Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy

Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.