Distinct epitope structures of defensin-like proteins linked to proline-rich regions give rise to differences in their allergenic activity.

BACKGROUND: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens. METHODS: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro. RESULTS: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 125-36 was observed. CONCLUSIONS: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis.

Immunologic characterization of isoforms of Car b 1 and Que a 1, the major hornbeam and oak pollen allergens.

BACKGROUND: Birch pollen allergy is one of the most common causes of spring pollinosis often associated with hypersensitivity reactions to pollen of other Fagales species. Yet, only the major disease eliciting allergens of alder and hazel have been fully characterized. Therefore, the aim of this study was to perform cloning, expression and immunologic characterization of the Bet v 1 homologues from oak (Que a 1) and hornbeam (Car b 1). METHODS: The isoform pattern of Car b 1 and Que a 1 was analyzed by proteomics using 2D gel electrophoresis and LC ESI-QTOF MS. Isoallergens showing high IgE-binding were cloned and expressed in Escherichia coli. IgE-binding activity of the recombinant proteins was determined by enzyme-linked immunosorbent assay (ELISA) and basophil mediator release assays using serum samples from patients mainly exposed either to oak and hornbeam or to birch pollen. Cross-reactivity of the allergens was further investigated at the T-cell level. RESULTS: Dominant isoforms of Car b 1 and Que a 1, identified by mass spectrometry, showed different IgE-binding properties when testing Fagales pollen-allergic patients living in birch-free areas as compared to birch-sensitized individuals. CONCLUSION: Tree pollen-allergic patients who are primarily exposed to Fagales pollen other than birch reacted stronger with rCar b 1 and rQue a 1 than with rBet v 1, as determined by inhibition ELISA and basophil mediator release assays. Thus, rCar b 1 and rQue a 1 allergens should be considered for improving molecule-based diagnosis and therapy of tree pollen allergies manifesting in birch-free areas.

Array-based profiling of ragweed and mugwort pollen allergens.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA). METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

Prevalence of IgE-binding to Art v 1, Art v 4 and Amb a 1 in mugwort-allergic patients.

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract. METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA. RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera. CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

Artemisia and Ambrosia hypersensitivity: co-sensitization or co-recognition?

BACKGROUND: Ragweed and mugwort have nearly identical flowering periods. Clinical and serological studies showed that ragweed and mugwort sensitization are often associated and this poses relevant clinical problems in patients for whom specific immunotherapy is warranted. OBJECTIVE: To establish whether the concurrent ragweed and mugwort pollen hypersensitivity is the result of co-sensitization or of co-recognition by using purified recombinant allergens. METHODS: Sensitization to ragweed and mugwort pollen was assessed by skin prick test (SPT) in all patients reporting allergic symptoms in August and September. IgE reactivity of sera from 42 patients (26 Amb+/Art+, 14 Amb+/Art-, and two Amb-/Art+) to ragweed and mugwort pollen extract as well as to several recombinant ragweed (rAmb a 1, rAmb a 5, rAmb a 6, rAmb a 8, rAmb a 9, and Amb a 10) and mugwort (rArt v 1, rArt v 4, rArt v 5, rArt v 6, and three EF-hand calcium-binding protein) allergens was detected by dot-blot and ELISA analyses. RESULTS: IgE reactivity of 372 weed pollen-allergic patients was studied. Mugwort reactivity was strongly associated with ragweed hypersensitivity: only 10/147 (7%) mugwort-hypersensitive patients were not sensitized to ragweed, whereas 225/362 (62%) ragweed-hypersensitive patients were not sensitized to mugwort. In vitro, 90% of ragweed-allergic patients reacted with rAmb a 1. Reactivity to other ragweed allergens ranged between 20% and 35%. Forty-six percent of the mugwort-sensitized patients recognized rArt v 1%, 25% reacted to Art v 4, Art v 5, and Art v 6, and 7% recognized the three-EF hand calcium-binding protein. Immunoblot inhibition experiments showed that pre-incubation with ragweed pollen extract only weakly decreased IgE reactivity to mugwort allergens. CONCLUSION: Patients showing both ragweed- and mugwort-positive SPT and/or RAST are co-sensitized. Future studies will establish whether IgE reactivity translates into clinical symptoms and, hence, if co-sensitized patients should undergo specific immunotherapy with extracts of both mugwort and ragweed pollen.