A Preliminary Study on Cross-Reactivity of Heat-Treated Quail and Hen's Egg White Proteins in Young Children.

We investigated the effects of different types of heat treatments on hen's egg white (HEw) and quail egg white (QEw) proteins and their cross-reactivity in young children. Crude extracts of raw and water-boiled HEw and QEw and commercially developed stone-baked HEw were prepared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed. Patients diagnosed with HEw allergy were enrolled, and pooled sera were tested with each extract in an enzyme-linked immunosorbent assay (ELISA)-inhibition test. A skin prick test (SPT) and oral food challenge (OFC) were also performed. The median age of 12 patients was 2.5 years. SDS-PAGE results revealed strongly stained bands for the ovomucoid of boiled HEw and QEw, while stone-baked HEw displayed remarkable changes for all protein fractions. In the ELISA-inhibition test, pre-incubation of the sera led to a profound decrease, moderate decrease, and minimal decrease in the amount of IgE binding to boiled and raw HEw, boiled and raw QEw, and stone-baked HEw proteins, respectively. SPTs and OFC demonstrated cross-reactivity values of 41.7% (5/12) and 16.7% (2/12) for boiled QEw and stone-baked HEw, respectively. We observed moderate cross-reactivity between QEw and HEw. Boiling had a limited effect on altering egg allergenicity. Commercially developed, stone-baked HEw can be an alternative food for children with HE allergy.

Effects of deglycosylation and the Maillard reaction on conformation and allergenicity of the egg ovomucoid.

Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (ß-turn, strang, and random coils) to α-helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products. PRACTICAL APPLICATION: In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products.

Component resolved diagnosis of egg yolk is an indispensable part of egg allergy.

INTRODUCTION AND OBJECTIVES: It was urgent to explain the role of egg yolk allergen sensitization to the egg allergic population and we would evaluate the diagnostic value of allergen components in whole eggs, including egg white and egg yolk. MATERIALS AND METHODS: Firstly, we collected 99 positive and 21 negative sera against egg allergy. Then we used modified enzyme linked immunosorbent assay (ELISA) to survey specific IgE (sIgE) to all-proven and single component in eggs, Ovomucoid (Gal d 1), Ovalbumin (Gal d 2), Ovotransferrin (Gal d 3), Lysozyme C (Gal d 4), Serum Albumin (Gal d 5), and YGP42(Gal d 6) in allergic and non-allergic populations. Last but not least, we studied the sIgE reactivities to egg allergen components by receiver operating characteristic (ROC) analysis. RESULTS: Among egg-allergic individuals, nearly 10% were sensitized to five of six egg allergen components, and the cross-reaction frequency between two egg yolk allergens with Gal d 1 was about 30% in the groups diagnosed with egg allergy or non-allergy. The best component-combination diagnosis in egg allergy of Gal d 1+ Gal d 6 demonstrated the largest area under curve (AUC) of 0.994. CONCLUSIONS: Our results suggested that there were individual differences in allergenicity of different egg allergen components, especially in the samples negative to egg allergy diagnosed but sensitive to egg yolk components. It was indicated that component resolved diagnosis of egg yolk improved the value for egg allergy management indispensably.

Detection of specific IgE against linear epitopes from Gal d 1 has additional value in diagnosing hen's egg allergy in adults.

BACKGROUND: Although hen's egg allergy is more prevalent in children, up to 0.6% of adults from different European countries suffer from a persistent or newly onset hen's egg allergy, making accurate diagnosis in adults necessary. However, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children. METHODS: Hen's egg allergic (n = 16) and tolerant (n = 19) adults were selected by sensitization towards recombinant components rGal d 1 and/or 3. Sensitization profiles towards egg white and yolk extract and the native components Gal d 1, 2, 3 and 4 were respectively evaluated with the ImmunoCAP or the EUROLINE system. Characterization of linear epitopes was performed with a peptide microarray containing 15mer peptides representing the entire sequence of mature Gal d 1 and 3. RESULTS: Overall, sIgE titres against hen's egg extracts and single components overlapped largely between allergic and tolerant adults. Although the median sIgE/sIgG4 ratio to Gal d 1 was increased in allergic adults, the range was comparable between both groups. Clinically relevant sensitization to Gal d 1 was confirmed by sIgE-binding to the linear epitopes aa30-41, aa39-50 or aa84-95 in 6/13 allergic adults, mainly suffering from objective symptoms. In comparison, these epitopes were recognized by 1/15 tolerant patient. Only a few linear epitopes were detected for Gal d 3, suggesting a greater importance of conformational epitopes for the recognition of Gal d 3. CONCLUSION AND CLINICAL RELEVANCE: Specific IgE-binding to linear epitopes of Gal d 1 is highly specific in identifying hen's egg allergic adults with objective symptoms.

Potential role of ovomucin and its peptides in modulation of intestinal health: A review.

Intestinal dysfunction, which may cause a series of metabolic diseases, has become a worldwide health problem. In the past few years, studies have shown that consumption of poultry eggs has the potential to prevent a variety of metabolic diseases, and increasing attention has been directed to the bioactive proteins and their peptides in poultry eggs. This review mainly focused on the biological activities of an important egg-derived protein named ovomucin. Ovomucin and its derivatives have good anti-inflammatory, antioxidant, immunity-regulating and other biological functions. These activities may affect the physical, biological and immune barriers associated with intestinal health. This paper reviewed the structure and the structure-activity relationship of ovomucin，the potential role of ovomucin and its derivatives in modulation of intestinal health are also summarized. Finally, the potential applications of ovomucin and its peptides as functional food components to prevent and assist in the pretreatment of intestinal health problems are prospected.

Diets Supplemented with 1% Egg White Induce Oral Desensitization and Immune Tolerance in an Egg White-Specific Allergic Mouse Model.

BACKGROUND: The objective of this study was to determine the required concentration of egg white (EW) in the diet to induce oral desensitization and/or immune tolerance within 4 weeks of oral immunotherapy (OIT) in an EW allergic mouse model. METHODS: Female BALB/c mice were systemically sensitized to EW by intraperitoneal injections and subsequently subjected to oral allergen gavage. Sensitized mice were provided 4 weeks of OIT by supplementing with 0 (non-OIT), 0.01, 0.1, or 1% EW in a 20% casein diet. Nonsensitized mice served as the nonallergy group. We performed oral and intraperitoneal EW challenges, assessed vascular permeability in the dorsal skin, and measured allergic biomarkers. RESULTS: The change in rectal temperature after oral challenge was not significantly different between the nonallergy and 1% EW groups, and the frequency of diarrhea in the 1% EW group was lower than that in the non-OIT group. The levels of plasma ovomucoid-specific IgE, IgA, and IgG2a in the 1% EW group at the study endpoint were significantly lower than those in the non-OIT group. IFN-γ and IL-10 secretions of spleen lymphocytes in the 1% EW group were significantly higher than those in the non-OIT group, and the percentage of CD4+Foxp3+ cells in the 1% EW group was higher than that in the non-OIT group. CONCLUSION: These results suggested that diet supplemented with 1% EW can induce oral desensitization and immune tolerance in the EW allergic mouse model.

Assessment of the Allergenic Potential of the Main Egg White Proteins in BALB/c Mice.

This work aimed to assess the contribution of the major egg white proteins, ovalbumin, ovomucoid, and lysozyme, to the induction and elicitation of allergenic responses. For this purpose, BALB/c mice were orally administered either the individual egg allergens or a mixture of the three proteins in the same proportion, to evaluate their relative allergenicity avoiding their different abundance in egg white. Cholera toxin was used as a T helper 2 (Th2)-polarizing adjuvant. Ovomucoid and lysozyme triggered the most severe anaphylaxis reactions upon oral challenge. In comparison to ovalbumin and ovomucoid, lysozyme was a more active promotor of early immunoglobulin E and immunoglobulin G1 production and stimulated stronger Th2-biased responses from both mesenteric lymph node and spleen cells. These results indicate that lysozyme is highly immunogenic and should be considered as a major allergen, whose clinical usefulness in the diagnosis, prognosis, and therapeutic approaches of egg allergy deserves further consideration.

Advances on the impact of thermal processing on structure and antigenicity of chicken ovomucoid.

BACKGROUND: Ovomucoid (OVM) is the dominant allergen found in egg white. The heat-induced changes on chicken OVM structure and antigenic properties were assessed at acidic, neutral and alkaline pH values. RESULTS: The fluorescence spectroscopy measurements indicated changes in the conformation of OVM caused by both pH and thermal treatment. The OVM molecule exhibited higher exposure of hydrophobic residues at 7.0, as indicated by the synchronous spectra, intrinsic fluorescence and quenching experiments. When heating the protein at pH 9.5, the molecular structure appeared more compact. The antigenic properties of OVM, estimated through the enzyme-linked immunosorbent assay, appeared not to be sensitive to heat at pH 7.0 and 4.5. Single molecule level investigations indicated that the secondary and tertiary structure of OVM was affected by the thermal treatment. CONCLUSIONS: Experimental results indicated over 90% reduction of the antigenicity at pH 9.5 and temperature of 100 °C. Significant changes of the linear epitopes exposure and location of the conformational epitopes were highlighted after performing heating molecular dynamics simulations of OVM from 25 °C to 100 °C. © 2017 Society of Chemical Industry.

Intake of Diet Including 1% Ovomucoid for 4 Weeks Induces Oral Desensitization in Ovomucoid-Specific Allergic Mouse Model.

We propose a new oral immunotherapy (OIT) method that includes a small amount of a food allergen in the diet. However, it is not clear whether this method will induce oral desensitization and immune tolerance. Therefore, we investigated the therapeutic effectiveness using a 1% food allergen diet in an allergic mouse model. C3H/HeJ mice were sensitized to ovomucoid (OM) in alum four times at 12-d intervals. Sensitized mice were divided into two groups: the OIT group (19% casein diet with 1% OM) and the non-treated group (20% casein diet without OM). The non-sensitized mice served as the non-allergy group. The OIT treatment was performed for 4 wk. To assess desensitization and immune tolerance, we performed oral and intraperitoneal OM challenges, assessed vascular permeability of the dorsal skin, and measured allergic biomarkers. The OIT group exhibited significantly lower oral symptom scores and vascular permeability than the non-treated group, but the two groups did not differ in intraperitoneal allergy symptom scores. Furthermore, the OIT group had significantly higher OM-specific IgA levels in their plasma than the non-treated group. However, the plasma levels of OM-specific IgE, IgG1, and IgG2a were not significantly different between the OIT and the non-treated groups. These results suggest that the proposed OIT using an OM-supplemented diet may induce desensitization, but not immune tolerance, in an OM allergic mouse model.

Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges.

BACKGROUND: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl ß-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients' sera. A pool of non-allergic patients' sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis.

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

BACKGROUND: Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. METHODS: We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. RESULTS: The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. CONCLUSIONS: Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE.

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Is the Quantification of Antigen-Specific Basophil Activation a Useful Tool for Monitoring Oral Tolerance Induction in Children With Egg Allergy?

OBJECTIVES: To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared before and after the children completed specific oral tolerance induction (SOTI) with egg. PATIENTS AND METHODS: We studied 28 egg-allergic children who completed SOTI with egg. The basophil activation test and specific IgE determinations with egg white, ovalbumin, and ovomucoid were performed in all 28 children. RESULTS: A decrease in antigen-specific activation with egg white, ovalbumin, and ovomucoid was observed only at the 2 lowest concentrations used (5 and 0.05 ng/mL). Baseline activation was higher in patients with multiple food allergies and in those who developed anaphylaxis during SOTI; this activation decreased in both groups after completion of SOTI. A significant decrease was also observed in specific IgE values for egg white, ovalbumin, and ovomucoid after tolerance induction. CONCLUSIONS: Food tolerance induction is a specific process for each food that can be mediated by immunologic changes such as a decrease in specific IgE values and in specific and spontaneous basophil activation.

Is the quantification of antigen-specific basophil activation a useful tool for monitoring oral tolerance induction in children with egg allergy?

Objectives: To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared before and after the children completed specific oral tolerance induction (SOTI) with egg. Patients and Methods: We studied 28 egg-allergic children who completed SOTI with egg. The basophil activation test and specific IgE determinations with egg white, ovalbumin, and ovomucoid were performed in all 28 children. Results: A decrease in antigen-specific activation with egg white, ovalbumin, and ovomucoid was observed only at the 2 lowest concentrations used (5 and 0.05 ng/mL). Baseline activation was higher in patients with multiple food allergies and in those who developed anaphylaxis during SOTI; this activation decreased in both groups after completion of SOTI. A significant decrease was also observed in specific IgE values for egg white, ovalbumin, and ovomucoid after tolerance induction. Conclusions: Food tolerance induction is a specific process for each food that can be mediated by immunologic changes such as a decrease in specific IgE values and in specific and spontaneous basophil activation (AU)

Objetivos: Valorar los cambios en la IgE específica y en la activación de basófilos basal, mediada por Anti-IgE y antígeno específica en niños con alergia a huevo, antes y después de finalizar el proceso de inducción oral de tolerancia. Métodos: Se estudiaron 28 niños con alergia a huevo que finalizaron una inducción oral de tolerancia con este alimento. En todos ellos se realizó test de activación de basófilos e IgE específica con clara de huevo, ovoalbúmina y ovomucoide. Resultados: Se produjo una reducción en la activación antígeno específica con clara de huevo, ovoalbúmina y ovomucoide únicamente con las dos concentraciones más bajas (5 y 0,05 ng/ml) empleadas. La activación basal era más alta en los pacientes con alergia alimentaria múltiple y en aquellos que desarrollaron anafilaxia en el proceso de inducción de tolerancia, disminuyendo esta activación en ambos grupos tras la finalización del proceso de inducción de tolerancia. Se observó igualmente una reducción significativa en los valores de IgE específica frente a clara de huevo, ovoalbúmina y ovomucoide tras finalizar la inducción de tolerancia. Conclusiones: La inducción de tolerancia con alimentos es un proceso específico para cada alimento que puede estar mediado por cambios inmunológicos como un descenso en los valores de IgE específica, así como en la activación de basófilos tanto específica como espontánea (AU)

Rush Oral Immunotherapy Does Not Reduce Allergic Response in Mice with Mild Allergy to Egg White Ovomucoid.

