Major Mugwort Pollen Allergen Research Articles

Utilization of Component-Resolved Diagnosis in Precision Allergology

It is very important to identify the causal allergen of each patient in the diagnosis and treatment of allergic diseases. An experimental method of specific IgE testing for single clinically meaningful antigens is called component resolved diagnosis (CRD). Since the introduction of CRD, many single antigens with clinical significance from animal and plant allergenic source have become known. Bet v 1, the major allergen of white birch pollen, can be an indication marker for allergen-specific immunotherapy if the patient is sensitized. Phl p 1, the group I allergen of timothy grass pollen, is an important marker that suggests that patients are truly sensitized to grass pollen. The major allergen of ragweed pollen, Amb a 1 and Art v 1, which is the major allergen of mugwort pollen, are clinically important to distinguish true sensitization. House dust mites have several clinically useful component allergens. Der p 1 and Der p 2 are the initiation molecules in the molecular spreading of allergic diseases caused by house dust mites. Among food allergens, Ara h 2 from peanut, which is closely related to anaphylaxis among several clinical manifestation of peanut allergy. Tri a 19, well known to omega-5 gliadin is important in patients with wheat-dependent exercise-induced anaphylaxis. It is conceivable that the molecular biological concept of CRD methods should lead to ‘patient-customized treatment’ beyond ‘diagnosis’ in the future. Primary prevention of the allergic diseases will apply, which will conceptually be called ‘allergen immunprophylaxis’. This is the right direction of future precision allergology.

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

BackgroundIn 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.MethodsFull‐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.ResultsVirus‐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.ConclusionVirus‐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.

Genetic restriction of antigen-presentation dictates allergic sensitization and disease in humanized mice

BackgroundImmunoglobulin(Ig)E-associated allergies result from misguided immune responses against innocuous antigens. CD4+ T lymphocytes are critical for initiating and perpetuating that process, yet the crucial factors determining whether an individual becomes sensitized towards a given allergen remain largely unknown. ObjectiveTo determine the key factors for sensitization and allergy towards a given allergen. MethodsWe here created a novel human T cell receptor(TCR) and human leucocyte antigen (HLA)-DR1 (TCR-DR1) transgenic mouse model of asthma, based on the human-relevant major mugwort (Artemisia vulgaris) pollen allergen Art v 1 to examine the critical factors for sensitization and allergy upon natural allergen exposure via the airways in the absence of systemic priming and adjuvants. ResultsAcute allergen exposure led to IgE-independent airway hyperreactivity (AHR) and T helper(Th)2-prone lung inflammation in TCR-DR1, but not DR1, TCR or wildtype (WT) control mice, that was alleviated by prophylactic interleukin(IL)-2-αIL-2 mAb complex-induced expansion of Tregs. Chronic allergen exposure sensitized one third of single DR1 transgenic mice, however, without impacting on lung function. Similar treatment led to AHR and Th2-driven lung pathology in >90% of TCR-DR1 mice. Prophylactic and therapeutic expansion of Tregs with IL-2-αIL-2 mAb complexes blocked the generation and boosting of allergen-specific IgE associated with chronic allergen exposure. ConclusionsWe identify genetic restriction of allergen presentation as primary factor dictating allergic sensitization and disease against the major pollen allergen from the weed mugwort, which frequently causes sensitization and disease in humans. Furthermore, we demonstrate the importance of the balance between allergen-specific T effector and Treg cells for modulating allergic immune responses.

Identification of a 62-kDa major allergen from Artemisia pollen as a putative galactose oxidase.

