Research Project: Reducing the Development and Severity of Allergy to Peanuts and Tree Nuts

Location: Food Processing and Sensory Quality Research

2023 Annual Report

Objectives
Objective 1: Decipher the molecular, structural and immunological properties of purified native and recombinant allergens that contribute to allergenic potency towards development of therapeutic products. [NP306, C1, PS1C] Objective 2: Use serum from verified nut allergic and non-allergic individuals to identify and compare IgE and IgG binding sites (or epitopes) of known nut allergens with peptide microarrays to understand cross-reactivity between multiple allergens and improve diagnosis of nut allergy. [NP306, C1, PS1A] Objective 3: Characterize, quantify and monitor allergen characteristics and levels pre- and post-harvest, and pre- and post-processing of commercial nuts and nut-containing foods, and during nut seed development to produce hypoallergenic, prophylactic or therapeutic food products. [NP306, C1, PS1B] The immunoglobulin E (IgE) binding sites (epitopes) 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 and immunoglobulin G4 (IgG4, thought to act as an IgE-blocking antibody) epitopes will be identified for the most potent nut allergens. These will be modeled on the surface of allergen structures to identify location and common or cross-reactive (or potentially blocked) sequences and structures of allergens among nuts and pollens. Simultaneously, the changes in peanut and tree nut extracts or purified allergens thereof (recombinant or native) will be assessed before and after processing treatments for changes in allergenic properties. Proteins found to be immunologically altered by processing will be assessed within a total nut extract or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various serum IgE and allergen-specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and thought to contribute to altered allergenic properties, will be identified by mass spectrometry. The studies above will be combined to identify the influence of the processing-induced alteration in relationship to the immunoglobulin binding sites of nut allergens. This will guide the development of better diagnostics and therapeutics as well as processing technologies to reduce allergenicity of nuts and products thereof. Early intervention methods to reduce the allergenic potential of nuts, the natural variation in allergen gene sequence, expression, post-translational modification, stability and accumulation patterns in a model tree nut (pecan) will be studied. Less allergenic variants and factors that can be used to interfere with allergen accumulation in plants will also be characterized in detail. Collectively our studies will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers.

Approach
The immunoglobulin E (IgE) binding sites (epitopes) 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 and immunoglobulin G4 (IgG4, thought to act as an IgE-blocking antibody) epitopes will be identified for the most potent nut allergens. These will be modeled on the surface of allergen structures to identify location and common or cross-reactive (or potentially blocked) sequences and structures of allergens among nuts and pollens. Simultaneously, the changes in peanut and tree nut extracts or purified allergens thereof (recombinant or native) will be assessed before and after processing treatments for changes in allergenic properties. Proteins found to be immunologically altered by processing will be assessed within a total nut extract or they will be purified and analyzed for alterations in size, structure, digestibility, binding to various serum IgE and allergen-specific antibodies. The specific amino acid residues, or peptides thought to be modified during different processing events, and thought to contribute to altered allergenic properties, will be identified by mass spectrometry. The studies above will be combined to identify the influence of the processing-induced alteration in relationship to the immunoglobulin binding sites of nut allergens. This will guide the development of better diagnostics and therapeutics as well as processing technologies to reduce allergenicity of nuts and products thereof. Early intervention methods to reduce the allergenic potential of nuts, the natural variation in allergen gene sequence, expression, post-translational modification, stability and accumulation patterns in a model tree nut (pecan) will be studied. Less allergenic variants and factors that can be used to interfere with allergen accumulation in plants will also be characterized in detail. Collectively our studies will also contribute to the development of better detection tools and labeling practices for industry and regulatory agencies resulting in better protection of consumers.

