MURINE MODELS FOR EVALUATING THE ALLERGENICITY OF GENETICALLY MODIFIED FOODS

Transcription

1 MURINE MODELS FOR EVALUATING THE ALLERGENICITY OF GENETICALLY MODIFIED FOODS, Ph.D University of Paris XI ILSI New Methods Workshop October 2007 Nice France.

2 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

3 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

4 Critical parameters for establishing a robust, cost effective and reproducible animal model * Animals Strain, age, sex, exposure to food, etc. * Test material: Allergens and non-allergens Determination of matrix effect: pure protein vs. whole plant Effects of food processing or digestion Presence of adjuvants: : modified immune responses * Experimental design: Route of administration (iv, ip, oral, td, etc.) Frequency and dose of administration: graded allergenicity * Biomarkers: IgE, IgG,, cytokines, cell surface markers, etc. * Reliability of the model: inter-laboratory comparisons * Relevance to human data: (e.g. Active Systemic Anaphylaxis)

5 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

6 MOUSE MODELS OF FOOD ALLERGY X-M. Li et al (Oral, Peanut) K. Adel-Patient et al (I.P., Milk) R. Dearman et al (I.P., OVA, BSA, PNA, STA, PAP) V. Morafo et al (I.G., CM, PNA) C.J. Betts et al (I.D., PNA & STA) F. vanwijk et al (Oral, Peanut) S-Y. Lee et al (I.G., Buckwheat) N. Birmingham et al & 2007 (I.P. and T.D., Hazelnut) K. Vaali et al (T.D., OVA) Gizzarelli et al (I.G., WT and GM-Soybean) etc

7 Oral gavage sensitisation models d Li et al Peanut Extract + Adjuvant d w-old C3H/HeJ van Wijk et al d Buckwheat Extract + Adjuvant Lee et al. 2005

8 Oral gavage sensitisation models (continued) BALB/c 4-8-w-old C3H/HeJ Peanut or Milk + Adjuvant d Morafo et al Adel-Patient et al d w-old BALB/c Wild type Soybean or GM-Soybean + Adjuvant Gizzarelli et al. 2006

9 Dermal & Intra-dermal sensitisation models d BALB/c OVA PNA or PAP d Betts et al Intra-dermal Vaali et al Transdermal BALB/c + Hazelnut extract + / - Adjuvant w1 w2 w3 w4 w5 w6 +d10 Birmingham et al & 2007 Transdermal C57BL/6

10 I.P. sensitisation models d-3 1 d11 w5 w6 +d10 BALB/c Hazelnut extract 1h Birmingham et al / - Adjuvant d C57BL/6 Milk Adel-Patient et al BALB/c OVA, BSA, PNA, STA & PAP d Dearman et al w-old BALB/c

11 Conclusion Many different models developed All based on IgE detection Adjuvant necessary for oral sensitisation Limited number of allergens and non- allergens tested

12 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

13 ILSI ring trial Animal models IP; no adjuvant; BALB/c IP; no adjuvant; BALB/c, A/J, C3H IP; no adjuvant; BALB/c IP; +/- adjuvant; BALB/c, A/J, C3H, C57BL6 IP; +/- adjuvant; BALB/c Laboratories Bayer CropScience Dupont Syngenta Monsanto Dow Test Proteins Ara h 1 (peanut allergen) Ara h 2 (peanut allergen) Β-lactoglobulin (cows milk allergen) Rubisco (non-allergen) Soy lipoxygenase (non-allergen)

14 ILSI ring trial Endpoints measured: Clinical scoring IgG levels IgE levels => Active Systemic Anaphylaxis (ASA) => ELISA => Passive Cutaneous Anaphylaxis (PCA)

15 Passive Cutaneous Anaphylaxis (PCA) Phase I: Sensitisation Protein i.p. injections D0 Blood withdrawal D7 D14 Naïve animal Sensitised animals IgG

16 Passive Cutaneous Anaphylaxis (PCA) Phase II: Elicitation Ear of a naïve mouse Serum from sensitised mouse e.g. PNA 48 h PNA + Evans Blue Mouse Tail

17 PCA (continued) Ear of a naïve mouse IgE Receptor IgE Serum from sensitised mouse e.g. PNA Mast cell 48h after PNA + Evans Blue

18 Passive Cutaneous Anaphylaxis (PCA) Phase II: Elicitation Ear of a naïve mouse Serum from sensitised mouse e.g. PNA Mouse ear 48 h PNA + Evans Blue 30 min after measure the diameter of the blue mark > 3mm => IgE (+) Mouse Tail < 3mm => IgE (-)

19 BCS Results STRONG HUMAN ALLERGENS Beta-Lactoglobulin (mm) 0 0.1% 2.5% 5% Ara h 1 IgE response (mm) % 0.01% 0.05% 0.1% 2.5% 5% Ara h 2 IgE response (mm) 0 No IgG!!! 0,1 % 2,5 % 5 % WEAK HUMAN ALLERGENS Rubisco IgE response (mm) 0 0.1% 0.3% SoyLipoxygenaseIgEresponse(mm) % 0.5% 1% 1.5% 2.5% 5% But high LPS contamination and potential cross-reactivity!

