Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection
Daisuke Tokuhara, Beatriz Álvarez, Mio Mejima, Tomoko Hiroiwa, Yuko Takahashi, Shiho Kurokawa, Masaharu Kuroda, Masaaki Oyama, Hiroko Kozuka-Hata, Tomonori Nochi, Hiroshi Sagara, Farah Aladin, Harold Marcotte, Leon G.J. Frenken, Miren Iturriza-Gómara, Hiroshi Kiyono, Lennart Hammarström, Yoshikazu Yuki
Daisuke Tokuhara, Beatriz Álvarez, Mio Mejima, Tomoko Hiroiwa, Yuko Takahashi, Shiho Kurokawa, Masaharu Kuroda, Masaaki Oyama, Hiroko Kozuka-Hata, Tomonori Nochi, Hiroshi Sagara, Farah Aladin, Harold Marcotte, Leon G.J. Frenken, Miren Iturriza-Gómara, Hiroshi Kiyono, Lennart Hammarström, Yoshikazu Yuki
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Research Article Infectious disease

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

Abstract

Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.

Authors

Daisuke Tokuhara, Beatriz Álvarez, Mio Mejima, Tomoko Hiroiwa, Yuko Takahashi, Shiho Kurokawa, Masaharu Kuroda, Masaaki Oyama, Hiroko Kozuka-Hata, Tomonori Nochi, Hiroshi Sagara, Farah Aladin, Harold Marcotte, Leon G.J. Frenken, Miren Iturriza-Gómara, Hiroshi Kiyono, Lennart Hammarström, Yoshikazu Yuki

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Figure 1

Expression and localization of water-soluble ARP1 in transgenic rice.

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Expression and localization of water-soluble ARP1 in transgenic rice. (A...
(A) Inserted plasmid for overexpression of ARP1 in rice seeds. P35S, CaMV35S promoter; mHPT, modified hygromycin phosphotransferase; P131P, 13-kDa prolamin promoter; 10N, signal sequence of 10-kDa prolamin; P131TL, 13-kDa prolamin terminator; T-Nos, nos terminator; RAP intron, rice aspartic protease intron; P-mUbi, ubiquitin promoter; LB, T-DNA left border; RB, T-DNA right border. (B) Production of MucoRice-ARP1. SDS-PAGE showed predominant expression of the transgenic protein with a molecular weight of approximately 12 kDa (arrowhead). Original rice proteins (arrows; 22- to 23-kDa and 34- to 37-kDa subunits of glutelin and 13-kDa prolamin) in nontransformed WT rice were markedly suppressed in MucoRice-ARP1. Western blotting revealed that a transgenic protein of 12 kDa was specifically detected using the anti-ARP1 antibody. ARP1 E. coli, ARP1 purified from E. coli. (C) Solubility of MucoRice-ARP1. MucoRice-ARP1 (PBS), extracts of MucoRice-ARP1 in PBS; MucoRice-ARP1 (urea), extracts of MucoRice-ARP1 in 8 M urea. (D) Immunohistochemistry showed that ARP1 had accumulated throughout the whole MucoRice-ARP1 seed, whereas it was not detected in a WT rice seed. Scale bars: 1 mm. (E) Immune electron microscopy showed that ARP1 is observed as black spots (left and middle panels). ARP1 is predominantly localized in the PB-II and the interspace between the PB-I and PB-II (left and middle panels). ARP1 is also slightly found at the surface of PB-I (left panel and lower middle panel). ARP1 is not detected in a WT rice seed used as a negative control (right panel). Scale bars: 1 μm.