Regulatory Toxicology and Pharmacology 54 (2009) S52–S57

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Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Murine models for evaluating the allergenicity of novel proteins and foods
Hatice Aldemir a,b,*, Rémi Bars b, Corinne Herouet-Guicheney b
a
University of Paris Sud XI, Faculty of Pharmacy, 5 rue J.B. Clément, 92290 Châtenay Malabry, France
b
Bayer CropScience, 355 rue Dostoïevski, 06903 Sophia-Antipolis, France

a r t i c l e i n f o a b s t r a c t

Article history: Genetically modified crops convey many benefits to world population. However, a rigorous safety assess-
Received 1 September 2008 ment procedure, including an evaluation of the allergenic potential, is fundamental before their release
Available online 7 December 2008 into the food chain. As an integral part of the safety assessment process, regulatory authorities worldwide
strongly recommend the use of tests that can predict the allergenic potential of the novel proteins. All
Keywords: guidance documents are based on an array of tests that have been proposed in 2003 by the Codex Alimen-
Mouse model tarius. Although the animal model is not a requirement of the Codex Alimentarius weight of evidence
Food allergy
approach, allergenic hazard of novel proteins could only be evaluated by an in vivo model that can poten-
Allergenicity
Protein
tially identify and distinguish commonly allergenic proteins from rarely allergenic proteins. Therefore,
Genetically modified food food allergy experts encourage its development. During the 2007 International Life Science Institute (ILSI)
workshop (Nice, France), worldwide experts shared their latest research results on rodent models to eval-
uate the allergenic potential of proteins and foods. This review presents the most promising rodent mod-
els for assessing food protein allergenicity that were evaluated during this ILSI workshop.
Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction sponse by mediator release (Gould et al., 2003; Sampson, 2004).

In recent years, a number of agricultural crops have been devel-
oped with biotechnology methods. The modifications in these
crops target deficiencies in nutrients, improve insect or salt resis-
tance so that less pesticide can be used or plants can grow under
non-optimal conditions and can often be produced in larger quan-
tities on less land. However, introduction of a novel gene into the
plants implies supplementary safety appraisals. Crucial parameters
of the risk assessment process of a novel product are nutritional
value, environmental impact, toxicity, and allergy.
Food allergy is characterized by sensitization to harmless food
proteins with production of allergen-specific immunoglobulin
(Ig) E, which binds to receptors on circulating basophils and mast
cells in mucosal tissues. Upon recurrent exposure to the same
allergen, cross-linking of cell-bound IgE induces an allergic re-
Abbreviations: Ara h, allergen from Arachis hypogea (peanut); ASA, active
systemic anaphylaxis; BW, buckwheat; CFA, complete Freund’s adjuvant; CT,
cholera toxin; EFSA, European Food Safety Agency; d, day; ELISA, enzyme-linked
immuno-sorbent assay; EPA, Environmental Protection Agency; FAO, Food and
Agriculture Organization; GM, genetically-modified; HESI, Health and Environmen-
tal Sciences Institute; ILSI, International Life Sciences Institute; Ig, immunoglobulin;
i.d., intra-dermal; i.p., intra-peritoneal; MCP-1, mast cell protease-1; OVA, ovalbu-
min; PATC, Protein Allergenicity Technical Committee; PCA, passive cutaneous
anaphylaxis; WHO, World Health Organization.
* Corresponding author. Address: University of Paris Sud XI, Faculty of Pharmacy,
5 rue J.B. Clément, 92290 Châtenay Malabry, France.
E-mail address: Aldemir.HA@gmail.com (H. Aldemir).
The clinical picture of food allergy is pleomorphic and can range
from chronic gastrointestinal symptoms to severe anaphylaxis
(Eigenmann et al., 2008). This adverse hypersensitivity response
to food poses an increasing public health problem (Kagan, 2003;
Roehr et al., 2004). Together with the risk of introducing novel
allergens to food or modifying existing proteins, allergenicity of
novel proteins needs more tools to re-assure the safety for people
and animals.
The importance of the correct evaluation of biotechnology-de-
rived foods by different means has been emphasized in great detail
(Metcalfe et al., 1996; Malarkey, 2003; Metcalfe, 2005). The aller-
genicity evaluation of transgenic foods mainly focuses on the
source of the gene, sequence homology to known allergens, sero-
logical identity and assessment of pepsin stability (Taylor, 2006;
Goodman et al., 2008). In addition, as an issue of the complex se-
quence of events that results in an allergic reaction, other safety
elements are compulsory for a complete evaluation of allergenic
potential of proteins and should be provided on the case by case
basis (Ladics, 2008).
The necessity to develop new tests for testing proteins was
highlighted in several guidance documents. In particular, only
validated animal models may help to address the risk that involves
expression of novel proteins that may become allergens de novo.
Therefore, the guidance from the Food and Agriculture Organiza-
tion (FAO) and the World Health Organization (WHO) suggested
to develop in vivo models for assessing the allergenicity potential
(FAO/WHO, 2001). The Codex Alimentarius (CAC, 2003) and

0273-2300/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.yrtph.2008.11.004
 H. Aldemir et al. / Regulatory Toxicology and Pharmacology 54 (2009) S52–S57 S53

European Food Safety Authority (EFSA, 2006) have also published of the protein as well as the frequency of exposure can orientate
the interest of the development of an in vivo model as part of the the immune reaction towards different directions (sensitization
safety assessment. More recently, the United States of America versus tolerance pathways). A reliable animal model should be able
Environmental Protection Agency (US EPA)’s Office of Research to discriminate between an allergen and a non-allergenic protein.
and Development has awarded two grants on the development of Therefore, including non-allergenic proteins as controls is central
animal models and has also developed an in-house mouse model for the validation of a model.
