Research Project: Reducing Peanut and Tree Nut Allergy

Location: Food Processing and Sensory Quality Research

2019 Annual Report

Objectives
Obective 1: Enable new commercial methods to reduce or eliminate the allergenic properties of peanut and tree nut products. Objective 2: Integrate overall oral and food allergenic properties of native and recombinant allergens with cross-reactivity among nuts, between nuts and pollens, and with pre and postharvest processing. Objective 3: Integrate allergenic properties and molecular changes with commercial tree nut development. Objective 4: Enable new commercial immunoassays for detection of allergens in processed foods.

Approach
The immunoglobulin E (IgE) binding sites that are responsible for the symptoms of allergic disease and cross-reactivity among peanut, tree nut and pollen allergens will be identified with peptide microarray technology. The IgE epitopes will be modeled on the surface of allergen structures to identify location and common or cross-reactive sequences and structural regions of allergens among nuts and pollens. Simultaneously, peanuts, tree nuts or purified allergens thereof (recombinant or native) will be subjected to existing and novel processing techniques (i.e. heat, chemical and enzymatic treatment). New allergens or changes in allergenic properties of existing allergens due to the processing methods will be identified by immunoassays with serum (containing IgE antibodies) from peanut and or tree nut allergic individuals. Proteins found to be immunologically altered by processing will be assessed within the food matrix or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various antibodies, including, serum IgE, known anti-processing reaction products, and allergen specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and to contribute to altered allergenic properties will be identified by mass spectrometry. Understanding the molecular basis of processing-induced alterations of allergens with respect to the IgE binding sights will guide the development of processing technologies towards reduced allergenicity of nuts and products thereof. This knowledge will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers. As a possible early interventional method to reduce the allergenic potential of nuts, the expression and accumulation patterns of allergens in a model tree-nut (pecan) will be studied under various conditions, which may allow interference with their accumulation in the future.

Progress Report
Progress was made on all 4 objectives, all of which fall under National Program 306, “Plant Product Development, Quality, and Marketability.” The project will address NP 306 Action Plan Component 1 Food: Problem Statement 1.A Define, Measure, Preserve/Enhance/Reduce Attributes that Impact Quality and Marketability, 1.B New Bioactive Ingredients and Functional Foods and 1.C New and Improved Food Processing Technologies. Under Objective 1, progress in methods of fermentation of cashew nut extracts with lactic acid (LAC) bacteria (Lactobacillus plantarum and Streptococcus thermophilus PrtS+) was made. LAC fermentation of cashew nut extract was shown to make 2 of the main cashew allergens more susceptible to degradation, which is promising for reduction in the allergic properties of cashew and other nut extracts. Also, Aspergillus niger, a type of food-grade fungus, was grown in peanut or cashew nut flour containing media and ribonucleic acid (RNA, genetic material) sequencing and transcriptome analysis was used to identify differentially induced genes, particularly food-grade enzymes that can be used to degrade allergenic proteins. Many previously uncharacterized genes encoding uncharacterized enzymes were identified in addition to previously characterized peptidases such as oryzin and the aspartic endopeptidase aspergillopepsin. Under Objective 2, immunoglobulin E (IgE)-binding sites have been identified for multiple nut allergens and computationally predicted immunogenic peptides are being compared with empirical IgE binding with serum antibodies from nut allergic patient blood. Cross-reactive IgE binding sites between vicilin and 2S albumin proteins, which are important for food allergy have been determined by different immunoassays. To meet the goals of Objective 2 & 4, major allergenic proteins of walnut (Jug r 1) and cashew ( Ana o 1 and Ana o 3) were purified from cashew nut and used to generate antibodies (immunological molecules derived from blood) in rabbits and mice. Recombinant walnut allergen (rJug r 2) has been purified from walnut and will be used to generate antibodies (immunological molecules derived from blood). Almond allergen (Pru du 1) has been expressed in recombinant form and purified for structural characterization and antibody production. Native and recombinant Ara h 1 leader sequence, a peanut allergen, has been cloned and purified. Allergen purification, antibody production and immunologically-based assay development and improvement are constantly ongoing toward the detection of multiple nut allergens in foods. Under Objective 3, understanding genetic differences will allow us to identify pecan nuts from a particular plant variety that has reduced IgE antibody binding with reduced allergic reactivity. Also, the description and naming of genes discovered in a library of expressed pecan nut genes from pecan varieties such as the Sumner cultivar continues to be updated and compared to other cultivars such as Desirable, MX87, and Pawnee cultivars to identify significant differences in pecan allergen sequences. A preliminary list of pecan nut protein accumulation has been generated and preliminary differences in the timing of allergen accumulation among the Sumner and Desirable pecan cultivars have been identified and these findings will be extended to other cultivars.

Accomplishments
1. Initiated characterization of food (egg) proteins for development as a pharmaceutical for the first time. Collaborative work between ARS researchers in New Orleans, Louisiana and industry has contributed to the initial characterization steps in the development of the first food allergy treatment/therapeutic drug for the treatment of egg allergy (AR201). This research is in the pre-investigational new drug (IND) stage of the FDA application for the use of egg as a pharmaceutical in oral immunotherapy (OIT), which has been shown to desensitize egg allergic individuals, in much the same way as inhaled allergy shots.

