title: Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant.
abstract: We have produced biologically active recombinant (r) LTB, the nontoxic B subunit of heat-labile toxin (LT) of Escherichia coli in tobacco mosaic virus (TMV)-infected Nicotiana benthamiana plants. We amplified the LTB encoding sequence with its leader and introduced a hexahistidyl tag and an endoplasmic reticulum retention signal. The resulting product was ligated into a TMV-based plant viral expression vector that was used for the generation of recombinant viral RNA. Eighty-nine percent of N. benthamiana plants inoculated with the recombinant viral RNA were systemically infected as determined by anti-TMV enzyme-linked immunosorbent assay (ELISA) experiments. The rLTB monomer was identified by LT-specific as well as by histidyl-tag-specific immunoblots. rLTB from plant extracts of TMV-infected N. benthamiana leaves was purified to give 75 microg rLTB pentamers per gram fresh plant material and was capable of binding G(M)1 ganglioside. The immunogenicity of the plant-produced rLTB was tested in mice and showed that intranasal application of rLTB (15 microg per mouse) induced LTB-specific IgG1 antibodies. To prove its adjuvanticity, rLTB was intranasally co-administered with the Hevea latex allergen Hev b 3, leading to allergen-specific IgG1 and IgG2a antibody production. The fact that intranasal application of rLTB and Hev b 3 prior to systemic challenge with the allergen enhanced the Th2 responses at the humoral and cellular level indicated that rLTB promoted immune responses that were naturally induced by the antigen/allergen. In conclusion, these results indicate that the plant viral expression system is suitable for the rapid large-scale production of biologically active LTB with strong mucosal adjuvant capacity.
authors.affiliation: Department of Pathophysiology, Medical University of Vienna, General Hospital Vienna EBO 3.Q, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Wagner
authors.forename: Karin
authors.initials: K
authors.lastname: Hufnagl
authors.forename: Christian
authors.initials: C
authors.lastname: Radauer
authors.forename: Stefan
authors.initials: S
authors.lastname: Wagner
authors.forename: Karin
authors.initials: K
authors.lastname: Baier
authors.forename: Otto
authors.initials: O
authors.lastname: Scheiner
authors.forename: Ursula
authors.initials: U
authors.lastname: Wiedermann
authors.forename: Heimo
authors.initials: H
authors.lastname: Breiteneder
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins; D016133:Polymerase Chain Reaction; D012313:RNA; D014026:Nicotiana / Q000821:virology*; D014027:Tobacco Mosaic Virus / Q000502:physiology*; D014614:Vaccines, Synthetic
chemical_list: D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C039919:RNA, recombinant; D014614:Vaccines, Synthetic; D012313:RNA; C027538:heat-labile enterotoxin, E coli