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Hypoallergenic mutants with reduced IgE-binding capacity but which show a similar T-cell response to the corresponding natural allergen are ideal tools for immunotherapy, for preventing a possible anaphylactic shock. An IgE conformational epitope has been identified in Cuc m 2, the major allergen and profilin from melon. Since this epitope is highly conserved in most pollen profilins, it may contribute to an explanation of cross-reactivity between pollen and food profilins. Mutants (Mut 1 and Mut 2) were generated by changing specific residues of the Cuc m 2 epitope to alanine, produced in Escherichia coli, and purified by chromatographic methods. Mut 1 showed a slight reduction in IgE binding but an allergenic activity that was similar to recombinant Cuc m 2, as measured by basophil activation test (BAT) and skin prick test (SPT). By contrast, Mut 2 displayed a substantial reduction in IgE-binding capacity (57%) and positive responses, as determined by BAT (33%) and SPT (50%), when compared to those of rCuc m 2. However, the T-cell proliferation and cytokine production induced by Mut 2 and rCuc m 2 were similar. Thus, this mutant represent potential candidate for immunotherapy of profilin allergies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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 S76 I77 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.

Background: Plant profilins are actin-binding proteins that form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. Melon profilin, Cuc m 2, is the major allergen of this fruit., Objective: We sought to map IgE epitopes on the 3-dimensional structure of Cuc m 2., Methods: IgE binding to synthetic peptides spanning the full Cuc m 2 amino acid sequence was assayed by using a serum pool and individual sera from 10 patients with melon allergy with significant specific IgE levels to this allergen. Three-dimensional modeling and potential epitope location were based on analysis of both solvent exposure and electrostatic properties of the Cuc m 2 surface., Results: Residues included in synthetic peptides that exerted the strongest IgE-binding capacity defined 2 major epitopes (E1, consisting of residues 66-75 and 81-93, and E2, consisting of residues 95-99 and 122-131) that partially overlapped with the actin-binding site of Cuc m 2. Two additional epitopes (E3, including residues 2-10, and E4, including residues 35-45) that should show weaker putative antigen-antibody associations and shared most residues with synthetic peptides with low IgE-binding capacity were predicted on theoretical grounds., Conclusions: Strong and weak IgE epitopes have been uncovered in melon profilin, Cuc m 2., Clinical Implications: The different types of IgE epitopes located in the 3-dimensional structure of melon profilin can constitute the molecular basis to explain the sensitization and cross-reactivity exhibited by this panallergen family.

Background: Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro., Objective: We sought to locate major IgE-binding epitopes of Pru p 3., Methods: A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection., Results: Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes., Conclusions: Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.

Based on our I in silico i structural analyses, the IgE-specific effects may be due to epitope masking by the VD3 ligand of two previously described IgE-binding B-cell epitope regions in the molecular surface of Bet v 1 (Figure 1C). Keywords: Bet v 1; birch pollen allergy; immunomodulation; lipocalin-like; vitamin D EN Bet v 1 birch pollen allergy immunomodulation lipocalin-like vitamin D 3801 3804 4 12/01/21 20211201 NES 211201 Abbreviations ANS 1-anilino-8-naphthalene sulfonate BCA bicinchoninic acid BP birch pollen E aff affinity energy FCS foetal calf serum IL interleukin K D binding constant kDa kilodalton MFI mean fluorescence intensity OD optical density PBMCs peripheral blood mononuclear cells PDB protein data bank RA retinoic acid RFUs relative fluorescence units VD3 Vitamin D3 VDRE vitamin D response element ACKNOWLEDGEMENTS We thank Bart R. Blokhuis for excellent technical assistance. [Extracted from the article]

Retinoic acid-loading of the major birch pollen allergen Bet v 1 may improve specific allergen immunotherapy: In silico, in vitro and in vivo data in BALB/c mice Even if this was not due to altered endolysosomal stability of Bet v 1a due to RA binding (Figure S3), it seemed so far like RA-loading transforms the hyperallergen Bet v 1a to a hypoallergen with improved tolerogenic capacity of potential implications for allergen immunotherapy. Mice were first made allergic against the major birch pollen allergen Bet v 1a and subsequently treated intranasally with either the I apo i -Bet v 1a, or with the RA-loaded I holo i -Bet v 1a, or as a control with RA alone. [Extracted from the article]

Plant lipid transfer proteins are a large family that can be found in all land plants. They have a hydrophobic cavity that allows them to harbor lipids and facilitates their traffic between membranes. However, in humans, this plant protein family is responsible for the main food allergies in the Mediterranean area. Nevertheless, not only the protein itself but also its ligand is relevant for allergic sensitization. The main aim of the present work is to analyse the natural ligands carried by four allergenic LTPs (Tri a 14, Art v 3, Par j 2, and Ole e 7), compared with the previously identified ligand of Pru p 3 (CPT-PHS ligand), and clarify their role within the immunological reactions. Results showed that the ligands of the LTPs studied shared a chemical identity, in which the presence of a polar head was essential to the protein-ligand binding. This ligand was transported through a skin cellular model, and phosphorylated phytosphingosine could be detected as result of cell metabolism. Since sphingosine kinase 1 was overexpressed in keratinocytes incubated with the LTP-ligand complex, this enzyme might be responsible for the phosphorylation of the phytosphingosine fraction of the CPT-PHS ligand. This way, phytosphingosine-1-phosphate could be mimicking the role of the human inflammatory mediator sphingosine-1-phosphate, explaining why LTPs are associated with more severe allergic responses. In conclusion, this work contributes to the understanding of the chemical nature and behavior of lipid ligands carried by allergens, which would help to gain insight into their role during allergic sensitization. [Display omitted] • Tri a 14, Art v 3, Par j, and Ole e 7 carry a similar ligand to that of Pru p 3. • The polar head of camptothecin is essential to stabilize the ligand-binding. • PHS tail from the LTP ligands can be phosphorylated by SphK1 in epithelial cells. • Natural LTP ligands are able to mimic sphingosine-1-phosphate function. [ABSTRACT FROM AUTHOR]