In al., 1997) and FASTA (Pearson & Lipman,

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.

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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).

 

 

 

 

Figure 1:- Safety Assessment guidelines used for GM
food testing

 

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. 

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