"Allergen engineering": Variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T cell reactivity

One problem of conventional allergen-specific immunotherapy is the risk of anaphylactic reactions. A new approach to make immunotherapy safer and more efficient might be the application of engineered allergens with reduced Ig E-binding capacity but retained T cell reactivity, Using overlapping dodeca -peptides, the dominant T cell epitopes of the timothy grass pollen allerge n Phl p 5b were identified. By site-directed mutagenesis outside these regi ons, point and deletion mutants were generated. Allergen variants were anal yzed for IgE-binding capacity with sera of different grass pollen allergic patients by Western blotting, Dot blotting, and EAST inhibition test, and f or histamine releasing capacity with peripheral blood basophils from differ ent patients. The deletion mutants revealed significantly reduced IgE react ivity and histamine releasing capacity, compared with the wild-type Phl p 5 b, Furthermore, in vivo skin prick tests showed that the deletion mutants h ad a significantly lower potency to induce cutaneous reactions than the wil d-type Phl p 5b, On the other hand, T cell clones and T cell lines from dif ferent allergic patients showed comparable proliferation after stimulation with allergen variants and wild-type Phl p 5b, Considering their reduced an aphylactogenic potential together with their conserved T cell reactivity, t he engineered allergens could be important tools for efficient and safe all ergen-specific immunotherapy.