Deliverables Test vaccine for wasp venom allergy Up to 5% of the population suffer from systemic, 19% from local allergic hypersensitivity reactions to stinging insects. Even though specific immunotherapy is very effective in treating allergy to insect venom, new concepts of treatment strategies with only the disease eliciting allergen in recombinant form, along with antigen application via a less invasive route might be suggested for enhanced treatment efficacy and compliance. In the present study we aimed - To establish a mouse model of wasp venom allergy, mimicking the natural mode of sensitisation, and - To develop a prophylactic treatment strategy based on mucosal tolerance induction, using one major wasp venom allergen in recombinant form, i.e. recombinant (r)Ves v 5. Immunisation with wasp venom - with or without the use of the adjuvant aluminium hydroxide - led to comparable Th2-like immune responses in vivo and in vitro. Intranasal administration of rVes v 5 prior to sensitisation with wasp venom resulted in a significant reduction of wasp venom-specific antibody levels (IgE/IgG2a), type I hypersensitivity reactions in vivo and cytokine production in vitro. Pre-treatment with the whole venom was less effective and caused toxic side reactions in higher concentrations, suggesting a favourable use of the recombinant venom allergen for mucosal application. Increased mRNA levels of TGF-b and IL-10, along with adoptive cell transfer experiments indicated that the immunosuppression after intranasal rVes v 5-application has been mediated by regulatory mechanisms. This is further supported by the fact that the immunosuppression to rVes v 5 was associated with a bystander suppression to the unrelated aero-allergen Bet v 1. In conclusion, we demonstrated that the intranasal application of recombinant Ves v 5 prevented subsequent allergic sensitisation to all components of the whole wasp venom. As allergy to insect venom develops in dependence of the frequency of insect stings, a prophylactic treatment based on mucosal tolerance induction with recombinant allergens might be of interest for people at high risk to frequent exposure to the stinging insects. Expression and folding of Ves v 2 A protocol has been developed to produce milligram quantities of recombinant VesV2 allergen. This protein was used by another partner of the TRAFSA project to determine the molecular structure of the allergen. This structure will facilitate the design of VesV2 derivatives, with reduced allergenicity, for assessment as potential vaccines. A paternability evaluation is currently being performed on this result. Mapping of B cell epitopes Allergic patients IgE recognises allergen epitopes located on the molecular surface of the allergen. This recognition event is crucial for the triggering of allergic symptoms as rhinitis and asthma. The allergen epitopes can only be identified after revealing the architecture of the allergen molecular surface. Using the structures determined with-in this project, in combination with amino acid alignment of relevant allergens and information about cross-reactivity, possible IgE binding epitopes have been identified for two important allergens. This new information has pin-pointed possible mutation sites on the surface of the allergens in order to obtain allergen mutants with decreased IgE binding capacity. This could lead to new products with lower potential to trigger IgE-mediated side-effects during vaccination treatment. Mapping of human T cell epitopes The cellular immune response to the recombinant antigen 5 from yellow jacket, rVes v 5, was characterized using poly-, oligo-, and monoclonal T lymphocyte cultures from vespid- allergic and non-allergic individuals. Using a panel of 65 synthetic peptides representing the complete amino acid sequence of Ves v 5, 16 different T cell epitopes covering all regions of the protein were delineated in Ves v 5-specific T cell lines (TCL) from allergic individuals. Thirteen epitopes were observed in TCL from non-allergic subjects. The peptide Ves v 5181-192 was identified as dominant T cell epitope in allergic individuals whereas Ves v 5181-192 was not prevalent in Ves v 5-specific TCL from non-allergic controls. According to their cytokine secretion pattern in response to allergen-specific stimulation the majority of Ves v 5-specific T cell clones isolated from allergic individuals belonged to the Th0 and not to the Th2 subset, which is normally, observed in Type I allergy. Clones derived from non-allergic individuals belonged to the Th1 phenotype. Our findings provide further evidence that Th0-like lymphocytes dominate the specific cellular response to Hymenoptera allergens in allergic patients, which might in part contribute to the high efficacy of specific venom immunotherapy. Allergen crystal structures Allergic patients IgE recognises allergen epitopes located on the molecular surface of the allergen. This recognition event is crucial for the triggering of allergic symptoms as rhinitis and asthma. The allergen epitopes can only be identified after revealing the architecture of the allergen molecular surface by X-ray crystallography. During the project, two important allergens have been crystallised and their molecular surface structure determined. This knowledge forms the basis for identifying IgE binding epitopes and for designing allergen mutants with decreased IgE binding capacity, and thus a lower potential to trigger IgE-mediated side-effects during vaccination treatment. Current status is that a patentability evaluation is being performed to secure rights for using these allergen structures for future vaccine design. Cat allergy vaccine Cat dander contains a number of allergenic molecules. However, Fel d 1 is the only one that fulfils the criteria of a major allergen. Majority of cat allergic individuals (60 to 90%) show specific IgE responses to Fel d 1. The dominant quality has made Fel d 1 a good candidate in efforts to develop novel vaccines for treatment of cat allergy. As with all recombinant proteins intended for human therapy it is necessary to rigorously characterize the structure of recombinant Fel d 1. In the present study use of mass spectrometry and Edman degradation was combined to characterize amino acid sequence and disulfide-bridge formation in rFel d 1 expressed in a BAC to BAC - Baculovirus expression system. Enzymatic digestion of the rFel d 1 and the further analyses by use of matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) resulted a complete coverage of the amino acid sequence of the rFel d 1. In addition, all the three disulfide bridges at the positions N70-O7, N44-O48 and N3-O73 were verified. The N-glycan structure of rFel d 1 was investigated by combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometrie detection (HPAEC PAD). The N-glycosylation analyses showed pattern of hybrid and paucimannosidic glycoforms with a presence of core N1.3- and/orn Ni|l-fucosylation. Further molecular characterization by use of human serum IgE, basophil histamine release and lymphocyte proliferation assays confirmed that the immunlogical characteristics of rFel d 1 are compatible with nFel d 1. The present study shows that the higher eukaryotic expression system is providing a solid ground and a number of potential tools for further molecular engineering of allergens that are intended for human therapy. Test vaccine for house-dust mite allergy A clinically relevant model of allergen induced airway inflammation has been developed. Mice sensitised with native Der p 1 or Der p 2 developed elevated serum IgE and Th2 mediated airway eosinophilia, goblet cell hyperplasia/metaplasia with increased mucus production, and parameters of airway remodelling including subepithelial collagen deposition and thickening of the basement membrane on chronic exposure of the airways to allergen. To reflect the natural route of exposure and sensitisation we have generated a transgenic strain expressing the TCR ¿N¿O chain specific for an immunodominant domain of the major allergen of Der p1 species of house dust mite. Due to the increased frequency of reactive T cells in the periphery as compared with wild type, mice develop airway inflammation characterized by a marked lymphocytic and eosinophilic infiltrate, goblet cell hyperplasia and mucin production on exposure of the airways to Der p 1 in the absence of prior systemic sensitisation. In reflecting the normal route of sensitisation to environmental inhalant allergens, this model may prove to be extremely valuable in investigating specific allergen desensitisation therapy. Models for investigating mucosal delivery of allergens as a means of induction of peripheral tolerance (unresponsiveness) have been developed, with a view to addressing the use of different vehicles for antigen release. One of the current limitations of mucosal delivery is the rapid enzymatic degradation of proteins. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
