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29 Protein Allergy and GMOs

GS Ladics, DuPont Company, Haskell Global Centers for Health and Environmental Sciences, Newark, DE, United States
© 2018 Elsevier Ltd. All rights reserved.

11.29.1 Introduction 639

11.29.2 Food Allergy 641 Protein Families That Become Food Allergens 641 Food allergy prevalence 642
11.29.3 IgE-Mediated Food Allergy 643 IgE-Mediated Food Allergy Diagnosis 644 Food allergy thresholds 645
11.29.4 Celiac Disease 645
11.29.5 Safety Assessment of GM Crops 645 Guidance Documents 646
11.29.6 Evaluating Potential Allergenicity Risk 647
11.29.7 First Category of Potential Allergenic Risk: Transfer of a Known Allergen or Crossreacting Allergen into a Food
Crop 647 History of Safe Use: Evaluation of the Gene Source 647 Bacillus thuringiensis (Bt) 648
11.29.8 In Silico Bioinformatic Tools 649 Allergen Databases 650 Bioinformatic criteria for IgE-mediated allergy 650
11.29.9 Specific IgE Sera Screening Studies 653
11.29.10 Second Category of Potential Allergenic Risk 654 Abundance of Protein in the Crop and Stability to Pepsin In Vitro 654 Heat stability 655
11.29.11 Third Category of Potential Allergenic Risk: Assessment of Endogenous Allergens 656 Analytical Methods to Measure Endogenous Allergens 656
11.29.12 Endpoints Requiring Further Evaluation/Validation 657 Animal Models to Evaluate Potential Protein Allergenicity 657 Targeted serum screens 658
11.29.13 GM Crops: Is There Evidence of New Sensitization and Allergies? 659 The Brazil Nut 2S Albumin Protein and Soybean 659 StarLink maize 659
11.29.14 Summary 660
References 661
Relevant Websites 667

Adjuvant Any substance which nonspecifically enhances the immune response to antigen.
Allergen An antigen which causes allergy.
Allergy A misguided reaction by the immune system to harmless foreign substances.
Anaphylaxis An often fatal hypersensitivity reaction, triggered by IgE or anaphylatoxin-mediated mast cell degranulation,
leading to anaphylactic shock due to vasodilatation and smooth muscle contraction.
Antibody A protein on the surface of B cells that is also secreted in large amounts into the blood or lymph in response to an
antigen, a component within an invader such as a bacterium, virus, parasite, or transplanted organ. Antibodies neutralize the
antigen, and thereby the invader, by binding to it, often directing it toward a macrophage for destruction. Also called an
Antigen Any molecule capable of being recognized by an antibody or T-cell receptor.
Antigen-presenting cell (APC) A term most commonly used when referring to cells that present processed antigenic peptide
and MHC class II molecules to the T-cell receptor on CD4 þ T-cells, for example, dendritic cells, macrophages, B-cells.
Atopic allergy IgE-mediated hypersensitivity, that is, asthma, eczema, hay fever, and food allergy.
Autoimmune disorders Conditions in which the body’s own immune system acts against it.
B-cell A type of lymphocyte that produces antibodies, which bind to free-floating microbes circulating in the blood so that they
cannot infect other cells.

Protein Allergy and GMOs 639

Basophil A type of granulocyte found in the blood and resembling the tissue mast cell.
Cell-mediated immunity (CMI) Refers to T-cell-mediated immune responses.
Chemokines A family of structurally related cytokines which selectively induces chemotaxis and activation of leukocytes. They
also play important roles in lymphoid organ development, cell compartmentalization within lymphoid tissues, Th1/Th2
development, angiogenesis, and wound healing.
Class switching The process by which a B-cell changes the class but not specificity of a given antibody it produces, for example,
switching from an IgM to an IgG antibody.
Cytokines Low molecular weight proteins that stimulate or inhibit the differentiation, proliferation, or function of immune
Cytotoxic T lymphocyte (CTL, Tc) T-cells (usually CD8 þ ) which kill target cells following recognition of foreign peptide–
MHC molecules on the target cell membrane.
Dendritic cell Refers to a cell which is MHC class II-positive, Fc receptor-negative, and presents processed antigens to T-cells in
the T-cell areas of secondary lymphoid tissues.
De novo starting from the beginning; anew.
ELISA (enzyme-linked immunosorbent assay) Assay for detection or quantitation of an antibody or antigen using a ligand
(e.g., an antiimmunoglobulin) conjugated to an enzyme which changes the color of a substrate.
Endogenous From within.
Epitope That part of an antigen recognized by an antigen receptor.
FASTA A suite of programs for searching nucleotide or protein databases with a query sequence.
Helper T lymphocyte (Th) A subclass of T-cells which provide help (in the form of cytokines and/or cognate interactions)
necessary for the expression of effector function by other cells in the immune system.
Histamine Vasoactive amine present in basophil and mast cell granules which, following degranulation, causes increased
vascular permeability and smooth muscle contraction.
HLA (human leukocyte antigen) The human major histocompatibility complex (MHC).
Hypersensitivity Excessive immune response which leads to undesirable consequences, for example, tissue or organ damage.
Immunoglobulin E (IgE) A class of antibodies that functions in allergic reactions.
In silico Performed on computer or via computer simulation.
In vitro Studies performed with microorganisms, cells, or biological molecules outside their normal biological context.
In vivo Studies performed on whole, living organisms usually animals including humans and plants.
Mass spectrometry An analytical chemistry technique that helps identify the amount and type of chemicals present in a sample
by measuring the mass-to-charge ratio and abundance of gas-phase ions.
MHC (major histocompatibility complex) A genetic region encoding molecules involved in antigen presentation to T-cells.
Class I MHC molecules are present on virtually all nucleated cells, and class II MHC molecules are expressed on antigen-
presenting cells (primarily dendritic cells, macrophages, and B-cells).
MHC restriction The necessity that T-cells recognize processed antigen only when presented by MHC molecules of the original
haplotype associated with T-cell priming.
Multiple reaction monitoring A multistage/mass spectrometry (MS/MS) experiment that uses two sequential stages of
independent mass analysis.
Plasma cell Terminally differentiated B lymphocyte which actively secretes large amounts of antibody.
Regulatory T-cell T-cells, mostly CD4 þ, which suppress the functional activity of lymphocytes and dendritic cells.
T-cell A type of lymphocyte that possesses highly specific cell surface antigen receptors; types include CD4 þ helper T-cells,
regulatory T-cells, and killer T-cells.
Tolerance Specific immunological unresponsiveness.

11.29.1 Introduction

By the year 2050, the United Nations predicts there will be an additional 3 billion people on the planet, approximately 9 billion in
total. In order to feed these many people, food production must double in the next 40 years (i.e., grow at a rate of 70% beyond
current levels). The majority of the arable land, however, is already being used for agriculture; therefore, the yield of each acre
farmed must be significantly increased. To increase productivity, farmers will need to adopt new technologies.
Genetically modified (GM) crops are developed using the tools of modern biotechnology where precise methods are used to
introduce the desirable traits into a plant. The intended change in a new GM crop is the desired phenotype brought about by
the introduced transgene (Fig. 1). In contrast, in traditional plant breeding, genes from two parents are mixed in many different
combinations often bringing undesirable traits (Herman and Ladics, 2011; Ladics et al., 2015) (Fig. 2). Some solutions for
640 Protein Allergy and GMOs

Fig. 1 Genetically Modified (GM) Crops are Developed Using the Tools of Modern Biotechnology.

Fig. 2 Conventional Breeding Versus Agricultural Biotechnology.

improving global sustainable agriculture are and will continue to be contributed through agricultural biotechnology, with current
and future GM crops. Crops improved through biotechnology have shown benefits of increased harvest yields, reduced plant path-
ogen impacts, or the production of crops with a healthier seed oil profile (e.g., high oleic soybeans). The high oleic GM soybean
product carries the trait for high oleic fatty acid production due to silencing of the endogenous omega-6 desaturase gene, which
leads to increased production of a monounsaturated fatty acid (oleic) and decreased production of polyunsaturated fatty acids
(linoleic and linolenic). A number of GM crops have already improved agricultural practices in many ways. GM crops have achieved
success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of pesti-
cides, decreased carbon dioxide emissions, and decreased soil erosion, energy, or water consumption. The most widely grown GM
crops contain genes for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal
(Cry) insecticidal proteins has been the primary way to impart insect resistance in GM crops. Bt is a common bacterium present in
soils, on grains, and in environmental habitats including the phylloplane and water (Martin and Travers, 1989). Not only is Bt
found in the environment but it is also found in many animals including voles, deer, rodents, and insectivorous mammals, as
well as in processed food products, such as pasta, bread, and other foods that contain flour (OECD, 2007). Numerous Bt strains
produce insecticidal Cry proteins or inclusion bodies that are effective in controlling certain species of insect pests (Aronson and
Shai, 2001). Bt biopesticides have been adopted for use in commercial agriculture, forestry, and mosquito control (OECD,
2007). Because of their robust insecticidal activity, preparations of whole Bt organisms have been used by farmers since the
1920s, being sold commercially in France (as Sporine) as early as 1938, and registered as an insecticide in the United States begin-
ning in 1961 (Ibrahim et al., 2010). As of 2011, more than 100 microbial Bt products have been registered to provide effective
control of insect pests (US-EPA, 2011). There is now a history of nearly 20 years of production and/or consumption of GM crops,
for example, insect-protected corn and cotton containing a specific protein(s) from Bt, herbicide-tolerant soybeans with a gene from
a soil bacterium, and virus-resistant papaya.
As GM crops produced using biotechnology have been introduced to the marketplace, they have been carefully evaluated for
their overall safety from an agronomic, environmental, performance, equivalence perspective, and the safety of the newly expressed
or novel protein. No harmful or adverse effects have been demonstrated after occupational exposure to Bt products, and no adverse
effects have been reported in the consumer population exposed to these products in the form of spray residues on conventional or
organic crops (WHO, 1999). The crystal proteins that confer insecticidal properties to Bt sprays are highly specific to a subset of
immature insects and have been widely used in GM crops to confer insect protection. The mammalian and environmental safety
of Cry proteins have been well studied, and they are as safe in Bt spray products used in conventional and organic farming and
forestry as they are in Bt-based GM crops (Federici and Siegel, 2008). Extensive uses of Cry protein-containing products and safety
studies have provided robust evidence of vertebrate safety. In fact, foods derived from GM crops have undergone more testing than
any other food in history (Cockburn, 2002). Furthermore, since the first GM crops were marketed in the mid-1990s, there have been
no reports of any adverse effects associated with them. One aspect of the protein safety evaluation is the question concerning
Protein Allergy and GMOs 641

potential allergenicity. Although transgenic modification of crops has many advantages over more conventional approaches, there is
some concern that introduction of a novel protein into the food supply could increase the risk of food allergy in susceptible

11.29.2 Food Allergy

The most common endogenous risks of food consumption are food allergies (Johansson et al., 2001; Sicherer and Sampson, 2006;
Burks et al., 2012). Food allergy encompasses other diseases, such as celiac disease (CD) or rare conditions such as food-induced
nickel allergy (systemic contact dermatitis) (Menne et al., 1994) in addition to immunoglobulin E (IgE)-mediated food allergy. A
number of conditions, such as eosinophilic esophagitis or gastroenteritis IgE-mediated food allergy, may play a role in worsening of
symptoms as well as driving the pathology. By definition, food allergy is a food hypersensitivity that has an immunological back-
ground, whereas nonimmunological food hypersensitivity (i.e., food intolerances) depends on other mechanisms (Fig. 3). Some
examples of food intolerances include scombroid poisoning where the active substance is histamine from the decarboxylation
of the amino acid, histidine in fish during putrefaction (Demoncheaux et al., 2012). Another differential diagnosis comprises
genetic or acquired deficiencies in metabolism, of which lactase deficiency (Jarvela et al., 2009) is probably the most common. There
are also idiosyncratic food intolerances (e.g., asthma and sulfites, headaches and chocolate, cheese, or aspartame). In adult and
adolescent patients, alcohol intolerance may be another important diagnosis in this respect. It is also valuable to note, however,
that the most common and severe risks of food ingestion are from contamination of food with exogenous materials. Contamination
can occur on the farm or during storage in restaurants or homes. Bacteria, viruses, fungi, parasites, and chemicals including myco-
toxins, heavy metals, and pesticides are relatively common food contaminants. The most significant acute risks are presented by
bacteria including Escherichia coli O157 and other toxin producing strains: Listeria monocytogenes, Salmonella sp., Campylobacter
sp., and Clostridium perfringens. The Center for Disease Control (CDC) and USDA FSIS estimate that there will be approximately
3000 deaths in 2015 in the US population of 310 million and approximately 128,000 hospitalizations (
poisoning/causes). Since the latter disease states are not well described in terms of pathophysiology, it is therefore essential to estab-
lish a clinically proven diagnosis of food allergy (e.g., a positive challenge with the offending food) (Ortolani et al., 1999;
Bindslev-Jensen et al., 1994). Importantly, if more than one person has reacted to the same exposure for food, it is likely to be
another mechanism than allergy. Toxins and antinutrients often affect nearly every consumer, while protein allergens in foods
only affect a small percentage of the population. Protein Families That Become Food Allergens

Approximately 200 of the hundreds of thousands of proteins in food are identified as food allergens (Day, 1996). The remaining
10% of food allergies are caused by less commonly allergenic proteins or minor allergenic foods and affect a relatively small number
of people (Hefle et al., 1996). The specific properties that make peanut and some tree nuts potent allergenic foods, and particular
proteins such as some of the 2S albumins (e.g., Arah 2 of peanut), vicilins (e.g., Ara h 1), or glycinins (e.g., Ara h 3) dominant aller-
gens, are not fully understood. Interestingly, very similar proteins in other legumes are not as potent as allergens (fewer allergic
subjects, typically less severe reactions). Are there subtle differences in physical properties of the more potent allergenic proteins
(e.g., stability to digestion or processing), or are other factors in the matrix (e.g., specific oils, polyphenols, or lectins) important
modulators of the immune response? Importantly, food, whether developed by conventional means or through biotechnology,
is a potential source of allergens.
Only a restricted number of protein families accounts for the majority of food allergic reactions in predisposed individuals. In
fact, only 2% of all known protein families to date contain allergenic proteins (Radauer et al., 2008). The most important allergenic
protein families from plant foods are the prolamin superfamily, including the nonspecific lipid transfer proteins (nsLTPs) and the
2S albumins, as well as the cupin superfamily with the 11S and 7S globulins (Hoffmann-Sommergruber and Mills, 2009). nsLTPs

Fig. 3 Differernt Types of Food Hypersensitivities.

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are relevant plant food allergens with a robust structure due to four disulfide bridges, which make them less susceptible to gastro-
intestinal digestion. Especially in fruits from the Rosaceae family, nsLTPs account for severe allergic reactions in patients. In addi-
tion, nsLTPs have been identified in pollen, tree nuts, peanut, and vegetables. Although the overall same protein fold is present in all
these nsLTPs, their crossreactivity with clinical relevance is related to sequence similarity and follows the botanical relationship
(Gadermaier et al., 2011). 2S albumins also are proteins with a distinct three-dimensional (3D) structure, formed by four disulfide
bridges. 2S albumins from peanut and tree nuts are recognized as allergens that induce rather severe allergic reactions, while for
other plant species (e.g., sunflower 2S albumin), allergenic reactivity is weaker. Reports identified crossreactivity among 2S albu-
mins from peanut and lupine, mustard and rapeseed, and sesame and poppy seeds, respectively. The 7/8S and 11S seed storage
globulins are the major components of seeds of dicotyledonous species. Typical allergens from the 7/8S protein family are Ara h
1 from peanut and Jug r 2 from walnut. The 11S globulins are allergenic proteins in peanut (Ara h 3), soybean (Gly m 6), and Brazil
nut (Ber e 2). Cupins share a similar overall fold but sequence similarities among proteins from different botanical families are low
and thus crossreactivity is limited. The panallergen profilin and the Bet v 1-related proteins have been identified in various plant
tissues and are mostly responsible for the pollen-food crossreactivity. Profilins are ubiquitous proteins, involved in various cell
signaling pathways such as cytokinesis. Despite rather high sequence similarity among different plant profilins (around 75%)
and frequent IgE crossreactivity, their clinical significance as allergens seems to be restricted to certain plant foods such as melon
and citrus fruits. Usually pollen profilins are regarded as the primary sensitizers, and the protein displays an intermediate stability
when subjected to gastro-duodenal digestion. Regarding the sensitization patterns to profilins, a North–South difference in Europe
has been identified with a higher relevance in Southern Europe versus a lower sensitization range in Northern parts of Europe
(Fernandez-Rivas et al., 2006). Bet v 1-related proteins all share a similar 3D structure and display rather high sequence similarity.
These proteins are identified as allergens in a range of pollens and plant foods usually evoking mild symptoms, whereas in some
cases, as in soybean, the Bet v 1 homologous allergen accounts for severe reactions in patients.
Among the animal food allergens, parvalbumins, tropomyosins, and the caseins are the most important protein families.
Parvalbumins are the major allergens in fish and are characterized by an EF-hand motif. They are Ca2 þ binding proteins (composed
of two helixes, E and F) with resistance to heat treatment and proteolysis. Their characteristic fold contributes to the IgE-binding
activity and, together with sequence similarity, accounts for crossreactivity with clinical significance. Tropomyosins, highly
conserved proteins with a central intracellular function, are major allergens from crustaceans and mollusks. They are also identified
as allergens in mites and cockroaches. Their alpha helical structure remains unaffected when subjected to heat treatment and enzy-
matic digestion. Caseins are proteins restricted to mammalian species and are abundant in milk. They bind Ca2 þ and typically form
clusters with a random coil structure. IgE crossreactivity between caseins from different species is related to sequence similarity. In
summary, it seems that conserved structures and certain biological activities contribute to allergenic activity. Within a protein
family, the presence of highly conserved surface structures and sequence identities above 50% account for clinically relevant
crossreactivity (Aalberse, 2000). Efforts have been undertaken to study the physicochemical properties of allergens and to identify
relevant IgE-binding epitopes, which in turn helped to discriminate between hypo- and hyperallergenic molecules. These
well-defined proteins can now be used to study molecular mechanisms involved in their uptake across mucosal barriers and their
interaction with the immune system (Smole et al., 2010). Improved knowledge on the specific uptake and processing of allergens
will contribute to understanding the factors that make a protein an allergen. Food allergy prevalence

Allergic reactions to food are relatively rare. The incidence of food allergy in the United States and other westernized countries is an
approximation and ranges from 1% to 3% in adults and 4% to 8% in children (Metcalfe et al., 1996; Sampson, 1997; Ladics et al.,
2003; Prescott et al., 2013) (Fig. 4). Thus, the incidence of allergy to specific foods is relatively low and to specific proteins even
lower compared to cases of food intolerances and food poisonings. The prevalence of food allergy is rising in industrialized coun-
tries (Prescott and Allen, 2011). However, there is uncertainty about the magnitude of the rate of increase and the cause. Part of the
increase may be due to increased consumer awareness of allergy as well as more awareness and testing by doctors. There is much
misinformation about prevalence, and people are often incorrectly diagnosed. Many individuals reported being food allergic, but
a clinical evaluation demonstrates they are not food allergic but rather have a food intolerance. From prospective birth cohort
studies, early sensitization to food allergens has been found to be an important factor predicting subsequent development of other
forms of allergies (Illi et al., 2006; Guilbert et al., 2004). There are a number of proposed mechanisms, including the “hygiene”

Fig. 4 Food Allergy Prevalence in Westernized Countries.

Protein Allergy and GMOs 643

Fig. 5 The Most Common Food Allergy-Inducing Foods.

hypothesis (lack of certain bacterial types from the environment or within the gastrointestinal tract) for sensitization (induction of
specific IgE) and tolerance (suppression of IgE and allergy) (Berin and Sampson, 2013). Most likely multiple factors interact at the
time of introduction of foods in the developing child, such as food processing methods, reduced vitamin D levels due to sedentary
indoor lifestyles, and reduced exposure to certain microorganisms or reduced parasite burden that together are contributing to
increases in allergic disease. Relatively few foods are responsible for the vast majority of significant food-induced allergic reactions;
however, what makes such foods unique is not entirely clear. Individuals are usually sensitive to between one and five allergenic
foods. Allergens pose a significant risk to those who are already allergic to the specific proteins, while they do not pose a risk for
nonallergic consumers. A research consortium, EuroPrevall, funded by the European Union was developed to evaluate the epide-
miology of food allergies in many European and several other non-European countries (Kummeling et al., 2009; Wong et al., 2010).
The exact causes of food allergies are unknown and the only current preventive treatment for food allergies is avoidance of the
offending food (Smith and Munoz-Furlong, 1997).
Typically, people describe food allergy as being to a whole food (e.g., milk, eggs, or peanuts). However, research over the past
two decades has identified specific proteins in the foods as the causes of allergy. In plants, specific seed storage proteins (certain
cupins and prolamins) are the dominant allergens along with a few pathogenesis-related proteins (lipid transfer proteins and
PR-10 family proteins) (Osterballe et al., 2005; Radauer and Breiteneder, 2007). Overall, approximately 90% of all food allergies
are associated with a small number of specific proteins represented by eight major allergenic foods: peanuts, tree nuts, cow’s milk,
hen’s eggs, fish, crustacean (e.g., shrimp), wheat, and soybeans (Fig. 5). Some allergenic foods such as peanuts, some tree nuts, cow’s
milk, eggs, crustacean shellfish (shrimp), and fish account for more severe reactions than fruits and vegetables. Certain risk factors
associated with some proteins or foods are partly understood (Poulsen, 2005; Lack, 2005). The prevalence of food allergy to specific
foods can vary geographically (e.g., buckwheat in Asia; celery in Europe; walnut and pecan in the United States; shrimp allergy in
Singapore, Thailand, and Hong Kong (Leung et al., 2009; Metcalfe et al., 1996). Interestingly, peanut allergy is very uncommon in
the Chinese populations despite widespread consumption of peanuts in China (Wong et al., 2010). As the dietary intake of food
varies widely among countries, it is not surprising that the patterns of food allergies are different among different ethnic groups.
However, the prevalence of food allergy to common specific food sources is low (e.g., < 0.01% for maize and approximately
0.4% for soybean) based on published reviews of studies using food challenges and detailed clinical histories (Zuidmeer et al.,
2008; Sicherer and Sampson, 2010).

11.29.3 IgE-Mediated Food Allergy

Food allergy responses are most commonly associated with production of protein-specific IgE and are characteristic of type-1 hyper-
sensitivity reactions. A food allergy is a reaction of the immune system to an otherwise harmless food or food component and is an
important health problem (Verhoeckx et al., 2015). All the antigens involved in eliciting allergic responses to food are proteins.
Most dietary proteins stimulate the immune system to become tolerant to contact with the protein. However, for those prone to
allergies, their T-helper (Th) cells and B-cells may become educated to develop protein-specific IgE immunoglobulin production
because of the mixture of cytokines and cell surface signals provided by T-helper type 2 cells. Food allergic reactions occur typically
in atopic individuals who are genetically predisposed to allergy and who have been previously sensitized to the allergen (Sicherer,
2000). Food allergies are specific because the individual has been sensitized to one or more specific proteins in the offending food.
The sensitizing properties of proteins are due to intrinsic, structural, and physicochemical properties of the particular allergenic
protein which interact with the host genetics, physiology, and with environmental conditions. Assessment of a food allergic patient
also includes factors which might have influenced the severity of the allergic response. Factors which might enhance the allergic
reactions to foods are physical exercise, concomitant intake of nonsteroidal antiinflammatory drugs, beta-blocking agents, and
alcohol (Sicherer and Sampson, 2010). Extrinsic factors that include dose, route, and mode of administration, as well as the
food matrix and presentation of the protein are also important and influence the polarization of the immune response (Th1-,
Th2-, Th17-, or a T regulatory (reg)-type response) leading to either oral tolerance or an allergic response.
The normal immune response to dietary proteins is associated with the induction of oral tolerance, a state of active inhibition of
immune responses to an antigen by means of prior exposure to that antigen via the oral route. The mechanisms responsible for the
644 Protein Allergy and GMOs

Fig. 6 The Generation of Type 1: IgE Mediated Allergic Reactions.

development of oral tolerance are still the subject of research but involve the presentation of antigen via dendritic cells to T lympho-
cytes and the development of various types of Treg cells (Chehade and Mayer, 2005). Currently, it is not clear why some individuals
become allergic to specific proteins in certain foods rather than becoming tolerant to these generally innocuous proteins, although
there are genetic risk factors for IgE-mediated allergy. In infancy, food allergy is most frequently the result of primary sensitization to
food allergens over the gastrointestinal tract and directed to digestion-resistant food allergens. A hallmark of adult food allergy is the
high prevalence of secondary food allergy, where the primary sensitization is directed to an inhalant allergen (i.e., pollen). The food
allergen is recognized by these inhalant allergen-specific IgEs due to a high structural homology between the food and the inhalant
allergen on the basis of crossreaction. Once sensitized, the individual may remain allergic throughout their life; however, young
children often become tolerant to their allergenic food (milk, soybeans, or egg) five or more years after initial reactions through
a process leading to immune tolerance. There is evidence, however, that the natural history of food allergy has changed. In the
past, most children with milk allergy would be able to tolerate milk intake by the time they reached school age. Data from the
United States suggest that cow’s milk allergy is more likely to persist into adolescents (Skripak et al., 2007).
During sensitization, the production of IgE by B lymphocytes that have undergone an isotype switch to IgE-producing plasma
cells (Poulsen and Hummelshoj, 2007) is believed to be mediated by the CD4 þ T-cell in allergy: the Th2-cell which expresses the
cytokines IL-4, IL-5, IL-9, IL-13, and IL-22 (Fig. 6). In IgE-mediated food allergy, reactions occur because the individual has devel-
oped specific IgE antibodies to at least two epitopes (IgE-binding sites) on a relatively abundant protein in the food. Their IgE anti-
bodies are bound to FcεR1 receptors on the surface of mucosal mast cells and blood basophils. Upon subsequent ingestion of the
offending food containing the allergenic protein(s) (i.e., the elicitation phase), the protein or fragments of the protein are absorbed
and crosslink IgE on the surface of mast cells or basophils. This causes the release of histamine and leukotrienes from the mast cells
and basophils, inducing vascular leakage and symptoms due to angioedema and nerve stimulation. The symptomatology of food
allergy is quite variable, and often symptoms originate from more than a single organ, including the oral cavity (oral allergy
syndrome), the skin (urticaria and exacerbation of atopic eczema), the respiratory system (rhinitis and asthma), the gastrointestinal
system (nausea, vomiting, abdominal pain, diarrhea), and additional symptoms such as conjunctivitis and angioedema. The most
frequent symptom of food allergy is the oral contact urticaria (i.e., a swelling and itching of the oral mucosa immediately after the
contact with the allergenic food), which is a mild reaction. This is known as the Oral Allergy Syndrome. In the worst case, individ-
uals may experience anaphylaxis, which is a severe, life-threatening systemic reaction that includes hypotension and difficulty
breathing and typically requires immediate medical attention, including the injection of epinephrine (Burks and Sampson,
1997; Sampson et al., 2006). In the United States, there are more than 30,000 episodes of food-induced anaphylaxis resulting
in more than 150 deaths every year (Bock et al., 2001; Sampson, 2003). Peanuts, a number of tree nut species, milk, and eggs
are the most common causes of fatal anaphylaxis from food. In most countries including the United States, there has not been a stan-
dard reporting system for food allergy anaphylaxis. Allergy to a particular food may give rise to differentially severe symptoms
depending on which precise allergen component(s) the individual is sensitized to (Lidholm et al., 2006; Ballmer-Weber and
Hoffmann-Sommergruber, 2011). IgE-Mediated Food Allergy Diagnosis

Diagnosis of food allergy must establish a reliable link between the clinical history of an adverse reaction to one or several foods as
reported by the patient and the immunological basis of this reaction. The assessment of patients with adverse reactions to foods
comprises first a careful case history. The case history is not reliable as a sole criterion to establish the diagnosis of food allergy,
but it can provide the clinician with an estimation of the severity of the allergic response. While total IgE may be an indicator of
general atopy, it is rarely helpful in discriminating persons with or without food allergy (Boyce et al., 2010). Likewise, acute
measurements during challenge such as plasma-histamine or tryptase have not been demonstrated to be of clinical value (Sampson
et al., 2012). An important factor in diagnosing food allergy is the specific IgE directed against the protein allergens in food. IgE and
the mast cells in the skin also form the basis of diagnosis made by the skin prick test (SPT), which is the major diagnostic tool, along
with measuring specific IgE levels in blood samples. SPT and the measuring of specific IgE levels in blood samples, however, do not
prove allergy; rather, they only prove sensitization or crossreactivity. As a next step, measurement of specific IgE antibodies to the
suspected foods by in vitro or skin testing try to link the clinical reaction with the IgE-mediated pathophysiology. Thus, these diag-
nostic tests only indicate the presence of food-specific IgE antibodies, but they do not establish the diagnosis of food allergy
Protein Allergy and GMOs 645

(Poulsen et al., 2006). To prove the clinical relevance of the reported history and the detected food-specific IgE, often a positive food
challenge is needed. In fact, the “gold standard” for diagnosing true food allergies is the double-blind placebo-controlled food chal-
lenge (DBPCFC) study (Rona et al., 2007). Titrated DBPCFC studies have provided important knowledge on the dose effect on the
development of allergic symptoms in tested individual patients. Starting with very low doses of the investigated food not leading to
allergic symptoms indicate that there is a no-observed-adverse-effect level, that is, an amount of the allergenic food that is safe for
the individual patient (Taylor et al., 2002). With increasing doses, patients often develop subjective or mild symptoms as the first
manifestation of the food-allergic response, whereas more severe and systemic symptoms usually occur at higher doses (Ballmer-
Weber et al., 2007; Mackie et al., 2012). Data from DBPCFC assays with unprocessed and processed foods, however, are very limited
and ethical considerations in addition to logistics may preclude their widespread use, even though they are considered the most
reliable method for clinical evaluations (Poulsen et al., 2006). Food allergy thresholds

While there are limited data to suggest the existence of thresholds below which allergic individuals will not react adversely to offend-
ing foods, these data are poorly documented even for well-known food allergens (Taylor et al., 2002; Crevel et al., 2008). Further-
more, the range of protein levels provoking allergic symptoms is very wide (from a 10th of a milligram up to grams). Regarding
thresholds of sensitization or the relationship between exposure to a protein and the frequency of sensitization, even less is known
(Poulsen et al., 2014; Ladics et al., 2014a). Probabilistic modeling may be used to validate established health-based intake limits
and to determine the statistical confidence and level of protection that such limits provide. Such models derive a probabilistic
estimate of the frequency of allergic reactions in a population by integrating the distribution of a range of variables including
the prevalence of food allergy, subpopulations (children; adults), thresholds for different allergens and severity of reactions, as
well as consumption figures for the population of interest or subgroups. These models maybe promising tools for assessing possible
differences in risks in term of the incidence of allergic reactions in a given population, as well as its severity at certain exposure levels.
However, such models need to be validated before they are implemented (Crevel et al., 2008).

11.29.4 Celiac Disease

CD is an immune disorder in which people cannot tolerate gluten because it damages the inner lining of their small intestine and
prevents it from absorbing nutrients. Gluten is a protein found in wheat, rye, and barley and occasionally in some products (e.g.,
vitamin and nutrient supplements, lip balms, and certain medications). CD is elicited by a limited number of glutenins and gliadins
proteins (i.e., glutens) from wheat, barley, rye, and possibly oat. CD is a genetically restricted autoimmune disease initiated by sensi-
tization to specific wheat, barley, and rye proteins by activation of Th 1-type CD4 þ T-cells (Di Sabatino and Corazza, 2009). CD is
hereditary, meaning that it runs in families. In 50% of people who have CD, a family member, when screened, also has the disease
(Rubio-Tapia et al., 2008). As many as one in 141 Americans have CD, although most remain undiagnosed (Rubio-Tapia et al.,
2012). CD, therefore, is a multifactorial disease caused by interactions between genes and environmental factors. CD patients
develop specific antibodies that bind to connective tissue in the intestine and T-cells that were activated upon binding wheat
peptides from glutens in the context of specific Major Histocompatibility Class II antigen receptors (MHC DQ 2.5 and MHC
DQ 8) (Tye-Din et al., 2010; Abadie et al., 2011). When people with CD eat foods or use products containing gluten, their immune
system responds by damaging or destroying villi, the tiny, fingerlike projections on the inner lining of the small intestine. Villi nor-
mally absorb nutrients from food and pass the nutrients through the walls of the small intestine and into the bloodstream. Without
healthy villi, people can become malnourished. CD occurs in less than 0.5% of the population. The disease is chronic and leads to
the flattening of the villi in the upper small intestine, wasting disease and sometimes to specific cancers. The genetic restriction is due
to unusual protein sequences that are presented most effectively by those with major histocompatibility complex loci HLA-DQ2.5
or HLA DQ8 (May-Ling et al., 2010). However, while more than 25% of the US population has either HLA-DQ 2.5 or DQ-8, only an
estimated 1% of US consumers are clearly diagnosed with CD, which is similar to the rate in Europe (Rubio-Tapia et al., 2012).
Specific peptides of glutens and gliadins have been identified as stimulating Th1 CD4 þ T-cell clones from MHC-restricted CD
patients (Vader et al., 2003; Molberg et al., 2005; Tye-Din et al., 2010; May-Ling et al., 2010). The only way CD patients manage
their disease is through avoiding consumption of foods containing gluten proteins from wheat, barley, rye, and, possibly, oats
(Leonard and Vasagar, 2014).

11.29.5 Safety Assessment of GM Crops

The safety of foods produced from GM crops is mandated by most countries including the United States, China, Brazil, as well as
countries who are members of the Codex Alimentarius Commission (Codex), an international food standards program within the
World Health Organization and the Food and Agricultural Organization of the United Nations ( The
Codex includes 185 member countries plus the European Union (EU) and has 224 official observers (nonmember countries plus
nongovernmental organizations (NGOs)) and outlines guidelines for many important questions regarding food safety and inter-
national trade. The safety standard used with food derived from GM crops is substantial equivalence: Is the food derived from
a GM crop substantially equivalent to the food derived from a non-GM crop? Absolute safety is unattainable for any food; thus,
646 Protein Allergy and GMOs

substantial equivalence is used. It is centered on the premise that existing products used as foods can serve as basis for comparison.
The safety assessment is therefore based on a comparison of the GM crop to its traditional (non-GM) counterpartdfocusing on
composition, toxicology, allergenicity, and nutritional data. The substantial equivalence principle was originally proposed in
1991 and has been endorsed by Codex and the Organization of Economic Cooperation and Development and adopted by
many countries and regions as the main principle for biosafety assessment of GM crops. Foods from GM crops undergo many scien-
tific studies to demonstrate that they are substantially equivalent to those from non-GM crops. Proteins expressed by GM crops
undergo extensive analysis to demonstrate that they are not allergenic or toxic. Before marketing GM crops, such products are
required to undergo an evaluation of the potential allergenic activity of the protein(s) that are produced from the introduced genes
(Metcalfe, 2003; Thomas et al., 2009). A major focus of the safety assessment is to evaluate and ensure that the gene transferred into
a GM crop does not produce an allergen or a CD eliciting gluten. The objectives of this evaluation are twofold: (1) protect allergic
consumers from exposure to known allergenic or crossreactive proteins that may trigger an adverse reaction in those already allergic
to such proteins and (2) protect atopic individuals from risks of allergic sensitization associated with the introduction of genes
encoding proteins that are likely to become food allergens. GM food crops undergo an extensive safety assessment that has a record
of producing safe consumer products.
The potential allergy risk to consumers from crops enhanced through biotechnology may be placed into one of three categories.
The first category that represents a potential risk to the allergic consumer is the transfer of a known allergen or crossreacting allergen
into a food crop. For example, a risk for consumers with allergy to peanuts would be the transfer of a gene encoding a major peanut
allergen into rice or corn. The second potential risk category involves expression of novel proteins that may become allergens de
novo (i.e., a new allergen) and the last, and likely least concerning category, is the potential for enhancing the allergenicity of
a GM crop (e.g., soybean) versus it non-GM counterpart by increasing the expression of endogenous allergens. Approaches to iden-
tifying potential food allergens for purposes of safety assessment have been developed and described in only a few documents. Over
the last 20 years, several guidance documents have been written to provide recommendations for assessing the potential allerge-
nicity of transgenic proteins (Codex Alimentarius Commission, 2003, 2009; FAO/WHO, 2001; Metcalfe et al., 1996). Guidance Documents

There have been three key documents published that have provided recommendations for evaluating the potential allergenicity of
transgenic proteins. The first systematic approach to address the potential allergenic concerns with GM crops was published by the
International Food Biotechnology Council (IFBC), in collaboration with the Allergy and Immunology Institute of the International
Life Sciences Institute (ILSI) in 1996 (Metcalfe et al., 1996). The IFBC/ILSI report suggested the use of a decision tree approach and
introduced the use of bioinformatics (i.e.,  8 contiguous identical amino acids to identify “theoretical” IgE epitopes) and pepsin
resistance (Herman et al., 2006) for the assessment of potential allergenicity. Central to the IFBC/ILSI assessment was a consider-
ation of the source of the transgene (i.e., allergenic or nonallergenic source). If the protein was derived from an allergenic source, an
IgE-binding study using sera from well-characterized patients allergic to the source was suggested, followed, if necessary, by addi-
tional clinical studies (e.g., skin prick testing, food challenge studies). If from a nonallergenic source, an eight or greater contiguous
identical amino acid search and a pepsin resistance study were suggested. If a significant bioinformatics match occurred, an IgE-
binding study and additional clinical studies were recommended. In 2001, the Joint Food and Agriculture Organization/World
Health Organization of the United Nations (FAO/WHO) Consultation on Foods Derived from Biotechnology developed a new
decision tree approach and included a number of additional recommendations (FAO/WHO, 2001). Similar to the IFBC/ILSI
approach, the FAO/WHO approach initially considered the source of the transgene (allergenic or nonallergenic) and subsequent
bioinformatics and pepsin resistance analysis. Specific IgE-binding studies using well-characterized sera from individuals allergic
to the identified allergenic source were also recommended. In contrast, the FAO/WHO eliminated human testing and modified
the bioinformatics parameters (i.e., decreased the threshold from  8 to  6 contiguous identical amino acids and introduced
a new parameter of  35% amino acid identity over an 80 amino acid window to identify potential crossreactive allergenic proteins
using FASTA or other equivalent programs). FASTA (Pearson and Lipman, 1988) is a program used for amino acid sequence (or
nucleotide) comparisons and database searches. Subsequent studies, however, have indicated that the use of a six contiguous iden-
tical amino acid match occurs too commonly across proteins resulting in an unacceptably high number of false-positive findings
and should not be utilized (Hileman et al., 2002; Stadler and Stadler, 2003; Ladics et al., 2006; Silvanovich et al., 2006).
Further, published data indicate that a conventional FASTA analysis (overall sequence alignments) produced fewer false-positive
findings (i.e., fewer nonallergenic proteins identified as allergens) and equivalent false-negative rates (i.e., allergenic proteins iden-
tified as nonallergens) compared to the 80 amino acid sliding window search suggested by FAO/WHO (2001), (Ladics et al., 2007;
Cressman and Ladics, 2009). In addition, Ladics et al. (2007) observed generally more significant expectation (E) values, which
reflect the potential random occurrence of aligned sequences and can be used to evaluate the significance of an observed alignment;
a more relevant identity to the query protein; and a better reflection of functional similarity with a conventional FASTA analysis. The
FAO/WHO also included a number of new recommendations. These included (1) targeted serum screening (assessment of IgE
binding in sera from subjects with allergic responses to broadly related categories of foods) of proteins from nonallergenic sources,
(2) targeted serum screening of proteins with no amino acid sequence homology to known allergens, and (3) use of animal models.
To date, these additional recommendations have not been validated for their specificity, sensitivity, or reproducibility (Ladics et al.,
2003; McClain and Bannon, 2006; Thomas et al., 2007b).
Protein Allergy and GMOs 647

Fig. 7 Weight-of-the-Evidence for Assessing the Potential of a Protein to Induce Food Allergy.

As noted earlier, the approach by which allergy assessment was initially recommended to be conducted involved the use of a step-
wise, decision tree (Metcalfe et al., 1996; FAO/WHO, 2001). Currently, however, there is no single, definitive test for determining
the allergenic potential of novel proteins. Therefore, a “weight-of-the-evidence” (WOE) approach, which accounts for a variety of
factors and experimental approaches for an overall assessment of the allergenic potential of the new protein, has been recommen-
ded by the Codex Alimentarius Commission (Codex, 2003). Currently, most global agencies responsible for the regulation of foods
derived from modern biotechnology use the WOE approach. This approach is consistent with the Annex to the Codex Alimentarius
Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants (Codex, 2003, 2009; Ladics,
2008). The recommended assessments include consideration of the source of the introduced protein (i.e., whether the gene source
for the new protein is known to induce allergy), the host crop’s propensity to cause allergy, similarity of the introduced protein to
known allergens (in silico amino acid sequence similarity comparisons to known human allergens), physicochemical properties
(e.g., susceptibility to acid and enzymatic digestion in vitro, heat stability, and glycosylation status), and protein abundance in
the crop (Fig. 7). When appropriate (i.e., a positive amino acid sequence match to a known allergen is observed or the transgenic
gene is derived from a known allergenic source), specific IgE-binding studies are considered. These studies require the use of well-
characterized sera from individuals known to be allergic (or SPT positive) to the identified source and present ongoing challenges in
terms of standardization of test materials, lack of available sera, and/or validation of procedures. Codex also recognized that certain
methods previously recommended (e.g., animal models; targeted serum screening) were not validated but may prove useful in the
future in assessing the allergenic potential of transgenic proteins “as scientific knowledge and technology evolves” (Codex, 2003,
2009). Codex has encouraged that all suggested methods be scientifically supportable and that additional work be done to clarify
uncertainties associated with the methods.
Thus, over the last 20 years, there have been multiple documents that have provided differing recommendations for assessing the
potential protein allergenicity of transgenic proteins (Metcalfe et al., 1996; FAO/WHO, 2001; Codex, 2003, 2009). These differing
recommendations have often resulted in confusion and arbitrary inclusion of tests across different geographies and subsequent
safety assessment evaluations based on nonvalidated (e.g., animal models) or refuted (i.e., six contiguous amino acid matches)
tests. Ideally, in the future, there needs to be better harmonization of testing requirements for assessing potential protein allerge-
nicity across geographies and inclusion of only those endpoints that have been “validated” based on “sound science” (peer-reviewed
published data).

11.29.6 Evaluating Potential Allergenicity Risk

To investigate the first category of risk to the allergic consumer, the transfer of a known allergen or crossreacting allergen into a food
crop, the history of safe use (HOSU) of the gene source and recipient is examined and in silico (i.e., bioinformatic) procedures are
employed. The second risk category involving the expression of novel proteins that may become allergens de novo (i.e., the poten-
tial to become a new allergen) is assessed by evaluating the physical/chemical properties and abundance of the protein in the crop.
The last category of risk regarding the potential for enhancing the allergenicity of a GM crop (e.g., soybean) versus it non-GM coun-
terpart by increasing the expression of endogenous allergenic proteins is evaluated using several different analytical tools (e.g.,
specific IgE-binding studies, enzyme-linked immunosorbant assay [ELISA], or mass spectrometry [MS]) (Fig. 8). Each of the latter
WOE assessment parameters will be discussed in detail in the following section.

11.29.7 First Category of Potential Allergenic Risk: Transfer of a Known Allergen or Crossreacting Allergen into
a Food Crop History of Safe Use: Evaluation of the Gene Source
The scope of the HOSU evaluation of the gene source and the gene recipient includes determining whether the gene source(s) for the
GM crop is a common cause of allergy. If this is the case, additional tests are likely to be required compared to sources without any
history of allergy. For example, peanuts and certain tree nuts (almond, hazelnut, walnut, and pecan) are considered common causes
648 Protein Allergy and GMOs

Fig. 8 Categories of Potential Health Risks for GM Crops Relative to Food Allergy and the Technology Utilized to Reduce Any Potential Food Allergy

of food allergy, while birch or grass pollen or house dust mite are causes of respiratory allergy and latex contact allergy. If a gene is
transferred from one of the commonly allergenic sources, specific serum IgE-binding studies will be required similar to the study
performed by Nordlee et al. (1996) for the Brazil nut 2S albumin. Specific IgE-screening studies utilize sera from subjects allergic to
the identified allergenic source of the gene and are described in detail later in the article. Bacillus thuringiensis (Bt)

Furthermore, human volunteers ingested or inhaled large quantities of Btk formulations but experienced no adverse health effects
(WHO, 1999).
Currently, the most widely grown GM crops are soybeans, maize, canola, and cotton containing one or more targeted genes that
bestow insect resistance or herbicide tolerance or a combination of these traits (Konig et al., 2004). Insect-resistant plants are desir-
able because of enhanced pest control, reduced need for chemical insecticides, and increased yields. Some insect-resistance crops
have been produced by genetic modification with genes from the environmentally ubiquitous, gram-positive bacterium, Bt. The
gram-positive bacterium Bt is ubiquitously found throughout the environment in soil, on leaf surfaces, and aquatic environments.
The distribution of various Bt subspecies is relatively uniform throughout the world (Martin and Travers, 1989). There is little infor-
mation, however, on levels of Bt found in food. Given the widespread prevalence of Bt, its presence in food is likely quite common
and is not always related to its use as a biopesticide on food products (WHO, 1999). Noble et al. (1992) reported that 5 out of 10
vegetable samples were positive for Btk. The positive samples were obtained from both supermarkets and from organically grown
products. These data may account for the recovery of Bt from fecal and urinary samples during occupational studies and may indi-
cate a general community exposure through food. Importantly, with the exception of case reports on ocular and dermal irritation,
no adverse health effects have been documented after occupational exposure to Bt products. Spores of this species have been used as
microbial pesticides for 70 years without demonstration that they cause allergies or toxicity in mammals.
A number of different strains of Bt have been found to produce crystal proteins (e.g., Cry1A, Cry2A, Cry3A, Cry34Ab1,
Cry35Ab1, and Cry1F) or inclusion bodies that are very effective in controlling certain species of insect pests (Aronson and Shai,
2001). The safety of the plant-expressed Cry proteins has been supported by the experience of decades of safe use of these same
proteins in microbial sprays. In over 40 years of commercial use as biopesticides, Bt insecticidal proteins have been used safely
with no adverse reports of human health or environmental effects (McClintock et al., 1995; Siegel, 2001). Furthermore, in USEPA’s
1998 Registration Eligibility Decision, the agency concluded that microbial Bt products pose no unreasonable adverse effects to
humans or the environment and that all uses of those products are eligible for reregistration (USEPA, 1998). In addition, transgenic
crops containing Bt proteins have been grown and consumed since 1996 with no documented reports of adverse health effects (Betz
et al., 2000; Society of Toxicology, 2003; USEPA, 2005; Federici and Siegel, 2008). The lack of toxicity of Bt spores has been estab-
lished by other studies: one clearly demonstrated that human acute oral exposure to live spores (1 g/day for 5 days) resulted in no
toxic effects nor did inhalation of 100 mg of Btk powder daily for 5 days (Fisher and Rosner, 1959). Another showed a lack of corre-
lation between the presence of Bt in feces collected from greenhouse workers after exposure and with any gastrointestinal ill effects
(Jensen et al., 2002). Based on these and additional studies, the World Health Organization and others have concluded that Bt
sprays containing Cry proteins have demonstrated no adverse health effects in workers who apply them nor in the general public
in the areas where the sprays are applied (WHO, 1999; Federici and Siegel, 2008). Because of their demonstrated safety, Bt formu-
lations are often sprayed on crops such as broccoli, tomatoes, cucumbers, cauliflower, and lettuce plants for insect control just prior
to harvest (Frederiksen et al., 2006). These crops are eaten raw and typically with only minimal washing, meaning humans have
been safely consuming Bt spores and Cry proteins (Federici and Siegel, 2008). Thus, in over 75 years of commercial use as bio-
pesticides, Bt insecticidal proteins have been used and consumed safely with no reported adverse effects on human health or the
environment (McClintock et al., 1995; Siegel, 2001). The historical safe use of the organic pesticides provides assurance of
HOSU for some Bt toxins, although that is true only for proteins that are demonstrated to be expressed by the bacteria used as
microbial pesticides and not from all varieties of the species.
Protein Allergy and GMOs 649

In accordance with suggested recommendations for assessing potential allergenicity (FAO/WHO, 2001; CODEX, 2003), Bt
proteins in general have been demonstrated to have no amino acid sequence similarity to known allergens, are rapidly digested
utilizing in vitro pepsin resistance assays, are heat labile, have a low prevalence in food, and low-to-no glycosylation (Betz et al.,
2000). Bt does not have a history of causing clinical allergy, including occupational allergy associated with the manufacture of prod-
ucts containing Bt (USEPA, 2000a). Batista et al. (2005) performed SPTs with protein extracts prepared from a number of GM and
non-GM maize and soy samples and reported no differences in terms of allergenicity between the GM and non-GM samples. In
addition, the USEPA states that “after decades of widespread use of Bt as a pesticide, there have been no confirmed reports of imme-
diate or delayed allergic reactions to the delta-endotoxin itself despite significant oral, dermal and inhalation exposure to the micro-
bial product” (USEPA, 2000a). The lack of allergenic concern for Bt was reconfirmed in an April 2005 USEPA Scientific Advisory
Panel Report (USEPA, 2005) in which the panel states it “.was not aware of any report of Bt being an allergen.” Taken together,
these data indicate a lack of allergenic concern for Bt in general.
There have been two reports in the literature (Bernstein et al., 1999; Doekes et al., 2004), which have suggested that occupational
exposure to Bt containing microbial pesticide products may be associated with a risk of biopesticide-specific IgE sensitization (i.e.,
the presence of serum IgE without the elicitation of clinical signs of allergy). There are, however, several aspects of these studies that
warrant further discussion. In either study, there was no link identified between exposure to Bt sprays and the elicitation of occu-
pationally related clinical allergy, such as respiratory symptoms, in the various cohorts examined. As indicated previously, in over
40 years of commercial use as biopesticides, Bt insecticidal proteins have been used safely with no adverse reports of human health
or environmental effects (McClintock et al., 1995; Siegel, 2001; USEPA, 2005). Specificity of the reported IgE data: Crude extracts
were utilized in these studies. The observed IgE binding may be directed against non-Bt media contaminants. As pointed out by the
authors of these studies, analysis of the extracts utilized and the identification and characterization of the actual IgE-binding compo-
nents are important and necessary next steps. In addition, as stated by Doekes et al. (2004), the optical density units of their ELISA
measures of IgE against the bacterium Bt are very low, < 0.2. Therefore, it is unclear whether a definitive positive IgE titer was
measured in the study by Doekes et al. (2004). Additional data are needed on the degree of IgE sensitization in nonexposed control
groups. Is the reported IgE binding also observed with other commonly found bacteria? If this is the case, then sensitization in the
general population would also be expected as well. Specifically, the Doekes et al. (2004) study did not include testing of a “true
negative” unexposed population which is necessary for statements as to the assay sensitivity and specificity. Further study is required
to determine the assay sensitivity and specificity of the measured IgE in the study patient’s serum to Bt. The results of both studies
were deemed preliminary by the authors and further analysis is required before definitive conclusions regarding risks of occupa-
tional allergy can be drawn. In summary, Bt products “are unlikely to pose any hazard to humans or other vertebrates provided
they are free from non-Bt microorganisms and from biologically active products other than insecticidal crystal proteins” (WHO,
1999). Bt does not have a history of causing clinical allergy, including occupational allergy associated with the manufacture of prod-
ucts containing Bt (U.S. Environmental Protection Agency, 2000a, 2005). CP4 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium tumefaciens

Roundup Ready Soybeans are a series of genetically engineered varieties of glyphosate-resistant soybeans produced by the Mon-
santo Company. Glyphosate kills plants by interfering with the synthesis of the essential amino acids phenylalanine, tyrosine,
and tryptophan. These amino acids are called “essential” because animals cannot make them; only plants and microorganisms
can make them and animals obtain them by eating plants. Plants and microorganisms make these amino acids with an enzyme
that only plants and lower organisms have, called 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). EPSPS is not present
in animals, which instead obtain aromatic amino acids from their diet (Steinrücken and Amrhein, 1980; Funke et al., 2006).
Roundup Ready Soybeans express a version of EPSPS from the CP4 strain of the bacteria, Agrobacterium tumefaciens (Padgette
et al., 1995). The donor organism, Agrobacterium sp. strain CP4, is not a known human or animal pathogen and is not known
to induce allergenic responses in humans. Additionally, the safety of the Agrobacterium sp. strain CP4 as the donor organism has
been reviewed previously as a part of the safety assessment for other Roundup Ready crops by regulatory agencies worldwide. A
HOSU of CP4 EPSPS protein has been demonstrated, based on the similarity of the CP4 EPSPS protein in Roundup Ready Soybeans
to EPSPS proteins naturally present in food crops (e.g., soybean and corn) and in microbial food sources such as baker’s yeast
(Saccharomyces cerevisiae). The CP4 EPSPS protein is functionally equivalent to native plant EPSPS proteins except for the lack of
affinity for the herbicide, glyphosate. In addition, there is experience of safe use of the CP4 EPSPS protein since the introduction
of Roundup Ready crops in 1996, which include Roundup Ready soybean, cotton, and corn. No biologically relevant structural
similarities were observed between the CP4 EPSPS protein and allergens, toxins, and pharmacologically active proteins, which
suggests that CP4 EPSPS is not likely to pose a human health concern. This conclusion is also supported by the rapid degradation
of CP4 EPSPS protein in simulated digestive fluids (Harrison et al., 1996).

11.29.8 In Silico Bioinformatic Tools

An area that has seen significant progress over the last decade is the adaptation of in silico bioinformatic tools to evaluate the aller-
genicity potential of a novel protein. Bioinformatics is the comparative analysis of protein sequences intended to evaluate structural
and functional relationships. Bioinformatics has several core principles: (1) protein structure is determined by amino acid sequence,
(2) similar amino acid sequences have similar structure, and (3) similar sequence and structure infers a common ancestor gene and
650 Protein Allergy and GMOs

related function. Most of the major food, dermal, and respiratory allergens have been identified and cloned. Subsequently, the
protein sequences for most, if not all of these allergens, have been incorporated into various databases. As a result, novel proteins
can be routinely screened for amino acid sequence homology with, and structural similarity to, known human allergens using an
array of bioinformatic tools early on in the product development process. Importantly, bioinformatics allows one primary question
to be asked: Is the novel protein an existing allergen? Bioinformatics also allows a secondary question to be asked: Is the novel
protein likely to crossreact with an existing allergen? Sequences sharing a high degree of identity often share immunologically rele-
vant topology (Aalberse, 2000; Goodman et al., 2008). Bioinformatics is not intended to answer whether a novel protein will
“become” an allergen de novo. Knowledge of the protein structures responsible for inducing sensitization is still lacking even for
known allergens (Ladics et al., 2014a). Current bioinformatic analyses involve two recommended criteria: a search for continuous,
identical stretches of amino acids eight residues or greater in length and an identity search using the FASTA (Pearson and Lipman,
1988) or BLAST (Altschul et al., 1997) local alignment algorithms to search amino acid sequences of known allergens contained in
databases (e.g., the AllergenOnline database) for alignments of 80 residues or longer possessing a sequence identity of greater than
or equal to 35% (FAO/WHO, 2001; Codex, 2003; Cressman and Ladics, 2009; Mirsky et al., 2013). The  35% identity match over
80 or greater amino acids criteria was considered more relevant than the short contiguous identical amino acid (i.e., theoretical
epitope) search based on data indicating protein crossreactivity occurring between Bet v 1 and vegetable proteins at approximately
40% protein identity (Scheurer et al., 1999). Allergen Databases

Today, many different allergen databases are developed and maintained. These databases differ in their content, accessibility, level
of descriptive information, data (biological or molecular data, update date, number of sequences), degree of curation, and the pres-
ence of informatics applications for comparing the query novel sequence to public-annotated sequences (Gendel and Jenkins,
2006). The AllergenOnline database, however, is a peer-reviewed allergen database containing food, inhalation (e.g., pollen, house
dust mites, and mold spores), dermal (e.g., latex), and injection (e.g., venom, saliva of biting insects) allergens maintained by the
Food Allergy Research and Resource Program (FARRP) ( at the University of Nebraska, Lincoln, since 2004. The
database is peer reviewed by clinical and research allergists from around the world and updated once per year. The inclusion of
protein allergens is based on available data in the public literature. The sequences of the proteins with published proof of IgE
binding using sera from appropriately allergic subjects have been included in the database. A number of the
proteins included in the AllergenOnline database have also been demonstrated to cause biological reactivity by SPTs of allergic
subjects, basophil histamine release, or basophil activation. The references used to categorize each allergenic protein group are listed
in the database along with an explanation of the process of classification. The database also provides sequence comparison algo-
rithms to evaluate potential new novel food proteins for potential risks of allergic crossreactivity. The database is available free to the
public at Version 15 of the database was released in January 2015 and includes 1897 allergen
sequences. Bioinformatic criteria for IgE-mediated allergy

