egg allergy: new allergens and molecular diagnosis

Egg Allergy

Suparat Sirivimonpan,MD.

7/9/2012

Egg allergy

Introduction Prevalence Egg allergen : new allergen Diagnosis : molecular diagnosis

Introduction

• Egg allergy may be defined as an adverse reaction of immunologic nature induced by egg proteins

– IgE ,non-IgE , mixed IgE-mediated disorders

• hen’s egg allergy is the second most common food allergy in infants and young children

• closely associated with atopic dermatitis • Increase risks of sensitization to aeroallergens and

asthma in children with egg allergy

Pediatr Clin N Am 2011;58:427–443

Prevalence

• estimated prevalence : varies depending on method of data collection or definition

• A meta-analysis of the prevalence of egg allergy in children – Self-reported prevalence : up to 7% – challenge-confirmed egg allergy : up to 1.7%

Allergy 2010; 65: 283–289

Prevalence

In Thailand• Santadusit S, et al.

– Egg allergy : 1.52%

• Unpublished data 2012, Chulalongkorn university– egg allergy : 1.1% (parent reported and physician diagnosis) in young

children

J Med Assoc Thai 2005;88:S27-32

Egg allergen

Egg allergens

• 5 major allergenic proteins : Gal d 1 to Gal d 5 (Gallus domesticus)

Curr Allergy Asthma Rep 2006;6(2):145–52

Egg White allergen

Egg white allergens

Allergy 2010; 65: 283–289

Ovomucoid

• dominant allergen in egg• unique characteristics

– relative stability against heat and digestion with proteinases– presence of strong disulfide bonds that stabilize protein

• children with persistent egg allergy had significantly higher specific IgE levels to OVM than children who outgrew their egg allergy

• A favorable prognosis was associated with the absence or a decline in OVM-specific IgE titers

Allergy 2007; 62:758–765Curr Opin Allergy Clin Immunol 2011, 11:210–

215

Ovalbumin

• heat-labile and less allergenic

• IgE-binding epitopes on OVA might be destroyed after heating : children who have specific IgE primarily to OVA are likely to tolerate heated egg

Curr Opin Allergy Clin Immunol 2011, 11:210–215

Ovalbumin

• The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods

• It produces chemically glycated proteins termed advanced glycation end products (AGEs) T-cell immunogenicity of chemically glycated ovalbumin

• The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy

J Allergy Clin Immunol 2010;125:175-83.

Newly identified egg white allergen

Allergology International. 2010;59:175-183

• Diluted egg white proteins were separated by 2-dimensional (2-D) gel electrophoresis


63 spots

• Immunolabeling was performed on individual patient sera • 19 child patients with egg white allergy • 11 negative control subjects


19 patients -Aged 2.2 ± 1.7 years-Total IgE 434.1 ± 424.7 Iuml-Diagnosis was based on clinical history and CAP-RAST results to hen’s egg white-Open food challenges : 9/19 patients

11 negative control subjects - Aged 4.0 ± 3.7 years- no clinical history of any allergic

symptoms

• Spots of egg white proteins that bound to the patients’ IgE were identified by mass spectrometry-based proteomics


15/25

10/25

Identification of IgE-reactive spots

• spots were excised• digested in-gel with trypsin• The resulting peptide mixtures were analyzed by

– LC-MS/MS (Liquid chromatographytandem mass spectrometry)

– MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry)


16% pt (1 anaphylaxis)

53% pt

26% pt

L-PGDS

• Reaction to LPGDS and cystatin was confirmed using each purified protein


L-PGDS

Cystatin

Ch21 protein and L-PGDS

• Ch21 protein and L-PGDS belong to the lipocalin Family • has been reported as allergenic

• Ch21 protein and L-PGDS were minor allergens

• However, the sera from patient (MT,HS), which reacted to spot no. 28 did not seem to react to the major spots, such as nos. 3, 4, 5, 48, or 49

not only the hidden allergen components, but there are some patients who are preferentially reacting to those minor egg allergens

