Analyses of IgE-epitope profiles from legume allergens as approach towards the development of novel diagnostic and therapeutic reagents in food allergy

Legumes are an important source of high-value proteins and edible oils in the human diet. However, they are also a known and frequent source of severe allergic reactions to foods. Immediate-type allergic reactions are elicited by allergenic proteins (allergens), mediated by their interaction with the immune system via allergen-specific antibodies of the isotype immunoglobulin E (IgE). These specific IgE antibodies to total protein of the allergenic food, e.g. peanut, can be measured in vitro to diagnose an allergic sensitization. While specific IgE to total protein extracts indicates a sensitization, it is not necessarily a proof for clinical reactivity. In contrast, the measurement of specific IgE to selected allergen components, such as the peanut 2S albumin Ara h 2, has been proposed to improve the in vitro diagnostic specificity for a clinical reactivity. In this context, specific recognition patterns of IgE binding at the protein level, and further at the peptide level, may allow the development of advanced diagnostic approaches, and innovative therapeutic reagents. However, knowledge about the relevance of allergenic storage proteins, such as 2S albumins and 7S globulins, of the legumes peanut, pea and soybean and their IgE-binding peptides is in part controversial or limited. Therefore, this study aimed to determine the serum IgE binding of legume-allergic versus sensitized but clinically tolerant children to homologous 2S and 7S legume allergens from peanut, pea and soybean at the protein and peptide level. In this study, sera from legume-allergic as well as sensitized but clinically tolerant children with legume extract-specific IgE ≥ 0.35 kUA/L were included. Total protein extracts and recombinant 7S globulins and 2S albumins of peanut, pea, and soybean, respectively, were prepared. Afterwards, serum IgE binding to legume extracts and recombinant legume allergens was individually determined by means of densitometric immunoblot analysis. Overlapping peptides (15 AA; offset 4 AA) representing the full-length allergens were synthesized and analyzed for their ability to bind serum IgE on CelluSpotTM multipeptide microarrays. The influence of post-translational hydroxylation of proline residues in peanut Ara h 2 on the capacity to bind serum IgE and thus on the diagnostic value was additionally investigated on the peptide level. Potential candidate diagnostic peptides specific for peanut and pea allergy were identified, according to predefined selection criteria that were established in this study. Finally, the diagnostic value of the investigated recombinant legume allergens and of the potential candidate diagnostic peptides was determined as area under curve (AUC) by receiver operating characteristic (ROC) curve analysis. By doing so, it should be determined whether peptides may serve as additional or alternative reagents in the in vitro diagnosis of legume allergy. However, due to the very limited number of available and included soybean-allergic patients, soybean was excluded from a detailed analysis of candidate diagnostic peptides and ROC curve analysis. According to immunoblot analysis and the specific limit of detection, serum IgE of 48%, 79% and 50% of peanut-, pea- and soybean-allergic children bound to the 7S globulins rAra h 1, rPis s 1 and rGly m 5.03, respectively. Of the peanut-, pea- and soybean-sensitized but tolerant children 8%, 20% and 20% showed a serum IgE binding to the respective 7S globulins. The most striking difference between the three legumes could be observed regarding the relevance of the 2S albumins. In immunoblot analysis, 65% and 78% of peanut-allergic children showed a serum IgE binding to rAra h 2.01 and rAra h 2.02, respectively. In contrast, serum IgE of peanut-sensitized but tolerant patients did not bind to any of both Ara h 2 isoforms. Hence, the relevance and the diagnostic value of both 2S albumin isoforms, especially of rAra h 2.02, in peanut allergy were further highlighted. Based on immunoblot analyses, such clinical relevance of 2S albumins could not be found in pea and soybean. Here, negligible or no serum IgE binding to 2S albumins was detectable in pea- as well as soybean-allergic children. Based on the ROC curve analysis of the investigated recombinant proteins analyzed in immunoblot, rAra h 2.02 (AUC 0.86) and rPis s 1 (AUC 0.86) had the highest diagnostic value in peanut and pea allergy, respectively, also compared to the respective total legume protein extract. With regard to potential diagnostic peptides, two Ara h 2-derived peptide pairs (AUC 0.87-0.90) with a diagnostic value comparable to that of full-length rAra h 2.02 (AUC 0.86) could be identified for peanut allergy. Each of the two peptide pairs contains at least one peptide with hydroxylated proline residues. In addition, it could be observed that hydroxylation of proline residues in Ara h 2-derived peptides increased the sensitivity while maintaining the specificity which makes this post-translational modification an interesting target for future diagnostic approaches. Moreover, for pea allergy eleven candidate diagnostic peptides of Pis s 1 could be identified. Compared to pea extract and rPis s 1, the candidate peptides had, with an AUC of 0.99, the highest diagnostic value in this pea study population. In this pediatric study population, general differences in allergenic potency of the 2S and 7S storage proteins of the three investigated legumes were apparent. In peanut, Ara h 1 and Ara h 2 could be identified as relevant allergens. However, based on immunoblot analysis, the 2S albumin Ara h 2, in particular the isoform Ara h 2.02, was the most relevant allergen with the highest diagnostic value in this peanut study population. In contrast, in pea and soybean 2S albumins were excluded as relevant allergens in this study. In pea, the 7S globulin Pis s 1 was identified as a major and immunodominant allergen. A similar trend seemed to be observed for Gly m 5.03 and soybean, but due to the limited number of soybean-allergic patients included in this study, no further conclusions can be drawn. In addition, the investigation of IgE binding at the linear peptide level revealed that peptides with a diagnostic value that is comparable or even better than that of the respective full-length recombinant proteins could be identified. These peptides may potentially serve as additional or alternative reagents in the in vitro diagnosis of legume allergy; however, this requires verification in a prospective study. Moreover, based on this study, additional knowledge about relevant allergens and their IgE-binding sites was obtained. This knowledge can support the development of novel therapeutic reagents with improved characteristics. In this context, IgE binding to short peptides of allergens was investigated to localize IgE-binding sites in approximation to linear (continuous) IgE epitopes. It could be shown that linear IgE-binding epitopes of natural Ara h 2, as represented by a 27-mer peptide, still induced relevant mast cell degranulation. This finding underpins the potential relevance of linear IgE-binding sites with regard to total allergenic potency, and should thus be considered in the development of novel reagents for the immunotherapeutic treatment of peanut allergy as otherwise patients might be at high risk of unintended side effects. In addition, identified immunodominant IgE-binding sites of Pis s 1 may allow the development of hypoallergenic substitution variants for allergen-specific immunotherapy of pea allergy. The relevance of these findings with regard to efficacy and safety can be further addressed in preclinical models of peanut and pea allergy, respectively.