As recommended by FAO/WHO (2001), IgE crossreactivity between a novel protein and a known allergen is considered a possibility
when there is more than 35% identity over a segment of 80 or greater amino acids. It should also be pointed out, however, that for
crossreactivity to occur, Aalberse (2000) has reported that a high degree of homology is needed, likely in excess of 50–60%, over
significant spans of the novel protein and allergen. Goodman et al. (2008) noted that high risk of crossreactivity exists among
proteins with > 70% identity. Radauer and Breiteneder (2006) reviewed sequence identities among allergenic and nonallergenic
homologs of pollen allergens and reported that the prerequisite for allergenic crossreactivity between proteins was a sequence iden-
tity of at least 50% across the length of the protein. In general, just considering the primary structure of the protein, < 50% amino
acid identity among proteins rarely results in antigenic crossreactivity. The step-wise contiguous, identical amino acid segment
searches are performed to identify amino acid sequences that may represent “theoretical” linear IgE-binding epitopes. IgE-
binding epitopes, however, have only been identified for a few allergens (Bannon and Ogawa, 2006). Therefore, in the absence
of an IgE-binding epitope database, potential epitopes may be evaluated by producing all overlapping peptides of the allergens con-
tained in a particular database and comparing them in a pair-wise manner to all same-size potential peptides of a novel protein
using bioinformatic tools as recommended by Metcalfe et al., 1996. Eight contiguous identical amino acid matches between a novel
protein and a known allergen(s) are required by some regulators to identify sequences that may represent linear IgE epitopes. The
value of short, contiguous matching amino acid searches, originally intended to identify “theoretical” shared epitopes between
a query sequence and one or more allergenic proteins, has been called into question. The original (FAO/WHO, 2001) recommen-
ded criteria, specifying a  6 amino acid continuous identity, has largely been discredited as producing too many false positives to be
meaningful (Hileman et al., 2002; Stadler and Stadler, 2003; Thomas et al., 2005; Ladics et al., 2006; Silvanovich et al., 2006;
Goodman, 2008). Furthermore, numerous other publications (EFSA, 2010; Goodman, 2008; Herman et al., 2009; Ladics et al.,
2011; Goodman and Tetteh, 2011; Harper et al., 2012; Young et al., 2012, Mirsky et al., 2013) have argued that the standard search
for a sequence of eight or more contiguous amino acids found in both the query protein and a known allergen provides little value
in predicting potential protein allergenicity.
The other criteria, involving the use of the FASTA local alignment algorithm (Pearson and Lipman, 1988) to identify regions
between a query sequence and an allergen sequence displaying identity above 35% over an 80 or greater residue “threshold,”
Protein Allergy and GMOs 651

Fig. 9 FASTA Local Alignment Algorithm Analysis to Evaluate Regions of Identity between a Query Sequence and an Allergen Sequence (example of
no similar identity).

Fig. 10 FASTA Local Alignment Algorithm Analysis between the Allergens Mal d 1 and Bet v 1 (example of significant identity).

appears to be a more robust method (Codex, 2003, 2009; EFSA, 2011). The purpose is to identify proteins sufficiently similar to
allergens to suspect potential crossreactivity. This criterion addressed the fact that the molecular basis of crossreactivity is not
only in the primary structure of the proteins but in the tertiary structure as well. Therefore, it takes into account the existence of
conformational B-cell epitopes. The algorithm employs a word search as an initial step, identifying all matching words between
two sequences (the default word size, or ktup, for protein sequences is 2). Matching words are joined, extended, and assigned
a numerical “score” using both a scoring matrix to give appropriate weight to conserved regions, as well as preset penalties for
the insertion/extension of gaps (Figs. 9 and 10). The resulting scores generated from comparisons between the query and all dataset
proteins are then used to establish a linear relationship between alignment score and protein length. The score of a particular align-
ment with respect to the calculated distribution of all scores gives an indication of whether the alignment is meaningful; this is
reflected in the expectation value (E-value, a measure of the potential random occurrence of aligned sequences used to evaluate
the significance of an observed alignment) assigned to the alignment. A small E-value (e.g., 10 4) indicates a potential biologically
relevant similarity in the context of potential allergenic crossreactivity; large E-values (e.g., > 1.0) represent random alignments that
do not possess biologically relevant similarity (Pearson, 2000; Silvanovich et al., 2009; Mirsky et al., 2013). Thus, the lower an E-
value for a particular alignment, the more likely the comparison between the proteins reflects a true structural similarity, although
the value is influenced by protein length and database size (Baxevanis and Ouellette, 1998). Such built-in methods for checking the
nature of an alignment results make the FASTA package helpful in determining the significance of protein sequence matches.
When FAO/WHO published recommendations for the determination of potential crossreactivity to known allergens, they also
included a “standardized methodology” or “sliding window” approach for performing the searches. The four step procedure
includes a step where one is to “prepare a complete set of 80-amino acid length sequences derived from the expressed protein”
(FAO/WHO, 2001) followed by subsequent comparison of each 80 residue protein to the allergen dataset. The potential for cross-
reactivity was to be considered when there was “more than 35% identity in the amino acid sequence of the expressed protein (i.e.
without the leader sequence, if any), using a window of 80 amino acids and a suitable gap penalty.” (FAO/WHO, 2001). As a result
of this suggestion, scripts were written that generated all possible 80 amino acid fragments or “sliding windows” from a given query
protein and submitted each one for FASTA analysis. Above threshold matches ( 35% over an 80 amino acid window) were auto-
matically identified for closer scrutiny. However, the positive predictive value of the 35% or greater identity over an 80 amino acid
sequence using a “sliding window” algorithm (FAO/WHO, 2001) has been questioned (Ladics et al., 2007; Cressman and Ladics,
2009). False-positive and -negative rates were evaluated using the “sliding window” of 80 amino acids versus conventional (i.e., full-
length sequence) FASTA analyses. Data indicated that a conventional FASTA analysis across the whole protein sequence produced
fewer false positives and equivalent false-negative rates compared to the 80 amino acid “sliding window” FASTA search. Moreover,
Silvanovich et al. (2006) noted that using the current 35% identity match over 80 amino acids sliding FASTA window criteria, the
same rate of false positives could be achieved using either random selected protein sequences or the same sequences after being
subjected to 1000 rounds of sequence shuffling. In addition, conventional FASTA derived E-values, which reflect the potential
random occurrence of aligned sequences, and which can be used to evaluate the significance of an observed alignment, were gener-
ally more significant (i.e., lower in value) than the sliding window-derived E-values. These data also suggest that a conventional
FASTA search provides more relevant identity to the query protein and better reflects functional similarities between proteins
652 Protein Allergy and GMOs

than a “sliding window” search. In many cases, the sliding window analysis resulted in identity matches to a variety of proteins from
different families with diverse functions. The positive results obtained with the conventional FASTA analysis, however, still exceeded
what would be predicted based on the expected percentage of real or true allergens in the clinic. This finding is likely due to the use
of the currently recommended, very conservative, threshold criteria of 35%. When Ladics et al. (2007) raised the threshold to 50%
when evaluating corn seed protein sequences, the number of positive findings decreased by half using the conventional FASTA anal-
ysis. By imposing a defined threshold of  35% sequence identity over an alignment length  80 amino acids, the default local align-
ment search criteria are constrained (Silvanovich et al., 2006; Ladics et al., 2007; Cressman and Ladics, 2009). This constraint
neglects many of the FASTA features that help to define relevant homologies between sequences, features incorporated into the algo-
rithms themselves (e.g., E-value). As a result, regulators currently do not consider E-value and thus the significance of an observed
alignment. Therefore, if an alignment of 35.5% is observed between two protein sequences with a very large E-value of 2.0, for
example, the alignment would likely still be considered significant by most regulators.
Rather than using a specific percent identity cutoff value, a methodology for determining an E-value threshold has been
proposed. For instance, by applying the E-value threshold methodology to 7695 corn protein sequences, a FASTA E-value cutoff
of 4.7  10 7 was determined. The cutoff was 100% effective at identifying known allergens but was sufficiently conservative as to
have a 95% false-positive rate (i.e., only 1 out of 20 positives would reflect a true potential for crossreactivity) such that no poten-
tial positive matches would be overlooked (Silvanovich et al., 2006). More recently, Mirsky et al. (2013) conducted a comprehen-
sive large-scale in silico evaluation of the various assessment criteria, including searches for: alignments between a query protein
and an allergen having  35% amino acid identity over a length  80; any sequence (of some minimum length) found in both
a query protein and an allergen; and any alignment between a query protein and an allergen with an E-value below some
threshold. The most effective criterion found declares a query protein potentially allergenic if there is either (1) an alignment
between it and an allergen having  35% amino acid sequence identity over an alignment length  80 or (2) some sequence
of 13 or more amino acids found both in it and an allergen, and (3) there is an alignment between it and an allergen with an
E-value  10 4. These data suggest that a combination of amino acid alignments and E-values should be employed when eval-
uating the potential allergenic crossreactivity between two proteins. Currently, the criterion of greatest emphasis is any
match  35% identity over any segment of 80 or more amino acids. It can be concluded that using greater than 35% shared iden-
tity over any 80 amino acid section (based on FASTA or other equivalent programs) is a highly conservative estimate (i.e., many
false positives) of the potential for crossreactivity (Thomas et al., 2005; Ladics et al., 2007; Cressman and Ladics, 2009; Mirsky
et al., 2013). Hence, although these sequence analogy approaches are useful, it is likely that many potentially beneficial protein
products are unnecessarily eliminated early in the evaluation process. Additional approaches to sequence/structure analysis
would be welcome.
Allergens are found in only a small subset of all known protein families (Radauer et al., 2008). Furthermore, protein families that
contain allergens also include hundreds of nonallergenic proteins. There are currently no known unique motifs that identify
a protein as an allergen; however, a better understanding of structural attributes and conformational epitopes could prove valuable
for assessing allergenic potential. A research need in this area is to identify additional sequences of allergenic IgE epitopes (Bannon
and Ogawa, 2006). In recent years, conformational epitopes in seven allergens have been identified by solving the X-ray crystal
structure of the allergen in complex with antibody fragments (Fab or Fab) (Pomés, 2010). Studies that take into account the 3D
structure of food allergens are needed to fully understand the B-cell repertoire and gain new insight into the molecular basis of cross-
reactivity for allergenicity prediction (Pomés, 2010). Although conformational epitopes are common in inhaled allergens, food
allergens may also contain them if the allergen is not completely cleaved in the digestive tract and digestion-resistant fragments
are absorbed. New approaches based on the protein conformational structure may be useful for refining the WOE approach in
the future, when proven predictive. For example, there is a structural database of allergenic proteins (SDAP) database (http:// SDAP is a web server that provides rapid, crossreferenced access to the sequences, structures, and IgE
epitopes of allergenic proteins (Oezguen et al., 2008; Ivanciuc et al., 2009a,b). The SDAP core is a series of scripts that process
the user queries, interrogate the database, perform various computations related to protein allergenic determinants, and prepare
the output Internet pages. The database component of SDAP contains information about the allergen name, source, sequence, struc-
ture, IgE epitopes and literature references, as well as easy links to the major protein databases (PDB, SWISS PROT/TrEMBL, PIR-
ALN, NCBI Taxonomy Browser) and literature (PubMed, MEDLINE) online servers. The computational component in SDAP
uses an original algorithm based on conserved properties of amino acid side chains to identify regions of known allergens similar
to user-supplied peptides or selected from the SDAP database of IgE epitopes. These types of bioinformatic tools may have the
potential to rapidly determine a potential crossreactivity between allergens and to screen novel proteins for the presence of IgE
epitopes they may share with known allergens. However, major limitations to these methods suggest they are not yet predictive.
In particular, there are relatively few allergens for which the crystal structure is known and the protein structure may be more related
to the intrinsic function of the protein and less to the form in which the protein may sensitize a person or elicit an allergenic
response. Therefore, it is useful to know the 3D structures of allergens in order to locate antibody recognition sites. However, there
are approximately 715 allergens in the official database for the systematic allergen nomenclature that is approved by WHO and the
International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Subcommittee (; WHO/
IUIS). The 3D structure has been solved for only  75 allergens, that is,  10% of the allergens in the database, from which only
 24 are food allergens (as assessed from the WHO/IUIS Allergen Nomenclature database and the Protein Data Bank). Therefore,
further data are required to assess the utility of protein structure in predicting protein crossreactivity. In particular, the identification
of sequence motifs that could characterize protein allergenicity may be useful.
Protein Allergy and GMOs 653

Overall, bioinformatics techniques based on linear sequence comparisons could be improved by using more advanced tools,
such as E-value thresholds. In addition, to increase the power of the bioinformatics analyses, the determination of the degree of
similarity between proteins and known allergens by using a structural database and appropriate comparison scripts may prove
useful in the future. Information derived from the 3D structure of allergens and conformational epitopes may be beneficial to assess
the allergenicity of new proteins on a case-by-case basis. Specifically, the location of IgE antibody-binding epitopes in allergens can
be important for the identification of potential allergens among novel allergen-homologous proteins. Bioinformatic criteria for celiac disease

Codex (2003) does require an evaluation for proteins derived from wheat or wheat relatives (barley, rye, and, possibly, oats) in
regard to their potential to induce CD; however, Codex does not provide guidance on the process (e.g., the use of bioinformatic
tools) to do so. Nevertheless, a number of proteins have been identified in the scientific literature as potential CD-inducing proteins
(Stepniak et al., 2005, 2008; Camarca et al., 2009; Mitea et al., 2010; Dørum et al., 2010). A database of peptides from wheat, barley,
and rye that cause T-cell stimulation or intestinal epithelial pathology ( has been con-
structed. The database is part of the database for bioinformatics evaluation of potential IgE-mediated aller-
genicity for GM proteins. Currently the CD database includes 1016 peptides with published evidence of T-cell reactivity using cells
from CD patients in the context of MHC Class II DQ 2.5 or DQ8 or toxic effects in intestinal epithelial cells or pathology in intestinal
villi from those with CD. The amino acids of proteins introduced into GM crops may be searched for exact matches to the peptides
in the database, or the proteins can be searched by FASTA for matches to whole proteins known to stimulate CD, using the proposed
criteria of > 45% identity over alignments of at least 100 amino acids or more having an E-value of < 1 10 15 as potentially stim-
ulating CD. Similar to the IgE allergenicity assessment, bioinformatic tools and databases should be able to identify proteins that
might represent a potential risk of causing CD. Genes taken from plants outside of the Pooideae subfamily of grasses represent little
risk of causing CD and therefore even if they are homologues of glutens that cause CD, they are highly unlikely to result in disease.
Proteins that do not exceed these criteria should present little or no risk of inducing CD. If a significant match is found, the protein
should be tested using cells or challenges in CD subjects to evaluate risks using cell-based assays or possibly food challenges to
ensure minimal risk to the CD population.

11.29.9 Specific IgE Sera Screening Studies

Specific IgE antibodies are implicated in the course of allergic diseases. IgE-binding capacity of proteins or foods is a prerequisite of
clinical reactivity, that is, ability to elicit an allergenic reaction in sensitized populations. Therefore, the Codex recommendations
(2003, 2009) include specific, IgE serum screening as part of the weight-of-the-evidence approach to identifying potential allergens.
When a gene is taken from a known allergenic source, specific IgE sera screening studies are required. Likewise, when significant
amino acid identity is observed between a novel protein and an allergenic protein (e.g.,  35% amino acid identity over  80 amino
acids), specific IgE sera screening studies are also conducted (Goodman, 2006; 2008; Ladics, 2008). A “specific” serum screen
involves testing a protein of interest with sera from patients with documented clinical food allergy to a specific allergen to confirm
that the tested protein is not crossreactive with the protein to which the patient produces IgE antibodies. IgE-specific Western blots
and ELISAs are typically used to identify allergenic proteins. Conducting “scientifically meaningful” IgE serum screens, however, is
problematic due in part to the lack of standardized methods and the availability of well-characterized human serum samples from
a sufficient number of patients since the incidence of individuals with specific clinically relevant allergies is quite low (Thomas et al.,
2007a; Goodman, 2008). There is a risk in performing such tests, as in vitro IgE-binding results can be ambiguous, with false-
positive binding that may inappropriately implicate a protein as a possible allergen (Goodman and Leach, 2004). In the case of
a major allergen (i.e., one to which more than 50% of individuals sensitive to that material respond in IgE-specific immunoassays),
FAO/WHO (2001) indicated that a minimum of eight relevant sera is required to achieve a 99% certainty that a new protein is not
an allergen. FAO/WHO (2001) further indicated that a minimum of 24 relevant sera might be required to achieve a 99% certainty
with regard to a minor allergen. Such quantities of well-characterized, clinically relevant sera may not be available for testing
purposes. Standard criteria are also needed for selection of allergic individuals (Eigenmann and Sampson, 1997). This begins
with documentation that the serum donor is allergic to the allergen of interest. A positive response to oral food challenge is the
most definitive test for specificity, but this test is often not performed for practical or ethical reasons. Careful clinical history and
positive SPTs results are viable alternatives for establishing specificity. It is also imperative that during assay development,
conditions be optimized (e.g., detection methods, blocking reagents) in order to establish assay sensitivity and specificity and to
minimize false-positive IgE binding. A number of issues must be addressed to ensure the integrity of the assay including appropriate
blocking agents to prevent nonspecific binding, overcoming the much larger higher concentrations of IgG relative to IgE that are
generally present in sera and can interfere with IgE detection, and demonstrating specificity of the anti-IgE component (secondary
antibody) in the ELISA (Holzhauser et al., 2008).
In addition to direct-binding studies, inhibition Western blot assays and ELISAs should also be conducted to demonstrate the
specificity of any identified IgE binding and to further minimize false-positive findings. These studies involve holding the solid-
phase protein target constant, while varying the concentration of soluble inhibitors (e.g., pure specific proteins, extracts, or nonre-
lated control proteins) mixed with allergic subject sera to generate an inhibition curve in a quantitative assay. Alternatively, such
studies can be conducted in a qualitative or semiquantitative fashion by employing a fixed concentration of inhibitor (Goodman
654 Protein Allergy and GMOs

and Leach, 2004; Scheurer et al., 1999). Following the incubation of allergic subject serum with the various inhibitors, the disap-
pearance of previously observed IgE binding indicates a lack of specificity/affinity of the IgE for the target protein. Conducting
inhibition studies is important, as the broad range of IgE binding that sometimes occurs may not be clinically relevant (Taylor
and Hefle, 1996; Bindslev-Jensen and Poulsen, 1997; Pasini et al., 2000), particularly for crossreactivity associated with carbohy-
drate determinants (Vieths et al., 2002). Generally, IgE binding to carbohydrate domains is irrelevant to clinical allergy, unless there
are multiple carbohydrates on the potentially allergenic protein (Altmann, 2007; Marie et al., 2008). Additional details on key
factors of experimental design and methodology for serum IgE tests, especially strategies to minimize false negatives and false posi-
tives, are published elsewhere (Goodman, 2008). If there is evidence of IgE binding in vitro and there is a desire to continue with
development of the GM crop, the biological relevance of binding should be tested using basophil activation or basophil histamine
release (Asero et al., 2007). Alternatively, SPT or DBPCFCs may be employed using highly characterized test materials and subjects
who are well informed and have consented to the challenge. The latter assessments, however, are unlikely to be conducted due to
the associated ethical concerns. Importantly, if there is no evidence the protein is likely to be an allergen based on source and a lack
of a bioinformatics match, there is no justification for performing serum IgE tests. There is a low probability of risk and there is no
at-risk population.

11.29.10 Second Category of Potential Allergenic Risk

Potential de novo sensitization: abundance of protein in crop, stability to pepsin in vitro heat stability, and glycosylation status. Abundance of Protein in the Crop and Stability to Pepsin In Vitro

To evaluate the second potential allergy risk associated with agricultural biotechnology, the creation of food allergens de novo,
biochemical and physical properties of the protein are evaluated as well as its abundance in the GM crop. The biochemical and
physical endpoints include stability to pepsin and trypsin digestion in vitro, glycosylation status, and stability to heating (i.e., pro-
cessing effects). As suggested by Metcalfe et al. (1996), proteins that are stabile in pepsin in an in vitro digestion assay and are abun-
dant have a higher probability of being an important food allergen. However, the correlation is low even though many major food
allergens are stable or fractions of the protein are stable and abundant (Fu et al., 2002; Bannon et al., 2003; Ofori-Anti et al., 2008).
There is also not a consensus on abundance, although it is clear that the abundance of a number major allergenic proteins in plants
used for foods is greater than 1% of the protein in the food fraction (Astwood and Fuchs, 1996). Moreover, novel proteins in GM
crops are expressed at very low levels, in the ppm or ppb range. For example, Cry 1Ab expression level in MON 810 GM corn event
was detected to be 0.83  0.15 ppm in the grain (Szekacs et al., 2010). Similarly, Cry1F expression levels in Herculex 1 GM corn was
between 71 and 115 ppm (Mendelsohn et al., 2003). Mean expression levels of Cry1Ac in DBT418 corn kernels ranged from 36 to
42.8 ng/g dry weight (FSANZ 20013 safety assessment of DBT418 corn). According to the WHO/GEMS database, the consumption
rate of corn in human is 4.98 g/kg/day; therefore, 42.8 ng/g Cry1Ac corresponds to around 213 ng/kg of Cry1Ac for humans.
Many food allergens share certain properties, notably stability as defined using denaturants (such as heat) and biochemical
measures of stability, such as resistance to pepsinolysis (Breiteneder and Mills, 2005). Indeed, resistance to digestion is a property
common to many, although not all, dietary proteins thought to sensitize by the human gastrointestinal tract (GIT) (Mills et al.,
2004). In order to sensitize an individual via the GIT, an allergen must have properties which preserve its structure from degradation
(such as resistance to low pH, bile salts, and proteolysis), thus allowing enough allergen to survive in a sufficiently intact form to be
taken up by the gut and sensitize the mucosal immune system (Taylor and Hefle, 2001; Mills et al., 2004). Investigations into the
role of digestion in allergenicity of proteins have been hampered by a lack of common approaches and protocols for modeling
gastrointestinal digestion in vitro. The first published application of an in vitro pepsin digestion assay to address the question of
food allergen stability was by Astwood et al. (1996). In this study, a number of food allergens or their peptide fragments were shown
to be resistant to digestive proteolysis in vitro. Since this initial report, there have been several studies repeating the pepsin digestion
assay for a variety of proteins (Buchanan et al., 1997; Kenna and Evans, 2000; Okunuki et al., 2001; Fu, 2002; Herman et al., 2007;
Mandalari et al., 2009). A number of variations in SGF assay parameters have been reported and include differences in the pH of the
assay, the purity of the pepsin, the pepsin to target protein ratio, the target protein purity, and, finally, the method of detection. A
standardized protocol for evaluating the in vitro pepsin resistance to proteins has been established in the context of an international
interlaboratory study (Thomas et al., 2004) (Fig. 11). Although a correlation between resistance to pepsin digestion and allergenic
potential has been proposed (Astwood et al., 1996), the correlation is low (Fu et al., 2002). There are examples of proteins in food
that are not digestible and do not illicit food allergy. Likewise, it cannot be concluded that allergenic food proteins are necessarily
more resistant to digestion. There is work in kiwi that suggests that these unstable allergens may be protected from pepsin digestion
by components of the food matrix (Polovic et al., 2007). Therefore, measurement of protein digestibility should not be regarded as
a stand-alone endpoint for the safety assessment of novel proteins. Instead, a weight-of-the-evidence approach, which accounts for
a variety of factors and experimental approaches for an overall assessment of the allergenic potential of the new protein, should be
utilized, as no single factor has been recognized as predictive of protein allergenicity. Novel proteins used in GM crops (e.g., Bt
proteins in corn or CP4 EPSPS in herbicide tolerant soybeans) are in general rapidly digested in pepsin in less than 1 minute
(Herman et al., 2003; Herouet et al., 2005; Herman et al., 2006).
Protein Allergy and GMOs 655

Fig. 11 Assay to Measure Protein Stability to Pepsin In Vitro. Heat stability

Stability of some protein allergens in the presence of heat is considered an important feature for the retention or increase in the
allergenicity of some foods after cooking or processing (Breiteneder and Mills, 2008). For most proteins, function is linked to their
native folded conformation (Berg et al., 2002). Therefore, loss of protein function strongly correlates with loss of native structure. A
protein function assay (e.g., enzyme activity) is the method of choice to assess thermal stability in the context of an allergenicity risk
assessment since a protein’s function is a relevant biophysical feature of the protein (Indian Ministry of Science and Technology,
2008). While a functional assay is commonly used to test the heat stability of novel food proteins, some regulatory authorities
also require the inclusion of an immunodetection assay using polyclonal IgG antibodies generated in animals as a prospective
surrogate for IgE-binding assessments. However, animal IgG binding is not considered an appropriate substitute when assessing
allergenicity of novel proteins as this is an indication of immunogenicity rather than allergenicity (Davis et al., 2001). Furthermore,
antibody binding does not necessarily correlate with loss of protein function, so assessing these two different endpoints together are
not additive to the weight-of-the-evidence approach used for identifying potential allergenicity, and, in fact, they can be contradic-
tory to one another. This is exemplified by the safely consumed phosphinothricin acetyltransferase (PAT) protein (Herouet et al.,
2005), which is inactivated at 40–45 C (15 min) or 60 C (10 min) but is clearly detectable even after heat treatment at 100 C.
These results show that the immunoreactivity is still detectable even if the PAT protein loses its enzymatic activity. In addition,
the same recognition of the heat-treated and the native proteins by anti-PAT antibodies indicates that the conformational changes
associated with denaturation do not affect the epitope accessibility by IgG. Therefore, a correlation between the loss of functional
activity upon heating or immunodetection and the allergenic status of a protein has not been consistently demonstrated. There is
a distinct difference between the maintenance of allergenicity in cooked food (i.e., the ability to elicit IgE binding in vivo) and the
retention of endogenous protein function (enzymatic or biological activity) (Privalle et al., 2011).
Heat-mediated unfolding may cause a loss of function and this could occur in conjunction with a change in immunological
status such as a loss of conformational IgE-binding sites (e.g., where sensitization occurred to native protein); unfolding could
be associated with no effect because only linear epitopes are relevant; or unfolding can reveal hidden allergenic epitopes (Mills
et al., 2009; Sathe and Sharma, 2009). These immunological impacts, however, are not known to correlate with or be caused by
a loss of protein function itself. Heat treatment has been shown to completely eliminate the allergic potential of some allergens
such as patatin protein in potato (Koppelman et al., 2002), chitinases present in fruits (Sanchez-Monge et al., 2000), and the hazel-
nut Cor a 1.04 allergen (Pastorello et al., 2002; Skamstrup Hansen et al., 2003). These are primarily incomplete allergens that
cannot sensitize but can elicit an allergenic reaction after sensitization to a crossreactive protein. Kiwi fruit has several allergens,
including crossreactive lipid transfer protein and chitinase, and most appear sensitive to heat (Fiocchi et al., 2004). The production
of soybean meal, a process which involves heat treatment, dramatically changes the profile of the extractible proteins and their
immunological properties (Franck et al., 2002). However, the allergenicity of the heat-treated soybean meal is not significantly
altered (Besler et al., 2001; Franck et al., 2002). Neoallergen formation has been known for at least four decades; it may be part
of the reason why some individuals can tolerate a raw food or raw food ingredient but will react to the same food when it is pro-
cessed. Neoallergens have been identified from pecans, wheat flour, roasted peanuts, lentil, almond, cashew nut and walnut,
soybean, shrimp, scallop, tuna, egg, apple, plum, milk, and potato (Vojdani, 2009).
In summary, measured loss of function and changes in protein conformation have no consistent association with changes in the
clinical allergenicity of protein allergens: structural changes to proteins can have no effect on allergenicity, may increase allergenicity,
or reduce allergenicity (Mills et al., 2009; Paschke, 2009). For a novel food protein, there is no way to predict which might occur.
Since the thermal stability of novel food proteins as assessed by a functional assay or immunodetection does not necessarily or
consistently correlate with allergenic risk, it does not provide any safety information as part of the allergy risk assessment of trans-
genic crops. Heat stability results with novel food proteins have no known predictive value in the allergenicity risk assessment. In
a limited approach to supporting the broader safety assessment of novel proteins, thermal deactivation may be relevant to the toxi-
cological risk assessment for cooked or processed food if a protein has some known adverse toxicological effect associated with its
function (Hammond and Jez, 2011).
656 Protein Allergy and GMOs Glycosylation status

A number of protein allergens are glycosylated, suggesting the possibility that the glycosyl groups may contribute to their allerge-
nicity (Huby et al., 2000; Breiteneder and Mills, 2005). Oligosaccharides are naturally added to many proteins during or after their
synthesis in eukaryotic cells. Glycosylation involves the covalent attachment of oligosaccharides most commonly to asparagine (N-
linked) or serine/threonine (O-linked) amino acids. Glycosylation can influence the physical properties of proteins such as stability,
hydrophobicity, solubility, and electrical charge, which may then affect its antigenic and allergenic potential. For example, antigen-
presenting cells have been shown to have enhanced uptake of glycosylated proteins compared to their nonglycosylated counterparts
(Sallusto et al., 1995). The latter uptake may be due to the presence of specific sugar receptors on the surface of antigen-presenting
cells (Condaminet et al., 1998). In addition, it has been reported that the receptor-mediated uptake of proteins by antigen-
presenting cells produced a quantitative increase in the antigenicity of proteins (Tan et al., 1997; Agnes et al., 1998). These data
suggest that antigen-presenting cells effectively process glycosylated proteins and subsequently mediate an enhanced immune
response. Garrido-Arandia et al. (2014) investigated the role of N-glycosylation in kiwi allergy. These investigators reported that
the sugar moiety induced the activation of antigen-presenting cells, thus playing a role in sensitization. Garrido-Arandia et al.
(2014), however, also indicated that it was the kiwi protein fraction, and not the sugar moiety, that was responsible for the allergic
reactions. Approximately 20% of allergic patients generate specific antiglycan IgE. Despite antibody-binding glycoproteins being
common in foods, insect venoms, and pollen, crossreactive carbohydrate determinants do not appear to cause clinical symptoms
in patients (Altmann, 2007). Nevertheless, there are many glycosylated proteins that are not allergenic. Therefore, it is important to
not consider glycosylation by itself, but rather in the context of the overall weight-of-the-evidence data in regard to the allergenic
potential of a novel protein as described earlier.

11.29.11 Third Category of Potential Allergenic Risk: Assessment of Endogenous Allergens

The question of whether transformation of a gene might increase endogenous allergenicity has been raised. The measurement of
endogenous allergens, however, represents the least likely potential risk associated with GM crops. It is mentioned as part of the
“compositional analysis of key components” in GM crops including nutrients, antinutrients, and toxicants in the Codex Alimentar-
ius document (Codex, 2009). Additionally, a requirement from the EU Commission to measure endogenous allergen levels in
soybean as part of the compositional assessment of GM crops has recently been implemented (European Commission
Implementing Regulation No. 503/2013). This recommendation, however, has generated much debate on the relevance of such
data in the risk assessment for GM crops (Goodman et al., 2008; Doerrer et al., 2010; Herman and Ladics, 2011; Panda et al.,
2013; Fernandez et al., 2013; Ladics et al., 2014b). There are several reasons why the measurement of endogenous allergens in
GM crops does not add to their risk assessment. First, allergic individuals will attempt to avoid offending foods completely, whether
GM or non-GM. Since soybean-containing products are labeled, based on labeling regulations including the regulation in the EU,
this risk is manageable by allergenic patients. Therefore, the level of allergen(s) is irrelevant when the food is already known to be
allergenic and is regulated as such. Second, the measurement of endogenous allergens in non-GM crops demonstrates a wide range
in seed concentrations due to differences in the genetics of commercial varieties (Houston et al., 2011) and the interactions of those
varieties with the environment (i.e., temperature, moisture, nutrients, plant pathogens, insect loads) (Sancho et al., 2006a,b;
Goodman et al., 2008; Doerrer et al., 2010; Stevenson et al., 2012). These data are not surprising given the finding that the insertion
of a small number of genes via transgenesis is less impactful on crop composition compared with traditional breeding methods
(Herman et al., 2009; Parrott et al., 2010; Ricroch et al., 2011; Herman and Ladics, 2011; Herman and Price, 2013). In addition,
several significant studies have evaluated IgE binding between non-GM and GM soybean varieties and found no significant differ-
ences between the GM and non-GM varieties (Sten et al., 2004; Hoff et al., 2007). Lastly, data are lacking in regard to thresholds for
individual proteins for either the sensitization or elicitation phase of an allergic reaction which prevents the interpretation of any
differences between the GM and non-GM crop (Taylor et al., 2002; Taylor and Hourihane, 2008; Crevel, 2010; Chassy, 2010).
Furthermore, there is no quantitative association between the exposure to an individual amount of a protein allergen(s) and the
risk for sensitization and/or clinically relevant reaction (Panda et al., 2013). For the earlier reasons, it is not clear what relevance
the measurement of endogenous allergens has to the risk assessment of GM crops (Rouquie et al., 2010; Ladics et al., 2014b).
Importantly, conventional breeding tactics, such as chemical and radiation mutation, can also alter existing endogenous levels
of allergenic proteins. Analytical Methods to Measure Endogenous Allergens

The ability to measure small differences in allergens among crop varieties is increasing; however, the challenge remains on how to
place these data within the context of assessing food safety risk. Historically, endogenous allergen data for extracts of GM soybean
(versus its non-GM counterpart control and commercially available non-GM reference varieties) were generated using two-
dimensional (2D) gel electrophoresis. This approach separates proteins according to their isoelectric point (pI) in the first dimen-
sion and molecular mass (Mr) in the second dimension and is coupled with antibody (i.e., IgE immunoblots that utilize sera from
well-characterized, clinically relevant soybean allergic subjects) or dye-based detection methods (Rouquie et al., 2010). Achieving
separation allows for quantification of the various spots using densitometry methods. Ultimately, these gel methods rely on MS to
Protein Allergy and GMOs 657

Fig. 12 Quantification of Proteins by MS Using Multiple Reaction Monitoring where Signals from the Endogenous Protein are Compared to Those
of a Synthetic Heavy Isotope-Labeled Internal Standard.

distinguish the highly related proteins present in the various spots. The multiplicity of 2D spots that must each be accounted for in
a quantitative study coincides with the biggest problem of 2D gel-based quantificationdlow throughput (Rabilloud et al., 2010).
Furthermore, the immunoblot studies to evaluate endogenous allergens are difficult to conduct due to the lack of availability of
well-characterized, clinically relevant serum samples from a sufficient number of allergic subjects because the prevalence of individ-
uals with food allergies in general is quite low (i.e., approximately 1–2% of adults and 4–6% of children) (Thomas et al., 2007;
Ladics, 2008). For specific food allergies (e.g., soybeans), the prevalence is estimated to be 0.4% in children and 0.3% in adults
in North America and even lower in the EU (Zuidmeer et al., 2008; Sicherer and Sampson, 2010).
More recently, a number of analytical tools, such as quantitative MS (Stevenson et al., 2010, 2012; Lee et al., 2010; Houston
et al., 2011) and ELISAs (Liu et al., 2012; Ma et al., 2010; You et al., 2008), have been employed. These methods are used to quan-
titatively measure protein expression level. Limitations of the ELISA method are access to validated methods, reagents, and stan-
dards. Quantification of proteins by MS can be achieved using a technique called multiple reaction monitoring (MRM), during
which signals from the endogenous protein are compared to those from a synthetic heavy isotope-labeled internal standard
(Kirkpatrick et al., 2005; Brun et al., 2009; Houston et al., 2010; Stevenson et al., 2010) (Fig. 12). More specifically, MRM analyzes
monitor peptides from proteins of interest, which are specific products of proteolysis often generated using the enzyme trypsin. The
internal standards are synthetic peptides identical to the endogenous peptides of interest that have been labeled by the addition of
a heavy isotope-containing amino acid, thereby changing its mass but nothing else. Because MRM is a form of tandem MS, peptides
(precursors) are fragmented during the analysis. Fragmentation is used to verify the sequence of the specific peptide of interest,
which provides an additional level of specificity to the analysis. In order to comply with the recent EU regulation, it is recommended
that analytical tools such as ELISAs and/or MS be employed to quantitatively measure endogenous allergens. The main challenge for
routine application of proteomics techniques in safety assessment of transgenic crops is to validate and standardize methodologies
to ensure its reproducibility and robustness and to interpret the significance of the large quantity of data generated. Therefore, it is
necessary to evaluate the natural variability of those allergens in non-GM crops (Houston et al., 2010; Stevenson et al., 2012; Ladics
et al., 2014b).

11.29.12 Endpoints Requiring Further Evaluation/Validation Animal Models to Evaluate Potential Protein Allergenicity
There has been research with animal models that shows some promise for evaluating mechanisms of allergy and immunotherapy
(Kulis et al., 2012) and for preliminary ranking of allergenic sources (Sun et al., 2013; Ahrens et al., 2014). Animal models are
frequently used for confirming the hypoallergenicity of foods (e.g., milk formulas for infants). Researchers have also investigated
the use of animal models to predict the allergenic potential of novel proteins in food as first suggested by Metcalfe et al. (1996).
The use of animal models for predicting food allergy gained further attention by the FAO/WHO recommendations (FAO/WHO,
2001), which called for testing each novel protein in two animal models based on two different sensitization routes, even though
the advisory publication recognized that there were no validated animal models for predicting food allergenicity. A number of
investigators have worked on developing models for predicting or ranking the potential allergenicity of food proteins in several
different species as reviewed in Ladics et al. (2010). Several models have been proposed using rats (Knippels et al., 1998,
1999a,b; Knippels et al., 2000; Ladics et al., 2003), mice (Dearman et al., 2000; Dearman and Kimber, 2001; Bowman and Selgrade,
658 Protein Allergy and GMOs

2008; Herouet-Guicheney et al., 2008; Aldemir et al., 2009), swine (Helm et al., 2002, 2003), or dogs (Ermel et al., 1997; Buchanan
and Frick, 2002; Teuber et al., 2002). Most of these models are based on the assessment of induced antibody (i.e., IgE or IgG)
responses and the frequency of responders in the test groups.
Presently, none of the animal models have been tested with a wide range of allergens and nonallergens, and there is a lack of data
on the reproducibility and predictive value (sensitivity and specificity) of any of the models.
A number of relevant questions still remain and include: (1) What is the most appropriate endpoint or design for an animal
model? Different species have been tested (e.g., mouse, rat, swine, and dog) and clear strain differences have been observed that
significantly impact results. This includes the potential use of various adjuvants, dosing regimen (i.e., number and timing of antigen
doses) and the route(s) of exposure for sensitization and challenge (Dearman et al., 2001). For instance, it may appear that the oral
(po) or intragastric (ig) route are the most relevant for testing food proteins; but the complication of oral tolerance by prior exposure
to the protein and uniformity of dosing must be overcome (e.g., via coadministration of adjuvant). Yet, while coadministration of
adjuvant will increase the sensitivity of detection of IgE antibody responses to proteins particularly by the ig route, there is concern
that this may result in some loss of specificity. On the other hand, an intraperitoneal (ip) injection may represent the most direct
assessment of the allergic potential for a novel protein, and it has been demonstrated that ip injections may overcome the tolerance
that may occur if the protein is administered orally (Dearman et al., 2003) as the antigen-presenting cells and lymphatic routes are
different for ip compared to ig. In addition, sensitization by routes other than ip or ig (e.g., dermal, subcutaneous, or inhalation)
may need to be considered. For example, Nelde et al. (2001) reported that dermal application of ovalbumin (OVA) in BALB/c mice
induced antigen-specific IgE production more efficiently than via the ip route, although anaphylactic symptoms could be induced in
all mice independently of the route of antigen application. Hsieh et al. (2003) reported successful induction of clinically active IgE
to OVA (based on oral challenge) by 4 week epicutaneous applications of 0.1 mg antigen on the shaved backs of BALB/c mice.
Administration of food extracts by transdermal application to mice has also been investigated (Navuluri et al., 2006; Birmingham
et al., 2007). (2) What constitutes the measurement of a positive allergic response? Given that IgE is the most common marker of
allergic sensitization and mechanistically it is essential for inducing the most common type of allergic reactions in humans
(Bruijnzeel-Koomen et al., 1995), it seems valid to choose this endpoint to measure. However, the choice of the test(s) that are
relevant is not as obvious. For example, in vivo measurement of protein-specific bioactive IgE by passive cutaneous anaphylaxis
(PCA) or active cutaneous anaphylaxis reactions, or active systemic anaphylaxis and/or in vitro measurement of serum levels of
antigen-specific IgE (i.e., antibody titers by ELISA) are not always equivalent. It is also known that the abundance of antigen-
specific IgE, as measured by in vitro antigen binding is not absolutely correlated with symptoms of food allergy (McClain and
Bannon, 2006). More generally, what constitutes negative or positive allergic responses in an animal model, like the level of anti-
body titer, the number of positive responders in the PCA assay, and/or frequency and severity of clinical signs of allergy, could
impact the concordance of an animal model (i.e., the ability to correctly identify both positives and negatives). Ideally, the validity
of any animal model will be based on the demonstration of a rank order of potency, as suggested by Dearman and Kimber (2001),
for a number of allergens, comparable to what is known regarding their prevalence and severity of responses in humans (Osterballe
et al., 2005; Rona et al., 2007; Bjorksten et al., 2008). Importantly, in regard to the potency of food allergens/allergenic foods in
humans, there is only information available on their severity in challenge reactions, as nothing is known on the sensitizing potential
of these food items in humans (Taylor and Hefle, 2001; Crevel et al., 2008; Ladics et al., 2014a). (3) What is the most appropriate
form of protein to test (i.e., isolated pure protein vs. protein in the food matrix)? Given all the questions raised earlier, it is not
surprising that to date, no animal model has been extensively evaluated and validated with pure proteins or unprocessed or pro-
cessed foods (Ladics et al., 2010). Targeted serum screens