Around 20years ago, a 60- to 70-kDa protein was reported as a major allergen of mugwort (Artemisia vulgaris) pollen. This study was to identify and characterize its molecular properties. Sera from 113 Chinese and 20 Dutch Artemisia-allergic/sensitized subjects (and pools thereof) were used to identify the 60- to 70-kDa allergen. Pollen extracts of seven Artemisia species were compared by immunoblotting. Transcriptomics and proteomics (mass spectrometry) of A.annua pollen were used to identify the putative 60- to 70-kDa Artemisia allergen. Both the natural purified and recombinant allergens were evaluated for IgE reactivity by ImmunoCAP. Fourteen Chinese Artemisia-allergic patients were tested intradermally with purified natural allergen. Immunoblots revealed two major bands at 12 and 25kDa, and a weak band at 70kDa for all seven Artemisia species. Using a combined transcriptomic and proteomic approach, the high molecular mass allergen in A.annua pollen was shown to be a 62-kDa putative galactose oxidase, with a putative N-glycosylation site. More than 94% of Artemisia pollen-allergic patients had IgE response to this allergen. Although recognition of a nonglycosylated recombinant version was only confirmed in a minority (16%) and at much lower IgE levels, this discrepancy cannot be explained simply by reactivity to the carbohydrate moiety on the natural allergen. Intradermal testing with the natural allergen was positive in five of nine sensitized patients. The previously reported 60- to 70-kDa allergen of Artemisia pollen is most likely a 62-kDa putative galactose oxidase here designated Art an 7.

Hypoallergenic acid-sensitive modification preserves major mugwort allergen fold and delivers full repertoire of MHC class II-binding peptides during endolysosomal degradation

Hypoallergenic acid-sensitive modification preserves major mugwort pollen allergen fold and delivers a full repertoire of MHC class II-binding peptides during endolysosomal degradation.

Interleukin-2/anti-interleukin-2 antibody complexes expand T regulatory cells and protect against allergen-induced airway hyperreactivity (HYP7P.256)

Abstract T-cells play a key role in the development and maintenance of allergic diseases and thus represent a promising target for therapeutic interventions. Evidence from SIT studies suggest that regulatory T-cells (Treg) might counterbalance the Th2-biased immune response. We here created double transgenic (tg) allergy mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1 to study the response to the aero-allergen in vivo. After allergen-specific sensitization, only double tg allergy mice, but not single tg or wt control mice showed enhanced airway-hyperreactivity (AHR) upon challenge, which was associated with airway and lung inflammation. Significantly, treatment of allergy mice with three daily i.p. injections of IL-2/anti-IL-2 mAb complexes induced a 6-fold increase of CD25+Foxp3+ Treg among CD3+CD4+T-cells in peripheral blood (17.9±6.2% versus 3.1±1.4%) compared to a 8-fold increase of CD39+ and KLRG1+ Treg subsets, respectively (17.2±7.9% versus 2.1±1.5%; 1.6±0.2% versus 0.2±0.2%). Furthermore, expansion of Treg by IL-2/anti-IL-2 mAb complexes before sensitization prevented mice from allergen-induced AHR upon challenge and significantly reduced Art v 1-specific serum IgE, when compared to sham-treated mice. Such in vivo expanded Treg might have similar effects in therapeutic settings. In summary, expansion of Treg by IL-2/anti-IL-2 mAb complexes seems to be a promising strategy to interfere with allergic diseases.

Synthesis of oligomers of β-l-arabinofuranosides of (4R)-4-hydroxy-l-proline relevant to the mugwort pollen allergen, Art v 1.

An efficient, convergent solution phase synthesis of monomer, dimer, trimer and tetramer of the β-l-arabinofuranosylated hydroxyproline (β-l-Araf-Hyp) glycocluster is described. This motif constitutes the carbohydrate-specific epitope of Art v 1, the major allergen of mugwort pollen. While a single monomeric unit was proposed at the outset, poor yields for the seemingly trivial steps of end-capping to replace protecting groups with N-terminal acetamides and C-terminal methyl amides led to the introduction of N-terminal, central and C-terminal β-l-Araf-Hyp building blocks. Dimer 2 was obtained in 60% yield by coupling of two monomers, followed by hydrogenolysis of benzyl ether protecting groups. Trimer 3 was obtained in 35% yield via a [2 + 1] coupling and tetramer 4 in 15% yield via a [2 + 2] fragment condensation. Circular dichroism spectra show that monomer 1 displays no organized structure, whereas compounds 2-4 show a strong negative band at 200 nm and a weak positive band at ∼220 mn, as is characteristic of the polyproline II helix.

Characterization of allergen-specific T-cell responses in a humanized mouse model relying on a human mugwort-specific T-cell receptor and HLA-DR1 (P6208)

Abstract While mice expressing an I-Ad restricted murine T-cell receptor (TCR) specific for chicken ovalbumin (Ova) are widely used to mimic allergic diseases induced by aeroallergens in vivo, implications for human allergic diseases remain questionable, since Ova is not a human-relevant aeroallergen. We here created double tg mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1. In tg mice ninety percent of peripheral blood CD4+ T lymphocytes expressed the Art v 1-specific TCR, while CD14+ monocytes and B220+ B lymphocytes revealed HLA-DR1 expression. Splenocytes of tg mice specifically proliferated upon incubation with the human-relevant immuno-dominant Art v 125-36 peptide or whole Art v 1 protein when compared to control mice. In vivo, after allergen-specific sensitization, only tg mice showed enhanced airway-hyperreactivity (AHR) as assessed by methacholine or allergen-specific challenge. Three consecutive exposures to nebulized allergen were found to be sufficient for induction of AHR. The effects on bronchoalveolar lavage fluids, lung histology and specific antibody titers will be described and discussed. Humanized allergy models are amenable to the evaluation of human-relevant allergen formulations or cellular intervention protocols and will thus contribute to the further understanding of allergic diseases and their cure.

Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background

BackgroundMugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4.ResultsThe majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1–peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1.ConclusionsThe predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.

Structural glycobiology of the major allergen of Artemisia vulgaris pollen, Art v 1: O-glycosylation influence on the protein dynamics and allergenicity

Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen. It is formed by an N-terminal globular defensin-like part and a C-terminal proline-rich domain. As the structure and the dynamics of Art v 1 have been mostly described for its recombinant, non-glycosylated form, which does not occur in normal plant physiology, the present work intends to obtain a three-dimensional model for Art v 1 native O-glycosylation structure and to evaluate the influence of such glycans over the protein dynamics and allergenicity through molecular dynamics simulations in triplicates. Structural insights into the mutual recognition of Art v 1 protein and carbohydrate moieties recognition by antibodies were obtained, in which glycan chains remained close to the previously identified epitopes in the defensin-like domain, thus pointing to potential interferences with antibodies recognition. To our knowledge, this is the first structural report of an entire furanose-containing glycoprotein. As well, together with the previously determined NMR structures, the obtained results contribute in the comprehension of the effect of glycosylation over both proline-rich and defensin-like domains, providing an atomic representation of such alterations.

Expression of the major mugwort pollen allergen Art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants

An economic and cheap production of large amounts of recombinant allergenic proteins might become a prerequisite for the common use of microarray-based diagnostic allergy assays which allow a component-specific diagnosis. A molecular pharming strategy was applied to express the major allergen of Artemisia vulgaris pollen, Art v 1, in tobacco plants and tobacco cell cultures. The original Art v 1 with its endogenous signal peptide which directs Art v 1 to the secretory pathway, was expressed in transiently transformed tobacco leaves but was lost in stable transformed tobacco plants during the alternation of generations. Using a light-regulated promoter and “hiding” the recombinant Art v 1 in the ER succeeded in expression of Art v 1 over three generations of tobacco plants and in cell cultures generated from stable transformed plants. However, the amounts of the recombinant allergen were sufficient for analysis but not high enough to allow an economic production. Although molecular pharming has been shown to work well for the production of non-plant therapeutic proteins, it might be less efficient for closely related plant proteins.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-011-1199-3) contains supplementary material, which is available to authorised users.

