A hypoallergenic cat vaccine based on Fel d 1–derived peptides fused to hepatitis B PreS Katarzyna Niespodziana, MSc, Margarete Focke-Tejkl, PhD, Birgit.

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Presentation transcript:

A hypoallergenic cat vaccine based on Fel d 1–derived peptides fused to hepatitis B PreS Katarzyna Niespodziana, MSc, Margarete Focke-Tejkl, PhD, Birgit Linhart, PhD, Vera Civaj, Katharina Blatt, Peter Valent, MD, Marianne van Hage, MD, Hans Grönlund, PhD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 127, Issue 6, Pages 1562-1570.e6 (June 2011) DOI: 10.1016/j.jaci.2011.02.004 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Construction of PreS fusion proteins. A, Chains 1 and 2 of Fel d 1 with peptides indicated. aa, Amino acid. B, Ribbon representation of Fel d 1 with peptides highlighted in different colors. C, PreS fusion proteins containing peptide 1 (P1; PreS-2xP1) or peptide 5 (P5; PreS-2xP5) at the N- and C-termini, as well as PreS-P1-P5 with an N-terminal P1 and C-terminal P5 peptide. Hexahistidine tags (6xHis) are at the C-terminal end. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Purity and immune reactivity of recombinant proteins. Coomassie-stained SDS-PAGE (A) and nitrocellulose blots (B) containing PreS (lane 1), PreS-2xP1 (lane 2), PreS-2xP5 (lane 3), PreS-P1-P5 (lane 4), and rFel d 1 (lane 5) were probed with rabbit preimmune serum (Fig 2, B, right, upper part), anti-P1, anti-P5, anti-PreS (Fig 2, B, left, bottom part), or anti–rFel d 1 (Fig 2, B, right, bottom part) antibodies. Molecular weights in kilodaltons are indicated in the left margin. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Comparison of allergenic activity of rFel d 1 and PreS fusion proteins in the CD203c basophil activation test. Blood cells from 7 patients with cat allergy (Table 1, nos. 4, 7, 13, 14, 17, 18, and 23) were exposed to PBS (unstim.), anti-IgE, and serial dilutions (10-0.01 μg/mL) of rFel d 1, PreS-2xP1, PreS-2xP5, or PreS-P1-P5 (x-axes). CD203c expression on basophils was determined by means of FACS analysis. CD203c upregulation is displayed as the stimulation index (y-axes). Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 PreS- (A) and rFel d 1–specifc (B) IgG1 responses in mice. Groups of 8 mice each were immunized subcutaneously with the antigen shown at the top of the boxes (PreS-2xP1, PreS-2xP5, PreS-P1-P5, PreS, rFel d 1, and PBS). Serum samples were taken 1 day before each immunization in 3-week intervals (0-12 weeks; x-axes). IgG1 reactivities (OD values, y-axes) were determined by means of ELISA and are displayed as box plots, where 50% of the values are within the boxes and nonoutliers are between the bars. The lines within the boxes indicate the median values. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Immunization with PreS fusion proteins does not induce allergic sensitization to Fel d 1. A, Sera from mice obtained before (PI) and 3 (1. IS), 6 (2. IS), and 12 (4. IS) weeks after immunization with rFel d 1 (x-axis) were heated at 56°C for different times (30 minutes, 2 hours, or 4 hours) or left untreated (0). These sera were loaded onto RBL and exposed to rFel d 1. B, RBL cells were sensitized with mouse antigen (Ag)–specific sera and stimulated with increasing concentrations of rFel d 1 (x-axis). C, RBL cells were sensitized with different dilutions (1:50, 1:25, and 1:10) of allergen-specific murine sera (x-axis) and stimulated with rFel d 1. D, Sensitized RBL cells (x-axis) were stimulated with PreS-2xP1, PreS-P1-P5, and rFel d 1. In each figure β-hexosaminidase releases are displayed as a percentage of total β-hexosaminidase release on the y-axes. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Inhibition of rFel d 1–induced CD203c upregulation on basophils from patients with cat allergy by rabbit IgG antibodies. Increasing concentrations of rFel d 1 (0.00001-0.001 μg/mL) were preincubated with rabbit anti–rFel d 1 or anti–PreS-P1-P5 IgG antibodies or the corresponding preimmune serum. Basophils from 2 patients with cat allergy (Table I, nos. 4 and 7) were then exposed to PBS (unstim.), anti-IgE, and the preincubated mixtures of rFel d 1 (x-axes). Upregulation of CD203c expression on basophils determined by means of FACS analysis is displayed as the stimulation index (y-axes). Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Lymphoproliferative responses of PBMCs from 4 patients with cat allergy to the rFel d 1–derived peptides (1-5). Mean stimulation indices are shown for 5 antigen concentrations: 1 μg/well, 0.5 μg/well, 0.25 μg/well, 0.125 μg/well, and 0.0625 μg/well. Stimulation indices (SIs) of greater than 2.0 are displayed in gray. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Boosting of Fel d 1–specific IgG responses in vaccinated mice with isolated peptides. A, Groups of 8 mice each were preimmunized with PreS, followed by 3 immunizations either with PreS-2xP1 (B), PreS-P1-P5 (C), or PreS-2xP5 (D). At week 21, when Fel d 1–specific IgG1 responses decreased, mice were randomized into 2 groups of 4 mice each and boosted either with peptide 1 (P1; white bars) alone or with the PreS-2xP1 fusion protein (black bars). Bars represent Fel d 1–specific IgG1 antibody levels (y-axes; OD values). Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Proliferative responses of splenocytes Proliferative responses of splenocytes. Displayed are proliferative responses (y-axes; stimulation indices) of splenocytes from immunized mice (top of the boxes, immunogens) that had been boosted with P1 (A) or PreS-2xP1 (B) and were stimulated with medium alone, P1, P5, rFel d 1, or PreS (x-axes). Mean values of triplicates are shown as box plots, where 50% of the values are within the boxes and nonoutliers are between the bars. The lines within the boxes indicate the median values of the groups. Journal of Allergy and Clinical Immunology 2011 127, 1562-1570.e6DOI: (10.1016/j.jaci.2011.02.004) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions