Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant
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. A combinatorial approach consisting of computational prediction and a peptide based immunoassay identified two peptides (44TGEYLTQKYFNSQRNN and 311GAEKNWAGQYVVDCNK) 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 surface exposed epitope conferring optimal IgE binding capacity to the folded allergen. Site directed alanine substitution identified four residues of the IgE epitope that were crucial 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 release by 10 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.
The Journal of Biological Chemistry