Safety of Oral Food Challenge for Individuals with Low Egg White and Ovomucoid-Specific IgE Antibodies.

INTRODUCTION: During an oral food challenge (OFC), there is a risk of adverse reactions, including anaphylaxis. Therefore, the physician should carefully conduct the OFC. This study aimed to evaluate the OFC results in individuals with low levels of egg white (EW)- and ovomucoid (OVM)-specific immunoglobulin E (sIgE) and the safety of a hen's egg (HE) OFC in these individuals. METHODS: A total of 2,058 individuals with low EW- or OVM-sIgE underwent HE-OFC at two institutions in Kumamoto prefecture, located in the western area of Japan, between January 1, 2017, and December 31, 2021, within 1 year of recorded sIgE measurements. The ImmunoCAP systems were used to measure sIgEs. The HE-OFC test was performed according to the 2017 Food Allergy Guidelines in an open and unblinded method. RESULTS: Five hundred and one individuals (24.3%) had low EW-sIgE levels (class 2 or lower), and 926 (45.0%) had low OVM-sIgE levels (class 2 or lower). Individuals with low EW-sIgE had lower total IgE and OVM-sIgE than did those with high EW-sIgE (greater than class 2). Those with low OVM-sIgE had lower total IgE and EW-sIgE than did those with high OVM-sIgE (greater than class 2). Among the individuals with low EW-sIgE, 86.4% (433/501 cases) passed the OFC without symptoms. Among the individuals with low OVF-sIgE, 82.6% (765/926 cases) passed the OFC without symptoms. CONCLUSION: More than 80% of individuals with suspected IgE-dependent HE allergy and low levels of EW- or OVM-specific IgE were able to consume at least a small amount of HE. As the OFC results are independent of the loading dose in cases with low EW- or OVM-sIgE, a medium-dose HE-OFC may be performed safely in individuals with no history of anaphylaxis.

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.

Production and characterization of eggs from hens with ovomucoid gene mutation.

Ovomucoid is a major egg white protein which is considered as the most dominant allergen in chicken eggs. Owing to the difficulty of separating ovomucoid from egg whites, researchers have adopted genetic deletion for development of hypoallergenic eggs. Previously, we used CRISPR/Cas9 to establish chickens with ovomucoid gene (OVM) mutations, but it remained unknown whether such hens could produce eggs at maturity. Here, we have reported on eggs laid by OVM-targeted hens. Except for watery egg whites, the eggs had no evident abnormalities. Real-time PCR revealed alternative splicing of OVM mRNA in hens, but their expression was limited. Immunoblotting detected neither mature ovomucoid nor ovomucoid-truncated splicing variants in egg whites. Sixteen chicks hatched from 28 fertilized eggs laid by OVM-targeted hens, and fourteen of the sixteen chicks demonstrated healthy growth. Taken together, our results demonstrated that OVM knockout could almost completely eliminate ovomucoid from eggs, without abolishing fertility. Thus, the eggs developed in this study have potential as a hypoallergenic food source for most patients with egg allergies.

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.

Dietary baked egg accelerates resolution of egg allergy in children.

BACKGROUND: Baked egg is tolerated by a majority of egg-allergic children. OBJECTIVE: To characterize immunologic changes associated with ingestion of baked egg and evaluate the role that baked egg diets play in the development of tolerance to regular egg. METHODS: Egg-allergic subjects who tolerated baked egg challenge incorporated baked egg into their diet. Immunologic parameters were measured at follow-up visits. A comparison group strictly avoiding egg was used to evaluate the natural history of the development of tolerance. RESULTS: Of the 79 subjects in the intent-to-treat group followed for a median of 37.8 months, 89% now tolerate baked egg and 53% now tolerate regular egg. Of 23 initially baked egg-reactive subjects, 14 (61%) subsequently tolerated baked egg and 6 (26%) now tolerate regular egg. Within the initially baked egg-reactive group, subjects with persistent reactivity to baked egg had higher median baseline egg white (EW)-specific IgE levels (13.5 kU(A)/L) than those who subsequently tolerated baked egg (4.4 kU(A)/L; P= .04) and regular egg (3.1 kU(A)/L; P= .05). In subjects ingesting baked egg, EW-induced skin prick test wheal diameter and EW-, ovalbumin-, and ovomucoid-specific IgE levels decreased significantly, while ovalbumin- and ovomucoid-specific IgG(4) levels increased significantly. Subjects in the per-protocol group were 14.6 times more likely than subjects in the comparison group (P< .0001) to develop regular egg tolerance, and they developed tolerance earlier (median 50.0 vs 78.7 months; P< .0001). CONCLUSION: Initiation of a baked egg diet accelerates the development of regular egg tolerance compared with strict avoidance. Higher serum EW-specific IgE level is associated with persistent baked and regular egg reactivity, while initial baked egg reactivity is not.

