A hypoallergenic variant of Der p 1 as a candidate for mite allergy vaccines David Walgraffe, PhD, Christel Mattéotti, PhD, Mohamed el Bakkoury, PhD,

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A hypoallergenic variant of Der p 1 as a candidate for mite allergy vaccines David Walgraffe, PhD, Christel Mattéotti, PhD, Mohamed el Bakkoury, PhD, Lida Garcia, MD, Céline Marchand, MD, Dominique Bullens, PhD, Michel Vandenbranden, PhD, Alain Jacquet, PhD Journal of Allergy and Clinical Immunology Volume 123, Issue 5, Pages 1150-1156 (May 2009) DOI: 10.1016/j.jaci.2008.11.038 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Characterization of ProDer p 1 coli. Coomassie-stained SDS-PAGE (A) and Western blotting (B) of natural Der p 1 (lane 1), recombinant ProDer p 1 CHO (lane 2), and ProDer p 1 coli (lane 3) incubated with anti–Der p 1 polyclonal antibodies. C, Gel filtration of ProDer p 1 coli or CHO. The Superdex 200-HR column was calibrated by using blue dextran (Ve, 2.000 kd), bovine IgG (I, 150 kd), BSA (B, 67 kd), ovalbumin (O, 43 kd), and cytochrome C (C, 14 kd). D, Infrared absorbance spectra of ProDer p 1 coli or CHO. Spectra are normalized at full scale on the amide I band. AU, Absorbance unit. Journal of Allergy and Clinical Immunology 2009 123, 1150-1156DOI: (10.1016/j.jaci.2008.11.038) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 ProDer p 1 coli displays very weak IgE reactivity but retains the Der p 1 T-cell reactivity. A, IgE reactivity of natural Der p 1 and recombinant ProDer p 1 coli or CHO evaluated in direct ELISA and using ImmunoCAP positive sera to D pteronyssinus (N = 23). ∗P < .001 compared with Der p 1 or ProDer p 1 CHO. B, ELISA inhibition assays. The binding of human IgE to natural Der p 1 was inhibited by using natural Der p 1 (•), recombinant ProDer p 1 coli (▴), or CHO (▪).The % inhibition is shown as 100 – (percentage of IgE binding in the presence of inhibitor / percentage of IgE binding in the absence of inhibitor). C, Mediator release of natural Der p 1 or recombinant ProDer p 1 coli or CHO cells with humanized RBL SX-38 cell line. Results were expressed as percentage of the total release of β-hexosaminidase minus the spontaneous release ± SD. ∗P < .001 compared with Der p 1. ∗∗P < .001 compared with Der p 1 or ProDer p 1 CHO. D, Induction of T-cell proliferation. PBMCs from individual patients (n = 3) were stimulated with Der p 1, ProDer p 1 CHO, and ProDer p 1 coli (concentration as indicated). Results are expressed as the means ± SEMs of quadruplicate stimulation index. Mean absolute thymidine counts are indicated between brackets. Journal of Allergy and Clinical Immunology 2009 123, 1150-1156DOI: (10.1016/j.jaci.2008.11.038) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Prevention of experimental house dust mite allergy by vaccinations with recombinant ProDer p1 coli. Mice (n = 7) were vaccinated with saline (PBS), recombinant ProDer p 1 coli or CHO, sensitized to natural Der p 1, and challenged (A). Antibody titers (IgG1, B; IgG2a, C; IgE, D), splenocytes cytokine production (E), airway eosinophilia (F), and airway hyperresponsiveness (G) were plotted as means ± SDs. One representative experiment out 3 is shown. ∗P < .05, ∗∗P < .001 compared with saline-vaccinated and ProDer p 1 CHO–vaccinated mice (C-E, G), with saline-vaccinated mice (F), and with ProDer p 1 CHO–vaccinated mice (B). nDer p 1, Natural Der p 1. Journal of Allergy and Clinical Immunology 2009 123, 1150-1156DOI: (10.1016/j.jaci.2008.11.038) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions