Toward reducing the immunogenic potential of wheat flour: identification and characterization of wheat lines missing omega-5 gliadins encoded by the 1D chromosome.

KEY MESSAGE: Eleven wheat lines that are missing genes for the 1D-encoded omega-5 gliadins will facilitate breeding efforts to reduce the immunogenic potential of wheat flour for patients susceptible to wheat allergy. Efforts to reduce the levels of allergens in wheat flour that cause wheat-dependent exercise-induced anaphylaxis are complicated by the presence of genes encoding omega-5 gliadins on both chromosomes 1B and 1D of hexaploid wheat. In this study, we screened 665 wheat germplasm samples using gene specific DNA markers for omega-5 gliadins encoded by the genes on 1D chromosome that were obtained from the reference wheat Chinese Spring. Eleven wheat lines missing the PCR product corresponding to 1D omega-5 gliadin gene sequences were identified. Two of the lines contained the 1BL·1RS translocation. Relative quantification of gene copy numbers by qPCR revealed that copy numbers of 1D omega-5 gliadins in the other nine lines were comparable to those in 1D null lines of Chinese Spring, while copy numbers of 1B omega-5 gliadins were like those of Chinese Spring. 2-D immunoblot analysis of total flour proteins from the selected lines using a specific monoclonal antibody against the N-terminal sequence of omega-5 gliadin showed no reactivity in regions of the blots containing previously identified 1D omega-5 gliadins. Interestingly, RP-UPLC analysis of the gliadin fractions of the selected lines indicated that the expression of omega-1,2 gliadins was also significantly reduced in seven of the lines, implying that 1D omega-5 gliadin and 1D omega-1,2 gliadin genes are tightly linked on the Gli-D1 loci of chromosome 1D. Wheat lines missing the omega-5 gliadins encoded by the genes on 1D chromosome should be useful in future breeding efforts to reduce the immunogenic potential of wheat flour.

Allergic reactions to hydrolysed wheat proteins: clinical aspects and molecular structures of the allergens involved.

Wheat gluten can be chemically or enzymatically hydrolysed to produce functional ingredients useful in food and cosmetics. However severe allergies to hydrolysed wheat proteins (HWP) have been described in Europe and Japan since the early 2000's. Triggering proteins and IgE epitopes were described both for French and Japanese cohorts and appeared remarkably similar leading to define a new wheat allergic entity. Deamidation induced by functionalisation generate neo-allergens responsible for this particular allergy. This article aims to review the processes leading to deamidation and the clinical features of the patients suffering from this allergy. Then the molecular determinants involved in HWP-allergy were exhaustively described and hypothesis regarding the sensitizing mechanism of HWP-allergy are discussed. Finally, current regulation and tools aiming at managing this risk associated with HWP are presented.

Reduction of Allergenic Potential in Bread Wheat RNAi Transgenic Lines Silenced for CM3, CM16 and 0.28 ATI Genes.

Although wheat is used worldwide as a staple food, it can give rise to adverse reactions, for which the triggering factors have not been identified yet. These reactions can be caused mainly by kernel proteins, both gluten and non-gluten proteins. Among these latter proteins, α-amylase/trypsin inhibitors (ATI) are involved in baker's asthma and realistically in Non Celiac Wheat Sensitivity (NCWS). In this paper, we report characterization of three transgenic lines obtained from the bread wheat cultivar Bobwhite silenced by RNAi in the three ATI genes CM3, CM16 and 0.28. We have obtained transgenic lines showing an effective decrease in the activity of target genes that, although showing a higher trypsin inhibition as a pleiotropic effect, generate a lower reaction when tested with sera of patients allergic to wheat, accounting for the important role of the three target proteins in wheat allergies. Finally, these lines show unintended differences in high molecular weight glutenin subunits (HMW-GS) accumulation, involved in technological performances, but do not show differences in terms of yield. The development of new genotypes accumulating a lower amount of proteins potentially or effectively involved in allergies to wheat and NCWS, not only offers the possibility to use them as a basis for the production of varieties with a lower impact on adverse reaction, but also to test if these proteins are actually implicated in those pathologies for which the triggering factor has not been established yet.

Relative reactivity to egg white and yolk or change upon heating as markers for baked egg tolerance.

