Anna Twardosz

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Calcium–Binding Allergens: From Plants to Man

Calcium–binding proteins contain a variable number of motifs, termed EF–hands, which consist of two perpendicularly placed α–helices and an interhelical loop forming a single calcium–binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium–dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF–hand fish allergen, calcium–binding allergens were discovered in pollens of trees, grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium–binding allergens, these allergens may be important because of their ability to cross–sensitize allergic individuals. Conformation and stability as well as IgE recognition of calcium–binding allergens greatly depend on the presence of protein–bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium–binding allergens can be engineered by recombinant DNA technology for immunotherapy of sensitized patients.

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind‐pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF‐hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen‐specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7‐homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium‐bound and apo‐rPhl p 7 indicated that differences in IgE recognition may be due to calcium‐induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation‐dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.—Niederberger, V., Hayek, B., Vrtala, S., Laffer, S., Twardosz, A., Vangelista, L., Sperr, W. R., Valent, P., Rumpold, H., Kraft, D., Ehrenberger, K., Valenta, R., Spitzauer, S. Calcium‐dependent immunoglobulin E recognition of the apo‐ and calcium‐bound form of a cross‐reactive two EF‐hand timothy grass pollen allergen, Phl p 7. FASEB J. 13, 843–856 (1999)

Genetically engineered and synthetic allergen derivatives: candidates for vaccination against type I allergy.

Abstract Type I allergy, a hypersensitivity disease affecting almost 20% of the population worldwide, is based on the IgE recognition of otherwise harmless antigens (i.e., allergens). Allergen-induced crosslink of effectorcell-bound IgE antibodies leads to the release of biological mediators and thus to immediate disease symptoms (allergic rhinitis, conjunctivitis and asthma). Specific immunotherapy, the only causative treatment of Type I allergy, is based on the administration of increasing doses of allergens to allergic patients in order to yield allergen-specific non-responsiveness. Major disadvantages are 1. that current forms of allergen immunotherapy are performed with allergens difficult to standardize which cannot be matched to the patients reactivity profile and 2. that the administration of active allergen preparations can cause anaphylactic side effects. Through the application of molecular biological techniques many relevant environmental allergens have been produced as active recombinant proteins which allow component-resolved allergy diagnosis and thus represent the basis for patient-tailored forms of immunotherapy. Here we review molecular strategies which have been recently applied to generate genetically engineered and synthetic hypoallergenic allergen derivatives for patient-tailored and safe vaccination against Type I allergy.

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.

Calcium-Binding Proteins in Type I Allergy: Elicitors and Vaccines

Type I allergy is an immunologically-mediated hypersensitivity disease with complex genetic background affecting almost 25% of the population (Kay, 1997; Lockey and Bukantz, 1998). As a major feature of their disease, allergic patients produce IgE antibodies against per se mostly harmless antigens (i.e., allergens) which, after allergen-binding, can activate effector and inducer cells of the atopic immune response (Ravetch and Kinet, 1991; Beaven and Metzger, 1993; Bieber, 1996; Stingi and Maurer, 1997). Depending on the site and duration of allergen contact and the type of immune cells involved, the manifestations of Type I allergy may greatly vary (e.g., allergic rhinitis, conjunctivitis, asthma, dermatitis, gastrointestinal disease). Progress made in the field of molecular allergen characterization has revealed that calcium-binding proteins from many sources are frequent targets for IgE antibodies of allergic patients and thus can act as widely spread elicitors of Type I allergy (Valenta et al., 1998). Calcium-binding allergens from different sources share sequence and structural similarities. Therefore, patients who are cross-sensitized to calcium-binding allergens can exhibit allergic symptoms after exposure to many allergen sources. Calcium-depletion experiments indicate that IgE antibodies of sensitized individuals recognize preferentially the calcium-bound forms of the allergens whereas the apoforms are poorly recognized. This fact opens possibilities to employ genetic engineering and synthetic peptide chemistry for the production of hypoallergenic apoforms which may be used for therapeutic vaccination against Type I allergy with a reduced rate of anaphylactic side effects.