Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy Margarete Focke-Tejkl, PhD, Milena.

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Presentation transcript:

Development and characterization of a recombinant, hypoallergenic, peptide-based vaccine for grass pollen allergy Margarete Focke-Tejkl, PhD, Milena Weber, MSc, Katarzyna Niespodziana, PhD, Angela Neubauer, PhD, Hans Huber, PhD, Rainer Henning, PhD, Gottfried Stegfellner, MSc, Bernhard Maderegger, PhD, Martina Hauer, MSc, Frank Stolz, PhD, Verena Niederberger, MD, Katharina Marth, MD, Julia Eckl-Dorna, MD, PhD, Richard Weiss, PhD, Josef Thalhamer, PhD, Katharina Blatt, MSc, Peter Valent, MD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 135, Issue 5, Pages 1207-1217.e11 (May 2015) DOI: 10.1016/j.jaci.2014.09.012 Copyright © 2014 The Authors Terms and Conditions

Fig 1 Scheme for the preclinical development of a hypoallergenic grass pollen allergy vaccine based on carrier-bound allergen peptides. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 2 Overview of recombinant fusion proteins consisting of PreS-fused allergen peptides made for Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Names of the fusion proteins are shown at the right margin. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 3 Comparison of the IgE reactivity of recombinant grass pollen allergens, fusion proteins, and PreS. IgE reactivity of sera from patients with grass pollen allergy and from a nonallergic person (N) to dot blots of rPhl p 1, Phl p 1-A, Phl p 1-B, and PreS (A); rPhl p 2, Phl p 2-A, Phl p 2-B, and PreS (B); rPhl p 5, Phl p 5-A, Phl p 5-B, Phl p 5-C, Phl p 5-D, and PreS (C); and rPhl p 6, Phl p 6-A, Phl p 6-B, and PreS (D) are shown. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 4 Comparison of the allergenic activity of an equimolar mix of recombinant grass pollen allergens (Phl p 1, 2, 5, and 6) with PreS and an equimolar mix of recombinant BM proteins (BM321-6). Basophils from patients with grass pollen allergy (n = 31; A: S1-S16; B: S17-S31) were exposed to different concentrations of the proteins (x-axes; white bars, BM mix; dark gray bars, allergen mix; light gray bars, PreS), anti-IgE, or buffer (controls; black bars). Allergen-induced upregulation of CD203c was calculated from mean fluorescence intensities (MFIs) obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells and is expressed as the stimulation index (MFIstim: MFIcontrol; means ± SDs of triplicates; y-axes). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 5 Comparison of a mix of recombinant BM proteins with natural grass pollen extract regarding the induction of proliferation and cytokine production in PBMCs of patients with grass pollen allergy. Lymphocyte proliferation responses (stimulation indices [SI]) and cytokine levels (in picograms per milliliter; y-axes) are displayed for cultured PBMCs from 50 patients with grass pollen allergy after exposure to the mix of BM proteins (left, open circles, 0.5 μg per well of each BM32 protein) or grass pollen extract (right, open diamonds, 50 μg per well of total proteins). Significant differences between the mix of BM proteins and grass pollen are indicated. ns, Not significant. *P < .05 and **P < .0001. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 6 Inhibition of allergen-induced upregulation of CD203c on basophils of patients with grass pollen allergy by anti-BM32 IgG antibodies. Shown are representative results for basophils from 2 patients (upper and lower panels) with grass pollen allergy. Patients' PBMCs were incubated with different concentrations of the mix of recombinant grass pollen allergens that had been preincubated with rabbit anti-BM32 IgG antibodies (gray bars) or IgG from rabbits before immunization (Pre IgG: white bars), with anti-IgE or buffer alone (black bars; x-axes). Upregulations of CD203c expression are shown as mean stimulation indices ± SD (y-axes). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig 7 A, Scheme of treatment with BM32 in a murine model of grass pollen allergy. Mice were sensitized by means of subcutaneous (s.c.) injection and by means of intranasal challenge with recombinant grass pollen allergens split into 2 groups, one of which was treated with BM32 and the other with PBS alone. Allergen-induced basophil activation testing was done on day 188. B, Basophils from grass pollen–sensitized mice treated with BM32 (n = 6) or PBS (n = 6, x-axis) were stimulated with either Phl p 1, Phl p 5, or Phl p 6 (top line). Shown are the mean fluorescence intensities (MFIs; y-axis) of CD200R expression for the mice in each group. Medians are indicated as horizontal bars for each group, and significant differences between the BM32 and PBS groups are indicated. *P < .05. The horizontal line indicates mean baseline expression of CD200R expression for the medium controls. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E1 Comparison of IgE reactivity to rPhl p 2 and Phl p 2–derived peptides in patients. Displayed are IgE levels (y-axis: OD values; horizontal bars = medians) specific for Phl p 2 or Phl p 2–derived peptides (P1-2 to P4-2; x-axis) measured by means of ELISA in sera from patients with grass pollen allergy (n = 22). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E2 Comparison of IgE reactivity to rPhl p 6 and Phl p 6–derived peptides in patients. Displayed are IgE levels (y-axis: OD values; horizontal bars = medians) specific for Phl p 6 or Phl p 6–derived peptides (P1-6 to P4-6; x-axis) measured by means of ELISA in sera from patients with grass pollen allergy (n = 22). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E3 IgG reactivity of sera from peptide-immunized rabbits with Phl p 2 (A) and Phl p 6 (B). IgG reactivities (mean ± SD OD levels of triplicates; y-axes) to Phl p 2 (Fig E3, A) or Phl p 6 (Fig E3, B) determined for different dilutions (x-axes) of sera from rabbits immunized with the complete allergens or KLH-coupled allergen-derived peptides are shown (Fig E3, A: Phl p 2 and Phl p 2 peptides P1-P4; Fig E3, B: Phl p 6 and Phl p 6 peptides P1-P4). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E4 Coomassie-stained SDS-PAGE. Lanes contain purified fusion proteins or markers (lanes M). Molecular weights (in kilodaltons) are indicated on the left margin. Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E5 Allergen-specific IgG reactivity of rabbits immunized with aluminum hydroxide–adsorbed allergens or fusion proteins. IgG reactivities (y-axes, mean OD levels of triplicates) of rabbits immunized with the complete recombinant allergens or the fusion proteins to Phl p 1 (A), Phl p 2 (B), Phl p 5 (C), or Phl p 6 (D) determined for different serum dilutions (x-axes) are shown. For each protein, 2 different rabbits were immunized (I and II). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions

Fig E6 Lymphocyte proliferation and IgG1 responses in BM32-immunized mice. Shown are lymphocyte proliferation responses at week 22 (stimulation indices [SI] as box plots: y-axis; whiskers = minimum and maximum; horizontal bars = medians, boxes = 25th to 75th percentiles) to PreS and grass pollen allergen extract (x-axis) (A) and IgG1 reactivities in preimmune sera (pre) and immune sera (IS: week 22; median ± SD OD levels, y-axis) measured for 6 BM32-immunized mice (B). Journal of Allergy and Clinical Immunology 2015 135, 1207-1217.e11DOI: (10.1016/j.jaci.2014.09.012) Copyright © 2014 The Authors Terms and Conditions