Macropinocytosis Is the Principal Uptake Mechanism of Antigen-Presenting Cells for Allergen-Specific Virus-like Nanoparticles.

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

New light on an old syndrome: Role of Api g 7 in mugwort pollen-related celery allergy.

BACKGROUND: Celery root is known to cause severe allergic reactions in patients sensitized to mugwort pollen. OBJECTIVE: We studied clinically well-characterized patients with celery allergy by IgE testing with a comprehensive panel of celery allergens to disentangle the molecular basis of what is known as the celery-mugwort syndrome. METHODS: Patients with suspected food allergy to celery underwent a standardized interview. Main inclusion criteria were a positive food challenge with celery or an unambiguous case history of severe anaphylaxis. IgE to celery allergens (rApi g 1.01, rApi g 1.02, rApi g 2, rApi g 4, nApi g 5, rApi g 6, rApi g 7) and to mugwort allergens (rArt v 1, rArt v 3, rArt v 4) were determined. IgE levels ≥0.35 kUA/L were regarded positive. RESULTS: Seventy-nine patients with allergy to celery were included. Thirty patients had mild oral or rhinoconjunctival symptoms, and 49 had systemic reactions. Sixty-eight percent had IgE to celery extract, 80% to birch pollen, and 77% to mugwort pollen. A combination of Api g 1.01, 1.02, 4, 5, and 7 increased the diagnostic sensitivity for celery allergy to 92%. The lipid transfer proteins Api g 2 and Api g 6 were not relevant in our celery-allergic population. IgE to Api g 7, detected in 52% of patients, correlated closely (r = 0.86) to Art v 1 from mugwort pollen. Eleven of 12 patients with monosensitization to Api g 7 were IgE negative to celery extract. The odds ratio for developing a severe anaphylactic reaction rather than only mild oral symptoms was about 6 times greater (odds ratio, 5.87; 95% confidence interval, 1.08-32.0; P = .0410) for Api g 7-sensitized versus -nonsensitized subjects. CONCLUSION: There is an urgent need for routine diagnostic tests to assess sensitization to Api g 7, not only to increase test sensitivity but also to identify patients at risk of a severe allergic reaction to celery.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules.

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin-proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

Clinical utility of basophil activation test in diagnosis and predicting severity of mugwort pollen-related peach allergy.

BACKGROUND: Cross-reactivity between pollen and plant foods results in low specificity of food-IgE and skin prick testing, which may cause over-diagnosis. A test that can accurately diagnose pollen-related food allergy and identify patients at risk of developing severe reactions is needed. This study evaluates basophil CD63 expression as a biomarker for diagnosis and predicting severity of mugwort pollen-related peach allergy. METHODS: Based on their allergic reactions to peach, an oral allergy symptom group (OAS, n â€‹= â€‹15), a systemic reaction group (SR, n â€‹= â€‹23), a peach-sensitized but tolerant group (PST, n â€‹= â€‹21) and a non-peach-sensitized nonallergic group (NSE, n â€‹= â€‹10) were identified among mugwort pollen allergic patients. Measurements of specific IgE to peach and its components, and basophil activation test (BAT) were performed. RESULTS: Upon stimulation with peach extract, BAT in peach-allergic patients (OAS and SR groups) showed a significant dose-dependent upregulation of CD63 compared with PST patients, but showed no difference between SR and OAS groups. BAT to Pru p 3 could discriminate not only between sensitization and clinical allergy, but also between OAS and systemic reactions. BAT to Pru p 3 revealed 92% sensitivity, 95% specificity, 92% positive predictive value, and 92% negative predictive value. Receiver operating characteristic curves showed that BAT to Pru p 3 had the largest area under the curve. CONCLUSIONS: In the diagnosis of mugwort pollen-related peach allergy, BAT to Pru p 3 is superior to testing for IgE specific for peach and its components. Additionally, basophil activation can predict clinical severity.

Mugwort Pollen-Related Food Allergy: Lipid Transfer Protein Sensitization and Correlation With the Severity of Allergic Reactions in a Chinese Population.

