A DNA replicon system for rapid high‐level production of virus‐like particles in plants

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Abstract
Recombinant virus‐like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low‐level antigen accumulation and long‐time frame to produce transgenic plants are the two major roadblocks in the practical development of plant‐based VLP production. In this article, we describe the optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs. Co‐delivery of bean yellow dwarf virus (BeYDV)‐derived vector and Rep/RepA‐supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co‐expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built‐in Rep/RepA cassette without P19 drove protein expression at similar levels as the three‐component system. These results demonstrate the advantages of fast and high‐level production of VLP‐based vaccines using the BeYDV‐derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706–714.