Induction of toxin‐specific neutralizing immunity by molecularly uniform rice‐based oral cholera toxin B subunit vaccine without plant‐associated sugar modification |
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| Authors: | Yoshikazu Yuki Mio Mejima Shiho Kurokawa Tomoko Hiroiwa Yuko Takahashi Daisuke Tokuhara Tomonori Nochi Yuko Katakai Masaharu Kuroda Natsumi Takeyama Koji Kashima Michiyo Abe Yingju Chen Ushio Nakanishi Takehiro Masumura Yoji Takeuchi Hiroko Kozuka‐Hata Hiroaki Shibata Masaaki Oyama Kunisuke Tanaka Hiroshi Kiyono |
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| Affiliation: | 1. Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, , Tokyo, Japan;2. International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, , Tokyo, Japan;3. Corporation for Production and Research of Laboratory Primates, , Ibaraki, Japan;4. Crop Development Division, NARO Agricultural Research Center, , Niigata, Japan;5. MG Pharma Inc., , Osaka, Japan;6. Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, , Kyoto, Japan;7. Biotechnology Research Department, Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Research Center, , Kyoto, Japan;8. Hytoculture Control Co. Ltd., , Osaka, Japan;9. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, , Tokyo, Japan;10. Tsukuba Primate Research Center, National Institute of Biomedical Innovation, , Ibaraki, Japan |
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| 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. |
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| Keywords: | cholera cholera toxin oral vaccine rice‐based vaccine MucoRice‐CTB |
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