Structural characteristics and immunogenicity of allergen vaccines

Recent clinical trials convincingly demonstrated that recombinant preparations based on will type allergens can effectively substitute extracts in allergen-specific immunotherapy (SIT). However, the problem of IgE-mediated side effects still remains and in principle could be an obstacle for a wider use of recombinant allergens in SIT. Thus, efforts are being undertaken to develop hypoallergenic molecules in order to diminish the risk of IgE- mediated side effects. Several strategies have been used to generate variants with reduced or abolished IgE antibody binding capacity. Such structural modifications might have different effects on allergen structure and consequently not only on the allergenicity but also on the immunogenicity of the molecules. Five major groups of Bet v 1 derivatives have been generated by different research groups, including the applicant`s group: (1) monomeric folded molecules obtained by multiple point mutations or gene shuffling, (2) monomeric unfolded molecules obtained by single point mutation, multiple point mutations, or by physicochemical treatment, (3) unfolded fragments, (4) folded oligomers and (5) unfolded oligomers. Presently, it is unclear which of these structural variants would be more effective as the active ingredient of an allergy vaccine. The first clinical trial with engineered hypoallergenic preparations of Bet v 1 displaying different structural features did no provide obvious conclusions. Thus, guidelines for designing molecule-based hypoallergenic vaccines are clearly lacking. In this project we aim to investigate the immunological features of engineered allergens displaying different structural characteristics as candidates for allergy vaccines. The major question addressed concerns on how structural manipulations of allergens impact immune responses. We will focus on the major allergen Bet v 1 and homologous proteins as model allergens and the birch pollen-food syndrome as the specific clinical condition. For this purpose, we will produce 7 vaccine candidates encompassing the following characteristics: folded and unfolded versions of monomeric and oligomeric derivatives of the Bet v 1 family of allergens (birch Bet v 1, apple Mal d 1 and hazelnut Cor a 1). All molecules will be subject to detailed physicochemical characterization, including the following parameters: primary structure, folding, surface exposed hydrophobic portions, aggregation status, stability, and adjuvant formulation. The engineered molecules will be evaluated in vitro concerning their human IgE binding properties and human T cell recognition. These experiments will be indicative of immunogenicity in man. Degradation assays using lysosomes from murine APCs will be used as predictors of in vivo immunogenicity. Immunization of BALB/c mice and measurement of allergen-specific Ig classes and subclasses will serve as a physiological measure of the engineered allergens` ability to be presented on MHC class II molecules and to prime CD4+ T cells in vivo. In addition, ex vivo experiments will be carried out to investigate T cell proliferative responses. The mouse experiments will allow the correlation between the vaccines` structural features and their in vivo immunogenic activity. The proposed comparative analysis of a representative panel of structural variants of a major allergen in terms of immunogenicity could provide directions for designing allergy vaccines and has implications for the field of vaccines in general.

The project addressed a major issue concerning the design of vaccines suitable for the treatment of adverse food reactions associated with birch pollen allergy. Available vaccines for the treatment of birch pollen allergy were shown not to be beneficial for patients suffering from associated food allergies, in particularly those caused by apple and hazelnut. Therefore, our work focused on engineering molecules for a combination therapy of birch pollen, apple, and hazelnut allergies. Derivatives of the major allergens from birch, apple, and hazelnut were designed, synthetically produced and shown to display very low allergenic activity both in vitro and in vivo in pre-clinical animal models. Thus, the developed molecules are promising candidates for safer treatment forms of food allergies associated with birch pollen sensitization.