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1.  Glutathione-S-Transferase: A Minor Allergen in Birch Pollen due to Limited Release from Hydrated Pollen 
PLoS ONE  2014;9(10):e109075.
Recently, a protein homologous to glutathione-S-transferases (GST) was detected in prominent amounts in birch pollen by proteomic profiling. As members of the GST family are relevant allergens in mites, cockroach and fungi we investigated the allergenic relevance of GST from birch (bGST).
bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. Sera from 217 birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The mediator-releasing activity of bGST was analysed with IgE-loaded rat basophil leukaemia cells (RBL) expressing human FcεRI. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses to either protein were assessed. IgE-cross-reactivity between bGST with GST from house dust mite, Der p 8, was studied with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen were assessed in water, simulated lung fluid, 0.9% NaCl and PBS. Eluted proteins were quantified by ELISA and analysed by immunoblotting.
Principle findings
Only 13% of 217 birch pollen-allergic patients showed IgE-reactivity to bGST. In RBL assays bGST induced mediator release. Immunization of mice with bGST induced specific IgE and a Th2-dominated cellular immune response comparably to immunization with Bet v 1. bGST did not cross-react with Der p 8. In contrast to Bet v 1, only low amounts of bGST were released from pollen grains upon incubation in water and the different physiological solutions.
Although bGST is abundant in birch pollen, immunogenic in mice and able to induce mediator release from effector cells passively loaded with specific IgE, it is a minor allergen for birch pollen-allergic patients. We refer this discrepancy to its limited release from hydrated pollen. Hence, bGST is an example demonstrating that allergenicity depends mainly on rapid elution from airborne particles.
PMCID: PMC4183528  PMID: 25275548
2.  Molecular Approach to Allergy Diagnosis and Therapy 
Yonsei Medical Journal  2014;55(4):839-852.
Presently, allergy diagnosis and therapy procedures are undergoing a transition phase in which allergen extracts are being step-by-step replaced by molecule-based products. The new developments will allow clinicians to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thus improved patients' management. In this respect, recombinant technology has been applied to develop this new generation of molecule-based allergy products. The use of recombinant allergens allows full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency of the products. In contrast, such parameters are extremely difficult to assay and standardize for extract-based products. In addition to the possibility of bulk production of wild type molecules for diagnostic purposes, recombinant technology opened the possibility of developing safer and more efficacious products for allergy therapy. A number of molecule-based hypoallergenic preparations have already been successfully evaluated in clinical trials, bringing forward the next generation of allergy vaccines. In this contribution, we review the latest developments in allergen characterization, molecule-based allergy diagnosis, and the application of recombinant allergens in therapeutic setups. A comprehensive overview of clinical trials using recombinant allergens as well as synthetic peptides is presented.
PMCID: PMC4075386  PMID: 24954310
Recombinant allergens; allergen characterization; molecule-based allergy diagnosis; allergen-specific immunotherapy
9.  Stabilization of the Dimeric Birch Pollen Allergen Bet v 1 Impacts Its Immunological Properties* 
The Journal of Biological Chemistry  2013;289(1):540-551.
Background: Frequently reported dimerization of allergens may contribute to their allergenicity.
Results: Polysulfide-bridged allergen dimers exhibit different allergenic properties compared with the monomer.
Conclusion: The N-terminal region has a distinct susceptibility for modifications and impacts its protein-protein interaction characteristics.
Significance: The crystal structures well mimic transient dimerization of the allergens in solution, providing a rational for effective IgE cross-linking on effector cells.
Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.
PMCID: PMC3879576  PMID: 24253036
Allergen; Crystal Structure; Mass Spectrometry (MS); Post-translational Modification; Protein Assembly; Dimerization; Noncanonical Amino Acid Incorporation; Polysulfide Linking; Position-specific Alteration of Genetic Code
10.  Proteomic and Immunochemical Characterization of Glutathione Transferase as a New Allergen of the Nematode Ascaris lumbricoides 
PLoS ONE  2013;8(11):e78353.
Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules.
Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.
PMCID: PMC3817249  PMID: 24223794
11.  The Fold Variant BM4 Is Beneficial in a Therapeutic Bet v 1 Mouse Model 
BioMed Research International  2013;2013:832404.
Background. Specific immunotherapy using recombinant allergens is clinically effective; still wild-type allergens can provoke treatment-induced side effects and often show poor immunogenicity in vivo. Thus, we tested the low IgE-binding, highly immunogenic fold variant BM4 in a Bet v 1 mouse model. Methods. Recombinant BM4 was used as active vaccine ingredient to treat mice sensitized to Bet v 1. As controls, mice were treated with either Bet v 1 or sham, and the humoral as well as cellular immune response was monitored. Moreover, lung function and lung inflammation were analysed. Results. BM4 was more effective than wild-type Bet v 1 in inducing Bet v 1-specific blocking antibodies as well as IFN-γ and IL-10 producing T cells. Further, birch pollen induced lung inflammation could be ameliorated significantly by BM4 treatment as demonstrated by a reduction of airway hyperresponsiveness and drastically decreased eosinophil counts in bronchoalveolar lavage fluids. Conclusion. The study outlines the high potential of BM4 as vaccine candidate for the treatment of Bet v 1-mediated birch pollen allergies.
PMCID: PMC3794650  PMID: 24175303
13.  Molecular and Immunological Characterization of Ragweed (Ambrosia artemisiifolia L.) Pollen after Exposure of the Plants to Elevated Ozone over a Whole Growing Season 
PLoS ONE  2013;8(4):e61518.
Climate change and air pollution, including ozone is known to affect plants and might also influence the ragweed pollen, known to carry strong allergens. We compared the transcriptome of ragweed pollen produced under ambient and elevated ozone by 454-sequencing. An enzyme-linked immunosorbent assay (ELISA) was carried out for the major ragweed allergen Amb a 1. Pollen surface was examined by scanning electron microscopy and attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR), and phenolics were analysed by high-performance liquid chromatography. Elevated ozone had no influence on the pollen size, shape, surface structure or amount of phenolics. ATR-FTIR indicated increased pectin-like material in the exine. Transcriptomic analyses showed changes in expressed-sequence tags (ESTs), including allergens. However, ELISA indicated no significantly increased amounts of Amb a 1 under elevated ozone concentrations. The data highlight a direct influence of ozone on the exine components and transcript level of allergens. As the total protein amount of Amb a 1 was not altered, a direct correlation to an increased risk to human health could not be derived. Additional, the 454-sequencing contributes to the identification of stress-related transcripts in mature pollen that could be grouped into distinct gene ontology terms.
PMCID: PMC3630196  PMID: 23637846
15.  Heat-Induced Structural Changes Affect OVA-Antigen Processing and Reduce Allergic Response in Mouse Model of Food Allergy 
PLoS ONE  2012;7(5):e37156.
Background and Aims
The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy.
Methodology/Principal Findings
Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes.
Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.
PMCID: PMC3357411  PMID: 22629361
16.  Response to Detection and analysis of unusual features in the structural model and structure-factor data of a birch pollen allergen  
A response to the article by Rupp (2012), Acta Cryst. F68, 366–376.
The authors of J. Immunol. 184, 725–735 respond to the article by Rupp (2012), Acta Cryst. F68, 366–376.
PMCID: PMC3325801  PMID: 22505401
response; protein structure; Bet V 1 birch pollen allergen
17.  Nitration of the Pollen Allergen Bet v 1.0101 Enhances the Presentation of Bet v 1-Derived Peptides by HLA-DR on Human Dendritic Cells 
PLoS ONE  2012;7(2):e31483.
Nitration of pollen derived allergens can occur by NO2 and ozone in polluted air and it has already been shown that nitrated major birch (Betula verrucosa) pollen allergen Bet v 1.0101 (Bet v 1) exhibits an increased potency to trigger an immune response. However, the mechanisms by which nitration might contribute to the induction of allergy are still unknown. In this study, we assessed the effect of chemically induced nitration of Bet v 1 on the generation of HLA-DR associated peptides. Human dendritic cells were loaded with unmodified Bet v 1 or nitrated Bet v 1, and the naturally processed HLA-DR associated peptides were subsequently identified by liquid chromatography-mass spectrometry. Nitration of Bet v 1 resulted in enhanced presentation of allergen-derived HLA-DR-associated peptides. Both the copy number of Bet v 1 derived peptides as well as the number of nested clusters was increased. Our study shows that nitration of Bet v 1 alters antigen processing and presentation via HLA-DR, by enhancing both the quality and the quantity of the Bet v 1-specific peptide repertoire. These findings indicate that air pollution can contribute to allergic diseases and might also shed light on the analogous events concerning the nitration of self-proteins.
PMCID: PMC3279363  PMID: 22348091
18.  30 Ragweed Allergy – What Role Does It Play in Bavaria? 
The World Allergy Organization Journal  2012;5(Suppl 2):S27-S28.
Ragweed (Ambrosia artemisiifolia), is increasingly spreading in Southern Germany and Central Europe. Little is yet known about the sensitization and allergy rates in Bavaria.
In 2008 to 2010 patients from a Bavarian university allergy unit were enrolled into the study. The patient's history was recorded by a standardised questionnaire concerning allergies. Sensitization rates were measured by skin prick test (SPT) for seasonal aeroallergens including ragweed. Patients sensitized to ragweed were further characterized by measuring specific serum immunoglobulin E (IgE) for ragweed specific allergens (by ImmunoCAP and ELISA). To determine the clinical relevance challenge tests (nasal/conjunctival) with ragweed were performed.
1022 patients were enrolled in the study (665 female, 357 male). 289 patients were sensitized to ragweed (SPT positive). In ragweed sensitized patients the sensitization rate to mugwort was 61.8% whilst in patients not sensitized to ragweed it was 7.4%. The sensitization to birch was 78.1% resp. 36.4%. In 120 ragweed sensitized patients challenge tests with ragweed extract were performed (nasal n = 110; conjunctival n = 60) with positive results in 29 (26%) resp. 12 (20%) patients. In 232 ragweed sensitized patients specific IgE to nArt v 1 was observed significantly more frequently than to nAmb a 1.
