title: Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity But Retained Immunogenicity.
abstract: 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.
  
  
  OBJECTIVE
  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 >1000-fold, which was translated into a >10(6)-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 < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen.
  
  
  CONCLUSIONS
  Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.
authors.affiliation: Department of Experimental Immunology, Academic Medical Center, Amsterdam, the Netherlands.
authors.forename: Serge A
authors.initials: SA
authors.lastname: Versteeg
authors.forename: Ingrid
authors.initials: I
authors.lastname: Bulder
authors.forename: Martin
authors.initials: M
authors.lastname: Himly
authors.forename: Toni M
authors.initials: TM
authors.lastname: van Capel
authors.forename: R
authors.initials: R
authors.lastname: van den Hout
authors.forename: Stef J
authors.initials: SJ
authors.lastname: Koppelman
authors.forename: Esther C
authors.initials: EC
authors.lastname: de Jong
authors.forename: Fatima
authors.initials: F
authors.lastname: Ferreira
authors.forename: Ronald
authors.initials: R
authors.lastname: van Ree