18 Proteomic and Immunological Characterization of Ragweed Allergens

BACKGROUND: The prevalence of sensitization to ragweed has risen in North America and across Europe. Although the pectate lyase Amb a 1, the major allergen of ragweed, was identified as long ago as the 1960s, little is known about the allergenicity of the 5 Amb a 1 isoallergens and other allergens present in ragweed pollen. Ragweed extracts and purified Amb a 1 isoallergens have now been characterized for their allergenic potential to determine whether a single Amb a 1 isoallergen, several isoallergens or a combination with other allergens should be included in a recombinant SIT vaccine. METHODS: Extracts from North American short ragweed (Ambrosia artemisiifolia) pollen were investigated by mass spectrometry (MS), 2D-PAGE and immunoblotting. Furthermore, Amb a 1 isoallergens were purified and IgE reactivity determined by immunoblotting and IgE inhibition. RESULTS: 2D-PAGE and MS of ragweed extract proved the presence of all 5 known Amb a 1 isoallergens, of which Amb a 1.01 represents the dominant form. Additionally all other ragweed allergens known by sequence (Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10) were identified. The highest IgE reactivity by immunoblotting was observed for Amb a 1.01 followed by Amb a 1.03; other Amb a 1 isoallergens as well as other detected ragweed allergens showed only weak IgE reactivity. All isoallergens with the exception of Amb a 1.04, which is only of low abundance in ragweed extract, were purified. Similar to the immunoblot analysis with crude extract, the purified isoallergens Amb a 1.02 and Amb a 1.05 showed weak IgE binding, whereas Amb a 1.01 and Amb a 1.03 had high IgE reactivity. First IgE inhibition experiments suggest that Amb a 1.01 contains all relevant IgE epitopes. CONCLUSIONS: Amb a 1.01 is the most abundant Amb a 1 isoallergen, and presumably the most important ragweed allergen. However, a larger panel of ragweed-allergic subjects has to be analyzed with regard to IgE and T cell reactivities, to be able to choose a candidate for a recombinant vaccine for specific immunotherapy of ragweed allergy.