Design of an Ara h 2 hypoallergen from conformational epitopes

Abstract

IntroductionAdverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA).MethodsX‐ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum.ResultsA ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site‐specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant.ConclusionThese studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Mutation of Disulfide Bond Sites Reduces the Immunoreactivity of Cra a 4 by Changing the Structural Characteristics

“…Site-directed mutation provided a new train of thought for studying the structure–allergenicity relationship of allergens by mutating critical amino acids that affected the structure . Min et al demonstrated that mutations in critical amino acids in conformational epitopes affect the structure and immunobinding activity of Ara h 2 . In addition, the hypoallergenic mutant obtained by site-directed mutation may be used to prevent allergy caused by allergen .…”

“…22 conformational epitopes affect the structure and immunobinding activity of Ara h 2. 23 In addition, the hypoallergenic mutant obtained by site-directed mutation may be used to prevent allergy caused by allergen. 24 It had been reported that hypoallergenic mutants of pollen allergen (Parietaria judaica) had lower IgE-binding activity and allergenicity in mice by sitedirected mutation.…”

A novel IgE epitope-specific antibodies-based sandwich ELISA for sensitive measurement of immunoreactivity changes of peanut allergen Ara h 2 in processed foods

“…Analytical methods currently used to detect peanut allergens, such as real-time polymerase chain reaction (7), reversedphase high-performance liquid chromatography (RP-HPLC) (8), liquid chromatography coupled mass spectrometry (9), enzyme-linked immunosorbent assay (ELISA) (10)(11)(12), and lateral flow immunoassay (10), lack the ability to specifically detect allergenic epitopes of the allergens. Traditionally, the method for measurement of IgE-binding capacity variations of peanut allergens is based on patients' IgE antibodies (13)(14)(15). However, the limited and variable sera from peanut-allergic patients makes the standardization of the detection method very difficult for commercial purposes.…”