Engineering of structural variants of the major peanut allergens Ara h 2 and Ara h 6 for allergen-specific immunotherapy

Authors: BUBLIN, MERIMA - Medical University Of Veinna; KOSTADINOVA, MARIA - Medical University Of Veinna; RADAUER, CHRISTIAN - Medical University Of Veinna; VARGA, EVA-MARIA - Medical University Of Veinna; HAFNER, CHRISTINE - Karl Landsteiner Institute Of Dermatological Research; SCHMIDTHALER, KLARA - Medical University Of Veinna; SAIDOVA, AZIZA - Medical University Of Veinna; Maleki, Soheila; EIWEGGER, THOMAS - Medical University Of Veinna; BREITENDER, HEIMO - Medical University Of Veinna; SZEPFALUSI, ZSOLT - Medical University Of Veinna

Submitted to: Journal of Allergy Clinical Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/9/2018
Publication Date: 3/9/2019
Citation: Bublin, M., Kostadinova, M., Radauer, C., Varga, E., Hafner, C., Schmidthaler, K., Saidova, A., Maleki, S.J., Eiwegger, T., Breitender, H., Szepfalusi, Z. 2019. Engineering of structural variants of the major peanut allergens Ara h 2 and Ara h 6 for allergen-specific immunotherapy. Journal of Allergy Clinical Immunology. 143:3. https://doi.org/10.1016/j.jaci.2018.10.039.
DOI: https://doi.org/10.1016/j.jaci.2018.10.039

Interpretive Summary: Peanut allergy is a persistent food allergy and a potentially life-threatening condition. Although clinical trials with oral immunotherapy are encouraging there is a strong need to develop novel hypoallergenic, pro-tolerogenic vaccine components. The Aim is to generate modified variants of the major peanut allergens Ara h 2 and Ara h 6 with reduced IgE-binding that also maintain the desensitizing potential and preserved the T-cell epitopes for treatment of peanut allergy. The hypoallergens were engineered by swapping neighbouring segments of 5-10 amino acids residues in surface exposed loops. The recombinant native and modified Ara h 2 and Ara h 6 proteins were produced in E. coli. IgE binding,cross-linking capacity (basophil activation test) and T-cell stimulatory capacity was evaluated with sera and cells from peanut allergic and non-peanut allergic individuals. Results: Modified Ara h 2 and Ara h 6 showed significantly (p <0.001) less IgE-binding capacities as compared to their natural counterparts. Compared to the natural allergens, thousand-fold higher concentrations of the modified proteins were required for basophil activation. Modified Ara h 2 and Ara h 6 were fully capable in inducing allergen-specific T-cell proliferation compared to the natural allergens. Hypoallergenic variants with significantly reduced IgE-binding and preserved T-cell activation may be a valuable option for a safer immunotherapy.

Technical Abstract: Introduction: Peanut allergy is a persistent food allergy and a potentially life-threatening condition. Although clinical trials with oral immunotherapy are encouraging there is a strong need to develop novel hypoallergenic, pro-tolerogenic vaccine components. Aim: To generate modified variants of the major peanut allergens Ara h 2 and Ara h 6 with reduced, but residual IgE-binding to maintain the desensitizing potential and preserved the T-cell epitopes for treatment of peanut allergy. Methods: The hypoallergens were engineered by swapping neighbouring segments of 5-10 amino acids residues in surface exposed loops. Expression of recombinant wild type and modified Ara h 2 and Ara h 6 proteins was carried out in E. coli SHuffle T7 cells. IgE binding (ELISA, immunoblot), IgE cross-linking capacity (basophil activation test) and T-cell stimulatory capacity (3H-thymidine incorporation assay, multiplex cytokine measurement) was evaluated in with sera and cells from peanut allergic and non-peanut allergic individuals. Results: Modified Ara h 2 and Ara h 6 showed significantly (p <0.001) less IgE-binding capacities as compared to their recombinant wild types and their natural counterparts. Compared to the natural allergens, thousand-fold higher concentrations of the modified proteins were required for basophil activation. Modified Ara h 2 and Ara h 6 were fully capable in inducing allergen-specific T-cell proliferation with reduced Th2 cytokine induction as compared to the natural allergens. Conclusions: Hypoallergenic variants with significantly reduced but not completely absent IgE-binding and preserved T-cell activation may be a valuable option for a safer immunotherapy.