• minor allergen does not always mean that it is allergenically poor


Ch21 protein and L-PGDS

• PGD2 synthesized by L-PGDS is related to allergic inflammation in vivo and that L-PGDS from chicken shows PGDS activity

• These reports suggest that L-PGDS induces allergic reactions in addition to binding to IgE, and that PGD2 may exist in eggs


Cystatin• cysteine protease inhibitor• allergen in cats and dogs

• considered to be a major allergen because it showed high reactivity to IgE from patients with egg allergy

• 2 possible reasons for the lack of literature on cystatin as an allergen– similarity in molecular weight of cystatin and lysozyme, a

major allergen, makes it difficult identify cystatin without using 2-D gel electrophoresis

– presence of only a small amount of cystatin in eggs makes it difficult to identify without development of LC-MSMS


Egg yolk allergen

Egg yolk allergens

• alpha-livetin or chicken serum albumin (Gal d 5) is the major allergen and is involved in the bird-egg syndrome

• Several other allergens have been identified in egg yolk– vitellenin (apovitellenin I) and – apoprotein B (apovitellenin VI)– roles in food allergy remain unclear


Bird-egg syndrome

• primary sensitization is to airborne bird allergens (feathers, droppings, serum) and there is secondary sensitization or cross-reactivity with albumin in egg yolk (Gal d 5)

• respiratory symptoms such as rhinitis and/or asthma with bird exposure

• allergic symptoms (respiratory and gastrointestinal symptoms) when egg is ingested

• In children with allergy to birds and egg, egg allergy is usually more persistent

• Sensitization to other aeroallergens is also greater in individuals with allergy to birds and egg

Allergy 2001: 56: 754-762Allergol et Immunopathol 2003;31(3):161-5

J. Agric. Food Chem. 2010, 58, 7453–7457

• Sera from 27 patients (ages 2-74 yrs) with egg allergy (clinical history and both positive-specific IgE and SPTs to egg)

• sera from 2 non-allergic subjects as negative controls

• The study was performed by – sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and – IgE-immunoblotting and – IgEimmunoblotting- inhibition assays

• egg yolk extract was fractioned by reverse-phase high performance liquid chromatography (RP-HPLC)

J. Agric. Food Chem. 2010, 58, 7453–7457

• A new allergen with an apparent molecular weight around 35 kDa was identified from the yolk extract and enriched by RPHPLC (Figure 1A)

• 5/27 patients studied (18%of patients) showed specific IgE binding to the allergen (Figure 1B)

J. Agric. Food Chem. 2010, 58, 7453–7457

J. Agric. Food Chem. 2010, 58, 7453–7457

• new allergen detected was characterized by N-terminal amino acid analysis

• revealed that allergen was the yolk glycoprotein 42 (YGP42) protein, a fragment of the vitellogenin-1 (VTG-1) precursor (positions 1628-1912)

• The allergen has been designated Gal d 6.0101 by the World Health Organization (WHO)/International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee

J. Agric. Food Chem. 2010, 58, 7453–7457

• heat-resistant but digestible by pepsin

J. Agric. Food Chem. 2010, 58, 7453–7457

• Conformational epitopes can be destroyed with heating or partial hydrolysis

• Egg-specific IgE molecules that identify sequential or conformational epitopes of OVM and OVA can distinguish different clinical phenotypes of egg allergy– patient with IgE antibodies reacting against sequential epitopes

tend to have persistent allergy– IgE antibodies primarily to conformational epitopes tend to

have transient allergy

J Allergy Clin Immunol 2004;113:805-19


Diagnosis

Diagnosis

• Double-blind, placebo-controlled food challenge : gold standard

• open food challenges (OFCs) – Less resource-intensive– are generally considered sufficient in clinical

practice

Curr Opin Allergy Clin Immunol2009;9:244–250

Diagnosis

• detailed history and physical examination • in vitro and/or in vivo allergy tests