Oral immunotherapy (OIT) is a promising therapeutic approach for treating food allergy. Past studies have shown that OIT reduces allergic response only in severe allergy model mice. We worked to establish mild allergy model mice, and investigated whether 'rush' OIT for 10 d improved the allergic response and biomarkers in these mice. Balb/c mice were sensitized to ovomucoid (OM) in alum. The rush OIT was done for 10 d. Oral OM challenge was used to determine the impact of OIT on the allergic response. We measured allergic biomarkers, such as vascular permeability in the skin, plasma levels of total IgE, OM-specific IgE, IgG1 and IgG2a and cytokines in splenocyte culture supernatant. OIT for 10 d did not improve allergy symptoms and increased vascular permeability. Total IgE in the plasma of OIT-treated mice was significantly higher than in that of non-treated mice. OM-specific IgG1 and IgG2a plasma levels were not significantly different between OIT-treated and non-treated mice. Among the cytokine secretion of splenocyte from OIT-treated mice, IFN-γ and IL-10 were significantly lower than in non-treated mice, and IL-4 and IL-5 were significantly higher. Total TGF-ß in the OIT-treated group was not detected. The IFN-γ/IL-4 ratio of the OIT-treated group was about 1/8 that of the non-treated group. OIT for 10 d was not effective and some biomarkers showed negative responses in the mild allergy model mice. We suggest OIT should be used very carefully as this treatment carries a risk of worsening allergy symptoms for mice with mild allergy.

Molecular diagnosis of egg allergy: an update.

Hen's egg allergy affects up to 2.5% of young children and is potentially life-threatening. Several phenotypes of egg allergy have been identified, including those who tolerate extensively heated egg in bakery products. Diagnosis and monitoring for resolution often requires oral food challenges, which can result in anaphylaxis. Newer approaches, such as component-resolved diagnostics, microarray analysis and epitope mapping, are being evaluated to determine if these strategies can replace or reduce the need for oral food challenges. Studies suggest that elevated levels of ovomucoid IgE indicate an inability to tolerate extensively heated forms of egg. Egg protein-specific IgE/IgG4 ratios may be helpful in predicting tolerance. Additionally, patients with conformational epitopes to hen's egg are more likely to resolve their allergy compared with those with IgE binding to sequential epitopes. The pairing of microarray technology to epitope mapping is a potential tool to improve diagnosis. This review examines the current body of literature on these tools.

Production and immunological analysis of IgE reactive recombinant egg white allergens expressed in Escherichia coli.

IgE-mediated allergy to chicken egg affects a large number of children and adults worldwide. The current management strategy for egg allergy is strict avoidance, however this is impractical due to the presence of eggs in a range of foods and pharmaceutical products including vaccines. Strict avoidance also poses nutritional disadvantages due to high nutritional value of eggs. Allergen specific immunotherapy is being pursued as a curative treatment, in which an allergic individual is gradually exposed to the allergen to induce tolerance. Use of recombinant proteins for immunotherapy has been beneficial due to the purity of the recombinant proteins compared to natural proteins. In this study, we produced IgE reactive recombinant egg white proteins that can be used for future immunotherapy. Using E. coli as an expression system, we successfully produced recombinant versions of Gal d 1, 2 and 3, that were IgE reactive when tested against a pool of egg allergic patients' sera. The IgE reactivity indicates that these recombinant proteins are capable of eliciting an immune response, thus being potential candidates for immunotherapy. We have, for the first time, attempted to produce recombinant versions of all 4 major egg white allergens in E. coli, and successfully produced 3, with only Gal d 4 showing loss of IgE reactivity in the recombinant version. The results suggest that egg allergy in Australian populations may mainly be due to IgE reactivity to Gal d 3 and 4, while Gal d 1 shows higher IgE reactivity. This is the first report of a collective and comparative immunological analysis of all 4 egg white allergens. The significance of this study is the potential use of the IgE reactive recombinant egg white proteins in immunotherapy to treat egg allergic patients.