Evaluating traditional plant breeding. To date, over

Evaluating
potential allergenicity of genetically modified (GM) food crops

Preface

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Genetically modified food crops
developed by recombinant DNA technology aims at improving food quantity and
quality. Transgenic proteins expressed by GM crops improve crop characteristics
like nutritional value, taste, texture and endow the host plant with resistance
against fungus, pests and insects.  With
the advancements in the field of agricultural biotechnology, the number of GM
varieties ready for commercialization is increasing in proportion with the area
under GM crop cultivation and the number of countries consuming GM crops.
However, prior to release of a GM crop in the market it must pass through a
rigorous safety evaluation tests to ensure its safety for feed, fodder and the
environment. In past few decades several international regulatory authorities’
have defined guidelines for the safety assessment of transgenic food products.
These protocols have been modified from time to time and efforts are underway
to ensure complete safety of the consumers. FAO/WHO, 2001 and Codex, 2003
guidelines for safety assessment of genetically modified foods are the most
widely accepted amongst all of the available guidelines. Safety evaluation
studies do not rely on a single parameter, therefore, we take into account
results from different test to define the safety of a GM crop on a relative
scale.

 

Introduction

Recent
advances in the field of r-DNA technology and improved agricultural equipments
have paved the way for large scale cultivation of genetically modified crops. Molecular biology methods are very
precise and aim at introducing only one or, at most, a few
well defined genes; rather than inserting parts of chromosomes as in
traditional plant breeding. To date, over 30 million hectares of transgenic
crops are being grown for human consumption. These are high yielding varieties resistant to pathogen attack and
tolerant to environmental imbalances like drought, cold and salinity (Huang et
al., 2005; Raman, 2017). Transgenes
expressed in GM crops encode for proteins foreign to the wild type varieties.
These proteins must be evaluated for their potency to act as novel allergens,
before using GM crops as feed or fodder. Transgenic proteins expressed in host
plant may share homology with the host plant proteins or these may share
identity with the reported food allergens (Eizaguirre et al., 2006; Romeis et
al., 2006). Therefore, prior to the release of GM crops in
the consumer market it is essential to conduct safety assessment of the GM
crops and/or transgenic proteins (Singh et al., 2008; Lu et al., 2018; Andrew
et al., 2018). Along with this GM food labeling should also be made mandatory
for all new and existing varieties.

Food
allergy is defined as an adverse health effect arising from a hypersensitivity reaction induced upon
exposure to a particular food allergen that occurs reproducibly on subsequent
exposures. Type 1 hypersensitivity food allergic reactions are
characterized by activation of mast cells and basophils, and
results in histamine release followed by synthesis of other inflammatory
molecules like leukotrienes, prostaglandins, cytokines etc.  These events manifest into severe allergic
symptoms like eczema,
hives, allergic rhinitis, asthma and gastrointestinal (GI) tract allergies. Food
allergy is reported by individuals of all age groups; however, the current
prevalence reported is 10% of children and 6% of the adults in Western countries (Sicherer &
Sampson, 2018). In general population – self reported prevalence of food
allergy ranges from 5% in Korea, 3.5% in France and 22.2% in Australia (Falcáo
et al., 2004). Food allergy is a major
health issue, and food related allergic reactions can often be life threatening
like food induced anaphylaxsis (Allen and Koplin, 2012). Food allergic
reactions may be IgE-mediated, non–IgE-mediated, both IgE/non-IgE mediated, or
cell-mediated immune reactions, induced upon exposure to the allergenic food.

In Europe and
most of the Western countries, 90% of the food allergic reactions are elicited
due to exposure to one or more of the following commodities like – milk, egg,
wheat, soy, peanut, tree nut, shellfish and fish (Sampson, 1999). However, the
prevalence of food allergy is greatly influenced by age and dietary
habits. 

 

Regulatory guidelines for safety assessment
of transgenic proteins 

Protocols
commonly used for safety assessment of GM food crops are drafted in compliance
with internationally acclaimed guidelines released by Organization for Economic
Cooperation and Development (OECD) (OECD, 1993), Food and Agriculture
Organization (FAO) of the United Nations/World Health Organization (WHO)
(FAO/WHO, 2001) and the Codex Alimentarius Commission (Codex, 2003).

