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Prédire l’allergénicité d’une protéine ?

dimanche 22 mars 2009, par Allerdata

Peut-on prédire l’allergénicité d’une protéine ?

Parmi les milliers de sortes de protéines connues, seules certaines semblent douées d’IgE-réactivité.

Et si des protéines réputées comme n’étant jamais allergisantes peuvent s’avérer un jour contredire ce dogme , et que la diversité des familles de protéines IgE-réactives s’agrandit chaque année, il est raisonnable de penser qu’une vaste proportion des protéines restera sans pouvoir allergisant démontrable.

Pourquoi ? On ne sait pas : on n’a pas encore trouvé d’explication générale à la question « pourquoi certaines protéines sont des allergènes ? ».

Pourtant il serait bien utile de pouvoir comprendre les fondements de l’allergénicité. Et en dehors d’un simple intérêt théorique, pouvoir prédire si une protéine pose un risque d’allergénicité permettrait diverses actions pour limiter ou éviter le contact des patients, voire de la population générale.

Dans ce cadre, on pense bien sûr aux protéines introduites par génie génétique, c’est-à-dire au cas des OGM. Mais posséder de moyens de prédiction de l’allergénicité peut également rendre des services s’agissant d’aliments en passe d’être introduits sur le marché (ex. fruits ou graines « exotiques » ) ou de procédés techno-alimentaires nouveaux (ex. colle biologique comme la transglutaminase ).


C’est dans ce but que des protocoles d’évaluation de l’allergénicité ont été publiés par la FAO et l’OMS

Ils ont été relayés par des avis du Codex Alimentarius (ex. ) et repris par les agences de sécurité sanitaire

Quelques études mettant en pratique ces protocoles ont été publiées , avec plus ou moins d’impartialité . Et de multiples travaux ont cherché à valider ou améliorer ces tests de prédiction d’allergénicité, notamment chez l’animal .

A l’heure actuelle, il subsiste de nombreux points non résolus, ainsi que l’attestent les opinions nuancées de beaucoup d’experts et de groupes de travail .

C’est pourquoi le protocole FAO/OMS, qui était prévu au départ comme un arbre décisionnel, a été assoupli : il est admis à présent qu’aucun des tests prédictifs n’est suffisant en soi pour accepter ou rejeter une protéine candidate ; et il est recommandé de se fonder sur les résultats d’ensemble des tests.

On pourra relever qu’il est généralement difficile de récupérer une validité statistique en additionnant des éléments qui en manquent déjà eux-mêmes …

Ceci étant dit, quels sont ces tests ? :

  • rechercher une homologie de séquence entre la protéine à tester et des allergènes connus
  • soumettre la protéine à des expériences de digestion simulée
  • tester son éventuelle réactivité croisée avec des protéines déjà allergisantes dans la population à l’aide de sérums de patients
  • accessoirement tenter de montrer que la protéine peut être allergisante chez l’animal

Bien sûr, il serait plus pertinent de recourir à des tests in vivo chez l’homme, mais ceci n’est éthiquement pas possible.

Il ne sera abordé ici (pour l’instant) que le premier point, à savoir la prédiction basée sur une homologie structurale entre la protéine et des allergènes. Comme cette méthode de prédiction repose sur des outils informatiques (bases de données, algorithmique), elle est dénommée « in silico », en référence aux expressions in vitro ou in vivo. C’est une des applications de la branche des mathématiques appelée bio-informatique.

La prédiction in silico

Pour diverses applications, comme les études de phylogénèse, il a été constitué des bases de données (BDD) accessibles par l’internet et stockant des centaines de milliers de séquences d’acides nucléiques (ADN) et/ou d’acides aminés (protéines). Les méthodes permettant d’obtenir expérimentalement une séquence d’acides nucléiques sont à présent robotisées et, comme la séquence en acides aminés (AA) se déduit de la séquence en acides nucléiques, le nombre de séquences protéiques mémorisées dans les BDD a grandi rapidement.

S’agissant des BDD de séqueces protéiques, on peut citer, par exemple, la base Interpro, à partir de laquelle on accède également à un grand nombre d’autres BDD.

Il est plus difficile d’établir la structure dans l’espace d’une protéine. Si l’on peut calculer assez bien la structure secondaire (hélices, etc..) à partir de l’enchaînement des AA, le repliement d’une protéine sur elle-même met en jeu des facteurs (ex. la glycosylation) qui ne sont pas accessibles à partir de la simple séquence en AA.

On doit faire appel à des méthodes physiques souvent lourdes (RMN, cristallographie X), ce qui explique que le nombre de protéines pour lesquelles on possède une structure tertiaire bien établie soit très limité : environ une cinquantaine d’allergènes avaient une structure 3D disponible dans la base PDB au 01/03/09.

Ceci est regrettable dans le cadre d’une prédiction d’allergénicité car les épitopes sont souvent conformationnels et leur structure en 3D influe également .

Pour tenter de pallier ce déficit de données 3D pour de nombreux allergènes, il est fait souvent appel à un « décalquage » : on part d’une protéine de la même famille et dont on connaît la structure 3D ; et à partir de ce modèle on recalcule les changements 3D suscités par le remplacement de tel ou tel AA sur la protéine à tester comparativement au modèle.

Si des enseignements peuvent être tirés de ces comparaisons 3D (ex. ), cette approche ne peut s’appliquer pour le moment à une protéine quelconque, ni tirer parti d’une estimation statistique de la probabilité d’IgE-réactivité de cette protéine car on connaît trop peu de structures 3D.

On doit donc se « replier » sur une estimation plus éloignée de la réalité, le degré d’homologie séquentielle.

C’est c’ailleurs ce que préconisent les protocoles officiels : rechercher si la protéine à tester renferme des portions de séquence susceptibles de ressembler totalement (sur 6 à 8 AA contigus) ou « exagérément » (plus de 35% d’AA identiques sur une suite de 80 AA) à un ou plusieurs allergènes connus.

De nombreux auteurs ont critiqué ces critères prédictifs d’IgE-réactivité car produisant trop de faux positifs : par exemple, avec une identité sur 6 AA contigus près des 2/3 de toutes les protéines seraient classés comme des allergènes potentiels !

C’est pourquoi plusieurs équipes ont cherché des moyens plus sophistiqués de prédiction in silico, dont certains sont exposés ci-dessous avec leurs résultats s’agissant des tropomyosines.

