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

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

doi:10.3389/fpls.2021.639953

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.

Highlights

Human norovirus infection is common in both developed and developing countries and is associated with severe complications in children younger than 5 years, elderly adults, and immunocompromised patients (Lopman et al, 2016)
We transformed the plasmids into japonica rice plants (Nipponbare) by using Agrobacterium-mediated transformation and established the lines with the highest expression of each MucoRice-VHH
SDS-PAGE analysis showed that the monomeric 7C6 MucoRice-VHH generated a band with a molecular weight of 13.6 kDa on Coomassie Brilliant Blue (CBB)-stained gels; the molecular weight of the heterodimeric MucoRice-VHH band was 27.3 kDa; these molecular weights were confirmed through western blotting with rabbit antibody against 7C6-VHH (Figure 2A)

Summary

Introduction

Human norovirus infection is common in both developed and developing countries and is associated with severe complications in children younger than 5 years, elderly adults, and immunocompromised patients (Lopman et al, 2016). Vaccination with the GII. virus-like particle (VLP) vaccine elicits antibodies (Abs) against several GII. strains, but the vaccine fails to induce any antibodies that block GII noroviruses other than those within the GII. genotype, including non-vaccine GII. strains (Kim et al, 2018; Leroux-Roels et al, 2018). The strategy for developing VLP-based vaccines needs further refinement, whether VLP vaccination is effective and safe for inducing antibodies against human norovirus in immunocompromised patients, infants, and the elderly remains unknown (Leroux-Roels et al, 2018)

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