Oral immunotherapy induces local protective mechanisms in the gastrointestinal mucosa.

BACKGROUND: Oral immunotherapy (OIT) is a promising treatment for food allergy. Studies are needed to elucidate mechanisms of clinical protection and to identify safer and potentially more efficacious methods for desensitizing patients to food allergens. OBJECTIVE: We established a mouse model of OIT to determine how the dose or form of antigen may affect desensitization and to identify mechanisms of desensitization. METHODS: Increasing doses of egg white or ovomucoid as OIT were administered orally to sensitized mice. The impact of OIT on anaphylaxis elicited by oral allergen challenge was determined. Allergen-specific antibody and cytokine responses and mast cell and basophil activation in response to OIT were measured. Gene expression in the small intestine was studied by microarray and real-time PCR. RESULTS: OIT resulted in desensitization but not tolerance of mice to the allergen. OIT did not result in desensitization of systemic effector cells, and protection was localized to the gastrointestinal tract. OIT was associated with significant changes in gene expression in the jejunum, including genes expressed by intestinal epithelial cells. Extensively heated ovomucoid that does not trigger anaphylaxis when given orally to sensitized mice was as efficacious as native ovomucoid in desensitizing mice. CONCLUSIONS: OIT results in clinical protection against food-induced anaphylaxis through a novel mechanism that is localized to the intestinal mucosa and is associated with significant changes in small intestinal gene expression. Extensively heating egg allergen decreases allergenicity and increases safety while still retaining the ability to induce effective desensitization.

Significance of ovomucoid- and ovalbumin-specific IgE/IgG(4) ratios in egg allergy.

BACKGROUND: The role of specific IgG(4) antibodies in natural tolerance acquisition remains a matter of debate; the specific IgE/IgG(4) ratio might add value to the measurement of absolute amounts of IgE for assessing the ongoing status of egg reactivity. OBJECTIVE: We sought to determine the significance of IgG(4) antibodies to ovalbumin (OVA) and ovomucoid (OVM) in egg-allergic children. METHODS: One hundred seven egg-allergic children (mean age 6.9 years; range 1.6-18.6 years) were challenged to baked egg. The outcomes of the challenges were related to the level of specific IgE and IgG(4) to OVM and OVA, component IgE/IgG(4) ratios, and mediator release in a functional assay based on the rat basophil leukemia cell line. RESULTS: Baked egg-reactive children had significantly higher OVA and OVM ratios of IgE/IgG(4) and mediator release in the rat basophil leukemia-based assay than did tolerant children (P < .05 for both). The OVA- and OVM-specific IgE/IgG(4) ratios and mediator release were correlated. In the receiver operating characteristic analysis, the areas under the curve for a logistic regression model including specific IgE and IgG(4) to OVA and OVM were significantly greater compared with the areas under the curve for egg white-specific IgE and OVM-specific IgE. CONCLUSIONS: The balance between IgE and IgG(4) to OVA and OVM has functional consequences. A model that includes the interactions between IgE and IgG(4) to OVA and OVM accurately predicts reactivity to baked egg and warrants further investigation.

Recognition and uptake of free and nanoparticle-bound betalactoglobulin--a food allergen--by human monocytes.

SCOPE: To improve our understanding of the interaction of food allergens with cells of the immune system, the endocytosis by human monocytes of bovine ß-lactoglobulin (BLG) and ovomucoid (OM)--two major food allergens--and human serum albumin (HSA) was studied. METHODS AND RESULTS: BLG was covalently conjugated to dextran-coated magnetic nanoparticles (MNPs) without affecting its structure and immunoreactivity. BLG-conjugated MNPs were taken up by human monocytes much more efficiently than non-conjugated MNPs, allowing easy magnetic separation of cells that had adsorbed the allergen. BLG, OM, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG-conjugated MNPs versus non-conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG-MNP conjugates into the cytoplasm. CONCLUSIONS: These results contribute to the current understanding of the interaction between food allergens and antigen-presenting cells, and demonstrate that the BLG is readily endocytosed by monocytes both as the single protein and as a conjugate.

Mechanisms underlying differential food allergy response to heated egg.

BACKGROUND: Egg white proteins are usually subjected to heating, making them edible for the majority of children with egg allergy. OBJECTIVE: We sought to investigate the underlying mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergens. METHODS: C3H/HeJ mice were orally sensitized with ovalbumin (OVA) or ovomucoid and challenged with native or heated proteins to evaluate their allergenicity. Immunoreactivity was assessed by immunoblotting using sera from children with egg allergy. In vitro gastrointestinal digestion of native and heated OVA and ovomucoid was studied by SDS-PAGE and liquid chromatography. Intestinal uptake of intact native and heated OVA and ovomucoid by human intestinal epithelial (Caco-2) cells was investigated. Rat basophil leukemia cells passively sensitized with mouse serum and human basophils passively sensitized with serum from children with egg allergy were used to assess the effector cell activation by heated, digested, and transported OVA and ovomucoid. RESULTS: Heated OVA and ovomucoid did not induce symptoms of anaphylaxis in sensitized mice when administered orally. Heating did not completely destroy IgE-binding capacity of OVA or ovomucoid but enhanced in vitro digestibility of OVA. Digestion of both OVA and ovomucoid diminished mediator release in rat basophil leukemia assay and basophil activation. Heating of allergens prevented transport across human intestinal epithelial cells in a form capable of triggering basophil activation or T-cell activation. CONCLUSION: Heat treatment reduces allergenicity of OVA and ovomucoid. This is partially a result of the enhanced gastrointestinal digestibility of heated OVA and the inability of heated OVA or ovomucoid to be absorbed in a form capable of triggering basophils.

Prophylaxis of experimentally induced ovomucoid allergy in neonatal pigs using Lactococcus lactis.

Probiotic Lactococcus lactis (LL) is immunomodulatory and may prevent allergy by biasing from type-2 to a type-1 immune response. We hypothesized that newborn pigs pre-treated orally with LL are protected against allergy to ovomucoid (Ovm). Pigs were assigned to two treatment groups. Piglets were pretreated orally on days of age 1-7, 10, 12, 14, 21, 28 and 35 with LL (n=30) or medium (control, n=32) and sensitized to Ovm by intraperitoneal injection together with cholera toxin on days 14, 21 and 35. Pigs were orally challenged with egg white (day 46) and assigned scores for allergic signs. Outcomes were measured as direct skin tests, serum antibody to Ovm [IgG (H+L); IgE; IgG(1) and IgG(2)] and cytokine production by mitogen-stimulated blood mononuclear cells (BMC). Clinical signs and skin test positivity were less frequent in the LL group (p ≤ 0.0001). Serum antibody associated with IgG (H and L), IgE, IgG(1) or IgG(2) was significantly increased on day 46 (post-sensitization) compared to day 14 (pre-sensitization) (p ≤ 0.0001). The LL-treated pigs had more IgE and IgG(2)-related antibody activity and lower IgG(1)/IgG(2) and IgE/IgG(2) ratios indicating a type-1 bias in immune response (p ≤ 0.05). Concentration of type-2 cytokines interleukin IL-4 and IL-10 were significantly lower in supernatants of stimulated BMC of LL-treated pigs (p ≤ 0.0001). Interferon-γ, TGF-ß and IL-13 were not detected in control or treated animals. Thus, oral treatment of neonatal pigs with LL significantly reduced subsequent frequency of allergy to Ovm associated with reduced type-2 immune response correlates hence supporting the "hygiene hypothesis" and potential use of LL as a neonatal immunoregulator.

Immunologic changes in children with egg allergy ingesting extensively heated egg.

