Molecular based allergic diagnosis

Molecular-based allergy

diagnostics Suparat Sirivimonpan, MD.

18/10/2013

A WAO - ARIA - GA2LEN consensus

document on molecular-based allergy

diagnostics

World Allergy Organization Journal 2013, 6:17

Topic outline

Introduction Definitions and concepts Clinical application

Increase accuracy and resolve cross-reactivity

Assess the risk and type of reaction

Specific immunotherapy

Micro-array technology

Diagnosis

• Clinical : History, Physical examination • Allergic test

• Skin prick test

• Blood test

• Specific IgE

• Component resolve diagnosis (CRD)

crude extracts

Currently available tests detect only sensitization,

not clinical allergy, cannot predict prognosis and severity

Nature

Allergenic

source

contain a mixture of allergenic and

non-allergenic molecules

Introduction

• Molecular allergy (MA) diagnostics is increasingly entering routine care • More than 130 allergenic molecules are commercially available for in vitro sIgE testing • Clinicians and immunologists specifically trained in allergology must keep abreast of the new and rapidly evolving evidence available for MA diagnostics


Introduction

• WAO - ARIA - GA2LEN consensus document : practical guide for the indication, determination, and interpretation of MA diagnostics aimed for clinicians specifically trained in allergology


Definitions and concepts


• Allergen extract • A crude, unfractionated mixture of allergenic and non-

allergenic proteins, polysaccharides, and lipids

• extraction from an allergen source

•Allergenic molecule (allergen component) • A molecule (i.e., protein or glycoprotein) bind sIgE antibodies “allergen”

• Allergens can be isolated from

• natural allergen sources (native, purified allergen)

• recombinant DNA technology (recombinant allergen)


Almost anything containing proteins can be an allergen source

- each allergen source contains many different allergenic protein (allergen component)

- each allergen component commonly has several different epitopes

- epitope is the actual three-dimensional binding site for an antibody

Allergen component

Clinical and Experimental Allergy;2010 ,40: 896-904


Stability of allergens

•Acid pH : • susceptible to acid pH (peptic digestion) not able to cross

gastric barrier

(except possibly in patients treated with antacid drugs)

•Temperature (cooking or boiling) : • Temperature susceptibility does not maintain its

allergenicity after cooking/heating procedures


Protein stability

- allergen that stable to heat and digestion are more likely to cause severe reaction

- allergen that heat and digestion labile are more likely to be tolerated or only cause mild/local symptom

Allergen component



• Primary allergen : • original sensitizing molecule (i.e., driving trigger)

•Secondary sensitization : • cross-reactivity



Cross-reactivity • phenomenon of an IgE antibody recognizing, binding,

and inducing an immune response to similar allergenic molecules (homologues) present in different species

(reacts to both Bet v 1 in birch pollen and Cor a 1 in hazelnut PR10)

• ≥ 50-70% sequence homology between primary

structures of proteins



Cross-reactivity • IgE cross-reactivity often occurs between the following:

a) Allergenic molecules in closely related species

b) Well preserved molecules with similar function present in widely different species that belong to the same protein family (tropomyosin : Der p 10 in house dust mite ,Pen m 1 in black tiger shrimp)



Co-sensitization • Genuine sensitization to more than one allergen source (e.g., timothy grass and birch) • sensitization is not due to cross-reactivity



Co-sensitization • Genuine sensitization to more than one allergen source (e.g., timothy grass and birch) • where the sensitization is not due to cross-reactivity



sIgE sensitization • presence of allergen-specific IgE (sIgE) antibodies in the blood

• presence or absence of clinical symptoms

a) Mono-sensitization: - Sensitization to one allergen source (D. pteronyssinus) or to a

closely related taxonomical family or group of allergen sources (i.e., mites)

b) Poly- (or multi-) sensitization: - Sensitization to three or more allergen sources (e.g., mite,

birch, and grass pollen).



sIgE detection based on allergen extracts • Singleplex or multiplex platforms

• such as CAP, radioallergosorbent test (RAST)

• interchangeably

•

sIgE detection based on allergenic molecules • Singleplex or multiplex platforms

• identify cross-reacting molecules


•World Health Organization and International Union of Immunological Species (WHO/IUIS)

• Using their Latin family name (genus and species)

• Arachis hypogaea : Ara h (peanut) • A number : distinguish various allergens from the same species (e.g., Ara h 1, Ara h 2, etc.)

• The numbers are assigned to the allergens in the order of their identification

• Allergenic molecules are classified into protein families, according to their structure and biological function

•www.allergen.org


Systematic allergen nomenclature


http://www.allergen.org/

Clinical application

Clinical application

• increased accuracy in allergy diagnosis and prognosis • plays an important role in three key aspects of allergy diagnosis: (1) Increase accuracy and resolve cross-reactivity

(2) Assess the risk and type of reaction

(3) Specific immunotherapy (patients and triggering allergens)


• Allergic individuals may produce IgE antibodies to allergens that are either unique to a single species or common to many

• Proteins within the same family share common epitopes

same IgE antibody can bind to similar structures present in allergens from different allergen sources

• These cross-reactive allergens give valuable information on potential sensitization and clinical reactions to several different sources



•




Stab le


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Lab ile


Lab ile


• While, MA studies have not fully elucidated the underlying mechanism governing cross-reactivity and symptom presentation analysis of the epitopes of key allergens will provide insight into this issue • Currently, a large number of purified or recombinant allergens are commercially available


World Allergy Organization Journal 2013, 6:17 Food



World Allergy Organization Journal 2013, 6:17 Aeroa lle rgens



World Allergy Organization Journal 2013, 6:17 O ther alle rgens


Clinical uses of MA : • identify the offending allergenic molecule • distinguish specific molecules from markers of cross-reactivity



• In the field of pollen-related food allergy : increase accuracy of diagnosis

* peanut allergic patients,

- Ara h 2 genuine marker,systemic reactions

-Ara h 8 cross-reactivity among food allergens ,Fagales tree pollen mild, oral reactions

•measuring IgE responses to certain food allergens may reduce the need for food challenges

•



• In patients sensitized to different pollen species

• detecting new relevant sensitizations

• by ruling out clinically irrelevant sensitizations caused by non-symptomatic cross-reactive allergens

• For example, MA diagnostics can help to distinguish baker’s asthma from pollen or wheat allergy



• In conventional SPTs, some allergens can be poorly represented in extracts because of the biological variability of allergen source

* Dog allergic patients • Can f 5 prostate-derived allergen produced by male dogs

38% of dog allergic patients

• Allergen extracts used in skin tests : dog hair

skin tests routinely fail to identify patients’ sensitivity to

Can f 5 (low concentration in dog hair)

• IgE response to Can f 5 using MA diagnostics may enhance accuracy of dog allergy diagnosis



• All allergy diagnostics, including MA, should be evaluated within framework of a patient’s clinical history

• IgE sensitization not necessarily imply clinical responsiveness

• This is of particular importance, since allergic patients respond in individualized manner to exposure to allergens from various sources

•i.e., every individual produces their own unique IgE antibody profile at molecular level



• Ability to improve risk assessment, particularly for food allergies

• Different foods contain unique allergenic molecules that are stable or labile to heat & digestion

• stability of a molecule & patient’s clinical history : risk of systemic versus local reactions

• Labile local reactions (typically oral symptoms) and cooked food is often tolerated

• Stable allergens systemic reactions

• MA diagnostics may decrease the need for provocation testing and improve recommendations for allergen avoidance



• Information may only be applicable to the specific population which has been studied

• Disease expression differ according to local exposures patterns characteristic of the geographical region

• ex. Peanut (USA, Spain, Sweden)


Food allergens


Allergens resistant to heat and digestion often trigger more severe

allergic reactions

Storage protein, LTP

Food allergens

• In addition, the amount of a molecule present in a food source is also a parameter to take into consideration


Food allergens

Peanut

• Ara h1,2,3 (storage proteins) genuine peanut reaction

• Ara h 8 (PR-10,Bet v 1-homologue) milder or local symptoms

• LTP (Ara h 9) southern Europe, prevalent sensitizing allergen

systemic and more severe reactions

• Further studies in other geographical regions

• Profilin or CCD sensitization no or local oral symptoms

heated peanuts may be

tolerated


Food allergens

Soy • Gly m 5 and/or Gly m 6 severe reactions • Gly m 4 (PR-10) OAS

• In birch pollen-allergic individuals, the combination of Gly m 4 sensitivity and intake of large amounts of mildly processed soy, such as soy drinks, can induce a severe reaction

• Profilin or CCD no, or local oral symptoms heated soy may be tolerated


Food allergens

Hazelnut • Cor a 1 (PR-10) local reactions like OAS • Cor a 8 (LTP) , storage proteins (Cor a 9,Cor a 14) severe symptoms • Profilin (Cor a 2) or CCD alone no or local oral symptoms heated hazelnuts may be tolerated.


Food allergens

Walnut • Storage protein (Jug r 1, Jug r 2) or LTP (Jug r 3) severe reactions • Walnut allergens have not been available on the market until recently, as is reflected by the lack of recent clinical studies • Profilin or CCD alone no, or local oral, symptoms heated walnut may be tolerated


Food allergens

Wheat • ω-5-gliadin (Tri a 19) immediate allergic reactions in children

systemic exercise-induced reactions in adults

• LTP (Tri a 14) some degree of cross-reactivity with other food LTPs

more knowledge is needed about its prevalence and clinical implication

• Profilin or CCD no, or local oral, symptoms

heated wheat may be tolerated


Food allergens

Rosaceae fruits • Apple, peach, Appricot, Plum • PR-10 proteins (Mal d 1, Pru p 1) or profilins (Pru p 4) local, oral reactions (sensitive to heat & digestion)

• LTP (Pru p 3) typical of the Mediterranean area

wide range of clinical expressions (from asymptomatic to anaphylaxis)

risk marker for severe reactions including cofactor (e.g., exercise, alcohol or drugs) dependent anaphylaxis


Food allergens

Egg • High levels of sIgE antibodies to ovomucoid (Gal d 1) risk factor for persistent egg allergy, including reactions to cooked/heated egg • Undetectable levels indicate tolerance to cooked egg


Food allergens

Milk • Casein (Bos d 8) and beta-lactoglobulin (Bos d 5) sIgE antibodies persistent allergy to milk, including heated milk • undetectable levels indicate tolerance to baked milk products


Food allergens

Fish • Parvalbumins (e.g., Gad c1 and Cyp c 1)

• major allergens in fish • typically stable to heat and digestion • high degree of cross-reactivity whereby patients sensitized to one parvalbumin may also

react to parvalbumins from other fish, including carp, cod, herring, plaice, mackerel, tuna, salmon, perch, and eel


Food allergens

Shellfish • Tropomyosin - high degree of cross-reactivity across a wide variety of species, including mites • Shrimp and other shellfish also contain other clinically relevant allergens - sarcoplasmic calcium-binding protein - arginine kinases


Food allergens

Meat allergy Galactose-α-1,3-galactose (α-Gal) • sugar structure found on glycoproteins and glycolipids of non-primate mammals and new world monkeys, but not on humans

• It is assumed that sensitization to α-Gal can be induced by tick bites or certain parasite infections

• α-Gal– sensitized patients delayed immediate type reactions to red meat (beef, pork, goat, deer)

• Anti-α-Gal-IgE severe allergic symptoms and with delayed-type anaphylaxis


Food allergens

Meat allergy • α-Gal : present on the chimeric antibody cetuximab

• Patients sensitized to α-Gal may react with anaphylactic reaction after the administration of cetuximab

• Testing for α-Gal before administration of cetuximab

• Bovine serum albumin (e.g. Bos d 6)

• heat labile allergen

• present both in milk and beef

• may cause cross-reactivity between different mammalian meat


Inhalants

Pet dander • Fel d 1 : asthma in cat-allergic individuals

• Lipocalins (Mus m 1, Equ c 1, Fel d 4, Can f 1, 2), kallikrein (Can f 5), and secretoglobin (Fel d 1) : severe asthma in Swedish children

• More knowledge is needed in the area of pet allergy

• many of the patients are poly-sensitized to several pets

• clinical history is often inconclusive

• cross-reactivities between e.g. cat, dog and horse is not fully clarified at the MA level


Inhalants

Pollen

• Research in pollen allergy has focused on distinguishing genuine allergens from those that are cross-reactive

• Little is known regarding specific markers of severe reactions

• Some sensitivities to specific allergens (ex.Ole e 9 ,LTP Ole e 7)

• may be markers of more severe symptoms in pollen allergy

• increasing the risk of systemic reactions during immunotherapy


Inhalants

Pollen • Profilin sensitization

• common among pollen allergic patients

• associated with mild or no clinical symptoms

• minority of patients: risk factor for more severe reactions in olive pollen-allergic individuals and in patients allergic to certain plant foods like melon or citrus


Inhalants

Mites • No specific sensitization profile has been described as a risk factor for lower airway disease or disease severity • Higher sIgE/IgG4 ratio for Der p 2 has been associated with asthma

• Der p 10 (tropomyosin)

• minor allergen in mite-allergic patients

• risk for allergic reactions to shellfish or snail, which can be severe


Inhalants

Molds Aspergillus fumigatus • Asp f 2, 4, and 6 may suggest ABPA

• Asp f 1 and/or Asp f 3 may be more indicative of allergic asthma

• These associations must still be confirmed in other patient populations


Inhalants

Cockroach • Per a 2 correlates with severity of airway allergy in cockroach-allergic patients

• Per a 2 is currently not commercially available for in vitro testing

• Per a 2 homologue Bla g 2 is available

• Cross-reactive tropomyosin (Bla g 7)

risk for allergic reactions to shellfish or snail, which can be severe


Other allergens

Latex • Sensitization to Hev b 8 (profilin)

• seems to be clinically irrelevant

• not related to clinical latex reactions

• The other latex allergens are linked to clinical reactions

• No association between allergens and severity of reactions

• The cross-reactive allergen responsible for the so called latex-fruit syndrome are not fully clarified

• although data indicate that Hev b 5, 6 and 11 play a role


Other allergens

Hymenoptera venoms

• Most hymenoptera venom allergens possess CCDs

responsible for a portion of clinically irrelevant IgE antibody cross-reactivity between bee and wasp venom

• Detection of recombinant venom allergens can discriminate between genuine venom sensitization and cross-reactivity due to CCDs in patients with double-positive IgE results from traditional venom tests that are based on allergen extract


Specific immunotherapy (SIT)

• Correct diagnosis, selection of truly eligible patients, and identification of primary sensitizing allergen(s) are important for optimal and cost-effective patient management

• In some patients, a detailed clinical history and traditional extract-based IgE testing (SPT and/or in vitro sIgE) is sufficient to identify the relevant allergen(s)

• Patient demonstrates polysensitization : traditional diagnostic tests based on allergen extracts and their clinical history is not sufficient to clarify the nature of the sensitization

• may occur in a relatively high proportion of patients



• Mixing numerous allergens appears to achieve good clinical efficacy

• there may be an inability to identify the responsible allergen in the case

of adverse events

• Certain proteins (e.g., profilins, polcalcins, LTPs, PR10, tropomyosins) are highly conserved in a wide variety of species

• The use of recombinant/purified allergens would allow for the discrimination between genuine sensitizations and cross reactivities


Grass, Pollen


In the example mentioned above a patient with sIgE antibodies against Phl p 1 and Phl p 5 but no sIgE to Bet v

1 is truly sensitized to grass

If sIgE antibodies to Phl p 12 (profilin) were also detected, profilin sensitization would probably be responsible

for the positive SPT result obtained with birch extract, which contains profilin as well.

SIT would be prescribed for grass only

• if a patient is sensitized to a traditional house dust mite extract

• but their IgE antibodies against Der p 10 (tropomyosin) and not to Der p 1, 2/

Der f 1, 2

• SIT for mites should not be given, because mite extracts mainly contain Der

p 1, 2/Der f 1, 2 and have variable or low amounts of Der p 10


HDM

Hymenoptera venom

• Sensitization to the major allergens Api m 1 of honeybee and Ves v 5 and/or

Ves v 1 of yellow jackets may be helpful in discriminating between true double

bee and wasp sensitization and cross-reactivity due to CCDs



• Most commercial allergen extracts used in SIT are well standardized for major allergens • contain only minimal or variable amounts of minor allergens • Patients with sensitization to minor allergens alone will likely not receive sufficient amounts of allergen to achieve a successful outcome by SIT



• Theoretically, identification of molecules tailored SIT?? • In practice, this does not seem feasible 1. number of possible combination of sensitization profiles is large when taking into consideration all allergenic sources 2. recombinant vaccines do not perform better than traditional allergen extracts, as observed in some studies 3. each single recombinant/purified allergen would need to be individually tested and registered, which carries a substantial financial burden for manufacturers • reality of patient-tailored SIT is still a distant prospect




• MA diagnostics has been available on singleplex platforms

• ImmunoCAP, ImmuLite, and HyTech platforms

• single allergens together with the corresponding allergen extract

• Multiplex technology :

• measure sIgE antibodies against multiple allergens in a single assay

• testing of a large number of allergens using a small amount of serum


ImmunoCAP Immuno-Solid Phase Allergen Chip (ISAC) (Phadia AB)

World Allergy Organization Journal 2013, 6:17 Fluorescence is measured with a laser scanner and results are evaluated using a Microarray Image Analysis (MIA) software, which provides an automatic readout of the results.

