Analysis of the IgE epitope profile of the soybean allergen Gly m 4

Individuals with birch pollinosis may show allergic reactions after consumption of soybean-containing food. This is caused by cross-reaction of IgE directed against the major birch pollen allergen Bet v 1 with the structurally homologous allergen Gly m 4 from soybean. Hypersensitivity reactions in birch-soy allergy range from mild reactions to severe systemic reactions. Sera of birch pollen-allergic subjects may contain IgE to Gly m 4, even though no allergy to soy is present. Thus Gly m 4-specific IgE per se is not a suitable biomarker for birch-related soy allergy. To develop novel approaches for improved diagnosis and therapy of birch-soy allergy, knowledge on epitopes and IgE epitope profile of Gly m 4 for is needed. To date, data on epitopes of Bet v 1 and its homologous allergen Gly m 4 is very limited. In this study, birch pollen-allergic patients with (27 subjects) and without (20 subjects) clinically confirmed allergy to soybean were included and analyzed regarding Gly m 4-specific serum IgE/IgG levels and epitope profiles of Gly m 4 for IgE. Specific IgE levels against Bet v 1, Gly m 4 and further soy allergens Gly m 5 and Gly m 6 were determined by ImmunoCAP™ and IgE binding to rGly m 4 and soy extract was tested in western blot. To analyze putative IgE epitopes of Gly m 4, non-allergenic Norcoclaurine synthase (NCS) from meadow rue was used as a model protein. NCS is structurally homologous to Gly m 4 but exhibits none to very little binding of Gly m 4-specific IgE antibodies enabling grafting of Gly m 4 epitopes onto the model protein. Potential candidate residues of Gly m 4 were selected by bioinformatic analysis of antibody-binding phage-displayed peptides and mapping of segments of Gly m 4 primary structure onto the molecular surface of the allergen. As a result a library of recombinant NCS variants with potential IgE binding was generated. In addition, five multiple substitutional variants of rGly m 4 with a total number of 18 amino acid substitutions crucial for IgE binding were generated and analyzed for antibody binding with patients’ sera. Furthermore a misfolded variant of each rBet v 1a and rGly m 4 was generated and defined molar ratios of folded/misfolded variants were compared in different immunological and physicochemical assays. Gly m 4-specific median IgE and IgG levels of allergic (IgE: 9.3 kUA/L, IgG: 8.1 mgA/L) and non-allergic (IgE: 4.5 kUA/L, IgG: 8.3 mgA/L) subjects were comparable. The specific IgE levels did not correlate to the (severity of) clinical phenotypes. 51 candidate residues of Gly m 4 were selected for IgE epitope analysis and 46 potential functional IgE epitopes, single residues within a structural IgE epitope which dominate the energetics of allergen-IgE binding, were identified with IgE binding to ΔNCSN42/P49 variants. The putative functional IgE epitope pattern was individual for each patient and not distinguishable between allergic and non-allergic subjects. Using five rGly m 4 variants parts of the results of the NCS-based analyses could be confirmed with 18 potential functional IgE epitopes identified. 46 potential functional IgE epitopes clustered into six distinct putative IgE-binding areas on Gly m 4 and eleven NCS variants (ΔNCSN42/P49_1-11) presenting parts of these epitope areas were purified, showing a Gly m 4-type secondary structure according to CD measurements. Densitometric analysis for binding of IgE antibodies was performed via dot blot with sera of the study population but no characteristic IgE epitope pattern could be found. In contrast ΔNCSN42/P49_9 was identified as most suitable marker to distinguish soy allergic from tolerant patients in birch-related soybean allergy with a sensitivity and specificity of 68% and 93%, respectively. Using a pool of ΔNCSN42/P49 variants a depletion of Gly m 4-specific IgE binding to about 80% and 70% in pooled sera of patients with and without soybean allergy, respectively, was possible. Therefore identified putative functional IgE epitopes are specific for interaction with IgE antibodies in study population. With ΔNCSN42/P49_9 a differentiation between sensitization to Gly m 4 and clinically confirmed soybean allergy might be possible. The usage of a Gly m 4-specific epitope library might be a more promising tool for evaluating birch-related soybean allergy compared to ImmunoCAP™ and screening of substitutional Gly m 4 variants alone. However no correlations between patient’s IgE epitope profile and specific symptoms to soy or severity of allergic reactions could be found using NCS-based epitope library. Rather patient-specific epitope pattern might be relevant for birch-related soybean allergy. Therefore a thorough diagnosis by the combination of component-resolved diagnosis and profound epitope analysis might be mandatory in the future. Using two misfolded variants of rBet v 1a and rGly m 4 the impact of unstructured allergens in rBet v 1a/rGly m 4 preparations was addressed with physico- and immunological assays. Both rBet v 1aS112P/R145P and rGly m 4S111P/L150P showed a highly disordered protein conformation and reduced IgE binding frequencies with analyzed patients’ sera. With CD spectroscopy, immunoblot, ELISA and RBL cell release assay defined combinations of native and unstructured allergen were assessed concerning secondary structure and IgE binding. Correlation of rBet v 1a content with secondary structure and IgE binding was suitable only at high rBet v 1aS112P/R145P levels in mixtures. CD spectroscopy and ELISA performed more precise compared to immunoblot and rat basophil cell mediator release assay where larger deviations between native and unstructured allergen were necessary. In addition, quantification of IgE-binding allergen was difficult for concentrations of rBet v 1a ≤10% in all assays. Overall, CD, ELISA and RBL cell release assay underestimated while immunoblot overestimated the actual level of rBet v 1a. Results of both misfolded variants rBet v 1aS112P/R145P and rGly m 4S111P/L150P might be used within screening of hypoallergenic molecules with potential use in treatment of allergies or in quality assessment of recombinant allergen preparations

Indirect Network Effects and Trade Liberalization

Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins

Analysis of the IgE epitope profile of the soybean allergen Gly m 4

Individuals with birch pollinosis may show allergic reactions after consumption of soybean-containing food. This is caused by cross-reaction of IgE directed against the major birch pollen allergen Bet v 1 with the structurally homologous allergen Gly m 4 from soybean. Hypersensitivity reactions in birch-soy allergy range from mild reactions to severe systemic reactions. Sera of birch pollen-allergic subjects may contain IgE to Gly m 4, even though no allergy to soy is present. Thus Gly m 4-specific IgE per se is not a suitable biomarker for birch-related soy allergy. To develop novel approaches for improved diagnosis and therapy of birch-soy allergy, knowledge on epitopes and IgE epitope profile of Gly m 4 for is needed. To date, data on epitopes of Bet v 1 and its homologous allergen Gly m 4 is very limited. In this study, birch pollen-allergic patients with (27 subjects) and without (20 subjects) clinically confirmed allergy to soybean were included and analyzed regarding Gly m 4-specific serum IgE/IgG levels and epitope profiles of Gly m 4 for IgE. Specific IgE levels against Bet v 1, Gly m 4 and further soy allergens Gly m 5 and Gly m 6 were determined by ImmunoCAP™ and IgE binding to rGly m 4 and soy extract was tested in western blot. To analyze putative IgE epitopes of Gly m 4, non-allergenic Norcoclaurine synthase (NCS) from meadow rue was used as a model protein. NCS is structurally homologous to Gly m 4 but exhibits none to very little binding of Gly m 4-specific IgE antibodies enabling grafting of Gly m 4 epitopes onto the model protein. Potential candidate residues of Gly m 4 were selected by bioinformatic analysis of antibody-binding phage-displayed peptides and mapping of segments of Gly m 4 primary structure onto the molecular surface of the allergen. As a result a library of recombinant NCS variants with potential IgE binding was generated. In addition, five multiple substitutional variants of rGly m 4 with a total number of 18 amino acid substitutions crucial for IgE binding were generated and analyzed for antibody binding with patients’ sera. Furthermore a misfolded variant of each rBet v 1a and rGly m 4 was generated and defined molar ratios of folded/misfolded variants were compared in different immunological and physicochemical assays. Gly m 4-specific median IgE and IgG levels of allergic (IgE: 9.3 kUA/L, IgG: 8.1 mgA/L) and non-allergic (IgE: 4.5 kUA/L, IgG: 8.3 mgA/L) subjects were comparable. The specific IgE levels did not correlate to the (severity of) clinical phenotypes. 51 candidate residues of Gly m 4 were selected for IgE epitope analysis and 46 potential functional IgE epitopes, single residues within a structural IgE epitope which dominate the energetics of allergen-IgE binding, were identified with IgE binding to ΔNCSN42/P49 variants. The putative functional IgE epitope pattern was individual for each patient and not distinguishable between allergic and non-allergic subjects. Using five rGly m 4 variants parts of the results of the NCS-based analyses could be confirmed with 18 potential functional IgE epitopes identified. 