Prédiction in silico : application aux tropomyosines

Les tropomyosines se prêtent bien à la prédiction in silico :

  • beaucoup de séquences connues, tant d’allergènes que de « non-allergènes »
  • structure tertiaire non globulaire et donc épitopes a priori linéaires

L’équipe de Li a recherché des motifs particuliers d’AA parmi un collectif d’allergènes et de non-allergènes en utilisant une fenêtre d’exploration de 30 AA et la prise en compte des propriétés physico-chimiques de ces AA. Les motifs retrouvés à la fois sur des allergènes et des non-allergènes sont éliminés. Après quoi la même recherche de motif est effectuée avec la protéine x.

Cette méthode a été appliquée par leurs auteurs à des tropomyosines  : les motifs n° 13, 14 et 15, dénommés « tropomyosin », sont bien retournés par le calcul pour des tropomyosines de crevette ou d’acarien, mais le motif n°15 est aussi donné en réponse pour la tropomyosine de poulet.

Mari et Stadler ont développé un algorithme permettant de classer les allergènes en calculant des motifs également. L’approche est différente car le calcul se base uniquement sur des allergènes (mémorisés dans la BDD Allergome). Tous les allergènes n’ont pas reçu de motif, certains restant donc non classés. On peut voir sur le site Allergome si tel allergène a un motif et consulter la liste des autres allergènes partageant le même motif. La fenêtre de calcul est de 50 AA et donc les motifs sont de 50 AA.

Pour montrer que leur méthode était efficace, les auteurs ont synthétisé le peptide de 50 AA correspondant au motif auquel sont rattachées les tropomyosines . Testée chez des patients positifs pour la crevette, l’IgE-réactivité de ce peptide s’est montrée très bien corrélée avec celle de rPen a 1. De plus, la tropomyosine de poulet ne parvenait pas à inhiber ce peptide. Les auteurs concluaient donc que leur méthode de prédiction in silico marchait très bien.

Ivanciuc est parti d’un autre point de vue : constituer une BDD incluant non seulement les séquences des allergènes mais aussi celles des épitopes. Un indice est calculé pour estimer si la protéine x à tester est susceptible de contenir un épitope croisant potentiel. L’indice tient compte également de différentes propriétés physico-chimiques des AA .

Cette méthode, dite SDAP, a été appliquée en testant la séquence d’AA 143-151 d’un allergène connu, la tropomyosine de crevette Pen i 1. On obtient bien en retour de nombreuses propositions d’allergènes croisants : des tropomyosines de crustacés, de mollusques, d’acariens, etc.. La méthode semble donc marcher et si la séquence d’AA était sur une protéine x, il faudrait retenir pour cette protéine un risque d’allergénicité.

Cependant, plusieurs écueils rendent cette méthode prédictive peu utile :

  • la séquence 143-151 de Pen i 1 renvoie aussi Der p 1 (D. pteronyssinus) comme proposition d’allergène croisant. Der p 1 est une cystéine protéase et non une tropomyosine : sa configuration 3D est tout à fait différente et une réactivité croisée entre Der p 1 et des tropomyosines est quasi-exclue
  • la méthode est limitée par le nombre très faible des séquences connues d’épitopes. Le risque est grand de ne pas trouver de réponse SDAP positive faute d’épitope croisant connu et inclus dans la BDD
  • de plus, les séquences mémorisées dans la BDD correspondent à des épitopes linéaires et non à la majorité des épitopes, c’est-à-dire aux épitopes conformationnels.

Soeria-Atmadja a développé une approche différente : peu importe si telle portion de séquence est un épitope ou non, la question est de savoir si cette séquence est plus souvent rencontrée sur un allergène que sur un non-allergène.

Le calcul, dit FLAP, part donc d’un double collectif de séquences : une base contenant tous les allergènes connus et une autre base comprenant toutes les protéines de certains organismes (moins les protéines homologues d’allergènes connus). D’abord principalement constituée de séquences de protéines de riz , cette base des non-allergènes est dorénavant à plus de 90% constituée de séquences de protéines humaines .

Des « séquences représentatives d’allergènes » (d’au moins 22 AA) sont calculées et mémorisées dans la base. C’est à ces séquences que sera comparée la protéine x à tester.
Le site internet Evaller permet de tester une séquence protéique quelconque et de calculer sa probabilité d’être un allergène .

Les auteurs ont appliqué cette méthode aux tropomyosines . Elle affiche une bonne efficacité globale, bien que certains allergènes ne sont pas retrouvés dans la liste des concordances (ex. Lep d 7, la tropomyosine de Lepidoglyphus ).

Le site Evaller a été interrogé (accès le 19/03/09) avec la séquence de la tropomyosine de poulet. Les réponses ont été :

  • 8,8% de chances pour que cette protéine soit un allergène
  • commentaire : « probablement non allergénique ».
  • des séquences représentatives sont listées : elles correspondent bien à des portions de tropomyosines (moule, turban)
  • mais les % d’identité de ces séquences avec les portions correspondantes sur la tropomyosine de poulet restent modestes (48 à 68%), et a priori insuffisants pour permettre une réactivité croisée.

La méthode semble donc relativement efficace pour les tropomyosines.

Mais elle ne fonctionne pas aussi bien, de l’avis même de ses auteurs, pour des protéines qui ont une conformation globulaire : c’est le cas pour les profilines .

Mari et Stadler ont été confrontés au même problème : leur méthode de prédiction ne donnait pas de bons résultats avec une famille de protéines globulaires, les Mn-SOD (superoxyde dismutases) .

Satisfaisant pour des protéines non globulaires comme les tropomyosines, les calculs basés sur une pure séquence primaire auront donc bien du mal à être efficaces avec des protéines repliées sur elles-mêmes et où les épitopes sont majoritairement discontinus. Et la grande majorité des allergènes connus sont sous forme globulaire …

Au total, les approches bio-informatiques de prédiction d’allergénicité ont encore beaucoup de progrès à faire, même si certaines d’entre elles ont quand même le mérite de produire moins de « faux positifs » que l’approche FAO/OMS.

Ceci étant, une autre dimension du problème, encore plus important, est rarement évoquée dans tous ces travaux qui, souvent, semblent satisfaits de leurs résultats : est-on sûr que les allergènes placés dans les BDD qui effectuent ces calculs prédictifs sont bien des allergènes ?!!

La question peut surprendre. Mais elle est essentielle. En effet, ce qui importe au patient c’est de ne pas (plus) présenter de symptômes. Il lui bien égal que telle protéine migre à x kDa sur une bande de nitrocellulose (et même que son propre sérum l’y détecte) si le contact avec cette protéine dans la vraie vie ne lui provoque aucun symptôme.

Il faut donc, avant toute chose, s’intéresser aux vrais allergènes, ceux qui ont prouvé leur capacité à induire une réponse clinique en conditions réalistes.

Vous avez dit allergène ?

Qu’est-ce qu’un allergène ? Un comité d’experts réuni par l’OMS a défini ainsi ce qu’est un allergène : « An allergen is an antigen causing allergic disease » .

Un allergène conduit donc jusqu’à la maladie.

Une molécule pouvant positiver des tests diagnostiques chez certains sujets mais pour laquelle il n’a pas encore été vérifié qu’elle provoquait également une réponse clinique visible en situation réaliste ne devrait pas être qualifiée d’allergène.

Bien sûr, on peut admettre qu’un test cutané positif est plus proche de la clinique qu’une tache révélée en blot. Mais on sait également qu’une proportion non négligeable des tests cutanés trouvés positifs restera sans implication clinique.

Dans la mesure où les tests cutanés à lecture immédiate font appel à une réaction impliquant des IgE, on pourrait regrouper sous le vocable de « protéines IgE-réactives » toutes les protéines ayant montré une IgE-réactivité in vitro (blots, « RASTs », dégranulation des basophiles,..) et/ou une réactivité cutanée. Le statut d’ « allergène » étant réservé à celles parmi elles qui ont une implication clinique prouvée.

Pour tenter de connaître la proportion de ces allergènes parmi l’ensemble des protéines IgE-réactives connues, une interrogation de la base Allergome a été opérée (accès 01/04/07). Cette BDD est très minutieusement mise à jour et contient, entre autres, une échelle d’allergénicité renseignée pour toutes les molécules mémorisées : IgE-immunoblotting, (quantitative) IgE, basophil test, skin test, provocation test.

Seul le niveau « provocation test » peut être assimilé à une condition réaliste et la molécule qui satisfait à ce niveau être déclarée « allergène ». Sinon, on est seulement en présence d’indices d’IgE-réactivité et donc de « protéines IgE-réactives ».

Le graphique ci-dessous montre la répartition des niveaux maxima atteints par les molécules mémorisées dans Allergome (bilan au 01/04/07) :

Le constat est simple : près de 98% des molécules dans Allergome ne sont pas des allergènes !! Même en incluant les réactivités cellulaires (TC, basophiles), on aurait encore près de 80% de molécules pour lesquelles une dénomination de « protéines IgE-réactives » serait plus honnête.

La conséquence la plus immédiate de ce constat est que les BDD qui alimentent les calculs de prédiction d’allergénicité ne sont pas constituées d’allergènes mais plutôt de protéines IgE-réactives.

Et une prédiction d’allergénicité se référant à un collectif au sein duquel très peu de protéines sont des allergènes prouvés cliniquement ne peut aboutir qu’à une conclusion d’utilité très relative : la protéine est plus ou moins à risque d’IgE-réactivité (notamment in vitro) … mais sans que l’on puisse en déduire que des patients en souffriront !

Sachant que les autres tests prévus dans les protocoles de type FAO sont également très critiquables (digestibilité, banques de sérums), le moyen le plus pertinent pour se rapprocher de la réalité clinique serait de mettre au point un modèle animal pour tester les protéines et estimer leur risque de se comporter comme des allergènes .

