murine models for evaluating the allergenicity of

MURINE MODELS FOR MURINE MODELS FOR EVALUATING THE EVALUATING THE

ALLERGENICITY OF ALLERGENICITY OF GENETICALLY MODIFIED FOODSGENETICALLY MODIFIED FOODS

Hatice Aldemir, Hatice Aldemir, Ph.DPh.DUniversity of Paris XIUniversity of Paris XI

ILSI New Methods WorkshopILSI New Methods Workshop2323--25 October 200725 October 2007

Nice Nice –– France.France.

Hatice Aldemir

PlanPlan

•• Critical parameters of animal modelsCritical parameters of animal models

•• Mouse modelsMouse models•• Literature review Literature review

•• ILSI ring trial (2005)ILSI ring trial (2005)

•• SyngentaSyngenta--Bayer CS Collaborative workBayer CS Collaborative work

•• A new promising mouse modelA new promising mouse model

•• ConclusionConclusion

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** AnimalsAnimals–– Strain, age, sex, exposure to food, etc.Strain, age, sex, exposure to food, etc.

** Test material:Test material:–– Allergens and nonAllergens and non--allergens allergens –– Determination of matrix effect: pure protein Determination of matrix effect: pure protein vs.vs. whole plantwhole plant–– Effects of food processing or digestionEffects of food processing or digestion–– Presence of Presence of adjuvantsadjuvants: modified immune responses: modified immune responses

** Experimental design:Experimental design:–– Route of administration (iv, ip, oral, td, etc.)Route of administration (iv, ip, oral, td, etc.)–– Frequency and dose of administration: graded allergenicityFrequency and dose of administration: graded allergenicity

** Biomarkers: Biomarkers: IgEIgE, , IgGIgG, cytokines, cell surface markers, etc., cytokines, cell surface markers, etc.** Reliability of the model: interReliability of the model: inter--laboratory comparisonslaboratory comparisons** Relevance to human data: (e.g. Active Systemic Anaphylaxis)Relevance to human data: (e.g. Active Systemic Anaphylaxis)

Critical parameters for establishing a robust,Critical parameters for establishing a robust,cost effective and reproducible animal modelcost effective and reproducible animal model

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PlanPlan







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MOUSE MODELS OF FOOD ALLERGYMOUSE MODELS OF FOOD ALLERGY

•• XX--M. Li et al. 2000 (Oral, Peanut)M. Li et al. 2000 (Oral, Peanut)

•• K. AdelK. Adel--Patient et al. 2000 (I.P., Milk)Patient et al. 2000 (I.P., Milk)

•• R. Dearman et al. 2003 (I.P., OVA, BSA, PNA, STA, PAP)R. Dearman et al. 2003 (I.P., OVA, BSA, PNA, STA, PAP)

•• V. V. MorafoMorafo et al. 2003 (I.G., CM, PNA)et al. 2003 (I.G., CM, PNA)

•• C.J. Betts et al. 2004 (I.D., PNA & STA)C.J. Betts et al. 2004 (I.D., PNA & STA)

•• F. F. vanWijkvanWijk et al. 2004 (Oral, Peanut)et al. 2004 (Oral, Peanut)

•• SS--Y. Lee et al. 2005 (I.G., Buckwheat)Y. Lee et al. 2005 (I.G., Buckwheat)

•• N. Birmingham et al. 2005 & 2007 (I.P. and T.D. , Hazelnut)N. Birmingham et al. 2005 & 2007 (I.P. and T.D. , Hazelnut)

•• K. K. VaaliVaali et al. 2006 (T.D., OVA)et al. 2006 (T.D., OVA)

•• GizzarelliGizzarelli et al. 2006 (I.G., WT and GMet al. 2006 (I.G., WT and GM--Soybean)Soybean)

•• etc…etc…

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Oral Oral gavagegavage sensitisation modelssensitisation models

♀

4-8-w-old

C3H/HeJ

Peanut Extract

+ Adjuvant

d0 7 21 35 42

Li et al.Li et al.20002000

d12 3 8 15 22 29 36 43 49

van van WijkWijk et al.et al.20042004

Buckwheat Extract

+ Adjuvant

d0 1 2 7 21 35

Lee et al.Lee et al.20052005

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Oral Oral gavagegavage sensitisation models (continued)sensitisation models (continued)