(Bowman et al., 2008). The presence of adjuvant plays also an important role by stimu-
This review paper presents in details the most promising mouse lating the immune system. IgE levels, for example, were found to
models that has been presented and challenged by food allergy ex- be higher in a mouse model of food allergy when alum was used as
perts, during one of the most recent International Life Science an adjuvant while IgG1 antibody levels did not differ with or without
Institute (ILSI) Protein Allergenicity Technical Committee (PATC) alum (Silva et al., 2006). The mucosal adjuvant CT induced Th2 cells
workshops, in presence of eminent academic, regulatory, and and IL-4 secretion in mice and caused biased results in a few models
industrial representative members (Thomas et al., 2008). (Marinaro et al., 1995). Alum adjuvant boosted adaptive immunity
by inducing uric acid and activating inflammatory dendritic cells
(Kool et al., 2008). In general, the impact of adjuvant on the immune
2. Choice of the mouse species for testing food and protein
system was major and, interestingly, the detailed mechanisms of ac-
allergenicity
tion of all the existing adjuvants remain unknown.
Likewise, the food matrix can alter the immunogenicity and
Mouse models are favorable since they possess certain advanta-
allergenicity as demonstrated by a study showing that whole pea-
ges compared to other species. Their small size and their short breed-
nut extract possessed a higher immune-stimulating capacity in
ing cycle are certainly important. More specifically, their
comparison to purified peanut allergens (van Wijk et al., 2005).
immunology is well-characterized. In particular, IgE is considered
Similarly, in a separate study, the plant-derived bioactive com-
to be a critical biological marker of allergenicity of proteins and sev-
pound saponin increased intestinal permeability and co-adminis-
eral strains are inbred high IgE responders as shown in this article.
tration of saponin with OVA induces higher IgE production
Moreover, various immunological and molecular reagents are
(Atkinson et al., 1996). More recently, the allergen Ber e 1 was
available for studying this species (Dearman et al., 2003a). Many
shown to induce differential IgE responses when administered
biomarkers involved in the specific IgE production sequence can
with different plant lipid compositions during the sensitization
be enlightening to reveal the allergenic potential. In the recent lit-
phase (Dearman et al., 2007). In contrast to the dose correlated
erature, several mouse allergy models were described for testing
IgG levels against an antigen, lower doses of allergens were shown
the allergenic potential by use of distinct markers and are reported
to result in more vigorous IgE responses (Li et al., 2000; Lee et al.,
later in this manuscript.
2005). This indicated the necessity of dose-response analysis for
Although only a relatively limited number of proteins have been
each protein or food tested in animal models.
examined to date in mice, marked and very significant differences
Other components like presence of lipopolysaccharides (LPS) or
in the ability of allergens and non-allergens to stimulate IgE re-
endotoxins are important compounds with regard to the immunity
sponses have been demonstrated over a wide dose range, and un-
status. For most strains of animals, LPS may alter immune re-
der conditions where the same proteins are of equivalent overall
sponses through Toll-like receptor 4 (Eisenbarth et al., 2002; Bashir
immunogenicity in terms of IgG antibody production (Atherton
et al., 2004; Berin et al., 2006). It is possible that the different re-
et al., 2002; Dearman et al., 2003a).
sponses to an allergen are a result of varying levels of endotoxins
Moreover, the sequence of events resulting in IgE in mice can be
(Eisenbarth et al., 2000; Delayre-Orthez et al., 2004; Thomas,
briefly described as antigen presentation by dendritic cells (DC) to
2005). Also post-translational modifications of the proteins can
T helper (Th) cells, Th2-type cytokine secretion and isotype class
play a role in the allergenicity (Hoffmann-Sommergruber, 2002).
switching to from IgG to IgE in B lymphocytes (Geha et al.,
Lectins are major constituents of many plant proteins and they
2003). It is identical to the sequence observed in humans (Eigen-
can interact with specific carbohydrates on cell-bound receptors.
mann et al., 2008). Furthermore, as in humans, two separate
For example, potato lectin was shown to interact with the chitobi-
phases are required for eliciting the clinical reaction: the sensitiza-
ose core of IgE glycans (Pramod et al., 2007).
tion phase followed by the challenge phase (Helm, 2002).
In conclusion, the quality of the material tested is critical as this
Overall, mouse models provide powerful tools for dissection of
may strongly impact the outcome of each study. Of course, the po-
pathogenic mechanisms of human disease and for testing new
tential impact of adjuvant should be studied in a given animal
therapies. In particular, differences in strain genetic backgrounds
model as this may alter the interpretation of the results by hyper-
have been suggested to parallel a genetic predisposition in humans
stimulating the immunity. There is also a need for further charac-
(Helm, 2002). Nevertheless, its use for predicting any allergenic po-
terizing each individual protein (with regards to the potential post-
tential of novel proteins, including those which are expressed in
translational modifications, folding for a correct immune recogni-
the genetically modified crops, should be demonstrated. In partic-
tion, etc). When not purified, protein extracts should also be deeply
ular, the route of antigen exposure, dose, response characteristics
evaluated with regards to the presence of other components that
including factors that may impact them, may mimic only partially
could impact the allergenic reaction like endotoxins, LPS, lipids,
the human disease. In addition, each model presents opportunities
carbohydrates, other proteins, etc.
for and barriers to a fuller understanding of the allergic response.
The conditions inherent to each model and the intended purpose
of the study should therefore be considered prior to its use (McC-
4. Biological markers
lain and Bannon, 2006).
4.1. IgE and IgG1 markers
3. Test material and adjuvants
Although there is not an absolute correlation, IgE and IgG1 are
For establishing an in vivo model, test material has an undeni- the only isotypes that potentially cause the anaphylactic reactions
able importance (McClain and Bannon, 2006). Animal models de- in mice (Baeza and Zubeldia, 2007). IgE is the most commonly used
scribed in the next sections have shown that the nature and dose marker of allergenicity. In animal models, total or specific IgE is
S54 H. Aldemir et al. / Regulatory Toxicology and Pharmacology 54 (2009) S52–S57
very often measured by ELISA (Birmingham et al., 2003). A strong
correlation between total and allergen-specific IgE responses in
different mice species has been shown during both primary and re-
call immune responses (Lewkowich et al., 2004). However, compe-
tition with IgG may mask the IgE detection in ELISA. Indeed, the
quality of detection can be enhanced by removal of IgG without
any significant impact on the assay sensitivity (Adel-Patient
et al., 2005; Birmingham et al., 2003). Reliable protein-specific
IgE measurements can also be performed by ELISA that makes
use of digoxigenin- (van Wijk et al., 2004) or biotin-labelled pro-
teins (Strid et al., 2004). Passive cutaneous anaphylaxis (PCA) is
also used for specific and biologically active IgE measurement in
mice. However, a comparison between of PCA and ELISA results
is cumbersome since different outputs are obtained. By using the
PCA method, induction of IgE antibody by food proteins with vary-
ing allergenic potentials was shown to be a relatively robust meth-
od (Dearman et al., 2003; Dearman and Kimber, 2007; Herouet-
Guicheney et al., 2008).
Food-specific IgG1 antibody levels that indicate relative immu-
nogenicity, do not distinguish commonly allergenic foods from
rarely allergenic or non-allergenic foods (Birmingham et al.,
2002; Tay et al., 2007). Mouse IgG1 is comprised of two function-
ally distinct and independently regulated types of antibodies. One
type possesses anaphylactic activity and its synthesis is completely
dependent on IL-4 synthesis, however, the other one lacks this
activity and its synthesis is stimulated by IL-12 and IFN-c (Fa-
quim-Mauro et al., 1999). Higher amounts of IL-4 are required to
drive IgE synthesis than those that are sufficient for the synthesis
of IgG1 (Fear et al., 2004). These two types of IgG1 should be fur-
ther identified in animal models for potentially identifying the
allergenic potential of GM proteins.
Therefore, for the development of an animal model, besides the
induction of specific IgE against a protein, the utility of the IgG1
subtype may be explored. In addition, the presence of IgE is essen-
tial for inducing an anaphylactic response but not sufficient. It may
be very important, without additional molecular end-points, to
show clinical effects after challenge.
4.2. Cytokines and other markers
Th2 cells play a role both during the sensitization and during
the effector phases of the allergic inflammatory response (Arps
et al., 1998; Toellner et al., 1998). Several cytokines, which are re-
leased by this subset of cells, are involved in the mechanism of the
allergic response by inducing IgE class-switch and secretion of IgE
antibodies (Gould, 2003). In animal models, cytokine analyzes
were shown to be useful as an alternative to serological analysis
(Kagan, 2003). For instance, IL-4 is the main cytokine released from
Th2 cells and it is also critical for the development of the allergenic
reaction together with IL-13 (Geha et al., 2003). However, inter-
feron gamma is known to antagonize the allergenic reaction (Betts
et al., 2004). Another Th2-type cytokine, IL-5 is also often used as
allergy marker. Very recently, significant IL-5 responses in re-
sponse to major peanut allergen Ara h 1 in comparison to a non-
allergenic potato protein induced strong Th1-type cytokine re-
sponse in our i.p.-sensitization BALB/c animal model (unpublished
results).
More generally, other components that are up-regulated or
down-regulated in hypersensitivity reactions can be potential
end-points if they are shown to be differentially regulated in re-
sponse to an allergen in comparison to a non-allergen. For in-
stance, the accumulation of mast cell granule neutral proteases
in peripheral blood could be used as a specific marker of mast cell
activation after an allergic response (Sagi-Eisenberg, 2007). In
addition, cell surface molecules (Wong et al., 2005; van Wijk
et al., 2007a,b) or transcription factors (Birmingham et al., 2007)
that are involved in differentiation into Th2 cells should be further
explored as alternative markers.
In conclusion, research is very active for finding new early and
accurate biological markers for predicting potential allergenicity of
proteins. Time of observation is very critical and kinetics should be
conducted before considering these markers as putative end-
points. Nowadays, none of these early markers can predict an aller-
genic potential.
5. Review of the mouse models
5.1. Oral sensitization models
For the evaluation of food allergy, oral dosing is the rational
choice, which explains the high number of oral sensitization mod-
els. However, in the absence of an adjuvant, a normal immune re-
sponse to dietary proteins is associated with the development of
mucosal tolerance rather than allergenicity (Chen et al., 1995;
van Wijk and Knippels, 2007). This state of non-responsiveness
of the immune system to soluble antigen depends on the dose,
exposure frequency and the nature of the antigen. Therefore, use
of an adjuvant e.g. cholera toxin (CT) is fairly common when this
route of exposure is chosen.