BACKGROUND: Hen's egg food allergy is frequent in childhood and phenotypically heterogeneous. Some children can tolerate extensively heated egg. We investigated whether individual relative responses could differentiate children who tolerate baked egg. METHODS: Reactivities to raw, pasteurized or hard-boiled egg (E), egg white (EW), and egg yolk (EY) fractions were tested by skin prick test (SPT) in 54 egg-allergic children. IgE-sensitization to EW and EY was determined by ImmunoCAP and IgE-binding to EW and 8 EW proteins and to EY and 4 EY sub-fractions by ELISA. Population heterogeneity was assessed by hierarchical ascending classification upon individual variations of reactivity and links between classifications and clinical features by analyzing the contingency tables. RESULTS: All children had positive SPT to raw E and raw EW and 72% to raw EY. Heating decreased SPT-reactivity for some children, pasteurization being less effective than hard-boiling. Children were classed into three classes from relative SPT-reactivity to raw fractions, two from variations of SPT-reactivity with each thermal processing or EW/EY ratio of sensitization, and four from their sensitization pattern. Classifications according to heating were found independent of each other. SPT variations with hard-boiling, IgE-sensitization (ratio or pattern) were linked to allowance by the physicians of egg in baked products. CONCLUSIONS: Egg-allergic children were often both sensitized to EY and EW, and heterogeneous patterns of relative responses were evidenced. Irrespective of age and level of sensitization, a low EW/EY ratio or SPT getting null with hard-boiling was found in children allowed to eat baked egg.

Towards reducing the immunogenic potential of wheat flour: omega gliadins encoded by the D genome of hexaploid wheat may also harbor epitopes for the serious food allergy WDEIA.

BACKGROUND: Omega-5 gliadins are a group of highly repetitive gluten proteins in wheat flour encoded on the 1B chromosome of hexaploid wheat. These proteins are the major sensitizing allergens in a severe form of food allergy called wheat-dependent exercise-induced anaphylaxis (WDEIA). The elimination of omega-5 gliadins from wheat flour through biotechnology or breeding approaches could reduce the immunogenic potential and adverse health effects of the flour. RESULTS: A mutant line missing low-molecular weight glutenin subunits encoded at the Glu-B3 locus was selected previously from a doubled haploid population generated from two Korean wheat cultivars. Analysis of flour from the mutant line by 2-dimensional gel electrophoresis coupled with tandem mass spectrometry revealed that the omega-5 gliadins and several gamma gliadins encoded by the closely linked Gli-B1 locus were also missing as a result of a deletion of at least 5.8 Mb of chromosome 1B. Two-dimensional immunoblot analysis of flour proteins using sera from WDEIA patients showed reduced IgE reactivity in the mutant relative to the parental lines due to the absence of the major omega-5 gliadins. However, two minor proteins showed strong reactivity to patient sera in both the parental and the mutant lines and also reacted with a monoclonal antibody against omega-5 gliadin. Analysis of the two minor reactive proteins by mass spectrometry revealed that both proteins correspond to omega-5 gliadin genes encoded on chromosome 1D that were thought previously to be pseudogenes. CONCLUSIONS: While breeding approaches can be used to reduce the levels of the highly immunogenic omega-5 gliadins in wheat flour, these approaches are complicated by the genetic linkage of different classes of gluten protein genes and the finding that omega-5 gliadins may be encoded on more than one chromosome. The work illustrates the importance of detailed knowledge about the genomic regions harboring the major gluten protein genes in individual wheat cultivars for future efforts aimed at reducing the immunogenic potential of wheat flour.

Meta-analysis of IgE-binding allergen epitopes.

IgE-binding epitopes are related to allergic symptoms by eliciting degranulation of special cells and release of molecules that trigger the hypersensitivity reaction. Little is known about what characterises allergen IgE-binding epitopes, although advances in analytical methods have led to the identification of a large number of them. To assess if a binary classification of allergen regions into epitopes or non-epitopes may accurately reflect biological reality, we computed the fraction of allergen amino acids that are involved in epitopes. A relationship between this fraction and the increasing number of literature references was modelled. Due to the wide variety of methods that are used in the literature, a peak in the number of matches between an allergen sequence and its epitopes confirms their validity. Accordingly, our graphical representation of positive assays along sequences provides an overview of epitope localisation, which should help to highlight major positions for IgE binding to allergens.

Tolerance to heated egg in egg allergy: Explanations and implications for prevention and treatment.

Hen's egg allergy is the second most frequent food allergy found in children. Allergic symptoms can be caused by raw or heated egg, but a majority of egg-allergic children can tolerate hard-boiled or baked egg. Understanding the reasons for the tolerance towards heated egg provides clues about the molecular mechanisms involved in egg allergy, and the differential allergenicity of heated and baked egg might be exploited to prevent or treat egg allergy. In this review, we therefore discuss (i) why some patients are able to tolerate heated egg; by highlighting the structural changes of egg white (EW) proteins upon heating and their impact on immunoreactivity, as well as patient characteristics, and (ii) to what extent heated or baked EW might be useful for primary prevention strategies or oral immunotherapy. We describe that the level of immunoreactivity towards EW helps to discriminate patients tolerant or reactive to heated or baked egg. Furthermore, the use of heated or baked egg seems effective in primary prevention strategies and might limit adverse reactions. Oral immunotherapy is a promising treatment strategy, but it can sometimes cause significant adverse events. The use of heated or baked egg might limit these, but current literature is insufficient to conclude about its efficacy.

Hemp seed: An allergen source with potential cross-reactivity to hazelnut.

The increasing exposure of the population to Cannabis sativa has revealed allergies to different parts of the plant, among which hemp seed. Nonetheless, the major hemp seed allergens remain to be identified. Several known families of allergens are present in hemp seed, including notably seed storage proteins. We therefore aimed to investigate the potential allergenicity of the hemp seed storage proteins and their potential cross-reactivity to different seeds and nuts. For this, we extracted hemp seed proteins sequentially using buffers with increasing levels of salinity (H2O, T2 and T3) to yield extracts differentially enriched in storage proteins. We used these extracts to perform immunoblots and ELISAs using sera of patients either sensitized to hemp seeds or sensitized/allergic to other seeds and nuts. Immunoblots and proteomics analyses identified vicilins and edestins as potential hemp seed allergens. Moreover, ELISA analyses revealed a correlation between sensitization to hazelnut and the hemp seed T3 extract (enriched in storage proteins). The possible cross-reactivity between hazelnut and hemp seed proteins was further strengthened by the results from inhibition ELISAs: the incubation of sera from hazelnut-sensitized individuals with increasing concentrations of the T3 extract inhibited serum IgE binding to the hazelnut extract by about 25-30%. Our study thus identifies vicilins and edestins as potential hemp seed allergens and highlights a possible cross-reactivity with hazelnut. The clinical relevance of this cross-reactivity between hemp seed and hazelnut needs to be further investigated in hazelnut-allergic individuals.

Fermentation of Gluten by Lactococcus lactis LLGKC18 Reduces its Antigenicity and Allergenicity.

Wheat is a worldwide staple food, yet some people suffer from strong immunological reactions after ingesting wheat-based products. Lactic acid bacteria (LAB) constitute a promising approach to reduce wheat allergenicity because of their proteolytic system. In this study, 172 LAB strains were screened for their proteolytic activity on gluten proteins and α-amylase inhibitors (ATIs) by SDS-PAGE and RP-HPLC. Gliadins, glutenins, and ATI antigenicity and allergenicity were assessed by Western blot/Dot blot and by degranulation assay using RBL-SX38 cells. The screening resulted in selecting 9 high gluten proteolytic strains belonging to two species: Enterococcus faecalis and Lactococcus lactis. Proteomic analysis showed that one of selected strains, Lc. lactis LLGKC18, caused degradation of the main gluten allergenic proteins. A significant decrease of the gliadins, glutenins, and ATI antigenicity was observed after fermentation of gluten by Lc. lactis LLGKC18, regardless the antibody used in the tests. Also, the allergenicity as measured by the RBL-SX38 cell degranulation test was significantly reduced. These results indicate that Lc. lactis LLGKC18 gluten fermentation can be deeply explored for its capability to hydrolyze the epitopes responsible for wheat allergy.

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis.

Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient's sera and with mouse monoclonal antibodies. Disruption of the C28-C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13-C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

Comparative analysis of eliciting capacity of raw and roasted peanuts: the role of gastrointestinal digestion.

This study investigated the simultaneous impact of food matrix and processing on the food allergy eliciting capacity of peanuts in a physiologically relevant context. Whole raw and roasted peanuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. SDS-PAGE and HPLC analysis showed that roasting increased digestibility of peanuts and this trend was even more evident after BBM degradation. The eliciting properties of raw and roasted peanuts were assessed by Rat Basophil Leukemia assay in the presence of sera from peanut-allergic patients. As general features, the BBM digestion reduced allergenicity of roasted peanuts compared to the raw counterpart, suggesting that intestinal peptidases effectively contribute to further destroy specific domains of peanut allergens. These findings provide new and more realistic insights in the stability of peanut allergens within their natural matrix.