46 potential functional IgE epitopes clustered into six distinct putative IgE-binding areas on Gly m 4 and eleven NCS variants (ΔNCSN42/P49_1-11) presenting parts of these epitope areas were purified, showing a Gly m 4-type secondary structure according to CD measurements. Densitometric analysis for binding of IgE antibodies was performed via dot blot with sera of the study population but no characteristic IgE epitope pattern could be found. In contrast ΔNCSN42/P49_9 was identified as most suitable marker to distinguish soy allergic from tolerant patients in birch-related soybean allergy with a sensitivity and specificity of 68% and 93%, respectively. Using a pool of ΔNCSN42/P49 variants a depletion of Gly m 4-specific IgE binding to about 80% and 70% in pooled sera of patients with and without soybean allergy, respectively, was possible. Therefore identified putative functional IgE epitopes are specific for interaction with IgE antibodies in study population. With ΔNCSN42/P49_9 a differentiation between sensitization to Gly m 4 and clinically confirmed soybean allergy might be possible. The usage of a Gly m 4-specific epitope library might be a more promising tool for evaluating birch-related soybean allergy compared to ImmunoCAP™ and screening of substitutional Gly m 4 variants alone. However no correlations between patient’s IgE epitope profile and specific symptoms to soy or severity of allergic reactions could be found using NCS-based epitope library. Rather patient-specific epitope pattern might be relevant for birch-related soybean allergy. Therefore a thorough diagnosis by the combination of component-resolved diagnosis and profound epitope analysis might be mandatory in the future. Using two misfolded variants of rBet v 1a and rGly m 4 the impact of unstructured allergens in rBet v 1a/rGly m 4 preparations was addressed with physico- and immunological assays. Both rBet v 1aS112P/R145P and rGly m 4S111P/L150P showed a highly disordered protein conformation and reduced IgE binding frequencies with analyzed patients’ sera. With CD spectroscopy, immunoblot, ELISA and RBL cell release assay defined combinations of native and unstructured allergen were assessed concerning secondary structure and IgE binding. Correlation of rBet v 1a content with secondary structure and IgE binding was suitable only at high rBet v 1aS112P/R145P levels in mixtures. CD spectroscopy and ELISA performed more precise compared to immunoblot and rat basophil cell mediator release assay where larger deviations between native and unstructured allergen were necessary. In addition, quantification of IgE-binding allergen was difficult for concentrations of rBet v 1a ≤10% in all assays. Overall, CD, ELISA and RBL cell release assay underestimated while immunoblot overestimated the actual level of rBet v 1a. Results of both misfolded variants rBet v 1aS112P/R145P and rGly m 4S111P/L150P might be used within screening of hypoallergenic molecules with potential use in treatment of allergies or in quality assessment of recombinant allergen preparations

Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.

Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a.To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations.rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared.1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%.Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected

Amino acids critical forIgE and IgG cross-reactivity in a subgroup of Bet v 1-related proteins.

<p>(A) Binding of serial dilutions of pool serum IgE to surface-coated equimolar amounts of Bet v 1 and Bet v 1_4x (Bet v 1_{N43I/E45S/N47D/K55A}) in ELISA. (B) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of either Bet v 1 or Bet v 1_4x in ELISA. (C) Binding of serial dilutions of monoclonal BV16 to surface-coated equimolar amounts of Bet v 1 and Bet v 1_4x in ELISA. (D) Mediator release induced by recombinant NCS and Bet v 1 variants. Humanized RBL cells were sensitized with a pool of human birch-specific sera. Cross-linking of membrane-bound human IgE by IgE-protein interaction and subsequent release of β-hexosaminidase was determined with serial dilutions of the proteins.</p

Δ29NCS_5x presents a cross-reactive Ig epitope.