ISAC

• Using a standard calibration curve, results are reported within a range of 0.3 - 100 ISAC Standardized Units (ISU-E)

• giving a semi-quantitative indication of IgE antibody levels


ISAC

• ImmunoCAP

• results : kU/L

• not interchangeable

• correlate well

• ImmunoCAP technology measures IgE binding under conditions of excess of immobilized allergen

• ISAC uses low amounts of immobilized allergen allowing for competition with allergen-specific isotypes other than IgE



When comparing ISAC with other sIgE measuring assays, concordance of results vary between allergens tested

ISAC

• Results of the ISAC assay are reproducible at a level that is generally accepted and agreed upon

• Special attention : samples contain low levels of sIgE

(0.3–1 ISU-E), higher degree of variability

• Comparative data with the ImmuLite or HyTech platforms are not available in the literature


at low sIgE levels ImmunoCAP is more sensitive than ISAC

ISAC

• ISAC is generally not recommended for monitoring quantitative IgE levels over time in clinical routine

•Because there is a higher degree of between assay variability for ISAC

compared to ImmunoCAP

• The interpretation of the results may be challenging 1. clinical relevance of the different allergens must be considered 2. results must be evaluated in relation to traditional diagnostic tests 3. results must be evaluated with regard to the patient’s clinical history (most important)



• ISAC results for some allergen sources such as cashew nut, sesame, dog, mugwort, and ragweed can be negative, even when the extract-based test is positive

triggering allergen is not present on the chip


•


Patients most likely to benefit from MA diagnostics

• Patients who are poly-sensitized, have an unclear symptom and/or sensitization pattern, or who do not respond to their treatment

• Patients with documented poly-sensitization to one or more inhalants, but also suffering from food allergy (i.e., from less severe manifestations such as OAS to more severe, including anaphylaxis, asthma or eczema)


Mono-sensitized patients with a clear case history and symptom profile may not benefit from MA diagnostics compared to traditional diagnostic tests

Molecular-based allergy diagnostics

• MA diagnostics may offer additional information for - early diagnosis of allergies - aid in the monitoring of the evolution of the allergic disease - useful for preventive indications to the patient


Unmet needs!

1) Large-scale, population-based multicenter studies : categories of patients

2) Large studies that include well-characterized patients and healthy, sensitized controls representative of different geographical regions

3) Cost-utility studies :

compare effectiveness of MA diagnostics with the traditional in vitro sIgE or SPT


Unmet needs!

4) Identification and clinical evaluation of the most relevant allergens have to be further investigated in many allergen sources.(e.g., nuts, molds, tree and weed pollen)

5) Training efforts in both the clinical and research settings

6) Development of clinical decision support is needed to prevent misinterpretation and improve knowledge as the amount of information obtained from MA diagnostics may be complex, especially as the evidence for MA is rapidly progressing.


• International guidelines recommendation

For the majority of patients, first- and second-line investigation is sufficient to define the nature of a

patient’s allergy

For experienced users MA may be included in second-line testing


1st Step : a thorough clinical case history

2nd Step : allergen extract-based IgE tests (in vitro specific IgE or skin prick test) identification of the allergen source

SPT and in vitro sIgE tests provide similar information associated advantages and disadvantages of both types of tests are dependent on the clinical case

3rd Step : Molecular-based allergy (MA) diagnostics used for select patients in whom first- and second-line investigations were inconclusive

• MA diagnostics is increasingly entering routine care and can improve management of allergic patients Benefit (1) Increase accuracy and resolve cross-reactivity (2) Assess the risk and type of reaction (3) Specific immunotherapy


Conclusions

- small serum or blood sample - broad spectrum analysis of a patient’s IgE profile

• Sensitivity compared to ImmunoCAP is still often lower • ISAC can have high clinical relevance by detecting sensitization patterns to important allergens and cross-reacting groups


Conclusions

Disadvantage • Interpretation of such sensitization in clinically unresponsive patients is difficult or even impossible • Relatively expensive compared with traditional tests economic consideration or budget limitations

• the search for more, clinically relevant molecules is needed and ongoing


Conclusions

• Although MA diagnostics is a complex area, it provides novel and relevant information for the allergist and will soon become a standard tool in the allergist’s armamentarium • Educational programs training allergists on the use and interpretation of MA are highly needed •


Conclusions

Thank you

Health & Medicine

Molecular based allergic diagnosis