Epitope mapping of Rhi o 1 and generation of a hypoallergenic variant

Abstract

Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent.Acombinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides (44TGEYLTQKYFNSQRNN and 311,GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surfaceexposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope thatwerecrucial for antibody binding.A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70%and suppressed allergen-mediated histamine releaseby10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing protective antibody response for immunotherapeutic applications.