Dirk Schiller

Kinetics, cross-reactivity, and specificity of Bet v 1-specific IgG4 antibodies induced by immunotherapy with birch pollen

IgE antibodies specific for the major birch pollen allergen frequently cross‐react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen (BP‐SIT) induces IgG4 antibodies that inhibit IgE binding to Bet v 1. However, information on cross‐reactivity of BP‐SIT‐induced Bet v 1‐specific IgG4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross‐reactivity, and IgE‐blocking activity of Bet v 1‐specific IgG4 antibodies emerging during conventional BP‐SIT and whether IgG4‐epitopes overlapped with IgE epitopes.

Prevention of Intestinal Allergy in Mice by rflaA:Ova Is Associated with Enforced Antigen Processing and TLR5-Dependent IL-10 Secretion by mDC

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.

Enlarging the Toolbox for Allergen Epitope Definition with an Allergen-Type Model Protein

Background 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. Method 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. Results 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. Conclusion 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.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

Birch pollen‐related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown.

IgE and allergen-specific immunotherapy-induced IgG4 recognize similar epitopes of Bet v 1, the major allergen of birch pollen

Allergen‐specific immunotherapy (AIT) with birch pollen generates Bet v 1‐specific immunoglobulin (Ig)G4 which blocks IgE‐mediated hypersensitivity mechanisms. Whether IgG4 specific for Bet v 1a competes with IgE for identical epitopes or whether novel epitope specificities of IgG4 antibodies are developed is under debate.

Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent

Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination.

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

Background 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. Objective 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. Methods 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. Results 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%. Conclusion 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.

Soybean allergy: IgE epitopes of glycinin (Gly m 6), an important soybean allergen

Methods 11 subjects with clinically confirmed soybean allergy and sIgE against rGly m 6 were enrolled in this study. Synthetic overlapping peptides (15-mers, 4 aa offset) representing the amino acid sequences of G1 and G5, two representative subunits of Gly m 6, were spotted onto CelluSpotTM slides and analyzed for IgE-binding, respectively. The sequences of IgE-binding peptides were allocated to the respective surface areas of the 3D-structures of G1 and G5. Allergenspecific IgE-binding to Gly m 6 peptides was verified by inhibition with rG1, rG5, and soy extract, respectively.

Analysis of the IgE epitope profile of 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.

Find the match! A tool for residue-specific analysis of epitopes in Bet v 1-like allergens

Background Birch pollen-allergic subjects often develop Bet v 1-specific IgE that cross-reacts with homologous food allergens. Bet v 1 and its homologs in pollen and food display exclusively conformational epitopes. We established a system to specifically analyze epitope cross-reactivity of Bet v 1-related allergens. The enzyme norcoclaurine synthase (NCS) from Thalictrum flavum is structurally homologous to Bet v 1 but does not bind IgE reacting with Bet v 1-like allergens. By substituting amino acids in a variant of NCS, the impact of individual residues of Bet v 1-like allergens in IgE binding can be studied.