Cosensitization to the 3 Nonhomologous Major Cashew Allergens Ana o 1, Ana o 2, and Ana o 3 Is Caused by IgE Cross-reactivity

Background: Cashew nuts often cause strong allergic reactions, which are even more severe than those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin), and Ana o 3 (2S albumin) are major cashew allergens. Cosensitization to all 3 nonhomologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. Methods: IgE cross-inhibitions were performed with Ana o 1-3 using serum samples from cashew nut–allergic patients. The related hazelnut allergens Cor a 11, 9, and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using serum samples from hazelnut-allergic patients. Results: The median percentages of cross-inhibition between Ana o 1, 2, and 3 were 84%-99%. In comparison, the median cross- inhibition values between hazelnut allergens were 33%-62%. The IC50 values revealed the highest IgE affinity to be to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE binding to Ana o 1, 2, and 3, with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. Potentially cross-reactive peptides of Ana o 3 identified in silico overlapped with previously reported IgE epitopes of all 3 allergens. Conclusion: IgE with high affinity to Ana o 3 that cross-reacts with the other 2 major nonhomologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE types comprise the major fraction of specific IgE in cashew-allergic patients and might be responsible for cross-reactivity between unrelated tree nuts (AU)

Antecedentes: Los anacardos suelen provocar fuertes reacciones alérgicas, que son incluso más graves que las del maní. Ana o 1 (vicilina), Ana o 2 (legumina) y Ana o 3 (albúmina 2S) son los principales alérgenos del anacardo. Se ha observado cosensibilización a los 3 alérgenos no homólogos del anacardo. Nuestra hipótesis es que esto podría deberse a la reactividad cruzada de la IgE. Métodos : Se realizaron inhibiciones cruzadas de IgE con Ana o 1-3 utilizando muestras de suero de pacientes alérgicos al anacardo. Como controles se utilizaron los alérgenos de avellana relacionados Cor a 11, 9 y 14. A modo de comparación, se investigó la reactividad cruzada de IgE entre los alérgenos de la avellana utilizando muestras de suero de pacientes alérgicos a la avellana. Resultados : Los porcentajes medios de inhibición cruzada entre Ana o 1, 2 y 3 fueron del 84% al 99%. En comparación, los valores medios de inhibición cruzada entre alérgenos de avellana fueron del 33% al 62%. Los valores de IC50 revelaron que la mayor afinidad de IgE era Ana o 3 y Cor a 14. La legumina de avellana Cor a 9 inhibió la unión de IgE a Ana o 1, 2 y 3, con porcentajes medios de 75%, 56% y 48%. , respectivamente. No se observó reactividad cruzada entre vicilinas alergénicas ni entre albúminas 2S de anacardo y avellana. Los péptidos potencialmente de reacción cruzada de Ana o 3 identificados in silico se superpusieron con epítopos de IgE previamente informados de los 3 alérgenos. Conclusión : La IgE con alta afinidad por Ana o 3 que reacciona de forma cruzada con los otros 2 alérgenos principales no homólogos del anacardo podría ser responsable de la alta potencia alergénica del anacardo. Estos tipos de IgE de reacción cruzada comprenden la fracción principal de IgE específica en pacientes alérgicos al anacardo y podrían ser responsables de la reactividad cruzada entre frutos secos no relacionados (AU)

Co-sensitization to the three non-homologous major cashew allergens Ana o 1, Ana o 2 and Ana o 3 is caused by IgE cross-reactivity.

BACKGROUND: Cashew nuts often cause strong allergic reactions, even exceeding those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) are major cashew allergens. Co-sensitization to all three non-homologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. METHODS: IgE cross-inhibitions were performed with Ana o 1-3 using sera from cashew nut allergic patients. Related hazelnut allergens Cor a 11, 9 and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using sera from hazelnut allergic patients. RESULTS: Median percentages of cross-inhibitions between Ana o 1-3 were 84-99%. In comparison, medians of cross-inhibitions between hazelnut allergens were 33-62%. The IC50 values revealed the highest IgE affinity to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE-binding to Ana o 1, 2, and 3 with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. In silico identified potentially cross-reactive peptides of Ana o 3 overlapped with previously reported IgE epitopes of all three allergens. CONCLUSIONS: IgE with high affinity to Ana o 3 that cross-reacts with the other two major non-homologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE comprises the major fraction of specific IgE in cashew allergic patients, and might be responsible for cross-reactivity between unrelated tree nuts.

Concomitant sensitization to legumin, Fag e 2 and Fag e 5 predicts buckwheat allergy.

BACKGROUND: Buckwheat (Fagopyrum esculentum) has become increasingly popular as a healthy food in Europe. However, for sensitized individuals, consumption can cause anaphylactic reactions. The aim of this study was to identify individual well-characterized buckwheat allergens for component-resolved diagnosis. METHODS: Patients were selected by positive skin prick test to buckwheat and divided into two groups: (1) sensitized to buckwheat without clinical symptoms and (2) buckwheat allergy. Buckwheat proteins were extracted from raw buckwheat seeds, purified applying a combination of protein precipitation and chromatographic methods, and analyzed by IgE immunoblotting and ELISA. RESULTS: Buckwheat-allergic patients had a significantly larger median skin prick test weal diameter for buckwheat than the sensitized group and the positive control. Also, IgE immunoblotting clearly showed a distinct pattern in sera from allergic patients when compared to sensitized individuals. Several IgE-reactive proteins were purified from crude buckwheat extract, namely legumin (Fag e 1 plus its large subunit), Fag e 2 (2S albumin), and newly identified Fag e 5 (vicilin-like) as well as hevein-like antimicrobial peptides, designated Fag e 4. All four allergens showed superior diagnostic precision compared to extract-based ImmunoCAP with high sensitivity as well as high specificity. CONCLUSIONS: Patients with clinical symptoms clearly show a distinct allergen recognition pattern. We characterized a buckwheat vicilin-like protein as a new relevant marker allergen, designated Fag e 5. Additionally, another new allergen, Fag e 4, potentially important for cross-reactivity to latex was added to the allergen panel of buckwheat. Further, our data show that the full-length legumin comprising both, large and small subunit should be applied for component-resolved diagnosis. Our data indicate that concomitant sensitization to legumin, Fag e 2 and Fag e 5, predicts buckwheat allergy.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

BACKGROUND: Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. METHODS: Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. RESULTS: Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. CONCLUSION: Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.

Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences.

The IUIS Allergen Nomenclature Sub-Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, www.allergen.org. In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cow's milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub-Committee encourages researchers to use these updated allergen names in future publications.

The performance of a component-based allergen microarray for the diagnosis of kiwifruit allergy.

BACKGROUND: Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. OBJECTIVE: To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. METHODS: With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. RESULTS: The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. CONCLUSIONS: Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests.

Bet v 1 and its homologous food allergen Api g 1 stimulate dendritic cells from birch pollen-allergic individuals to induce different Th-cell polarization.

BACKGROUND: Bet v 1 is the most relevant sensitizing protein for birch pollen (BP)-allergic individuals. Its homologues from plant foods are mainly involved in allergic reactions caused by IgE cross reactivity. We aimed to evaluate the polarizing effect of dendritic cells (DCs) pulsed with Bet v 1, Mal d 1, Api g 1 or Dau c 1 on Th-cell responses. METHODS: Immature DCs were generated from peripheral blood monocytes of BP-allergic and healthy donors by culture with GM-CSF and IL-4 and subsequently pulsed with allergens in combination with maturation factors. Cell surface markers were analysed by FACS. Mature DCs were co-cultured with autologous Th cells and T-cell proliferation and cytokine profiles were determined. RESULTS: In co-culture, mature allergen-pulsed DCs induced autologous Th cells of BP-allergic donors to proliferate significantly more than those of healthy individuals. Exposure of DCs from BP-allergic donors to Bet v 1 resulted in a robust Th2 skewing with significantly higher quantities of IL-5 and elevated IL-13 compared to maturation factors. In contrast, Api g 1-primed DCs from BP allergics significantly enhanced the production of the Th1 cytokine IFN-γ and significantly down-regulated IL-13 compared to maturation factors. In healthy donors, no significant cytokine production could be detected. CONCLUSION: Bet v 1 in contrast to homologous food allergens seems to possess distinct molecular features that enable it to condition DCs from BP-allergic donors to induce allergen-specific T-cell proliferation and Th2 polarization.

Expression levels of parvalbumins determine allergenicity of fish species.

BACKGROUND: Parvalbumins are the most important fish allergens. Polysensitization to various fish species is frequently reported and linked to the cross-reactivity of their parvalbumins. Studies on cross-reactivity and its association to the allergenicity of purified natural parvalbumins from different fish species are still lacking. In addition, some studies indicate that dark muscled fish such as tuna are less allergenic. METHODS: Total protein extracts and purified parvalbumins from cod, whiff, and swordfish, all eaten frequently in Spain, were tested for their IgE-binding properties with 16 fish allergic patients' sera from Madrid. The extent of cross-reactivity of these parvalbumins was investigated by IgE ELISA inhibition assays. Additionally, the cDNA sequences of whiff and swordfish parvalbumins were determined. RESULTS: Extractable amounts of parvalbumins from cod were 20 times and from whiff 30 times higher than from swordfish. Parvalbumins were recognized by 94% of the patients in extracts of cod and whiff, but only by 60% in swordfish extracts. Nevertheless, a high cross-reactivity was determined for all purified parvalbumins by IgE inhibition. The amino acid sequence identities of the three parvalbumins were in a range of 62-74%. CONCLUSIONS: The parvalbumins of cod, whiff and swordfish are highly cross-reactive. The high amino acid sequence identity among cod, whiff and swordfish parvalbumins results in the observed IgE cross-reactivity. The low allergenicity of swordfish is due to the low expression levels of its parvalbumin.

Cross-reactive and species-specific immunoglobulin E epitopes of plant profilins: an experimental and structure-based analysis.

BACKGROUND: Profilins are cross-reactive plant allergens responsible for multiple pollen sensitization and pollen-associated food allergy. While it is assumed that profilins from different species are immunologically equivalent, some studies suggest partial or even lacking IgE cross-reactivity between certain profilins. OBJECTIVE: We aimed to obtain a semi-quantitative assessment of the contributions of conserved and species-specific epitopes to IgE binding of plant profilins. METHODS: We compared model structures of profilins from timothy, mugwort, celery and bell pepper with crystal structures of birch and latex profilins. We predicted potential conformational epitopes that consisted of contiguous patches of at least 20% surface-exposed residues. Celery and timothy profilins were purified from their natural sources, and profilins from birch, mugwort, bell pepper and latex were expressed in Escherichia coli. The structural integrity of all purified proteins was confirmed by circular dichroism spectroscopy. IgE ELISAs and ELISA inhibitions using sera from 22 profilin-sensitized allergic patients were carried out. RESULTS: Peptide backbone conformations of all six profilins were highly similar. Nine variable epitopes and two containing high proportions of conserved residues were predicted. IgE from all sera bound to all tested profilins and the amounts were highly correlated. However, IgE inhibition experiments revealed that up to 60% of total IgE binding was mediated by species-specific epitopes. The extent of cross-reactivity among profilins from timothy, birch, latex and celery was greater than cross-reactivity to mugwort and bell pepper profilins. This pattern was mirrored by sequence similarities among one of the predicted variable epitopes. Patients with IgE to cross-reactive epitopes displayed allergic reactions to a greater number of plant foods than patients having IgE directed to species-specific epitopes. CONCLUSION: The large extent of cross-reactivity among plant profilins justifies using a single profilin for diagnosis. However, the fine specificity of IgE directed to variable epitopes may influence the clinical manifestation of profilin sensitization.

Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity.

BACKGROUND: Cherry allergy is often reported in the context of allergy to other fruits of the Rosaceae family and pollinosis to trees because of cross-reactive allergens. Allergic reactions to cherry are reported by 19-29% of birch pollen-allergic patients. Pru av 2, identified as a thaumatin-like protein (TLP) from sweet cherry, was recognized by the majority of cherry-allergic patients in immunoblotting. OBJECTIVES: In order to investigate the structural characteristics and the immunoglobulin (Ig)E- and T cell reactivity of cherry-derived TLP, recombinant Pru av 2 was expressed in Escherichia coli and natural Pru av 2 was purified. METHODS: Parallel-His and FLAG expression vectors were used for recombinant production of Pru av 2 in the cytoplasm and the periplasm of E. coli. Natural Pru av 2 was purified from fresh cherries and verified by N-terminal sequencing. Structural characterization was performed using circular dichroism (CD) measurements, and the biologic activity was measured in a glucanase assay. Using cherry-specific sera, the IgE-binding ability of recombinant and natural Pru av 2 was investigated in IgE-ELISA and the T cell reactivity was studied in proliferation assays. Results Natural Pru av 2 revealed thaumatin-like structural features and bound IgE of 50% of cherry-allergic patients. It was demonstrated to be enzymatically active. Recombinant Pru av 2 expressed in the cytoplasm of E. coli exhibited a slightly different folding compared with the natural protein. It was not recognized by IgE from cherry-allergic subjects, but retained the ability to stimulate T lymphocytes. Periplasmic recombinant Pru av 2 was able to bind an anti-grape TLP antibody and cherry-specific IgE. CONCLUSIONS: We prepared two recombinant model TLPs from cherry, and compared their molecular characteristics as well as their IgE-binding activity and T cell interactions in relation to the natural counterpart. The cytoplasmic recombinant Pru av 2 can be used as a hypoallergenic variant in allergen-specific immunotherapy, whereas the periplasmic protein can be included in a component-resolved diagnosis.

Characterization of cross-reactive bell pepper allergens involved in the latex-fruit syndrome.

BACKGROUND: Between 30% and 50% of individuals who are allergic to latex products are also allergic to specific plant foods, a fact that is well documented as the latex-fruit syndrome. Simultaneous sensitization to latex and bell pepper has been previously reported. Although bell pepper fruits are frequently consumed raw, cooked or as a spice, little is known about the cross-reactive allergens. OBJECTIVE: In this study we wished to identify bell pepper allergens involved in the latex-fruit syndrome. METHODS: Sera of four patients who displayed clinical symptoms to latex and bell pepper were used in immunoblot studies on protein extracts of three different cultivars of fresh bell pepper and fresh Hevea latex. Cross-reactive allergens were identified by inhibition experiments using recombinant Hev b 8 (latex profilin), and natural Hev b 2 (latex beta-1,3-glucanase) in addition to the protein extracts. A novel cross-reactive IgE-reactive 30 kDa protein was subjected to sequence analysis. RESULTS: Three patients displayed IgE to profilins from bell pepper fruits and latex. Two patients possessed IgE to Hev b 2, a latex beta-1,3-glucanase, and a homologous protein in bell pepper. One patient possessed IgE reactive with a protein of 30 kDa identified by N-terminal sequencing as an l-ascorbate peroxidase and another patient to a protein of 38 kDa. Additionally, IgE binding proteins in two higher molecular weight ranges showed cross-reactive capacities. CONCLUSION: Our findings show on the molecular level that bell pepper is part of the latex-fruit syndrome. For the first time we have identified the major latex allergen Hev b 2, a beta-1,3-glucanase, and the bell pepper l-ascorbate peroxidase as cross-reactive allergens. We were also able to show that profilins are responsible for some of the IgE cross-reactivity.

Type I allergy to elderberry (Sambucus nigra) is elicited by a 33.2 kDa allergen with significant homology to ribosomal inactivating proteins.

BACKGROUND: Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra. OBJECTIVE: Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort. METHODS: Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed. RESULTS: 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs). CONCLUSION: We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.

Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase.

BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes. OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02). METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay. RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture. CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.

