• Open Access

Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification

Authors

  • Yoshikazu Yuki,

    Corresponding author
    1. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
    • Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Mio Mejima,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Shiho Kurokawa,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Tomoko Hiroiwa,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Yuko Takahashi,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Daisuke Tokuhara,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Tomonori Nochi,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Yuko Katakai,

    1. Corporation for Production and Research of Laboratory Primates, Ibaraki, Japan
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  • Masaharu Kuroda,

    1. Crop Development Division, NARO Agricultural Research Center, Niigata, Japan
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  • Natsumi Takeyama,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Koji Kashima,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Michiyo Abe,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
    2. MG Pharma Inc., Osaka, Japan
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  • Yingju Chen,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Ushio Nakanishi,

    1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
    2. MG Pharma Inc., Osaka, Japan
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  • Takehiro Masumura,

    1. Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
    2. Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, Kyoto, Japan
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  • Yoji Takeuchi,

    1. Hytoculture Control Co. Ltd., Osaka, Japan
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  • Hiroko Kozuka-Hata,

    1. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Hiroaki Shibata,

    1. Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan
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  • Masaaki Oyama,

    1. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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  • Kunisuke Tanaka,

    1. Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
    2. Hytoculture Control Co. Ltd., Osaka, Japan
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  • Hiroshi Kiyono

    Corresponding author
    1. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
    • Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
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Correspondence (Tel +81-3-5449-5274; fax +81-3-5449-5411; emails yukiy@ims.u-tokyo.ac.jp and kiyono@ims.u-tokyo.ac.jp)

Summary

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.

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