Sasou Ai, Yuki Yoshikazu, Kurokawa Shiho, Sato Shintaro, Goda Yuki, Uchida Masao, Matsumoto Naomi, Sagara Hiroshi, Watanabe Yuji, Kuroda Masaharu, Sakon Naomi, Sugiura Kotomi, Nakahashi-Ouchida Rika, Ushijima Hiroshi, Fujihashi Kohtaro, Kiyono Hiroshi
Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Division of Mucosal Vaccines, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Front Plant Sci. 2021 Feb 23;12:639953. doi: 10.3389/fpls.2021.639953. eCollection 2021.
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.
人诺如病毒是全球所有年龄段人群急性非细菌性胃肠炎的主要病因。目前，尚无用于控制诺如病毒感染的获批诺如病毒疫苗、药物或治疗方法。在此，我们使用水稻转基因系统MucoRice来生产针对人诺如病毒的羊驼重链抗体片段（VHH）的可变结构域（MucoRice-VHH）。VHH是一种小型的耐热和耐酸稳定蛋白，类似于单克隆抗体。因此，VHH已成为用于肠道传染病口服免疫治疗的有吸引力且有用的抗体（Ab）。通过使用带有RNA干扰的过表达系统抑制水稻主要内源性贮藏蛋白的产生，在水稻种子中高产生成了MucoRice-VHH构建体。作为磷酸盐缓冲盐水（PBS）可溶性VHH，水稻种子中针对GII.4诺如病毒的单体VHH（7C6）以及针对GII.4和GII.17诺如病毒的异二聚体VHH（7C6-1E4）的平均产量水平分别为0.54%和0.28%（w/w）。通过在人诱导多能干细胞衍生的肠上皮细胞（IEC）中使用人诺如病毒繁殖系统，我们证明了表达单体VHH（7C6）的MucoRice对GII.4诺如病毒以及异二聚体VHH（7C6-1E4）对GII.4和GII.17诺如病毒均具有高中和活性。此外，MucoRice-VHH（7C6-1E4）即使在90°C热处理20分钟后仍保留中和活性。这些结果为继续开发MucoRice-VHH异二聚体奠定了基础平台，该异二聚体可作为口服免疫治疗以及预防GII.4和GII.17诺如病毒的候选药物，不仅适用于健康的成人和儿童，也适用于免疫功能低下的患者和老年人。
