Allergen Valency, Dose, and Fc{varepsilon}RI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens [ALLERGY AND OTHER HYPERSENSITIVITIES]

Ag-mediated crosslinking of IgE–FcRI 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 rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat–based gene editing, human RBL^rαKO cells were created that exclusively express the human FcRIα subunit. Pen a 1–specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1–specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1–10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ~2700 IgE–FcRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60–79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE–FcRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

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