♀

BALB/cPeanut or Milk

♀

4-8-w-old

C3H/HeJ

♀

4-w-old

BALB/c

Wild type Soybean or GM-Soybean

+ Adjuvant

GizzarelliGizzarelli et al.et al.20062006

d0 7 14 21 28

MorafoMorafo et al.et al.20032003

AdelAdel--Patient et al.Patient et al.20052005

d0 7 14 21 28 35 42

+ Adjuvant

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Dermal & IntraDermal & Intra--dermal sensitisation modelsdermal sensitisation models

PNA

or

PAP

d0 7 14

Betts et al.Betts et al.20042004

IntraIntra--dermaldermal

C57BL/6

BALB/c+

Hazelnutextract

+ / - Adjuvant

Birmingham et al.Birmingham et al.2005 & 20072005 & 2007

TransdermalTransdermal

w1 w2 w3 w4 w5 w6 +d10

♀

BALB/c

OVA

d1 4 8 22 35 40 … 51

VaaliVaali et al.et al.20062006

TransdermalTransdermal

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I.P. sensitisation models I.P. sensitisation models

♀

8-12-w-old

BALB/c

OVA, BSA, PNA,STA & PAP

d0 7 14

DearmanDearman et al.et al.20032003

♀

BALB/cHazelnutextract

+ / - Adjuvant

C57BL/6

Birmingham et al.Birmingham et al.20052005

1h

d-3 1 d11 w5 w6 +d10

♀

BALB/c

AdelAdel--Patient et al.Patient et al.20002000

Milk

d1 11 18

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•• Many different models developedMany different models developed

•• All based on All based on IgEIgE detection detection

•• Adjuvant necessary for oral sensitisationAdjuvant necessary for oral sensitisation

•• Limited number of allergens and nonLimited number of allergens and non--

allergens testedallergens tested

ConclusionConclusion

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PlanPlan







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ILSI ring trialILSI ring trial

Test ProteinsAra h 1 (peanut allergen)Ara h 2 (peanut allergen)Β-lactoglobulin (cows’ milk allergen)Rubisco (non-allergen)Soy lipoxygenase (non-allergen)

Animal modelsIP; no adjuvant; BALB/cIP; no adjuvant; BALB/c, A/J, C3HIP; no adjuvant; BALB/c IP; +/- adjuvant; BALB/c, A/J, C3H, C57BL6IP; +/- adjuvant; BALB/c

LaboratoriesBayer CropScienceDupontSyngentaMonsantoDow

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ILSI ring trialILSI ring trial

Endpoints measured:

Clinical scoring => Active Systemic Anaphylaxis (ASA)

IgG levels => ELISA

IgE levels => Passive Cutaneous Anaphylaxis (PCA)

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IgGIgG

Passive Passive CutaneousCutaneous Anaphylaxis (PCA)Anaphylaxis (PCA)Phase I: Phase I: SensitisationSensitisation

D0 D7 D14D7 D14Protein Protein i.p. injectionsi.p. injections

Naïve animal Sensitised animals

Blood withdrawalBlood withdrawal

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Passive Passive CutaneousCutaneous Anaphylaxis (PCA)Anaphylaxis (PCA)Phase II: Phase II: ElicitationElicitationEar of a naïve mouse Serum from

sensitised mouse e.g. PNA

PNA+

Evans Blue

48 h

Mouse Tail

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Mast cellMast cell

IgEIgE ReceptorReceptor IgEIgEEar of a naïve mouse

Serum from sensitised mouse

e.g. PNA

48h after PNA +

Evans Blue

PCA PCA (continued)(continued)

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Passive Passive CutaneousCutaneous Anaphylaxis (PCA)Anaphylaxis (PCA)Phase II: Phase II: ElicitationElicitationEar of a naïve mouse Serum from

sensitised mouse e.g. PNA

PNA+

Evans Blue

48 h

< 3mm => IgE (-)

30 min after measure the diameter of

the blue mark

> 3mm => IgE (+)

Mouse ear

Mouse Tail

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Beta-Lactoglobulin (mm)

0 0.1% 2.5% 5%0123456789101112

STRONG HUMAN ALLERGENS

0 0.005% 0.01% 0.05% 0.1% 2.5% 5%

Ara h 1 IgE response (mm)

789101112

0123456

Ara h 2 IgE response (mm)

789101112

0123456

0,1 % 2,5 % 5 %0

Rubisco IgE response (mm)

6789101112

012345

0 0.1% 0.3%

WEAK HUMAN ALLERGENS

6789101112

5

01234

0 0.1% 0.5% 1% 1.5% 2.5% 5%

Soy Lipoxygenase IgE response (mm)

But high LPS contamination and potential cross-reactivity!

BCS ResultsBCS Results

No No IgGIgG !!!!!!

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(+) specific IgG and IgE

Positive Clinical Score


Not examined.(+) specific IgG and IgE

(+) specific IgE

(+) specific IgE

SoybeanLipo-oxygenase




Not examined.Not examined.(+) specific IgE

(+) specific IgE

Ara h1

(-) specific IgG



Not examined.Not examined.(-) specific IgG and IgE

(-) specific IgG and IgE

Ara h2


Negative Clinical Score



Not examined.(-) specific IgE

(-) specific IgE

β-lacto-globulin




Not examined.(+) specific IgG and IgE

(+) specific IgE

(+) specific IgE

Rubisco

Lab DSensitize days 1, 7 IgG =>ELISAIgE => PCA d14

Lab CSensitize days 0, 7, 28IgE => het. PCA day 28 Clinical Symptoms d45

Lab BSensitize days 1, 7IgG => ELISAIgE => PCA d14

Lab ASensitize days 1, 7, 14IgG => ELISAIgE => PCA d21

Protein

BALB/cC3H/HeJA/JBDF1C3H/HeJBDF1BALB/c

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.

Combined ResultsCombined Results

Hatice Aldemir

• 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.

ILSI ring trial: ConclusionILSI ring trial: Conclusion

Hatice Aldemir

PlanPlan







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R.J. R.J. DearmanDearman et al. => et al. => Intraperitoneal sensitisation modelIntraperitoneal sensitisation model 20032003

Ovalbumin

Peanut agglutinin

Potato agglutinin

d0 d7 d14

I.P. I.P. Sacrifice

♀

8-12-w-old

BALB/c

Dearman RJ, Stone S, Caddick HT, Basketter DA, Kimber I. Evaluation of protein allergenic potential in mice: dose-response analyses. Clin Exp Allergy. 2003 Nov;33(11):1586-94.Clinical and Experimental Allergy 33: 1586, 2003.

NO Adjuvant

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Results: Phase IResults: Phase I 20032003

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): 1509-1516, 2003.

PNA 0.1% 2% OVA 5% STAPeanut Egg Potato

Serum levels of specificSerum levels of specific--IgGIgG and IgG1and IgG1

Syn BCS Syn BCS Syn BCS

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Results: Phase IResults: Phase I 20032003


Serum levels of specific Serum levels of specific IgEIgE

PNA 0.1% 2% OVA 5% STAPeanut Egg Potato

Syn BCS Syn BCS Syn BCS

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Apparent spectrum based on Apparent spectrum based on IgEIgE levelslevels

1

10

100

0 1 2 3 4 5 6

PNA 0.1%

2% OVA

5% STA

Protein concentration (%)

Rec

ipro

calP

CA

titr

e

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Conclusions: Phase IConclusions: Phase I 20032003


=> 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.

Hatice Aldemir C. Herouet-Guicheney, H. Aldemir, R. Bars, D. de Barbeyrac, D. Rouquié, P. Kennel, B. Stahl, R. Dearman, I. Kimber. Manuscript ready for submission.

Results: Phase IIResults: Phase II 20072007

IgEIgE levelslevels

0

1

2

3

4

5

6

OVA 2% OVA 1% OVA 0.2%R

ecip

roca

l PCA

titre

64

32

16

8

4

2

1

2% 1% 0.2%2% 1% 0.2%OvalbuminOvalbumin

0

1

2

3

4

5

6

PNA 0.1% PNA 0.05% PNA 0.01%

Reci

proc

al P

CA ti

tre

64

32

16

8

4

2

1<1 ND

0.1% 0.05% 0.01%0.1% 0.05% 0.01%Peanut AgglutininPeanut Agglutinin

Syngenta BCS

Hatice Aldemir C. Herouet-Guicheney, H. Aldemir, R. Bars, D. de Barbeyrac, D. Rouquié, P. Kennel, B. Stahl, R. Dearman, I. Kimber. Manuscript ready for submission.

Conclusions: Phase IIConclusions: Phase II 20072007

=> Confirmation of the strength and repeatability of the model.