In an oral anaphylaxis model, IgE antibodies to the major pea-
nut (Arachis hypogea) allergens Ara h 1 and Ara h 2 were detected
in the serum of female C3H/HeJ mice (Li et al., 2000). Splenocytes
also showed proliferative responses to both whole peanut extract
and pure proteins. Of interest, low doses (5 mg) in comparison to
high doses (25 mg) resulted in higher IgE levels. Mast cell prote-
ase-1 (MCP-1) and histamine levels, together with active anaphy-
laxis scoring, indicated the allergenic potential. This model
closely reflected the clinical characteristics of peanut allergy in hu-
man subjects. In particular, specific Ara h 2 epitopes were shown to
be identical between two species. In a slightly modified version of
this model, specific polyisotypic antibodies were observed together
with a mixed Th1 and Th2 cytokine effect in the spleen at 1 and 7
weeks after the onset of exposure (van Wijk et al., 2004). IgE re-
sponses to the major peanut allergens Ara h 1 and Ara h 3 and to
a lesser extent Ara h 2 and Ara h 6 were also reported.
Moreover, regulatory T lymphocytes (CD4+CD25+ T cells) play an
important role in the control of tolerance induction and regulation of
the intensity of allergic responses to peanut (van Wijk et al.,
2007a,b). For instance, a murine model was developed to investigate
immunoregulatory processes after ingestion of peanut protein and
compared it to a model of oral tolerance in the presence of complete
Freund’s adjuvant (CFA) (Strid et al., 2004). It was demonstrated that
oral tolerance induction was highly dose-dependent and differed for
the allergenic proteins peanut and chicken egg ovalbumin (OVA).
Tolerance to peanut required a significantly higher oral dose than
tolerance to OVA and low doses of peanut were more likely to induce
oral sensitization and increased production of IL-4 and specific IgE
upon challenge. In this model, tolerance was in concert with sup-
pressed Th1 and Th2 responses. Of importance, these results showed
that oral tolerance to peanut can be induced experimentally
although peanut proteins have a potent sensitizing effect.
Furthermore, a murine model for testing buckwheat (Fagopy-
rum esculentum) was set up by intragastric (i.g.) sensitization and
challenge (Lee et al., 2005). IgE response was higher in the low dose
group (1 and 5 mg) although the same profile was observed at IgG1
and IgG2a levels in all treatment groups. Highest dose levels
(25 mg) did not induce any Ig production and this was in line with
systemic anaphylaxis scores. Buckwheat extract also stimulated
splenocyte proliferation and cytokine secretion. IL-4 and IL-5 re-
sponses were significantly increased at all dose levels and, again,
were highest at the lowest dose level (1 mg).
 H. Aldemir et al. / Regulatory Toxicology and Pharmacology 54 (2009) S52–S57
More recently, soybean (Glycine max) was shown to induce
allergic responses in a BALB/c model when administered by
the oral route in the presence of CT (Gizzarelli et al., 2006).
Wild-type and genetically modified (GM) soybean extract were
compared in soy-free diet-fed female mice. Following i.g.
administration of 1 mg of soybean extract per mouse together
with 10 mg of CT at days 0, 7, and 21, wild-type and GM de-
rived soybean extracts were highly comparable in terms of
the Ig and cytokine responses. Predominant Th2-type re-
sponses with high IgE and IgG1 versus low IgG2a and con-
comitant production of IL-4 and IL-5 were observed
(Gizzarelli et al., 2006).
As shown by local anaphylactic reactions and specific IgE re-
sponses, cow’s milk and peanut allergens can orally sensitize
BALB/c mice in the presence of CT (Adel-patient et al., 2005).
In a milk allergy model, early feedings with b-lactoglobulin (b-
LG) were shown to lead to active tolerisation, with a specific
pattern of cytokine secretion in Peyer’s patches (Cong et al.,
2004). IL-10 cytokine, which is secreted by a subset of regulatory
T cells, was shown to be involved in the modulation of tolerance
in a similar manner to observed clinical tolerance to foods that
involves IL-10-secreting cells in Peyer patches in another animal
model (Frossard et al., 2004).
The importance of strain selection was strongly emphasized
in an oral sensitization model (Morafo et al., 2003). In particular,
C3H/HeJ mouse strain has a propensity for high IgE production
compared to BALB/c (Reese et al., 2004). Homogenized cow’s
milk and crude peanut extract with CT were used for sensitiza-
tion of BALB/c and C3H/HeJ. BALB/c mice produced specific IgE
against peanut only; however, no clinical allergic reactions were
observed. On the other hand, C3H/HeJ mice showed both allergic
responses to cow’s milk and peanut. Surprisingly, in splenocyte
cultures of BALB/c mice, IFN-c production (Th1 cytokine) was
very high while IL-4 (Th2 cytokine) and IL-10 levels (T regula-
tory cytokine) were found to be increased in C3H/HeJ mice.
These results confirmed that development of food allergy de-
pends in part on cytokine responses driven by genetic variables
(Morafo et al., 2003).
Finally, another promising oral mouse model using C3H/HeJ
mice tested several protein extracts from peanut, Brazil nut, egg
white, spinach, or turkey, in presence of 10 lg CT (Bowman and
Selgrade, 2008). Mice were gavaged two or four times at a weekly
interval with 1, 2, or 5 mg (total protein) of food. This model was
able to distinguish allergenic from non-allergenic foods at the
higher 2 mg dose. However, again there was no validation of the
transferability and robustness by other independent laboratories
and the range of tested food extracts was limited. Interestingly,
by using the same model, findings thus far suggest that there is
good correlation between digestive lability of foods and failure to
elicit an IgE response via the oral route, supporting the use of pep-
sin resistance in the current guidelines.
In conclusion, a multitude of oral animal model was devel-
oped. The results seem to be very dependent on the study de-
sign. In particular, the routes of exposure (oral, gavage),
together with the presence/absence of adjuvant, dose adminis-
tered and frequency of administration, are of importance. The
oral route appears to be relevant for testing food proteins; how-
ever, the interpretation of findings is complicated by the oral tol-
erance process, driven by the regulatory T cells and other
tolerance mechanisms. Several models used adjuvant for over-
coming this difficulty; however, the hyperstimulation of the im-
mune system may not reflect perfectly the type I allergic
mechanisms as observed in humans. This aspect should be fur-
ther evaluated with a large panel of proteins and/or food ex-
tracts as well as other strains with different genetic
backgrounds. More generally, the nature of the allergens admin-
S55
istered to animals play an important role and it is critical to
evaluate further the impact of allergen modification through
food processing, including digestion, as well as the clinical
importance of allergen cross-reactivity, before adopting one of
these models for the regulatory evaluation of GM foods.
5.2. Intraperitoneal sensitisation models
Intraperitoneal (i.p.) sensitization has been shown to be a
reliable, specific and sensitive method for inducing IgE antibod-
ies compared to oral exposure (Atherton et al., 2002).
In a BALB/c strain mouse model, which allowed discrimina-
tion between proteins on the basis of their relative ability to in-
duce IgE antibody responses, animals were treated with various
concentrations of the test protein without adjuvant (Dearman
and Kimber, 2007). The test sample was injected subcutaneously
into the mouse ears. After an incubation period of sufficient time
to allow the binding of IgE to its specific receptor on the surface
of the mast cells, the animals were administered again the anti-
gen by intravenous (i.v.) injection, together with a high molecu-
lar weight dye, Evans blue. IgE levels were evaluated by
homologous PCA in a pool of serum samples. The PCA test made
use of the classical anaphylaxis mechanism. Receptor-bound IgE
were cross-linked by the antigen and degranulation of mast cells
took place locally in the ears. As a consequence, vasodilatation
permitted the large molecular weight dye to extravasate to the
interstitial environment. The diameter of the blue mark on the
skin was measured and used as semi-quantitative measure of
IgE level for the corresponding dilution of serum sample. In par-
allel, immunogenic potential was assessed by ELISA as a function
of the induction of individual protein-specific IgG antibody re-
sponses, since IgG1 can interfere with the IgE reaction (Inagaki
et al., 1988).
Dissimilar sensitizing potentials of various proteins from pea-
nut, egg, spinach, potato, soybean, milk, and bacteria have been
illustrated with this protocol (Dearman et al., 2001, 2003b; Tho-
mas, 2005). For a more extensive evaluation of the accuracy and
repeatability of this approach, two inter-laboratory comparison
studies were conducted (Dearman et al., 2003, Herouet-Guicheney
et al., 2008). The first one compared the sensitization phase and the
latter aimed at comparing the elicitation phase of the PCA. Collec-
tively, the results obtained with these studies have revealed that
this method, although technically demanding, was accurately
transferred between laboratories. This model could be used to
evaluate the allergenicity if additional results demonstrate that
the model can discriminate known allergens from non-allergenic
proteins.
In an attempt to develop a hazelnut (Corylus avellana) allergy
model, immune responses in BALB/c and C57BL/6 mice were also
compared after i.p. sensitisation (Birmingham et al., 2005). Th2-
type cytokine responses were observed both in the presence or
absence of adjuvant alum. Direct elicitation of hazelnut-binding
IgE antibodies in both mice strains studied indicated an intrinsic
IgE inducing capacity of hazelnut extract.
In conclusion, the intraperitoneal (i.p.) injection models may
permit the most direct assessment of the allergic potential for
a novel protein, and it has been demonstrated that i.p. injections
may overcome the tolerance that may occur if the protein is
administered orally. Experience to date suggests that the mea-
surement of antibody (IgE) responses in mice shows some prom-
ise for the discrimination between allergens and those materials
that apparently lack allergenicity. Nevertheless, these studies
have been conducted with a limited number of proteins and fur-
ther experience with a wider range of proteins of differing aller-
genic potential is required before their use in the risk
assessment evaluation.
S56 H. Aldemir et al. / Regulatory Toxicology and Pharmacology 54 (2009) S52–S57
5.3. Transdermal sensitisation models
The dermal route of sensitization was also used for testing the
potential allergenic potential of proteins. The animal models that
use this route have an important role for exploring the mecha-
nisms of the immune reactions.
In an intradermal (i.d.) exposure model, treatment by peanut
over a 14 days protocol was sufficient to prime draining lymph
node cells for Th2 cytokine production (Betts et al., 2004). By using
non-allergen potato acid phosphatase, an enzyme from potato
(Solanum tuberosum), no shift to Th2-type cytokine secretion was
detected. This approach confirmed the utility of a large cytokine ar-
ray investigation to obtain more information on the inherent pro-
tein allergenicity.