For
the first time, when the need to evaluate the safety of the GM crop was felt,
it was compared to its native counterparts. The aim was to identify differences
between the two types of varieties (GM/non-GM) and whether the differences
would account for any unintended allergies. Later, FAO & WHO in 1991, in a
joint consultation proposed the idea of substantial equivalence (SE), and later
in 1993 OECD further elaborated on the principles of “SE” (OECD, 1993). Joint
FAO/WHO Expert Consultation on ‘Safety Aspects of Genetically Modified Foods of
Plant Origin’ proposed the usefulness of the concept of substantial equivalence
and concluded that “there were presently no alternative strategies that
would provide a better assurance of safety of GM foods than the appropriate use
of the concept of substantial equivalence”. Substantial equivalence (SE)
measures whether the genetically modified variety is safe as its traditional
counterpart, if such counterpart exists or to an earlier approved variety. SE
is evaluated on a scale of three – where one (1) refers to
“complete”, two (2) means “partial” and three (3) stands
for “not at all”. SE (1) implies that the GM food is similar to the
native counterpart; SE (2) explains that GM food is substantially equivalent
except for the inserted gene and SE (3) i.e. ‘not at all’ means that the GM
food is not at all equivalent to its counterpart and thorough evaluation of the
transgenic food is mandatory (Kupier et al., 2002). However, the comparison of
the GM & non-GM varieties is bound by certain limitations; like both the
varieties should be grown under similar environmental conditions and the choice
of the statistical designs for field trials should be similar for both the
varieties. A detailed safety assessment strategy involves step by step
evaluation of the transgenes expressed in the host plant (Metcalfe et al.,
1996; Panda et al., 2013).

Most commonly used safety assessment protocols primarily
evaluate the source of the transgenes, followed by bioinformatic studies to
compare the transgenic protein sequences with the reported allergens. Depending
on the results obtained for the preliminary tests, the transgenic protein is
assessed for pepsin resistance (simulated gastric fluid digestion), thermal
stability, and IgE binding with food allergic patients’ sera (serum screening
by ELISA) (Mishra et al., 2010; Arora and Mishra, 2011).

            As per the decision tree proposed
by FAO/WHO, 2001 (Figure 1), weight of evidence approach by Codex, 2003
(FAO/WHO, 2001), and guidelines given
by ICMR, 2008 (ICMR, 2008) below are mentioned some of the parameters to be
assessed for safety testing of GM crops:

 

Source
of the gene: Use of recombinant DNA
technology for development of genetically modified food crops involves transfer
of genes from a wide variety of organisms (donors). However, the conventional
breeding programs involved crossing between closely related species or genera.
Therefore, regulatory bodies have laid emphasis on evaluating the source of the
transgene, because diversity in selection of the donor species may account for
unforeseen allergenicity. Nordlee and group in 1996, reported
that transgenic soybeans expressing 2S albumin
(major allergen) protein from Brazil nut, displayed allergenic properties. And
this was one of the landmark studies, emphasizing on the importance of
evaluating the source of the transgene for developing GM food crops. Depending
on the information available transgene donors have been categorized as
allergic/non-allergic or moderately allergic. Events where the GM food crops,
contains genes from sources with known allergenic properties, rigorous in vitro
and in vivo safety testing procedures are employed to ensure safety of the GM
product  (Nordlee et al., 1996).

 

In silico – Sequence homology studies

Allergens are
proteins recognized by the immune system and specific antibodies are generated
in response to different proteins. Although, the human diet comprises of
several different types of proteins, but allergic reactions are elicited only
for some specific proteins in predisposed individuals. Hence, it is interesting
to know what renders the proteins as allergic/non-allergic. Presence of
epitopes on the proteins accounts for allergenicity. Epitopes may be linear
and/or conformational; these may be on the surface or cryptic. Therefore, in
order to evaluate the allergenic potential of a novel protein it is important
to investigate epitopic regions on the protein structure.