20 Combined Results Protein Lab A Sensitize days 1, 7, 14 IgG => ELISA IgE => PCA d21 Lab B Sensitize days 1, 7 IgG => ELISA IgE => PCA d14 Lab C Sensitize days 0, 7, 28 IgE => het. PCA day 28 Clinical Symptoms d45 Lab D Sensitize days 1, 7 IgG =>ELISA IgE => PCA d14 BALB/c BDF1 C3H/HeJ BDF1 A/J C3H/HeJ BALB/c Ara h1 (+) specific IgE (+) specific IgE Not examined. Not examined. Positive Clinical Score Positive Clinical Score (+) specific IgG and IgE Ara h2 β-lactoglobulin (-) specific IgG and IgE (-) specific IgE (-) specific IgG and IgE (-) specific IgE Not examined. Not examined. Not examined. (+) specific IgG and IgE Positive Clinical Score Negative Clinical Score Positive Clinical Score Negative Clinical Score (-) specific IgG (+) specific IgG and IgE Rubisco Soybean Lipooxygenase (+) specific IgE (+) specific IgE (+) specific IgE (+) specific IgE (+) specific IgG and IgE (+) specific IgG and IgE Not examined. Not examined. Negative Clinical Score Positive Clinical Score Negative Clinical Score Positive Clinical Score (+) specific IgG and IgE (+) specific IgG and IgE EVALUATION OF IP MOUSE MODELS FOR ASSESSING THE ALLERGENIC POTENTIAL OF PROTEINS. Thomas, K., Hérouet, C., Bannon, G., Ladics, G., MacIntosh, S., Privalle, L., Woolhiser, M., Hefle, S. ILSI, Bayer CropScience, Monsanto, Dupont Co., Bayer Bioscience, BASF, The Dow Chemical Company, University of Nebraska. AAAAI Congress, San Antonio, 2005.

21 ILSI ring trial: Conclusion Difficult to differentiate between allergens and non-allergens consistently with various models. Adequate purification (e.g. LPS) and good characterization of proteins are important. The mechanisms of action for each protein (allergy vs. tolerance) need to be explored.

22 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

23 R.J. Dearman et al. => Intraperitoneal sensitisation model w-old BALB/c Ovalbumin Peanut agglutinin Potato agglutinin d0 d7 d14 I.P. I.P. Sacrifice NO Adjuvant Dearman RJ, Stone S, Caddick HT, Basketter DA, Kimber I. Evaluation of protein allergenic potential in mice: doseresponse analyses. Clin Exp Allergy Nov;33(11): Clinical and Experimental Allergy 33: 1586, 2003.

24 Results: Phase I 2003 Serum levels of specific-igg and IgG1 Syn BCS Syn BCS Syn BCS PNA 0.1% 2% OVA 5% STA Peanut Egg Potato Dearman RJ, Skinner RA, Herouet C, Labay K, Debruyne E, Kimber I. Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons. Food Chem Toxicol. 41(11): , 2003.

25 Results: Phase I 2003 Serum levels of specific IgE Syn BCS Syn BCS Syn BCS PNA 0.1% 2% OVA 5% STA Peanut Egg Potato Dearman RJ, Skinner RA, Herouet C, Labay K, Debruyne E, Kimber I. Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons. Food Chem Toxicol. 41(11): , 2003.

26 Apparent spectrum based on IgE levels 100 PNA 0.1% Reciprocal PCA titre 10 2% OVA 5% STA Protein concentration (%)

27 Conclusions: Phase I 2003 => Measurement of IgE responses by PCA in BALB/c mice appears to accurately identify allergens. => This model is relatively robust and can be transferred successfully between laboratories. Dearman RJ, Skinner RA, Herouet C, Labay K, Debruyne E, Kimber I. Induction of IgE antibody responses by protein allergens: inter-laboratory comparisons. Food Chem Toxicol. 41(11): , 2003.

28 Results: Phase II 2007 IgE levels Syngenta BCS Reciprocal PCA titre Recip rocal P CA titre <1 ND 0.1% PNA PNA PNA 0.05% 0.01% Peanut Agglutinin % 1% 0.2% Ovalbumin OVA OVA OVA 2% 1% 0.2% C. Herouet-Guicheney, H. Aldemir, R. Bars, D. de Barbeyrac, D. Rouquié, P. Kennel, B. Stahl, R. Dearman, I. Kimber. Manuscript ready for submission.

29 Conclusions: Phase II 2007 => Confirmation of the strength and repeatability of the model. => The model needs to be standardized by testing more proteins. C. Herouet-Guicheney, H. Aldemir, R. Bars, D. de Barbeyrac, D. Rouquié, P. Kennel, B. Stahl, R. Dearman, I. Kimber. Manuscript ready for submission.

30 Plan Critical parameters of animal models Mouse models Literature review ILSI ring trial (2005) Syngenta-Bayer CS Collaborative work A new promising mouse model Conclusion

31 Aim A new model of food allergy Strongly allergenic Weakly allergenic Peanut Ara h 1 Ara h 2 Peanut Agglutinin Potato PAP

32 Sensitisation protocol 6-12-w-old female Balb/c mice Different concentrations of Ara h 1, PAP or PNA were tested. Protein Concentrations 0.01 g/l ( mg/mouse) 0.04 g/l (0.01 mg/mouse) 0.16 g/l (0.04 mg/mouse) 0.64 g/l (0.16 mg/mouse) 2.50 g/l (0.64 mg/mouse)

33 The parameters: Tim-1, IL-5/IFN 5/IFN-γ, IgE IFN-γ IgG2a Th 1 Tim-1 IL-4 B Lymphocyte Th 2 Early IL-5 Intermediate IgG1 & IgE Late

34 Late Marker => Protein-specific IgE (ELISA) Protein-specific IgE levels Arah1 Pap PNA / 16 1 / 32 1 / 64 1 / / / / / / 16 1 / 32 1 / 64 1 / / / / / / 16 1 / 32 1 / 64 1 / / / / / / 16 1 / 32 1 / 64 1 / / / / / / 16 1 / 32 1 / 64 1 / / / / / / 16 1 / 32 1 / 64 1 / / / / / Protein sensitisation concentrations (g/l) PBS 0.03 mg/ml 0.15 mg/ml 0.27 mg/ml 0.83 mg/ml 2.5 mg/ml

35 Splenocyte culture for cytokine expression In the immune system of the sensitised- mice Ex vivo Cell Culture Allergen Cutting into pieces Filter APC Lymphocyte Th Allergen is added in culture Put in culture Supernatant collection Cell recovery 96-well plate

36 Intermediate Markers => IL-5 5 / IFN-γ Ratio Arah1 PNA PAP Protein sensitisation concentrations (g/l) H. Aldemir, R. Bars, C. Herouet-Guicheney. Partly presented in ITCASS, Lodi, Italy, 2006.

37 Early marker => Surface Tim-1 1 expression Control-Arah1 Allergen Ara h % 42.3 % Control-PAP Non-Allergen PAP 20.7 % 14.1 % Tim-1 surface expression

38 The parameters: Tim-1, IL-5 5 / IFN-γ, IgE IFN-γ IgG2a Th 1 Tim-1 IL-4 B Lymphocyte Th 2 Early IL-5 Intermediate IgG1 & IgE Late

39 New model: Conclusion This animal model with the selected parameters is promising to discriminate between Strong Allergen Weak allergen However: Standardisation of the model is necessary

40 General Conclusion => Standardisation / International validation Need for more proteins to be tested => Several animal models are needed * Clinical model ASA scoring * Protein vs. vs. Mechanistic model IgE, IgGs,, cytokines, etc. Food Purity & conformation Matrix & Processing => Weigh of evidence approach (Codex( Alimentarius 2003)

41 Thank you very much for your kind attention.

42 Sponsored by Bayer CropScience Special thanks to: Dr. C. Herouet-Guicheney (BCS) Dr. R. Bars (BCS) Prof. M. Pallardy (Univ. Paris XI) Prof. N. Glaichenhaus (Univ. Nice) BCS participants to the work Dr. de Barbeyrac Dr. P. Kennel Dr. D. Rouquié BCS Zootech & Technical Groups

43 Late Marker => Protein-specific IgG Ara h1 PAP mg/ml mg/ml Peanut Lectin RUBISCO mg/ml mg/ml

44 Molecular mechanism of allergy Primary contact Allergen Secondary contact Tim-1 Allergen Proliferation Differentiation DC T helper 0 Naive Th 2 Primary contact Non-Allergen Secondary contact Tim-3 Non-Allergen Differentiation DC T helper 0 Naive Th 1

45 ELISA (Igs( Igs,, Cytokines, etc..) Blood sample Centrifugation Serum recovery The protein Serum dilutions Evaluation of OD by spectrophotometre Antibody against mouse antibody

46 Cytometric Bead Array (CBA) Supernatant + + IFN- γ IL IL-5 +

47 Analysis of cytokines by Cytometric Bead Array (CBA)( Color of the beads = Differentes cytokines IL-4 IFN-γ Fluorescence Intensity = Quantity of cytokine IL-5 Becton Dickinson (BD) FACS In CNRS Lab.

48 CD4 => T helper cell marker Total splenocytes Total splenocytes are analysed according to size and granularity Cell granularty Non-stained cells Cell size CD4-FITC T helper cells are marked with the specific antibody CD4-FITC T helper marker-stained cells Non-Th cells T helper cells CD4-FITC CD4-FITC

49 Endotoxins (Endotoxin Units / mg protein) EU endotoxin / mg protein Endotoxin levels Before Purification After Purification < 10< < 10< Arah1 PAP PNA RUB SLP 100 EU/mg Threshold for cytokine response bias. Ref: Dearman 2007