[1] - Leduc V, de Lacoste de Laval A, Ledent C, Mairesse M. Allergie respiratoire aux protéines de feuilles, implication d'un nouvel allergène. Rev Fr Allergol Immunol Clin 2008;48:521-525
Les symptômes des sujets présentant une rhinite, une conjonctivite ou une urticaire de contact lors d‚une exposition ou d‚un contact avec des herbes, sont généralement attribués à une allergie aux pollens de graminées ou à certaines moisissures atmosphériques. Les quatre cas décrits ici présentent des symptômes allergiques lors de la tonte de pelouse. La suspicion d‚allergie aux feuilles de pelouse (graminées) a été vérifiée par des tests cutanés natifs. Un extrait de feuille d‚ivraie a été produit et les allergènes analysés par SDS-PAGE et immunoempreinte. Trois des quatre patients ont montré la présence d‚IgE spécifiques vis-à-vis d‚une seule molécule de 56 kDa. Cette protéine est majoritaire dans la feuille, et a été identifiée comme la sous-unité de la ribulose-1,5-diphosphate carboxylase/oxydase, enzyme majeure du règne végétal, impliquée dans la photosynthèse. Cette protéine est universellement présente dans les feuilles et est, de plus, utilisée comme modèle de non-allergénicité, dans les modèles expérimentaux étudiant l‚allergénicité des protéines alimentaires. En effet, elle est instantanément dégradée par les enzymes digestives, contrairement aux principaux allergènes alimentaires connus. L‚allergie respiratoire aux protéines de feuilles est donc montrée dans l‚étude de ces quatre cas, qu‚il y ait ou non existence d‚allergie ou de sensibilisation associée aux pollens de graminées.
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[2] - Gubesch M, Theler B, Dutta M, Baumer B, Mathis A, Holzhauser T, et al. Strategy for allergenicity assessment of "natural novel foods" : clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant). Allergy 2007;62:1243-1250
BACKGROUND: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant . METHODS: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC) . RESULTS: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients . CONCLUSIONS: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.
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[3] - Malandain H. Transglutaminases : a meeting point for wheat allergy, celiac disease, and food safety. Eur Ann Allergy Clin Immunol 2005;37:397-403
Wheat is the staple cereal in many countries and its uses in manufactured foods are ever growing due to the technological qualities of gluten proteins. Transglutaminases (TG) are ubiquitous enzymes with many functions. They are able to transform proteins by deamidation and/or transamidation. This last reaction can cross-link proteins together. Intestinal tissue TG has been shown to play an important role in two kinds of immune reactions to wheat: celiac disease and wheat-dependent exercise-induced anaphylaxis. In addition, new epitopes have been suspected in cases of anaphylaxis to wheat isolates, a food ingredient consisting mainly of deamidated gluten proteins. As a microbial TG is included in many food technological processes, its safe use should be checked. This assessment must cover not only the safety of the TG itself but also that of the deamidated/cross-linked proteins generated by this enzyme. This article aims at discussing the possible consequences of using TG in food industry in the light of today knowledge about immune reactions to wheat.
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[4] - Codex Alimentarius Commission - FAO / WHO Report of the 3rd session of the Codex ad-hoc intergovernmental Task Force on foods derived from biotechnology - Yokohama, Japan, 4-8 March 2002. www.fao.org/codex/alinorm03/Al03_34e.pdf
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[5] - Davies HV. GM organisms and the EU regulatory environment: allergenicity as a risk component. Proc Nutr Soc 2005;64:481-486
The European Food Safety Authority, following a request from the European Commission, has published a guidance document for the risk assessment of GM plants and derived food and feed to assist in the implementation of provisions of Regulation (EC) 1829/2003 of the European Parliament and Council on GM food and feed. This regulation has applied since 18 April 2004. In principle, hazard identification and characterisation of GM crops is conducted in four steps: characterisation of the parent crop and any hazards associated with it; characterisation of the transformation process and of the inserted recombinant DNA, including an assessment of the possible production of new fusion proteins or allergens; assessment of the introduced proteins (toxicity, allergenicity) and metabolites; identification of any other targetted and unexpected alterations in the GM crop, including changes in the plant metabolism resulting in compositional changes and assessment of their toxicological, allergenic or nutritional impact. In relation to allergenicity specifically, it is clear that this property of a given protein is not intrinsic and fully predictable but is a biological activity requiring an interaction with individuals with a predisposed genetic background. Allergenicity, therefore, depends on the genetic diversity and variability in atopic human subjects. Given this lack of complete predictability it is necessary to obtain, from several steps in the risk-assessment process, a cumulative body of evidence that minimises any uncertainty about the protein(s) in question.
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[6] - Gubesch M, Theler B, Dutta M, Baumer B, Mathis A, Holzhauser T, et al. Strategy for allergenicity assessment of "natural novel foods" : clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant). Allergy 2007;62:1243-1250
BACKGROUND: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant . METHODS: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC) . RESULTS: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients . CONCLUSIONS: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.
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[7] - Bindslev-Jensen C, Sten E, Earl LK, Crevel RW, Bindslev-Jensen U, Hansen TK, et al. Assessment of the potential allergenicity of ice structuring protein type III HPLC 12 using the FAO/WHO 2001 decision tree for novel foods. Food Chem Toxicol 2003;41:81-87
The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein. Therefore, decision trees for evaluation of the risk have been developed, the latest being proposed by WHO/FAO early in 2001. Proteins developed using modern biotechnology and derived from fish are being considered for use in food and other applications, and since allergy to fish is well established, a potential risk from such proteins to susceptible human beings exists. The overall aim of the study was to investigate the potential allergenicity of an Ice Structuring Protein (ISP) originating from an arctic fish (the ocean pout, Macrozoarces americanus) using the newly developed decision tree proposed by FAO/WHO. The methods used were those proposed by FAO/WHO including amino acid sequence analysis for sequence similarity to known allergens, methods for assessing degradability under standardised conditions, assays for detection of specific IgE against the protein (Maxisorb RAST) and histamine release from human basophils. In the present paper we describe the serum screening phase of the study and discuss the overall application of the decision tree to the assessment of the potential allergenicity of ISP Type III. In an accompanying paper [Food Chem. Toxicol. 40 (2002) 965], we detail the specific methodology used for the sequence analysis and assessment of resistance to pepsin-catalysed proteolysis of this protein. The ISP showed no sequence similarity to known allergens nor was it stable to proteolytic degradation using standardised methods. Using sera from 20 patients with a well-documented clinical history of fish allergy, positive in skin prick tests to ocean pout, eel pout and eel were used, positive IgE-binding in vitro to extracts of the same fish was confirmed. The sera also elicited histamine release in vitro in the presence of the same extracts. The ISP was negative in all cases in the same experiments. Using the proposed decision tree, we demonstrated the safety of the ISP to patients already sensitised to fish, as well as to individuals potentially susceptible to producing IgE responses to proteins. Furthermore, the practicability of the new decision tree was confirmed.
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[8] - Pedersen MH, Hansen TK, Sten E, Seguro K, Ohtsuka T, Morita A, et al. Evaluation of the potential allergenicity of the enzyme microbial transglutaminase using the 2001 FAO/WHO Decision Tree. Mol Nutr Food Res 2004;48:434-440
