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.
Highlights
Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination
As observed in an IgE dot blot (Fig. 1a), 17 of 45 patients (38%) showed strong reactions with rRhi o 1 but not with rBla g 2. These 17 patients had no detectable IgE antibodies cross-reacting between rRhi o 1 and rBla g 2
In ELISA with these 28 patients’ sera (Fig. 1b), the intensity of IgE antibody binding to rRhi o 1 was on average ϳ2.45-fold higher (p Ͻ 0.001) than to rBla g 2
Summary
IgE Antibody Reactivity Pattern of Rhi o 1 and Bla g 2 among R. oryzae-sensitized Patients—Co-sensitization to multiple allergens having conserved molecular surfaces can often give rise to unwanted cross-reactivity of low clinical importance. In ELISA with these 28 patients’ sera (Fig. 1b), the intensity of IgE antibody binding to rRhi o 1 was on average ϳ2.45-fold higher (p Ͻ 0.001) than to rBla g 2 Such a frequently strong IgE binding to Rhi o 1 suggests that IgE recognition of this allergen principally depends on non-cross-reactive epitope(s) and is not significantly influenced by the presence of cross-reactive epitope in these two less conserved allergens (28% sequence identity). After cooling down (descending scan), there was no significant increase in the CD signal at 215 nm, indicating that rRhi o 1 did not refold upon cooling This result led us to hypothesize that rRhi o 1 remained denatured on the membrane during Western blotting, and IgE binding under this condition could only be possible principally with continuous sequences.
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