title: Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification.
abstract: Plants have been used as expression systems for a number of vaccines. However, the expression of vaccines in plants sometimes results in unexpected modification of the vaccines by N-terminal blocking and sugar-chain attachment. Although MucoRice-CTB was thought to be the first cold-chain-free and unpurified oral vaccine, the molecular heterogeneity of MucoRice-CTB, together with plant-based sugar modifications of the CTB protein, has made it difficult to assess immunological activity of vaccine and yield from rice seed. Using a T-DNA vector driven by a prolamin promoter and a signal peptide added to an overexpression vaccine cassette, we established MucoRice-CTB/Q as a new generation oral cholera vaccine for humans use. We confirmed that MucoRice-CTB/Q produces a single CTB monomer with an Asn to Gln substitution at the 4th glycosylation position. The complete amino acid sequence of MucoRice-CTB/Q was determined by MS/MS analysis and the exact amount of expressed CTB was determined by SDS-PAGE densitometric analysis to be an average of 2.35 mg of CTB/g of seed. To compare the immunogenicity of MucoRice-CTB/Q, which has no plant-based glycosylation modifications, with that of the original MucoRice-CTB/N, which is modified with a plant N-glycan, we orally immunized mice and macaques with the two preparations. Similar levels of CTB-specific systemic IgG and mucosal IgA antibodies with toxin-neutralizing activity were induced in mice and macaques orally immunized with MucoRice-CTB/Q or MucoRice-CTB/N. These results show that the molecular uniformed MucoRice-CTB/Q vaccine without plant N-glycan has potential as a safe and efficacious oral vaccine candidate for human use.
authors.affiliation: Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Yuki
authors.forename: Mio
authors.initials: M
authors.lastname: Mejima
authors.forename: Shiho
authors.initials: S
authors.lastname: Kurokawa
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hiroiwa
authors.forename: Yuko
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Daisuke
authors.initials: D
authors.lastname: Tokuhara
authors.forename: Tomonori
authors.initials: T
authors.lastname: Nochi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Katakai
authors.forename: Masaharu
authors.initials: M
authors.lastname: Kuroda
authors.forename: Natsumi
authors.initials: N
authors.lastname: Takeyama
authors.forename: Koji
authors.initials: K
authors.lastname: Kashima
authors.forename: Michiyo
authors.initials: M
authors.lastname: Abe
authors.forename: Yingju
authors.initials: Y
authors.lastname: Chen
authors.forename: Ushio
authors.initials: U
authors.lastname: Nakanishi
authors.forename: Takehiro
authors.initials: T
authors.lastname: Masumura
authors.forename: Yoji
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kozuka-Hata
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shibata
authors.forename: Masaaki
authors.initials: M
authors.lastname: Oyama
authors.forename: Kunisuke
authors.initials: K
authors.lastname: Tanaka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyono
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002772:Cholera Toxin / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D022121:Cholera Vaccines; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007114:Immunization / Q000379:methods; D008251:Macaca; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry
chemical_list: D057134:Antibodies, Neutralizing; D022121:Cholera Vaccines; D002772:Cholera Toxin
keywords: MucoRice-CTB; cholera; cholera toxin; oral vaccine; rice-based vaccine