2. Development of a rapid method for purification of peanut and tree nut allergens. Methods and access to rapid, large-scale production and detection of allergens is in demand by the food industry, regulatory agencies, and other research partners (i.e. universities). ARS scientists in New Orleans, Louisiana, have developed methods for rapid, large-scale allergen purification from walnut (Jug r 1) and cashew (Ana o 1 and Ana o 3), which not only allows ARS scientists to develop antibodies and immune assays, and to study the structure and function of individual allergic proteins. Large amounts of allergens are used by university and industry partners to develop various types of assays that are relevant to understanding and mitigating food allergy, such as characterization of the types and mechanisms of action of immune cells that are involved in the immunological responses in food allergy.

3. Identification of the Immunoglobulin E (IgE) binding sites of major nut allergens. Biomarkers for following the progression and resolution of food allergy are needed. As part of a CRADA agreement with industry partners, ARS scientists in New Orleans, Louisiana, have identified biomarkers of allergic disease to study the interaction of the immune system molecules, such as IgE, with allergenic proteins. Identification of the sites of contact or IgE binding sites on allergic proteins has been shared with our industry partners to help monitor patient response to therapeutic treatment and to improve diagnostics and therapeutics. As a dual approach, Immunoglobulin E (IgE) binding sites for 100s of peanut and tree nut allergic individuals have been identified for multiple nut allergens, which have enhanced computer-based prediction tools for the accuracy in predicting IgE binding peptides of allergens.

4. Development of antibodies and immunoassays against the major food allergens. The food industry, allergists, scientists and regulators need methods to detect allergenic proteins (allergens) in foods, on product lines, on surfaces, in pharmaceuticals, etc. In response to stakeholder needs, ARS researchers in New Orleans, Louisiana, have developed antibodies against specific food and purified allergenic proteins, as part of our project plan and agreements with our industry partners, which allowed the development of various assays to detect and characterize the individual allergens. The antibodies and immunological methods are used to detect allergens in foods, on shared food product manufacturing lines and the pharmaceutical industries who require normalization and detailed characterization of allergen content and levels for diagnostics and therapeutic treatments material.

Review Publications
Plundrich, N.J., Cook, B.T., Maleki, S.J., Fourches, D., Lila, M.A. 2019. Binding of peanut allergen Ara h 2 with Vaccinium fruit polyphenols. Journal of Food Chemistry. 284:287-295.
Mohamadi, M., Falak, R., Emameh, R.Z., Maleki, S.J., Ali Kardar, G. 2018. Computational analysis of specific IgE epitopes responsible for allergy to fish. Current Immunology Reviews. 14(2):130-136. https://doi.org/10.2174/1573395514666180622121750.
Meng, S., Li, J., Chang, S., Maleki, S.J. 2019. Quantitative and kinetic analyses of peanut allergens as affected by food processing. Journal of Food Chemistry. 1:100004. https://doi.org/10.1016/j.fochx.2019.100004.
Mank, N., Pote, S., Majorek, K., Arnette, A., Klapper, V., Hurlburt, B.K., Chruszcz, M. 2018. Structure of aspartate semialdehyde dehydrogenase from Francisella tularensis. Acta Crystallographica Section F: Structural Biology and Crystallization Communications. F74:14-22. https://doi.org/10.1107/S2053230X17017241.
Mattison, C.P., Khurana, T., Tarver, M.R., Florane, C.B., Grimm, C.C., Pakala, S., Cottone, C.B., Riegel, C., Bren-Mattison, Y., Slater, J.E. 2017. Cross-reaction between Formosan termite (Coptotermes formosanus) proteins and cockroach allergens. PLoS One. 12(8):e0182260. https://doi.org/10.1371/journal.pone.0182260.
Xu, S., Liu, G., Xiao, A., Maleki, S.J., Alcocer, M., Gao, Y., Cao, M., Liu, G. 2017. Eucheuma cottonii sulfated oligosaccharides decrease food allergic responses in animal models by up-regulating regulatory T (Treg) cells. Journal of Agricultural and Food Chemistry. 65:3212-3222. https://doi.org/10.1021/acs.jafc.7b00389.
Orgel, K.A., Duan, S., Wright, B.L., Maleki, S.J., Wolf, J.C., Vickery, B.P., Burks, W., Paulson, J.C., Kulis, M.D., MaCauley, M.S. 2018. Exploiting CD22 on antigen-specific B-cells to prevent allergy to the major peanut allergen Ara h 2. Journal of Allergy Clinical Immunology. 139:366-369.e2. https://doi.org/10.1016/j.jaci.2016.06.053.
Thyssen, G.N., Fang, D.D., Turley, R.B., Florane, C.B., Li, P., Mattison, C.P., Naoumkina, M.A. 2017. A Gly65Val substitution in an actin, GhACT_LI1, disrupts cell polarity and F-actin organization resulting in dwarf, lintless cotton plants. Plant Journal. 90(1):111-121. https://doi.org/10.1111/tpj.13477.
Scott, D., Nesbit, J.B., Cabanillas, B., Cheng, H., Hurlburt, B.K., Maleki, S.J. 2018. Contribution of chemical modifications and conformational epitopes to IgE binding by Ara h 3. Foods. 7(11):189. https://doi.org/10.3390/foods7110189.