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

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 effector cell-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.

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.

Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition.

Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.

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 alpha-helics and an inter-helical 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. Confrontation 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.

Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4.

Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated from a birch pollen expression cDNA library constructed in phage lambda gt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen. Recombinant Bet v 4, expressed as beta-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals. Depletion of allergenbound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes. The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity. By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen. Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies.