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Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization

MONIKA KREBITZ^*, URSULA WIEDERMANN^*, DAGMAR ESSL, HERTA STEINKELLNER, BIRGIT WAGNER^*, THOMAS H. TURPEN, CHRISTOF EBNER^*, OTTO SCHEINER^* and HEIMO BREITENEDER^*1

^* Department of Pathophysiology, University of Vienna, Vienna 1090, Austria;
Centre of Applied Genetics, University of Agricultural Sciences, Vienna 1190, Austria; and
Large Scale Biology Corp., Vacaville, California 95688, USA

¹Correspondence: Department of Pathophysiology, University of Vienna, AKH-EBO-3Q, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: Heimo.Breiteneder{at}akh-wien.ac.at

Type I allergies are immunological disorders that afflict a quarter of the world’s population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 µg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.—Krebitz, M., Wiedermann, U., Essl, D., Steinkellner, H., Wagner, B., Turpen, T. H., Ebner, C., Scheiner, O., Breiteneder, H. Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Key Words: plant expression system • tobacco mosaic virus • recombinant allergen • BALB/c • Th2 response

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