=> The model needs to be standardized by testing more proteins.

Hatice Aldemir

PlanPlan







Hatice Aldemir

AimAim

Ara h 1Ara h 2Peanut Agglutinin

A new model of food allergyA new model of food allergy

Peanut

Strongly allergenicStrongly allergenic

Potato

PAP

Weakly allergenicWeakly allergenic

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6-12-w-old female Balb/c mice

Sensitisation protocolSensitisation protocol

Different concentrations of Ara h 1, PAP or PNA were tested.Different concentrations of Ara h 1, PAP or PNA were tested.

Protein ConcentrationsProtein Concentrations0.01 0.01 g/lg/l (0.0025 mg/mouse)(0.0025 mg/mouse)0.04 0.04 g/lg/l (0.01 mg/mouse)(0.01 mg/mouse)0.16 0.16 g/lg/l (0.04 mg/mouse)(0.04 mg/mouse)0.64 0.64 g/lg/l (0.16 mg/mouse)(0.16 mg/mouse)2.50 2.50 g/lg/l (0.64 mg/mouse)(0.64 mg/mouse)

Hatice Aldemir

The parameters: TimThe parameters: Tim--1, 1, ILIL--5/IFN5/IFN--γγ, , IgEIgE

IL-4

IgEIgEIgG1IgG1

&

B LymphocyteB Lymphocyte

IgG2aIgG2a

IFN-γ

Th 1Th 1

Th 2Th 2

TimTim--11

IL-5

IntermediateEarly Late

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Late Marker => ProteinLate Marker => Protein--specific IgEspecific IgE (ELISA)(ELISA)

Protein-specific IgE levels

0.000

0.500

1.000

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

1 / 1

61

/ 32

1 / 6

41

/ 128

1 / 2

561

/ 512

1 / 1

024

1 / 2

048

PBS 0.03 mg/ml 0.15 mg/ml 0.27 mg/ml 0.83 mg/ml 2.5 mg/ml

Arah1 Pap PNA

0 0.01 0.04 0.16 0.64 2.5 Protein sensitisation concentrations (g/l)

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Splenocyte culture for cytokine expressionSplenocyte culture for cytokine expression

Cell recovery

Put in culture

96-well plate

Allergen is Allergen is added in added in cultureculture

Filter

Cutting into pieces

Ex vivoEx vivo Cell CultureCell Culture

Supernatant collection

In the immune system of the sensitisedIn the immune system of the sensitised--micemice

Lymphocyte ThLymphocyte Th

AllergenAllergen

APCAPC

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Intermediate Markers => ILIntermediate Markers => IL--5 / IFN5 / IFN--γγ RatioRatio

H. Aldemir, R. Bars, C. Herouet-Guicheney. Partly presented in ITCASS, Lodi, Italy, 2006.

0

20

40

60

80

100

120

140

0 10 50 250 0 10 50 250 0 10 50 250 0 10 50 250 0 10 50 250 0 10 50 250

0.0025 0.01 0.04 0.16 0.64 2.5

Arah1 PNA PAP

0 0.01 0.04 0.16 0.64 2.5Protein sensitisation concentrations (g/l)

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Early marker => Surface TimEarly marker => Surface Tim--1 expression1 expression

Control-Arah1 Allergen Ara h 1

Tim-1 surface expression

42.3 %20.4 %

14.1 %20.7 %

Control-PAP Non-Allergen PAP

Hatice Aldemir

The parameters: TimThe parameters: Tim--1, 1, ILIL--5 / IFN5 / IFN--γγ, , IgEIgE

IL-4

IgEIgEIgG1IgG1

&

B LymphocyteB Lymphocyte

IgG2aIgG2a

IFN-γ

Th 1Th 1

Th 2Th 2

TimTim--11

IL-5

IntermediateEarly Late

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New model: ConclusionNew model: Conclusion

This animal model with the This animal model with the

selected parameters selected parameters

is promising to discriminate between is promising to discriminate between

Strong AllergenStrong Allergen Weak allergenWeak allergen

However: Standardisation of the model is necessaryHowever: Standardisation of the model is necessary

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General ConclusionGeneral Conclusion

=> => Standardisation / International validation Standardisation / International validation

Need for more proteins to be testedNeed for more proteins to be tested

=> => Several animal models are neededSeveral animal models are needed

* * Clinical modelClinical model vs.vs. Mechanistic modelMechanistic model

ASA scoringASA scoring IgEIgE, , IgGsIgGs, cytokines, etc., cytokines, etc.

