A Novel Isoallergen Dau c 1.0401 in Carrot: Stability, Allergenicity, and Comparison with Other Isoallergens.

SCOPE: Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce. METHODS AND RESULTS: The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and ß-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays. CONCLUSION: Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing.

Der p 1-based immunotoxin as potential tool for the treatment of dust mite respiratory allergy.

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed.

Food Processing Does Not Abolish the Allergenicity of the Carrot Allergen Dau c 1: Influence of pH, Temperature, and the Food Matrix.

SCOPE: The major carrot allergen Dau c 1 belongs to the group of pathogenesis related class 10 (PR-10) proteins and is homologous to the birch pollen allergen Bet v 1. In contrast to most other PR-10 allergens, Dau c 1 can elicit Bet v 1 independent sensitization. Although Dau c 1 is considered heat labile, allergic reactions against cooked carrots are possible. METHODS AND RESULTS: The pH and temperature stability as well as the allergenic potential before and after treatment of purified natural (n) Dau c 1 and different recombinant (r) isoallergens is investigated: rDau c 1.0104, rDau c 1.0105, rDau c 1.0201, rDau c 1.0301. All proteins except rDau c 1.0201 are able to refold at physiological pH. pH conditions around the pI (4.4-5.5) or the presence of the carrot matrix reduce the refolding capacity. Below the pI, most isoallergens are heat resistant and still able to cause mediator release, indicating allergenicity. Moreover, cooked carrot extract is still able to provoke mediator release due to remaining soluble Dau c 1. CONCLUSION: Patients allergic to carrots should avoid processed carrot containing foodstuff because heating or pH treatment do not completely abolish the allergenicity of Dau c 1.

Pea (Pisum sativum) allergy in children: Pis s 1 is an immunodominant major pea allergen and presents IgE binding sites with potential diagnostic value.

BACKGROUND: Food allergy to pea (Pisum sativum) has been rarely studied in children at the clinical and molecular levels. OBJECTIVE: To elucidate the allergenic relevance and diagnostic value of pea 7S globulin Pis s 1, nsLTP, and 2S albumins PA1 and PA2 in children. METHODS: Children with pea-specific IgE ≥ 0.35 kUA /L and clinical evidence of pea allergy or tolerance were included in the study. IgE binding against pea total protein extract, recombinant (r) rPis s 1, rPA1, rPA2, and natural nsLTP was analysed using IgE immunoblot/inhibition. Mediator release potency was investigated in passively sensitized rat basophil leukaemia (RBL) 2H3-cells. IgE binding to synthetic overlapping peptides of Pis s 1 was detected on multipeptide microarrays. RESULTS: 19 pea-sensitized children were included, 14 with doctors' diagnosed allergy and 5 with tolerance to pea (median age 3.5 and 4.5 years, respectively). 11/14 (78%) pea-allergic and 1/5 (20%) tolerant children were sensitized to Pis s 1. Under the reducing conditions of immunoblot analysis, IgE binding to rPA1 was negligible, sensitization to rPA2 and nsLTP undetectable. Compared to pea total protein extract, rPis s 1 displayed on average 58% IgE binding capacity and a 20-fold higher mediator release potency. Selected Pis s 1-related peptides displayed IgE binding in pea-allergic but not in pea-tolerant children. CONCLUSIONS AND CLINICAL RELEVANCE: In this study group, Pis s 1 is a major immunodominant allergen in pea-allergic children. Evidence for sensitization to nsLTP and 2S albumins was low but requires further verification with regard to conformational epitopes. Recombinant Pis s 1 and related peptides which were exclusively recognized by pea-allergic children may improve in vitro diagnosis of pea allergy once verified in prospective studies with larger study groups.

New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7.

BACKGROUND: Ole e 7 is a nonspecific lipid transfer protein (nsLTP) from olive pollen, one of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no evidence of cross-reactivity, which explains this observed co-sensitization. Therefore, the purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. METHODS: A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. Inhibition assays were performed to determine the existence of cross-reactivity between both nsLTPs. Allergic response was analyzed ex vivo (basophil activation test) and in vitro (RBL-2H3 mast cell model). RESULTS: Common IgG and IgE epitopes were identified between both allergens. IgE-binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. CONCLUSIONS: Our results suggest that Ole e 7 could play a new role as primary sensitizer in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

BACKGROUND: To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. OBJECTIVE: To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. METHODS: Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. RESULTS: All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). CONCLUSION AND CLINICAL RELEVANCE: Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

Fresh water, marine and terrestrial cyanobacteria display distinct allergen characteristics.

During the last decades, global cyanobacteria biomass increased due to climate change as well as industrial usage for production of biofuels and food supplements. Thus, there is a need for thorough characterization of their potential health risks, including allergenicity. We therefore aimed to identify and characterize similarities in allergenic potential of cyanobacteria originating from the major ecological environments. Different cyanobacterial taxa were tested for immunoreactivity with IgE from allergic donors and non-allergic controls using immunoblot and ELISA. Moreover, mediator release from human FcεR1-transfected rat basophilic leukemia (RBL) cells was measured, allowing in situ examination of the allergenic reaction. Phycocyanin content and IgE-binding potential were determined and inhibition assays performed to evaluate similarities in IgE-binding epitopes. Mass spectrometry analysis identified IgE-reactive bands ranging between 10 and 160kDa as phycobiliprotein compounds. Levels of cyanobacterial antigen-specific IgE in plasma of allergic donors and mediator release from sensitized RBL cells were significantly higher compared to non-allergic controls (p<0.01). Inhibition studies indicated cross-reactivity between IgE-binding proteins from fresh water cyanobacteria and phycocyanin standard. We further addressed IgE-binding characteristics of marine water and soil-originated cyanobacteria. Altogether, our data suggest that the intensive use and the strong increase in cyanobacterial abundance due to climate change call for increasing awareness and further monitoring of their potential health hazards.

