Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly Raffaela Campana, PhD, Susanne Vrtala,

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Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly Raffaela Campana, PhD, Susanne Vrtala, PhD, Bernhard Maderegger, PhD, Peter Jertschin, PhD, Gottfried Stegfellner, PhD, Ines Swoboda, PhD, Margarete Focke-Tejkl, PhD, Katharina Blatt, Anna Gieras, MSc, Domen Zafred, BSc, Angela Neubauer, PhD, Peter Valent, MD, Walter Keller, PhD, Susanne Spitzauer, MD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 126, Issue 5, Pages 1024-1031.e8 (November 2010) DOI: 10.1016/j.jaci.2010.05.023 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Hypoallergenic rBet v 1 derivatives. A, Construction scheme of the rBet v 1 derivatives. The amino acids at the borders of the protein segments and the binding sites of 2 mAbs, mAb#2 and mAb#12, are indicated. B, SDS-PAGE of purified rBet v 1 and rBet v 1 derivatives. M, Molecular mass markers (kd). Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IgG reactivity of rBet v 1 and rBet v 1 derivatives with Bet v 1–specific antibodies. Nitrocellulose-blotted rBet v 1 and rBet v 1 derivatives were probed with rabbit anti–Bet v 1 antibodies (rαBet v 1) (A), mAb Bip 1 (B), mAb#2 (C), or mAb#12 (D). Molecular weights are displayed in the right margins in kilodaltons. M, Marker. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IgE reactivity of rBet v 1 and rBet v 1derivatives. Nitrocellulose-dotted rBet v 1, rBet v 1-rs1, rBet v 1-rs2, rBet v 1-mosaic, HSA, and BSA were exposed to sera from patients with birch pollen allergy (1-19), individuals without allergy (20-21), buffer (0), or anti–rBet v 1 antibodies (22). Bound IgE and rabbit IgG antibodies were detected and visualized by autoradiography. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Allergenic activity of rBet v 1 and rBet v 1 derivatives as determined by CD203c upregulation. Blood samples from birch-allergic patients (A-F) were exposed to increasing concentrations (0.005 to 50 pmol/L) of rBet v 1, an equimolar mixture of rBet v 1 fragments (F1+F2) or rBet v 1-rs1 (x-axes; A), and in additional patients (G-J) with rBet v 1, rBet v 1-rs1, rBet v 1-rs2, and rBet v 1-mosaic (x-axes; B). Anti-IgE served as a positive control. The stimulation indices (SI; y-axes) reflect the upregulation of CD203c expression compared with buffer (0). Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Gel filtration profiles of rBet v 1 (A), Bet v 1-rs2 (B), Bet v 1-mosaic (C), and Bet v 1-rs1 (D). The x-axes show the elution volumes (mL), and the y-axes the adsorbance of the proteins at 280 nm. Arrows indicate the approximate molecular masses (kd) as determined with the standards. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Allergenic activity of rBet v 1 and rBet v 1 derivatives as determined by CD203c upregulation. Blood samples from 3 patients with birch allergy (K-M) were exposed to increasing concentrations (0.005-50 pmol/L) of rBet v 1, rBet v 1-rs1, rBet v 1-rs2, and rBet v 1-mosaic (x-axes). Anti-IgE served as a positive control. The mean stimulation indices (SIs; y-axes) reflect the upregulation of CD203c expression compared with buffer (0). SDs are indicated. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Reactivity of IgG antibodies with rBet v 1 and rBet v 1 derivatives Reactivity of IgG antibodies with rBet v 1 and rBet v 1 derivatives. Different dilutions (x-axes) of rabbit anti–rBet v 1 (anti–Bet v 1: ●), anti-rBet v 1 derivative antibodies (anti–Bet v 1-rs1: ■; anti–Bet v 1-rs2: ▴; anti–Bet v 1-mosaic: x) or antibodies from a normal rabbit (—) were reacted with rBet v 1 (A), rBet v 1-rs1 (B), rBet v 1-rs2 (C), or rBet v 1-mosaic (D). OD values (y-axes) correspond to the amounts of bound antibodies. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Reactivity of rabbit antibodies raised with aluminum hydroxide–adsorbed proteins by ELISA. Different dilutions (x-axes) of rabbit anti–rBet v 1 (anti–Bet v 1: ●), anti–rBet v 1—derivative antibodies (anti–Bet v 1-rs1: ■; anti–Bet v 1-rs2: ▴ anti–Bet v 1-mosaic: x) or antibodies from a normal rabbit (—) reacted with rBet v 1. OD values (y-axes) correspond to the amounts of bound antibodies. Journal of Allergy and Clinical Immunology 2010 126, 1024-1031.e8DOI: (10.1016/j.jaci.2010.05.023) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions