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Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening  Theresa Thalhamer, PhD, Heidi Dobias, MSc, Tatjana.

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Presentation on theme: "Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening  Theresa Thalhamer, PhD, Heidi Dobias, MSc, Tatjana."— Presentation transcript:

1 Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening  Theresa Thalhamer, PhD, Heidi Dobias, MSc, Tatjana Stepanoska, PhD, Martina Pröll, PhD, Hanno Stutz, PhD, Oliver Dissertori, MSc, Peter Lackner, PhD, Fatima Ferreira, PhD, Michael Wallner, PhD, Josef Thalhamer, PhD, Arnulf Hartl, PhD  Journal of Allergy and Clinical Immunology  Volume 125, Issue 4, Pages e10 (April 2010) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 SDS-PAGE and CZE of the mutated proteins. Lane 1, Molecular weight marker (in kilodaltons); lane 2, rBet Mut 123; lane 3, rBet Mut 1234 (A). Electropherograms of rBet Mut 1234 (a), rBet Mut 123 (b), and ultrapure water (c). Arrows indicate 2 minor fractions. EOF, Signal for electro-osmotic flow. x-axis, Migration time (in minutes); y-axis, UV absorption (210 nm; B). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Structure and CD spectra analysis. Cartoon representation of the wild-type Bet v 1 (1BV1). The 4 mutation sites are indicated as spheres (A). Spectra analysis of the mutants rBet Mut 123 and rBet Mut 1234 in comparison with rBet v 1a indicate a structural change as a consequence of the mutations (B). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Antibody responses. ELISA of total IgG (A), IgG1 (B), and IgG2a (C) against rBet v 1a (rBet), rBet Mut 123, and rBet Mut Mice were immunized twice in a weekly interval, and sera were taken 2 weeks after the last immunization. The y-axis shows Δ preserum values in kilophoton counts per second (kpc) expressed as means ± SEMs. ∗∗P < Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 RBL cell release and release-inhibition assay. Mice were immunized twice with 5 μg of rBet v 1a in a weekly interval. RBL cells were sensitized with these sera, and cross-linking was performed with either rBet v 1a, rBet Mut 123, or rBet Mut 1234 (A). Similarly, sera of mice immunized with rBet, rBet Mut 123, or rBet Mut 1234 served for sensitization of RBL cells and cross-linking with the wild-type rBet v 1a (B). The same sera and rBet v 1a were used to assess IgE epitope blocking antibodies with an inhibition RBL assay (C). Data are shown as the percentage of β-hexosaminidase release. ∗∗P < .01. ∗P < .05. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Binding and inhibition assays with human IgE antibodies. Binding of IgE antibodies from a pool of 42 patients with birch pollen allergy to rBet v 1a or the mutants rBet Mut 123 or rBet Mut 1234 was assessed by means of ELISA (A). Inhibition ELISA was performed with human sera preincubated with wild-type and mutant proteins (B). IgE epitope blocking antibodies detected by means of inhibition ELISA (C). Data are expressed as absorption at 405 nm and means ± SEMs. ∗∗P < .01. ∗P < .05. Significances are related to rBet (Fig 5, A and B) and murine preserum (Fig 5, C). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 IFN-γ– and IL-5–producing cells in spleens of immunized mice. ELISPOT assays of IFN-γ–producing (A) and IL-5–producing (B) cells are shown. Data are expressed as means ± SEMs. ∗∗P < .01. ∗P < .05. Significances were calculated compared with ovalbumin-stimulated cells. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Binding of human IgE antibodies to rPhl p 5b wt and rPhl p 5b Mut. Binding of IgE antibodies from individual and pooled sera of 7 patients with grass pollen allergy to rPhl p 5b and rPhl p5b Mut was assessed by means of ELISA. Sera were diluted as shown. Data are expressed as mean values at 405 nm absorption. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Sequence alignment of the Phl p 5 wild-type and mutant.
Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Inhibition ELISA with a pool of sera from patients with grass pollen allergy. The inhibition ELISA was performed with a human serum pool preincubated with serial dilutions of either wild-type or mutant rPhl p5b. Data are shown as means ± SEMs at 405 nm absorption. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Genetic immunization with pCMV Bet and pCMV Bet Mut 123
Genetic immunization with pCMV Bet and pCMV Bet Mut 123. Bet v 1–specific total IgG response after 4 injections of plasmid DNA (A). Allergen-specific proliferation (B) and ELISPOTs (C) after in vitro restimulation of splenocytes. RBL assay detecting functional cross-linking IgE antibodies (D), IL-5 ELISPOT assay (E), and eosinophilia as percentage eosinophils in bronchoalveolar lavage fluid (F). DNA (↑) and protein (↓) immunizations are indicated by arrows. Data are expressed as means ± SEMs. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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