Robust production of virus‐like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

Abstract

Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing.

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

“…For N. benthamiana leaves that were entirely infiltrated with Agrobacteria containing geminiviral vectors, GFP expression was observed over the entire leaf area under UV light starting from as early as 2 dpi and reached peak accumulation at 4 dpi ( Figure 4C). This early GFP expression by geminiviral vector is consistent with previous results of other recombinant proteins 14,16,18,19 . In contrast, leaves infiltrated with MagnICON vector-containing Agrobacterium combinations showed GFP fluorescence only after 5 dpi and reached its maximum accumulation at 7 dpi (Figure 4D).…”

“…Both protocols are described here using N. benthamiana, which is closely related to the common tobacco plant, as the host plant for transient expression of two fluorescent proteins: the green fluorescent protein (GFP) from jellyfish Aequorea victoria and the red fluorescent protein from Discosoma coral (DsRed) 12,13 . N. benthamiana is the most common host plant for recombinant protein because it is amenable to genetic transformation, can yield high amounts of biomass rapidly, and is a prolific seed producer for scale-up production 14 . Another advantage of using N. benthamiana as hosts for protein expression is the availability of a variety of expression vectors 2,5 .…”

“…Three DNA constructs are also needed for expression with geminiviral vectors. In addition to vectors containing the replicon of the target gene ( pBYGFP or pBYDsRed), a vector coding for the replication protein (pREP110) is required for the amplification of the target replicon 11,14,16 . Furthermore, the inclusion of a vector encoding the silencing suppressor p19 from tomato bushy stunt virus is desired for high level target gene expression 11,16 .…”

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

“…These vaccine and therapeutic mAb candidates can be readily purified in high-yield from lettuce leaves, without the extra steps required for elimination of plant secondary metabolites. These lettuce-produced pharmaceutical agents not only are produced in large quantities, but also exhibit fully functional activity [34]. Significantly, our feasibility studies with grocery storepurchased lettuce also opened a new avenue for obtaining biomass for commercial scale agroinfiltration.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…The system requires co-expression of the silencing suppressors. Some geminivirus systems have been adapted to express recombinant proteins in non-Nicotiana species, including lettuce [71].…”