The Joint FAO/WHO (2001) consultation also recommended using targeted human serological screening in an attempt to deter-
mine whether a protein that is not similar to any known allergen might pose a risk due to existing sensitization or crossreactivity.
Therefore, targeted sera screening was recommended in order to help identify potentially crossreactive proteins using sera from
patients allergic to substances broadly related (i.e., dicots, monocots, fungi/yeast, vertebrates, invertebrates) to the source of the
gene. For example, for genes from a dicotyledonous plant, individuals allergic to one or more other dicot species would be used
for sera testing. There was an exemption for proteins from bacteria since there are almost no known allergies to bacteria. As noted
earlier, an issue of critical importance to any sera screening study is the availability of well-characterized, clinically relevant human
sera from a sufficient number of subjects. Furthermore, the positive and negative predictive value of the targeted sera screening
method has not been demonstrated and the method may have no clinical significance. For example, some types of IgE binding
found in these assays may not be clinically relevant (affinity, single vs. multiple epitopes) and thus can lead to a high degree of false
positives or false negatives (Taylor and Hefle, 1996; Bindslev-Jensen and Poulsen, 1997; Pasini et al., 2000). In addition, the occur-
rence of crossreactive IgE antibodies is not always correlated with clinical food allergy, particularly for crossreactivity for carbohy-
drate determinants (Vieths et al., 2002). Thus, the predictive value or clinical relevance of using targeted serum screens has not been
established and was identified by Codex (2003, 2009) as one technique requiring further development and validation before being
implemented. The targeted serum testing has never been tested in a way that would demonstrate its predictive power and it is coun-
terintuitive based on our knowledge of crossreactivity. Homologous proteins from even moderately related sources (family level)
are rarely crossreactive by in vitro tests and clinical reactivity is rarely shared. The only proteins that are so broadly crossreactive in
laboratory tests are profilins, PR-10 proteins (Bet v 1 homologues), lipid transfer proteins, and tropomyosins from crustaceans and
other invertebrates. Such proteins are all easily identified by in silico bioinformatic tools.
Protein Allergy and GMOs 659 Adjuvanticity and physiological-based pepsin digestion assay

EFSA guidance (EFSA, 2011) includes a number of suggestions for unproven tests including: evaluation of potential adjuvanticity of
the new protein and the use of more physiological-based pepsin digestion assay. Adjuvants are immunostimulatory compounds
that can increase and/or modulate the immunogenicity of an available antigen, leading to stronger and longer-lasting immune
responses (Brunner et al., 2010). Many naturally derived materials have been used as immunization adjuvants, and these are often
found in the diet. Some examples of adjuvants include saponins (Rajput et al., 2007); lectins (Cordain et al., 2000); chitin (Svind-
land et al., 2012); taurine (Hayes and Trautwein, 1994); lithium (Ishizaka et al., 1990; cholera toxin (Lycke and Holmgren, 1986);
and lipopolysaccharide (Wang and Singh, 2011). The adjuvanticity of Cry proteins has also been investigated to explore their poten-
tial as vaccine adjuvants (Vazquez-Padron et al., 1999; Moreno-Fierros et al., 2003; Rojas-Hernandez et al., 2004; Esquivel-Perez and
Moreno-Fierros, 2005; Verdin-Teran et al., 2009; Legorreta-Herrera et al., 2010). Some studies of the potential adjuvanticity of Bt
Cry insecticidal proteins have prompted questions about whether ingestion of certain Cry proteins could result in immunomodu-
lation in humans. These studies evaluated potential Bt Cry protein adjuvanticity within laboratory animal vaccine models; models
not appropriate for assessing oral exposure. Study design is highlighted as a critical aspect in discerning the potential use of animal
models to identify adjuvant effects from Cry protein exposure. The main concern with these studies appears to be the coexposure to
unwanted toxicants or known immune-modulating molecules such as LPS or mycotoxins when preparing purified Cry protein or
test diets (Inagawa et al., 2011), the use of antacids which are themselves adjuvants (i.e., aluminum hydroxide) (Vazquez-Padron
et al., 1999), the use of routes other than oral exposure (Moreno-Fierros et al., 2003; Rojas-Hernandez et al., 2004; Verdin-Teran
et al., 2009), and/or very large doses of Cry protein (Guimaraes et al., 2008). As noted earlier, Cry proteins are in GM crops at
very low ppm and ppb levels. In those studies that best control for unwanted contaminants and also use positive, immune-
modulating controls, the results clearly support the hypothesis that Cry proteins likely have low or no immune effects through
the oral route (Kroghsbo et al., 2008; Adel-Patient et al., 2011; Reiner et al., 2014). The known susceptibility of Cry proteins to
gastric digestion in combination with low dietary exposure levels (i.e., ppm or ppb) supports a conclusion that it is highly unlikely
that Cry proteins, as expressed in GM crops, have any potential to act as an adjuvant. Since there is no evidence that the Cry proteins
have any sequence similarity to known mitogens or lectins and the host plants (expressing the Cry proteins) are not commonly
allergenic; there is no reason to consider the test for adjuvanticity of the Cry protein or the GM crop. Furthermore, evidence of repro-
ducibility of animal models that evaluate adjuvanticity is lacking, as there are no studies employing the same model with the same
protocol. Thus, there is no validated method currently available to test for adjuvanticity of proteins to demonstrate how they would
improve the risk assessment of a novel protein. Investigations into the utility of a more physiological-based pepsin digestion assay
are currently underway. For example, the International Life Science Institute’s Health and Environmental Sciences Institute is eval-
uating a combined gastric and duodenal phase in vitro digestion assay based on the paper of Mandalari et al. (2009) using various
allergenic and nonallergenic proteins. However, there is no validated method currently available to demonstrate how such a more
physiologically based digestion assay would improve the risk assessment of a novel protein.

11.29.13 GM Crops: Is There Evidence of New Sensitization and Allergies?

Some scientists and the general public question whether there is evidence that GM crops have increased food allergy since they were
first commercialized in 1996 or whether individuals may become allergic to the novel proteins encoded by the inserted DNA
(Buchanan, 2001). There is no proof that the introduced protein in any approved and commercialized GM crop has caused
food allergy. The Brazil Nut 2S Albumin Protein and Soybean

More than 20 years after regulatory approval of the first GM plants, only one potential product was demonstrated to present a real
risk of food allergy. That possibility was demonstrated by the experience of Pioneer Hi-Bred when they transferred a gene encoding
the 2S albumin from Brazil nut into soybean to improve feed quality for animals. Soybeans have a high concentration of protein but
are deficient in sulfur containing amino acids. The 2S albumin of Brazil nut is a small protein with a high concentration of methi-
onine and cysteine amino acids. In 1995, the allergenic proteins in Brazil nuts were not known; however, upon testing for serum IgE
binding using samples from the at-risk population of Brazil nut allergic subjects, IgE binding was observed. In addition, SPT posi-
tivity for Brazil nut allergic subjects was also observed (Nordlee et al., 1996). That potential product was terminated by the
company, not submitted to regulators, and never commercialized. To date, no currently approved and commercialized product
has received a gene from a commonly allergenic source. Thus, the Brazil nut 2S albumin is the only example that would have pre-
sented a major risk of food allergy to a subset of consumers. The experience with the Brazil nut protein helped to formulate the
evaluation process outlined by Metcalfe et al. (1996) for evaluating potential allergenicity of GM proteins and eventually the Codex
Alimentarius Commission guideline initially published in 2003. StarLink maize

StarLink maize, expressing approximately 50 ppm of Cry 9C protein from Bt that was toxic to certain insect pests (i.e., the European
corn borer moth larvae) of maize and acts as a pesticide, was developed in the mid-1990s. As noted previously, GM proteins in
660 Protein Allergy and GMOs

general have been found to be digested rapidly in pepsin at pH 1.2 or 2. In contrast, the Cry 9C protein was found to be stable to
digestion in pepsin, a characteristic that is considered a potential risk factor for either sensitization or elicitation of food allergy
(US EPA, 2000b,c). However, at such low concentrations, it is an unlikely candidate as a potential new food allergen. Furthermore,
there are a number of nonallergenic food proteins that are highly stable to pepsin digestion (Fu et al., 2002). Food approval in the
United States was withheld because the protein was stable in the pepsin digestion assay and regulators felt there was some risk the
protein might eventually sensitize someone, predisposing them to allergic responses to Cry 9C. StarLink maize was grown
on  122,000 hectares in the United States in 1999, and some grain from the corn was accidentally, but illegally, included in
some human food products (corn chips and taco shells). Tests by an anti-GM nongovernment organization discovered the inclusion
of Cry 9C in some food products and notified the US government and news media. The question asked was whether people might
become allergic to the Cry 9C protein; however, time would be needed before people became sensitized. There was no indication that
individuals were preexposed to Cry 9C, so sensitization would have been from exposure in the contaminated taco shells and chips.
However, within 2 weeks of the announcement, some consumers complained they had experience food allergic reactions following
consumption of taco shells or corn chips. Since corn is one of the least allergenic of grains and the quantity of Cry9C was very low in
the ppm range in corn grain and the grain was grown for only one year, it is highly unlikely that individuals were sensitized to the
protein. Nevertheless, the US CDC investigated each consumer report. Some individuals who claimed reactions that might be consis-
tent with food allergy provided blood samples. None of those individuals had IgE specific for the Cry 9C protein (Raybourne et al.,
2003). Since the grain and corn seeds were released and consumed in food without approval, the US government demanded a food
recall and subsequent monitoring. Foods, ingredients, and corn seed were screened and those containing the Cry 9C protein or the
transgene were pulled from the market. It took approximately 6 years to completely remove all traces of the Cry 9C protein from seed
and grain stores. Importantly, there is no scientific proof that anyone was harmed or sensitized by consuming the Cry 9C protein. CP4 EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)

Serum IgE binding to the CP4 EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) protein in Monsanto’s herbicide tolerant
soybean was tested as a postmarket monitoring evaluation (Hoff et al., 2007). The study was performed to determine whether
soybean allergic subjects might have IgE binding to the CP4 EPSPS enzyme that was introduced into soybean to provide tolerance
to the herbicide glyphosate. This was not a regulatory study but was rather performed for product stewardship purposes to deter-
mine if there was any evidence of sensitization many years after the product was initially introduced into the market. Serum samples
were collected from soybean allergic subjects in Europe and South Korea and tested using common protocols and highly charac-
terized test materials. The study did not find evidence of IgE binding to purified CP4 EPSPS or to the protein in extracts of GM
soybeans (Hoff et al., 2007). Postmarket monitoring could be performed based on consumer complaint communication and
follow-up or by direct testing of selected populations. The intent of either is to sample the population of new consumers to measure
sensitization rates and provide data for considering risk (Hepburn et al., 2008). However, it is important to consider the technical
challenges of measuring specific sensitization in real populations. The sample size and selection of subjects are critical, an estimate
of exposure is essential, and baseline (preexposure) serum samples are important, as well as postexposure measurement and clinical
evaluation. New proteins expressed in the GM crops approved so far have been expressed and accumulated at low levels (i.e., ppm
or ppb levels) in the food materials of the crop, often in the range of or less than a few micrograms per gram dry weight of seed
(CERA GM Crop database, 2014; Thus all of the GM proteins accumulate at
levels markedly below the concentration of most of the important dietary allergens (typically > 1% of total protein) (Astwood
and Fuchs, 1996). There is no published evidence that an approved GM crop has caused allergies due to the presence of the trans-
genic protein. Alpha-amylase inhibitor protein The alpha-amylase inhibitor (άAI) protein is expressed at up to 4% of protein in
many rarely allergenic common beans (Phaseolus vulgaris). The gene was transferred into field peas (Pisum sativum) to protect the
seeds from storage beetles which can cause 100% loss of product.
The GM pea was tested in a nonvalidated animal model, triggering sensitization and eosinophilia during airway provocation
(Prescott et al., 2005). The results have blocked further development of GM legumes with transferred άAI in areas that would benefit
from such products (Goodman et al., 2008). A more recent study was unable to reproduce the Prescott et al. (2005) findings and
reported that the GM pea was no more allergenic than non-GM peas and other legumes (Lee et al., 2013). Evaluation of the άAI
protein following Codex guidelines (personal communication, Goodman) indicated a need to test serum IgE binding with peanut
sera due to low-level sequence identity of άAI with peanut agglutinin, a protein rarely reported to cause allergy. Sera from 34 peanut
allergic subjects failed to demonstrate crossreactivity but did uncover specific IgE binding to asparagine-linked carbohydrate deter-
minants (CCD) on άAI in common with binding to CCD on other legume proteins (Campbell et al., 2011; Ladics et al., 2014a).
However, basophil tests using the same sera failed to demonstrate activation, suggesting there is little likelihood of a risk of food
allergy to this product. In fact, the risk would be no different than that posed by common beans.

11.29.14 Summary

The introduction of whole new foods (e.g., kiwi fruit) in a population, or the introduction of commonly consumed foods in the diet
of any individual, may lead to sensitization and food allergy. Although eating is a risk factor for food allergy, there are only certain
Protein Allergy and GMOs 661

Fig. 13 The Codex Guideline (2009) Recommends a Weight of Evidence Approach to Maximize the Probability of Identifying Proteins with Potential
Risks of Inducing Food Allergy.

foods that can cause significant food allergies, with only a few of the many proteins in food accounting for such allergic reactions.
The Codex (2009) guideline for evaluating food safety was designed to maximize the probability of identifying significant risks of
food allergy (Fig. 13). The assessment process is efficient for identifying proteins that are likely to present a significant risk of food
allergy, which would be the transfer of a known allergen or a likely crossreactive protein. There is less certainty trying to predict
whether a new protein with no obvious risks factors might sensitize de novo, but risks are clearly low in those cases where the
protein is rapidly digested by pepsin in vitro and/or low in abundance in the food component. The potential that a GM crop
has significantly higher expression of endogenous allergens is quite low compared to non-GM varieties, but, in addition, the risk
is for those consumers who should be avoiding consumption of food from the host plant in the first place. Thus, there is no practical
increase in risk even if the content of endogenous allergens was increased. The primary risk would be introducing a major allergen
from one source into a new food source or transferring a protein that is nearly identical to a major allergen and capable of causing
crossreactions. Those high risks are for individuals who are already sensitized and the methods for assessing such risks are relatively
straightforward (Goodman et al., 2008). There is much to learn about factors influencing sensitization (Ladics et al., 2014a; Van Ree
et al., 2014; McClain et al., 2014) and allergy in general; however, at this time, those GM crops in development with the potential to
cause allergy can be identified and their introduction stopped before commercialization.
How do we know GM crops are safe? There is an extensive safety assessment process in place that evaluates GM crops for poten-
tial allergenicity and toxicity issues and employs a weight-of-the-evidence approach. In fact, GM crops have undergone more testing
than any other food in history (Cockburn, 2002). Since GM crops were first commercialized 20 years ago, there is no evidence that
the introduced novel protein(s) in any approved and commercialized GM crop has caused food allergy.


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Relevant Websites – The US Department of Health and Human Services. – The University of Nebraska. – The University of Nebraska. – The University of Texas Medical Branch.