BACKGROUND: Prior studies have suggested that heated egg might be tolerated by some children with egg allergy. OBJECTIVE: We sought to confirm tolerance of heated egg in a subset of children with egg allergy, to evaluate clinical and immunologic predictors of heated egg tolerance, to characterize immunologic changes associated with continued ingestion of heated egg, and to determine whether a diet incorporating heated egg is well tolerated. METHODS: Subjects with documented IgE-mediated egg allergy underwent physician-supervised oral food challenges to extensively heated egg (in the form of a muffin and a waffle), with tolerant subjects also undergoing regular egg challenges (in a form of scrambled egg or French toast). Heated egg-tolerant subjects incorporated heated egg into their diets. Skin prick test wheal diameters and egg white, ovalbumin, and ovomucoid IgE levels, as well as ovalbumin and ovomucoid IgG4 levels, were measured at baseline for all subjects and at 3, 6, and 12 months for those tolerant of heated egg. RESULTS: Sixty-four of 117 subjects tolerated heated egg, 23 tolerated regular egg, and 27 reacted to heated egg. Heated egg-reactive subjects had larger skin test wheals and greater egg white-specific, ovalbumin-specific, and ovomucoid-specific IgE levels compared with heated egg- and egg-tolerant subjects. Continued ingestion of heated egg was associated with decreased skin test wheal diameters and ovalbumin-specific IgE levels and increased ovalbumin-specific and ovomucoid-specific IgG4 levels. CONCLUSIONS: The majority of subjects with egg allergy were tolerant of heated egg. Continued ingestion of heated egg was well tolerated and associated with immunologic changes that paralleled the changes observed with the development of clinical tolerance to regular egg.

A neonatal swine model of allergy induced by the major food allergen chicken ovomucoid (Gal d 1).

BACKGROUND: Food allergy is a serious health problem for which a validated outbred large animal model would be useful in comparative investigations of immunopathogenesis and treatment and in testing hypotheses relevant to complex host-environmental interactions in predisposition to and expression of food allergy. OBJECTIVE: To establish a neonatal swine model of IgE-mediated allergy to the egg protein ovomucoid (Ovm) that may mimic human allergy. METHODS: In order to induce Ovm sensitivity, piglets at days 14, 21 and 35 of age were sensitized by intraperitoneal injection of 100 microg of crude Ovm and cholera toxin (50, 25 or 10 microg). Controls received 50 microg of cholera toxin in phosphate-buffered saline. The animals were challenged orally on day 46 with a mixture of egg white and yoghurt. Outcomes were reported as direct skin tests, clinical signs, IgG-related antibody and passive cutaneous anaphylaxis. RESULTS: Sensitized pigs developed immediate wheal and flare reactions, and after oral challenge, sensitized but not control animals displayed signs of allergic hypersensitivity. Serum IgG-related, Ovm-specific antibodies were detected only in the sensitized pigs and IgE-mediated antibody response to Ovm was confirmed by positive passive cutaneous anaphylaxis reactions induced by sera of sensitized but not by heat-treated sera from Ovm-sensitized pigs or sera of unsensitized control pigs. CONCLUSION: The present results confirm induction of Ovm-specific allergy in pigs and provide opportunity to investigate allergic predisposition and immunopathogenesis of IgE-induced Ovm allergy using outbred neonatal swine. This may better simulate allergic disease in humans and allow investigation of candidate prophylactic and therapeutic approaches.

Maintenance of tolerance to cow's milk in atopic individuals is characterized by high levels of specific immunoglobulin G4.

BACKGROUND: The central role of specific IgE in cow's milk allergy (CMA) is well documented. However, less is known about the function of other immunoglobulin isotypes in allergy and tolerance to cow's milk proteins (CMPs). OBJECTIVE: To determine differences in the antibody responses that are associated with allergy and tolerance to cow's milk in allergic, atopic and non-atopic individuals of different age groups. METHODS: Nineteen infants (<1 year), 18 children (6-14 years) and 41 adults (21-68 years) were included. Each age group was comprised of subjects with CMA, atopic individuals without a history of CMA and non-atopic subjects. Levels of specific IgE, IgG4, IgG1 and IgA to whole cow's milk and the six most abundant individual CMPs were determined in plasma by ELISA. For comparison, specific IgE and IgG4 were measured to ovomucoid and house dust mite (HDM) in individuals allergic for the respective allergens, and in atopic and non-atopic subjects without allergy. RESULTS: In infants and children with CMA, alphas1-casein and beta-lactoglobulin induced the highest specific IgE response, whereas alphas1-casein was the most allergenic CMP in adult patients. Specific IgG4 and IgG1 responses were the highest to alphas1-casein and beta-lactoglobulin in all age groups, while kappa-casein and alpha-lactalbumin induced the highest levels of IgA. CMP-specific IgG4 was higher in atopic children and adults without CMA, as compared with non-atopic individuals. A similar difference between tolerant atopic and non-atopic subjects was observed for IgG4 specific to ovomucoid, whereas HDM-specific IgG4 was not detectable in these subjects. CONCLUSION: Maintenance of tolerance to cow's milk in atopic children and adults without CMA is associated with elevated levels of specific IgG4, in combination with low specific IgE. The up-regulation of specific IgG4 in tolerant atopic individuals may be related to the type of allergen and its regular dose of exposure.

