Abstract
Epitope mapping of Der p 2, a clinically important dust-mite allergen is the first step in designing immunotherapy hypoallergen vaccine candidates. Twenty-one single alanine mutants of Der p 2 were generated and their secondary structure was analysed using circular dichroism spectra. Only one mutant, K96A resulted in a misfolded protein. All mutants were tested for serum IgE reactivity using serum from dust mite allergic individuals by immuno dot-blots. Mutations to five residues, N10, E25, K77, K96 and E102 consistently showed reduced IgE reactions compared to wild-type Der p 2, and therefore these residues constitute the major IgE epitopes of Der p 2. Two mutants with consistent low IgE binding, K96A and E102A, were subsequently evaluated as hypoallergen candidates. IgG antibodies raised in mice against both mutants could inhibit human IgE-binding to WT Der p 2. Both mutants had intact T-cell epitopes as they were able to stimulate peripheral blood mononuclear cell proliferation similar to WT Der p 2. However, a switch in Th1:Th2 cytokine profile was not observed. In summary, we have identified the major conformational epitopes of Der p 2, and evaluated two Der p 2 hypoallergen vaccine candidates for immunotherapy.
Highlights
The only curative treatment for allergies currently known is by allergen specific immunotherapy (SIT)[5]
We evaluated the serum IgE of five Der p 2 allergic individuals to its structural homologue, human Niemann-Pick C2 protein, and found that hNPC2 did not show IgE binding (Fig. 1a)
We rationalized that the surface residues of Der p 2 that were different from hNPC2 most likely contained the IgE binding epitopes
Summary
The only curative treatment for allergies currently known is by allergen specific immunotherapy (SIT)[5]. Immunotherapy was performed using crude allergen extracts. This method risked allergic side effects, including anaphylaxis which could be fatal. To improve the safety of SIT, biologist are generating modified allergen proteins which have lesser IgE binding capacity, but retained T-cell proliferation[7]. Maintaining T-cell proliferation is important as studies on the mechanisms of immunotherapy have highlighted the role of allergen-specific CD4+ T-cells in tolerance mechanisms and control of allergic responses[8]. We identify the conformational IgE epitopes of Der p 2 using site-directed mutations, and test these mutants for their reduction in IgE-binding capacity using serum from dust-mite allergic individuals. We test two site directed mutants of Der p 2 for their potential as prophylactic hypoallergen vaccine in a murine model
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