Development of allergen vaccines by gene shuffling

The oral allergy syndrome (OAS), an association of food allergies to fruits, nuts, and vegetables in patients with pollen allergy, is a frequent clinical manifestation caused by cross-reactive IgE antibodies. OAS in patients suffering from tree pollen-allergy is caused in most cases by IgE cross-reactivity of Bet v 1, the major birch pollen allergen, and its homologous proteins. Several studies indicated that although specific immunotherapy with tree pollen preparations effectively improves hayfever symptoms in OAS patients, a parallel reduction of clinical sensitivity to foods is not always observed. Therefore we decided to use the concept of molecular breeding to generate molecules which are suitable for SIT not only against tree pollen allergens but also against cross-reactive food allergens. In principle, the features of the DNA family shuffling method would allow the generation of allergen chimaeras having T cell epitopes derived from several family members, but at the same time with reduced anaphylatic potential and improved immunogenicity. Such molecules could be used for immunotherapy of patients suffering from OAS. To generate such allergen chimaeras, the coding sequences of Bet v 1 family members will be randomly recombined by gene shuffling and the resultant products expressed in E. coli. This chimaeric library will then be screened for proteins having the following properties: 1) Low/no IgE-binding activity when tested in vitro with sera from allergic patients and low allergenicity in vivo in skin tests: this property is important for minimizing side effects during specific immunotherapy and, thus, allowing the application of sufficiently high doses of the therapeutic agent. 2) Recognition by allergen-specific T helper cells: since modulation of allergen-specific T cells is an important aspect in the clinical improvement achieved by immunotherapy, these proteins should be able to activate these cells. In addition to that, best candidates are those that also display a maximum of T-cell stimulatory activity against a maximum number of the parental allergens of the Bet v 1 family.

Inhalant allergies caused by pollen affect at least 10% of the world population and up to one-third of the affected individuals displaying hay fever symptoms will later develop allergic asthma. Spring pollinosis is mainly caused by Bet v 1, the major birch pollen allergen, and homologous allergens from related trees. This family of allergens is estimated to cause allergy in 10-20 million individuals worldwide. In this project, we have randomly recombined in vitro 14 genes of the Bet v 1 family and expressed the chimeric products in bacteria. Two chimeric proteins were selected displaying low capacity to induce release of inflammatory mediators but with T cell immunogenicity higher than of the original allergens. These hypoallergenic chimeras combine epitopes from different trees, and thus might efficiently substitute extract mixtures used for treating tree pollen allergy, thereby increasing the efficacy of the treatment and reducing the risk of side effects for the patient.