All novel proteins must be assessed for their potential allergenicity before they are introduced into the food market. One method to achieve this is the 2001 FAO/WHO Decision Tree recommended for evaluation of proteins from genetically modified organisms (GMOs). It was the aim of this study to investigate the allergenicity of microbial transglutaminase (m-TG) from Streptoverticillium mobaraense. Amino acid sequence similarity to known allergens, pepsin resistance, and detection of protein binding to specific serum immunoglobulin E (IgE) (RAST) have been evaluated as recommended by the decision tree. Allergenicity in the source material was thought unlikely, since no IgE-mediated allergy to any bacteria has been reported. m-TG is fully degraded after 5 min of pepsin treatment. A database search showed that the enzyme has no homology with known allergens, down to a match of six contiguous amino acids, which meets the requirements of the decision tree. However, there is a match at the five contiguous amino acid level to the major codfish allergen Gad c1. The potential cross reactivity between m-TG and Gad c1 was investigated in RAST using sera from 25 documented cod-allergic patients and an extract of raw codfish. No binding between patient IgE and m-TG was observed. It can be concluded that no safety concerns with regard to the allergenic potential of m-TG were identified.
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[9] - Adel-Patient K, Wal JM. Apport et limites des modèles animaux pour l'évaluation de l'allergénicité des OGM. Eur Ann Allergy Clin Immunol 2004;36:88-91
Incidence of IgE-mediated allergic reactions to foods is increasing as well as the severity of associated symptoms and numerous foods are now incriminated, probably in relation with modifications of dietary habits and increased exposure to new or modified food ingredients. Therefore, the introduction on the market of food composed of or derived from genetically modified organisms (GMOs) raised the question of their potential allergenicity. Particularly with regards to the allergenicity of a newly expressed protein, it is necessary to obtain, from several steps in the risk assessment process, a cumulative body of evidence which minimises any uncertainty. This may include the use of animal model despite no fully reliable validated model is available yet. Such animal models should allow to address 3 major issues: Is the novel protein a sensitizer, i.e. does it possess intrinsic properties that allow to sensitize a predisposed individual? Is the protein an elicitor i.e. is it able to elicit an allergic reaction in a sensitised individual? And is the protein an adjuvant, i.e. can it facilitate or enhance the sensitisation to an other protein? Animal models under investigation currently include mice, rats and guinea pigs but models such as dogs and swine also appeared a few years ago. The aim is to mimic the mechanism and characteristics of the sensitisation phase and/or the elicitation phase of the allergic reaction as it occurs in atopic humans. They are necessary because sensitisation studies can obviously not be done in human and because in vitro tests cannot reproduce the complexity of the immune system. We propose a mouse model which mimics both phases of the allergic reaction. It has permitted to evidence that biochemical and clinical manifestations occuring during the active phases of the allergic reaction differ according to the structure of the allergen used for the challenge. This may allow to compare the allergenic potential of a genetically modified protein with that of the conventional one and to identify possible unintended effects. However, pathogenesis of food allergy in human is very complex and multifactorial, including individual differences in susceptibility, environmental factors, conditions of exposure, ... No animal model can take into account all these factors and allow a reliable prediction of the prevalence and severity of allergic reactions which would result from the exposure to a (novel) protein. Nevertheless, point by point analysis using the different models available may provide useful informations on the potential allergenicity of a novel protein.
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[10] - Wal JM. Apport et limites des modèles animaux pour l'évaluation de l'allergénicité des OGM. Alim'Inter 2003;8:91-94
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[11] - Aalberse RC. Presentation of the decisional tree for GMO allergenic safety as proposed by the FAO/WHO Expert Committee. Alim'Inter 2003;8:102-105
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[12] - Spök A, Gaugitsch H, Laffer S, Pauli G, Saito H, Sampson H, et al. Suggestions for the Assessment of the Allergenic Potential of Genetically Modified Organisms. Int Arch Allergy Immunol 2005;137:167-180
The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.
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[13] - Moneret-Vautrin DA. Les plantes transgéniques (OGM végétaux) : connaissances et inconnues sur les risques d'allergénicité…. Rev Fr Allergol Immunol Clin 2006;46:85-91
Les OGM alimentaires d'origine végétale suscitent beaucoup d'attention en raison du postulat d'un risque allergénique. Aucun risque allergique n'a été documenté pour les OGM de première génération conférant aux variétés végétales une résistance aux herbicides et aux larves d'insecte. Les expérimentations actuelles sur des OGM hypoallergéniques sont rapportées et discutées. La seconde génération d'OGM portant sur des améliorations nutritionnelles correspondra à des protéines d'intérêt d'origine végétale, présentes dans une fourchette prévisible de 4 à 8 % du contenu protéique total. L'évaluation d'une potentialité allergénique différente de celle des variétés naturelles devra être examinée au niveau des produits alimentaires ˜ risque d'allergie alimentaire ˜ et au niveau des pollens ˜ risque d'allergie respiratoire pour les populations vivant en régions de culture. Les directives de l'OMSˆFAO du Codex Alimentarius et de l'EFSA prévoient, pour les protéines transgéniques, la recherche d'homologie in silico puis une recherche de réactivité croisée avec les allergènes actuellement identifiés, ainsi qu'une étude précise d'éventuelles modifications du protéome de la plante hôte. Une immunogénicité potentielle devra faire l'objet d'études animales in vivo. Aucune étape ne permet de statuer formellement sur une absence de potentialité allergénique, C'est pourquoi l'ensemble de ces données orientera plus vers une absence de commercialisation de plantes transgéniques ne répondant pas à des critères de sécurité correspondant au poids de l'évidence, qu'elle ne permettra d'affirmer l'absence certaine de risque de produits qui seront commercialisés. La surveillance des OGM végétaux commercialisés sera donc indispensable. Cette revue précise les nécessités de sérothèques publiques de référence, et complète les propositions de sélection des sérums issus de l'OMSˆFAO par des critères précis. Elle propose la mise en place de systèmes d'allergovigilance alliant les agences nationales et européennes de sécurité sanitaire alimentaires et des centres de référence hospitalo-universitaires cliniques et biologiques pour la constitution de ces sérothèques, en partenariat avec des réseaux d'allergologues cliniciens. Ces derniers seront en mesure d'évaluer dans la population le risque de nouvelles sensibilisations des variétés transgéniques comme de signaler les réactions allergiques aux aliments nouveaux, transgéniques en particulier. Un tel projet est en cours de réalisation en France.
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[14] - Prescott VE, Hogan SP. Genetically modified plants and food hypersensitivity diseases: Usage and implications of experimental models for risk assessment. Pharmacol Ther 2006;111:374-383
The recent advances in biotechnology in the plant industry have led to increasing crop production and yield that in turn has increased the usage of genetically modified (GM) food in the human food chain. The usage of GM foods for human consumption has raised a number of fundamental questions including the ability of GM foods to elicit potentially harmful immunological responses, including allergic hypersensitivity. To assess the safety of foods derived from GM plants including allergenic potential, the US FDA, Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), and the EU have developed approaches for evaluation assessment. One assessment approach that has been a very active area of research and debate is the development and usage of animal models to assess the potential allergenicity of GM foods. A number of specific animal models employing rodents, pigs, and dogs have been developed for allergenicity assessment. However, validation of these models is needed and consideration of the criteria for an appropriate animal model for the assessment of allergenicity in GM plants is required. We have recently employed a BALB/c mouse model to assess the potential allergenicity of GM plants. We have been able to demonstrate that this model is able to detect differences in antigenicity and identify aspects of protein post-translational modifications that can alter antigenicity. Furthermore, this model has also enabled us to examine the usage of GM plants as a therapeutic approach for the treatment of allergic diseases. This review discusses the current approaches to assess the allergenic potential of GM food and particularly focusing on the usage of animal models to determine the potential allergenicity of GM foods and gives an overview of our recent findings and implications of these studies.
