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1.  Structural Integrity of the Antigen Is a Determinant for the Induction of T-Helper Type-1 Immunity in Mice by Gene Gun Vaccines against E.coli Beta-Galactosidase 
PLoS ONE  2014;9(7):e102280.
The type of immune response is critical for successful protection and typically determined by pathogen-associated danger molecules. In contrast, protein antigens are usually regarded as passive target structures. Here, we provide evidence that the structure of the antigen can profoundly influence the type of response that is elicited under else identical conditions. In mice, gene gun vaccines induce predominantly Th2-biased immune reactions against most antigens. One exception is E. coli beta-galactosidase (βGal) that induces a balanced Th1/Th2 response. Because both, the delivered material (plasmid DNA-coated gold particles) as well as the procedure (biolistic delivery to the skin surface) is the same as for other antigens we hypothesized that Th1 induction could be a function of βGal protein expressed in transfected cells. To test this we examined gene gun vaccines encoding structural or functional variants of the antigen. Employing a series of gene gun vaccines encoding individual structural domains of βGal, we found that neither of them induced IgG2a antibodies. Even disruption of the homo-tetramer association of the native protein by deletion of a few N-terminal amino acids was sufficient to abrogate IgG2a production. However, enzymatically inactive βGal with only one point mutation in the catalytic center retained the ability to induce Th1 reactions. Thus, structural but not functional integrity of the antigen must be retained for Th1 induction. βGal is not a Th1 adjuvant in the classical sense because neither were βGal-transgenic ROSA26 mice particularly Th1-biased nor did co-administration of a βGal-encoding plasmid induce IgG2a against other antigens. Despite this, gene gun vaccines elicited Th1 reactions to antigens fused to the open reading frame of βGal. We interpret these findings as evidence that different skin-borne antigens may be differentially handled by the immune system and that the three-dimensional structure of an antigen is an important determinant for this.
doi:10.1371/journal.pone.0102280
PMCID: PMC4099185  PMID: 25025197
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.
doi:10.3349/ymj.2014.55.4.839
PMCID: PMC4075386  PMID: 24954310
Recombinant allergens; allergen characterization; molecule-based allergy diagnosis; allergen-specific immunotherapy
4.  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.
doi:10.1155/2013/832404
PMCID: PMC3794650  PMID: 24175303
5.  Crystallographically Mapped Ligand Binding Differs in High and Low IgE Binding Isoforms of Birch Pollen Allergen Bet v 1 
Journal of Molecular Biology  2012;422(1):109-123.
The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.
Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.
The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.
Graphical Abstract
Highlights
► Ligand binding to Bet v 1 may contribute to explain its allergenicity. ► High-resolution structures reveal the binding mode of diverse ligands to Bet v 1. ► Residue 30 starkly influences the binding properties of different Bet v 1 isoforms. ► Ternary complexes with diverse ligands explain anomalous fluorescence binding curves. ► Betv1 isoforms differ in ligand binding, which may translate into their allergenicity.
doi:10.1016/j.jmb.2012.05.016
PMCID: PMC3422537  PMID: 22634284
ANS, 1-anilino-8-naphthalene sulfonate; BRA, brassinolide; DXC, deoxycholate; iDXC, inner deoxycholate; oDXC, outer deoxycholate; LPS, lipopolysaccharide; MPD, 2-methyl-2,4-pentanediol; NDSB-256, non-detergent sulfobetaine 256; PR-10, pathogenesis-related protein 10; PDB, Protein Data Bank; molecular allergenicity; ANS displacement assay; structure–allergenicity relationship; binding specificity and promiscuity; dressed allergens
7.  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.
Conclusions
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.
doi:10.1371/journal.pone.0037156
PMCID: PMC3357411  PMID: 22629361
8.  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.
doi:10.1107/S1744309112008433
PMCID: PMC3325801  PMID: 22505401
response; protein structure; Bet V 1 birch pollen allergen
10.  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.
Background
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.
Methodology
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.
Conclusion/Significance
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.
doi:10.1371/journal.pone.0019062
PMCID: PMC3084253  PMID: 21552565
11.  Assessing Protein Immunogenicity with a Dendritic Cell Line-Derived Endolysosomal Degradome 
PLoS ONE  2011;6(2):e17278.
Background
The growing number of novel candidate molecules for the treatment of allergic diseases imposed a dramatic increase in the demand for animal experiments to select immunogenic vaccines, a pre-requisite for efficacy. Because no in vitro methods to predict the immunogenicity of a protein are currently available, we developed an in vitro assay that exploits the link between a protein's immunogenicity and its susceptibility to endolysosomal proteolysis.
Methodology
We compared protein composition and proteolytic activity of endolysosomal fractions isolated from murine bone marrow- and human blood- derived dendritic cells, and from the dendritic cell line JAWS II. Three groups of structurally related antigen variants differing in their ability to elicit immune responses in vivo (Bet v 1.0101 and Bet v 1.0401, RNases A and S, holo- and apo-HRP) were subjected to in vitro simulated endolysosomal degradation. Kinetics and patterns of generated proteolytic peptides were evaluated by gel electrophoresis and mass spectrometry.
