Background: The incidence of allergic diseases in developed countries has been increasing constantly in the last 2 decades. The only curative treatment available thus far is specific immunotherapy (SIT). The problematic side effects that can occur during SIT with allergen extracts led to the search for safer alternatives, such as recombinant hypoallergenic proteins with reduced allergenic potential and preserved T-cell immunogenicity.
Objective: We created hypoallergenic variants of the allergens Bet v 1a and Phl p 5b by using in silico mutation and screening and characterized the biochemical and immunologic properties of selected mutant proteins.
Methods: Knowledge-based potentials were used to estimate structural changes of the protein structures under sequence variation. IgE antibodies and their cross-linking capacity were determined by using a basophil release assay, binding of human birch pollen-specific IgE was investigated by means of ELISA, and ELISPOT assays were performed to examine the T-cell immunogenicity.
Results: Selected mutated proteins showed significantly reduced IgE-binding and cross-linking ability but retained their T cell-stimulating properties. Immunization with the hypoallergenic mutants induced blocking antibodies against murine and human IgE epitopes.
Conclusion: In silico calculation and selection of mutations that render a protein hypoallergenic represents a novel and rapid tool to create candidate molecules for SIT with recombinant allergens.
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