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

Abstract 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.


| INTRODUC TI ON
Hen's egg is known as a major cause of food allergic reactions in children, and this allergy is often outgrown by the age of five.
Nevertheless, the prevalence of hen's egg allergy in adults average 0.02 to 0.6% across European countries. 1,2 Although hen's egg allergy in adulthood is predominantly a persistent allergy developed in childhood, it can also be newly developed later in life. 3 In a study conducted in the United States, 29% of all hen's egg allergic adults suffered from an adult-onset allergy. 4 Diagnosis of hen's egg allergy is comprised of anamnesis, skin prick test, measuring sIgE and food challenges. Food challenges are the gold standard, but they are burdensome, expensive and require dedicated hospital facilities and personnel. To avoid or replace food challenges, intensive research has been performed to improve the diagnostic value of sIgE measurements in hen's egg allergic children. In a systematic review, an evaluation of sIgE measurements towards egg white extract in children ranging from infants to adolescents showed an overall sensitivity of 0.93, but only a specificity of 0.49. 5 Component-resolved diagnostics improved the accuracy of sIgE measurement in several food allergies. 6 Major allergenic components of hen's egg white, which is responsible for most of its allergenicity, are ovomucoid (Gal d 1), a thermo-stable allergen, ovalbumin (Gal d 2), ovotransferrin (Gal d 3) and lysozyme (Gal d 4).
Ovomucoid, the major allergen of egg white, is by far the most studied component in relation to hen's egg allergy in children and although ovomucoid is classified as a prognostic marker for persistent hen's egg allergy, its superior role compared to egg white extract has been debated. 7 Patients' sera contain polyclonal IgE antibodies recognizing a broad range of epitopes comprised of either sequential residues of the amino acid sequence (linear) or amino acids closely located upon folding (conformational). Epitope mapping approaches aim to identify clinically relevant epitopes which are undetectable by measuring sIgE against extracts or full-length single components. In hen's egg allergy, linear epitope mapping of Gal d 1 identified epitopes (aa 1-10, aa 11-20, aa 47-56 and aa 113-122) exclusively recognized by children with persistent hen's egg allergy. 8 Comparable allergenic parts (aa 1-10, aa 11-20 and aa 47-56) were described as immunodominant linear epitopes by several other studies. [9][10][11] So far, the impact of sIgE titres to hen's egg components and sIgE-binding to their linear epitopes on discriminating between clinically relevant and irrelevant sensitization is poorly studied in hen's egg allergic and tolerant adults. To this end, we evaluated sensitization patterns and sIgE titres to hen's egg components (Gal d 1, 2, 3 and 4) in allergic and tolerant, but sensitized adults. Since Gal d 1 is known as the most important single component for diagnosing hen's egg allergy in children, recognition of linear epitopes derived from Gal d 1 was evaluated by peptide chip analysis. Since the role of Gal d 2 is controversially discussed, 7 we decided to additionally map the linear epitopes of Gal d 3, another major egg white allergen of which little information is known so far.

| Heterologous expression of hen's egg components
The mature hen's egg components Gal d 1 (accession number: P01005) and Gal d 3 (accession number: P02789) were heterologously expressed as fusion proteins with N-terminal-His (6x)-tag in E coli and purified as previously described. 12,13 All heterologously expressed proteins were purified by immobilized metal ion chromatography under denaturing conditions. Purified Gal d 1 and 3 were separated by gel electrophoresis and blotted onto a nitrocellulose membrane.

| Determination of sIgE and sIgG4 sensitization
Sensitization to egg white and yolk extract was determined using the commercially available ImmunoCAP system and sIgE and sIgG4 sensitization to the native components Gal d 1, 2, 3 and 4 were measured using the EUROLINE-immunoblot strip "Paediatrics' 1" (DP 3812-1601-1 E, EUROIMMUN AG, Germany) according to manufacturer's instructions. Briefly, the immunoblots were manually incubated overnight at room temperature with serum diluted 1:11 (IgE) or 1:51 (IgG4) in working strength universal buffer (WSUB).
After extensive washing with WSUB, bound IgE and IgG4 antibodies were detected with anti-human IgE or IgG4 conjugate coupled with alkaline phosphatase. Upon another extensive washing step, visualization was provided by applying nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolyphosphate substrate for ten minutes and specific IgE levels were evaluated as EUROLINE (EL)-intensities and expressed as response units. Specific IgE levels to the heterologously expressed hen's egg components were determined under the same conditions.

| Microarray design
A microarray with synthetic 15mer peptides, comprising the sequence of the mature Gal d 1 (accession number: P01005) and Gal d 3 (accession number: P02789) (offset = 3 due to limited space), was commercially obtained (PEPperPRINT). The peptide length of 15 amino acids was in accordance with the experience of PEPperPRINT to provide sufficient sensitivity without significant formation of secondary structures. All peptides were printed in triplicates with a linker consisting of 2 ß-alanine and one aspartic acid. This linker was chosen to circumvent the binding of negatively charged fluorescent dyes to positively charged amino acids which are close to the array surface.

| Microarray incubation
The microarray incubation was performed as previously described. 14 Briefly, patient sera were diluted 1:4 in WSUB and incubated overnight. For detecting bound-specific IgE and IgG4, a biotinylated anti-IgE antibody (clone MHE-18 1:5000, BioLegend) and simultaneously a biotinylated anti-human IgG4 coupled with Neutravidin DyLight 680 (clone HP6025, 1:5000, Southern Biotech) were applied on the microarray and incubated for one hour at room temperature. Bound biotinylated human anti-IgE antibodies were visualized by adding Neutravidin DyLight 800 (1:5000, Thermo Fisher) for one hour at room temperature. After extensive washing and drying, the microarray slides were scanned at a wavelength of 700 nm for IgG4 and 800 nm for IgE (intensity: 8.5) and the focus was set to 0.8 mm and the resolution to 21 µm.

| Microarray evaluation
For data evaluation, the fluorescent signals for each peptide were obtained using the Pepslide Analyzer Software (SICASYS) with the fixed-spot adjustment and the logarithmic signal-to-noise ratios (S) were computed according to the following quotation: For normalization, the S-values were compared to the S-values of blank spots, resulting in z-scores defined as: Epitopes were defined as recognition of 2-4 contiguous peptides with a median z-score ≥ 3.0 and the amino acid residues are counted based on the amino acid sequence without signal peptide.

| Determination of surfaced exposed epitopes
Surface-exposed residues of an epitope were determined by submitting the 3D structure (Gal d 3, PDB ID: 1OVT) to the http://curie. utmb.edu/getar ea.html interface. 15 Under the conditions (default settings) as radius of the water probe set to 1.4 and no gradient in calculations, the algorithm calculates the probability of each residue to be solvent accessible. For an epitope, at least 25% of its residues must have a greater probability than 50% to be solvent accessible for calling this epitope "surface-exposed." The definition was confirmed by mapping the linear epitopes onto the 3D structure of Gal d 3 (pdb: 1OVT) using PyMol 1.3 (Schrödinger, Inc, USA). The corresponding images are shown in Figure S1.

| Statistical analyses
Statistical differences between the hen's egg allergic and tolerant adults regarding their sensitization profiles were evaluated with the non-parametric Mann-Whitney U test and visualized by GraphPad Prism 8.3. For peptides and epitopes derived from Gal d 1, their recognition by IgE was evaluated by principle component analyses in R. Heat maps were generated in R using the "ComplexHeatmap" package. 16

| Patient characteristics
Patients sensitized to the recombinant components rGal d 1 and/ or rGal d 3 were divided into (a) allergic (75% female) and (b) tolerant patients (52% female) based on food challenge outcome or convincing history. Patients with subjective symptoms were more often diagnosed by a food challenge (4/6:67%) compared to patients with objective symptoms (3/7:43%), reducing at least the risk of misclassification. Allergic patients showed a median age of 25 and were overall younger than the tolerant patients with a median age of 28, although the age range was comparable (P = .63). Even though the majority of patients were co-sensitized to nGal d 1 and nGal d 3, one allergic and one tolerant patient were mono-sensitized to nGal d 1 and one tolerant patient was mono-sensitized to nGal d 3. Interestingly, up to 94% of all included patients, irrespective of allergy or tolerance, suffered from atopic dermatitis. Besides, more than 60% of all included patients experienced symptoms related to allergic asthma (allergic 63%, tolerant 75%) and allergic rhinitis (allergic 81%, tolerant 60%). All characteristics are shown in Table 1 and File S1.

