Carrier-bound, nonallergenic Ole e 1 peptides for vaccination against olive pollen allergy Teresa E. Twaroch, MSc, Margit Focke, PhD, Vera Civaj, Milena.

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Carrier-bound, nonallergenic Ole e 1 peptides for vaccination against olive pollen allergy Teresa E. Twaroch, MSc, Margit Focke, PhD, Vera Civaj, Milena Weber, MSc, Nadja Balic, Adriano Mari, MD, Rosetta Ferrara, MD, Santiago Quirce, MD, Susanne Spitzauer, MD, Ines Swoboda, PhD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 128, Issue 1, Pages 178-184.e7 (July 2011) DOI: 10.1016/j.jaci.2011.03.011 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Amino acid sequence alignment of Ole e 1, Fra e 1 (ash), Lig v 1 (privet), and Syr v 1 (lilac). The N-glycosylation motif of Ole e 1 (underlined) and identical amino acids (dashes) are shown. Synthetic Ole e 1–derived peptides are shown in boxes. Bars indicate major T-cell epitopes of Ole e 1. A surface accessibility plot of Ole e 1 is shown at the bottom. Regions (x-axis) with high surface accessibility show scores of greater than 5.5 (y-axis). Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IgE reactivity of nOle e 1 and Ole e 1–derived peptides. A, Dotted nOle e 1 and peptides probed with sera from allergic patients, a nonatopic subject (N), or buffer (B). B, Box plot of OD values corresponding to bound IgE from 63 allergic patients. Patients are identified as in Tables E1 to E3. Horizontal bars, Median values; asterisks, extreme values. Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Lymphoproliferative responses to nOle e 1 and Ole e 1-derived peptides. Stimulation indices (SI; y-axis) are shown as box plots (horizontal bars, median values; circles and asterisks, outliers and extreme values, respectively) for PBMCs from 6 allergic patients for medium, peptides, and nOle e 1 (x-axis). Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Anti-peptide and anti–rOle e 1 antibodies cross-react with nFra e 1. Nitrocellulose-blotted ash pollen extract was incubated with the anti-peptide antisera (S1-S4, anti-peptide 1 to anti-peptide 4), the anti–rOle e 1 antiserum (SrOl), and, for control purposes, a mixture of the anti-peptide preimmune sera (P1-4) or the anti-rOle e 1 pre-immune serum (PrOl). Molecular weights are indicated in the left margin. Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Rabbit anti-peptide and anti–rOle e 1 antibodies inhibit Oleaceae-sensitized patients' IgE binding to Ole e 1. Percentage inhibitions of IgE binding to Ole e 1 by Ole e 1– or peptide-specific antibodies for sera of 14 Oleaceae-sensitized patients are shown as box plots. Horizontal bars, Median values. Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Induction of Ole e 1–specific IgG antibodies in rabbits. Rabbits were immunized with rOle e 1 or with the KLH-coupled Ole e 1–derived peptides. IgG reactivities of the antisera were measured by means of ELISA. Four different serum dilutions (x-axis: 1:1,000, 1:5,000, 1:10,000, and 1:50,000) are displayed as mean OD values (y-axis). Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Rabbit anti-peptide and anti–rOle e 1 antibodies inhibit Oleaceae-sensitized patients' IgE binding to Ole e 1. nOle e 1 preincubated with increasing dilutions (x-axis) of the antisera or with the corresponding preimmune sera was incubated with serum IgE from an Oleaceae-sensitized patient. IgE reactivity is displayed as OD values. Journal of Allergy and Clinical Immunology 2011 128, 178-184.e7DOI: (10.1016/j.jaci.2011.03.011) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions