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 peanut-allergic patients is available. Previous trials were associated with severe side effects. Objective To determine the relative importance of conformational and linear IgE-binding epitopesof the major peanut allergen Ara h 2 and to produce a hypoallergenicvariant with abolished anaphylactogenic activity. Methods Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopeswere produced in insect cells. Conformational IgE epitopeswere removed by unfolding these proteins by reduction and alkylation. IgEbinding was tested by ELISA with sera from 48 Ara h 2-sensitized, peanut-allergic patients. Basophil activationand T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by intraperitoneal challenge of mice sensitized intragastrically to peanut extract. Results Patients’ IgErecognized conformational and linear epitopesin a patient-specific manner. The unfolded mutant lacking both types of epitopesdisplayed a significantly lower IgEbinding (median ELISA OD 0.03, interquartile range0.01-0.06) than natural Ara h 2 (median 0.99, interquartile range0.90-1.03; p Conclusions By removing conformational and linear IgE epitopes, a hypoallergenicAra h 2 mutant with abolished IgEbinding and anaphylactogenic potency but retained T cell activation was generated.