PURPOSE: Little is known about the importance of lipid transfer protein (LTP) sensitization in China. In this study, we investigated the relationship between LTP sensitization and the severity of clinical symptoms in a population of patients with mugwort pollen-related food allergy. METHODS: Food-induced symptoms were evaluated in 148 patients with mugwort pollen allergy by a standardized questionnaire. Specific immunoglobulin E (IgE) to Art v 1, Art v 3, Pru p 3, Ara h 9 and Cor a 8 were quantified by ImmunoCAP. Immunoblotting of peach extracts were performed with sera from peach-allergic patients. RESULTS: In total, 72% (107/148) of the study population experienced food allergy. Forty-eight percent (51/107) of patients with mugwort pollen-related food allergy experienced at least 1 episode of food-induced anaphylaxis. Food allergy correlated with IgE reactivity to Art v 3, but not to Art v 1. Sensitization to Pru p 3, Ara h 9 or Cor a 8 was prevalent (80%, 69 or 63%, respectively) among individuals with food allergy. Food allergic patients with systemic reactions (SR) had higher values for Pru p 3, Ara h 9 and Cor a 8 than patients with oral allergy syndrome (OAS). Furthermore, the strong IgE reactivity detected in immunoblots of peach extracts indicated that Pru p 3 was the major allergen and was more prevalent in patients with SR than in patients with OAS (100% vs. 55%). CONCLUSIONS: LTPs are major food allergens for mugwort pollen-related food allergy in China, and may contribute to SR.

Prevention of allergy by virus-like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model.

BACKGROUND: In high-risk populations, allergen-specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus-like nanoparticles (VNP) expressing surface-exposed or shielded allergens. METHODS: Full-length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. RESULTS: Virus-like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface-exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1-specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen-specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface-exposed but not shielded allergen induced allergen-specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen-protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg-dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. CONCLUSION: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

Mugwort Pollen-Related Food Allergy: Lipid Transfer Protein Sensitization and Correlation With the Severity of Allergic Reactions in a Chinese Population

PURPOSE: Little is known about the importance of lipid transfer protein (LTP) sensitization in China. In this study, we investigated the relationship between LTP sensitization and the severity of clinical symptoms in a population of patients with mugwort pollen-related food allergy. METHODS: Food-induced symptoms were evaluated in 148 patients with mugwort pollen allergy by a standardized questionnaire. Specific immunoglobulin E (IgE) to Art v 1, Art v 3, Pru p 3, Ara h 9 and Cor a 8 were quantified by ImmunoCAP. Immunoblotting of peach extracts were performed with sera from peach-allergic patients. RESULTS: In total, 72% (107/148) of the study population experienced food allergy. Forty-eight percent (51/107) of patients with mugwort pollen-related food allergy experienced at least 1 episode of food-induced anaphylaxis. Food allergy correlated with IgE reactivity to Art v 3, but not to Art v 1. Sensitization to Pru p 3, Ara h 9 or Cor a 8 was prevalent (80%, 69 or 63%, respectively) among individuals with food allergy. Food allergic patients with systemic reactions (SR) had higher values for Pru p 3, Ara h 9 and Cor a 8 than patients with oral allergy syndrome (OAS). Furthermore, the strong IgE reactivity detected in immunoblots of peach extracts indicated that Pru p 3 was the major allergen and was more prevalent in patients with SR than in patients with OAS (100% vs. 55%). CONCLUSIONS: LTPs are major food allergens for mugwort pollen-related food allergy in China, and may contribute to SR.

Immunoelectron-microscopic localization of IgE binding site of mugwort pollen

To elucidate the IgE binding site of mugwort (Artemisia vulgaris r.) pollen, pollen grains were frozen and fixed using a cryocut. They were incubated with antibodies according to the following sequence: Sera pool of individuals who showed mugwort-RAST class 3 or 4, biotin-labeled goat anti-human IgE antibody, streptavidin-peroxidase and diaminobenzidine. Then, they were observed under electron microscopy. The control section was incubated with the sera pool from individuals who showed a negative result on a skin prick test to mugwort pollen. Antigenic activity (electrondense line) was noted on the surface of the exine. There was no activity in cytoplasm or the intine layer. The control section was completely free of activity. It was suggested that the IgE binding site of mugwort pollen was present on the surface of the exine.