| sIgE levels towards hen's egg extracts overlapped largely between allergic and tolerant but sensitized patients
All patients of this study were included based on their sensitization to at least one heterologously expressed hen's egg component (rGal d 1 and/or rGal d 3) and hence, detectable sensitization to hen's egg extracts was detected in all tested patients. Specific IgE titres towards egg white and yolk extract overlapped greatly between allergic and tolerant adults, resulting in low specificity even at increased cut-off levels (0.53 at 5 kU/L). Overall, tolerant patients tend to have lower sIgE titres to egg white extract than allergic patients (median 4.6 kU/L vs 7.0 kU/L). On the other hand, sIgE levels to yolk extract were even higher in tolerant patients (median 3.3 kU/L) compared to allergic patients (median 0.9 kU/L), suggesting greater relevance of hen's egg white proteins compared to yolk-derived ones ( Figure 1A). No statistically significant difference was observed for egg white and egg yolk extract.

| Gal d 1 sIgE/sIgG4 ratios were higher in allergic patients
As specific IgE levels towards hen's egg white extract were on average higher in allergic than in tolerant patients, we next analysed

| Linear epitope recognition of Gal d 1 confirms clinically relevant sensitization in allergic patients with objective symptoms
As linearization of rGal d 1 only marginally affected IgE-binding, we next analysed linear epitope recognition with a peptide microarray.

| Most patients with objective symptoms also recognized linear epitopes of Gal d 3
Additionally, a linear epitope mapping was also performed for Gal d 3.
Regarding Gal d 3, only a small number of different epitopes (n = 11) were recognized by IgE in relation to its molecular mass (78 kDa) as already indicated by the reduction in sIgE-binding upon linearization.

| D ISCUSS I ON
So far, sensitization to hen's egg extracts, components and linear epitopes is solely studied in children although persistent and newly onset hen's egg allergy do appear in adults with a prevalence of 0.02 to 0.6% across European countries. 1,2 In the present study, we showed great overlap in sIgE levels to hen's egg extracts or single components between allergic and tolerant, but sensitized adults.
Clinically relevant sensitization to Gal d 1 was confirmed by sIgEbinding to the linear epitopes aa 30-41, aa 39-50 or aa 84-95 in 6 out of 13 hen's egg allergic adults, mainly suffering from objective symptoms. In contrast, patients with mild subjective symptoms showed no binding to linear epitopes of Gal d 1.
This is, to our knowledge, the first study focussing on sensitization patterns in hen's egg allergic and tolerant adults. While largely overlapping sIgE titres to egg white extract were not able to clearly discriminate between allergy and tolerance in adults, the definition of clinically relevant cut-off levels appeared to be supportive in diagnosing raw or heated hen's egg allergy in children. 5 Although these epitopes were described independently in different studies with hen's egg allergic children, 9,11,23 they did not belong to the so-called "informative" epitopes (aa 1-10, aa 11-20, aa 47-56 and aa 113-122) which showed great potential to predict persistent hen's egg allergy in children. 8 A1  A2  A3  A4  A5  A6  A7  A8  A9  A10  A1 1  A12  A13  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T1 1  T12  T13  T14  T15   *   aa 1-11  aa 6-17  aa 9-20 A1  A2  A3  A4  A5  A6  A7  A8  A9  A10   A12  A13  T1  T2  T3  T4  T5  T6  T7  T8  T9  T10  T1 1  T12  T13  T14  As a step forward, the promising results of the present study should be validated in a prospective cohort exclusively diagnosed by food challenge, minimizing the risk of misclassification. Moreover, inclusion based on suspicion of egg allergy provides a broader population with more distinct sensitization patterns such as mono-sensitization to Gal d 2.
In conclusion, sIgE-binding to linear epitope of Gal d 1 (aa 30-41, aa 39-50 or aa 84-95) is highly specific to identify hen's egg allergic adults, mainly suffering from objective symptoms and may improve sIgE diagnostics as an additional tool to conventional testing using egg extracts and single allergen components.

ACK N OWLED G M ENTS
We would like to thank B. Brix and M. Klinge for critical and fruitful discussions. Line blots and corresponding reagents were kindly provided by EUROIMMUN AG, Lübeck, Germany. This study was funded by a grant from the European Regional Development Fund of the European Union (TBI-V-1-098-E).

I N FO R M E D CO N S E NT
This study was carried out in accordance with the University Medical Center Utrecht, Biobank Regulations, which are in compliance with the applicable national and international laws and regulations. These regulations permit the use of "residual material from diagnostic testing" for research, unless the patient objects (Article 8, "no objection" procedure).
None of the included patients objected the use of their serum. The protocol was approved by the Biobank Research Ethics Committee of the University Medical Center Utrecht under the protocol number 18-428.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article and its supplementary information files.