Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens

Abstract

Antigen-mediated cross-linking of IgE-FcεRI 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 FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using CRISPR-based gene editing, rat basophilic leukemia (hRBLrαKO) cells were created that exclusively express the hFcεRIα 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. Mathematical modeling provided estimates of receptor aggregation kinetics based upon FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ~2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of 8+), although measurable responses were achieved with only a few hundred FcεRI 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 amino acids), that 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-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Development of a novel Ara h 2 hypoallergen with no IgE binding or anaphylactogenic activity

“…The more pronounced reaction to mtAra h 2 compared with wtAra h 2 can be explained by the increased aggregation of the mutant protein (see Fig E1, D) and therefore more efficient cross-linking of mast cell-bound IgE. 36 The administered amount of 16.3 mg corresponds to 56 mg in a human weighing 65 kg. This is slightly lower than the maximum maintenance doses used in peanut oral immunotherapy (4000 mg of peanut protein, 18 corresponding to 400 mg of Ara h 2 37 ) and much greater than allergen doses used in subcutaneous immunotherapy, which are in the microgram range.…”

IgE Activates Monocytes from Cancer Patients to Acquire a Pro-Inflammatory Phenotype

“…This observation is consistent with our previous findings showing maximal activation of human macrophages by IgE stimulation even in the absence of FcεR saturation [ 22 ]. Cross-linking of a proportion of IgE-FcεRI complexes on the cell surface has been shown to be sufficient for maximum mast cell and basophil activation and mediator release [ 45 , 46 , 47 ]. Monocytes already have FcεRs partly occupied by endogenous IgE and thus exogenous provision of MOv18 IgE will result in a fraction of FcεRs bearing tumour-specific IgE.…”

Overcoming Shellfish Allergy: How Far Have We Come?

“…Despite the fact that low-dose immunotherapy was well tolerated, medium-dose AIT triggered mild allergic symptoms during the treatment phase, severe anaphylactic responses (convulsion and tremor) or even death were observed in the high-dose group. This might be attributed to the well-spaced epitopes and exceptional IgE crosslinking capacity of tropomyosin [59,60]. These observations indicate that recombinant shellfish tropomyosin in AIT should be used with much caution.…”

“…Another logical concept of engineering hypoallergens is to limit the structural features of an allergen in activating the IgE receptor FcεRI. Mahajan et al recently designed five sequential Pen a 1 recombinant polypeptides and dissected their capacities for inducing histamine release in basophils and RBL cells that exclusively express the human IgE receptor FcεRIα subunit (hRBL rαKO cells) [59]. Polypeptides are continuous and longer peptide bonds with more than fifty monomer units.…”