Evaluation of Potential Allergenicity of New Wheat Varieties
Evaluation of Potential Allergenicity of New Wheat Varieties
The goal of the study is to determine accurate blood test levels that can predict whether or
 not subjects are truly allergic to certain foods. In this study, subjects that are clinically
 documented to be wheat-allergic will participate in a double blind oral food challenge. Blood
 will be collected from the subject to evaluate the potential allergenicity of proteins
 introduced into genetically modified crops and evaluate wheat-specific immunoglobulin-e (IgE)
 antibody reactivity to biotech and conventional wheat varieties.
The prevalence of food allergy is increasing in the pediatric population. A diagnosis of food
 allergy carries significant medical, nutritional, and psychological implications for both
 patients and their families. Therefore, making an accurate diagnosis is critical. However,
 the diagnosis of symptomatic food allergy continues to be a challenge for allergists and
 other practicing physicians. The patient's clinical history remains a key component in
 diagnosing food allergy. Skin prick testing and food-specific immunoglobulin-e (IgE) testing
 act as secondary tools to aid in the evaluation process, but these tests also suffer from a
 high rate of false positivity. For specific IgE levels below the cutoffs to these foods,
 however, supervised oral food challenges (OFCs) may be necessary to establish the diagnosis
 of symptomatic food allergy, as the sensitivity of these values is relatively low. Currently,
 the double-blind, placebo-controlled food challenge (DBPCFC) remains the gold standard for
 the diagnosis of food allergy. OFCs have their own inherent drawbacks: they are not always
 readily available to all patients, they are time-consuming, and they are potentially
 dangerous due to their risk of anaphylaxis.

 Wheat is one of the eight allergenic foods that are responsible for approximately 90% of all
 food allergies. Wheat is less allergenic than other foods in this group and less likely to
 cause multi-organ, severe, life-threatening reactions. Allergy to wheat is more prevalent in
 children than adults and is considered a transient allergy of infancy/childhood. Wheat
 allergy is often outgrown in early childhood, in most cases by age 3-5 years, but 35% show a
 persistent allergy into adolescence. While previous studies using food-specific IgE
 antibodies have determined diagnostic decision points that indicate a high likelihood of
 clinical reactivity for certain foods, namely milk, egg, peanut, and fish, wheat-specific IgE
 levels do not seem to be useful predictors of food challenge outcome, enabling reduction in
 need for food challenges. Furthermore, false-positive IgE reactions to wheat and other
 cereals are frequently seen in grass-allergic patients due to insignificant cross-reactivity
 between water/salt-soluble proteins. Therefore, identification of wheat proteins associated
 with symptoms to wheat is of importance.

 Gliadins in particular have been implicated in allergy to wheat, both in IgE-mediatedallergy
 and the non-IgE mediated celiac allergy. IgE antibodies to ω-5-gliadin have been found in
 children with immediate reactions to ingested wheat, and published results show that
 increased levels of IgE to ω-5-gliadin correlate with the outcome of food challenges. In a
 Finnish study, all children with IgE antibodies to ω-5-gliadin reacted with immediate
 symptoms to wheat challenges, while all patients with delayed or negative challenge test
 results showed no detectable IgE antibodies to ω-5-gliadin.15 In a Japanese cohort, the
 presence of elevated ω-5-gliadin-specific IgE antibodies was associated with immediate
 symptoms with wheat food challenges and also in patients with a strong convincing history of
 wheat allergy who were not challenged due to risk of anaphylaxis. Furthermore, patients with
 severe reactions upon challenge had significantly higher levels of ω-5-gliadin-specific IgE
 antibodies compared to children with mild or moderate symptoms. In a multicenter Japanese
 study, Ebisawa et al. showed a significant relationship between the probability of wheat
 allergy and the concentration of ω-5-gliadin-specific IgE antibodies with 2.6-fold increased
 risk. In a study of mixed German and American subjects, ω-5-gliadin-specific IgE antibodies
 did not correlate with food challenge outcomes in patients with suspected allergy. However,
 the sample size in this study was much smaller than in others, and the German population
 included more patients with non-IgE mediated wheat allergy with delayed symptoms, almost half
 of the German cohort. Nonetheless, ω-5-gliadin-specific IgE antibodies may differ among
 populations in Asia, Europe, and the United States due to dietary habits and genetics, and
 further study is warranted, especially in a larger U.S. cohort.

