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

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

Serological and clinical information for the 34 peanut allergic patients recruited in different European centres, within the Europrevall European project, used in EAST studies, basophil histamine release tests and/or PBMC proliferation tests.

<p>HEP: histamine equivalent prick test; nd: not determined. Where known, other allergies of the subjects are indicated.</p><p>The origin of the sera is as follows: 2A: Zurich, 52A: Amsterdam, 54A-82: Arnhem, 1682-2305: Vienna; 03-0043 till 12-0048 EuroPrevall Serum Bank.</p><p>Grading is based on Brockow and Ring and on Sampson <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Brockow1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023998#pone.0023998-Sampson1" target="_blank">[42]</a>. Grade 1 = dermal symptoms; grade 2 = gastro-intestinal problems like nausea and or cramping; grade 3 = any of the former grades plus vomiting/diarrhoea and respiratory tract problems like throat pruritis or tightness, grade 4 = any of the former grades and respiratory arrest plus cardiovascular problems like hypotension.</p

Effect of heating on secondary structure and oligomeric state of Ara h 2/6.

<p>Far-UV CD spectra of Ara h 2/6 heated alone (<b>A</b>, H-Ara h 2/6) or in the presence of 100 mM glucose (<b>B</b>, G-Ara h 2/6) before heating (N-Ara h 2/6, —) and for heated-cooled protein after heating to 110°C for 15 min (−−−) and 60 min (---). Inset graphs show change in molar residue ellipticity at 228 nm with heating time. Size exclusion chromatography profiles (<b>C</b>) are shown of the Ara h 2/6 before and after heating for 15 min at 110°C in the presence or absence of glucose. Retention volumes of molecular weight standards (size indicated in kDa) are shown by arrow heads. <b>D</b>. Far-UV CD spectra of Ara h 6 purified from roasted peanut.</p

Effect of thermal treatment on cytokine induction capacity.

<p>Fresh PBMC were left unstimulated (Med) or stimulated with N-, H- or G-Ara h 2/6 and production of IL-5 (<b>A</b>), IL-13 (<b>B</b>), IL-10 (<b>C</b>) and IFN-γ (<b>D</b>) was measured after 7 days of culture. Symbols represent peanut-allergic patients #65 (▪), #66 (♦), #70 (▴), #67 (•) and #73 (×). Horizontal bars represent the mean of the five PA responder subjects (solid) and the mean of 7 NA controls (dotted). Asterisks indicate statistically significant differences between the PA and the NA group and # indicate statistically significant differences between the medium and allergen-stimulated cultures for the PA group (*, #: <i>P</i><0.05). No differences between the medium and allergen-stimulated cultures were observed for the NA group.</p

Effect of thermal treatment on the IgE binding capacity of Ara h 2/6.

<p><b>A.</b> IgE capture inhibition curves obtained for sera #05-0209 with N-Ara h 2/6 (•), H-Ara h 2/6 (⧫) or G-Ara h 2/6 (▴). IgE binding capacities of native and heated processed Ara h 2/6 was assessed by competitive assays in the IgE capture format. Inhibition was performed into microtiter plates coated with anti-IgE, and previously incubated with allergic patient sera at convenient dilution. Competition was then conducted by adding increasing concentrations of competitors at the same time as biotinylated N-Ara h 2/6. Competition obtained with Ara h 2/6 purified from roasted peanut is shown as a clear circle. <b>B</b>. Analysis of IC50 (ng/ml) values obtained using an IgE capture inhibition assay with native and heat processed Ara h 2/6 as competitors using 30 individual sera from peanut-allergic patients. Increase in IC50 value corresponds to a decrease in IgE-binding capacity. R-Ara h 2/6: mix of Ara h 2 and Ara h 6 purified from roasted peanut. Bars indicate a significant difference between the 2 corresponding treatments (<i>P</i><0.05, non-parametric Wilcoxon signed rank test).</p

Effect of thermal treatment on the activation of effector cells induced by Ara h 2/6.

<p>(<b>A</b>) Human stripped basophils were passively sensitized with individual sera (#2305) and then incubated with increasing concentrations of N-Ara h 2/6 (•), H-Ara h 2/6 (⧫), and G-Ara h 2/6 (▴), or whole peanut extract from raw (★) or roasted (○) peanut. Histamine release was assayed in corresponding supernatants. Twenty-three sera out of the 35 available corresponding to all sera from Zurich, Amsterdam, Vienna and EPSB, and sera 54A to 58A from Arnhem were used, giving similar results (not shown). No histamine release was induced by the different allergens when using a serum from patients not sensitized to peanut (data not shown). (<b>B</b>) Humanized RBL-2H3 cells were passively sensitized with sera from peanut-allergic patients (#70) and stimulated with increasing concentrations of native (N; ―•―), heated (H; – –⧫– –) or glycated (G; - - -▴- - -) Ara h 2/6. Error bars represent the SD of triplicate values. No β-hexosaminidase release was induced by the different allergens when using sera from non-allergic patients (data not shown). The table presented within the figure represents the average protein concentrations (ng/ml, n = 6) to obtain 50% of the maximum allergen release of the native allergen (EC50). Means without a common letter differ (P<0.05).</p