– used to support diagnosis – food-specific IgE antibodies– skin prick tests– diagnostic elimination diet– OFC


Skin prick tests

• used in screening for egg-specific IgE and should be performed by trained personnel

• diagnostic accuracy is dependent on quality of the extract, which should be standardized

• SPT shows a good sensitivity and NPV, but poor specificity and PPV – negative test essentially excludes an IgE-mediated egg

allergy– positive test does not predict clinical reactivity accurately

Allergy 2010; 65: 283–289

Skin prick test for diagnosisReferences Year Age group (yr) Method

of DxNumbers of patients

PPV SPT Wheal diameter (mm)

Sampson and Ho1

1997 Children and adolescents

DBC 100 85 ≥ 3 *

Sporik et al2 2000 <2>2

OC 3982

100 ≥ 5 EW≥ 7 EW

Boyano-Martinez et al 3

2001 <2 OC 81 93 ≥ 3 EW,EY

Monti et al4 2002 <19 mo (mean 16 mo) OC 107 100 ≥ 5 EW, EY

Hill et al5 2004 <2 OC 30 100 ≥ 5 *

Verstege et al6 2005 All(3 mo-14.5yr)<1>1

DBC/OC 16026134

95 ≥ 13 fresh mixed EW&EY

≥ 11.2 ≥ 13.3

1J Allergy Clin Immunol 1997;100(4):444–51 4Clin Exp Allergy 2002;32:1515–92Clin Exp Allergy 2000;30(11):1540–6 5Pediatr Allergy Immunol 2004;15(5):435–413Clin Exp Allergy 2001;31(9):1464–9 6Clin Exp Allergy 2005;35(9):1220–6

Serum specific IgEReferences Year Age group (yr) Method


PPV Egg-specific IgE (kUA/L)

Sampson and Ho1

1997 Children and adolescents

DBC 100 95 6 *

Sampson2 2001 3 mo-14 yrs (median 3.8yr)

DBC 75 98 7 *

Boyano-Martinez et al3

2002 11-24 mo (mean 16 mo)

OC 58 95 2 EW

Osterballe and Bindslev-Jensen4

2003 0.5-4.9 yr (median2.2yr)

OC 56 95 1.5 EW

Celik-Bilgili et al5 2005 0.1-16.1 yr (median13mo)

OC All 277≤ 1 yr 41> 1 yr 186

95 12.6 *10.913.2

1J Allergy Clin Immunol 1997;100(4):444–51 4 J Allergy Clin Immunol 2003; 112:196–2012J Allergy Clin Immunol 2001;107:891-6 5Clin Exp Allergy 2005; 35:268–733 J Allergy Clin Immunol 2002;110(2):304–9

Serum specific IgE (cont.)References Year Age group (yr) Method


PPV Egg-specific IgE (kUA/L)

Komata et al6

2007 0.2-14.6 yr (median2.2yr)

OC 764 95 ALL ≥ 25.5≤ 1 : 131-2 : 23≥ 2 : 30

Benhamou et al7

2008 16 mo – 11.9yr (median 3.9 yr)

OC/DBC 35 95 7 *

Ando et al 8 2008 14 mo – 13yr (median 34.5 mo)

DBC 108 95 (raw EW allergy)

84-88 (heated EW allergy)

EW 7.38OVA 9.84OVM 5.21

EW 30.7OVA 29.3OVM 10.8

6J Allergy Clin Immunol 2007; 119:1272–47Pediatr Allergy Immunol 2008; 19:173–98J Allergy Clin Immunol 2008; 122:583–8

MOLECULAR DIAGNOSIS IN FOOD ALLERGY

• specific responses at the level of individual allergenic proteins : component-resolved diagnosis [CRD]

or• IgE-binding epitopes of allergens : epitope mapping or profiling

Immunol Allergy Clin N Am 2012;32:97–109

Microarray-based component-resolved diagnosis

• ImmunoCAP-ISAC or Immuno Solid phase Allergen Chip (VBC Genomics-Vienna, Austria; Phadia, Uppsala, Sweden)

