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

Abstract

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 microg/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.

Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases

“…This prokaryotic expression system is still applied for the production of many different recombinant allergens [103, 104, 105, 106, 107, 108]. In more recent years, several allergens were produced in eukaryotic expression systems like the yeast Pichia pastoris [109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127], insect cells [128, 129, 130, 131, 132, 133, 134, 135, 136], and tobacco plants [137, 138]. These systems facilitate post-translational modifications that do not (easily) occur in E. coli , like disulfide-bridge formation, hydroxylation of prolines, glycosylation.…”

The expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems

“…This is the first report of apo-plastic extraction of a recombinant allergen. Previously expressed recombinant allergens (Krebitz et al 2000(Krebitz et al , 2003 have been extracted by grinding leaves in an extraction buffer. Upon using this extraction method for rJun a 3, several contaminating proteins were extracted as well, making vacuum infiltration a much more valuable tool for extraction from plants (Fig.…”

“…Several recombinant allergens have also been produced in ground leaves of N. benthamiana plants via a tobacco mosaic virus (TMV) vector (Krebitz et al 2000(Krebitz et al , 2003. However, extraction from ground leaves can make purification difficult.…”

A Model System to Study the Environment-Dependent Expression of the <i>Bet v 1a</i> Gene Encoding the Major Birch Pollen Allergen

“…Modern diagnostics of allergic diseases and new therapeutic approaches are based on the use of recombinant allergens [5]. It was shown that Escherichia coli and Nicotiana benthamiana expression systems could be used to produce recombinant allergens with the assigned properties [5, 6].…”

“…Modern diagnostics of allergic diseases and new therapeutic approaches are based on the use of recombinant allergens [5]. It was shown that Escherichia coli and Nicotiana benthamiana expression systems could be used to produce recombinant allergens with the assigned properties [5, 6]. Furthermore, analysis of the 3-dimensional structure identified potential B cell epitopes on the molecular surface of the Bet v 1 protein and thus provided a structural basis for the reactivity of the molecule with IgE antibodies [7].…”