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[15] - Cantani A. Benefits and concerns associated with biotechnology-derived foods: can additional research reduce children health risks ? Eur Rev Med Pharmacol Sci 2006;10:197-206
The development of techniques devised for the genetic manipulation of foods poses new risks for children with food allergy (FA). The introduction of foreign allergenic proteins from different foods into previously tolerated foods may trigger allergic reactions, often complicating with anaphylactic shock in a subset of allergic babies. Children with FA, even if subjected to preventative diets, always challenge the risk of developing allergic manifestations after unintentional intake of a non tolerated food in restaurant settings, with relatives or schoolmates, etc, where product labelling is necessarily lacking. The introduction of potentially allergenic proteins into foods generally considered safe for allergic children can be done deliberately, by either substantially altering the food ingredients, or by genetic manipulation which change the composition or transfer allergens, or unintentionally by quality-control failures, due to contaminations in the production process, or to genetic mismanipulation. There is a controversy between multinationals often favored by governments and consumer association resistance, thus an equidistant analysis poses some unprecedented impediments. The importance of FA and the potential of transgenic plants to bring food allergens into the food supply should not be disregarded. The expression in soybeans of a Brazil nut protein resulted in a food allergen expressed in widely used infant formulas, so paving the way to an often reported multinational debacle. Genetic engineering poses innovative ethical and social concerns, as well as serious challenges to the environment, human health, animal welfare, and the future of agriculture. In this paper will be emphasized practical concepts more crucial for pediatricians.
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[16] - Thomas K, Bannon G, Herouet-Guicheney C, Ladics G, Lee L, Lee SI, et al. The Utility of an International Sera Bank for Use in Evaluating the Potential Human Allergenicity of Novel Proteins. Toxicol Sci 2007;97:27-31
In the safety assessment of novel foods produced through biotechnology, careful consideration is given to determining the allergenic potential of newly introduced proteins. IgE serum screening is one tool for evaluating whether the protein in question has sequence identity to a known allergen or if the source of the gene encoding the protein is a known allergenic food. A specific serum screen involves testing a gene product with sera from patients with documented clinical allergy to a specific allergen to confirm that the gene product of interest is not the same protein to which the patient produces IgE antibodies. A targeted serum screen involves testing the gene product of interest with sera from patients sensitive to food or aeroallergens from the same broad group. The concept of a global sera bank with accessible, well-characterized sera for use in such assays is an appealing option. This paper summarizes the consensus elements from a workshop to evaluate the potential utility of an international sera bank for evaluating the allergenicity of novel proteins . Areas of agreement following the workshop included: 1) specific sera screens are appropriate for exploring potentially cross-reactive proteins that have been identified through bioinformatics analyses, however, additional validation is needed, particularly for targeted sera screens; 2) practical and ethical considerations may preclude the formation of a global sera bank; and therefore, 3) a regional network of clinicians that could serve as sources of patient sera or be approached to conduct sera studies would be the most practical alternative.
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[17] - Goodman RE, Vieths S, Sampson HA, Hill D, Ebisawa M, Taylor SL, et al. Allergenicity assessment of genetically modified crops—what makes sense ? Nat Biotechnol 2008;26:73-81
GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops.
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[18] - Thomas K, Herouet-Guicheney C, Ladics G, McClain S, MacIntosh S, Privalle L, et al. Current and future methods for evaluating the allergenic potential of proteins: international workshop report 23-25 October 2007. Food Chem Toxicol 2008;46:3219-3225
The International Life Science Institute's Health and Environmental Sciences Institute's Protein Allergenicity Technical Committee hosted an international workshop October 23-25, 2007, in Nice, France, to review and discuss existing and emerging methods and techniques for improving the current weight-of-evidence approach for evaluating the potential allergenicity of novel proteins. The workshop included over 40 international experts from government, industry, and academia. Their expertise represented a range of disciplines including immunology, chemistry, molecular biology, bioinformatics, and toxicology. Among participants, there was consensus that (1) current bioinformatic approaches are highly conservative; (2) advances in bioinformatics using structural comparisons of proteins may be helpful as the availability of structural data increases; (3) proteomics may prove useful for monitoring the natural variability in a plant's proteome and assessing the impact of biotechnology transformations on endogenous levels of allergens, but only when analytical techniques have been standardized and additional data are available on the natural variation of protein expression in non-transgenic bred plants; (4) basophil response assays are promising techniques, but need additional evaluation around specificity, sensitivity, and reproducibility; (5) additional research is required to develop and validate an animal model for the purpose of predicting protein allergenicity.
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[19] - Ofori-Anti AO, Ariyarathna H, Chen L, Lee HL, Pramod SN, Goodman RE. Establishing objective detection limits for the pepsin digestion assay used in the assessment of genetically modified foods. Regul Toxicol Pharmacol 2008;52:94-103
RATIONALE: Guidelines for assessing the potential allergenicity of genetically modified (GM) organisms recommend testing the digestibility of the introduced protein by pepsin. Previous studies detailed the digestion procedure but have not described a simple objective measurement of the extent of digestion nor evaluated the impact of variation in pepsin activity. METHODS: Samples of eight proteins were digested by pepsin at pH 1.2 and 2.0 using standard conditions (10,000 U of pepsin activity per mg test protein) as well as 5000 and 20,000 units per mg of test protein. An independent digestion assay of hemoglobin was used to verify pepsin activity for each assay. Digestion was stopped in timed samples between 0.5 and 60 min. Digestion samples and undigested protein (10% and 100%) were separated by SDS-PAGE. Residual stained protein bands were measured by image analysis. RESULTS: The differences in pH and pepsin concentration only had minor effects on digestion of intermediately stable proteins: concanavalin A, ovalbumin, and lysozyme, but not on rapidly digested or stable proteins. CONCLUSIONS: Verification of pepsin activity and measurement of an objective endpoint of digestion (e.g. (90%) should provide more comparable results for the safety assessment of novel food proteins.
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[20] - Schnell S, Herman RA. Should digestion assays be used to estimate persistence of potential allergens in tests for safety of novel food proteins ?. Clin Mol Allergy 2009;7:1
ABSTRACT: Food allergies affect an estimated 3 to 4% of adults and up to 8% of children in developed western countries. Results from in vitro simulated gastric digestion studies with purified proteins are routinely used to assess the allergenic potential of novel food proteins. The digestion of purified proteins in simulated gastric fluid typically progresses in an exponential fashion allowing persistence to be quantified using pseudo-first-order rate constants or half lives. However, the persistence of purified proteins in simulated gastric fluid is a poor predictor of the allergenic status of food proteins, potentially due to food matrix effects that can be significant in vivo. The evaluation of the persistence of novel proteins in whole, prepared food exposed to simulated gastric fluid may provide a more correlative result, but such assays should be thoroughly validated to demonstrate a predictive capacity before they are accepted to predict the allergenic potential of novel food proteins.
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[21] - Borges JP, Barre A, Culerrier R, Archimbaud N, Didier A, Rougé P. How reliable is the structural prediction of IgE-binding epitopes of allergens? The case study of plant lipid transfer proteins. Biochimie 2007;89:83-91
The linear IgE-binding epitopes of non-specific lipid transfer proteins (nsLTP) from plants were predicted using a combination of predictive tools including (1) the hydropathic profiles based on different scales of hydrophilicity, flexibility and exposure to the solvent, (2) the hydrophobic cluster analysis plots, (3) the occurrence of charged residues in the predicted amino acid sequence stretches and, (4) the exposition of the predicted linear IgE-binding epitopes checked on the three-dimensional models built for the nsLTP. A reliable prediction was obtained for nsLTP as compared with the previously characterized IgE-binding epitopes of various proteins. A consensual IgE-binding epitope occurring in other plant nsLTP and responsible for some IgE-binding cross-reactivity among fruit nsLTP has been identified and characterized. Despite some discrepancies, a fairly good prediction resulted in applying our combination of predictive methods to longer nsLTP or plant profilins.
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[22] - Li KB, Issac P, Krishnan A. Predicting allergenic proteins using wavelet transform. Bioinformatics 2004;20:2572-2578
MOTIVATION: With many transgenic proteins introduced today, the ability to predict their potential allergenicity has become an important issue. Previous studies are based on either sequence similarity or the protein motifs identified from known allergen databases. The similarity based approaches, although being able to produce high recalls, usually have low prediction precisions. Previous motif-based approaches have been shown to be able to improve the precisions on cross-validation experiments. In this study a system that combines the advantages of similarity-based and motif-based prediction is described. RESULTS: The new prediction system uses a clustering algorithm that groups the known allergenic proteins into clusters. Proteins within each cluster are assumed to carry one or more common motifs. After a multiple sequence alignment, proteins in each cluster go through a wavelet analysis program whereby conserved motifs will be identified. An HMM profile will then be prepared for each identified motif. The allergens that don't appear to carry detectable allergen motifs will be saved in a small database. The allergenicity of an unknown protein may be predicted by comparing it against the HMM profiles, and, if no matching profiles can be found, against the small allergen database by BLASTP. Over 70% of recall and over 90% of precision were observed using cross-validation experiments. Using the entire Swiss-Prot as the query, we predicted about two thousand potential allergens. AVAILABILITY: The software is available upon request from the authors.
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[23] - Marti P, Truffer R, Stadler MB, Keller-Gautschi E, Crameri R, Mari A, et al. Allergen motifs and the prediction of allergenicity. Immunol Lett 2007;109:47-55
We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.
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[24] - Ivanciuc O, Schein CH, Braun W. Data mining of sequences and 3D structures of allergenic proteins. Bioinformatics 2002;18:1358-1364
Motivation: Many sequences, and in some cases structures, of proteins that induce an allergic response in atopic individuals have been determined in recent years. This data indicates that allergens, regardless of source, fall into discreet protein families. Similarities in the sequence may explain clinically observed cross-reactivities between different biological triggers. However, previously available allergy databases group allergens according to their biological sources, or observed clinical cross-reactivities, without providing data about the proteins. A computer-aided data mining system is needed to compare the sequential and structural details of known allergens. This information will aid in predicting allergenic cross-responses and eventually in determining possible common characteristics of IgE recognition. Results: The new web-based Structural Database of Allergenic Proteins (SDAP) permits the user to quickly compare the sequence and structure of allergenic proteins. Data from literature sources and previously existing lists of allergens are combined in a MySQL interactive database with a wide selection of bioinformatics applications. SDAP can be used to rapidly determine the relationship between allergens and to screen novel proteins for the presence of IgE or T-cell epitopes they may share with known allergens. Further, our novel similarity search method, based on five dimensional descriptors of amino acid properties, can be used to scan the SDAP entries with a peptide sequence. For example, when a known IgE binding epitope from shrimp tropomyosin was used as a query, the method rapidly identified a similar sequence in known shellfish and insect allergens. This prediction of cross-reactivity between allergens is consistent with clinical observations. Availability: SDAP is available on the web at http://fermi.utmb.edu/SDAP/index.html Contact: werner@newton.utmb.edu
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[25] - Björklund AK, Soeria-Atmadja D, Zorzet A, Hammerling U, Gustafsson MG. Supervised identification of allergen-representative peptides for in silico detection of potentially allergenic proteins. Bioinformatics 2005;21:39-50
Motivation: Identification of potentially allergenic proteins is needed for safety assessment of genetically modified foods, certain pharmaceuticals and various other products on the consumer market. Current methods in bioinformatic allergology exploit common features among allergens for detection of amino acid sequences of potentially allergenic proteins. Features for identification still unexplored are motifs occurring commonly in allergens, but rarely in ordinary proteins. In this paper we present an algorithm for the identification of such motifs with the purpose of biocomputational detection of amino acid sequences of potential allergens. Results: Identification of allergen-representative peptides (ARPs) with low or no occurrence in proteins lacking allergenic properties is the essential component of our new method, designated DASARP (Detection based on Automated Selection of Allergen-Representative Peptides). This approach consistently outperforms the criterion based on identical peptide match for predicting allergenicity recommended by ILSI/IFBC and FAO/WHO and show comparable results to the alignment-based criterion as outlined by FAO/WHO. Availability: The detection software and the ARP set needed for the analysis of a query protein reported here are properties of the Swedish National Food Agency and are available upon request. The protein sequence sets used in this work are publicly available on: http://www.slv.se/templatesSLV/SLV_Page____9343.asp. Allergenicity assessment for specific protein sequences of interest is also possible via ulfh@slv.se.
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[26] - Soeria-Atmadja D, Lundell T, Gustafsson MG, Hammerling U. Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning. Nucleic Acids Res 2006;34:3779-3793
The placing of novel or new-in-the-context proteins on the market, appearing in genetically modified foods, certain bio-pharmaceuticals and some household products leads to human exposure to proteins that may elicit allergic responses. Accurate methods to detect allergens are therefore necessary to ensure consumer/patient safety. We demonstrate that it is possible to reach a new level of accuracy in computational detection of allergenic proteins by presenting a novel detector, Detection based on Filtered Length-adjusted Allergen Peptides (DFLAP). The DFLAP algorithm extracts variable length allergen sequence fragments and employs modern machine learning techniques in the form of a support vector machine. In particular, this new detector shows hitherto unmatched specificity when challenged to the Swiss-Prot repository without appreciable loss of sensitivity. DFLAP is also the first reported detector that successfully discriminates between allergens and non-allergens occurring in protein families known to hold both categories. Allergenicity assessment for specific protein sequences of interest using DFLAP is possible via ulfh@slv.se.
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[27] - Martinez Barrio A, Soeria-Atmadja D, Nistér A, Gustafsson MG, Hammerling U, Bongcam-Rudloff E. EVALLER: a web server for in silico assessment of potential protein allergenicity. Nucleic Acids Res 2007;35:W694-W700
Bioinformatics testing approaches for protein allergenicity, involving amino acid sequence comparisons, have evolved appreciably over the last several years to increased sophistication and performance. EVALLER, the web server presented in this article is based on our recently published 'Detection based on Filtered Length-adjusted Allergen Peptides' (DFLAP) algorithm, which affords in silico determination of potential protein allergenicity of high sensitivity and excellent specificity. To strengthen bioinformatics risk assessment in allergology EVALLER provides a comprehensive outline of its judgment on a query protein's potential allergenicity. Each such textual output incorporates a scoring figure, a confidence numeral of the assignment and information on high- or low-scoring matches to identified allergen-related motifs, including their respective location in accordingly derived allergens. The interface, built on a modified Perl Open Source package, enables dynamic and color-coded graphic representation of key parts of the output. Moreover, pertinent details can be examined in great detail through zoomed views. The server can be accessed at http://bioinformatics.bmc.uu.se/evaller.html.
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[28] - Soeria-Atmadja D, Lundell T, Gustafsson MG, Hammerling U. Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning. Nucleic Acids Res 2006;34:3779-3793
The placing of novel or new-in-the-context proteins on the market, appearing in genetically modified foods, certain bio-pharmaceuticals and some household products leads to human exposure to proteins that may elicit allergic responses. Accurate methods to detect allergens are therefore necessary to ensure consumer/patient safety. We demonstrate that it is possible to reach a new level of accuracy in computational detection of allergenic proteins by presenting a novel detector, Detection based on Filtered Length-adjusted Allergen Peptides (DFLAP). The DFLAP algorithm extracts variable length allergen sequence fragments and employs modern machine learning techniques in the form of a support vector machine. In particular, this new detector shows hitherto unmatched specificity when challenged to the Swiss-Prot repository without appreciable loss of sensitivity. DFLAP is also the first reported detector that successfully discriminates between allergens and non-allergens occurring in protein families known to hold both categories. Allergenicity assessment for specific protein sequences of interest using DFLAP is possible via ulfh@slv.se.
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[29] - Björklund AK, Soeria-Atmadja D, Zorzet A, Hammerling U, Gustafsson MG. Supervised identification of allergen-representative peptides for in silico detection of potentially allergenic proteins. Bioinformatics 2005;21:39-50
Motivation: Identification of potentially allergenic proteins is needed for safety assessment of genetically modified foods, certain pharmaceuticals and various other products on the consumer market. Current methods in bioinformatic allergology exploit common features among allergens for detection of amino acid sequences of potentially allergenic proteins. Features for identification still unexplored are motifs occurring commonly in allergens, but rarely in ordinary proteins. In this paper we present an algorithm for the identification of such motifs with the purpose of biocomputational detection of amino acid sequences of potential allergens. Results: Identification of allergen-representative peptides (ARPs) with low or no occurrence in proteins lacking allergenic properties is the essential component of our new method, designated DASARP (Detection based on Automated Selection of Allergen-Representative Peptides). This approach consistently outperforms the criterion based on identical peptide match for predicting allergenicity recommended by ILSI/IFBC and FAO/WHO and show comparable results to the alignment-based criterion as outlined by FAO/WHO. Availability: The detection software and the ARP set needed for the analysis of a query protein reported here are properties of the Swedish National Food Agency and are available upon request. The protein sequence sets used in this work are publicly available on: http://www.slv.se/templatesSLV/SLV_Page____9343.asp. Allergenicity assessment for specific protein sequences of interest is also possible via ulfh@slv.se.
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[30] - Soeria-Atmadja D, Lundell T, Gustafsson MG, Hammerling U. Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning. Nucleic Acids Res 2006;34:3779-3793
The placing of novel or new-in-the-context proteins on the market, appearing in genetically modified foods, certain bio-pharmaceuticals and some household products leads to human exposure to proteins that may elicit allergic responses. Accurate methods to detect allergens are therefore necessary to ensure consumer/patient safety. We demonstrate that it is possible to reach a new level of accuracy in computational detection of allergenic proteins by presenting a novel detector, Detection based on Filtered Length-adjusted Allergen Peptides (DFLAP). The DFLAP algorithm extracts variable length allergen sequence fragments and employs modern machine learning techniques in the form of a support vector machine. In particular, this new detector shows hitherto unmatched specificity when challenged to the Swiss-Prot repository without appreciable loss of sensitivity. DFLAP is also the first reported detector that successfully discriminates between allergens and non-allergens occurring in protein families known to hold both categories. Allergenicity assessment for specific protein sequences of interest using DFLAP is possible via ulfh@slv.se.
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[31] - Marti P, Truffer R, Stadler MB, Keller-Gautschi E, Crameri R, Mari A, et al. Allergen motifs and the prediction of allergenicity. Immunol Lett 2007;109:47-55
We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.
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[32] - Johansson SGO, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey RF, et al. Revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol 2004;113:832-836
The nomenclature proposed in the October 2003 report of the Nomenclature Review Committee of the World Allergy Organization is an update of the European Academy of Allergology and Clinical Immunology Revised Nomenclature for Allergy Position Statement published in 2001. The nomenclature can be used independently of target organ or patient age group and is based on the mechanisms that initiate and mediate allergic reactions. It is assumed that as knowledge about basic causes and mechanisms improves, the nomenclature will need further review
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[33] - Prescott VE, Hogan SP. Genetically modified plants and food hypersensitivity diseases: Usage and implications of experimental models for risk assessment. Pharmacol Ther 2006;111:374-383
The recent advances in biotechnology in the plant industry have led to increasing crop production and yield that in turn has increased the usage of genetically modified (GM) food in the human food chain. The usage of GM foods for human consumption has raised a number of fundamental questions including the ability of GM foods to elicit potentially harmful immunological responses, including allergic hypersensitivity. To assess the safety of foods derived from GM plants including allergenic potential, the US FDA, Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), and the EU have developed approaches for evaluation assessment. One assessment approach that has been a very active area of research and debate is the development and usage of animal models to assess the potential allergenicity of GM foods. A number of specific animal models employing rodents, pigs, and dogs have been developed for allergenicity assessment. However, validation of these models is needed and consideration of the criteria for an appropriate animal model for the assessment of allergenicity in GM plants is required. We have recently employed a BALB/c mouse model to assess the potential allergenicity of GM plants. We have been able to demonstrate that this model is able to detect differences in antigenicity and identify aspects of protein post-translational modifications that can alter antigenicity. Furthermore, this model has also enabled us to examine the usage of GM plants as a therapeutic approach for the treatment of allergic diseases. This review discusses the current approaches to assess the allergenic potential of GM food and particularly focusing on the usage of animal models to determine the potential allergenicity of GM foods and gives an overview of our recent findings and implications of these studies.
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