Cross-reactive epitopes and their role in food allergy.

Allergenic cross-reactivity among food allergens complicates the diagnosis and management of food allergy. This can result in many patients being sensitized (having allergen-specific IgE) to foods without exhibiting clinical reactivity. Some food groups such as shellfish, fish, tree nuts, and peanuts have very high rates of cross-reactivity. In contrast, relatively low rates are noted for grains and milk, whereas many other food families have variable rates of cross-reactivity or are not well studied. Although classical cross-reactive carbohydrate determinants are clinically not relevant, α-Gal in red meat through tick bites can lead to severe reactions. Multiple sensitizations to tree nuts complicate the diagnosis and management of patients allergic to peanut and tree nut. This review discusses cross-reactive allergens and cross-reactive carbohydrate determinants in the major food groups, and where available, describes their B-cell and T-cell epitopes. The clinical relevance of these cross-reactive B-cell and T-cell epitopes is highlighted and their possible impact on allergen-specific immunotherapy for food allergy is discussed.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Identification of Pru du 6 as a potential marker allergen for almond allergy.

BACKGROUND: Oral food challenges have demonstrated that diagnosis of almond allergy based on extract-sIgE tests displays low specificity. Molecular allergy diagnosis is expected to improve accuracy, but its value in diagnosing almond allergy remains unknown. The aim of this study was to identify relevant almond allergens and examine their ability to improve almond allergy diagnosis. METHODS: IgE-reactive proteins were purified from almond kernels. IgE binding to almond extract and the allergens was analyzed by quantitative ELISA using sera from 18 subjects with a proven almond allergy. The control group consisted of sera from 18 subjects allergic to peanut and/or tree nuts but tolerant to almond. RESULTS: Three IgE-binding proteins were identified: legumin (Pru du 6), alpha-hairpinin (Pru du 8), and mandelonitrile lyase (Pru du 10). Positive IgE (≥0.35 kU/L) to almond extract showed 94% sensitivity but only 33% specificity. IgE to Pru du 6 maintained high sensitivity (83%) and provided superior specificity (78%). Sera from almond-allergic subjects had significantly higher IgE levels to almond extract (P < .0001) and Pru du 6 (P < .0001) than sera from tolerant donors. Sensitization to Pru du 6 was highly specific for almond allergy, while frequencies of sensitization to legumins from peanut, walnut, hazelnut, and cashew were similar in both groups. IgE to Pru du 8 and Pru du 10 was less sensitive (41% and 67%), but showed specificities of 100% and 61%. CONCLUSION: The use of almond allergens markedly increases the diagnostic specificity compared to the extract. Pru du 6 is a potential new molecular marker for almond allergy.

Development of a novel Ara h 2 hypoallergen with no IgE binding or anaphylactogenic activity.

BACKGROUND: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects. OBJECTIVE: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity. METHODS: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract. RESULTS: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice. CONCLUSIONS: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated.

Evidence for a Role of TGF-ß-Activated Kinase 1 and MAP3K7 Binding Protein 3 in Peanut-Specific T-Cell Responses.

Peanut allergy is considered to be the most common cause for food-induced anaphylaxis. Currently, no approved treatment is available. Avoidance is the only measure to prevent anaphylactic reactions to peanuts. T-helper cells are of special importance for the sensitization process and the maintenance of allergic inflammation. Identifying markers of allergen-specific T-cell responses may help to develop novel treatment approaches. Therefore, we aimed to define new T-cell target genes in Ara h 2-specific T cells and to investigate the possibility of using them as biomarkers of peanut allergy in peripheral blood mononuclear cells (PBMCs). We performed whole mRNA array analysis (whole human genome oligo microarray) of in vitro expanded Ara h 2-specific T cells (CFSElowCD3+CD4+) from 5 peanut-allergic (PA) and 5 non-peanut-sensitized individuals. Expression of selected genes as a result of a two-step bioinformatic approach was confirmed in a second cohort by quantitative PCR. TGF-ß- activated kinase 1 and MAP3K7 binding protein 3 (TAB3), calcium/calmodulin-dependent protein kinase type IV (CAMK4) and HemK methyltransferase family member 1 (HEMK1) were significantly upregulated in Ara h 2-specific T cells of PA patients. In addition, the expression of these genes was also assessed in unstimulated PBMCs from a cohort (n = 43) of PA, atopic non-PA, and nonatopic controls. Interestingly, in unstimulated PBMCs, TAB3 expression was significantly downregulated in PA patients compared to atopic non-PA individuals. Thus, TAB3 may play a significant role at the level of T-cell activation and may also be a candidate biomarker for PA.