• It currently has 112 native/recombinant component allergens from 51 allergenic sources

• two main advantages:– assesses simultaneously specific IgE to different components and

requires small amounts of serum(relevant in children)– cost-efficient approach, as it delivers results for over 100 components

Curr Opin Allergy Clin Immunol 2011;11:210–215Immunol Allergy Clin N Am 2012;32:97–109

• infants and children ,suspected IgE-mediated food (CM or HE)– Skin (42%), respiratory (6%), GI (16%), combination of skin, RS,GI (32%),

anaphylactic shock (4%)– Only atopic eczema were excluded

• all patients SPT, sIgE (ImmunoCAP and microarray) and open food challenge test were performed

• Total 104 patients (62 males and 42 females), median age of 4.9 yrs (range 0.7–15.1)

• hen’s egg allergy 46 patient

Clinical & Experimental Allergy, 2010 (40) 1561–1570

• FCT resulted positive in 22/46 (48%) patients with suspected HE allergy


Clinical & Experimental Allergy, 2010 (40) 1561–157095% predicted probability (CDP)of a positive FCT


↓FCT≈15% ↓FCT≈37%

Sequential use of the two tests did not lead to a further improvement in clinical performance

False negative remains relatively high FCT should still be performed

• 68 children (47 male, 69.1%), • Age 1 - 11 years (median 4.1 years)• suspected HE allergy based on

– reported history of reactions like asthma, rhinitis, conjunctivitis, urticaria, worsening of eczema, vomiting, other gastro-intestinal tract symptoms, anaphylaxis, after ingestion or contact with HE,

– and positive SPT or IgE to HE white extracts

• 42/68 (62%) reported atopic dermatitis as the main allergic complain

Clinical & Experimental Allergy 2012 :42;441-451

• Skin prick test – Commercial extracts (Allergopharma, Reinbek, Germany) and fresh eggs using

the prick-prick technique– Fresh: white and yolk from both raw and processed boiled HE– Positive : wheal greater than 7 mm2

• Total and specific IgE– HE white and yolk : ImmunoCAP (Phadia AB, Uppsala, Sweden) – Specific IgE detection for Gal d 1, Gal d 2, Gal d 3 and Gal d5 : ISAC 103

microarray test (PMD, Vienna, Austria)

• Double- blind, placebo-controlled food challenge (DBPCFC)– First : boiled egg (totol HE protein 6 g)– Patients tolerating boiled HE were then challenged with raw HE– patients with positive response to boiled HE challenge were considered likely to

be allergic to both boiled and raw eggs



No significant differences

significant differences, P<0.01

significant differences

ImmunoCAP



ISAC20 of 21 Gal d 1 positive patients (95%) reacted to raw egg.

44/47 Gal d 1 negative patients (94%) could tolerate boiled egg

ISAC – Other sensitization

• 13 patients had no additional IgE detectable sensitizations on ISAC (103 microarray system)

– 10 were in ST group– 3 in PT group– none in A group

• 54 subjects : positive for at least one non-HE allergen– higher sensitization prevalence to other food and nonfood

allergens in group A



A

PT

ST

Allerrgen group sensitizationLTP: Cor a 8, Pru p 3Parvalbumin: Cyp c 1, Gad c 1PR-10: Act d 11, Api g 1, Ara h 8, Bet v 1, Cor a 1.0101, Cora1.0401, Dau c 1, Mald 1, Pru p 1, Gly m 4Profilin: Bet v 2, Hev b 8, Mer a 1, Ole e 2, Phl p 12Tropomyosin: Der p 10, Ani s 3, Pen a 1,Pen i 1, Pen m 1;


