{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T20:28:54Z","timestamp":1775766534387,"version":"3.50.1"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T00:00:00Z","timestamp":1614038400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:p>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\u00b0C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and <jats:strike>for<\/jats:strike> prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.<\/jats:p>","DOI":"10.3389\/fpls.2021.639953","type":"journal-article","created":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T14:19:34Z","timestamp":1614089974000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":18,"title":["Development of Antibody-Fragment\u2013Producing Rice for Neutralization of Human Norovirus"],"prefix":"10.3389","volume":"12","author":[{"given":"Ai","family":"Sasou","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yoshikazu","family":"Yuki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shiho","family":"Kurokawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shintaro","family":"Sato","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuki","family":"Goda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masao","family":"Uchida","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Naomi","family":"Matsumoto","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiroshi","family":"Sagara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuji","family":"Watanabe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Masaharu","family":"Kuroda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Naomi","family":"Sakon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kotomi","family":"Sugiura","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rika","family":"Nakahashi-Ouchida","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiroshi","family":"Ushijima","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kohtaro","family":"Fujihashi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiroshi","family":"Kiyono","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,2,23]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1016\/j.jbiotec.2014.02.005","article-title":"A rice-based soluble form of a murine TNF-specific llama variable domain of heavychain antibody suppresses collagen-induced arthritis in mice","volume":"175","author":"Abe","year":"2014","journal-title":"J. Biotechnol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"284","DOI":"10.1128\/CVI.05592-11","article-title":"Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus","volume":"9","author":"Czak\u00f3","year":"2012","journal-title":"Clin. Vaccine Immunol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"555","DOI":"10.1002\/prot.20378","article-title":"Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen","volume":"59","author":"Dolk","year":"2005","journal-title":"Proteins"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"161","DOI":"10.1002\/bit.22653","article-title":"Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity","volume":"106","author":"Giersberg","year":"2010","journal-title":"Biotechnol. Bioeng."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Hamers-Casterman","year":"1993","journal-title":"Nature"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1046\/j.1365-313X.1994.6020271.x","article-title":"Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of the boundaries of the T-DNA","volume":"6","author":"Hiei","year":"1994","journal-title":"Plant J."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"e121077","DOI":"10.1172\/jci.insight.121077","article-title":"Safety and immunogenicity of an oral tablet norovirus vaccine, a phase I randomized, placebo-controlled trial","volume":"3","author":"Kim","year":"2018","journal-title":"JCI Insight"},{"key":"ref8","doi-asserted-by":"publisher","first-page":"471","DOI":"10.1007\/BF00392095","article-title":"Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds","volume":"169","author":"Krishnan","year":"1986","journal-title":"Planta"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"2348","DOI":"10.1271\/bbb.100465","article-title":"A simple set of plasmids for the production of transgenic plants","volume":"74","author":"Kuroda","year":"2010","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1007\/s00299-013-1513-3","article-title":"RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds","volume":"1","author":"Kurokawa","year":"2014","journal-title":"Plant Cell Rep."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"593","DOI":"10.1093\/infdis\/jix572","article-title":"Safety and immunogenicity of different formulations of norovirus vaccine candidate in healthy adults: a randomized, controlled, double-blind clinical trial","volume":"217","author":"Leroux-Roels","year":"2018","journal-title":"J. Infect. Dis."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1007\/s00253-017-8644-3","article-title":"Expression of single-domain antibody in different systems","volume":"2","author":"Liu","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"e1001999","DOI":"10.1371\/journal.pmed.1001999","article-title":"The vast and varied global burden of norovirus: prospects for prevention and control","volume":"13","author":"Lopman","year":"2016","journal-title":"PLoS Med."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"220","DOI":"10.1007\/s12560-013-9127-z","article-title":"A food handler-associated, foodborne norovirus GII.4 Sydney 2012-outbreak following a wedding dinner, Austria, October 2012","volume":"5","author":"Maritschnik","year":"2013","journal-title":"Food Environ. Virol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"21173","DOI":"10.2807\/1560-7917.es2015.20.26.21173","article-title":"Genetic analyses of GII.17 norovirus strains in diarrheal disease outbreaks from December 2014 to March 2015 in Japan reveal a novel polymerase sequence and amino acid substitutions in the capsid region","volume":"20","author":"Matsushima","year":"2015","journal-title":"Eur. Secur."