Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6.

BACKGROUND: Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes. OBJECTIVE: To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library. METHODS: A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch. RESULTS: Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history. CONCLUSIONS: The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.

The value of specific IgE to peanut and its component Ara h 2 in the diagnosis of peanut allergy.

BACKGROUND: To avoid unnecessary oral food challenges, which are time consuming, stressful, and risky, improved in vitro diagnostic methods for food allergy such as component resolved diagnostics are still under investigation. OBJECTIVE: To investigate the role of whole peanut- and peanut-component (Ara h 1, Ara h 2, Ara h 3, Ara h 6 and Ara h 8)-specific IgE levels in the diagnostic procedure of peanut allergy as well as the diagnostic properties of peanut-specific IgG and IgG4. METHODS: Sixty-one children underwent oral peanut challenge tests for diagnostic purposes irrespective of their peanut-specific IgE levels. Peanut-specific serum IgE, IgG, and IgG4 levels were determined by ImmunoCAP FEIA and specific IgE against individual peanut proteins by Immuno Solid-phase Allergen Chip. RESULTS: Thirty-four of 61 patients (56%) had a peanut allergy. No significant difference was observed for peanut-specific IgG or peanut-specific IgG4 levels between patients who were allergic and tolerant patients, whereas peanut-specific IgE was significant higher in patients who were allergic than in tolerant patients (P < .005). Twenty-five of 61 children had peanut-specific IgE above a previously proposed cutoff level of 15 kUA/L; however, 7 of these 25 children (28%) were clinically tolerant. Ara h 2-specific IgE was significantly lower in tolerant than in patients with allergies (P < .0001). Interestingly, 94% of the patients with peanut allergies showed IgE-binding to Ara h 2. Unfortunately, 26% of the sensitized but tolerant patients have shown IgE binding to Ara h 2 too. CONCLUSIONS: Neither the level of specific IgE to peanut nor to Ara h 2 was able to clearly distinguish patients with clinical relevant peanut allergy from those who were clinical tolerant in our population. As expected, peanut-specific IgG and IgG4 did not improve the diagnostic procedure.

Lipids are required for the development of Brazil nut allergy: the role of mouse and human iNKT cells.

BACKGROUND: Lipids are required for mice sensitization to Ber e 1, Brazil nut major allergen. Here, we characterized different lipid fractions extracted from Brazil nuts and the lipid-binding ability of Ber e 1. Further, we determined their in vivo ability to induce Ber-specific anaphylactic antibodies and the role of invariant natural killer T (iNKT) cells in this process. METHODS: Wild-type (WT) and iNKT cell-deficient mice were sensitized with Ber e 1 and specific lipid fractions, and anaphylactic antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). The lipid-binding characteristic of Ber e 1 (Ber) was established by using fluorescent probes and (15) N-labeled NMR. In vitro production of IL-4 was determined in Ber/lipid C-stimulated mouse iNKT cells and human T-cell lines containing NKTs primed with CD1d+C1R transfectants by flow cytometry and ELISA, respectively. RESULTS: Only one specific lipid fraction (lipid C), containing neutral and common phospholipids, induced Ber anaphylactic antibodies in mice. Ber e 1 has a lipid-binding site, and our results indicated an interaction between Ber e 1 and lipid C. iNKT-deficient mice produced lower levels of anaphylactic antibodies than WT mice. In vitro, Ber/lipid C-stimulated murine iNKT cells produced IL-4 but not IFN-gamma. Human T-cell lines derived from nut-allergic patients produced IL-4 to Ber/lipid C in a CD1d- and dose-dependent manner. CONCLUSION: Lipid fraction C from Brazil nut presents an essential adjuvant activity to Ber e 1 sensitization, and iNKT cells play a critical role in the development of Brazil nut-allergic response.

Ber e 1 protein: the versatile major allergen from Brazil nut seeds.

Due mainly to its extremely high content of sulphur amino acids, Ber e 1 protein, the major allergen from Brazil nut, has attracted much scientific and press attention. Ber e 1 was the main target protein in early biotechnology transgenic work, in early processing studies of plant storage proteins, in plant vacuolar targeting studies and as the main protein in early nutritional supplementation experiments. Ber e 1 was also one of the first food allergens to be unintentionally transferred from one plant to another and was involved in the first reported case of systemic allergic reaction caused by a food allergen transferred in semen. In this review, many of the Ber e 1 unique biotechnological and structural functions are discussed with a particular emphasis on its use as model protein for studies of intrinsic allergenicity of food proteins.