Differential T-helper cell polarization after allergen-specific stimulation of autologous dendritic cells in polysensitized allergic patients.

BACKGROUND: Dendritic cells (DCs) play an important role in the induction and regulation of adaptive immune responses by polarizing T-helper (Th) cells. In allergic disease this response is dominated by Th2 cells. It is still unclear whether the activation of Th cells by DCs in atopic individuals is allergen specific. METHODS: Monocyte-derived DCs (MoDCs) obtained from polysensitized patients were stimulated with purified Bet v 1, Phl p 5 and Act d 10, and the surface marker expression was analysed. Proliferation and cytokine profiles of autologous naïve CD4+ T cells co-cultured with allergen-pulsed MoDCs were assessed. RESULTS: The addition of either Bet v 1 or Phl p 5 did not further increase the expression of surface markers from matured MoDCs in all study groups. In co-cultures, autologous naïve CD4+ T cells proliferated when DCs obtained from individuals allergic to birch and grass pollen were stimulated with Bet v 1 and Phl p 5, respectively. In the co-culture supernatants, significantly increased levels of IL-5 and IL-13 were detected. This effect correlated with the sensitization background and was absent when applying an unspecific allergen, Act d 10. The levels of IL-10 in supernatants of MoDCs and the levels of IL-10 and IFN-γ in supernatants of T cells remained unchanged upon stimulation with allergens. CONCLUSIONS: In this study we observed that allergen-specific stimulation of MoDCs induces T-cell proliferation and upregulation of Th2-type cytokines. Interestingly, this Th2 polarization was only observed in cells stimulated with the allergen to which the patients were sensitized.

Component-resolved IgE profiles in Austrian patients with a convincing history of peanut allergy.

BACKGROUND: Peanut allergy develops after primary sensitization to peanut allergens and/or IgE cross-sensitization with homologous allergens from various plants. Therefore, heterogeneous patterns of sensitization to individual peanut allergens are observed in different countries. The aim of this study was to examine the IgE sensitization patterns of Austrian peanut-allergic patients. METHODS: Sera from 65 peanut-allergic patients and 20 peanut-tolerant atopics were obtained in four Austrian allergy clinics. Sensitization patterns against peanut allergens Ara h 1-3, 6, 8 and 9 were identified by ImmunoCAP and ImmunoCAP ISAC. RESULTS: Austrian peanut-allergic patients were sensitized to Ara h 2 and 6 (71%), followed by Ara h 1 (62%), Ara h 8 (45%), Ara h 3 (35%) and Ara h 9 (11%). All sera containing Ara h 2-specific IgE were also positive for Ara h 6, with Ara h 6-specific IgE levels significantly (p < 0.05) higher compared with Ara h 2. Twelve percent displayed IgE reactivity exclusively to Ara h 8. Peanut extract and Ara h 8 showed low diagnostic specificities of 25 and 10%, respectively. The other peanut allergens showed 100% specificity. Diagnostic sensitivities determined by ImmunoCAP ISAC and ImmunoCAP were highly similar for Ara h 2, 3 and 8. CONCLUSIONS: The majority of symptomatic peanut-allergic patients are sensitized to Ara h 2 and Ara h 6. In peanut-symptomatic patients with additional birch pollen allergy, other peanut allergens, especially Ara h 8, should be tested when IgE reactivity to Ara h 2 is absent.

Applications of Molecular Diagnostic Testing in Food Allergy.

IgE-mediated food allergy is a relevant health problem inducing symptoms ranging from mild local reactions up to severe life-threatening situations. Currently, no immunotherapy is available and avoidance of the incriminating food is the method of choice. Therefore, reliable diagnostic tools to formulate dietary recommendations and to avoid unnecessary exclusion diets for the individual patient are urgently needed. This review provides an update on the current knowledge on food allergens and their application in various diagnostic approaches such as skin prick test, basophil activation test, and serum IgE testing. Furthermore, these new approaches are discussed and compared to conventional extract-based assays and correlated to the gold standard of food allergy diagnosis, the double-blind placebo-controlled food challenge. Finally, the application of food allergens for preventive measurements such as allergen detection assays and the determination of threshold levels for allergen levels are discussed.

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed.

Do lipids influence the allergic sensitization process?

Allergic sensitization is a multifactorial process that is not only influenced by the allergen and its biological function per se but also by other small molecular compounds, such as lipids, that are directly bound as ligands by the allergen or are present in the allergen source. Several members of major allergen families bind lipid ligands through hydrophobic cavities or electrostatic or hydrophobic interactions. These allergens include certain seed storage proteins, Bet v 1-like and nonspecific lipid transfer proteins from pollens and fruits, certain inhalant allergens from house dust mites and cockroaches, and lipocalins. Lipids from the pollen coat and furry animals and the so-called pollen-associated lipid mediators are codelivered with the allergens and can modulate the immune responses of predisposed subjects by interacting with the innate immune system and invariant natural killer T cells. In addition, lipids originating from bacterial members of the pollen microbiome contribute to the outcome of the sensitization process. Dietary lipids act as adjuvants and might skew the immune response toward a TH2-dominated phenotype. In addition, the association with lipids protects food allergens from gastrointestinal degradation and facilitates their uptake by intestinal cells. These findings will have a major influence on how allergic sensitization will be viewed and studied in the future.

Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.

BACKGROUND: Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. OBJECTIVE: We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. METHODS: Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1-sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. RESULTS: rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. CONCLUSION: The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific.

Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1.

Beta-parvalbumins from different fish species have been identified as the main elicitors of IgE-mediated reactions in fish-allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross-reactivity among fish parvalbumins. In addition, the high-pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca(2+) -free forms for potential use in immunotherapy.

Developing therapies for peanut allergy.

Peanut allergy is an IgE-mediated, persisting immune disorder that is of major concern worldwide. Currently, no routine immunotherapy is available to treat this often severe and sometimes fatal food allergy. Traditional subcutaneous allergen immunotherapy with crude peanut extracts has proven not feasible due to the high risk of severe systemic side effects. The allergen-specific approaches under preclinical and clinical investigation comprise subcutaneous, oral, sublingual and epicutaneous immunotherapy with whole-peanut extracts as well as applications of hypoallergenic peanut allergens or T cell epitope peptides. Allergen-nonspecific approaches include monoclonal anti-IgE antibodies, TCM herbal formulations and Toll-like receptor 9-based immunotherapy. The potential of genetically engineered plants with reduced allergen levels is being explored as well as the beneficial influence of lactic acid bacteria and soybean isoflavones on peanut allergen-induced symptoms. Although the underlying mechanisms still need to be elucidated, several of these strategies hold great promise. It can be estimated that individual strategies or a combination thereof will result in a successful immunotherapy regime for peanut-allergic individuals within the next decade.

Cross-reactivity of peanut allergens.

Peanut seeds are currently widely used as source of human food ingredients in the United States of America and in European countries due to their high quality protein and oil content. This article describes the classification and molecular biology of peanut seed allergens with particular reference to their cross-reactivities. Currently, the IUIS allergen nomenclature subcommittee accepts 12 peanut allergens. Two allergens belong to the cupin and four to the prolamin superfamily, and six are distributed among profilins, Bet v 1-like proteins, oleosins, and defensins. Clinical observations frequently report an association of peanut allergy with allergies to legumes, tree nuts, seeds, fruits and pollen. Molecular cross-reactivity has been described between members of the Bet v 1-like proteins, the non-specific lipid transfer proteins, and the profilins. This review also addresses the less well-studied cross-reactivity between cupin and prolamin allergens of peanuts and of other plant food sources and the recently discovered cross-reactivity between peanut allergens of unrelated protein families.

IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3.

BACKGROUND: Ara h 1, a vicilin; Ara h 2, a 2S albumin; and Ara h 3, a legumin, are major peanut allergens. Ara h 2 is an important predictor of clinical reactivity to peanut, but cosensitization to all 3 allergens is correlated with the severity of patients' symptoms. OBJECTIVE: We investigated whether cosensitization to these 3 allergens is caused by IgE cross-reactivity, despite the fact that they do not display obvious structural or sequence similarities. METHODS: IgE cross-inhibitions were performed with purified Ara h 1, Ara h 2, and Ara h 3 and IgG-depleted sera from 10 patients with peanut allergy. After an in silico search for similar peptides, IgE ELISA inhibition assays with synthetic peptides were performed. RESULTS: Ara h 2 inhibited IgE binding to Ara h 1 (average, 86% ± 13%) and Ara h 3 (average, 96% ± 6%). IgE binding to Ara h 2 was inhibited by Ara h 1 by 78% ± 15% and by Ara h 3 by 80% ± 6%. A subsequent sequence comparison showed that these nonhomologous allergens contained several similar surface-exposed peptides. IgE binding to Ara h 2-derived peptides was completely inhibited by Ara h 1 and Ara h 3. A mixture of these peptides reduced IgE binding to Ara h 1 and Ara h 3 by 20% to 60% and to Ara h 2 by 49% to 89%. CONCLUSION: Occurrence of similar sequences in the 3 major peanut allergens accounts for the high extent of cross-reactivity among them.