Previous
studies have reported that proteins sharing similarity among the primary and
tertiary structures may also share cross reactive epitopes (Aalberse, 2005;
Aalberse, 2006). And, these findings have formed the basis, for sequence
homology studies, to be used for assessing potential allergenicity of novel
proteins. A sequence homology study involves comparison of the primary amino
acid sequence, of the novel protein with that of the reported allergens. BLAST
(Altschul et al., 1997) and FASTA (Pearson & Lipman, 1988) are the most
commonly used alignment algorithms available online for bioinformatic studies.
Both the algorithms, predict functional similarity and clinically relevant
cross reactivity, on the basis of sequence similarity among proteins.
Therefore, FAO/WHO (2001) and Codex (2003), proposed that greater than 35%
identity over any stretch of 80 amino acids, between the GM protein and any
reported allergen, depicts that the GM protein may act as an allergen and
should be subjected to rigorous testing (Mishra and Arora, 2017; Compton et
al., 2017).

Hileman
and co-workers in 2002, reported that if proteins display significant linear
sequence similarity, they may share three dimensional structural motifs and
cross reactive epitopes (Hileman et al., 2002). Hence, high degree of
similarity among protein sequences i.e. transgenic protein and reported
allergens, requires IgE serum screening studies, to further validate the safety
of the protein in question (Goodman and Hefle, 2005; Mishra et al., 2012).
Several allergen databases like Food Allergy Research and Resource Program
(FARRP), Structural Database of Allergenic Protein (SDAP) (Ivanciuc et al., 2003), AlgPred (Saha and
Raghava, 2006), EVALLER (Martinez-Barrio et al., 2007) etc are
available online for homology studies.

 

Pepsin resistance, in vitro digestibility
assay and thermal stability

FAO/WHO (2001), Codex Alimentarius (2003) and ICMR
(2008) have proposed that transgenic proteins should be assessed for simulated
gastric fluid (SGF)/intestinal fluid (SIF) digestibility and thermal stability
before use in crop development. Thermal stability of food proteins are evaluated
over a broad temperature range i.e. 25°C to 95°C for up to 60 minutes (Metcalfe
et al., 1996). While, SGF/SIF tests are designed to mimic the physiological
conditions of gastric digestion and evaluate the allergenic potential of
foreign proteins.

Several studies suggest that a correlation exists
between the potential of a protein to act as an allergen and its resistance to
SIF digestion, pepsin degradation and thermal stability (Mishra et al., 2015).
For example food allergens that sensitize through the oral route demonstrate
stability during gastric and/or intestinal digestion under physiological
conditions (Singh et al., 2006; Taylor et al., 1987; Astwood et al., 1996).
Since, most of the foods are cooked (boiled, fried, roasted, baked etc.) before
consumption, therefore, heat labile proteins might not elicit allergic
reactions, due to lack of potential epitopes. However, the reverse might be
true for heat stable proteins, as these might possess intact epitopic regions.

In addition, numerous reports have also demonstrated
that this correlation is not an absolute parameter, and several proteins act
differently i.e. proteins resistant to pepsin degradation or stable at high
temperatures might not be allergenic on interaction with the gut lining, while
heat and/or pepsin labile proteins may act as allergens (Fu et al., 2002; Fu,
2002; Bannon et al., 2002).

            Some of the commonly used food
processing techniques like refrigeration/freezing, canning, dehydration,
freeze-drying, pickling (salting), pasteurizing, fermentation, moist
or dry heating (Sathe et al., 2005) etc disrupts the 3D
conformation of some of the food proteins. And this in turn affects the overall
allergenicity of the foods (Besler et al., 2000), due to loss of conformational
epitopes, activation of new epitopes or improving accessibility of cryptic
epitopes (Hefle, 1999). However, sometimes the processing methods may reduce antigenicity of the
food proteins, for example ?-irradiation has been reported to reduce the
antigenicity of ovalbumin, bovine serum albumin, milk protein and shrimp
tropomyosin (Kume & Matsuda, 1995; Lee et al., 2001; Byun et al., 2002).

 

IgE
serum screening studies

‘Decision
tree’ and ‘weight of evidence’ approach proposed by FAO/WHO, 2001 and Codex,
2003, respectively, involves IgE serum screening studies for safety assessment
of transgenic proteins. If the transgenic
proteins do not share significant sequence homology with any of the reported
allergens, under such circumstances random serum screening studies might not be
of any use. However, serum screening should be performed for
proteins showing greater than 35% homology with the reported allergens. Two
types of serum screening studies may be performed depending on the requirement
(Poulsen, 2004) i.e. specific or targeted serum screening.

