Toward Consensus Epitopes B and T of Tropomyosin Involved in Cross-Reactivity across Diverse Allergens: An In Silico Study.

Tropomyosin (TM) is a pan-allergen with cross-reactivity to arthropods, insects, and nematodes in tropical regions. While IgE epitopes of TM contribute to sensitization, T-cell (MHC-II) epitopes polarize the Th2 immune response. This study aimed to identify linear B and T consensus epitopes among house dust mites, cockroaches, Ascaris lumbricoides, shrimp, and mosquitoes, exploring the molecular basis of cross-reactivity in allergic diseases. Amino acid sequences of Der p 10, Der f 10, Blo t 10, Lit v 1, Pen a 1, Pen m 1, rAsc l 3, Per a 7, Bla g 7, and Aed a 10 were collected from Allergen Nomenclature and UniProt. B epitopes were predicted using AlgPred 2.0 and BepiPred 3.0. T epitopes were predicted with NetMHCIIpan 4.1 against 10 HLA-II alleles. Consensus epitopes were obtained through analysis and Epitope Cluster Analysis in the Immune Epitope Database. We found 7 B-cell epitopes and 28 linear T-cell epitopes binding to MHC II. A unique peptide (residues 160-174) exhibited overlap between linear B-cell and T-cell epitopes, highly conserved across tropomyosin sequences. These findings shed light on IgE cross-reactivity among the tested species. The described immuno-informatics pipeline and epitopes can inform in vitro research and guide synthetic multi-epitope proteins' design for potential allergology immunotherapies. Further in silico studies are warranted to confirm epitope accuracy and guide future experimental protocols.

A novel IgE epitope-specific antibodies-based sandwich ELISA for sensitive measurement of immunoreactivity changes of peanut allergen Ara h 2 in processed foods.

Background: Peanut is an important source of dietary protein for human beings, but it is also recognized as one of the eight major food allergens. Binding of IgE antibodies to specific epitopes in peanut allergens plays important roles in initiating peanut-allergic reactions, and Ara h 2 is widely considered as the most potent peanut allergen and the best predictor of peanut allergy. Therefore, Ara h 2 IgE epitopes can serve as useful biomarkers for prediction of IgE-binding variations of Ara h 2 and peanut in foods. This study aimed to develop and validate an IgE epitope-specific antibodies (IgE-EsAbs)-based sandwich ELISA (sELISA) for detection of Ara h 2 and measurement of Ara h 2 IgE-immunoreactivity changes in foods. Methods: DEAE-Sepharose Fast Flow anion-exchange chromatography combining with SDS-PAGE gel extraction were applied to purify Ara h 2 from raw peanut. Hybridoma and epitope vaccine techniques were employed to generate a monoclonal antibody against a major IgE epitope of Ara h 2 and a polyclonal antibody against 12 IgE epitopes of Ara h 2, respectively. ELISA was carried out to evaluate the target binding and specificity of the generated IgE-EsAbs. Subsequently, IgE-EsAbs-based sELISA was developed to detect Ara h 2 and its allergenic residues in food samples. The IgE-binding capacity of Ara h 2 and peanut in foods was determined by competitive ELISA. The dose-effect relationship between the Ara h 2 IgE epitope content and Ara h 2 (or peanut) IgE-binding ability was further established to validate the reliability of the developed sELISA in measuring IgE-binding variations of Ara h 2 and peanut in foods. Results: The obtained Ara h 2 had a purity of 94.44%. Antibody characterization revealed that the IgE-EsAbs recognized the target IgE epitope(s) of Ara h 2 and exhibited high specificity. Accordingly, an IgE-EsAbs-based sELISA using these antibodies was able to detect Ara h 2 and its allergenic residues in food samples, with high sensitivity (a limit of detection of 0.98 ng/mL), accuracy (a mean bias of 0.88%), precision (relative standard deviation < 16.50%), specificity, and recovery (an average recovery of 98.28%). Moreover, the developed sELISA could predict IgE-binding variations of Ara h 2 and peanut in foods, as verified by using sera IgE derived from peanut-allergic individuals. Conclusion: This novel immunoassay could be a user-friendly method to monitor low level of Ara h 2 and to preliminary predict in vitro potential allergenicity of Ara h 2 and peanut in processed foods.

Immunoglobulin E Epitope Mapping and Structure-Allergenicity Relationship Analysis of Crab Allergen Scy p 9.

Filamin C is an allergen of Scylla paramamosain (Scy p 9), and six IgE linear epitopes of the allergenic predominant region had previously been validated. However, the IgE epitope and structure-allergenicity relationship of Scy p 9 are unclear. In this study, a hydrophobic bond was found to be an important factor of conformation maintaining. The critical amino acids in the six predicted conformational epitopes were mutated, and the IgE-binding capacity and surface hydrophobicity of four mutants (E216A, T270A, Y699A, and V704A) were reduced compared to Scy p 9. Ten linear epitopes were verified with synthetic peptides, among which L-AA187-205 had the strongest IgE-binding capacity. In addition, IgE epitopes were mapped in the protruding surface of the tertiary structure, which were conducive to binding with IgE and exhibited high conservation among filamin genes. Overall, these data provided a basis for IgE epitope mapping and structure-allergenicity relationship of Scy p 9.

One-Step Purification of IgE Epitope-Specific Antibody Using Immunomagnetic Beads and Highly Sensitive Detection of Bovine ß-Lactoglobulin for the Prediction of Milk Allergenicity in Foods.

Bovine ß-lactoglobulin (BLG) is a common allergen found in milk, and the immunoglobulin E (IgE) epitope plays a crucial role in cow milk allergy. Therefore, targeting the IgE epitope could be useful in accurately detecting BLG and assessing its allergenicity. However, producing an IgE epitope-specific antibody (IgE-EsAb) through traditional methods requires complex and time-consuming procedures. Here, IgE-EsAb was purified from rabbit anti-BLG sera by immunomagnetic beads in one step. Then, a sandwich ELISA (sELISA) based on the IgE-EsAb was developed to detect BLG and predict the potential milk allergenicity in foods. The obtained IgE-EsAb could specifically recognize the target IgE epitope of BLG and exhibited high affinity and specificity. The developed IgE-EsAb-based sELISA demonstrated an ultra-wide linear range of 3.9-1.28 × 105 ng/mL, with a limit of detection of 0.49 ng/mL for BLG. Additionally, the proposed immunoassay showed high specificity and recoveries (91.24-109.61%). The ability of the IgE-EsAb-based sELISA to evaluate the potential milk allergenicity in foods was validated using sera from cow milk allergy patients. These results suggest that immunomagnetic beads are an effective tool for rapidly obtaining the IgE-EsAb, and our proposed sELISA could be a reliable and user-friendly method for monitoring trace amounts of BLG and predicting the potential milk allergenicity of food samples.

IgE Epitope Analysis and Hypo-Immunoreactivity Derivative of Arginine Kinase in Mantis Shrimp (Oratosquilla oratoria).

As the main allergenic food, shrimp can trigger allergic reactions in various degrees. In this study, arginine kinase (AK) was identified as an allergen in Oratosquilla oratoria by LC-MS/MS. The open reading frame of AK was obtained, which included 356 amino acids, and recombinant AK (rAK) was expressed in Escherichia coli. The results of immunological analysis and circular dichroism showed that rAK displayed similar IgG-/IgE-binding activity and structure as native AK. Besides, five IgE linear epitopes of AK were verified by serological analysis, on the basis of which an epitope-deleted derivative was obtained and named as mAK-L. It has been shown that mAK-L displayed hypo-immunoreactivity compared to rAK, and the contents of secondary structures were different. In conclusion, these discoveries enrich the overall understanding of crustacean allergens and epitopes and set the foundations for food allergy diagnosis and immunotherapy.

Updates on Databases of Allergens and Allergen-Epitopes.

The increasing prevalence of allergic diseases is of great public health concern. Environmental and food allergens are the major triggers of allergic diseases via respiratory or gastrointestinal routes, respectively. A major setback in the clinical management of allergies is the unavailability of purified allergens required for diagnostic purposes. Furthermore, manipulation of allergen sequences and structures by employing protein-engineering approaches is needed to design immunotherapeutic vaccines. All these approaches rely upon the sequence, structure, and epitope location of allergens. A number of databases have therefore been developed that serve as repositories of molecular information of allergens. In this chapter, we discuss the five most important widely used allergen databases that might be helpful for the research community working on molecular allergology.

IgE Epitope Analysis for Scy p 1 and Scy p 3, the Heat-Stable Myofibrillar Allergens in Mud Crab.

Tropomyosin (Scy p 1) and myosin light chain (Scy p 3) are investigated to be important heat-stable allergens in Scylla paramamosain. However, the epitopes of Scy p 1 and Scy p 3 are limited. In this study, recombinant Scy p 1 and Scy p 3 had similar IgE-binding capacity to natural proteins. Mimotopes of Scy p 1 and Scy p 3 were analyzed by bioinformatics, phage display, and one-bead-one-compound technology. Ten linear epitopes of Scy p 1 and seven linear epitopes of Scy p 3 were identified by synthetic peptides and inhibition dot blot. Meanwhile, three conformational epitopes of Scy p 1 and seven conformational epitopes of Scy p 3 were verified by site-directed mutagenesis and the serological test. Furthermore, strong IgE-binding epitopes of Scy p 1 and Scy p 3 were conserved in multiple crustaceans. Overall, these epitopes could enhance our understanding of crab allergens, which lay the foundation for a cross-reaction.

The Fate of IgE Epitopes and Coeliac Toxic Motifs during Simulated Gastrointestinal Digestion of Pizza Base.

Understanding how food processing may modify allergen bioaccessibility and the evolution of immunologically active peptides in the gastrointestinal tract is essential if knowledge-based approaches to reducing the allergenicity of food are to be realised. A soy-enriched wheat-based pizza base was subjected to in vitro oral-gastro-duodenal digestion and resulting digests analysed using a combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). The digestion profile of pizza base resembled that of bread crust where higher temperatures during baking reduced protein solubility but still resulted in the generation of a complex mixture of peptides. MS profiling showed numerous peptides carrying IgE epitopes, and coeliac toxic motifs were in excess of 20-30 residues long and were only released after either 120 min of gastric digestion or a combination of gastric and duodenal digestion. In silico prediction tools showed an overestimated number of cleavage sites identified experimentally, with low levels of atypical peptic and chymotryptic cleavage sites identified particularly at glutamine residues. These data suggest that such alternative pepsin cleavage sites may play a role in digestion of glutamine-rich cereal foods. They also contribute to efforts to provide benchmarks for mapping in vitro digestion products of novel proteins which form part of the allergenicity risk assessment.

Designing Next-Generation Vaccines Against Common Pan-Allergens Using In Silico Approaches.

Next-generation allergy vaccines refer to allergen-derived attenuated molecules that can boost allergen-blocking IgG response. These IgG antibodies are specifically directed toward the IgE epitope of allergens and interfere in allergen-IgE interaction. Our study is a computational approach to design such vaccines against four widespread pan-allergens families. Pan-allergens display extensive immunological cross-reactivity due to the presence of conserved IgE epitope and T cell epitope. In this study, the vaccine design is based on hapten-carrier concept in which the carrier protein is an immunogenic component providing T cell help. Either PreS protein of hepatitis B or cholera enterotoxin B (CTB) fused with three tetanus toxoid fragments (TTFrC) was used here as the carrier. The hapten components are nonanaphylactic peptides (NAPs) derived from experimentally determined antigenic regions of the allergens. The charged residues of NAPs are selectively modified to obliterate IgE, as well as T cell reaction, and hence, are safe to apply in allergy patients. Various combinations of vaccine constructs (PreS/CTB+TTFrC and NAPs) were designed with intermediate linker motifs. Screening of constructs was performed through a three-step method such as physicochemical parameters, secondary structures, and tertiary structures using various bioinformatic tools. The final construct with best quality and stability was selected for each allergen family. Suitability of these constructs for being expressed in recombinant form was checked at DNA, RNA, and protein level. Presence of putative epitopes inducing tolerogenic interleukin-10 was also predicted for these constructs. The present work led to the design of putative vaccines with immunotherapeutic potential and broad applicability for allergic diseases caused by a wide array of cross-reactive allergens.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Identification of an immunodominant IgE epitope of Der p 39, a novel allergen of Dermatophagoides pteronyssinus.

Background: House dust mites (HDMs) are the main source of indoor inhalatory allergens that cause IgE-mediated allergic diseases. The discovery and identification of HDM allergens are important for the diagnosis and treatment of allergic diseases. Objective: We sought to identify a Group 39 Dermatophagoides pteronyssinus (Der p) allergen, namely Der p 39, and explore its immunodominant IgE epitopes. Methods: Homology analysis of amino acid (aa) sequences in HDM and human troponin C (TnC)-like protein was performed. Total RNA of Der p was extracted and used to amplify Der p 39 cDNA with specific primers. Recombinant Der p 39 protein was expressed with a pET-His prokaryotic expression system and purified with Ni-NTA resins. IgE binding was evaluated with western blot, dot blot, and enzyme-linked immunosorbent assay (ELISA) experiments. The IgE binding epitopes of Der p 39 were identified by observing HDM-allergic sera interactions with truncated and hybrid proteins formed from Der p 39 and human TnC-like proteins. Results: The Der p 39 open reading frame (ORF) cDNA was found to be 462 base pairs and registered in the NCBI library (GenBank no. MZ336019.1). Der p 39, which encoded 153 aa, was found to have 35.63% and 99.35% homology with human TnC and Dermatophagoides farina (Der f) 39, respectively. IgE-ELISA showed IgE binding with expressed and purified recombinant Der p 39 (18 kDa) in 5/87 (5.75%) HDM-allergic sera samples. Analyses of IgE binding with Der p 39-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The data from this study were submitted to the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature database. Conclusion: Der p 39 was identified as a minor HDM allergen with a conformational IgE binding epitope. These findings could have important theoretical implications in the development of HDM allergy diagnostics and therapeutics.

Crystal Structure Analysis and IgE Epitope Mapping of Allergic Predominant Region in Scylla paramamosain Filamin C, Scy p 9.

Filamin C (FLN c) is a novel allergen in shellfish. In this study, FLN c from Scylla paramamosain was divided into three regions for recombinant expression based on the number of domains and amino acids. Using dot blot and basophil activation tests, the allergic predominant region of FLN c was determined to be 336-531 amino acid positions (named FLN c-M). It was confirmed that by X-ray diffraction, the crystal structure of FLN c-M with immunoglobulin-like folding at a resolution of 1.7 Å was obtained. The monomer was a barrel structure composed of 16 ß-strands and 2 α-helices. Three conformational epitopes were predicted, six linear epitopes were verified by serological test, and they were positioned on the crystal structure of FLN c-M. For the first time, the crystal structure of the allergic predominant region of FLN c was determined, and it provided an accurate template for the localization of IgE epitopes.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded ß-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Analysis of Immunoreactivity of α/α2-Tropomyosin from Haliotis discus hannai, Based on IgE Epitopes and Structural Characteristics.

Tropomyosin (TM) was reported to be a supercoil allergen of shellfish. However, little information is available about its link between structure and allergenicity. In this study, the subunit of TM (α-TM) and supercoil of TM (α2-TM) were identified from Haliotis discus hannai. α2-TM showed higher immunoreactivity than α-TM. Meanwhile, seven linear epitopes in α-TM and α2-TM were verified, and two conformational epitopes in α2-TM were predicted. The physicochemical properties and chemical bond assays confirmed the existence of the disulfide bond in α2-TM. According to spectroscopy and hydrophobicity analysis, α-TM showed higher α-helix features and blueshift of the fluorescence intensity peak compared with those of α2-TM. The structure analysis revealed the possibility of conformational epitopes in α2-TM, which could explain the immunoreactivity differences between α-TM and α2-TM further. These results improved the understanding of Haliotis discus hannai TM, which lay the foundation for the food processing of abalone.

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.

Epitope Mapping of Allergenic Lipid Transfer Proteins.

Food allergy is becoming a great problem in industrialized countries. Thus, there is the need for a robust understanding of all aspects characterizing IgE response to allergens. The epitope mapping of B-cell epitopes has the potential to become a fundamental tool for food allergy diagnosis and prognosis and to lead to a better understanding of the pathogenesis. Using this approach, we have worked on epitope mapping of the most important plant food allergens identified in the Mediterranean area. The final aim of this study is to define the immune response regarding B epitopes and its clinical relevance in LTP allergy. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of allergenic lipid transfer proteins.

IgE and IgG4 epitopes revealed on the major fish allergen Lat c 1.

BACKGROUND: The IgE- and IgG4-binding patterns of the major fish allergen parvalbumins are not clearly understood. IgE antibody-binding to parvalbumin from Asian seabass, Lat c 1.01, is implicated in up to 90 % of allergic reactions, although the region of IgE or IgG4 epitopes are unknown. In the present study, we characterized the specific IgE- and IgG4-binding regions of Lat c 1.01 using serum from pediatric and adult patients with clinically-confirmed fish allergy. METHODS: A comparative investigation of patient IgE- and IgG4-binding to recombinant Lat c 1.01 was performed by immunoblotting and indirect ELISA using serum from 15 children and eight adults with clinically confirmed IgE-mediated reactions to fish. The IgE- and IgG4-binding regions of Lat c 1.01 were determined by inhibition ELISA using seven overlapping peptides spanning the entire 102 amino acid sequence. Elucidated IgE-binding regions were modelled and compared to known antibody-binding regions of parvalbumins from five other fish species. RESULTS: Ninety five percent (22/23) patients demonstrated IgE-binding to rLat c 1.01, while fewer patients (10/15 children and 7/8 adults) demonstrated robust IgG4 binding when determined by immunoblots. IgE-binding for both cohorts was significantly higher compared to IgG4-binding by ELISA. All patients in this study presented individual IgE and IgG4 epitope-recognition profiles. In addition to these patient-specific antibody binding sites, general IgE epitopes were also identified at the C- and N-terminal regions of this major fish allergen. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings demonstrate two specific IgE epitopes on parvalbumin from Asian seabass, while IgG4 binding is much lower and patient specific. This study highlights the importance of advancement in epitope analysis regardless of the age group for diagnostics and immunotherapies for fish allergy.

AlgPred 2.0: an improved method for predicting allergenic proteins and mapping of IgE epitopes.

AlgPred 2.0 is a web server developed for predicting allergenic proteins and allergenic regions in a protein. It is an updated version of AlgPred developed in 2006. The dataset used for training, testing and validation consists of 10 075 allergens and 10 075 non-allergens. In addition, 10 451 experimentally validated immunoglobulin E (IgE) epitopes were used to identify antigenic regions in a protein. All models were trained on 80% of data called training dataset, and the performance of models was evaluated using 5-fold cross-validation technique. The performance of the final model trained on the training dataset was evaluated on 20% of data called validation dataset; no two proteins in any two sets have more than 40% similarity. First, a Basic Local Alignment Search Tool (BLAST) search has been performed against the dataset, and allergens were predicted based on the level of similarity with known allergens. Second, IgE epitopes obtained from the IEDB database were searched in the dataset to predict allergens based on their presence in a protein. Third, motif-based approaches like multiple EM for motif elicitation/motif alignment and search tool have been used to predict allergens. Fourth, allergen prediction models have been developed using a wide range of machine learning techniques. Finally, the ensemble approach has been used for predicting allergenic protein by combining prediction scores of different approaches. Our best model achieved maximum performance in terms of area under receiver operating characteristic curve 0.98 with Matthew's correlation coefficient 0.85 on the validation dataset. A web server AlgPred 2.0 has been developed that allows the prediction of allergens, mapping of IgE epitope, motif search and BLAST search (https://webs.iiitd.edu.in/raghava/algpred2/).

Identification of immunodominant IgE binding epitopes of Der p 24, a major allergen of Dermatophagoides pteronyssinus.

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 ß-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.

Two grass pollen tablets commercially available for allergy immunotherapy display different IgE epitope repertoires.

BACKGROUND: The distribution of Pooideae species varies across Europe. Especially, Timothy is less represented in Southern than in Northern Europe. Since allergenic cross-reactivity between pollens from different grasses is only partial, grass pollen-allergic patients are expected to display different sensitization profiles, with specific IgE directed against different combinations of allergenic epitopes, depending on their living places in Europe and the grasses they are exposed to. In this context, this study aimed at comparing two tablets commercially available for allergy immunotherapy, namely a 5-grass (Cocksfoot, Meadow-grass, Rye-grass, Sweet vernal-grass and Timothy) and a 1-grass (Timothy) pollen tablets, for their ability to represent the sensitization profiles of patients, depending on whether they live in Southern or Northern Europe. METHODS: Sera were collected from adult patients living in Spain (n = 19) and Sweden (n = 22). Tablets were compared for their ability to inhibit the binding of patient serum IgE to pollen allergens from twelve grasses commonly distributed throughout Europe, as determined by the areas under the curves obtained by ELISA-inhibition. Tablets were adjusted to an equivalent allergenic activity, based on the CBER/FDA bioequivalent allergy unit. RESULTS: Inhibition of the IgE binding to pollen allergens from twelve grasses was significantly stronger with the 5-grass than with the 1-grass pollen tablet (p < 0.0001), regardless of whether patients were considered as a whole or by geographical area. This difference between tablets was significantly greater for Southern than Northern European patients (p < 0.05). CONCLUSIONS: Compared to the 1-grass tablet, the 5-grass tablet generally covers better the sensitization profiles of European patients, especially patients from Southern Europe, in principle less exposed to pollen from Timothy than from other grasses. The 5-grass tablet is therefore expected to elicit larger spectra of blocking antibodies, which might have implications in light of the generally accepted mechanisms of allergy immunotherapy.