Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76177 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Keywords:Lipid transfer protein T-cell epitope Peptide immunotherapy Plant food allergy Peach Pru p3Lipid transfer proteins (LTPs), particularly peach Pru p 3, are the most relevant plant food allergens in the South of Europe, and, therefore, their allergic properties have been extensively studied. However, neither T-cell epitopes nor their effect on the patients' T-cell response has been investigated in any member of the LTP panallergen family. The objective of the present study was to map the major T-cell epitopes of Pru p 3, as well as to evaluate their induced T-cell response in peach-allergic versus control subjects. Thus, peripheral blood mononuclear cells (PBMCs) from 18 peach-allergic patients and Pru p 3-specific T-cell lines (TCLs) from 9 of them were cultured with Pru p 3 and with a panel of 17 derived peptides (10-mer overlapping in 5 amino acids representing the full sequence of Pru p 3). Proliferation in 5-day assays was carried out via tritiated-thymidine incorporation, while IL4 and IFN7 production was assessed via sandwich enzymelinked immunosorbent tests (ELISA) of TCL culture supernatants. The results were compared to those obtained from 10 non-peach allergic control volunteers. Two consecutive peptides showed the highest activation capacity. About 74% of PBMCs and TCLs recognized them, forming a single T-epitope: Pru p 365-80-Additionally, other specific T-cell epitopes were observed. Pru p 3 2 s-35 was detected by more than 60% of TCLs from peach-allergic patients, and Pru p 345-55 only activated PBMCs from control subjects. Interestingly, TCLs from patients were associated with a Th2-type, whereas control TCLs presented a Thltype cytokine response. The major immunogenic T-cell epitope identified in Pru p 3, Pru p 365-so, is a good candidate to develop new vaccines for hypersensitivity reactions associated with LTP allergens from Rosaceae fruits.

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BackgroundProfilin sensitisation is considered a diagnostic confounding factor in areas where patients are exposed to multiple pollens. The aim of this study is to assess pollen sensitisation profiles in adults and children and to evaluate, by means of component-resolved diagnosis (CRD) and skin prick testing (SPT), which pollens may be considered as risk factors of profilin sensitisation in order to establish the best diagnostic approach in polysensitised patients.MethodsA total of 231 pollen-allergic patients (adults and children) were included, out of the pollen season, from an area with similar levels of pollen exposure. Allergological diagnosis was performed by SPT and determination of specific IgE (sIgE) to major allergen components (ADVIA-Centaur™). Patients had not received immunotherapy in the last 5 years and had to reside in the area for 5 consecutive years before entering the study.ResultsThe relation between sensitisation measured by SPT and by sIgE was studied using a model of cases (patients with +sIgE to a specific allergen) and controls (patients with −sIgE to the same allergen). The outcome, in terms of odds-ratios (OR), was statistically significant for Olea (Ole e 1) (p = 0.0005), Salsola (Sal k 1) (p = 0.0118) and Platanus (Pla a 1+ 2) (p = 0.0372). While positivity of SPT to most pollens was statistically associated with a risk of profilin sensitisation, by CRD the association was statistically significant only for Ole e 1 (OR 3.5, CI 95 %, 1.6–7.6, p = 0.0014), and Phl p 5 (OR 11.9, CI 95 %, 4.1–35.2, p < 0.001). When analysing this association using a logistic regression model, Phl p 5 was the only allergen associated with the risk of being sensitised to profilin (p = 0.0023).ConclusionsIn patients sensitised to profilin, the concordance between SPT and CRD is much lower than in those not sensitised to profilin. CRD is able to provide refined information about which pollens increase the risk of sensitisation to profilin.