Michael Weichel

Screening the allergenic repertoires of wheat and maize with sera from double-blind, placebo-controlled food challenge positive patients.

Background:  Food allergy to wheat and maize is an increasing factor of deterioration of life quality, especially childhood and can, in rare cases, even induce anaphylaxis. Although omega‐5 gliadin from wheat and maize lipid transfer protein have been characterized as major cereal allergens on the molecular level, the list of food allergens is far to be complete.

Fungal Allergies: A Yet Unsolved Problem

Airborne fungal spores have been implicated as causative factors in respiratory allergy, particularly asthma. However, the prevalence of fungal sensitization is not known mainly due to the lack of standardized fungal extracts and to the overwhelming number of fungal species able to elicit IgE-mediated reactions. Recent work based on high-throughput cloning of fungal allergens revealed that fungi are able to produce extremely complex repertoires of species-specific and cross-reactive allergens. There is evidence that fungal sensitization also contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens. Detailed studies at structural and immunological level indicate molecular mimicry as a basic mechanism involved in perpetuation of severe chronic allergic diseases. The real challenge at present is not related to cloning or production of a large number of different fungal allergens but rather to the assessment of the clinical relevance of each single structure. To date, substitution of complex extracts presently used in the diagnosis of fungal allergy by single, perfectly standardized components seems feasible in contrast to specific immunotherapy which is still not developed. Recombinant fungal allergens might create new perspectives in diagnosis and therapy of fungal allergy.

Nuclear transport factor 2 represents a novel cross-reactive fungal allergen

Background: Ubiquitously occuring moulds are important allergenic sources known to elicit IgE‐mediated allergic diseases and to share cross‐reactive allergens. Limited information is available about the molecular structures involved in cross‐reactivity. We aimed to clone and characterize cross‐reactive mould allergens.

Immunoglobulin E-binding and skin test reactivity to hydrophobin HCh-1 from Cladosporium herbarum, the first allergenic cell wall component of fungi

Background For many years, fungal spores have been recognized as potential causes of respiratory allergies. All fungal allergens cloned so far represent either secreted or cytoplasmatic proteins, but nothing is known about the involvement of fungal surface proteins in allergic diseases.

IL-10 controls Aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis.

Up to 90% of patients with cystic fibrosis (CF) are chronically colonized with Pseudomonas aeruginosa, and 10% to 50% of CF patients are colonized with Aspergillus fumigatus. Despite an extensive inflammatory reaction, patients cannot eliminate the microorganisms. The present study demonstrates that an IL-10 mediated T-cell tolerance to major infectious agents A. fumigatus and P. aeruginosa plays an important role in the control of T-cell-mediated inflammatory responses in CF. Peripheral blood mononuclear cells of CF patients secreted significantly higher amounts of IL-10. T-cell response against recombinant A. fumigatus antigens rAsp f 3, rAsp f 4, rAsp f 6, and heatinactivated P. aeruginosa was controlled by IL-10. Proliferation and interferon-γ production was significantly increased when endogenous IL-10 was blocked in aspergillus and pseudomonas antigen-stimulated cells of CF patients. The role of IL-10 was further documented by increased spontaneous proliferation of peripheral blood mononuclear cells of CF patients after preincubation with antisense oligonucleotides blocking the synthesis of IL-10 receptor-associated kinases janus tyrosine kinase 1 and tyrosine kinase 2. Together, these data demonstrate an important role of IL-10-mediated peripheral T-cell tolerance to P. aeruginosa and A. fumigatus in the control of the intensity of the inflammatory T-cell response in CF.

Unfolding and Double-stranded DNA Binding of the Cold Shock Protein Homologue Cla h 8 from Cladosporium herbarum

The cloning, purification, and biophysical characterization of the first eukaryotic cold shock protein homologue, Cla h 8, expressed as single functional polypeptide is reported here. It was discovered as a minor allergen of the mold Cladosporium herbarum by phage display using a library selectively enriched for IgE-binding proteins. Based on the sequence homology of Cla h 8 with bacterial cold shock proteins (CSPs), a homology-based computer model of the allergen was computed indicating an all-β structure of Cla h 8. This major structural feature was confirmed by CD spectroscopy. Despite the structural similarities with bacterial CSPs, the DNA-binding and unfolding behavior of Cla h 8 exhibited unique and previously undescribed characteristics. High affinities of Cla h 8 for single-stranded DNA as well as for double-stranded DNA corresponding to the human Y-box were detected. The affinity for double-stranded DNA increased significantly with decreasing temperature, which was paralleled by an increase in the β sheet content of the protein. Temperature-dependent fluorescence anisotropy and far-UV CD measurements revealed different unfolding transitions at 28 and at 35.7 °C, respectively, indicating a multistate transition, which is uncommon for CSPs. The enhanced affinity for DNA at low temperatures together with the low unfolding transition refer to the functional significance of Cla h 8 at reduced temperatures.

Structural aspects and clinical relevance of Aspergillus fumigatus antigens/allergens

Robotics-based high throughput screening of Aspergillus fumigatus cDNA libraries displayed on phage surfaces revealed at last 81 different structures able to bind IgE from serum of patients sensitized to this fungus. Among these, species-specific as well as phylogenetically highly conserved structures and such with unknown function have been detected. A subset of cDNAs have been used to produce and characterize the corresponding recombinant allergens which have proven to be useful diagnostic reagents allowing specific detection of A. fumigatus sensitization and differential diagnosis of allergic bronchopulmonary aspergillosis. Phylogenetically highly conserved structures like manganese-dependent superoxide dismutase, P2 acidic ribosomal protein, cyclophilins and thioredoxins induce, beyond sensitization, IgE antibodies able to cross-react with the corresponding homologous self antigens. These reactions, likely to contribute to the exacerbation and perpetuation of allergic bronchopulmonary aspergillosis, can be traced back to shared conformational B-cell epitopes build up from conserved amino acid residues scattered over the surface of the molecules as shown by detailed analyses of the crystal structures.

Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin.

Coeliac disease (CD), a gastrointestinal illness characterized by intestinal malabsorption, results from gluten intolerance accompanied with immunological responses towards gliadin, an ethanol‐soluble protein fraction of wheat and other cereals. The role of gliadin in eliciting immune responses in CD is still partly unclear; however, the occurrence of anti‐gliadin in the sera of patients suffering from CD correlates well with clinical symptoms. In this work we report the construction of isotype‐specific, phage‐displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. VH and VL chains were amplified by reverse transcription–polymerase chain reaction (RT–PCR) using a set of oligonucleotides recognizing all human variable gene families. The three scFv libraries (IgA, IgG and IgM) were selectively enriched for gliadin‐binding phage. After four rounds of affinity selection, polyclonal enrichment of gliadin‐binding phage was observed in all libraries from the CD patient but in none from the healthy donor. Phagemid particles generated from single clones were demonstrated to be gliadin‐specific, as shown by strongly positive enzyme‐linked immunosorbent assay (ELISA) and BiaCore signals. The VH and VL chains from samples of these monoclonal isotype‐specific phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin.

Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells

Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens.

Identification of Borrelia Burgdorferi Ribosomal Protein L25 by the Phage Surface Display Method and Evaluation of the Protein's Value for Serodiagnosis

ABSTRACT The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.