Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus

Ai Sasou; Yoshikazu Yuki; Shiho Kurokawa; Shintaro Sato; Yuki Goda; Masao Uchida; Naomi Matsumoto; Hiroshi Sagara; Yuji Watanabe; Masaharu Kuroda; Naomi Sakon; Kotomi Sugiura; Rika Nakahashi-Ouchida; Hiroshi Ushijima; Kohtaro Fujihashi; Hiroshi Kiyono

doi:10.3389/fpls.2021.639953

Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus

Front Plant Sci. 2021 Feb 23:12:639953. doi: 10.3389/fpls.2021.639953. eCollection 2021.

Authors

Ai Sasou¹, Yoshikazu Yuki^{1

2}, Shiho Kurokawa¹, Shintaro Sato^{2

3

4}, Yuki Goda¹, Masao Uchida⁵, Naomi Matsumoto³, Hiroshi Sagara⁶, Yuji Watanabe⁶, Masaharu Kuroda⁷, Naomi Sakon⁸, Kotomi Sugiura¹, Rika Nakahashi-Ouchida^{1

2}, Hiroshi Ushijima⁹, Kohtaro Fujihashi^{10

11}, Hiroshi Kiyono^{1

2

12

13}

Affiliations

¹ Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
² Division of Mucosal Vaccines, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
³ Mucosal Vaccine Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
⁴ Department of Immunology and Genomics, Graduate School of Medicine, Osaka City University, Osaka, Japan.
⁵ Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Japan.
⁶ Medical Proteomics Laboratory, The Institute of Medical Science, University of Tokyo, Tokyo, Japan.
⁷ The National Agriculture and Food Research Organization, Tsukuba, Japan.
⁸ Department of Microbiology, Osaka Institute of Public Health, Osaka, Japan.
⁹ Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan.
¹⁰ Division of Clinical Vaccinology, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
¹¹ Department of Pediatric Dentistry, The University of Alabama at Birmingham, Birmingham, AL, United States.
¹² Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan.
¹³ Division of Gastroenterology, Department of Medicine, Chiba University - University of California San Diego Center for Mucosal Immunology, Allergy, and Vaccine, University of California, San Diego, San Diego, CA, United States.

Abstract

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.

Keywords: MucoRice; norovirus; transgenic rice; variable-domain llama heavy-chain antibody fragment; virus-like particle.