The correlation between ovomucoid-derived peptides, human leucocyte antigen class II molecules and T cell receptor-complementarity determining region 3 compositions in patients with egg-white allergy.

BACKGROUND: Food allergies are more prevalent in children, due to the immature gastrointestinal epithelial membrane barrier allowing more proteins through the barrier and into circulation. Ovomucoid (OM) is one of the major allergens that is found in egg white. OBJECTIVE: The aim of this study was to determine T cell epitopes, antigen-presenting human leucocyte antigen (HLA) class II molecules of the T cell lines (TCLs) and T cell clones (TCCs), and complementarity determining region (CDR) 3 loops of the T cell receptor (TCR) alpha and beta chains of the TCCs specific to OM. METHODS: We established TCLs and TCCs specific to OM from peripheral blood mononuclear cells (PBMCs) of four atopic patients with egg-white allergy using a mixture of a panel of overlapping synthetic peptides corresponding to the amino acid sequence of the entire OM. We identified the T cell epitopes by antigen-induced proliferative responses, antigen-presenting molecules using allogeneic PBMCs and CDR3 loops of the TCR alpha and beta chains by cloning and sequence analysis. RESULTS: The TCLs and TCCs responded to seven different peptides, and their antigen-presenting molecules were different from each other. Sequence analysis of the TCR alpha and beta gene usage of the TCCs showed marked heterogeneity, and the usage of the CDR3 loop of the TCCs involved heterogenous amino acid residues. Interestingly, TCCs 'IH3.3' and 'YT6.1' recognized the same OM peptides, and had the same TCR Vbeta-Jbeta gene usage. Considering that peptide motifs bind to HLA class II molecules, the electrically charged residue (positive or negative) on the CDR3alpha and the CDR3beta loops of TCR of TCC may form ionic bonds with a charged residue on the HLA class II molecules-peptide complex. CONCLUSIONS: TCCs that have the same TCR gene usage were established from patients who had shown similar hypersensitivity-type, indicating that antigen recognition by a specific TCR is closely associated with the characteristics of each patient's symptoms.

Allergenic activity of heated and ovomucoid-depleted egg white.

BACKGROUND: No egg white products have been clearly proven to be hypoallergenic. The role of egg white proteins in allergic reactions to eggs is still debatable. OBJECTIVE: This study was designed to determine the importance of ovomucoid, an egg white protein, in the development of allergies to egg white. METHODS: We performed a double-blind, placebo-controlled food challenge in subjects with high levels of IgE antibodies for egg white to compare the allergenicities of heated and ovomucoid-depleted egg white, freeze-dried egg white, and heated egg white. Levels of IgE antibodies for egg white, ovomucoid, ovalbumin, ovotransferrin, and lysozyme were measured in serum by RAST. RESULTS: Twenty-one of 38 subjects with positive challenge responses to freeze-dried egg white had negative challenge responses to heated egg white, whereas 16 of 17 subjects (94.1%) with positive responses to heated egg white did not respond to the heated and ovomucoid-depleted egg white challenge. The subjects with positive challenge responses to freeze-dried egg white tended to have higher IgE antibody values to ovomucoid than those with negative responses. IgE antibody levels to ovomucoid were significantly higher in subjects with positive responses to a challenge with heated egg white than in those with no response. There were no significant differences in the levels of IgE antibodies to the other proteins, except ovomucoid, in the negative-response and positive-response groups in challenge tests with freeze-dried and heated egg white. CONCLUSION: The heated and ovomucoid-depleted egg white preparation was less allergenic than heated or freeze-dried preparations. Ovomucoid has a more important role in the pathogenesis of allergic reactions to egg white than other proteins in egg white.