Progress Report
ARS researchers in New Orleans, Louisiana, made progress on all 3 National Program 306 objectives under, “Plant Product Development, Quality, and Marketability.” The project addresses National Program 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. To address Objective 1, ARS researchers demonstrated that small fragments of proteins called leader sequences, which were believed to be broken down during plant development, are in fact stable and can induce an allergic reaction. To create the leader sequence for vicilin proteins from peanut, walnut, pistachio, cashew, and tomato (a non-allergic control), ARS researchers produced them in bacteria. Nuclear magnetic resonance (NMR) was then utilized to analyze the structure of peanut and walnut vicilin leader sequences and assess their potential for causing an allergic response. The methods established with bacterial production of recombinant allergens will be employed in future studies. The goal of Objective 2 is to identify the sections of allergens that illicit an immune response in people with allergies. To do this, they studied how antibodies called IgE and IgG4 interact with allergens. IgE is believed to be the mediator of allergic reactions, while IgG4 might reduce them. ARS researchers tested this idea by attaching pieces of allergens on glass slides and mixing them with blood serum from people who are allergic to peanuts and tree nuts. This allowed ARS scientist at the SRRC in New Orleans, Louisiana to identify the specific parts of allergenic proteins recognized by antibodies in the blood of people with allergies to peanut and tree nut proteins. This data was then submitted to a ‘big data’ collection so that other scientists can test it’s usefulness for prediction of protein allergenicity and diagnostic purposes. Additionally, ARS scientists observed changes in specific IgE and IgG4 binding sites before and after oral immunotherapy treatments, which could help assess a patient’s response to oral immunotherapy. In support of Objective 3, ARS researchers evaluated the genetic diversity and protein accumulation among pecan cultivars and related hickory species. Overall protein and allergen accumulation during pecan nut development indicated very similar patterns among pecan cultivars. Although the sample size was relatively low, this information suggests a limited amount of genetic diversity among some allergen and other genes. Gene sequences from these analyses provide targets for breeding of improved cultivars. Scientists from ARS New Orleans, Louisiana studied how bacterial and fungal fermentation affects peanut and tree nut flours. Although fermentation was shown to reduce allergen content, it was not to an extent that would render these products safe for consumption by those with peanut or tree nut allergies. ARS scientists developed highly specialized immunoassays and methods for a thorough examination of how roasting affects peanut allergens. This knowledge and these methods allowed ARS scientists to classify peanut flour as a drug product that is manufactured and sold by pharmacies.

Accomplishments
1. ARS scientists collaborated with an industry partner to the development and marketing of roasted peanut flour as a manufactured, prescribed and marketed pharmaceutical named Palforzia®. Food allergy costs the US $25 billion a year, is the primary cause of FDA food recalls, and creates significant challenges for farmers, allergic consumers, the food industry, and regulators. Previous to Palforzia®, no approved oral dosed food treatments existed for food allergy. ARS scientists at New Orleans, Louisiana, assisted a company in developing methods for detecting, quantifying, and characterizing individual allergens in peanut flour. Exhaustive tests conducted by ARS scientists produced antibodies and immunoassays to measure the levels and structure of multiple allergens in roasted peanut flours. Today, Palforzia®, athis peanut- flour- derived pharmaceutical, is the only food that has been FDA approved for prescription by physicians. It is marketed under the name Palforzia®. Oral administration of increasing doses of the product Palforzia® over time can desensitize an individual so they might be able to consume several peanuts without severe reactions. This results in lower number of allergic reactions, emergency room visits, food recalls, and accidental ingestions in day cares, schools, restaurants, etc. This product established standards and paves the way for treatment of other food allergies and other methods of treatment using food in a similar way.

2. Spanish residents react differently to peanut allergens than individual in the U.S. Understanding what factors contribute to development of food allergy will help guide development of new treatments. Geographic, environmental, and cultural factors influence food allergy development, but it is not clear how they do this. Scientists from the Agricultural Research Service (ARS) in New Orleans, Louisiana, found that individuals from both Spain and the United States, who have peanut allergies, showed similar responses to Ara h 9 peptides. However, there was a notable difference. Only the Spanish individuals who experienced allergic reactions to Ara h 9 bound to the folded and intact allergen. On the other hand, allergic individuals in the U.S. only bound to unstructured Ara h 9 peptides. This finding sheds light on the variations in allergic responses to this major allergen between these two populations. This discovery not only improves our understanding of how environmental, geographic, and cultural considerations may contribute to development of food allergy, it will also help create more focused tools for diagnosing and managing food allergies.

3. Protein pieces, previously thought of as unimportant, ARS scientists uncover how leader sequences contribute to the cross reactivity of nut allergens. Improved recognition and understanding of what specific nut proteins, or parts of proteins, cause food allergy will allow increased safety and provide new therapeutic targets. A few peanut and tree nut allergens have been characterized, but a number of allergens remain to be identified. Vicilins are seed storage proteins from which a fragment is cleaved after it serves the purpose of directing the protein to the correct compartment in the seed. The scientific community has assumed the severed fragments degrade rapidly. However, ARS scientists at New Orleans, Louisiana, identified the severed fragments of vicilins from peanut, walnut, cashew, and pistachio in the seeds and found that they contained small sections (called binding sites) that could cause an allergic response. The scientists found that both the chemical make-up and the structures of these binding sites contribute to an individual's allergies to multiple nuts, known as cross-reactivity. This finding implies that the vicilin fragments in nuts are a new class of allergic proteins that contribute to cross-reactions to different nuts that result in multi-nut allergies. This discovery will enable scientists to develop more accurate detection, diagnosis, and treatment tools for nut allergies.

