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Peanut oleosins associated with severe peanut allergy—importance of lipophilic allergens for comprehensive allergy diagnostics  Christian Schwager, Dipl.

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Presentation on theme: "Peanut oleosins associated with severe peanut allergy—importance of lipophilic allergens for comprehensive allergy diagnostics  Christian Schwager, Dipl."— Presentation transcript:

1 Peanut oleosins associated with severe peanut allergy—importance of lipophilic allergens for comprehensive allergy diagnostics  Christian Schwager, Dipl Food Chem, Skadi Kull, PhD, Jochen Behrends, PhD, Niels Röckendorf, PhD, Frauke Schocker, PhD, Andreas Frey, PhD, Arne Homann, PhD, Wolf-Meinhard Becker, PhD, Uta Jappe, MD, MSc  Journal of Allergy and Clinical Immunology  Volume 140, Issue 5, Pages e8 (November 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Illustration of the modified recombinant Ara h 15 without hydrophobic domain (A) and the proposed oleosin structure (B) with respect to the 3 distinct domains (N-terminus, HCD, and C-terminus). The modified domain (MD) comprises of a His-tag linked to a Glycin-Alanin-Glycin-spacer on each side. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Western blot of mixed oleosin fractions Ara h 14/15 (A) and Ara h 10/11 (B) obtained from in-shell roasted peanuts. A1, Anti–Ara h 10/14 antibody; A2, anti–Ara h 15 antibody; C1, buffer control anti-human IgE antibody; C2, buffer control anti-rabbit IgG antibody; M, molecular mass marker; P1-P35, PA with severe allergic symptoms; S, protein staining; #, patients included in BAT. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 BAT of PA (n = 15, A), PS (n = 15, B), and NA (n = 15, C) using defined oleosin mixtures and the modified rAra h 15. The P value refers to comparisons of median percentages of CD63pos basophils at indicated doses of allergens between PA and PS: ****P < .0001, ***P < .001, and **P < .01. fMLP, Formyl-methionyl-leucyl-phenylalanine. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Location of the IgE epitope identified in the Ara h 15 sequence. Alignment of the C-terminal domains of selected oleosins registered as allergens of peanut, hazelnut, sesame, and 3 additional homologous oleosins of almond, soybean, and rapeseed. Dashes represent gaps introduced for best alignment. The boxed sequence indicates the determined IgE epitope of Ara h 15. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E1 Flow cytometric gating strategy for BAT displaying the basophil analysis of whole blood samples from PA (n = 15), PS (n = 15), and NA (n = 15). A representative flow cytometric gating strategy (P54) is shown using anti-IgE as stimulant. Cells were stained using labeled anti-human antibodies; FITC-lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56), PerCP/Cy5.5 HLA-DR, PE/Cy7 FcεRIα, BV421 CD123, BV510 CD45, PE CD203c and APC CD63. A, Forward scatter area (FSC-A) versus sideward scatter area (SSC-A) was used for gating leucocytes. Doublets were excluded by using FSC-area versus FSC-height (FSC-H). B, After gating CD45pos cells, lineage-negative cells were further assayed for CD123 and FcεRIα. CD123posFcεRIαpos cells were gated and further analyzed to exclude HLA-DRpos dendritic cells. C, Finally, HLA-DRnegCD45pos cells were used and assessed for their CD63 and CD203c expression. The percentage of CD63pos basophils (CD45pos, LINneg, CD123pos, FcεRIαpos, HLA-DRneg, CD203cpos) was used as result for the box blot diagram. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E2 Western blot of mixed oleosin fractions Ara h 14/15 (A) and Ara h 10/11 (B) from in-shell roasted peanuts probed with sera of PS, NA, and PA with mild allergic symptoms. A1, Anti–Ara h 10/14 antibody; A2, anti–Ara h 15 antibody; C1, buffer control anti-human IgE antibody; C2, buffer control anti-rabbit IgG antibody; M, molecular mass marker; P2, peanut-allergic individual with severe allergic symptoms (serving as positive control for IgE reactivity to oleosins); P36-P50, NA; P51-P65, PS; P66-P81, PA with mild allergic symptoms; P82, patient with Ara h 8-IgE-positive pollen-associated peanut allergy with severe allergic symptoms but without oleosin sensitization; S, protein staining. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E3 Western blot of mixed oleosin fractions Ara h 14/15 (A) and Ara h 10/11 (B) from raw peanuts probed with sera of PA with severe allergic symptoms. A1, Anti–Ara h 10/14 antibody; A2, anti–Ara h 15 antibody; C1, buffer control anti-human IgE antibody; C2, buffer control anti-rabbit IgG antibody; M, molecular mass marker; P1-P35, PA with severe allergic symptoms; S, protein staining; #, patients included in BAT. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E4 ROC curves (A) and characteristics of the BAT (B) performed with oleosins from raw and in-shell roasted peanuts as well as the modified rAra h 15 (n = 45). Values in parentheses represent the 95% CI. AUC, Area under the curve; ROC, receiver-operating characteristic. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E5 Prediction of the Ara h 15 protein sequence with regard to hydrophobicity (Kyte-Doolittle; A), antigenic index (Jameson-Wolf; B), and surface probability (Emini; C) of the 3 structural domains (N-terminal domain, HCD, and C-terminal domain). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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