In case if the transgene is
obtained from an allergic source or the transgene shares homology with any
of the reported allergens, specific serum screening is recommended (by
FAO/WHO, 2001). Specific serum screening studies, evaluates the IgE
binding potential of the transgenic protein, by ELISA, using patients’
sera from – individuals allergic to the source of the transgenic protein
and/or individuals allergic to other sources sharing cross reactive
epitopes with the source of the transgenic protein. These findings will
assess whether the allergen specific IgE antibodies present in patients’
sera react with the epitopes present on the novel proteins (Taylor and
Hefle, 2002).
While targeted serum screening,
involves evaluating the IgE binding potential of the transgenic protein by
ELISA, using sera from individuals allergic to a broad range of allergens.
For targeted serum screening studies, a wide range of allergen groups are
taken into consideration, because the information in context to the source
of transgene is limited.

 

The
major concern for conducting IgE serum screening studies is lack of proper
documentation of the serum donor and his/ her clinical history (Goodman, 2008).
If experiments demand the use of pooled serum, it is important to characterize individual
serum samples before use. This will limit a sample from dominating over others
and diluting the antibodies present in low abundance (Goodman, 2008). However,
the gold standard method for food allergy testing till date remains – a
positive response to oral food challenge, but is not commonly used for
practical and ethical reasons.

 

Animal
model studies

As
per FAO/WHO, 2001 decision tree approach, the allergenic potential of
transgenic proteins should be investigated by animal model studies, on
obtaining positive results from IgE serum screening studies. Different animal
models may be used for safety assessment studies like mice, rats, guinea pigs,
atopic dogs or neonatal swine (Penninks & Knippels, 2001; McClain &
Bannon, 2006; Van Gramberg et al., 2013; Bøgh et al., 2016). For safety testing
of transgenic proteins, food allergic conditions are developed in animal models
by administering well known allergens like ovalbumin, peanut allergens etc.
Followed by administration of the transgenic proteins in varying concentrations
(ranging from optimum to high) via different routes of sensitization like –
skin, oral, nasal, intra-peritoneal etc. and symptoms are scored as per the
immunization protocols. These models evaluate the allergic potency of the
transgenic proteins on the basis of IgE production, Th-2 cytokine release, and
clinical responses on re-exposure. Although, these studies have the potential
to predict allergenicity of foreign proteins, however, these are only
sensitization models and do not reflect all aspects of food allergies in
humans.

Among the various animal models available, murine
models are the most commonly used, for food allergy related studies. In
addition to the numerous advantages associated with the use of murine models, a
major drawback is that – animals develop oral tolerance to the ingested protein
and do not show allergic symptoms. However, oral tolerance can be avoided by
the use of adjuvants like cholera toxin etc.

Therefore, depending on the available scientific
information, further studies are necessary to develop better animal models with
improved immunization protocols, capable of predicting potential allergenicity
of transgenic proteins and imitating human food allergy conditions.

 

Summary

GM
crops are engineered with specific transgenes incorporated in the genome, to
endow the host plant with enhanced characteristics like – disease resistance,
improved yield, biotic and abiotic stress tolerance etc. Although, classical
breeding also aims at improving the crop characteristics by incorporating
beneficial genes, however, the methodology followed is labor intensive and time
taking. GM crops possess an edge over other conventionally developed varieties,
but concern is raised against the safety of the foreign gene used for GM crop
development.

Therefore, regulatory bodies have released guidelines for safety
assessment of genetically modified food crops. The foreign gene and/or the transgenic
protein should be assessed for intended and unintended effects, immediate and
long term effects via animal model studies etc. The
current protocols used for allergenicity assessment of GM food crops are not
completely predictive of the safety of the foreign proteins. Consequently,
further refinement is required based on scientific research and findings.
Future protocols should not only target safety assessment of foreign proteins,
instead should also focus on limiting the use of allergenic components in
development of GM crops.

Some of the non-government organizations and
environmentalists hold an opinion that until there are validated and accepted
methods for detection of potential allergenicity, there should be no further
approvals of GM crops and foods, and existing approvals should be suspended.
Rather than simply increasing the use of animal testing, which will not
necessarily reflect human allergic reactions, there is a need to question the
actual need for a GMO before the testing phase is reached. The need for the
product must justify both the expense and the ethical issues involved in its
testing.

 

 References 

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