Bla g 2 Hypoallergens Retaining the Native Fold and Capacity to Modulate T Cell Reactivity Provide Candidates for Cockroach Immunotherapy

Abstract

Modified allergens that display reduced IgE reactivity along with T-cell activating properties, are strong candidates for immunotherapy, because of the potential to decrease side-effects due to IgE cross-linking, but still retain immunogenicity. Single and multiple Bla g 2 mutants were designed according to prior knowledge of the antigenic structure of the allergen and expressed in Pichia pastoris. Folding of the mutants was assessed by CD spectrometry or X-ray crystallography. IgE reactivity was measured by antibody binding and mast cell release assays. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. Single and multiple mutations of residues implicated in binding to monoclonal antibodies (K132A, K251A and/or F162Y) reduced IgE reactivity but did not influence the native molecular fold, as proven by comparing the triple mutant with wild type Bla g 2 by X-ray crystallography. As compared with wild type allergen, mutants KK and KKF showed from at least 100-fold to a total reduction in IgE antibody binding. Whereas similar T-cell activating capacity was retained based on CD25 expression, both mutants were weaker inducers of the Th2 cytokine, IL-13. Furthermore, both mutants induced high levels of IL-10 from a non-T-cell source, and levels induced by the triple mutant exceeded those induced by Bla g 2 (p=0.004). A rational design of site-directed mutagenesis was effective in producing candidate molecules for immunotherapy that maintain the same fold as wild type Bla g 2, but display reduced IgE reactivity with T-cell modulatory potential.