Introduction
Current food allergy diagnostics comprise careful history, skin prick tests, measuring specific IgE (sIgE) and double-blind placebo-controlled food challenges (DBPCFC) as the gold standard. However, DBPCFCs are burdensome for the patient, costly and require dedicated hospital facilities1, 2. On the other hand, current technologies to measure sIgE detect both clinically relevant and irrelevant sensitization, potentially leading to incorrect diagnosis and consequently unnecessary food restrictions3, 4. Clinically relevant peanut sensitization is associated with sIgE against the major peanut allergens belonging to the 2S albumin family, Ara h2 and 6. In previous studies in adults, 100% positive predictive values for sIgE against Ara h2 and 6 were found using sIgE positivity thresholds of respectively 1.75 kU/l and 1.8 kU/l. Specific IgE levels below these thresholds, however, overlapped between allergic and tolerant subjects, preventing precise diagnosis5, 6.
The occurrence of clinically irrelevant sensitization to Ara h2 and 6 might be explained by differences in peanut (Ara h2 and 6) specific antibody repertoires. These differences may include the presence of non-IgE antibodies blocking the binding of clinically relevant epitopes by sIgE of tolerant patients. Moreover, differences may also be based on antibody affinity and/or epitope recognition patterns. So far, no clear differences between allergic and tolerant subjects were observed by epitope mapping approaches7. These evaluations may have been hampered by the use of patient sera consisting of polyclonal IgE antibodies. Sera of allergic subjects usually contain mixtures of antibodies recognizing both clinically relevant and irrelevant epitopes. On the other hand, sera from tolerant subjects might contain antibodies recognizing clinically relevant epitopes with insufficient affinity for successful FcεRI receptor crosslinking, as well as antibodies recognizing clinically irrelevant epitopes8. Hence, deep analysis of monoclonal antibodies (mAbs) from specific B-cells may provide more insights into differences in specific antibody repertoires between allergic and tolerant subjects.
To this end, we analyzed gene sequences encoding the variable region of peanut 2S albumin-specific mAbs from 6 allergic and 6 sensitized but tolerant adults. In particular, sequence motifs of the HCDR3 region, the most important region for recognizing antigens9, were hierarchically clustered. Clustering of HCDR3 regions resulted in four motifs exclusively present in allergic donors and three motifs associated with tolerance.