Kerstin Westritschnig

A Combination Vaccine for Allergy and Rhinovirus Infections Based on Rhinovirus-Derived Surface Protein VP1 and a Nonallergenic Peptide of the Major Timothy Grass Pollen Allergen Phl p 1

Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients’ IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.

Generation of an Allergy Vaccine by Disruption of the Three-Dimensional Structure of the Cross-Reactive Calcium-Binding Allergen, Phl p 7

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients’ IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients’ IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen’s three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

A comparative analysis of the cross‐reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen

Background:  Polcalcins are pollen‐specific allergens with two EF‐hand calcium‐binding sites that exhibit strong cross‐reactivity. Our objective was to isolate and express the cDNA coding of the EF‐hand calcium‐binding allergen from lilac pollen and to study cross‐reactivity with other polcalcins from related and nonrelated pollen sources with different specific antibodies and sera from two different populations.

Immunoglobulin E antibody reactivity to bacterial antigens in atopic dermatitis patients

Cite this as: K. Reginald, K. Westritschnig, T. Werfel, A. Heratizadeh, N. Novak, M. Focke‐Tejkl, A. M. Hirschl, D. Y. M. Leung, O. Elisyutina, E. Fedenko and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 357–369.

Three-Dimensional Structure of the Cross-Reactive Pollen Allergen Che a 3: Visualizing Cross-Reactivity on the Molecular Surfaces of Weed, Grass, and Tree Pollen Allergens

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-Å crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an α-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.

Allergen cleavage by effector cell-derived proteases regulates allergic inflammation

The key event of allergic inflammation, allergen‐induced crosslinking of mast cell‐bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular β‐tryptase. We provide evidence that protease‐mediated cleavage of allergens represents a mechanism that regulates allergen‐induced mast cell activation. When used in molar ratios as they occur in vivo, purified β ‐tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase‐cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin‐dependent effector cell proteases, augmented allergen‐induced release of mediators from effector cells. Protease‐mediated allergen cleavage may represent an important mechanism for terminating allergen‐induced effector cell activation.—Rauter, I., Krauth, M.‐T., Flicker, S., Gieras, A., Westritschnig, K., Vrtala, S., Balic, N., Spitzauer, S., Huss‐Marp, J., Brockow, K., Darsow, U., Ring, J., Behrendt, H., Semper, H., Valent, P., Valenta, R. Allergen cleavage by effector cell‐derived proteases regulates allergic inflammation. FASEB J. 20, E61–E69 (2006)

Recombinant allergen-based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation.

To cite this article: Gadermaier E, Staikuniene J, Scheiblhofer S, Thalhamer J, Kundi M, Westritschnig K, Swoboda I, Flicker S, Valenta R. Recombinant allergen–based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation. Allergy 2011; 66: 1174–1182.

A Hypoallergenic Vaccine Obtained by Tail-to-Head Restructuring of Timothy Grass Pollen Profilin, Phl p 12, for the Treatment of Cross-Sensitization to Profilin

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive α-helical contents of the protein but retained its β-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients’ IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.

Staphylococcus aureus fibronectin-binding protein specifically binds IgE from patients with atopic dermatitis and requires antigen presentation for cellular immune responses.

BACKGROUND Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell-mediated skin inflammation by a superantigen effect. OBJECTIVE To characterize IgE-reactive proteins from S aureus. METHODS A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli, purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. RESULTS Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T(h)2/T(h)1 cytokine secretion. CONCLUSION Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus-induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.