Human monoclonal antibodies to Ara h 2 inhibit allergen-induced, IgE-mediated cell activation.
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ABSTRACT:
SUBMITTER: Liao S
PROVIDER: S-EPMC6682448 | biostudies-literature | 2019 Aug
REPOSITORIES: biostudies-literature
Publications
Human monoclonal antibodies to Ara h 2 inhibit allergen-induced, IgE-mediated cell activation.
Liao Sumei S Patil Sarita U SU Shreffler Wayne G WG Dreskin Stephen C SC Chen Xueni X
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 20190627 8
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Project description:Cockroach allergy is strongly associated with asthma, and involves the production of IgE antibodies against inhaled allergens. Reports of conformational epitopes on inhaled allergens are limited. The conformational epitopes for two specific monoclonal antibodies (mAb) that interfere with IgE antibody binding were identified by X-ray crystallography on opposite sites of the quasi-symmetrical cockroach allergen Bla g 2.Mutational analysis of selected residues in both epitopes was performed based on the X-ray crystal structures of the allergen with mAb Fab/Fab' fragments, to investigate the structural basis of allergen-antibody interactions. The epitopes of Bla g 2 for the mAb 7C11 or 4C3 were mutated, and the mutants were analyzed by SDS-PAGE, circular dichroism, and/or mass spectrometry. Mutants were tested for mAb and IgE antibody binding by ELISA and fluorescent multiplex array. Single or multiple mutations of five residues from both epitopes resulted in almost complete loss of mAb binding, without affecting the overall folding of the allergen. Preventing glycosylation by mutation N268Q reduced IgE binding, indicating a role of carbohydrates in the interaction. Cation-? interactions, as well as electrostatic and hydrophobic interactions, were important for mAb and IgE antibody binding. Quantitative differences in the effects of mutations on IgE antibody binding were observed, suggesting heterogeneity in epitope recognition among cockroach allergic patients.Analysis by site-directed mutagenesis of epitopes identified by X-ray crystallography revealed an overlap between monoclonal and IgE antibody binding sites and provided insight into the B cell repertoire to Bla g 2 and the mechanisms of allergen-antibody recognition, including involvement of carbohydrates.
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Project description:Mast cells and basophils have long been implicated in the pathogenesis of IgE-mediated hypersensitivity reactions. They express the high-affinity IgE receptor, FcϵRI, on their surface. Antigen-induced crosslinking of IgE antibodies bound to that receptor triggers a signaling cascade that results in activation, leading to the release of an array of preformed vasoactive mediators and rapidly synthesized lipids, as well as the de novo production of inflammatory cytokines. In addition to bearing activating receptors like FcεRI, these effector cells of allergy express inhibitory ones including FcγR2b, an IgG Fc receptor with a cytosolic inhibitory motif that activates protein tyrosine phosphatases that suppress IgE-mediated activation. We and others have shown that food allergen-specific IgG antibodies strongly induced during the course of oral immunotherapy (OIT), signal via FcγR2b to suppress IgE-mediated mast cell and basophil activation triggered by food allergen challenge. However, the potential inhibitory effects of IgA antibodies, which are also produced in response to OIT and are present at high levels at mucosal sites, including the intestine where food allergens are encountered, have not been well studied. Here we uncover an inhibitory function for IgA. We observe that IgA binds mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells. Binding to BMMCs is dependent on calcium and sialic acid. We also found that IgA antibodies inhibit IgE-mediated mast cell degranulation in an allergen-specific fashion. Antigen-specific IgA inhibits IgE-mediated mast cell activation early in the signaling cascade, suppressing the phosphorylation of Syk, the proximal protein kinase mediating FcεRI signaling, and suppresses mast cell production of cytokines. Furthermore, using basophils from a peanut allergic donor we found that IgA binds to basophils and that activation by exposure to peanuts is effectively suppressed by IgA. We conclude that IgA serves as a regulator of mast cell and basophil degranulation, suggesting a physiologic role for IgA in the maintenance of immune homeostasis at mucosal sites.
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Project description:Acute allergic symptoms are caused by allergen-induced crosslinking of allergen-specific immunoglobulin E (IgE) bound to Fc-epsilon receptors on effector cells. Desensitization with allergen-specific immunotherapy (SIT) has been used for over a century, but the dominant protective mechanism remains unclear. One consistent observation is increased allergen-specific IgG, thought to competitively block allergen binding to IgE. Here we show that the blocking potency of the IgG response to Cat-SIT is heterogeneous. Next, using two potent, pre-selected allergen-blocking monoclonal IgG antibodies against the immunodominant cat allergen Fel d 1, we demonstrate that increasing the IgG/IgE ratio reduces the allergic response in mice and in cat-allergic patients: a single dose of blocking IgG reduces clinical symptoms in response to nasal provocation (ANCOVA, p = 0.0003), with a magnitude observed at day 8 similar to that reported with years of conventional SIT. This study suggests that simply augmenting the blocking IgG/IgE ratio may reverse allergy.
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Project description:Purpose of reviewBound to its high affinity receptor on mast cells and basophils, the IgE antibody molecule plays an integral role in the allergic reaction. Through interactions with the allergen, it provides the sensitivity and specificity parameters for cell activation and mediator release that produce allergic symptoms. Advancements in human hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific human IgE monoclonal antibodies.Recent findingsA high-resolution structure of dust mite allergen Der p 2 in complex with Fab of the human IgE mAb 2F10 was recently determined using X-ray crystallography. The structure reveals the fine molecular details of IgE 2F10 binding its 750 Å2 conformational epitope on Der p 2. This review provides an overview of this major milestone in allergy, the first atomic resolution structure of an authentic human IgE epitope. The molecular insights that IgE epitopes provide will allow for structure-based design approaches to the development of novel diagnostics, antibody therapeutics, and immunotherapies.
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Project description:BackgroundPatients with eosinophilic esophagitis have increased numbers of mucosal mast cells. Administration of the proton pump inhibitor omeprazole can reduce both esophageal mast cell and eosinophil numbers and attenuate type 2 inflammation in these subjects.ObjectiveGiven that maintenance of an acidic environment within granules is important for mast cell homeostasis, we sought to evaluate the effects of omeprazole on mast cell functions including development, IgE:FcεRI-mediated activation, and responses to food allergen.MethodsMast cell degranulation, cytokine secretion, and early signaling events in the FcεRI pathway, including protein kinase phosphorylation and Ca2+ flux, were measured after IgE crosslinking in murine bone marrow-derived mast cells and human cord blood-derived mast cells. The effects of omeprazole on these responses were investigated as was its impact on mast cell-dependent anaphylaxis and food allergy phenotypes in vivo.ResultsMurine and human mast cells treated with omeprazole exhibited diminished degranulation and release of cytokines and histamine in response to allergen. In murine mast cells, phosphorylation of protein kinases, ERK and SYK, was decreased. Differentiation of mast cells from bone marrow progenitors was also inhibited. IgE-mediated passive anaphylaxis was blunted in mice treated with omeprazole as was allergen-induced mast cell expansion and mast cell activation in the intestine in a model of food allergy.ConclusionsOur findings suggest that omeprazole targets pathways important for the differentiation and activation of murine mast cells and for the manifestations of food allergy and anaphylaxis.
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Project description:IgE Abs drive the symptoms of allergic disease upon cross-linking allergens on mast cells or basophils. If the IgE binding sites on the allergens could be identified, it may be useful for creating new forms of immunotherapy. However, direct knowledge of the human IgE (hIgE) epitopes is limited because of the very low frequency of IgE-producing B cells in blood. A new hybridoma technology using human B cells from house dust mite-allergic patients was used to identify four Der p 2-specific hIgE mAbs. Their relative binding sites were assessed and compared by immunoassays with three previously studied murine IgG mAbs. Immunoassays showed that the recognition of Der p 2 by the first three hIgE was inhibited by a single murine IgG, but the fourth hIgE recognized a different epitope from all the other mAbs. The functional ability of the hIgE that bind different epitopes to cross-link Der p 2 was demonstrated in a mouse model of passive systemic anaphylaxis. Nuclear magnetic resonance analyses of Der p 2 in complex with IgG and IgE Abs were used to identify specific residues in the epitopes. To our knowledge, the combination of immunoassays to distinguish overlapping epitopes and nuclear magnetic resonance analyses to identify specific residues involved in Ab binding provided the first epitope mapping of hIgE mAbs to an allergen. The technologies developed in this study will be useful in high-resolution mapping of human epitopes on other Ags and the design of improved therapeutics.
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Project description:Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in E. coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general
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Project description:BackgroundPeanut 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.MethodsDEAE-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.ResultsThe 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.ConclusionThis 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.
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Project description:BackgroundAffinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation.ObjectiveWe sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity.MethodsTen IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments.ResultsThe shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen.ConclusionOur results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens.
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Project description: Not available
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