The transdermal exposure model of hazelnut could induce a re-
sponse without adjuvant in female BALB/c mice (Birmingham et al.,
2007) similar to systemic sensitization model (Birmingham et al.,
2005). Hazelnut protein extract at 5, 50, or 500 lg per mouse
weekly during 6 weeks followed by an oral challenge with 13 mg
extract ten days after were applied. Mice were sacrificed 1 h after
the oral challenge to collect blood and spleen. Immunoglobulins
in serum and cytokines in cell cultures were tested. Sensitization
with 50 and 500 lg induced dose-related IgE, Th2 cytokines (IL-
4, IL-5, IL-13) and Th2-specific transcription factor, GATA-3 re-
sponses (Birmingham et al., 2007).
To circumvent oral tolerance and evoke differential IgE re-
sponses to a panel of allergenic (peanut, Brazil nut, egg white)
and non-allergenic food (spinach, turkey) extracts, female C3H/
HeJ mice were exposed subcutaneously twice at a 3-week interval
at 60 or 300 lg of food extract (Bowman and Selgrade, 2008). All
foods elicited IgE by the subcutaneous route suggesting that it is
inadequate to distinguish allergens from non-allergens.
In conclusion, several animal models were developed for further
investigating the food allergy immune pathways. The exploration
of the immune mechanisms is certainly useful to refine the end-
points to be studied for the elaboration of animal models for pre-
dicting food allergic potential. In order to be considered predictive,
the model would have to predict the allergenicity of a high per-
centage of known allergenic proteins and to discriminate them
from non-allergenic proteins.
6. Discussion and conclusion
Because of their small size, short breeding cycle and available
information on their immunology, mice are the best species for
use in mechanistic food allergy animal models. Nevertheless, the
natural complexity of the allergic reactions makes difficult to find
a single reliable marker to detect the sensitization potential of a
protein.
An accurate design is critical for any model. Mainly, most of
the models require several administrations of well-characterized
test material. Use of adjuvant may have adverse effects in animal
models since it can modify inherent immunogenic potential of
the test proteins. The oral and i.p. routes of exposure may be
used and it is recommended to test several doses. Many param-
eters should be controlled (frequency of exposure, presence of
other components, impact of processing, measure of the re-
sponse, etc).
As a hallmark of allergy, specific IgE level is an important bio-
logical marker and its titer should be observed. Monitoring of the
clinical signs reflecting human food allergy would be also helpful
to classify proteins in terms of allergenicity. However, these signs
are observed after challenge with the allergen and would be late
phase markers of the model. On the other hand, markers of early
phase sensitization could be more valuable since once the sensiti-
zation has occurred the risk of a challenge, even from cross-reac-
tive products, is more likely.
Of importance, none of these animal models, which are cur-
rently developed, are part of the Codex Alimentarius weight of evi-
dence or other regulatory guidance document (EFSA, US EPA)
approaches. These models should be validated with a large range
of allergenic and non-allergenic proteins before their use in the
health-decision making process. Developing technology and in-
creased knowledge of the allergic mechanism will help to improve
the existing animal models and use them for predicting protein
allergenicity.
Acknowledgments
We thank Pr. Marc Pallardy for his contribution on the issues re-
lated to the animal models for predicting potential allergenicity of
protein. We thank Dr. Helen Tinwell for her critical reading of the
manuscript.
References
Adel-Patient, K. et al., 2005. Peanut and cow’s milk-specific IgE, Th2 cells and local
anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera
toxin. Allergy 60, 658–664.
Arps, V. et al., 1998. Antigen dose-dependent differences in IgE antibody production
are not due to polarization towards Th1 and Th2 cell subsets. Eur. J. Immunol.
28, 681–686.
Atherton, K.T. et al., 2002. Protein allergenicity in mice: a potential approach for
hazard identification. Ann. N.Y. Acad. Sci. 964, 163–171.
Atkinson, H.A. et al., 1996. Brown Norway rat model of food allergy: effect of plant
components on the development of oral sensitization. Food Chem. Toxicol. 34,
27–32.
Baeza, M.L., Zubeldia, J.M., 2007. Immunology of anaphylaxis: lessons from murine
models. Curr. Allergy Astma Rep. 7, 49–55.
Bashir, M.E.H., Louie, S., Shi, H.N., Nagler-Anderson, C., 2004. Toll-like receptor 4
signaling my intestinal microbes influences susceptibility to food allergens. J.
Immunol. 172, 6978–6987.
Berin, M.C., Zheng, Y., Domaradzki, M., Li, X.M., Sampson, H.A., 2006. Role of TLR4 in
allergenic sensitization to food proteins in mice. Allergy 61, 64–71.
Betts, C.J. et al., 2004. Intradermal exposure of BALB/c strain mice to peanut protein
elicits a type 2 cytokine response. Food Chem. Toxicol. 42, 1589–1599.
Birmingham, N.P. et al., 2002. Relative immunogenicity of commonly allergenic
foods versus rarely allergenic and nonallergenic foods in mice. J. Food Prot. 65,
1988–1991.
Birmingham, N .P. et al., 2003. An ELISA-based method for measurement of food-
specific IgE antibody in mouse serum: an alternative to the passive cutaneous
anaphylaxis assay. J. Immunol. Methods 275 (1–2), 89–98.
Birmingham, N.P. et al., 2005. Hazelnut allergy: evidence that hazelnut can directly
elicit specific IgE antibody response via activating type 2 cytokines in mice. Int.
Arch. Allergy Immunol. 137, 295–302.
Birmingham, N.P. et al., 2007. An adjuvant-free mouse model of tree nut allergy
using hazelnut as a model tree nut. Int. Arch. Allergy Immunol. 144, 203–210.
Bowman, C.C., Selgrade, M.-J., 2008. Differences in allergenic potential of food
extracts following oral exposure in mice reflect differences in digestibility:
potential approaches to safety assessment. Toxicol. Sci. 102, 100–109.
