"Plant-based expression is a cost-effective and convenient system for antigens to be used in mucosal delivery. Plants represent relatively safe platforms to express vaccines since they are free of mammalian pathogens that affect other production systems such as transgenic animals and cell lines  . Norwalk VLPs have been previously shown to accumulate in transgenic potato , tobacco  , and tomato  . "
[Show abstract][Hide abstract] ABSTRACT: Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.
BioMed Research International 05/2014; 2014:807539. DOI:10.1155/2014/807539 · 2.71 Impact Factor
"Plants are a cheap source of biomass and as such are an attractive alternative to conventional expression hosts for the manufacture of biologics with clinical, industrial, and research applications (Faye and Gomord, 2010; Fischer et al., 2012). However, the economic viability of plant-based protein production is strongly yield dependent, and considerable research has been dedicated to developing novel approaches to increasing transgene expression. "
[Show abstract][Hide abstract] ABSTRACT: In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.
The Plant Cell 07/2013; 25(7). DOI:10.1105/tpc.113.113944 · 9.58 Impact Factor
"Alternatively, plants (both whole plants and in vitro cultures) are gaining attention for the large-scale production of recombinant proteins and particularly, as a promising platform for vaccine production     although a higher number of clinical trials have to be performed in order to increase general acceptance. To date, there is more than a dozen of plant-made biopharmaceuticals in clinical developments . Some of the advantages of the plant-based systems are that they can rapidly be bulked to large biomass, its maintenance is relatively inexpensive, and they do not harbor mammalian proteins or pathogens. "
[Show abstract][Hide abstract] ABSTRACT: The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical
diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest
inductor of the immune response. There are several available commercial vaccines against bovine viral
diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative
productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the
BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium
binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into
the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification
and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was
introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens
was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using
specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts.
An estimated amount of 20 �g of tE2 per gram of fresh leaves was regularly obtained with this plant
system. Injection of guinea pigs with plant extracts containing 20 �g of rtE2 induced the production of
BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines.
This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the
advantage to be free of any eventual animal contaminant.
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