title: Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.
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. 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.
authors.affiliation: From the Division of Plant Biology, Bose Institute (Main Campus), 93/1 Acharya Prafulla Chandra Road, Kolkata-700009, India.
authors.forename: Gaurab
authors.initials: G
authors.lastname: Sircar
authors.affiliation: the Division of Molecular Medicines and.
authors.forename: Kuladip
authors.initials: K
authors.lastname: Jana
authors.affiliation: the Department of Chest Medicine, BR Singh Hospital and Centre for Medical Education and Research, Kolkata-700014, India.
authors.forename: Angira
authors.initials: A
authors.lastname: Dasgupta
authors.affiliation: the Bioinformatics Centre, Bose Institute (Centenary Building), P 1/12, C. I. T. Road, Scheme-VIIM, Kolkata-700054, India, and.
authors.forename: Sudipto
authors.initials: S
authors.lastname: Saha
authors.affiliation: From the Division of Plant Biology, Bose Institute (Main Campus), 93/1 Acharya Prafulla Chandra Road, Kolkata-700009, India, swati@jcbose.ac.in.
authors.forename: Swati
authors.initials: S
authors.lastname: Gupta Bhattacharya
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000818:Animals; D002460:Cell Line; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D005260:Female; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D008297:Male; D012233:Rhizopus / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014612:Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D018984:Epitopes, T-Lymphocyte; D005656:Fungal Proteins; D007074:Immunoglobulin G; D014612:Vaccines; D007073:Immunoglobulin E
keywords: allergen; asthma; epitope mapping; immunoglobulin E (IgE); immunotherapy; mold allergy; mutagenesis; vaccine