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

Location: Food Processing and Sensory Quality Research

2021 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
Progress was made by ARS scientists in New Orleans, Louisiana on all 3 National Program 306 objectives that fall 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. Objective 1, small fragments of proteins known as leader sequences that are thought to be degraded during plant development are actually conserved and can elicit allergic responses. The leader sequence from peanut, cashew, and hazelnut were produced in transgenic bacteria or purified directly from the allergen source for these experiments. Purification methods were developed for individual allergens from both bacteria and nuts to obtain the allergens for future studies. Objective 2, it is important to identify the parts of an allergen that illicit an immune response in allergic individuals. To accomplish this the target binding sites of specific antibodies, called immunoglobulin E (IgE) and immunoglobulin G4 (IgG4) known to be involved in food allergic reactions were identified by ARS scientists in New Orleans, Louisiana. This project involves identification of the allergen fragments that are target binding sites for IgE and IgG4. In IgE-mediated food allergy, IgE and IgG4 are known to compete for binding of the same allergenic proteins. Here, IgE facilitate the symptoms of allergic reactions, while IgG4 is thought to block IgE from binding to allergens. Consequently, promoting the binding of IgG4 through therapeutic treatments could reduce allergic responses. To accomplish this, it is necessary to identify what fragments of allergens are targeted by IgE and IgG4. Therefore, segments of allergens (peptides) were printed on the surface of glass slides and mixed with allergic patient blood serum. This allowed ARS scientists to identify the specific parts of allergenic proteins that are recognized by antibodies in blood of people with allergies to peanut and tree nut proteins. Antibody binding sites for 40 allergic proteins were identified by ARS scientists from 30 volunteers from the southern United States with peanut and tree nut allergies. The data from this project was submitted to a ‘big data’ collection and will be used to improve computer-based modeling and prediction of the allergic potential of the proteins studied and predict IgE binding sites of unknown peptides in other allergens. Objective 3, the genetic diversity of allergenic proteins from pecan trees was assessed by ARS scientists in New Orleans, Louisiana. This assessment was performed by determining the DNA sequences of allergen genes that are expressed in different types of pecan trees. Other information such as the accumulation of allergens and other proteins in pecan nuts was also evaluated. Multiple stages of pecan nut development were evaluated by computerized comparison of the proteins present in the soluble pecan nut extracts collected at different development stages. Using this type of proteomic analyses of developing pecan nut samples, the accumulation of individual proteins during nut development was observed. This analysis determined that rapid overall protein accumulation did not occur until mid-September, during a transition stage of development when the developing nut material begins to solidify. Using this protein comparison method, 1,267 protein spots were observed, and approximately 565 of them were identified. Pecan allergens Car i 1 and Car i 2 were first observed to accumulate during the solidifying stage in late September, and reliable markers for Car i 1 and Car i 2 that could be used as indicators for the presence of pecan nuts in foods were identified. Also, as part of Objective 3, the effects of fungal and bacterial fermentation on the allergenic properties of peanut and cashew allergens were assessed. Analysis of a commercially available cashew nut-based yogurt, a fermented product, with IgE antibodies from cashew allergic volunteers indicated that while much of the allergen content was degraded by bacterial enzymes, the yogurt still contained substantial allergen-IgE binding content and is not suitable for consumption by those with cashew nut allergy.

Accomplishments
1. Identification of allergic individual immunoglobulin E binding sites of lipid transfer proteins from peanut, walnut, and peach. ARS scientists in New Orleans, Louisiana, suggest Non-specific lipid transfer proteins (LTPs) are well studied allergens that often cause oral allergy syndrome (itching, blisters and burning of mouth and lips) in allergic individuals in the Mediterranean area and other European countries, but are less understood in the United States. The goal of this study was to compare the difference in the target binding sites (epitopes) of IgE antibodies obtained from the blood of allergic individuals form the United States and Spain. Therefore, the IgE binding locations of LTPS from peanut, walnut and peach were compared for peanut allergic individuals from the United States and Spain. ARS scientists found that certain regions of the LTPs are recognized more often in United States subjects indicating that they represent areas of the allergens that are most similar and may be responsible for different allergic reactions between the two countries. Also, these results show that both unfolded and folded parts of the allergen are recognized by IgE antibodies and are important for understanding why individuals with one nut allergy react to other nuts (known as cross-reactivity). This study allows ARS scientists to understand regional/global differences in allergic reactions to the same food, which in turn leads to more targeted therapeutics and diagnostics.

2. Fragments of major allergens in walnut and peanut contribute to allergic reactions to multiple nut seeds in some individuals (referred to as cross-reactivity). ARS scientists in New Orleans, Louisiana, suggests 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 severed fragments, referred to as a leader sequence (LS) were thought to be degraded rapidly. However, the LS of vicilins from peanut (Ara h 1 LS) and walnut (Jug r 2 LS) were identified in seed and shown to contain highly allergic IgE binding sites or epitopes. Both the common amino acid sequences and the protein structures containing these IgE epitopes were shown to contribute to allergic reaction of certain individuals to multiple nuts, also known as cross-reactivity. Understanding the interaction of the allergic human’s immune system with the allergic molecule, allows us to develop better, detection, diagnostic and therapeutic tools for allergic disease.

3. Completed pecan proteome reveals unique peptide markers of pecan allergens. ARS scientists in New Orleans, Louisiana, believe Pecan (Carya illinoinensis) nuts are an economically valuable crop native to the United States. Pecan kernels progress through multiple stages of development, and computerized comparisons of soluble proteins elucidated the accumulation of individual proteins during kernel development. Rapid overall protein accumulation occurred in mid-September during a time period when the nut material begins to solidify. Thorough analysis of samples provided a clear timeline of protein accumulation. In particular, pecan allergens (Car i 1 and Car i 2) were first observed to accumulate during the solidification of the nut material in late September. The information collected from these projects will enable the development of 1) reliable peptide markers that can be used for pecan nut detection in foods, and 2) may enable potential breeding strategies for the creation of new pecan cultivars with nuts containing reduced allergen content.

Review Publications
Mcbride, J.K., Cheng, H., Maleki, S.J., Hurlburt, B.K. 2019. Purification and characterization of pathogenesis related class 10 panallergens. Foods. 8:609-623. https://doi.org/10.3390/foods8120609.
Shah, F., Shi, A., Ashley, J., Kronfel, C., Wang, Q., Maleki, S.J., Adhikari, B., Zhang, J. 2019. Peanut Allergy: Characteristics and Approaches for Mitigation. Comprehensive Reviews in Food Science and Food Safety. 0:2019. https://doi.org/10.1111/1541-4337.12472.
Meng, S., Tana, Y., Chang, S., Li, J., Maleki, S.J., Naveen, P. 2019. Peanut allergen reduction and functional property improvement by means of enzymatic hydrolysis and transglutaminase crosslinking. Food Chemistry. 302:125186. https://doi.org/10.1016/j.foodchem.2019.125186.
Novak, N., Maleki, S.J., Cuadrado, C., Crespo, J.F., Cabanillas, B. 2020. Interaction of monocyte-dervied dendritic cells with Ara h 2 from raw and roasted peanut. Foods. 9:863. http://dx.doi.org/10.3390/foods9070863.
Nesbit, J.B., Schein, C.H., Braun, B.A., Gipson, S.A., Cheng, H., Hurlburt, B.K., Maleki, S.J. 2020. Epitopes with similar physicochemical properties contribute to cross reactivity between peanut and tree nuts. Molecular Immunology. https://doi.org/10.1016/j.molimm.2020.03.017.
Xie, M.M., Liu, H., Yang, K., Koh, B., Wu, H., Maleki, S.J., Hurlburt, B.K., Kaplan, M.H., Dent, A.L. 2020. Peanut-specific IgE produced in a mouse food allergy model requires help from T follicular regulatory cells and IL-10 signaling by B cells. Journal of Clinical Investigation. 130(7):3820-3832. https://doi.org/10.1172/JCI132249.
Santos, A., Nuno, B., Hurlburt, B.K., Sneha, R., Kwok, M., Bahnson, H., Cheng, H., James, L., Maleki, S.J., Lack, G., Gould, H., Sutton, B. 2020. IgE to epitopes of Ara h 2 enhance the diagnostic accuracy of Ara h 2-specific IgE. Allergy. https://doi.org/10.1111/all.14301
Ghasemi, A., Falak, R., Mohammadi, M., Maleki, S.J., Assarezadegan, M.A., Jafary, M. 2020. Incorporation of T-cell epitopes from tetanus and diphtheria toxoids into in-silico-designed hypoallergenic vaccine may enhance the protective immune response against allergens. Iranian Journal of Basic Medical Sciences. 23:636-644.
Mattison, C.P., Aryana, K.J., Clermont, K., Prestenburg, E., Lloyd, S.W., Grimm, C.C., Wasseman, R.L. 2020. Microbiological, Physiochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms21218267.
Mattison, C.P., Tungtrongchitr, A., Tille, K.S., Cottone, C.B., Riegel, C. 2020. Cloning, expression, and immunological characterization of formosan subterranean termite (blattodea: rhinotermitidae) arginine kinase. Journal of Insect Science. Volume 20; Issue 4. https://doi.org/10.1093/jisesa/ieaa071.
Ruiter, B., Smith, N.P., Flemming, E., Patil, S.U., Hurlburt, B.K., Maleki, S.J., Shreffler, W.G. 2021. Peanut protein acts as a Th2 adjuvant by inducing expression of RALDH2 in human antigen-presenting cells in a TLR2-dependent manner. Journal of Allergy Clinical Immunology. 148(1):182-194. https://doi.org/10.1016/j.jaci.2020.11.047.
Zamani Sani, M., Bargahi, A., Momenzadeh, N., Dehghani, P., Vakili Moghadam, M., Maleki, S.J., Nabipour, I., Shirkani, A., Akhtari, J., Hesamizadeh, K., Heidari, S., Omrani, F., Akbarzadeh, S., Mohamadi, M. 2020. Genetically engineered fusion of allergen and viral-like particle induces a more effective allergen-specific immune response than a combination of them. Applied Microbiology and Biotechnology. 105:77-91. https://doi.org/10.1007/s00253-020-11012-0.
Maleki, S.J., Teuber, S.S., Mustafa, S.S. 2021. Adult peanut allergy: What we know and what we need to learn. Journal of Allergy Clinical Immunology. https://doi.org/10.1016/j.jaci.2021.03.031.