1a. Objectives (from AD-416):
Objective 1: Identify and compare the structural, chemical, functional, and immunological characteristics of peanut with homologous; less allergenic legume (green pea and soy) and tree nut allergens in raw and processed forms towards delineating the clinically-relevant antibody-allergen interactions. Objective 2: Clone, express, and purify the major peanut and select tree nut and legume allergens, and fragments thereof, in recombinant form to further delineate clinically-relevant antibody-allergen interactions. Objective 3: Assess the role of processing-induced chemical or structural modifications on the individual allergens by systematically altering amino acids thought to be important in clinically-symptomatic allergic reactions. Objective 4: Develop computer models and/or determine NMR/crystal structures of native and recombinant peanut and select tree nut and legume allergens in raw and processed forms. Objective 5: Combine the structural information obtained with the empirical knowledge from Objectives 1, 2, and 3 to identify clinically-relevant allergen-antibody interactions in peanut and tree nut allergy. Objective 6: Develop processing technologies for peanut products with reduced allergenic properties. Objective 7: Develop and improve immunoassays for detection of peanut, select tree nut, and soy allergen residues before and after processing (i.e. roasting, baking into cookies, etc.). Objective 8: After establishing standardized protocols for determining threshold doses for peanut, select tree nut, and soy allergens, determine threshold (minimal eliciting) dose of reactivity for processed forms of peanut allergens and develop computational and statistical models to estimate population thresholds.
1b. Approach (from AD-416):
Specifically, peanuts and tree nuts will be subjected to thermal processing (i.e., roasting). New allergens or changes in allergenic properties of existing allergens due to the thermal processing will be identified by immunoassays, using serum (containing IgE antibodies) from peanut and/or tree nut allergic individuals. Proteins found to be immunologically altered by thermal processing will be purified by conventional chromatography and analyzed for alterations in size, structure, digestibility, and binding to various antibodies, including anti-Maillard reaction products and specific anti-allergen antibodies. The specific amino acid residues, thought to be modified during different processing events and to contribute to altered allergenic properties (i.e. IgE binding), will be identified. These amino acids will be identified by cloning and expression of select recombinant major allergens of peanut and tree nuts in E. coli followed by site directed mutagenesis, simulated processing and immunological analysis of previously identified IgE binding sites, specifically, sites thought to be modified by processing. Understanding the molecular basis of processing-induced alterations of allergens will guide development of processing technologies towards reduced allergenicity of nuts. This knowledge will also contribute to the development of better labeling practices and detection tools for industry and regulatory agencies resulting in better protection of consumers.
3. Progress Report:
The optimal concentration of tannic acid required to remove peanut allergens from solution at gastric and intestinal pH were determined. As a result, allergens would potentially pass through the gastrointestinal system without being absorbed, which could reduce allergic response. Also, a combined system of high pressure and polyphenol oxidase (an enzyme) were used to reduce peanut allergens. High pressure alone had no effect, and polyphenol oxidase alone was not as effective as the combination of both pressure and enzyme. High pressure was also used in conjunction with autoclaving to treat roasted peanuts, and was shown to denature peanut proteins, which has been shown to decrease allergic potential. Objective 3 and 6, which involve exploring and developing processing technologies in order to reduce the allergenic potential of peanuts, were addressed with these experiments. Additionally, allergenic capacity of shrimp extracts treated with pulsed ultraviolet light, and then with digestive enzymes was measured. Results showed that this treatment and digestive enzyme-treated shrimp extracts were less allergenic. D-aspartic and D-glutamic acids were identified and measured in both raw and roasted peanuts, as proposed in Objective 3, but no significant difference in the levels were found. To meet the goals of Objective 2, the major cashew allergens Ana o 3 (2S albumin), and Ana o 1 (7S vicillin) were purified from cashew; expressed in escherichia (E.) coli, and purified for structural, immunological, and biological studies. To address Objective 3 and 4, the specific chemical modifications to the peanut allergen Ara h 1 were identified in a simulated roasting model to determine immunologic and allergic consequences of roasting, and to explore methods to reduce the allergic potential. A fragment of Ara h 1 was cloned, expressed, and then purified to assist in these immunological evaluations. The crystal structure of Ara h 1 was also obtained (Objective 4). Combined structural and empirical information was used to identify clinically relevant allergen-antibody interactions in peanut and tree nut allergy, as stated in Objective 5. A peptide microarray assay was optimized to screen peanut and tree nut allergic sera for binding to allergenic peptides. A computational prediction method was used to predict immunoglobulin E (IgE), which is associated with allergenic responses binding sites (or peptide epitopes), that would be cross-reactive among peanuts and tree nut allergens. The microarray study will assist in identifying more of these IgE epitopes in peanuts and tree nuts, which is important for designing better detection, diagnostic and therapeutic tools, as well as processing methods, to reduce allergenic potential in nuts. Also, we have identified epitopes that cross-react among the different peanut allergens, which may be the reason that peanuts are so allergenic. Meanwhile, methods were developed and validated to measure peanut dust on various surfaces and locations; such as, the home and day care, which addresses Objective 7 of the project plan.
1. Tannic acid to remove peanut allergens. Peanut extracts were precipitated with tannic acid, and were tested to see if peanut allergens were released from the precipitates at gut pH. No release was observed, indicating that peanut allergens can pass through without being absorbed or causing an allergic response. The research could lead to development of less allergenic peanut-based products and beverages.
2. High pressure with polyphenol oxidase reduces peanut allergens. Peanut extracts were treated with high pressure in the presence of an enzyme polyphenol oxidase. As a result, the allergenic capacity of the treated peanut extracts was reduced. The research could lead to development of less allergenic peanut-based products and beverages.
3. Computational tools to predict cross-reactive binding sites among peanuts and tree nuts. Individuals with peanut or tree nut allergy are often allergic to multiple nuts, but ARS scientists at the Southern Regional Research Center in New Orleans, LA, are unable to predict which nuts based on existing diagnostic tests, with the exception of a food challenge (feeding the food to the patient). A computational prediction method was developed to predict clinically-relevant immunoglobulin E (IgE) binding sites (or peptide epitopes) that would be cross-reactive among peanuts and tree nuts. This method allowed previously unknown epitopes to be identified in walnut and other nuts. This finding is important for designing better detection, diagnostic and therapeutic tools, as well as processing methods, to reduce allergenic potential in nuts.
4. Specific roasting-induced modifications to the peanut allergen, Ara h 1, identified. Roasting has been shown to enhance the allergenic potency of Ara h 1 and other peanut proteins. Recombinant Ara h 1 was cloned, expressed in escherichia coli, and then purified. The allergen was then subjected to a simulated roasting system, and specific non-enzymatic chemical modifications that contribute to structural and immunonolgical alterations were identified. Understanding what modifications contribute to enhance IgE binding and immunological reactions to peanut, will enable ARS scientists at the Southern Regional Research Center in New Orleans, LA, to design better tools to reduce the allergenic potential.
5. Method to detect walnut residue in foods. Cross-contamination of product lines with allergenic nuts causes millions of dollars in recalls, other costs, and many accidental allergic reactions. An immunological method was optimized for detection of walnut residue in foods and on product lines. Once validated, this method will be developed into a tool for the industry and regulators for enhancing food labeling.
Cabanillas, B., Rodriguez, J., Crespo, J.F., Pedrosa, M.M., Cuadrado, C., Burbano, C., Maleki, S.J. 2011. Influence of enzymatic hydrolysis on the allergenicity of roasted peanut protein. International Archives of Allergy and Immunology. 157:41-50.