Hev b 8, the Hevea brasiliensis latex profilin, is a cross-reactive allergen of latex, plant foods and pollen.

BACKGROUND: Plant profilins are important pan-allergens. They are responsible for a significant percentage of pollen-related allergies. Limited information is available about their involvement in the latex-fruit syndrome and the cross-reactivities between latex and pollen. We aimed to clone and express the Hevea brasiliensis latex profilin to investigate its allergological significance and serological cross-reactivities to profilins from plant foods and pollens. METHODS: A DNA complementary to messenger RNA (cDNA) coding for the Hevea latex profilin, Hev b 8, was amplified by polymerase chain reaction from latex RNA. Recombinant (r)Hev b 8 was produced in Escherichia coli and used to screen sera from 50 latex- allergic health care workers (HCWs) with well-documented histories of food and pollen allergy and 34 latex-allergic spina bifida (SB) patients. The cross-reactivity of natural Hev b 8 and rHev b 8 with other plant profilins was determined by ELISA inhibition assays. A three-dimensional homology model of Hev b 8 was constructed based on known profilin structures. RESULTS: The cDNA of Hev b 8 encoded a protein of 131 amino acids with a predicted molecular mass of 14 kD. Twelve of the 50 HCWs and 2 of the 34 SB patients were sensitized to Hev b 8. All Hev b 8-sensitized patients showed allergic symptoms to pollen or plant foods. Cross-reactivities between profilins of latex, pollen and plant food were illustrated by their ability to inhibit IgE binding to rHev b 8. Homology modeling of Hev b 8 yielded a structure highly similar to Bet v 2, the birch pollen profilin, with the most distinct differences located at the N-terminus. CONCLUSIONS: We conclude that primary sensitization to latex profilin in the majority of cases takes place via pollen or food profilins. Additionally, pollinosis and food-allergic patients with profilin-specific IgE can be at risk of developing latex allergy.

Allergen mimotopes for 3-dimensional epitope search and induction of antibodies inhibiting human IgE.

There is no definite information available on the structural characteristics of IgE binding epitopes on allergenic molecules, although it is widely accepted that most of them are conformational. In the current study we aimed to characterize the IgE epitope of Bet v 1, the major birch pollen allergen, by the application of phage display peptide libraries. We purified IgE specific for Bet v 1 from allergic patients' sera to select mimotopes representing artificial IgE epitopes by biopanning of phage libraries. By linear alignment, it was not possible to attribute mimotope sequences to the primary structure of Bet v 1. We developed a computer-aided, 3-dimensional coarse-grained epitope search. The 3-dimensional search, followed by statistical analysis, revealed an exposed area on the Bet v 1 molecule (located between residues 9-22 and 104-123) as the IgE binding structure. The IgE epitope was located at a 30 A distance from a previously described IgG epitope and the respective mimotope, designated Bet mim E. Such mimotopes could potentially be used for the induction of IgG capable of interfering with the IgE/allergen interaction. To test this hypothesis, we immunized BALB/c mice with the phage-displayed Bet mim E. Immunizations resulted in the induction of Bet v 1-specific IgG, which was able to block the IgE binding to Bet v 1 in vitro. Based on these observations, we propose that immunotherapy with IgE mimotopes generated by biopannings result in formation of blocking IgG. We conclude that mimotope immunotherapy may represent a new and promising concept for treatment of type I allergic disease.

Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens.

BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.

N-terminal sequences of high molecular weight allergens from celery tuber.

BACKGROUND: Celery tuber is an important source of food allergens. Low molecular weight celery allergens were identified as homologues of Bet v 1 and profilin. Little is known about the relevant allergens with molecular weights between 45 and 60 kDa, which cross-react with other plant food and pollen allergens. OBJECTIVE: The aim of this study was to isolate cross-reactive, high molecular weight allergens from celery and to characterize them by N-terminal sequencing. METHODS: High molecular weight allergens of celery were identified by immunoglobulin (Ig) E immunoblotting with patients' sera, and the IgE-binding patterns were compared with those of the monoclonal antibirch pollen antibody BIP3, as well as of a polyclonal rabbit anti-Art v 1 antiserum. Two independent methods, elution from preparative SDS-PAGE or anion exchange chromatography, were used to purify the IgE-binding celery proteins of interest. The isolated proteins were examined by N-terminal sequencing and IgE-immunoblots. RESULTS: Celery allergens with molecular masses of 55, 58 and 63 kDa, which were also recognized by the monoclonal BIP3 antibody and a polyclonal anti-Art v 1 antiserum, were isolated. The 63-kDa allergen was N-terminally blocked. The 55- and 58-kDa compounds yielded the same N-terminus, which showed no homology to known proteins in the databases. CONCLUSION: The combination of two independent protein separation techniques, immunoblotting and N-terminal sequencing, identified an N-terminus of two allergens in the 60-kDa molecular weight region. Our data will be helpful for the definite molecular characterization of these important cross-reactive molecules.

Molecular and immunologic characterization of new isoforms of the Hevea brasiliensis latex allergen hev b 7: evidence of no cross-reactivity between hev b 7 isoforms and potato patatin and proteins from avocado and banana.

BACKGROUND: Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39% to 42% to patatins recently identified as potato allergens. The complementary DNAs encoding 2 different Hev b 7 isoforms were previously reported. OBJECTIVE: The aim of this study was to determine the sequence variation of Hev b 7 and to compare the IgE reactivity of individual isoforms in vitro and in vivo. A further objective was to evaluate possible cross-reactivities between Hev b 7 and patatins and proteins from banana and avocado. METHODS: An H brasiliensis lambda ZAP complementary DNA (cDNA) library was screened with use of a Hev b 7 cDNA probe. Four Hev b 7 isoforms were produced in recombinant form and their IgE-binding capacities were compared. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate the possible cross-reactivity between Hev b 7 and recombinant potato patatin and proteins from avocado and banana. RESULTS: Two new isoforms, S2 and D2, were identified by sequencing 32 cDNA clones with full-length coding regions. All 4 recombinant isoforms displayed esterase activity and identical IgE-binding capacities. The new isoforms S2 and D2 were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No cross-reactivity was observed between Hev b 7 isoforms and potato patatin and proteins from avocado and banana. CONCLUSIONS: All 4 recombinant Hev b 7 isoforms have equivalent IgE-binding capacity and therefore represent suitable reagents for the development of in vitro and in vivo diagnostic tests. Hev b 7, patatins, and their homologs appear not to contribute to cross-reactivity in the latex-fruit syndrome.

New Bet v 1 isoforms including a naturally occurring truncated form of the protein derived from Austrian birch pollen.

Bet v 1, the major pollen allergen from white birch, displays a considerable degree of heterogeneity. Until now, all molecular and immunological characterization studies of Bet v 1 isoforms have been performed with commercially available pollen of Swedish origin. In regard to clinical studies with Austrian birch pollen allergic individuals, knowledge about the isoform repertoire in Austrian birch pollen was necessary. cDNAs coding for Bet v 1 isoforms from Austrian birch pollen were cloned by PCR amplification and sequenced. Besides the Austrian variants of the Swedish isoforms Bet v 1a (62% of the clones), ALK167 (4%), and Bet v 1d/h, Bet v 1g, and Bet v 11 (24%), three sequences with a significantly lower homology to known isoforms and two Bet v 1a-homologous sequences with a 7 bp insertion coding for a truncated protein were detected. No Austrian variants of the majority of the Swedish isoforms were found. The isoforms coding for truncated proteins were expressed in Escherichia coli and tested by immunoblotting. They bound a polyclonal anti-Bet v 1 antibody but did not recognize birch pollen allergic patients' serum IgE and two Bet v 1-specific monoclonal antibodies. The similarity of the Bet v 1 isoform patterns of Swedish and Austrian birch pollen justifies the use of Bet v 1 derived from Swedish pollen for clinical studies with birch pollen allergic individuals from outside Northern Europe.