Anisakis: Ani s 1Kiwi: Act d 1, Act d 2,Act d 5Cow’s milk: Bos d 4, Bos d 5, Bos d 8Seeds: Ana o 2, Ara h1, Ara h 2, Ara h 3, Ber e 1, Cor a 9, Gly m 5,Gly m 6, Ses i 1;Wheat: Tri a 18, Tri a 19, Tri a Gliadin

statistically significant differences


Cat: Fel d 1,Cypress: Cry j 1, Cup a 1; Dog: Can f 1, Can f 3Grasses: Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 11Mites: Der p 1, Der p 2, Der f 1, Der f 2, Eur m 2Olive: Ole e 1

• IgE detection to Gal d 1 on the ISAC microarray system useful tool to predict oral tolerance to boiled eggs

• DBPCFC in egg allergic children should be performed first with boiled eggs to introduce at least well cooked eggs in HE allergic children’s diet

• Outgrowing clinical allergy leads to negativity of tests later on, and ISAC IgE detection reaches such condition before all other tests

– majority of the subjects tested negative for HE allergens on ISAC were in the PT and ST groups


• Patients without previous egg exposure were recruited between January 2005 and October 2011

• examined to determine their sIgE titers (mostly because of atopic dermatitis, other food allergies, or family history)

• n= 100, males/females = 69/31• Median age of 17 months (range 12-23 months)

J Allergy Clin Immunol 2012:129(6);168132

• sIgE titers to EW (n = 100) and OVM (n = 80, males/females = 55/25) : ImmunoCAP System (Phadia AB, Uppsala, Sweden)

• OVM was not determined in 20 patients because of inadequate sample volume or cost of the testing

• 2 children were sIgE-negative to EW, but they had previously tested positive to EW or had a positive skin prick test result


• open OFCs using boiled EW

• Each child ate EW, boiled for more than 20 minutes, in increasing amounts (trace, 1, 2, 5, and 10 g) at 20-minute intervals

• total amount : half a large egg, containing 2.0 g of EW protein

• OFC was not conducted in patients with extremely high sIgE titers to EW and OVM (50 kUA/L or higher [n = 17]) to decrease the risk of inducing reactions


• Positive challenges 33 patients – dermal (n = 31), RS (n = 8), and GI (n = 4) symptoms– sIgE to OVM was examined in 27 patients

• Multisystem reactions : 12 patients • 3 required intramuscular epinephrine injection• The median cumulative EW dose required to provoke allergic

symptoms was 10.6 g (range, 1.0-18.5 g), containing approximately 1.7 g of EW protein


• The median concentration of sIgE against EW was – 15.6 kUA/L (range, 1.58-51.3 kUA/L) in challenge-positive patients,

(significantly higher, P <0.01)– 4.34 kUA/L (range, 0.35-51.5 kUA/L) in challenge-negative patients

• A similar difference was observed for OVM (P < 0.01)– 8.12 kUA/L (range, 0.35-48.8 kUA/L) – 1.00 kUA/L (range, 0.35-32.0 kUA/L; P < .01)



sIgE to EW 10 kUA/L: 30% PPV

30 kUA/L:65% PPV

sIgE to OVM 10 kUA/L: 54% PPV

30 kUA/L:97% PPV

The calculated sIgE titer that indicated a 95% PPV was 61.8 kUA/L for EW 26.6 kUA/L for OVM


Patients with undetectable sIgE against OVM (<0.35 kUA/L; n = 24),

regardless of the sIgE titer to EW (range, 0.35-27.5 kUA/L), showed 88% (21 of 24) likelihood of a negative challenge

3 individuals demonstrating a positive OFC showed only mild cutaneous symptoms

indicate the advantage of using sIgE titers against OVM to identify patients who can safely consume boiled egg products

• OVM IgE : new predictive marker• sIgE titer against OVM has a better predictive value than does

a titer against EW

• But for safety, we recommend an office challenge to introduce egg products to the sensitized children, even if sIgE to OVM is negative


Treatment

• Standard therapy for egg allergy is strict avoidance

Oral immunotherapy (OIT) or specific oral tolerance induction (SOTI)

Thank you

egg allergy: new allergens and molecular diagnosis

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