4. Development of a new method monoclonal antibody-based assays to detect and measure allergens with high accuracy and precision. Accurate detection of peanut and tree nut allergens will provide increased public safety, reduce accidental allergen exposure, and prevent costly life-threatening medical emergencies. Serious food allergy reactions are one of the top causes of emergency room visits in the United States. Scientists are continuously working to develop methods to isolate single allergens from cashew, pecan, peanut, and walnut for antibody production. These antibodies undergo numerous tests for their specificity, sensitivity, and utility. Additionally, researchers create various types of immune assays to detect and measure the levels and properties of allergen in various food preparations (such as roasted or extruded nuts) and final products. For example, scientists developed two novel monoclonal antibody-based assays to detect the immunodominant peanut allergen Ara h 1 with high accuracy and precision. Apart from research purposes, these assays are also used by the food and pharmaceutical industry for detection and measurement of allergens in products.

Review Publications
Hnasko, R.M., Lin, A.V., McGarvey, J.A., Mattison, C.P. 2022. Sensitive and selective detection of peanut allergen Ara h 1 by ELISA and lateral flow immunoassay. Food Chemistry. 396. Article 133657. https://doi.org/10.1016/j.foodchem.2022.133657.
Clermont, K., Graham, C., Lloyd, S.W., Grimm, C.C., Randall, J., Mattison, C.P. 2023. Proteomic analysis of pecan (Carya illinoinensis) nut development. Foods. 12:866. https://doi.org/10.3390/foods12040866.
Mattison, C.P., He, Z., Zhang, D., Dupre, R., Lloyd, S.W. 2023. Cross-serological reaction of glandless cottonseed proteins to peanut and tree nut allergic IgE. Molecules. 28(4):1587. https://doi.org/10.3390/molecules28041587.
Minkiewicz, P., Mattison, C.P., Darewicz, M. 2022. Quantitative in silico evaluation of allergenic proteins from Anacardium occidentale, Carya illinoinensis, Juglans regia, and Pistacia vera and their epitopes as precursors of bioactive peptides. Current Issues in Molecular Biology. 44(7): 3100-3117. https://doi.org/10.3390/cimb44070214.
Lebar, M.D., Mack, B.M., Carter Wientjes, C.H., Wei, Q., Mattison, C.P., Cary, J.W. 2022. Small NRPS-like enzymes in Aspergillus sections Flavi and Circumdati selectively form substituted pyrazinone metabolites. Frontiers in Fungal Biology. 3:1029195. https://doi.org/10.3389/ffunb.2022.1029195.
Leonard, S.A., Ogawa, Y., Jedrzejewski, P.T., Maleki, S.J., Chapman, M.D., Tiles, S.A., Du Toit, G., Mustafa, S., Vickery, B.P. 2022. Manufacturing processes of peanut (arachis hypogaea) allergen powder-dnfp. Frontiers in Allergy. 3. https://doi.org/10.3389/falgy.2022.1004056.
Kronfel, C., Cheng, H., Mcbride, J.K., Krouse, R., Burns, P., Cabanillas, B., Crespo, J.F., Simon, R.J., Robert, R., Maleki, S.J., Hurlburt, B.K. 2023. IgE epitopes of Ara h 9, Jug r 3, and Pru p 3 in peanut-allergic individuals from Spain and the US. Frontiers in Allergy. https://doi.org/10.3389/falgy.2022.1090114.
Foo, A., Nesbit, J.B., Gipson, S., Cheng, H., Hurlburt, B.K., Kulis, M.D., Kim, E.H., Dreskin, S., Maleki, S.J., Mueller, G. 2022. Structure, immunogenicity, and IgE cross-reactivity among cashew, pistachio, and peanut vicilin-buried peptides. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.1c07225.
Hardy, L.C., Smeekens, J., Raghuwanshi, D., Sarkar, S., Dashkan, G.C., Rogers, S.I., Maleki, S.J., Burks, W.A., Paulson C, J.C., Macauley, M.S., Kulis, M.D. 2022. Targeting CD22 on Memory B cells to induce tolerance to peanut allergens. Journal of Allergy Clinical Immunology. 150(6):1476-1485. https://doi.org/10.1016/j.jaci.2022.06.022.