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A Recombinant Hypoallergenic Parvalbumin Mutant for Immunotherapy of IgE-Mediated Fish Allergy¹

Ines Swoboda^2,*,, Agnes Bugajska-Schretter^*, Birgit Linhart, Petra Verdino, Walter Keller, Ulrike Schulmeister^*, Wolfgang R. Sperr, Peter Valent, Gabriel Peltre^¶, Santiago Quirce^||, Nikolaos Douladiris^#, Nikolaos G. Papadopoulos^#, Rudolf Valenta and Susanne Spitzauer^*

^* Institute of Medical and Chemical Laboratory Diagnostics, Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria; Division of Structural Biology, Institute of Chemistry, University of Graz, Graz, Austria; Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria; ^¶ Laboratory of Environment and Analytical Chemistry, ESPCI, Paris, France; ^|| Allergy Department, Fundación Jiménez Díaz, Madrid, Spain; and ^# Second Department of Pediatrics, University of Athens, Athens, Greece

IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients’ IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants F01804, F01805, F01809, and F01815 from the Austrian Science Fund, a research grant from the Christian Doppler Association, and Biomay (Vienna, Austria).

² Address correspondence and reprint request to Dr. Ines Swoboda, Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Medical University of Vienna, AKH, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail address: ines.swoboda{at}meduniwien.ac.at

³ Abbreviation used in this paper: RBL, rat basophil leukemia.