Deamidation and Enzymatic Hydrolysis of Gliadins Alter Their Processing by Dendritic Cells in Vitro.

Gliadins are major wheat allergens. Their treatment by acid or enzymatic hydrolysis has been shown to modify their allergenic potential. As the interaction of food proteins with dendritic cells (DCs) is a key event in allergic sensitization, we wished to investigate whether deamidation and enzymatic hydrolysis influence gliadin processing by DC and to examine the capacity of gliadins to activate DCs. We compared the uptake and degradation of native and modified gliadins by DCs using mouse bone marrow-derived DCs. We also analyzed the effects of these interactions on the phenotypes of DCs and T helper (Th) lymphocytes. Modifying gliadins induced a change in physicochemical properties (molecular weight, hydrophobicity, and sequence) and also in the peptide size. These alterations in turn led to increased uptake and intracellular degradation of the proteins by DCs. Native gliadins (NGs) (100 µg/mL), but not modified gliadins, increased the frequency of DC expressing CD80 (15.41 ± 2.36% vs 6.81 ± 1.10%, p < 0.001), CCR7 (28.53 ± 8.17% vs 17.88 ± 2.53%, p < 0.001), CXCR4 (70.14 ± 4.63% vs 42.82 ± 1.96%, p < 0.001), and CCR7-dependent migration (2.46 ± 1.45 vs 1.00 ± 0.22, p < 0.01) compared with NGs. This was accompanied by Th lymphocyte activation (30.37 ± 3.87% vs 21.53 ± 3.14%, p < 0.1) and proliferation (16.39 ± 3.97% vs 9.31 ± 2.80%, p > 0.1). Moreover, hydrolysis decreases the peptide size and induces an increase in gliadin uptake and degradation. Deamidation and extensive enzymatic hydrolysis of gliadins modify their interaction with DCs, leading to alteration of their immunostimulatory capacity. These findings demonstrate the strong relationship between the biochemical characteristics of proteins and immune cell interactions.

Allergenicity of Fermented Foods: Emphasis on Seeds Protein-Based Products.

Food allergy is an IgE-mediated abnormal response to otherwise harmless food proteins, affecting between 5% and 10% of the world preschool children population and 1% to 5% adults. Several physical, chemical, and biotechnological approaches have been used to reduce the allergenicity of food allergens. Fermentation processes that contribute to technological and desirable changes in taste, flavor, digestibility, and texture of food products constitute one of these approaches. Lactic acid bacteria (LAB), used as starter cultures in dairy products, are a subject of increasing interest in fermentation of plant proteins. However, the studies designed to assess the impact of LAB on reduction of allergenicity of seed proteins are at an early stage. This review presents the current knowledge on food fermentation, with a focus on seed proteins that are increasingly used as ingredients, and its impacts on food potential allergenicity.

Digestion differently affects the ability of native and thermally aggregated ovalbumin to trigger basophil activation.

Ovalbumin (OVA), a major allergen from hen's egg albumen, tends to aggregate when heated. Depending on the balance of attractive and repulsive interactions, heat-induced OVA aggregates have various morphologies, which differ in digestibility. In the context of food allergy to egg, we investigated the ability of native and thermally aggregated OVA as well as their digests to induce the degranulation of a humanized rat basophil leukemia (RBL) cell line, which was sensitized with a pool of sera from egg-allergic children. Native and two thermally aggregated OVA forms were digested in vitro using a gastrointestinal digestion model based on the INFOGEST harmonized protocol including a final degradation with jejunal brush border membranes (BBM) enzymes. The course of digestion was monitored by the OPA method and by RP-HPLC. Digestibility was OVA small aggregates>OVA large aggregates>>native OVA and BBM peptidases only significantly hydrolyzed small-sized peptides from gastro-duodenal digests of the aggregates. The degranulation ability of the native OVA slightly changed during the gastric phase but mostly decreased during the duodenal digestion with no further change with BBM digestion. The degranulation ability of aggregates, which was significantly lower than the ability of native OVA, was not significantly affected by digestion. Digestibility and ability to induce basophil degranulation can thus not be straightforward linked.

Wheat ATI CM3, CM16 and 0.28 Allergens Produced in Pichia Pastoris Display a Different Eliciting Potential in Food Allergy to Wheat ‡.