* * ProteinProtein vs.vs. FoodFood

Purity & conformationPurity & conformation Matrix & ProcessingMatrix & Processing

=> => Weigh of evidence approach (Weigh of evidence approach (Codex Codex AlimentariusAlimentarius 2003)2003)

Thank you very much Thank you very much

for your kind attention.for your kind attention.

Sponsored bySponsored by

Bayer Bayer CropScienceCropScience

Special thanks to:Special thanks to:

Dr. C. Dr. C. HerouetHerouet--GuicheneyGuicheney (BCS)(BCS)

Dr. R. Bars (BCS)Dr. R. Bars (BCS)

Prof. M. Prof. M. PallardyPallardy (Univ. Paris XI)(Univ. Paris XI)

Prof. N. Prof. N. GlaichenhausGlaichenhaus (Univ. Nice)(Univ. Nice)

BCS participants to the workBCS participants to the work

Dr. de Dr. de BarbeyracBarbeyrac

Dr. P. KennelDr. P. Kennel

Dr. D. Dr. D. RouquiéRouquié

BCS BCS ZootechZootech & Technical Groups& Technical Groups

Hatice Aldemir

Ara h1

02

46

810

1214

1618

PAP

0

24

6

810

12

1416

18

Late Marker => ProteinLate Marker => Protein--specific specific IgGIgG

Peanut Lectin

0

2

4

6

8

10

12

14

16

18

RUBISCO

0

2

4

6

8

10

12

14

16

18

0.0025 0.01 0.04 0.16 0.64 2.5 mg/ml 0.0025 0.01 0.04 0.16 0.64 2.5 mg/ml

0.0025 0.01 0.04 0.16 0.64 2.5 mg/ml 0.0025 0.01 0.04 0.16 0.64 2.5 mg/ml

Hatice Aldemir

Molecular mechanism of allergyMolecular mechanism of allergy

Differentiation

ProliferationAllergenAllergenSecondary contactSecondary contact

T helper 0T helper 0NaiveNaive

AllergenAllergen

Primary contactPrimary contact

DCDC

T helper 0T helper 0NaiveNaive

NonNon--AllergenAllergen

Primary contactPrimary contact

DCDC

Th 2Th 2

TimTim--11

TimTim--33

Th 1Th 1

Differentiation

NonNon--AllergenAllergenSecondary contactSecondary contact

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Blood sample CentrifugationSerum recovery

The protein

Serum dilutions

Antibody against mouse antibodyEvaluation of OD by

spectrophotometre

ELISA (ELISA (IgsIgs, Cytokines, etc..), Cytokines, etc..)

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+

+

+

+

+

+

+

+

+

SupernatantSupernatant

IFN- γ

IL-4

IL-5

CytometricCytometric Bead Array (CBA)Bead Array (CBA)

Hatice Aldemir

Analysis of cytokines by Analysis of cytokines by Cytometric Bead Array (Cytometric Bead Array (CBA)CBA)

Fluorescence Intensity=

Quantity of cytokine

Col

or o

f the

bea

ds

=

Diff

eren

tes c

ytok

ines

ILIL--44ILIL--55

IFNIFN--γγ

FACS In CNRS Lab.

Becton Dickinson (BD)

Hatice Aldemir

CD4 => T helper cell markerCD4 => T helper cell marker

CD4-FITC

Total splenocytes are analysed according to size and granularity C

ell g

ranu

lart

y

Cell sizeC

ell g

ranu

lart

y

Total splenocytes

Non-stained cells

CD4-FITCCD4-FITC

T helper cells are marked with the specific antibody CD4-FITC

T helper marker-stained cells

T helper cells

Non-Th cells

Hatice Aldemir

Endotoxins (Endotoxin Units / mg protein)Endotoxins (Endotoxin Units / mg protein)

100 EU/mg

Threshold for cytokine response bias.Ref: Dearman 2007

Endotoxin levels

10

4105

10

7800

1028

10 30 10 89 150

1000

2000

3000

4000

5000

6000

7000

8000

9000

Arah1 PAP PNA RUB SLP

EU e

ndot

oxin

/ m

g pr

otei

n

Before Purification After Purification

< < < <

murine models for evaluating the allergenicity of

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