Chen, Y. et al., 1995. Peripheral deletion of antigen-reactive T cells in oral tolerance.
Nature 376, 177–180.
Codex Alimentarius Commission (CAC). 2003. Report of the sixth session of the
Codex Ad hoc intergovernmental task force on foods derived from
biotechnology. Chiba, Japan, 27 November–1 December 2006. CL 2006/54-FBT.
Cong, Y. et al., 2004. Early upregulation of T cell IL-10 production plays an important
role in oral tolerance induction. Ann. N. Y. Acad. Sci. 1029, 319–320.
Dearman, R.J. et al., 2001. Characterization of antibody responses induced in rodents
by exposure to food proteins: influence of route of exposure. Toxicology 167,
217–231.
Dearman, R.J. et al., 2003a. Evaluation of protein allergenic potential in mice: dose-
response analyses. Clin Exp Allergy. 33, 1586–1594.
Dearman, R.J. et al., 2003b. Induction of IgE antibody responses by protein allergens:
inter-laboratory comparisons. Food Chem Toxicol. 41, 1509–1516.
Dearman, R.J. et al., 2007. Influence of plant lipids on immune responses in mice to
the major Brazil nut allergen Ber e 1. Clin Exp Allergy. 37, 582–591.
Dearman, R.J., Kimber, I., 2007. A mouse model for food allergy using intraperitoneal
sensitization. Methods 41, 910–998.
Delayre-Orthez, C. et al., 2004. Dose-dependent effects of endotoxins on
allergen sensitization and challenge in the mouse. Clin Exp Allergy. 34,
1789–1795.
Eigenmann, P., Beyer, K., Wesley Burks, A.W., Lack, G., Liacouras, C.A., Hourihane,
J.O’B., Sampson, H.A., Sodergren, E., 2008. New visions for food allergy: An iPAC
summary and future trends. Pediatric Allergy Immunology. 19, 26–39.
 H. Aldemir et al. / Regulatory Toxicology and Pharmacology 54 (2009) S52–S57
Eisenbarth, S.C. et al., 2002. Lipopolysaccharide-enhanced, Toll-like Receptor 4
(TLR-4)-dependent T Helper Cell Type 2 Responses to Inhaled Antigen. J Exp
Med 196, 1645–1651.
European Food Safety Authority (EFSA). 2006. Guidance document for the risk
assessment of genetically modified plants and derived food and feed by the
Scientific Panel on Genetically Modified Organisms (GMO). The EFSA Journal 99,
1-100.
FAO/WHO. 2001. Evaluation of Allergenicity of Genetically Modified Foods.
Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods
Derived from Biotechnology. Rome: Food and Agriculture Organization of the
United Nations.
Faquim-Mauro, E.L. et al., 1999. Cutting edge: mouse IgG1 antibodies comprise two
functionally distinct types that are differentially regulated by IL-4 and IL-12. J
Immunol. 163, 3572–3576.
Fear, D.J. et al., 2004. Transcription of Ig germline genes in single human B cells and
the role of cytokines in isotype determination. J Immunol. 173, 4529–4538.
Frossard, C.P. et al., 2004. Antigen-specific secretory IgA antibodies in the gut are
decreased in a mouse model of food allergy. J Allergy Clin Immunol. 114, 377–
382.
Geha, R.S. et al., 2003. The regulation of immunoglobulin E class-switch
recombination. Nat Rev Immunol 3, 721–732.
Gizzarelli, F. et al., 2006. Evaluation of allergenicity of genetically modified soybean
protein extract in a murine model of oral allergen-specific sensitization. Clin
Exp Allergy. 36, 238–248.
Goodman, R.E., Vieths, S., Sampson, H.A., Hill, D., Ebisawa, M., Taylor, S.L., van Ree,
R., 2008. Allergenicity assessment of genetically modified crops—what makes
sense? Nature Biotech. 26, 73–81.
Gould, H.J., 2003. The biology of IgE and the basis of allergic disease. Ann. Rev.
Immunol. 21, 579–628.
Helm, R.M., 2002. Food allergy animal models- An overview. Ann. N. Y. Acad. Sci.
964, 139–150.
Herouet-Guicheney, C., Aldemir, H., Bars, R., Kennel, P., Rouquié, D., Stahl, B.U.,
Dearman, R., Kimber, I. 2008. Inter-laboratory comparisons of assessment of the
allergenic potential of proteins in mice. J. Appl. Toxicol. 20 October 2008 [Epub
ahead of print].
Hoffmann-Sommergruber, K., 2002. Pathogenesis-related (PR)-proteins identified
as allergens. Biochem. Soc. Trans. 30, 930–935.
Inagaki, N. et al., 1988. Comparative study of IgG1 and IgE antibody mediated
homologous PCA in the mouse ear. Lack of cross-desensitization of IgG1
antibody mediated PCA to IgE antibody mediated PCA. Int. Arch. Allergy Appl.
Immunol. 86, 325–330.
Kagan, R.S., 2003. Food allergy: an overview. Env. Health Perspect. 111, 223–225.
Kool, M., Soullié, T., van Nimwegen, M., Willart, M.A.M., Muskens, F., Jung, S.,
Hoogsteden, H.C., Hammad, H., Lambrecht, B.N., 2008. Alum adjuvant boosts
adaptive immunity by inducing uric acid and activating inflammatory dendritic
cells. J. Exp. Med. 205, 869–882.