 Food crops that have been developed through agricultural biotechnology for commercial use are
 thoroughly assessed for their safety. One of the key elements in the safety assessment of a
 genetically improved crop is an evaluation of potential changes in their allergenic
 properties. Allergenic properties of the crop can potentially be altered if a known allergen
 or a protein that has high potential to become an allergen is introduced. In addition, the
 level of expression of endogenous allergens might be altered as a result of transformation
 and insertion of the new gene into the plant genome.

 Since wheat is a known allergenic food crop, international guidelines require analyses to
 determine if the introduction of the genes and production of the recombinant proteins in
 wheat cause an unintended change in the levels of endogenous allergenic proteins. To address
 this question, levels of wheat-specific IgE binding observed in the biotechnology derived
 wheat varieties are compared to the set of binding values observed in reference wheat
 varieties that are already on the market. Determining the levels of direct IgE binding using
 an enzyme linked immuno-sorbent assay (ELISA) has been shown to be an appropriate method to
 perform such comparisons, especially when the assay is validated and calibrated prior to the
 production of data. This comparison is important as it will enable determinations of whether
 there is heterogeneity in wheat-allergic patients with wheat allergy in terms of a
 predominant trigger. Ultimately, crops may be engineered to eliminate culprit components if
 predominant.

 Food challenge outcomes and standard ImmunoCAP IgE assays to wheat will be compared to CRD
 wheat testing in this study (Thermo Fisher Scientific, Waltham, MA). When evaluated together
 with clinical history, skin test reactivity, and OFC outcomes, responses to individual
 components may be able be used to predict food challenge responsiveness. With respect to
 intact wheat-specific IgE levels, in two previous studies using these, a true 95% positive
 predictive value cutoff to predict reactivity at OFC could not be determined to wheat,
 although a physician challenge was suggested at an estimated wheat level of 26 kU/L to
 determine possible reactivity.6,7 However, these studies are now over a decade old and
 pre-dated component resolved diagnostics (CRD). CRD to major proteins ω-5-gliadin may prove
 to be more beneficial in determining predictive levels of IgE and positive OFC outcomes to
 wheat. Therefore, in addition to determining wheat component patterns that could predict OFC
 outcomes, the investigators hope to determine 95% predictive decision points for each of the
 individual components and compare these to the decision points estimated by the ImmunoCAP
 assay to wheat.
Inclusion Criteria:

 -  Age 1 year to 21 years of any sex and any race

 -  Physician-diagnosed food allergy to wheat with convincing clinical history of food
 allergy to wheat AND a skin prick test positive to wheat (diameter of wheal 3 mm or
 greater than negative control) or a detectable serum food-specific IgE level
 (ImmunoCAP > 0.35 kUA/L) to wheat within the previous 12 months or at the time of
 entry into the study

 -  If no history of clinical reactivity to wheat, then a positive skin prick test to
 wheat (diameter of wheal 3 mm or greater than negative control) OR a detectable serum
 food-specific IgE level to wheat within the previous 12 months or at the time of entry
 into the study

 -  Patients who meet any of the following criteria are eligible for enrollment as study
 participants if they meet all other criteria, but will not undergo a DBPCFC:

 -  Recent (within one year) failed open OFC

 -  Positive DBPCFC to wheat at CHCO

 -  Recent (within one year) exposure to wheat resulting in immediate IgE-mediated
 allergic symptoms (within 2 hours of ingestion; symptoms such as urticaria,
 angioedema, congestion, rhinorrhea, wheezing, respiratory compromise, hypoxia,
 hypotension)

 -  Written informed consent from parent/guardian and assent (when age appropriate).

 -  Willingness to submit specimen for laboratory serum IgE testing

 -  Willingness to submit lab specimen for ELISA testing

 Exclusion Criteria:

 -  Inability to discontinue antihistamines for skin prick testing and OFCs

 -  FEV1 value <80% predicted OR any clinical features of moderate or severe persistent
 asthma, as defined by the 2007 NHLBI guidelines table for classifying asthma, at the
 time of entry into the study

 -  Use of >500 μg/day fluticasone or equivalent

 -  Asthma requiring either:

 -  1 hospitalization in the past year for asthma, or

 -  1 ER visit in the past 6 months for asthma

 -  Use of steroid medications (IV, IM or oral) for asthma in the following manners:

 -  history of daily oral steroid dosing for >1 month during the past year, or

 -  steroid course/burst in the past 3 months (except for steroid dosing used in the
 treatment of an allergic reaction during the DBPCFC), or

 -  2 steroid courses/bursts in the past year.

 -  History of intubation due to allergies or asthma

 -  Diagnosis of active eosinophilic gastrointestinal disease in the past year

 -  Severe atopic dermatitis, as assessed by a Three-Item Severity Score of 6 or greater