Project Number: 6054-43440-052-015-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2021
End Date: Aug 31, 2025
IgE-mediated food allergy is a major health problem with allergy to peanuts (PN) and tree nuts (TN) affecting approximately 3% of children and up to 2% of adults in the US, the UK and Europe 1-10. Co-allergy between PN and TN and among TN is a significant clinical issue 9. Recent progress with early administration of peanuts should reduce new cases, although it cannot help already sensitized individuals 11-13. Oral immunotherapy (OIT) is currently approved and sublingual as well as epicutaneous immunotherapy hold promise 14-18. Omalizumab (anti-IgE) is a useful adjunct 19, 20. Unfortunately, these approaches have limitations regarding cost, compliance, lack of efficacy and unpredictable breakthrough 18, 21-23. There are significant, unmet needs to: 1) understand the immunologic details of IgE-mediated activation of mast cells by allergens from PN and TN, 2) understand the molecular basis for co-allergy among TN and between PN and TN, 3) develop improved diagnostics to predict relevant clinical outcomes and 4) design new approaches to interfere with allergic reactions.
The overarching concept of this proposal is that the 2S albumins are the most important allergens of peanuts and tree nuts and are the key to understanding PN and TN allergy as well as co-allergy and to developing potent diagnostic and potentially therapeutic reagents 4. Preliminary data show we have 1) developed a sensitive ELISA assay, 2) identified the critical amino acids within key IgE-binding peptides, 3) demonstrated that conformationally constrained (3D) peptides bind IgE strongly and can be resistant to digestion and 4) shown that patients with PN allergy alone and PN + TN allergy identify different patterns of peptides. Success in this project will establish a new intellectual framework regarding allergen/IgE interactions, describe, at least in part, the molecular basis for these co-allergies, design new diagnostics and move us along the path toward development of an oral, peptide based, treatment for peanut allergy. Specific Aim 1 (SA1). Test the hypothesis that we can optimize linear sequences and linear mimotopes of conformational epitopes of the 2S albumins of PN for binding to IgE. We will focus this SA on linear peptides of Ara h 2. We will a) perform positional aa screening and insertion of modified side chains to optimize binding of IgE (microarrays), b) analyze how these aa changes alter 3D structures (computer modeling) and c) use click chemistry, helical stapling and backbone stabilization to enhance IgE binding and/or resistance to proteases (ELISA). The affinity of peptides, mimotopes and their analogues will be evaluated using Surface Plasmon Resonance (SPR), inhibition ELISA and RBL-SX38 cell assays. Specific aim #2 (SA2). Identify IgE binding linear peptides mimotopes of IgE binding to the 2S albumins of CN and WN. We will test the hypothesis that specific linear epitopes and mimotopes of IgE binding to Ara h 2, Ana o 3 and Jug r 1 will cross-react and these cross-reactions will correlate with confirmed co allergy. We will confirm known linear epitopes and generate novel mimotopes of IgE binding to Jug r 1 (WN) and Ana o 3 (CN) with well-defined samples from patients with confirmed co-allergies to: a) WN/PecN, b) CN/PisN and c) PN/WN and PN/CN compared to patients allergic to PN alone. Critical peptides with the most frequent and/or the most intense binding will be analyzed by alanine scanning and positional aa substitution (as in SA1), tested in inhibition assays for peptides from other allergens and modeled to the 3D structures of the 2S albumins and of other allergens. Specific Aim 3. Test the hypothesis that IgE binding to existing and novel peptides and mimotopes will have potential predictive value for important clinical outcomes for PN allergy. Using microarray technology as well as streptavidin/biotin ELISA, we will use native peptides, mimotopes and novel peptides from SA1 to a) re-assay existing samples from UNC that we have previously studied, b) assay new samples from UNC and from Stanford and c) work to acquire new samples.