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1186\/s13567-015-0151-x","article-title":"Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies","volume":"46","author":"Moonens","year":"2015","journal-title":"Vet. Res."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"10986","DOI":"10.1073\/pnas.0703766104","article-title":"Rice-based mucosal vaccine as a global strategyfor cold-chain- and needle-free vaccination","volume":"104","author":"Nochi","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"875","DOI":"10.1080\/08820139.2019.1688828","article-title":"Nanobodies in human infections: prevention, detection, and treatment","volume":"49","author":"Sanaei","year":"2019","journal-title":"Immunol. Investig."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1097\/00005176-200101000-00009","article-title":"Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea","volume":"32","author":"Sarker","year":"2001","journal-title":"J. Pediatr. Gastroenterol. Nutr."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"1149","DOI":"10.1097\/00006454-199812000-00010","article-title":"Successful treatment of rotavirus diarrhea in children with immunoglobulin from immunized bovine colostrum","volume":"17","author":"Sarker","year":"1998","journal-title":"Pediatr. Infect. Dis. J."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"740.e8","DOI":"10.1053\/j.gastro.2013.06.053","article-title":"Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial","volume":"145","author":"Sarker","year":"2013","journal-title":"Gastroenterology"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"686.e685","DOI":"10.1016\/j.jcmgh.2018.11.001","article-title":"Human norovirus propagation in human induced pluripotent stem cell-derived intestinal epithelial cells","volume":"7","author":"Sato","year":"2019","journal-title":"Cell. Mol. Gastroenterol. Hepatol."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1056\/NEJMoa1806311","article-title":"Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura","volume":"4","author":"Scully","year":"2019","journal-title":"New Engl. J. Med."},{"key":"ref24","first-page":"1633","article-title":"Isolation and characterization of two types of protein bodies in the rice endosperm","volume":"44","author":"Tanaka","year":"1980","journal-title":"Agric. Biol. Chem."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1007\/s11248-009-9338-0","article-title":"High-level expression of Camelid nanobodies in Nicotiana benthamiana","volume":"19","author":"Teh","year":"2010","journal-title":"Transgenic Res."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1016\/j.meegid.2017.03.020","article-title":"Emergence of norovirus GII.2 and its novel recombination during the gastroenteritis outbreak in Japanese children in mid-2016","volume":"51","author":"Thongprachum","year":"2017","journal-title":"Infect. Genet. Evol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"3829","DOI":"10.1172\/JCI70266","article-title":"Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection","volume":"123","author":"Tokuhara","year":"2013","journal-title":"J. Clin. Invest."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"4592","DOI":"10.1128\/IAI.01033-13","article-title":"A single VHH-based toxin-neutralizing agent and an effector antibody protect mice against challenge with shiga toxins 1 and 2","volume":"81","author":"Tremblay","year":"2013","journal-title":"Infect. Immun."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1016\/S0167-4838(99)00030-8","article-title":"Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies","volume":"1431","author":"van der Linden","year":"1999","journal-title":"Biochim. Biophys. Acta"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1128\/JCM.01535-14","article-title":"Advances in laboratory methods for detection and typing of norovirus","volume":"53","author":"Vinj\u00e9","year":"2015","journal-title":"J. Clin. Microbiol."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"756","DOI":"10.1093\/pcp\/pcj049","article-title":"The correlation between expression and localization of a foreign gene product in rice endosperm","volume":"47","author":"Yamada","year":"2006","journal-title":"Plant Cell Physiol."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1094","DOI":"10.1104\/pp.70.4.1094","article-title":"Biosynthesis of storage proteins in developing rice seeds","volume":"70","author":"Yamagata","year":"1982","journal-title":"Plant Physiol."},{"key":"ref33","first-page":"135","article-title":"The site of synthesis and accumulation of rice storage proteins","volume":"27","author":"Yamagata","year":"1986","journal-title":"Plant Cell Physiol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"470","DOI":"10.1093\/infdis\/jiaa115","article-title":"A heterodimeric antibody fragment for passive immunotherapy against norovirus infection","volume":"222","author":"Yuki","year":"2020","journal-title":"J. Infect. Dis."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"799","DOI":"10.1111\/pbi.12071","article-title":"Induction of toxin-specific neutralizing immunity by molecularly uniform rice-based oral cholera toxin B subunit vaccine without plant-associated sugar modification","volume":"7","author":"Yuki","year":"2013","journal-title":"Plant Biotechnol. J."}],"container-title":["Frontiers in Plant Science"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpls.2021.639953\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,4,1]],"date-time":"2021-04-01T10:16:44Z","timestamp":1617272204000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fpls.2021.639953\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,2,23]]},"references-count":35,"alternative-id":["10.3389\/fpls.2021.639953"],"URL":"https:\/\/doi.org\/10.3389\/fpls.2021.639953","relation":{},"ISSN":["1664-462X"],"issn-type":[{"value":"1664-462X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,2,23]]},"article-number":"639953"}}