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* Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, and
Division of Hematology and Hemostaseology, Department of Internal Medicine I, Vienna General Hospital, Medical University of Vienna, Vienna, Austria;
Institute of Chemistry, University of Graz, Graz, Austria;
Allergy Unit, National Health Service, Rome, Italy;
¶ Institute of Physiology and Pathophysiology, Paracelsus Private Medical University, Salzburg, Austria;
# Department of Molecular Biology, University of Salzburg, Salzburg, Austria; and
** Paul-Ehrlich-Institut, Langen, Germany
Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive 
-helical contents of the protein but retained its β-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients’ IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.
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 Austrian Science Fund Grants F01805, F01809, F01814, F01815, and S8811, Austrian Research Promotion Agency Grant 810105-SCK/SAI, and a grant from Biomay and the Christian Doppler Research Association (Vienna, Austria).
2 Address correspondence and reprint requests to Dr. Rudolf Valenta, Christian Doppler Laboratory for Allergy Research, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Vienna General Hospital (Allgemeines Krankenhaus Wien), Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria. E-mail address: Rudolf.valenta{at}meduniwien.ac.at
3 Abbreviations used in this paper: rs, restructured; rPhI, recombinant profilins from timothy grass pollen; CD, circular dichroism; HAS, human serum albumin; n, natural; RBL, rat basophil leukemia.
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