Polymeric Structure and Host Toll-like Receptor 4 Dictate Immunogenicity of NY-ESO-1 Antigen in Vivo

In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.

Abstract 2696: Polymeric structure and host Toll-Like Receptor 4 dictate the immunogenicity of NY-ESO-1 in vivo

Abstract Introduction: NY-ESO-1 is a tumor-associated Ag (TAA) with distinctively strong immunogenicity. Our recent investigation of the specific interaction between NY-ESO-1 and complement C1q receptor (or Calreticulin, CRT) on the surface of immature DC, macrophages, and monocytes, indicated a unique interaction between NY-ESO-1 and the innate immune system (The Journal of Immunology, Zeng et al 2006). We further explore the molecular mechanism of the link between NY-ESO-1 and immature DC. Experimental Procedures: Cysteine-to-serine (C-to-S) substitutions were introduced into NY-ESO-1 proteins. Wild type NY-ESO-1 and mutant proteins were expressed in bacterial and mammalian cells and detected by western blot. The binding properties of wild type and mutant NY-ESO-1 with human and mouse immature DC were characterized. Bone marrow derived immature DC from the wild-type, TLR2-/-, and TLR4-/- mice were used for binding to NY-ESO-1. Mouse prostate cancer myc-cap cells with NY-ESO-1 and variants expression were co-cultured with human immature DC and mouse macrophage cell RAW264, and Th1/Th2 cytokines secretion were measured. Normal and the TLR4-/- mice were immunized with gene-gun delivery of plasmids encoding the wild type and variants of NY-ESO-1. Plasmids encoding NY-ESO-1 fused with a major mugwort pollen allergen Art v1 and a TAA carbonic anhydrase 9 were also delivered by gene gun to balb/c mice for immunization. Results: Wild Type NY-ESO-1 showed clear formation of dimers, trimers and even polymers due to inter-molecular disulfide bonds. Mutant NY-ESO-1 with all cysteines substituted by serines appeared only as monomer. C-to-S mutations significantly abolished the ability of NY-ESO-1 binding to human and mouse immature DC in vitro. The absence of TLR4 but not TLR2 partially impaired the binding of NY-ESO-1 to mouse bone marrow derived DC in vitro. Similarly, binding of recombinant NY-ESO-1 to human immature DC was blocked by anti-TLR4 monoclonal antibody. Mouse TLR4 co-precipitated with NY-ESO-1 by western blotting confirming the direct interaction between NY-ESO-1 and TLR4. Disrupting polymeric structure of NY-ESO-1 led to diminished immunogenicity and altered TLR4 dependence in the induced antibody responses. NY-ESO-1 expression in myc-cap cells impacted the immature DC inflammatory responses in co-culture system. Myc-cap cells bearing membrane-anchored NY-ESO-1 also affected tumor growth in vivo, correlated with in vitro studies. Gene gun-delivered plasmid DNA encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Conclusion: NY-ESO-1 binds to immature DC depending on its polymeric architectural structure and its interaction with immature DC surface receptor TLR4. The unique interaction between NY-ESO-1 and immature DC renders NY-ESO-1 serve as a molecular adjuvant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2696. doi:10.1158/1538-7445.AM2011-2696

Mapping the Interactions between a Major Pollen Allergen and Human IgE Antibodies

The interaction of specific IgE antibodies with allergens is a key event in the induction of allergic symptoms, thus representing an important target for therapeutic interventions in Type I allergies. We report here the solution NMR structure of Art v 1, the major mugwort pollen allergen. Art v 1 is the first protein structure with an allergenic defensin fold linked to a polyproline domain, which has not been identified in any reported allergen structure in the PDB. Moreover, the direct interaction of polyclonal IgE antibodies from an allergic patient has been mapped on the surface of an allergen for the first time. The data presented herein provide the basis for the design of tools for safe and effective vaccination against mugwort pollen allergy.

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 1×105-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.

Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules

T(H)2 lymphocytes play an important role in the induction and maintenance phase of type I allergy. Modulation of the responses of T(H)2 lymphocytes by novel forms of antigen-presenting platforms may help shape the immune response to allergen and palliate allergic diseases. To present HLA class II/allergen-peptide complexes on virus-like particles (VLPs) and to evaluate their potential to modulate allergen-specific T-cell responses. Virus-like particles that express the immunodominant T-cell epitope Art v 1(25-34) of the major mugwort pollen allergen in the context of HLA-DR1 and costimulatory molecules were produced by transfection of 293 cells. The effect of VLPs on IL-2 promoter activity, proliferation, and cytokine production of allergen-specific T cells derived from donors with and without mugwort pollen allergy was determined. Flow-cytometric analyses showed that HLA class II molecules, invariant chain::Art v 1 fusion proteins, and costimulatory molecules were expressed on 293 cells. Biochemical analyses confirmed that these molecules were efficiently targeted to VLPs. The engineered VLPs activated Art v 1-specific T cells in a costimulation-dependent manner. VLPs lacking costimulators induced T-cell unresponsiveness, which was overcome by addition of exogenous IL-2. Costimulation could be provided by CD80, CD86, or CD58 and induced distinct cytokine profiles in allergen-specific T cells. Unlike the other costimulatory molecules, CD58 induced IL-10/IFN-gamma-secreting T cells. Virus-like particles represent a novel, modular, acellular antigen-presenting system able to modulate the responses of allergen-specific T cells in a costimulator-dependent fashion. Allergen-specific VLPs show promise as tools for specific immunotherapy of allergic diseases.

Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

3-Layer-based analysis of peptide–MHC interaction: In silico prediction, peptide binding affinity and T cell activation in a relevant allergen-specific model

CD4+ T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells by their T cell receptor (TCR). Using a well-characterized allergen-specific model we studied peptide/MHC (pMHC) interactions by combining computational methods with experimental analyses. A 12-mer and an 18-mer peptide, both containing the human leukocyte antigen (HLA)-DR1-restricted, immunodominant T cell epitope of Art v 1, the major mugwort pollen allergen, were compared. A Molecular Dynamics simulation for a real time of 20 ns using GROMACS was performed. To this aim, the peptides were modelled into the binding groove of HLA-DRB1*0101 using different amino acid substitution tools. Binding of synthetic peptides to purified HLA-DRB1*0101 molecules was analysed in competition assays. The potency of the peptides to activate Art v 1-specific T cells was assessed using oligo- and monoclonal Art v 1-specific T cell cultures expanded from mugwort allergic individuals. All approaches revealed that the 18-mer peptide possessed higher HLA DR affinity as compared to the 12-mer. Computer modelling indicated that a loop-like structure within the additional N-terminal peptide flanking region of the 18-mer contributed to the pMHC interaction. Our approach, to combine computational methods validated by experimental results, demonstrates that Molecular Dynamics simulation may be a useful tool for the prediction of pMHC interactions in the future with possible applications in T cell-based immunotherapy e.g. in Type I allergy.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation

Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Chemical modification of Art v 1, a major mugwort pollen allergen, by cis-aconitylation and citraconylation

Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen, a significant cause of hay fever all over Europe. Specific immunotherapy is the only treatment modality for allergic disease. Application of modified allergens makes the treatment safer and more efficient. In this work, two out of three (citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride) tested anhydrides were proven to be suitable for chemical modifications of allergens. Art v 1 was modified by cis-aconitylation and citraconylation in order to obtain derivatives of Art v 1 that may be suitable for further immunological testing. Acylation of Art v 1 gave derivatives (caaArt v 1 and citArt v 1) with about 80 % modified amino groups. The derivatives were in the monomeric form and had dramatically reduced pI values. Both derivatives were relatively stable at neutral pH values, while the acyl groups undergo hydrolysis under acidic conditions. Modification of allergens by cis-aconitylation and citraconylation could be a new tool for obtaining allergoids.