A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice.

Previous studies have shown that the production of recombinant antibodies in plants is highly efficient and presents numerous therapeutic applications. It is, however, known that plant glycoproteins display different glycosylation patterns to those exhibited by mammalian glycoproteins. Thus, it is important to know if these plant recombinant antibodies could induce undesirable immune responses in mammals; and to date no report has documented the potential immunogenicity of parenterally administered plant recombinant antibodies in animals. In order to answer this question, mice were immunised subcutaneously with a recombinant mouse monoclonal antibody produced in tobacco plants, together with alum as adjuvant. Two control groups were immunised in the same way with either the original murine monoclonal antibody or horseradish peroxidase (a plant glycoprotein). Analyses by direct immunoassay, competition immunoassay and real-time surface plasmon resonance, showed undetectable levels of antibody directed against both the protein and the glycan part of the plant recombinant antibody. These results have a direct relevance for the application of plant recombinant proteins as therapeutic agents and vaccines in humans.

Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block.

White clover mosaic virus strain O (WClMV-O), species of the Potexvirus genus, contains a set of three partially overlapping genes (the triple gene block) that encodes nonvirion proteins of 26 kDa, 13 kDa, and 7 kDa. These proteins are necessary for cell-to-cell movement in plants but not for replication. The WClMV-O 13-kDa gene was mutated (to 13*) in a region of the gene that is conserved in all viruses known to possess triple-gene-block proteins. All 10 13* transgenic lines of Nicotiana benthamiana designed to express the mutated movement protein were shown to be resistant to systemic infection by WClMV-O at 1 microgram of WClMV virions per ml, whereas all plants from susceptible control lines became systemically infected. Of the 13* transgenic lines, 3 selected for their abundant seed supply were shown to be resistant to systemic infection when challenged by inoculation with three different WClMV strains (O, M, and J) or with WClMV-O RNA at 10 micrograms/ml. Most plants were also resistant to systemic infection at inoculum concentrations up to 250 micrograms of WClMV virions per ml. In addition, the three 13* transgenic plant lines were found to be resistant to systemic infection with two other members of the Potexvirus group, potato virus X and narcissus mosaic virus, and the Carlavirus potato virus S but not to be resistant to tobacco mosaic virus of the Tobamovirus group. These results indicate that virus resistance can be engineered into transgenic plants by expression of dominant negative mutant forms of triple-gene-block movement proteins.

The coat protein of white clover mosaic potexvirus has a role in facilitating cell-to-cell transport in plants.

Functions of the coat protein of white clover mosaic potexvirus (WCIMV) were investigated using C-terminal deletion mutants. Whereas plants inoculated with RNA transcripts of a full-length wild-type clone of WCIMV produced typical infections, plants inoculated with transcripts of each mutant did not produce symptoms, and viral RNA species were not detected by Northern analysis. The mutants were able to replicate in protoplasts, although, relative to the wild-type RNA profile, the level of genomic RNA, but not subgenomic RNA, was reduced. These results indicate a role for the coat protein in efficient cell-to-cell transport in plants. Virus-like particles were detected in protoplast extracts inoculated with transcripts of a mutant in which the coat protein was truncated by 31 amino acids. This result suggests that the lack of detectable transport in plants was not due solely to a failure of the mutants to form virus particles. Possible roles for the coat protein in transport and replication are discussed. A 6-kDa open reading frame, internal to the coat protein gene, was shown by mutational analysis not to be essential for replication or transport.