Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES

• Published in: The Journal of biological chemistry 2016-08, Vol.291 (34), p.18016-18029
• Main Authors: Sircar, Gaurab, Jana, Kuladip, Dasgupta, Angira, Saha, Sudipto, Gupta Bhattacharya, Swati
• Format: Article
• Language: English
• Subjects: Allergens - chemistry; Allergens - genetics; Allergens - immunology; Animals; Cell Line; Epitope Mapping; Epitopes, T-Lymphocyte - chemistry; Epitopes, T-Lymphocyte - genetics; Epitopes, T-Lymphocyte - immunology; Female; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - immunology; Humans; Immunoglobulin E - chemistry; Immunoglobulin E - immunology; Immunoglobulin G - chemistry; Immunoglobulin G - immunology; Male; Molecular Bases of Disease; Rhizopus - chemistry; Rhizopus - genetics; Rhizopus - immunology; Vaccines - chemistry; Vaccines - genetics; Vaccines - immunology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.732032

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 ((44)TGEYLTQKYFNSQRNN 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 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.