Allergen valency, dose and FcERI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens

Authors: MAHAJAN, AVANIKA - University Of New Mexico; YOUSSEFF, LAMA - Damascus University; CLEYRAT, CEDRIC - University Of New Mexico; GRATTAN, RACHEL - University Of New Mexico; LUCERO, SHAYNA - University Of New Mexico; Mattison, Chris; ERASMUS, FRANK - University Of New Mexico; JACOBSON, BRUNA - University Of New Mexico; TAPIA, LYDIA - University Of New Mexico; HLAVACEK, WILLIAM - University Of New Mexico; SCHUYLER, MARK - University Of New Mexico; WILSON, BRIDGET - University Of New Mexico

Submitted to: Journal of Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2016
Publication Date: 2/1/2017
Citation: Mahajan, A., Yousseff, L.A., Cleyrat, C., Grattan, R., Lucero, S., Mattison, C.P., Erasmus, F.M., Jacobson, B., Tapia, L., Hlavacek, W.S., Schuyler, M., Wilson, B.S. 2017. Allergen valency, dose and FcERI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens. Journal of Immunology. 198:1034-1046. doi:10.4049/jimmunol.1601334.

Interpretive Summary: Eight foods including tree nut, peanut, soy, wheat, milk, egg, fish and sea foods such as shrimp are responsible for the majority of food allergy. Tropomyosin is a common food allergen found in shrimp and other shellfish. Allergic responses require the cross-linking of a receptor with allergen molecules such as (tropomyosin) to activate a cellular response. We demonstrate that the cellular response to shrimp tropomyosin is linked to the number of antibody receptor-tropomyosin complexes, as well as the concentration of tropomyosin and its ability to be bound by antibodies. We modeled the concentration dependent response of immune cells to provide information on the provided information on receptor complex formation. We found that maximal degranulation occurred when approximately 1000 receptor-tropomyosin complexes were cross linked. Prolonged periods of protease digestion, to mimic what occurs in the stomach, were required to diminish cellular responses. Small tropomyosin fragments produced in the lab were relatively weak at cellular allergen activation. Our findings represent a starting point for the design of promising hypoallergens (weaker allergens) for immunotherapy.

Technical Abstract: Antigen-mediated cross-linking of IgE-Fc'RI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to recombinant Pen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of Fc'RI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using CRISPR-based gene editing, rat basophilic leukemia (hRBLr'ko) cells were created that exclusively express the hFc'RI' subunit. Pen a 1 specific-IgE (IgEpen a 1) was affinity purified from shrimp positive plasma. Cells primed with a range of IgEpen a 1 and challenged with Pen a 1 show a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. A rule-based mathematical model provided information on receptor aggregation kinetics based upon Fc'RI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ~1000 IgE-Fc'RI complexes were crosslinked with full length Pen a 1 (IgE epitope valency of 10+), although measurable responses were achieved with only a few hundred Fc'RI engaged. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 amino acids), that together span the entire length of tropomyosin, were relatively weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-Fc'RI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.