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]

Abstract

Ag-mediated crosslinking 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 rPen 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 clustered regularly interspaced palindromic repeat–based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα 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 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 when 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 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–FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Footnotes

  • This work was supported by National Institutes of Health Grant P50GM085273 (to B.S.W.) and National Science Foundation Career Award III-1553266 (to L.T.). This work was also supported in part by funds from the U.S. Department of Agriculture, Agricultural Research Service (to C.P.M.).

  • Mention of trade names, commercial products, or companies in this paper is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CD
    circular dichroism
    CRISPR
    clustered regularly interspaced palindromic repeat
    gRNA
    guide RNA
    h
    human
    hIgEAlexa-488
    Alexa Fluor 488–conjugated hIgE
    IgEPena1
    Pen a 1–specific IgE
    m
    mouse
    SGF
    simulated gastric fluid.
  • Received August 2, 2016.
  • Accepted November 30, 2016.

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