<p>(A) Inhibition of IgE binding to Δ29NCS_5x. Binding of serum IgE to Δ29NCS (lane 3) and to Δ29NCS_5x (lane 4) in the presence of increasing amounts of inhibitors Δ29NCS_5x (lanes 5–8) or Bet v 1 (lanes 9–12). No serum (lane 1) and negative serum pool (lane 2) served as control. (B) Dose-dependent inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of increasing concentrations of PR-10 allergens in ELISA. (C) Inhibition of IgE binding to surface-coated Δ29NCS_5x in the presence of both Bet v 1 and serial dilutions of Bet v 1-binding monoclonal mouse IgG antibody BV16 (–: no BV16; 10⁻⁵ to 10⁻³: dilutions of BV16) in ELISA. (D) Binding of mouse monoclonal IgG antibody BV16 to surface-coated Bet v 1-related proteins. 250 ng of protein (50 ng of Api g 1) was coated and incubated with dilution series of the monoclonal BV16 in ELISA.</p

Δ29NCS, Δ29NCS_4x, and Δ29NCS_5x structures are virtually identical to the Bet v 1 structure.

<p>(A) Structural sequence alignment. β-strands (underlined), helices (italic), and amino acid identities (green) to Bet v 1 (pdb: 1BV1) are 23.8% (Δ29NCS), 26.8% (Δ29NCS_4x), and 27.5% (Δ29NCS_5x), respectively. (B) Circular dichroism of Δ29NCS variants. (C) Overlay of three ¹H-¹⁵N-HSQC spectra of Δ29NCS variants. Black arrows, signals numbered and named according to Δ29NCS. Black: Δ29NCS, blue: Δ29NCS_4x, red: Δ29NCS_5x. The arrows highlight signals of selected amino acids that are expected to differ between the three Δ29NCS variants. Labeling is according to Δ29NCS scheme. (D) Overlay of secondary structure topologies of Δ29NCS (grey), Δ29NCS_4x (blue), Δ29NCS_5x (red), and Bet v 1 (yellow).</p

Structural alignment of the Bet v 1 epitope forBV16 and amino acids critical for Ig cross-reactivity in PR-10 allergens.

<p>Upper panel: model structures of Δ29NCS_5x and PR-10 allergens showing IgE cross-reactivity with Δ29NCS_5x. The amino acids comprising the BV16-binding epitope and lysine 55 of Bet v 1 and the corresponding residues of Δ29NCS_5x, Gly m 4, and Cor a 1 are listed. Residues of the epitope and of position 55 of Bet v 1 that are critical for Ig cross-reaction are highlighted in green. Lower panel: model structures of Δ29NCS and allergens that do not show IgE cross-reactivity with Δ29NCS_5x. The amino acids corresponding to the epitope in the cross-reactive proteins are listed and highlighted in green.</p

Grafting of an epitope onto Δ29NCS.

<p>Left panel: 13 of the 16 amino acids comprising the Bet v 1 epitope (Bet v 1_{E42/N43/I44/E45/G46/N47/G48/G49/P50/G51/T52/R70/D72/H76/I86/K97}) of mouse monoclonal IgG antibody BV16 (yellow). Six amino acids of the epitope are labeled. Lys55 of Bet v 1 located adjacent to the epitope is highlighted (green). Middle panel: the corresponding 13 residues of the Bet v 1 epitope for BV16 and a lysine corresponding to K55 of Bet v 1 have been grafted onto Δ29NCS to generate Δ29NCS_4x (Δ29NCS_{N57/I58E/D60N/V63P}) (not shown) and Δ29NCS_5x (Δ29NCS_{N57/I58E/D60N/V63P/D68K}). Right panel: corresponding surface area of Δ29NCS. Protein models were based on the structures of NCS (pdb 2VNE) and Bet v 1a (pdb 1BV1), and modelled with a confidence of 100% and a sequence coverage of at least 85%.</p