Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy Raphaela.

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Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy Raphaela Freidl, MSc, Antonia Gstoettner, MSc, Ulrike Baranyi, PhD, Ines Swoboda, PhD, Frank Stolz, PhD, Margarete Focke-Tejkl, PhD, Thomas Wekerle, MD, Ronald van Ree, PhD, Rudolf Valenta, MD, Birgit Linhart, PhD Journal of Allergy and Clinical Immunology Volume 139, Issue 6, Pages 1897-1905.e1 (June 2017) DOI: 10.1016/j.jaci.2016.10.018 Copyright © 2016 The Authors Terms and Conditions

Fig 1 Sensitization and treatment protocol for a mouse model of fish allergy. A, Mice were sensitized to rCyp c 1 5 times by intragastric gavage as indicated, treated by the intraperitoneal (i.p.) application of the mCyp c 1–specific rabbit antiserum or control antiserum and challenged with rCyp c 1. B, Mice were sensitized by 5 intragastric (i.g.) gavages of carp extract, treated by mouse mCyp c 1–specific or control antiserum and were challenged with carp extract. Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig 2 Cyp c 1–specific immune responses in rCyp c 1-sensitized mice. A and B, Induction of Cyp c 1–specific IgG1 and IgG2a, IgG3, IgA, and IgM as measured by ELISA. C, Position of synthetic Cyp c 1–derived peptides 1 to 7 in the Cyp c 1 molecule. aa, Amino acids. D and E, Epitope mapping of mouse IgE and allergic patients' IgE with Cyp c 1 peptides. Shown are mean antibody levels (y-axes: OD values ± SDs). F, Proliferation of splenocytes from Cyp c 1–sensitized and naive mice in response to Cyp c 1 peptides, rCyp c 1, and medium alone. Results are displayed as mean counts per minute ± SDs for the 2 mouse groups. Asterisks (*) indicate significant differences (P < .05) between mouse groups. Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig 3 Characterization of rabbit antiserum induced by immunization with mCyp c 1. A, Titration of rabbit antiserum induced by immunization with mCyp c 1 for IgM, IgA, and IgG reactivity to rCyp c 1. Mean OD values of triplicates ± SD corresponding to bound IgM, IgA, and IgG (y-axis) are displayed for different serum dilutions (x-axis). B, Inhibition of IgE binding to rCyp c 1 by mCyp c 1–induced IgG in patients who are allergic to fish. For these patients, IgE binding (patients 1-8) (y-axis: mean OD values) to rCyp c 1, which was preincubated with various dilutions of serum from a rabbit obtained before (Pre) or after immunization with mCyp c 1 (x-axis), is shown. C, Epitope mapping of mCyp c 1–induced rabbit IgG antibodies. IgG reactivity (y-axis: mean OD values ± SDs) of mCyp c 1–specific IgG to rCyp c 1 and Cyp c 1 peptides 1 to 7 (x-axis). D, Cross-reactivity of mCyp c 1–induced rabbit IgG antibodies to nitrocellulose-blotted fish allergen extracts. Bound mCyp c 1–specific rabbit IgG antibodies were detected with 125I-labeled anti-rabbit IgG antibodies and visualized by autoradiography. Molecular weights (kDa) are indicated on the left margin. Phl p 6, Recombinant Phl p 6. Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig 4 mCyp c 1–specific rabbit antiserum inhibits rCyp c 1–induced basophil degranulation in vitro and Cyp c 1–induced allergic symptoms in vivo. A, Rat basophil leukemia cells were loaded with pooled sera from rCyp c 1–sensitized mice in the presence or absence of mCyp c 1–specific rabbit antiserum or control antiserum (x-axis). The percentage of total ß-hexosaminidase release is displayed on the y-axis (triplicates ± SD). B, Significant (**P < .01) suppression of allergic symptoms in Cyp c 1–sensitized mice by mCyp c 1–specific rabbit antiserum after challenge with Cyp c 1. Allergic symptoms (y-axis: mean symptom scores ± SD) in Cyp c 1–sensitized mice having received mCyp c 1–specific or control antiserum (x-axis). Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig 5 Epitope mapping of mouse IgG antibodies induced by gavage with carp extract and vaccination with mCyp c 1. IgG1 (A and C) and IgG2a (B and D) antibody levels (y-axes: OD values) specific for Cyp c 1–derived peptides 1 to 7 and rCyp c 1 (x-axes) were measured: A and B, after immunization with mCyp c 1 (α-mCyp c 1 serum, white bars; preimmune serum, gray bars); C and D, after sensitization (day 43) with carp extract (gavage carp extract, black bars; preimmune serum, gray bars). Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig 6 Suppression of Cyp c 1–induced basophil degranulation and allergic symptoms with a mCyp c 1–specific mouse antiserum. A, Rat basophil leukemia cells loaded with pooled sera from rCyp c 1–sensitized mice (day 43) were challenged with rCyp c 1 in the presence or absence of mCyp c 1–specific mouse antiserum or control hP62 (a recombinant grass pollen hypoallergen)–specific antiserum (x-axis). The percentage of total ß-hexosaminidase release is displayed on the y-axis (triplicates ± SD). B, Allergic symptoms (y-axis: mean symptom scores ± SD) of carp extract-sensitized mice treated with either mouse α-mCyp c 1 serum or mouse α-hP62 serum or of untreated mice (x-axis) after challenge with carp extract (*P < .05; **P < .01). Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions

Fig E1 Reduced allergen-specific IgE reactivity of Cyp c 1–sensitized mice that had been treated with mCyp c 1–specific rabbit antiserum. A and B, Presence of Cyp c 1–specific rabbit IgG in mice before and after injection of mCyp c 1–specific antiserum or Phl p 1–specific antiserum. C and D, Cyp c 1–specific IgE reactivity of mice before and after injection of mCyp c 1–specific antiserum or Phl p 1–specific antiserum. Antibody reactivities are shown for each mouse (y-axes: OD values) before and after antiserum injection (y-axes). Journal of Allergy and Clinical Immunology 2017 139, 1897-1905.e1DOI: (10.1016/j.jaci.2016.10.018) Copyright © 2016 The Authors Terms and Conditions