Kiwifruit allergy across Europe: clinical manifestation and IgE recognition patterns to kiwifruit allergens.

BACKGROUND: Kiwifruit is a common cause of food allergy. Symptoms range from mild to anaphylactic reactions. OBJECTIVE: We sought to elucidate geographic differences across Europe regarding clinical patterns and sensitization to kiwifruit allergens. Factors associated with the severity of kiwifruit allergy were identified, and the diagnostic performance of specific kiwifruit allergens was investigated. METHODS: This study was part of EuroPrevall, a multicenter European study investigating several aspects of food allergy. Three hundred eleven patients with kiwifruit allergy from 12 countries representing 4 climatic regions were included. Specific IgE to 6 allergens (Act d 1, Act d 2, Act d 5, Act d 8, Act d 9, and Act d 10) and kiwifruit extract were tested by using ImmunoCAP. RESULTS: Patients from Iceland were mainly sensitized to Act d 1 (32%), those from western/central and eastern Europe were mainly sensitized to Act d 8 (pathogenesis-related class 10 protein, 58% and 44%, respectively), and those from southern Europe were mainly sensitized to Act d 9 (profilin, 31%) and Act d 10 (nonspecific lipid transfer protein, 22%). Sensitization to Act d 1 and living in Iceland were independently and significantly associated with severe kiwifruit allergy (odds ratio, 3.98 [P = .003] and 5.60 [P < .001], respectively). Using a panel of 6 kiwifruit allergens in ImmunoCAP increased the diagnostic sensitivity to 65% compared with 20% for skin prick tests and 46% ImmunoCAP using kiwi extract. CONCLUSION: Kiwifruit allergen sensitization patterns differ across Europe. The use of specific kiwifruit allergens improved the diagnostic performance compared with kiwifruit extract. Sensitization to Act d 1 and living in Iceland are strong risk factors for severe kiwifruit allergy.

Poppy Seed Allergy: Molecular Diagnosis and Cross-Reactivity With Tree Nuts.

BACKGROUND: Poppy seed (PS) can be a cause of severe allergic reactions, especially in individuals with concurrent allergy to tree nuts and other seeds, but diagnostic criteria and sensitization patterns are lacking. OBJECTIVE: To assess the role of PS extract and individual allergens in diagnosing PS allergy and their cross-reactivities with tree nuts and buckwheat. METHODS: Our retrospective study included 36 PS-sensitized patients; 10 with a positive and 26 with a negative oral food challenge (OFC). We identified individual PS allergens and compared the diagnostic performance of specific IgE (sIgE) to PS extract with its allergens. Cross-reactivities between PS and related allergens from other seeds were assessed by a competitive enzyme-linked immunosorbent assay. RESULTS: We identified 4 novel PS allergens: Pap s 1 (vicilin), Pap s 1 (27-424) (α-hairpinin), Pap s 2 (legumin), and Pap s 3 (small hydrophilic seed protein). A positive OFC correlated with higher PS-sIgE levels and elevated sIgE levels for the PS allergens, except for Pap s 3. PS and α-hairpinin-sIgE effectively differentiated allergic from tolerant patients, with area under the curve values of 0.95 and 0.94. PS-sIgE >10.00 kUA/L exhibited 90% sensitivity and 73% specificity, whereas α-hairpinin-sIgE >2.60 kUA/L showed 100% sensitivity and 77% specificity. PS vicilin and legumin highly cross-reacted with hazelnut and buckwheat homologs, whereas α-hairpinin-sIgE cross-reacted with the related almond allergen. CONCLUSIONS: This is the most extensive study on PS allergy to date. PS and α-hairpinin-sIgE are highly sensitive indicators of clinical reactivity to PS, whereas vicilin and legumin-sIgE contribute to concurrent sensitization to hazelnut and buckwheat.