Location: Food Processing and Sensory Quality Research
2024 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 at 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, we 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), we produced them in bacteria. We then utilized nuclear magnetic resonance (NMR) 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 cause an immune response in people with allergies. To do this, they normally study how antibodies called IgE and IgG4 interact with allergens. IgE is believed to be the mediator of allergic reactions, while IgG4 might reduce them. We 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 us 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, we 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, we 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 gained 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 the we 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.
We 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.
In support of agreement 6054-43440-052-012H: Determining the Levels and Potency of Allergenic Proteins in Differently Treated Peanut Flour, Extract, and Other Products the Cooperator Provides (MOONLIGHT THERAPEUTICS). The differently treated peanut flours and extracts sent by the company were fully characterized for the best extraction buffers and conditions, the most stable storage conditions and stability and potency of the individual allergens over time. Allergen stability and storage conditions were determined and transferred to the company.
In support of agreement 6054-43440-052-015R: Exploiting and Enhancing IgE-Binding Epitopes of the 2S Albumins of Peanuts and Tree Nuts (REGENTS OF THE UNIVERSITY OF COLORADO): peptides with systemic mutations were synthesized and added to peptide microarrays which will be probed with serum of allergic individuals to determine the preferences of immunoglobulin E (IgE) binding to mutant and non-mutated peptides.
In support of agreement 6054-43440-052-017H: Production of Antibodies to Detect Peanut and Tree Nut Allergens Aimmune Nestlé Health Science US R&D LLC. Monoclonal antibodies were screened to identify highly specific antibodies against individual allergens in a single nut that would not bind to any other allergens within the same nut or within other nuts.
In support of agreement 58-6054-3-0013: Antibodies and Allergens used to Detect Peanut Allergens (BLUEWILLOW BIOLOGICS INC.) Materials and protocols were exchanged.
In support of agreement 6054-43440-052-016R: Trees for the Future: Coordinated Use of Genetic Tools to Develop Geographic and Climate Adapted Pecan (NEW MEXICO STATE UNIVERSITY) Proteomic maps of several pecan cultivars allowed identification of distinct protein differences between them, and numerous heat resistant peptide markers for the Car i 1, 2, and 4 allergens were identified that can be applied to reliably detect pecan allergens in foods.
Accomplishments
1. Determining the changes in antibody binding patterns in individuals subjected to Oral Immunotherapy with roasted peanut flour.. 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. ARS scientists at New Orleans, Louisiana, assessed the serum immunoglobulin E (IgE) and IgG4 antibodies that had under gone Oral immunotherapy (OIT), which is oral administration of increasing doses of lightly roasted peanut flour over time to desensitize peanut allergic subjects. Serum was collected before and after a year of OIT in which the active group (receiving peanut treatment) were desensitized and able to consume several peanuts without severe reactions. The placebo group was not desensitized. Assessing the antibody binding to peanut allergens and peptides thereof allowed us to identify key peptide biomarkers that were significantly different between the active and placebo and pre and post OIT. These biomarkers may play an important role in future diagnosis and predicting treatment outcomes in peanut allergy
2. Protein pieces, previously thought of as unimportant, contribute to the cross reactivity to 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 and improve diagnostics. A fragment is cleaved from vicilins are seed storage proteins after it serves the purpose of directing the protein to the correct compartment in the seed. The scientific community has assumed the severed fragments, referred to as leader sequences (LS), degrade rapidly. However, ARS scientists at New Orleans, Louisiana identified the LS of vicilins from peanut (Ara h 1 LS) and walnut (Jug r 2LS) in the seeds and found that they contain Immunoglobulin E (IgE) binding sites. ARS scientists discovered that fragments of the protein vicilin in peanut, walnut, cashew, and pistachio, referred to as leader sequences, contain binding sites for IgE, which is an antibody involved in allergic reactions. Furthermore, they found that both common amino acid sequences and the protein structures containing these IgE-binding sites contribute to an individual's allergies to multiple nuts, known as cross-reactivity. We have determined that these factors a) can be major contributors in cross-reactivity to multiple nuts in food allergy and b) are important biomarkers that can distinguish between peanut, walnut or peanut and walnut allergic individuals. This discovery will enable scientists to develop more accurate detection, diagnosis, and treatment tools for nut allergies.
3. Development of a new method 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. ARS scientists at New Orleans, Louisiana developed methods to isolate single allergens from cashew, pecan, peanut, and walnut for antibody production. These antibodies were subjected to numerous tests for their specificity, sensitivity, and utility. Multiple types of immune assays to detect and measure the levels and properties of allergens in various food preparations (such as roasted or extruded nuts) and final food and drug 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 used by the food and pharmaceutical industry for detection and measurement of allergens in products
4. Artificial Intelligence (AI)/Machine Learning (ML). Our group collaborates with SciNet scientists and is currently using bioinformatics and machine learning to identify biomarkers of allergic status towards developing improved diagnostic tools for food allergy. Our collaborative project identifies immunoglobulin E (IgE) and IgG4 binding to allergenic proteins and peptides thereof using microarray technology. The binding targets of these antibodies from the serum of known nut allergic individuals are assessed for the ability to predict the allergic status of individuals via a simple blood test.
Review Publications
Rambo, I., Kronfel, C.M., Rivers, A.R., Swientoniewski, L., Mcbride, J.K., Cheng, H., Simon, R.J., Ryan, R., Tilles, S., Nesbit, J.B., Kulis, M.D., Hurlburt, B.K., Maleki, S.J. 2023. IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy. Frontiers in Allergy. 4: Article 1279290. https://doi.org/10.3389/falgy.2023.1279290.
Bruni, G.O., Qi, Y., Terrell, E., Dupre, R.A., Mattison, C.P. 2024. Characterization of levan fructan produced by a Gluconobacter japonicus strain isolated from a sugarcane processing facility. Microorganisms. 12(1). Article 107. https://doi.org/10.3390/microorganisms12010107.