Results
Antigens displaying weak capacity of T cell priming in vivo were highly susceptible to endolysosomal proteases in vitro. As proteolytic composition, activity, and specificity of endolysosomal fractions derived from human and murine dendritic cells were comparable, the JAWS II cell line could be used as a substitute for freshly isolated human or murine cells in in vitro degradation assays.
Conclusions
Endolysosomal fractions prepared from the JAWS II cell line provide a reliable tool for in vitro estimation of protein immunogenicity. The rapid and simple assay described here is very useful to study the immunogenic properties of a protein, and can help to replace, reduce, and refine animal experiments in allergy research and vaccine development in general.
doi:10.1371/journal.pone.0017278
PMCID: PMC3040223  PMID: 21359181
12.  Antigen Aggregation Decides the Fate of the Allergic Immune Response 
Previously, defined naturally occurring isoforms of allergenic proteins were classified as hypoallergens and therefore suggested as an agent for immunotherapy in the future. In this paper, we report for the first time the molecular background of hypoallergenicity by comparing the immunological behavior of hyperallergenic Betula verrucosa major Ag 1a (Bet v 1a) and hypoallergenic Bet v 1d, two isoforms of the major birch pollen allergen Betula verrucosa 1. Despite their cross-reactivity, Bet v 1a and Bet v 1d differ in their capacity to induce protective Ab responses in BALB/c mice. Both isoforms induced similar specific IgE levels, but only Bet v 1d expressed relevant titers of serum IgGs and IgAs. Interestingly, hypoallergenic Bet v 1d activated dendritic cells more efficiently, followed by the production of increased amounts of Th1- as well as Th2-type cytokines. Surprisingly, compared with Bet v 1a, Bet v 1d-immunized mice showed a decreased proliferation of regulatory T cells. Crystallographic studies and dynamic light scattering revealed that Bet v 1d demonstrated a high tendency to form disulfide-linked aggregates due to a serine to cysteine exchange at residue 113. We conclude that aggregation of Bet v 1d triggers the establishment of a protective Ab titer and supports a rationale for Bet v 1d being a promising candidate for specific immunotherapy of birch pollen allergy.
doi:10.4049/jimmunol.0902080
PMCID: PMC2968749  PMID: 19995902
13.  Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization 
Background
In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from Candida tenuis in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate qxylose (g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit qxylose of BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose.
Results
BP10001 and BP000, expressing C. tenuis xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L) were converted sequentially, the corresponding qsubstrate values being similar for each strain (glucose: 3.0; xylose: 0.05). The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g) and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g) and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g) as compared to BP000. Increase in xylose concentration from 10 to 50 g/L resulted in acceleration of substrate uptake by BP10001 (0.05 - 0.14 g/g CDW/h) and reduction of the xylitol yield (0.28 g/g - 0.15 g/g). In mixed substrate batches, xylose was taken up at low glucose concentrations (< 4 g/L) and up to fivefold enhanced xylose uptake rate was found towards glucose depletion. A fed-batch process designed to maintain a "stimulating" level of glucose throughout the course of xylose conversion provided a qxylose that had an initial value of 0.30 ± 0.04 g/g CDW/h and decreased gradually with time. It gave product yields of 0.38 g ethanol/g total sugar and 0.19 g xylitol/g xylose. The effect of glucose on xylose utilization appears to result from the enhanced flux of carbon through glycolysis and the pentose phosphate pathway under low-glucose reaction conditions.
Conclusions
Relative improvements in the distribution of fermentation products from xylose that can be directly related to a change in the coenzyme preference of xylose reductase from NADPH in BP000 to NADH in BP10001 increase in response to an increase in the initial concentration of the pentose substrate from 10 to 50 g/L. An inverse relationship between xylose uptake rate and xylitol yield for BP10001 implies that xylitol by-product formation is controlled not only by coenzyme regeneration during two-step oxidoreductive conversion of xylose into xylulose. Although xylose is not detectably utilized at glucose concentrations greater than 4 g/L, the presence of a low residual glucose concentration (< 2 g/L) promotes the uptake of xylose and its conversion into ethanol with only moderate xylitol by-product formation. A fed-batch reaction that maintains glucose in the useful concentration range and provides a constant qglucose may be useful for optimizing qxylose in processes designed for co-fermentation of glucose and xylose.
doi:10.1186/1475-2859-9-16
PMCID: PMC2847541  PMID: 20219100
14.  The Influence of Recombinant Production on the Immunologic Behavior of Birch Pollen Isoallergens 
PLoS ONE  2009;4(12):e8457.
Background
Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.
Methodology
Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.
Conclusion
The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.
doi:10.1371/journal.pone.0008457
PMCID: PMC2795169  PMID: 20041109

Results 1-14 (14)