The results of this 3-year study show that in a Bavarian allergy unit sensitization to ragweed is frequent. Often ragweed-sensitized patients have sensitivities to multiple seasonal aeroallergens. There is a coexistence of ragweed and mugwort specific allergens. One fourth of the challenged patients that are sensitized to ragweed show clinical allergy symptoms. With sufficient ambient allergen exposure, a prolonged allergy season can be expected for this at-risk population.
PMCID: PMC3513050
19.  Expression of the major mugwort pollen allergen Art v 1 in tobacco plants and cell cultures: problems and perspectives for allergen production in plants 
Plant Cell Reports  2011;31(3):561-571.
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 material
The online version of this article (doi:10.1007/s00299-011-1199-3) contains supplementary material, which is available to authorised users.
PMCID: PMC3325494  PMID: 22159963
Artemisia vulgaris; Molecular pharming; Pollen; Allergy; Recombinant allergen
20.  Ozone affects pollen viability and NAD(P)H oxidase release from Ambrosia artemisiifolia pollen 
Air pollution is frequently proposed as a cause of the increased incidence of allergy in industrialised countries. We investigated the impact of ozone (O3) on reactive oxygen species (ROS) and allergen content of ragweed pollen (Ambrosia artemisiifolia). Pollen was exposed to acute O3 fumigation, with analysis of pollen viability, ROS and nitric oxide (NO) content, activity of nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase, and expression of major allergens. There was decreased pollen viability after O3 fumigation, which indicates damage to the pollen membrane system, although the ROS and NO contents were not changed or were only slightly induced, respectively. Ozone exposure induced a significant enhancement of the ROS-generating enzyme NAD(P)H oxidase. The expression of the allergen Amb a 1 was not affected by O3, determined from the mRNA levels of the major allergens. We conclude that O3 can increase ragweed pollen allergenicity through stimulation of ROS-generating NAD(P)H oxidase.
► O3 reduces the viability of ragweed pollen. ► ROS and allergens of ragweed pollen were not affected by O3 exposure. ► O3 enhances the activity of the ROS-generating enzyme NAD(P)H oxidase. ► O3 increases ragweed pollen allergenicity through NAD(P)H-oxidase stimulation.
This study focuses on the effects of the atmospheric pollutant ozone on ROS content and NAD(P)H oxidase activity of ragweed pollen grains.
PMCID: PMC3173721  PMID: 21605929
NAD(P)H oxidase; Ozone; Pollen; Ragweed; ROS
21.  Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery 
PLoS ONE  2011;6(8):e24150.
Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model.
786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs.
IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides.
Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.
PMCID: PMC3163685  PMID: 21897872
24.  Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity But Retained Immunogenicity 
Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason.
To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity.
Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant Fel d 1 (rFel d 1) was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP-inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural Fel d 1 and recombinant unmodified Fel d 1 were used as controls.
rFel d 1 demonstrated similar IgE binding and biological activity as its natural counterpart. Upon modification, IgE-binding potency decreased to >1000-fold, which was translated into a >106-fold reduction in the biological activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen.
Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.
PMCID: PMC3488918  PMID: 23268458
allergoid; Felis domesticus; hypoallergen; immunotherapy; rFel d 1
25.  Is Aboriginal Food Less Allergenic? Comparing IgE-Reactivity of Eggs from Modern and Ancient Chicken Breeds in a Cohort of Allergic Children 
PLoS ONE  2011;6(4):e19062.
Hen's egg allergy ranks among the most frequent primary food allergies in children. We aimed to investigate sensitization profiles of egg allergic patients and compare in vitro IgE reactivities of eggs from ancient chicken breeds (Araucana and Maran) with those from conventional laying hen hybrids.
Egg allergic children (n = 25) were subjected to skin prick test, double blind placebo controlled food challenge, and sensitization profiles to Gal d 1–5 were determined by allergen microarray. IgE binding and biological activity of eggs from different chicken breeds were investigated by immunoblot, ELISA, and mediator release assays.
Principal Findings
We found that Gal d 1 and Gal d 2 are generally major egg allergens, whereas Gal d 3–5 displayed high sensitization prevalence only in patients reacting to both, egg white and yolk. It seems that the onset of egg allergy is mediated by egg white allergens expanding to yolk sensitization in later stages of disease. Of note, egg white/yolk weight ratios were reduced in eggs from Auraucana and Maran chicken. As determined in IgE immunoblots and mass analysis, eggs from ancient chicken breeds did not differ in their protein composition. Similar IgE-binding was observed for all egg white preparations, while an elevated allergenicity was detected in egg yolk from Araucana chicken.
Our results on allergenicity and biological activity do not confirm the common assumption that aboriginal food might be less allergenic. Comprehensive diagnosis of egg allergy should distinguish between reactivity to hen's egg white and yolk fractions to avoid unnecessary dietary restrictions to improve life quality of the allergic child and its family.
PMCID: PMC3084253  PMID: 21552565

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