A Fusion Protein Consisting of the Vaccine Adjuvant Monophosphoryl Lipid A and the Allergen Ovalbumin Boosts Allergen-Specific Th1, Th2, and Th17 Responses In Vitro.

Background. The detoxified TLR4-ligand Monophosphoryl Lipid A (MPLA) is the first approved TLR-agonist used as adjuvant in licensed vaccines but has not yet been explored as part of conjugated vaccines. Objective. To investigate the immune-modulating properties of a fusion protein consisting of MPLA and Ovalbumin (MPLA : Ova). Results. MPLA and Ova were chemically coupled by stable carbamate linkage. MPLA : Ova was highly pure without detectable product-related impurities by either noncoupled MPLA or Ova. Light scattering analysis revealed MPLA : Ova to be aggregated. Stimulation of mDC and mDC : DO11.10 CD4(+) TC cocultures showed a stronger activation of both mDC and Ova-specific DO11.10 CD4(+) TC by MPLA : Ova compared to the mixture of both components. MPLA : Ova induced both strong proinflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine responses from mDCs while also boosting allergen-specific Th1, Th2, and Th17 cytokine secretion. Conclusion. Conjugation of MPLA and antigen enhanced the immune response compared to the mixture of both components. Due to the nonbiased boost of Ova-specific Th2 and Th17 responses while also inducing Th1 responses, this fusion protein may not be a suitable vaccine candidate for allergy treatment but may hold potential for the treatment of other diseases that require a strong stimulation of the host's immune system (e.g., cancer).

Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.

BACKGROUND: Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a. OBJECTIVE: To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations. METHODS: rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared. RESULTS: 1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%. CONCLUSION: Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected.

Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent.

Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination.

Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

BACKGROUND: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens. METHOD: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1. RESULTS: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation. CONCLUSION: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

Vig r 6, the cytokinin-specific binding protein from mung bean (Vigna radiata) sprouts, cross-reacts with Bet v 1-related allergens and binds IgE from birch pollen allergic patients' sera.

SCOPE: Birch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity. METHODS AND RESULTS: Bet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients' sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients' sera. CONCLUSION: We demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy.

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.

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

BACKGROUND: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. OBJECTIVE: In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. METHODS: Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. RESULTS: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. CONCLUSION: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.

BACKGROUND: Hypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug- and/or metabolite-specific antibodies in selective DF hypersensitivity. METHODOLOGY/PRINCIPAL FINDINGS: DF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG. CONCLUSIONS/SIGNIFICANCE: We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded.

Targeting the cysteine-stabilized fold of Art v 1 for immunotherapy of Artemisia pollen allergy.

Plants of the genus Artemisia domestic in Northern and Central Europe, USA and parts of Asia are a major cause of allergic symptoms from late summer to autumn. Art v 1, the major mugwort pollen allergen appears as two-domain glycoprotein, consisting of an N-terminal defensin-like and a proline/hydroxyproline-rich C-terminal part. Patients sensitized to Art v 1 commonly display IgE antibodies against the cysteine-stabilized defensin fold. Site-directed mutagenesis of eight cysteines was used to disrupt disulfide bonds to generate molecules with altered IgE-binding capacity. Engineered constructs were expressed in E. coli and recombinant proteins were tested for their allergenic and T cell reactivity as well as for their physicochemical characteristics. Three cysteine variants (C22S, C47S, and C49S) exhibited extremely low IgE-binding activity in immunoblot and ELISA using sera from Art v 1-allergic patients. Mediator release assays using rat basophil leukemia cells showed that these variants displayed a 1x10(5)-fold reduced allergenic potency as compared to wild-type protein. All variants were able to activate allergen-specific T cells in PBMC, as well as Art v 1-specific T cell lines and clones. Variant C49S displayed an increased hydrophobic surface potential which correlated with an advanced activation of allergen-specific T cells. The low allergenicity and high immunogenic activity of Art v 1 variant C49S renders the molecule an attractive candidate for hypoallergen-based immunotherapy of Artemisia pollen allergy.

Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen.

Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.

The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level.

Ragweed is one of the most important pollen allergens in North America and parts of Europe. Although the major allergen Amb a 1 was isolated and cloned in 1991, recombinant Amb a 1 was not explored further to improve diagnosis and specific immunotherapy of ragweed-pollen allergy. In the present study the immunological properties of natural Amb a 1 and its proteolytical cleavage products was investigated in detail and compared with recombinant produced Amb a 1 variants. Characterization of natural Amb a 1 and the identification of its proteolytic fragments, designated Amb a 1 alpha and Amb a 1 beta, was performed by N-terminal sequencing and mass spectroscopy. Amb a 1 and fragments were further produced in Escherichia coli, purified, and immunologically characterized. Amb a 1-specific T-cell cultures were used to compare the T-cell response to the different Amb a 1 variants. Divergent immunological properties of Amb a 1 alpha (aa 181-396) and Amb a 1 beta (aa 26-180) were revealed. Amb a 1 beta contained important IgE epitopes, whereas Amb a 1 alpha showed low IgE binding. When compared to natural Amb a 1, all recombinant variants possessed >100-fold reduced IgE-mediated mediator release activity. At the T-cell level recombinant and natural Amb a 1 stimulated comparable T-cell responses and the T-cell reactivity was largely directed to the C-terminal part. The results demonstrated that recombinant Amb a 1 alpha behaves as hypoallergen with reduced IgE binding but preservation of the major T-cell reactivity. In addition, recombinant Amb a 1 alpha can be easily purified to homogeneity in large quantity and therefore represents an ideal candidate for specific immunotherapy.

Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1.

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1-6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.