Evaluation of Potential Allergenicity of New 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