title: Rice endosperm produces an underglycosylated and potent form of the HIV-neutralizing monoclonal antibody 2G12.
abstract: Protein microbicides against HIV can help to prevent infection but they are required in large, repetitive doses. This makes current fermenter-based production systems prohibitively expensive. Plants are advantageous as production platforms because they offer a safe, economical and scalable alternative, and cereals such as rice are particularly attractive because they could allow pharmaceutical proteins to be produced economically and on a large scale in developing countries. Pharmaceutical proteins can also be stored as unprocessed seed, circumventing the need for a cold chain. Here, we report the development of transgenic rice plants expressing the HIV-neutralizing antibody 2G12 in the endosperm. Surprisingly for an antibody expressed in plants, the heavy chain was predominantly aglycosylated. Nevertheless, the heavy and light chains assembled into functional antibodies with more potent HIV-neutralizing activity than other plant-derived forms of 2G12 bearing typical high-mannose or plant complex-type glycans. Immunolocalization experiments showed that the assembled antibody accumulated predominantly in protein storage vacuoles but also induced the formation of novel, spherical storage compartments surrounded by ribosomes indicating that they originated from the endoplasmic reticulum. The comparison of wild-type and transgenic plants at the transcriptomic and proteomic levels indicated that endogenous genes related to starch biosynthesis were down-regulated in the endosperm of the transgenic plants, whereas genes encoding prolamin and glutaredoxin-C8 were up-regulated. Our data provide insight into factors that affect the functional efficacy of neutralizing antibodies in plants and the impact of recombinant proteins on endogenous gene expression.
authors.affiliation: Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA), University of Lleida-Agrotecnio Center, Lleida, Spain.
authors.forename: Evangelia
authors.identifier: http://orcid.org/0000-0002-9818-4404
authors.initials: E
authors.lastname: Vamvaka
authors.affiliation: TRM Ltd, York, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Twyman
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Laboratory of Synthetic Biology, Parque Estacao Biologica, Brasilia, Distrito Federal, Brazil.
authors.forename: Andre Melro
authors.initials: AM
authors.lastname: Murad
authors.affiliation: Department for Applied Genetics and Cell Biology, Molecular Plant Physiology and Crop Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Melnik
authors.affiliation: Molecular Immunology Unit, Division of Clinical Sciences, St. George's University of London, London, UK.
authors.forename: Audrey Yi-Hui
authors.initials: AY
authors.lastname: Teh
authors.affiliation: Department for Applied Genetics and Cell Biology, Molecular Plant Physiology and Crop Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.affiliation: Department of Chemistry, Muthgasse, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Department for Applied Genetics and Cell Biology, Molecular Plant Physiology and Crop Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Laboratory of Synthetic Biology, Parque Estacao Biologica, Brasilia, Distrito Federal, Brazil.
authors.forename: Elibio
authors.initials: E
authors.lastname: Rech
authors.affiliation: Molecular Immunology Unit, Division of Clinical Sciences, St. George's University of London, London, UK.
authors.forename: Julian K C
authors.initials: JK
authors.lastname: Ma
authors.affiliation: Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA), University of Lleida-Agrotecnio Center, Lleida, Spain.|Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain.
authors.forename: Paul
authors.initials: P
authors.lastname: Christou
authors.affiliation: Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA), University of Lleida-Agrotecnio Center, Lleida, Spain.
authors.forename: Teresa
authors.initials: T
authors.lastname: Capell
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D057134:Antibodies, Neutralizing / Q000096:biosynthesis*; D000080908:Broadly Neutralizing Antibodies; D015536:Down-Regulation / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D056625:Endosperm / Q000378:metabolism* / Q000648:ultrastructure; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006031:Glycosylation; D015483:HIV Antibodies / Q000096:biosynthesis*; D015488:HIV Antigens / Q000276:immunology; D012275:Oryza / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D059467:Transcriptome / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
chemical_list: C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015488:HIV Antigens; D012333:RNA, Messenger
keywords: 2G12; HIV; endosperm; glycosylation; microbicides; rice
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