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

Abstract

Background: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects.

Objective: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity.

Methods: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract.

Results: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice.

Conclusions: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated.

Keywords: Ara h 2; Peanut allergy; epitopes; hypoallergen; immunotherapy; mouse model.

Fig 1

Construction of Ara h 2 derivatives. A, Structural representations of native and red/alk proteins. B, Amino acid sequences of wtAra h 2 and mtAra h 2 showing the IgE-binding epitopes removed in mtAra h 2. Previously determined linear IgE epitopes are boxed, immunodominant T-cell epitopes are shown in boldface, and helices are shown in gray.

Fig 2

IgE binding to Ara h 2 derivatives tested by means of ELISA with 48 Ara h 2–sensitized patients with peanut allergy. A, Distributions of ELISA OD values in all patients. *P < .05 and ***P < .001. B, IgE ELISA and inhibition ELISA results of 5 representative patients. For the inhibition ELISA, nAra h 2 was immobilized, and residual IgE binding after preincubating sera with Ara h 2 derivatives was measured. C, Comparison of IgE binding to native and red/alk proteins (lacking conformational epitopes) in individual patients. D, Individual epitope recognition patterns demonstrated by comparing IgE binding to wtAra h 2, mtAra h 2 (lacking linear epitopes), and red/alk wtAra h 2 (lacking conformational epitopes).

Fig 3

Correlation of Ara h 2–specific IgE levels with measures of epitope recognition patterns derived from IgE ELISA data. A, The mtAra h 2 to wtAra h 2 ratio represents IgE binding to conformational epitopes. B, The red/alk to native wtAra h 2 ratio represents IgE binding to linear epitopes.

Fig 4

Basophil activation tests (n = 7) with native and red/alk proteins. Areas under the concentration-dependent activation curves are shown. *P < .05 and **P < .01.

Fig 5

T cell–stimulating abilities of Ara h 2 derivatives. A, In vitro proliferation assays with PBMCs from Ara h 2–sensitized patients with peanut allergy (n = 9). Data are presented as relative SIs normalized to the SIs of cells treated with nAra h 2. B, Flow cytometry–based proliferation assay with PBMCs of a representative patient. Data represent percentages of proliferating (carboxyfluorescein N-succinimidyl ester–low) cells among CD4⁺ T cells (CD3⁺CD8⁻ cells).

Fig 6

Anaphylactic response in C3H/HeOuJ mice with peanut allergy after challenge with native and red/alk proteins. A, Mice were sensitized intragastrically (i. g.) with PE and challenged intraperitoneally (i. p.) on day 37 with native and red/alk proteins. B, Ara h 2–specific IgE and IgG₁ levels of peanut-sensitized mice and control mice sensitized by PBS. **P < .01. C, Maximum core body temperature decrease after intraperitoneal challenge measured over the period of 1 hour every 10 minutes. *P < .05.