Ladics, G.S., 2008. Current codex guidelines for assessment of potential protein
allergenicity. Food Chem. Toxicol. 46 (Suppl. 10), S20–S23.
Lee, S.Y. et al., 2005. Murine model of buckwheat allergy by intragastric
sensitization with fresh buckwheat flour extract. J. Korean. Med. Sci. 20, 566–
572.
Lewkowich, I.P. et al., 2004. In vivo IgE levels in exogenous antigen stimulated
responses: measurement of total IgE as a valid, simple surrogate for Ag-specific
IgE. J. Immunol. Methods 286, 123–132.
Li, X.M. et al., 2000. A murine model of peanut anaphylaxis: T- and B-cell responses
to a major peanut allergen mimic human responses. J. Allergy Clin. Immunol.
106, 150–158.
Malarkey, T., 2003. Human health concerns with GM crops. Mutat. Res. 544, 217–
221.
S57
Marinaro, M. et al., 1995. Mucosal adjuvant effect of cholera toxin in mice results
from induction of T helper 2 (Th2) cells and IL-4. J. Immunol. 155, 4621–4629.
McClain, S., Bannon, G.A., 2006. Animal models of food allergy: opportunities and
barriers. Curr. Allergy Asthma Rep. 6, 141–144.
Metcalfe, D.D. et al., 1996. Assessment of the allergenic potential of foods derived
from genetically engineered crop plants. Crit. Rev. Food Sci. Nutr. 36 (suppl),
S165–S186.
Metcalfe, D.D., 2005. Genetically modified crops and allergenicity. Nat. Immunol. 6,
857–860.
Morafo, V. et al., 2003. Genetic susceptibility to food allergy is linked to differential
TH2–TH1 responses in C3H/HeJ and BALB/c mice. J. Allergy Clin. Immunol. 111,
1122–1128.
Pramod, S.N. et al., 2007. Potato lectin activates basophils and mast cells of atopic
subjects by its interaction with core chitobiose of cell-bound non-specific
immunoglobulin E. Clin. Exp. Immunol. 148, 391–401.
Reese, G., Schicktanz, S., Randow, S., Lehrer, S.B., Vieths, S., 2004. An animal model
for shrimp allergy: the influence of genetic background and adjuvant on the
allergen-specific IgE response. J. Allergy Clin. Immunol. 113, 233 (Abstract).
Roehr, C.C. et al., 2004. Food allergy and non-allergic food hypersensitivity in
children and adolescents. Clin. Exp. Allergy 34, 1534–1541.
Sagi-Eisenberg, R., 2007. The mast cell: where endocytosis and regulated exocytosis
meet. Immunol. Rev. 217, 292–303.
Sampson, H.A., 2004. Update on food allergy. J. Allergy Clin. Immunol. 113, 805–819.
Silva, A.S. et al., 2006. Regulation of anaphylactic IgG1 antibody production by IL-4
and IL-10. Int. Arch. Allergy Immunol. 141, 70–78.
Strid, J. et al., 2004. A novel model of sensitization and oral tolerance to peanut
protein. Immunology 113, 293–303.
Tay, S.S., Clarck, A.T., Deighton, J., King, Y., Ewan, P.W., 2007. Patterns of
immunoglobulin G responses to egg and peanut allergens are distinct:
ovalbumin-specific immunoglobulin responses are ubiquitous, but peanut-
specific immunoglobulin responses are up-regulated in peanut allergy. Clin.
Exp. Allergy. 37, 1512–1518.
Taylor, S.L., 2006. Review of the development of methodology for evaluating the
human allergenic potential of novel proteins. Mol. Nut. Food Res. 50, 604–609.
Thomas, K. et al. 2005. Evaluation of i.p. mouse models for assessing the allergenic
potential of proteins. AAAAI Congress, San Antonio.
Thomas, K., Herouet-Guicheney, C., Ladics, G., MacClain, S., MacIntosh, S., Privalle, L.,
Woolhiser, M., 2008. Current and future methods for evaluating the allergenic
potential of proteins: International workshop report 23–25 October 2007. Food
Chem. Toxicol. 46, 3219–3225.
Toellner, K.M. et al., 1998. T helper 1 (Th1) and Th2 characteristics start to develop
during T cell priming and are associated with an immediate ability to induce
immunoglobulin class switching. J. Exp. Med. 187, 1193–1204.
van Wijk, F. et al., 2004. Mixed antibody and T cell responses to peanut and the
peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 in an oral sensitization
model. Clin. Exp. Allergy 34, 1422–1488.
van Wijk, F. et al., 2005. The effect of the food matrix on in vivo immune responses
to purified peanut allergens. Toxicol. Sci. 86, 333–341.
van Wijk, F. et al., 2007a. CD4+CD25+ T cells regulate the intensity of
hypersensitivity responses to peanut, but are not decisive in the induction of
oral sensitization. Clin. Exp. Allergy 37, 572–581.
van Wijk, F. et al., 2007b. The CD28/CTLA-4-B7 signaling pathway is involved in
both allergic sensitization and tolerance induction to orally administered
peanut proteins. J. Immunol. 178, 6894–6900.
van Wijk, F., Knippels, L., 2007. Initiating mechanisms of food allergy: oral tolerance
versus allergic sensitization. Biomed. Pharmacother. 61, 8–20.
Wong, C.K. et al., 2005. Increased expression of plasma and cell surface co-
stimulatory molecules CTLA-4, CD28 and CD86 in adult patients with allergic
asthma. Clin. Exp. Immunol. 141, 122–129.