A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination

Background: Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity. Objective: To develop a novel type of allergy vaccine for complex allergen sources that combines defined amounts of the major allergens in the form of single hybrid molecules. Methods: A hybrid molecule was engineered by PCR-based mending and expression of the cDNAs coding for the 4 major grass pollen allergens and compared with its single components by circular dichroism analysis, T-cell proliferation, ELISA competition, and histamine release assays. Immune responses to the hybrid molecule were studied in BALB/c mice and rat basophil leukemia assays. Results: The hybrid contained most of the B-cell epitopes of grass pollen and could be used to diagnose allergy in 98% (n = 652) of patients allergic to grass pollen. Immunization of mice and rabbits with the hybrid induced stronger and earlier IgG antibody responses than equimolar mixtures of the components, which can be explained by the induction of stronger T-cell responses by the hybrid versus the individual components. IgG antibodies induced by vaccination with the hybrid blocked immediate allergic reactions, as demonstrated by rat basophil degranulation assays in a murine model of grass pollen allergy. Conclusion: We demonstrate for grass pollen allergy that recombinant hybrid molecules covering the spectrum of the disease-eliciting epitopes of complex allergen sources can be engineered.