Tobacco and related Nicotiana
species are the most common host plants for production of PMPs. Downstream processing of target PMPs from these plants, however, is limited by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. While low-alkaloid Nicotiana
varieties naturally exist and more have been created through breeding, these varieties are not always optimal hosts for PMP production due to the lack of correlation between varieties that accumulate high levels of target protein and those with low alkaloid content (Conley et al., 2010
). In this study, we explored the use of lettuce, a plant that grows rapidly yet produces low levels of toxic secondary metabolites, for viral vector-based transient expression system as a robust PMP production platform. Our results show that the BeYDV-based geminiviral replicon system can efficiently promote high-level expression of NVCP VLP vaccine and anti-EBV or WNV mAb therapeutic candidates in lettuce. Using the geminiviral-lettuce system, the VLP and the two therapeutic mAbs accumulated to levels that were comparable to that observed in tobacco (Huang et al., 2010
; Lai et al., 2010
), but higher than previously reported in lettuce using non-viral vectors (Kapusta, 1999
; Rosales-Mendoza et al., 2010
; Webster et al., 2006
). For example, hE16 and 6D8 mAbs accumulated to 0.27 mg/g fresh leaf weight 4 dpi (), up to 13.5-fold higher than that produced by non-viral expression vectors in lettuce (Negrouk et al., 2005
). To our knowledge, this is the highest level of mAb accumulation ever reported in lettuce. Recently, two novel transient expression vector systems that are based on a plastocyanin promoter or the 5′ and 3′-untranslated region of Cowpea mosaic virus RNA-2 have been developed. These vector systems do not require viral replication yet allow the high level expression of mAbs from a single plasmid in N. Benthamiana
(Sainsbury et al., 2009
; Vézina et al., 2009
). As such, it will be interesting to explore the utility of these vectors in lettuce as well.
In addition to targeting transgenes to the nuclear genome, certain pharmaceutical proteins can be expressed via a chloroplastic genome (Daniell, 2006
). As a result, higher level of target protein accumulation can be achieved by expression in transplastomic lettuce (Davoodi-Semiromi et al., 2010
). Nonetheless, because chloroplasts lack posttranslational modification machinery, transplastomic plant lines cannot efficiently produce complex hetero-oligomeric proteins such as mAbs or simple proteins that require posttranslational modifications (e.g., N-linked glycosylation) for pharmaceutical function (Chen, 2008
). In contrast, the geminiviral-lettuce system relies on transient expression of nuclear-targeted transgenes, and can efficiently express and assemble complex proteins including mAbs.
P19, a suppressor of gene silencing from tomato bushy stunt virus (TBSV), has been reported to enhance recombinant protein expression by suppressing post-transcriptional gene silencing (PTGS) (Voinnet et al., 2003
). In prior research with N. benthamiana
, we demonstrated that co-infiltration of pP19 with BeYDV replicons elevated target mRNA and protein accumulation (Huang et al., 2009
; Huang et al., 2010
). However, inclusion of the pP19 construct in the co-infiltration of replicon vector did not enhance the accumulation of the NVCP VLP in lettuce (see ) or the two target mAbs in lettuce (Q. Chen, unpublished results). It seems unlikely that this is caused by a poor interaction between P19 and the heterologous RNA-silencing machinery in lettuce. P19 prevents gene silencing by sequestering the small interfering RNAs (siRNAs) and microRNAs (miRNAs) (Lakatos et al., 2004
), and thus should not be plant species-specific. Indeed, P19 has been shown to inhibit RNA interference in a broad range of organisms including human cells (Dunoyer et al., 2004
). Moreover, lettuce is susceptible to TBSV infection, suggesting P19’s effectiveness as a suppressor of gene silencing in this plant (Obermeier et al., 2001
). It remains possible that under the conditions tested in lettuce, PTGS occurred at a low level or not at all. This hypothesis is corroborated by the results of a previous study in which P19 and beta-glucuronidase constructs were co-introduced into lettuce leaves by non-replicon expression vectors (Simmons and VanderGheynst, 2007
In addition to the geminiviral replicon system, we examined the ability of TMV and PVX-based MagnICON deconstructed viral vectors to promote transgene expression in lettuce. Notably, we did not observe accumulation of GFP in lettuce leaves infiltrated with TMV or PVX-based vectors or expression of NVCP or mAbs. Similar to other positive-stranded RNA viruses, TMV and PVX RNA viral genomes are templates for both translation and replication, which leads to complex interactions at multiple levels between plant host factors and viral RNA replication and translation (Ahlquist et al., 2005
). Such host-virus interaction is one of the critical factors in determining the host range of the infecting virus. Several such plant host factors have been identified for TMV and PVX (Park et al., 2009
). For example, a 56-Kda tomato protein was found to be related to the RNA-binding subunit of yeast translational initiation factor, eIF3 and important for the translation of RNA-dependent RNA polymerase (RdRp) of TMV (Buck, 1999
). Therefore, for MagnICON vectors to work in a plant species, host factors from this particular plant species must recognize and interact with TMV and/or PVX elements/factors to assist the translation of RdRp and the replication of the deconstructed viral genome. In contrast, geminiviruses have a small, single-stranded DNA genome that replicates using the well-conserved cellular apparatus and a virus-encoded protein Rep (Chen et al., 2011
). For some geminivirus species, they can even replicate in non-host plant cells, but their host-ranges are limited instead by their inability to move from cell to cell for systemic infection (Teng et al., 2010
). BeYDV has a broad host range in dicotyledonous plants and remains competent for replication as long as Rep and its cis-binding element (LIR) are present (Liu et al., 1999
). Thus, the BeYDV-based geminiviral vectors may be functional in a wide range of plant species. We speculate that our finding reflects the host specificity of the viral elements/factors of TMV and PVX; host factors in lettuce may be sufficiently different from those in tobacco and other Solanaceae
plants such that they cannot interact with the viral elements/factors and assist the translation of RdRp and/or for the replication of deconstructed TMV or PVX genome. In contrast, LIR and the gene coding for Rep are readily engineered as parts of the BeYDV replicon vectors, thereby, allowing its replication in lettuce and potentially a broader range of other host plant species.
Downstream processing can account for up to 80% of the total pharmaceutical protein production cost (Chen, 2011a
). The development of effective, scalable, economical and cGMP compliant purification methods for recovering target proteins from lettuce is crucial. We developed robust and effective downstream processing protocols to purify our target vaccine and mAbs from lettuce. Our procedures can efficiently isolate the NVCP vaccine candidate and the two therapeutic mAbs to high (>95%) purity, in a scalable and cGMP compatible format. Direct loading of clarified lettuce extracts onto Protein A affinity columns did not adversely affect the integrity of the resin over 20 purification cycles. Thus, in contrast to tobacco, the precipitation step for reducing phenolics and alkaloids from the feed-stream becomes unnecessary and can be eliminated. This simplifies purification processing, prolongs resin life, and reduces the cost of downstream processing, and the overall cost of goods. The ease of purifying PMPs from lettuce and its cost-saving benefit support the feasibility of the geminiviral-lettuce expression system as a viable large-scale PMP production platform.
The geminiviral-lettuce derived NVCP and mAbs had the expected structural and functional properties. Similar to NVCP produced in tobacco, our preliminary results suggest that it accumulates in the cytosol of lettuce leaf cells (Q. Chen, unpublished results), although its precise subcellular location requires further confirmation. Nevertheless, NVCP derived from lettuce assembled into 30 nm VLPs in a manner indistinguishable from NVCP VLP derived from insect cells. Moreover, lettuce-derived mAbs readily assembled into tetramers without degradation, establishing that the geminiviral-lettuce system can assemble intact mAbs efficiently. In general, the minor difference in protein N-glycosylation between plant and mammalian cells could be an issue for plant-derived mAbs, since the possibility of inducing plant-glycan specific antibodies could reduce stability and therapeutic efficacy by accelerating clearance from plasma, or cause potential adverse effects through immune complex formation. However, since the HCs of our mAbs were tagged with the ER-retention signal KDEL at their C-termini, we speculate that they would be retained in ER and have a high-mannose form of glycans at their Fc region. Our N-glycan analyses confirmed that both mAbs produced in lettuce have the expected high-mannose glycoform (Q. Chen, unpublished results). Since high-mannose is common between plant and mammalian cells, the concern for the immunogenicity of lettuce-derived mAbs in humans is diminished. Previously, we demonstrated that a tobacco-derived hE16 mAb with the same high-mannose N-glycosylation pattern had no in vivo
stability issue and showed potent therapeutic activity against lethal WNV challenge in a mouse model (Lai et al., 2010
). The structural equivalency of lettuce and tobacco-derived mAbs suggests their similarity in stability and functional activities. Indeed, these lettuce-derived mAbs showed specific binding to their respective antigens, and for hE16 retained equivalent functional activity. To the best of our knowledge, this is the first demonstration of functional activity for a lettuce-produced mAb. Our success in producing fully-assembled VLP and mAbs with the geminiviral-lettuce system strongly suggest its utility in producing a broad range of pharmaceutical proteins.
This study serves as a proof-of-principle for using commercially produced lettuce in high-level PMP production. While a greater variation in expression levels of mAbs was observed between different batches of lettuce possibly due to differences in growing or post-harvest storage conditions, surprisingly, the overall accumulation levels of mAbs was similar between grocery stored-purchased lettuce and the laboratory-grown lettuce. Lettuce is a fast-growing plant that is already cultivated year-round in commercial greenhouses for mass production. In addition, processing technologies for commercial-scale harvesting, washing, and processing of lettuce exist. Therefore, biomass production could be subcontracted to existing commercial growers to provide lettuce with consistent growth and post-harvest storage conditions, which likely would reduce variation in target protein accumulation. The existence of large-scale processing technology and commercial infrastructure in the agricultural industry will facilitate adaptation for PMP production. Our success in using commercially produced lettuce and geminiviral expression vectors for high-level PMP production allows access to potentially unlimited quantities of inexpensive plant material for large-scale production.
Since lettuce is a food crop, there may be environmental and regulatory concerns for its use in PMP production. While this is an issue for production systems using transgenic lettuce cultivated in open fields, our geminiviral transient expression system does not require transgenic lettuce, but rather, uses non-transgenic lettuce that can be purchased from commercial sources. In addition, operations for agroinfiltration and downstream processing can be performed in a contained cGMP controlled environment as required for all pharmaceutical production systems. As a result, there would be no exposure of transgenic lettuce or vectors to the environment, essentially eliminating the risk of contaminating food supply. This safety feature will minimize biosafety and regulatory concerns and costs associated with transgenic crops and genetically modified plants.
In summary, the use of geminiviral replicon expression system and commercially produced lettuce as plant production host provides a new platform for pharmaceutical protein production that is robust, low-cost, safe, and scalable to commercial manufacturing.