Although wheat is a staple food for most of the human population, some of its components trigger adverse reactions. Among wheat components, the alpha-amylase/trypsin inhibitors (ATI) are important triggers of several allergies and activators of innate immunity. ATI are a group of exogenous protease inhibitors and include several polypeptides. The three ATI polypeptides named CM3, CM16 and 0.28 are considered major allergens, and might also play a role in other common wheat-related pathologies, such as Non Celiac Wheat Sensitivity and even Celiac Disease. On this basis, we pointed to obtain high amounts of them in purity and to evaluate their allergenicity potential. We thus isolated the mRNA corresponding to the three ATI genes CM3, CM16 and 0.28 from 28 days post-anthesis wheat kernels and the corresponding cDNAs were used for heterologous expression in Pichia pastoris. The three purified proteins were tested in degranulation assay against human sera of patients with food allergy to wheat. A large range of degranulation values was observed for each protein according to the sera tested. All of the three purified proteins CM3, CM16 and 0.28 were active as allergens because they were able to induce basophils degranulation on wheat allergic patients' sera, with the highest values of ß-hexosaminidase release observed for CM3 protein.

Intestinal translocation capabilities of wheat allergens using the Caco-2 cell line.

Because intestinal absorption of food protein can trigger an allergic reaction, the effect of wheat proteins on intestinal epithelial cell permeability was evaluated and the abilities of these proteins in native or pepsin-hydrolyzed state to cross the epithelial cell monolayer were compared. Enterocytic monolayers were established by culturing Caco-2 cells, a model of enterocytes, on permeable supports that separate the apical and basal compartments. Proteins were added into the apical compartment, and the transepithelial resistance (TER) was measured; proteins that crossed the cell monolayer were detected in the basal medium by ELISA. Wheat proteins did not alter the cell monolayer. TER and Caco-2 cell viability were conserved, and the passage of dextran was prevented. Native and pepsin-hydrolyzed forms of omega5-gliadin and lipid transfer proteins were detected in the basal medium. The results suggest that these two major allergens in food allergy to wheat were able to cross the cell monolayer by the transcellular route.

Influence of the allelic variants encoded at the Gli-B1 locus, responsible for a major allergen of wheat, on IgE reactivity for patients suffering from food allergy to wheat.

Wheat presents an important genetic diversity that could be useful to look for cultivars with reduced allergencity. omega5-Gliadins have been described as major allergens for wheat allergic patients suffering from wheat-dependent exercise-induced anaphylaxis (WDEIA) and some cases of chronic urticaria (U). Our objective was to study the influence of genetic variability at the Gli-B1 locus encoding for omega5-gliadins on the reactivity of IgE antibodies from these patients. We selected cultivars expressing 13 alleles at Gli-B1 including a wheat/rye translocation and studied the reactivity to gliadins of a rabbit antiserum specific for omega5-gliadins and of IgE from 10 patients. The antiserum and IgE from nine patients with WDEIA and U strongly detected omega5-gliadins expressed by most of the Gli-B1 alleles but showed no or faint responses to the gliadins and secalins extracted from the translocated wheat. The selection of genotypes lacking the Gli-B1 locus may reduce wheat allergenicity.

How Proteins Aggregate Can Reduce Allergenicity: Comparison of Ovalbumins Heated under Opposite Electrostatic Conditions.

Heated foods are recommended for avoiding sensitization to food proteins, but depending on the physicochemical conditions during heating, more or less unfolded proteins aggregate differently. Whether the aggregation process could modulate allergenicity was investigated. Heating ovalbumin in opposite electrostatic conditions led to small (A-s, about 50 nm) and large (A-L, about 65 µm) aggregates that were used to sensitize mice. The symptoms upon oral challenge and rat basophil leukemia degranulation with native ovalbumin differed on the basis of which aggregates were used during the sensitization. Immunoglobulin-E (IgE) production was significantly lower with A-s than with A-L. Although two common linear IgE-epitopes were found, the aggregates bound and cross-linked IgE similarly or differently, depending on the sensitizing aggregate. The ovalbumin aggregates thus displayed a lower allergenic potential when formed under repulsive rather than nonrepulsive electrostatic conditions. This further demonstrates that food structure modulates the immune response during the sensitization phase with some effects on the elicitation phase of an allergic reaction and argues for the need to characterize the aggregation state of allergens.