Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity But Retained Immunogenicity

Background: Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason. Aim: To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity. Methods: Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant Fel d 1 (rFel d 1) was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP-inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural Fel d 1 and recombinant unmodified Fel d 1 were used as controls. Results: rFel d 1 demonstrated similar IgE binding and biological activity as its natural counterpart. Upon modification, IgE-binding potency decreased to \u3e1000-fold, which was translated into a \u3e106- fold reduction in the biological activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P Conclusions: Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity