An unfolded variant of the major peanut allergen Ara h 2 with decreased anaphylactic potential

Authors
Philipp Starkl, Ferdinand Felix, Durga Krishnamurthy, Caroline Stremnitzer, Franziska Roth-Walter, Sara R. Prickett et al.

Lab
Medical University of Vienna, Vienna, Austria / The Alfred Hospital and Monash University, Melbourne, Australia

Journal
Clinical & Experimental Allergy

Abstract
Background
Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis.

Objective
Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T-cell reactivity as well as anaphylactic potential.

Methods
The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2 specific T cell lines and splenocytes isolated from sensitized mice.

Results
Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13, and IFN? levels upon treatment with either Ara h 2 variant.

Conclusion and Clinical Relevance
Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients.