D1.283 - Endolysosomal degradation of major peanut allergens revealed distinct degradation kinetics and T cell epitope regions

Endolysosomal processing of antigens is fundamental for allergen sensitization. The in vitro endolysosomal degradation assay mimics this process and aims to link protein stability with antigenicity and to predict T cell epitopes. So far, data on endolysosomal degradation of type I food allergens are limited. Thus, we investigated the endolysosomal stability of major peanut allergens and compared the endolysosomal degradome with in silico and experimentally validated T cell epitopes.

Purified natural Ara h 1, Ara h 2 and Ara h 6 were investigated by a newly standardized endolysosomal degradation assay. Allergens were degraded with endolysosomal proteases isolated from the murine dendritic cell line JAWSII for up to 72 hours. Endolysosomal half-lives were calculated based on protein gel staining. The endolysosomal degradome was obtained by mass spectrometry. MHCII binding prediction was performed by NetMHCIIpan4.1 testing 18-mer peptides with all HLA alleles. Experimentally validated T cell epitopes positive for >25% of T cell lines or allergic patients were obtained from the Immune Epitope Database.

The endolysosomal half-life was 2 hours for Ara h 1 and Ara h 2 while Ara h 6 revealed a half-life of more than 72 hours. The endolysosomal degradome of Ara h 1 revealed prominent peptide clusters at Ara h 1_126-158, Ara h 1_212-245, Ara h 1_259-299, Ara h 1_331-376, Ara h 1_411-452, Ara h 1_518-559. Within those clusters, four in silico predicted MHCII-binding peptides were located, while three clusters encompassed experimentally validated T cell epitopes.  Dominant peptide clusters of Ara h 2 were located at the N-terminus. Notably, Ara h 2_1-37 represents a validated T cell epitope while Ara h 2_38-61 encompasses only in silico predicted region. Ara h 6 endolysosomal degradome revealed three prominent peptide clusters, Ara h 6_3-42, Ara h 6_50-70 and Ara h 6_102-124. Experimentally validated T cell epitopes are not available and in silico predictions spanned regions that were processed during endolysosomal degradation.  Preliminary testing of the Ara h 6_51-69 peptide, which was selected based on endolysosomal degradation, stimulated peanut specific T cells (SI=4.5). Endolysosomal degradome comparison of the homologous allergens Ara h 2 and Ara h 6 revealed highly similar peptide clusters at the N- and C-termini. Notably, the additional peptide region, Ara h 2_33-67, that is only present in Ara h 2 encompassed a prominent peptide cluster.

Endolysosomal degradation of Ara h 1 and Ara h 2 revealed rapid processing, while Ara h 6 was more resistant to degradation. The prominent peptide cluster present in the additional region of Ara h 2 and the divergent half-lives of Ara h 2 and Ara h 6 could provide insights on the distinct allergenicity pattern of these homologous allergens. Discovered endolysosomal peptide clusters of tested peanut allergens frequently incorporated validated and predicted T cell epitopes. The endolysosomal degradation assay is an in vitro tool for endolysosomal stability assessment and refined T cell epitope identification which can be used as an orthogonal method for antigenicity and allergenicity predictions of novel foods.