D2.194 - Norovirus-Like Particles as Novel Carriers of Hypoallergenic Artemisia vulgaris Allergen Art v 3 for Allergen-Specific Immunotherapy

The prevalence of allergic diseases has increased significantly over recent decades, with Artemisia vulgaris (common mugwort) being a major source of airborne pollen allergens in Europe. Up to 70% of A. vulgaris allergic patients are sensitized to the Art v 3 allergen. Allergen-specific immunotherapy (ASIT) is the only disease-modifying treatment for IgE-mediated allergies. Still, its effectiveness is limited due to the variability of natural allergen extracts and potential adverse reactions. New strategies, such as hypoallergens and chimeric virus-like particles (VLPs), are being developed to enhance ASIT. VLPs, which are self-assembling nanostructures resembling the structure of native viruses but lacking their genomic material, can effectively deliver allergens and enhance immune responses. Norovirus (NoV) VLPs are efficiently produced by yeast and are a promising platform for allergen delivery. This study aimed to create chimeric NoV VLPs fused with hypoallergenic Art v 3 variants for improved ASIT.

To develop hypoallergenic variants of the Art v 3, mutations were introduced via site-directed mutagenesis. The mutant proteins were synthesized in E. coli, purified using Ni-NTA chromatography, and tested with IgE-positive human blood serum samples through an indirect ELISA. The gene encoding the major NoV capsid protein VP1 was fused with genes of native-like and two mutant Art v 3 variants and cloned into a yeast expression vector. Chimeric VLPs were produced in Pichia pastoris and purified by ultracentrifugation through a sucrose cushion followed by CsCl density gradient separation. Protein samples were analyzed by SDS-PAGE and Western blot, and VLPs were visualized using transmission electron microscopy (TEM).

Eleven mutant variants of the Art v 3 allergen were created by substituting specific amino acids with alanine. Seven mutants showed a significant reduction in reactivity with specific IgE. Native-like and two mutant variants of Art v 3 were genetically fused with the major NoV capsid protein VP1. Chimeric VLPs were produced in P. pastoris and purified. The formation of chimeric VLPs was confirmed by visualization using TEM.

Mutant Art v 3 proteins with significantly reduced reactivity to specific IgE antibodies are promising for designing a new form of ASIT with fewer side effects. The development of Art v 3 hypoallergen-fused chimeric NoV VLPs is a promising approach for allergen delivery, supporting further investigation for ASIT applications.