Sabine Flicker

Renaissance of the blocking antibody concept in type I allergy.

Formation of IgE antibodies against per se harmless antigens (i.e. allergens) is the hallmark and key pathomechanism of type I allergy, a hypersensitivity disease affecting more than 25% of the population. Classical experiments performed more than 65 years ago demonstrated that allergen-specific IgG antibodies, termed blocking antibodies, can antagonize the cascade of allergic inflammation resulting from allergen recognition by IgE antibodies. However, controversial results have questioned the protective role of IgG antibodies in allergic diseases. Here, we review recent data demonstrating that blocking antibodies inhibit allergen-induced release of inflammatory mediators from basophils and mast cells as well as IgE-facilitated allergen presentation to T cells, thus leading to suppression of T cell activation. Furthermore, it has been reported that the development of blocking antibodies is associated with reduced boosts of allergen-specific IgE production in patients receiving allergen-specific immunotherapy. These findings suggest that blocking antibodies have protective activity by inhibiting immediate as well as late inflammatory responses and long-term ameliorating activity on the allergic immune response by antagonizing the underlying IgE production. Induction of blocking antibodies is thus an important mechanism underlying allergen-specific immunotherapy. In addition, passive administration of blocking antibodies may be considered as a potential therapeutic strategy for allergic diseases.

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.

High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

We report the three-dimensional structure of the complex between the major respiratory grass pollen allergen Phl p 2 and its specific human IgE-derived Fab. The Phl p 2-specific human IgE Fab has been isolated from a combinatorial library constructed from lymphocytes of a pollen allergic patient. When the variable domains of the IgE Fab were grafted onto human IgG1, the resulting Ab (huMab2) inhibited strongly the binding of allergic patients’ IgE to Phl p 2 as well as allergen-induced basophil degranulation. Analysis of the binding of the allergen to the Ab by surface plasmon resonance yielded a very low dissociation constant (KD = 1.1 × 10−10 M), which is similar to that between IgE and Fcε;RI. The structure of the Phl p 2/IgE Fab complex was determined by x-ray crystallography to 1.9 Å resolution revealing a conformational epitope (876 Å2) comprised of the planar surface of the four-stranded anti-parallel β-sheet of Phl p 2. The IgE-defined dominant epitope is discontinuous and formed by 21 residues located mostly within the β strands. Of the 21 residues, 9 interact directly with 5 of the 6 CDRs (L1, L3, H1, H2, H3) of the IgE Fab predominantly by hydrogen bonding and van der Waals interactions. Our results indicate that IgE Abs recognize conformational epitopes with high affinity and provide a structural basis for the highly efficient effector cell activation by allergen/IgE immune complexes.

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

Conversion of grass pollen allergen-specific human IgE into a protective IgG1 antibody

More than 100 million individuals exhibit IgE‐mediated allergic reactions against Phl p 2, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phl p 2‐specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen‐allergic patient. Recombinant Phl p 2‐specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N‐terminal amino acids of Phl p 2 and cross‐reacted with group 2 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG1 to obtain a complete, recombinant Phl p 2‐specific human IgG1. This antibody blocked the binding of grass pollen‐allergic patients IgE (n=26; mean inhibition: 58%) to Phl p 2 and caused a 100‐fold reduction of Phl p 2‐induced basophil histamine release. The recombinant human Phl p 2‐specific IgG1 may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.

Mapping of Conformational IgE Epitopes with Peptide-Specific Monoclonal Antibodies Reveals Simultaneous Binding of Different IgE Antibodies to a Surface Patch on the Major Birch Pollen Allergen, Bet v 1

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients’ IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients’ IgE binding to Bet v 1 (52–75%) were obtained with mAbs specific for two peptides comprising aa 29–58 (P2) and aa 73–103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

The immunoglobulin E-allergen interaction: a target for therapy of type I allergic diseases.

The interaction of immunoglobulin E and otherwise harmless antigens (allergens) leadsin sensitized individuals through effector cell activation to the immediate induction of acascade of inflammatory reactions, the hallmark of type I allergy. Recently, the molecularand structural characterization of allergens, specific IgE antibodies and their epitopes hasmade rapid progress. Here we discuss active and passive strategies for therapy of type Iallergy, which are based on interfering with the IgE–allergen interaction.

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.

Passive immunization with allergen-specific IgG antibodies for treatment and prevention of allergy

IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans.