RATIONALE: Previous studies revealed the major mugwort allergen Art v 1 to harbor one immunodominant T cell epitope, thus mugwort allergy may represent an ideal candidate for peptide-based immunotherapy. Due to the usage of synthetic peptides of pre-defined length within T cell epitope mapping the current artificial methods cannot reflect the quality of naturally occurring allergen derived peptides. We propose the isolation and characterization of naturally processed MHC peptides after exogenous pulsing of human B cells. Peptide sequencing will be performed by highly sensitive mass spectrometry. A prerequisite to yield sufficient peptide material for analysis is efficient antigen internalization, as promised by a lectin mediated antigen delivery system. Produced as biotinylated protein, Art v 1 will be incubated with tetravalent avidin and biotin-carrying lectin binding preferentially to carbohydrate chains on B cell surface immunoglobulins. This approach results in an enhanced allergen uptake. METHODS: The pHIS-Parallel2 expression vector was modified by insertion of the leader sequence of the 1.3S subunit of Propionobacterium shermanii transcarboxylase that can be biotinylated by bacterial biotin ligase holoenzyme. Downstream cloning of Art v 1 enabled the expression of biotinylated allergens in E. coli BL21(DE3)-RIL. Protein purification was performed by immobilized metal chelate affinity chromatography. RESULTS: Coomassie-staining and immunoblotting revealed sufficiently pure amounts of recombinant allergen. Additionally, biotinylation, as required for enhanced antigen delivery, was checked by streptavidin blotting. CONCLUSIONS: Biotinylated Art v 1 can be used for human B cell pulsing. The examination of naturally processed allergen derived MHC peptides has direct implications for vaccine development and peptide immunotherapy.

Rationale Art v 1 is the major mugwort (Artemisia vulgaris) pollen allergen and therefore represents one of the main causes of late summer pollinosis in Europe. Cross-reacting specific IgE antibodies can lead to clinically significant reactions with other members of the Compositae plant family (e.g. ragweed) in other geographic areas, such as Northern America. Furthermore, previous studies revealed cross-reactivity between mugwort pollen and certain food, known as the celery-mugwort-spice-syndrome. Methods In vitro cross-reactivity between Art v 1 and proteins from other Compositae-, grass- and tree pollen and common food allergen sources was investigated by immunoblot and IgE-inhibition experiments using the following antibodies: polyclonal rabbit anti-natural Art v 1, rabbit anti-recombinant Art v 1, moAb anti-Art v 1, produced by genetic immunization, and sera from mugwort and ragweed sensitized patients. Results The glycoallergen Art v 1 shares epitopes with proteins originating mainly from the botanically related plants of the Compositae family (Artemisia absinthium, Ambrosia artemisifolia, Ambrosia psilostachya, Ambrosia trifida and Helianthus annuus) and with the timothy grass (Phleum pratense) major allergen Phl p 1. We could not observe cross-reactivity between Art v 1 and proteins in extracts from tree pollen (Betula verrucosa) and food (apple and celery). Conclusions The Art v 1 cross-reactive epitopes seem to encompass both the Art v 1 polypeptide and O-glycans attached to its C-terminal domain. Cross-reactivity with the glycoprotein Phl p 1 seems to be due to structural similarities in the Art v 1 defensin like domain or to sugar chains linked to its proline rich domain.