Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen Masako Toda, PhD, Gerald Reese, PhD, Gabriele.

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Presentation transcript:

Protein unfolding strongly modulates the allergenicity and immunogenicity of Pru p 3, the major peach allergen Masako Toda, PhD, Gerald Reese, PhD, Gabriele Gadermaier, PhD, Veronique Schulten, MSc, Iris Lauer, PhD, Matthias Egger, PhD, Peter Briza, PhD, Stefanie Randow, Sonja Wolfheimer, Valencia Kigongo, BSc, Maria del Mar San Miguel Moncin, MD, Kay Fötisch, PhD, Barbara Bohle, PhD, Stefan Vieths, PhD, Stephan Scheurer, PhD Journal of Allergy and Clinical Immunology Volume 128, Issue 5, Pages 1022-1030.e7 (November 2011) DOI: 10.1016/j.jaci.2011.04.020 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Analysis of the purity and secondary structure of nPru p 3 and R/A Pru p 3. A, SDS-PAGE and Coomassie staining of nPru p 3 (lane 1) and after treatment by means of R/A (lane 2). B, Circular dichroism spectra of nPru p 3 and R/A Pru p 3. M, Molecular weight marker (in kilodaltons); θMRW, mean residue weight ellipticity. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Digestive stability analysis. Pepsin and trypsin digestion of nPru p 3 and R/A Pru p 3 were analyzed by using SDS-PAGE and Coomassie staining (A) and IgE immunoblotting (B) with pooled sera from patients with peach allergy (nos. 10 and 11 in Table E1). Pepsin digestion: Pru p 3 in H2O (lane 1) and digestion buffer for 2 hours (lane 2); reaction stopped after 5 minutes (lane 3), 15 minutes (lane 4), 30 minutes (lane 5), 1 hour (lane 6), and 2 hours (lane 7). Trypsin digestion: Pru p 3 in H2O (lane 1) and digestion buffer for 20 hours (lane 2); reaction stopped after 1 hour (lane 3), 2 hours (lane 4), 4 hours (lane 5), and 20 hours (lane 6). M, Molecular weight marker (in kilodaltons). Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IgE reactivity analysis. A, Serial dilutions of sera from patients with peach allergy (n = 13) sensitized to Pru p 3 were tested by means of ELISA for IgE binding to nPru p 3 and R/A Pru p 3. B, For IgE inhibition assays, diluted sera from patients with peach allergy (n = 3) were incubated with serial dilutions of nPru p 3 and R/A Pru p 3, respectively. Competition of IgE binding to nPru p 3 on the solid phase was quantified by means of ELISA. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Allergenic potency analysis. Allergenic potency of peach extract, nPru p 3, and R/A Pru p 3 were compared in vitro by means of histamine release assay with basophils from nonallergic donors passively sensitized with sera from 3 patients with peach allergy (nos. 2, 8, and 14 in Table E1). Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 T cell–activating capacity analysis. PBMCs (circles) and Pru p 3–specific T-cell lines (triangles) from 6 patients with peach allergy were stimulated with nPru p 3 (white) or R/A Pru p 3 (black). Proliferative responses are shown as stimulation indices. Black squares indicate the median values of the 6 subjects. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Antigenicity analysis. CBA/J mice were intraperitoneally immunized with 1 or 10 μg of nPru p 3 or R/A Pru p 3 six times. The levels of serum IgG1, IgG2a, and IgE binding to nPru p 3 (A) or R/A Pru p 3 (B) on the solid phase were determined by means of ELISA. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 T-cell immunogenicity analysis in mice. CBA/J mice were intraperitoneally immunized with 1 or 10 μg of nPru p 3 or R/A Pru p 3 four times. One week after the last immunization, splenic CD4+ T cells were isolated and cocultured with mitomycin-treated syngeneic splenocytes in the presence of nPru p 3 or R/A Pru p 3. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 Endolysosomal processing analysis. After 0.5 to 48 hours of incubation of nPru p 3 and R/A Pru p 3 with endolysosomal proteases, generated peptides were analyzed by means of mass spectrometry. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Antigenicity analysis. CBA/J mice were intraperitoneally immunized with 1 μg of nPru p 3 or R/A Pru p 3 six times. The levels of functional IgE antibodies against the allergens in sera were measured by using the RBL-2H3 mediator release assay. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 T-cell immunogenicity analysis in mice. CBA/J mice were intraperitoneally immunized with 1 or 10 μg of nPru p 3 or R/A Pru p 3 four times. One week after the last immunization, splenic CD4+ T cells were isolated and cocultured with mitomycin-treated syngeneic splenocytes in the presence of nPru p 3 or R/A Pru p 3. The levels of IFN-γ (A) and IL-4 (B) in the culture supernatant were determined by means of ELISA. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Endolysosomal processing analysis. After 0.5 to 48 hours of incubation of nPru p 3 and R/A Pru p 3 with endolysosomal proteases, the digestion reactions containing allergens were analyzed by means of SDS-PAGE and Coomassie staining. The degradation (as a percentage) is measured by using densitometric analysis. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 T-cell epitopes of Pru p 3 in CBA/J mice. CBA/J mice were subcutaneously immunized with 10 μg of nPru p 3 plus TiterMax adjuvant. One week after immunization, CD4+ T cells were isolated from lymph nodes and cocultured with mitomycin-treated syngeneic splenocytes in the presence of nPru p 3 (25 μg/mL) or peptides covering the Pru p 3 sequence (10 μg/mL). Proliferation of the cells was measured by means of uptake of tritiated methyl thymidine. Journal of Allergy and Clinical Immunology 2011 128, 1022-1030.e7DOI: (10.1016/j.jaci.2011.04.020) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions