Elisabeth Roesler, MSc, Richard Weiss, PhD, Esther E

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Immunize and disappear—Safety-optimized mRNA vaccination with a panel of 29 allergens Elisabeth Roesler, MSc, Richard Weiss, PhD, Esther E. Weinberger, MSc, Angelika Fruehwirth, MSc, Angelika Stoecklinger, PhD, Sven Mostböck, PhD, Fatima Ferreira, PhD, Josef Thalhamer, PhD, Sandra Scheiblhofer, PhD Journal of Allergy and Clinical Immunology Volume 124, Issue 5, Pages 1070-1077.e11 (November 2009) DOI: 10.1016/j.jaci.2009.06.036 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Experimental schedule. Numbers indicate days. Mice were immunized 3 times with mRNA vaccines and subsequently sensitized with recombinant allergen/alum. Sera were taken before (pre) and after (post) sensitization. Subsequently, mice were challenged by means of intranasal instillation of allergen, analyzed with whole-body plethysmography (WBP), and finally killed for cellular assays (cytokine profiles and bronchoalveolar lavage). i.n., Intranasal. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Phl p 5–specific IgG1, IgG2a, and IgE levels and total IgE levels after mRNA vaccination (A and C) and subsequent sensitization (B, C, and D). Fig 2, A, B, and D, show ELISA data; Fig 2, C, shows RBL assay data (n = 5). Sera were diluted 1:100 (Fig 2, A), 1:10,000 (Fig 2, B), and 1:100 (Fig 2, C, pre) or 1:200 (Fig 2, C, post, and D). Statistical significance was calculated compared with sera from naive mice (Fig 2, A) or sera from sensitization control animals (Fig 2, B, C, and D; control). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Number of IFN-γ–secreting (A), IL-4–secreting (B), and IL-5–secreting (C) splenocytes and levels of IL-13 (D) after in vitro restimulation with recombinant Phl p 5, as determined by means of ELISPOT assays and ELISAs. Data are shown as means ± SEMs (n = 5). Statistical significance was calculated compared with sensitization control animals (control). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Number of eosinophils (A) and CD8+ cells (B) in bronchoalveolar lavage fluid of sensitized mice after intranasal application of allergen. Values are shown as means ± SEMs (n = 5). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. ND, Not done. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Penh values of mice prevaccinated with P5-RNA (A) and P5-repRNA (B) after sensitization and intranasal allergen challenge. Mice were exposed to escalating doses of methacholine with a final recovery interval. BL, Baseline measurement. Values are shown as means ± SEMs (n = 5). P values are shown for the 20 mg/mL dose. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Comparison of mRNA and DNA vaccines at equal doses. IgG2a induction and IgG1 suppression (A), as well as IgE suppression after sensitization (B), are shown. C, IFN-γ and IL-5 levels in supernatants of restimulated splenocytes after sensitization. D, Number of eosinophils in bronchoalveolar lavage fluid of challenged mice. All data are shown as means ± SEMs (n = 4). Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

RNA quality control. A, Agarose gel electrophoresis of in vitro transcribed P5-RNA (approximately 990 bp, lane 1) and P5-repRNA (approximately 8900 bp, lane 2). B, Western blot of cell lysates of BHK-21 cells transfected with P5-RNA (lane 1), P5-repRNA (lane 2), or no RNA (lane 3). Four nanograms of recombinant Phl p 5 were applied to lane 4. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Stability of RNA preparations Stability of RNA preparations. mRNA encoding luciferase was stored for 32 days at the indicated temperatures either in water (A) or lyophilized (B). Integrity of RNA was assessed by using denaturing agarose gel electrophoresis (Fig E2, A and B) and luciferase expression in transfected BHK-21 cells (C). RT, Room temperature. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

In vivo RNA expression is short lived In vivo RNA expression is short lived. In vivo expression of luciferase in mice (n = 3) was quantitated on an Ivis Lumina imaging system (A) and expressed as a percentage of luminescence compared with that of naive mice injected with luciferin (B). Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Selected groups of mice (n = 3) from Table E1 representing Fagales (Bet v 1, Cas s 1, and Fag s 1), timothy grass (Phl p 6), and food (Api g 1 and ovalbumin [Ova]) allergens were reproduced as described. Suppression of allergen-specific IgE (A, RBL assay), induction of IFN-γ (B, ELISA), suppression of IL-4 (C, ELISPOT assay) in splenocytes restimulated with the respective allergen, invasion of eosinophils into bronchoalveolar lavage fluid (D), and IL-5 levels in bronchoalveolar lavage fluid (E) were measured after sensitization and intranasal challenge. ∗P < .05 and ∗∗P 0.01 compared with the sensitization control groups. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL -5 (A) and IFN-γ (B) levels in bronchoalveolar lavage fluid of mice (n = 5) prevaccinated with escalating doses (0.1-100 μg) of mRNA vaccines encoding Phl p 5 or β-galactosidase (mock control) after sensitization and intranasal challenge. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 compared with the sensitization control groups. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Lung resistance (A) and dynamic compliance (B) after sensitization and intranasal provocation of mice prevaccinated with 40 μg of P5-RNA or 8 μg P5-repRNA or sensitization control animals. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

In vivo expression of interferon regulatory factor 7 (IRF-7) and CXCL10 in draining lymph nodes of mice (n = 4) 0, 5, and 24 hours after vaccination with 40 μL of P5-RNA. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

TH1 immunogenicity of mRNA vaccines is reduced but still intact in MyD88−/− mice. Antibody subclasses (A and C) and IFN-γ secretion (B and D) from restimulated splenocytes 2 weeks after vaccination (Fig E8, A and B) and after the last sensitization (Fig E8, C and D) are shown. Fig E8, A: n = 4 to 6, serum dilution 1:500. Fig E8, C: n = 3, serum dilution 1:10,000. Statistical differences between wild-type (WT) and corresponding knockout groups: ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2009 124, 1070-1077.e11DOI: (10.1016/j.jaci.2009.06.036) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions