HCDR3 sequence motifs are related to allergy or tolerance
As HCDR3 regions have a great impact on antigen binding, their characteristics were more deeply analyzed. Detailed analysis of physiochemical properties, such as amino acid distribution, aliphatic index, Boman index and Kidera factors 1-10, are shown in the Supplementary, Figure S5 and 617 . The Kidera factor 5, expressing the preference to form double-bended structures, was significantly higher for the HCDR3 region of allergic donors compared with that of tolerant donors (p = 0.0226, Bonferroni correction: non-significant, Figure 3D). Nevertheless, the mean Boman index, describing the theoretical ability to bind proteins, was comparable between HCDR3 regions of allergic, tolerant, and non-atopic donors. For deeper analysis, HCDR3 sequence motifs associated with peanut allergy and/or tolerance, were evaluated by Levenshtein distances (≤5 replacements, deletions or insertions) and hierarchical clustering. Levenshtein distances were favored over clonal relationship analysis because of individual genetic variation regarding haplotype diversity, single nucleotide polymorphisms, gene copy number and preferred gene lineage27. For this analysis, all successfully amplified and sequenced heavy chain gene transcripts were used irrespectively of successfully sequenced corresponding light chain gene transcripts. Overall, four unique HCDR3 sequence motifs were associated with peanut allergy and three with peanut tolerance (Figure 3E, Table 3). The most dominant motif was ‘CARDSSALEIYNRFDPW’ (motif 1), which was derived from 36 different B-cells belonging to three different allergic donors (Pt-1 (n=32), Pt-5 (n=3), Pt-6 (n=1)) including 32 nearly identical B-cells of Pt-1 (monoclonal proliferation). This motif was formed by VH3-30, DH3-3 and JH5 genes. Besides motifs exclusively related to either allergy or tolerance, five motifs were shared between allergic, tolerant, and non-atopic donors. It must be mentioned that one motif (CARNVFDGYWLVYW) associated with tolerance was only found in Pt-11 and no motif was shared between all allergic or tolerant donors. Heterologously expressed mAbs corresponding to these motifs showed specific binding to Ara h2 or 6, supporting the relevance of the identified motifs and their potential role in diagnosing patients with suspected peanut allergy.