fragments regardless of the protease tested. Inhibition ELISA assays demonstrated that each of the hydrolysates had decreased capacity to bind peanut-specific IgE compared with nonhydrolyzed controls. Basophil activation tests revealed that all hydrolysates were comparable (p > 0.05) to nonhydrolyzed controls in IgE cross-linking capacity. Conclusions: These results indicate that hydrolysis of peanut flour reduced IgE binding capacity; however, IgE cross-linking capacity during hydrolysis was retained, thus suggesting such hydrolysates are not hypoallergenic.

Analysis of the Structural and Immunological Stability of 2S Albumin, Nonspecific Lipid Transfer Protein, and Profilin Allergens from Mustard Seeds

This work investigates the resistance to proteolysis and heating of the yellow mustard (Sinapis alba L.) allergens Sin a 1 (2S albumin), Sin a 3 (nonspecific lipid transfer protein, LTP), and Sin a 4 (profilin) to explain their potential capability to induce primary sensitization at the gastrointestinal level. Sin a 1 and Sin a 3 resisted gastric digestion showing no reduction of the IgE reactivity. Intestinal digestion of Sin a 1 and Sin a 3 produced a limited proteolysis but retained significant IgE-binding reactivity. Sin a 1 was stable after heating, and although Sin a 3 was modified, most of its structure was recovered after cooling back. These two allergens would be therefore able to sensitize by ingestion. Sin a 4 was completely digested by gastric treatment and its conformational structure markedly modified at 85 °C. Thus, this allergen can be described as a nonsensitizing mustard allergen.

Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China

We report the cloning and molecular characterization of a full-length cDNA encoding house dust mite allergen, Der f 6 from D. farinae isolated in China. The full-length Der f 6 cDNA was obtained with 840 nucleotides long. Nucleotide sequencing analyses showed a total of 36 mutations in five Der f 6 cDNA clones, corresponding to 23 incompatible amino acid residues. Recombinant Der f 6 (rDer f 6) protein was successfully expressed in and purified from E. coli BL21. Among 20 asthmatic patients, 45% was positive to rDer f 6 by ELISA. Bioinformatics analyses revealed that the mature Der f 6 was a hydrophobic and extracellular protein with chymotrypsin-like serine protease activity, its secondary structure was composed of alpha helix (7.69%), extended strand (34.62%), random coils (57.69%), and the similarity of Der f 6 to Blo t 6, Sui m 6, Der f 3 and Der f 9 was 64, 65, 35, and 38%, respectively.

Effects of different processing methods on digestibility of Scylla paramamosain allergen (tropomyosin)

Crustacean allergy is a significant health problem around the world, and tropomyosin (TM) represents the major allergen of crustaceans. The aim of this study was to evaluate three processing methods (boiling, CUB, HPS) and identify the one method that is most effective in the degradation of TM and reduction of its IgE-binding reactivity, and make it easier to be decomposed during gastrointestinal digestion. SDS–PAGE analysis indicated that boiling had little impact on the digestive stability of TM. In contrast, combined ultrasound and boiling (CUB), and high pressure steaming (HPS) both could accelerate the digestion of TM. Similarly, western blotting and inhibition ELISA also demonstrated that the reactivity of IgG/IgE-binding of TM that was extracted from processed crab was partially decreased after treating with CUB or HPS. Among the three processing methods, HPS was the most effective method to accelerate the digestion of TM in gastrointestinal digestion, and reduce the reactivity of IgG/IgE-binding of TM. These results suggest that proper processing of crab could promote the degradation of TM in simulated gastrointestinal digestion, reduce the reactivity of IgG/IgE-binding of TM, and decrease the incidence of crab hypersensitivity in humans.

IgE Binding Reactivity of Peptide Fragments of Bla g 4, a Major German Cockroach Allergen

Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.

Molecular Cloning and the Allergenic Characterization of Tropomyosin from Tyrophagus putrescentiae

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.

Relevance of the recombinant lipid transfer protein of Hevea brasiliensis: IgE-binding reactivity in fruit-allergic adults

Lipid transfer proteins (LTPs) are relevant allergens in certain plants. The role of the LTP of Hevea brasiliensis in the latex-fruit syndrome is widely unknown. To study IgE reactivity with recombinant Hevea LTP in sera of fruit-allergic adults with and without natural rubber latex (NRL) allergy. An LTP-specific complementary DNA of H brasiliensis leaves was amplified, subcloned into the pMAL expression system, and analyzed. The recombinant protein was coupled to ImmunoCAP, and the IgE-binding properties were studied in sera of 10 NRL-allergic patients without symptoms to fruit and 48 atopic patients with fruit allergy. Eleven of these 48 patients were also allergic to NRL, 14 displayed sensitization to NRL without symptoms on NRL exposure so far, and 23 had neither symptoms nor IgE antibodies to NRL. After expression in Escherichia coli, a soluble maltose-binding protein-rHev b 12 fusion protein was isolated and coupled to ImmunoCAP to determine rHev b 12 specific IgE reactivity. rHev b 12 specific IgE binding was found in 3 fruit-allergic patients with NRL sensitization (0.68, 0.88, and 0.96 kU/L) and in 3 fruit-allergic patients without NRL sensitization (1.58, 2.25, and 2.27 kU/L). The remaining 52 serum samples and all maltose-binding protein control test results were negative (< 0.35 kU/L). In these patients, rHev b 12 specific IgE reactivity seems to result from common cross-reactive epitopes with some of the fruit LTPs tested and underscores only an involvement in co-recognition. No clinical relevance of IgE binding to the LTP of H brasiliensis in association with NRL allergy was detected.

Ablation of ovomucoid-induced allergic response by desensitization with recombinant ovomucoid third domain in a murine model.

Attempts to modulate the allergenic response by hypoallergens aimed at eliminating IgE-binding epitopes have been established recently for allergen immunotherapy. Desensitization offers an alternative approach to mounting a protective immune response. We have shown previously that mutation of the decisive amino acids in the B cell epitope of the ovomucoid third domain suppresses IgE binding reactivity against human patient sera and we hypothesize that this hypoallergenic variant could be a potential candidate molecule for specific immunotherapy against an ovomucoid-induced IgE reaction. The aim of this study was to investigate whether hyposensitization with the ovomucoid-modified isoform could desensitize ovomucoid-sensitized mice. We mapped the immunodominant B cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid mutant isoform, having ablated allergenicity against patient sera, was used to desensitize ovomucoid-sensitized Balb/c mice by intraperitoneal injection. Female Balb/c mice were sensitized with intact ovomucoid molecule (Fovm) and desensitized with the modified isoform of the third domain of ovomucoid (GMFA). Intact ovomucoid-sensitized mice desensitized with phosphate-buffered saline (PBS) served as positive controls to maintain hypersensitivity. To gain insight into the efficacy of the modified ovomucoid variant in desensitization, effects on hypersensitivity reactions and histamine levels, followed by its association with antibody levels and cytokine profiles, were measured. Abrogation of the allergic response with complete suppression of anaphylactic symptoms and lower serum histamine levels was observed in the desensitized group by GMFA, accompanied by significantly reduced ovomucoid-specific IgE and IgG1 levels and enhanced specific IgG and IgG2a levels. The sensitized group showed severe anaphylactic symptoms, enhanced serum histamine concentrations and increased levels of specific IgE and IgG1. The level of interleukin (IL)-4 was decreased dramatically in the desensitized group and higher levels of interferon (IFN)-gamma were found, whereas mice sensitized with intact ovomucoid exhibited significantly higher levels of IL-4 favouring a Th2 skewed pathway. We demonstrate clearly that GMFA is able to ablate ovomucoid-induced allergic reactions in sensitized mice. This occurs via a suppression of specific IgE accompanied by an increase in suppressor T cell activity. This approach offers some promise for the development of treatment against ovomucoid-induced allergic response.

Molecular cloning and characterization of full‐length cDNAs encoding a novel high‐molecular‐weight Dermatophagoides pteronyssinus mite allergen, Der p 11

Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application. The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5'-3' rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children. Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73-80% at concentrations of 100 microg/ml. This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.