title: A DNA replicon system for rapid high-level production of virus-like particles in plants.
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.
authors.affiliation: Biodesign Institute, School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Huang
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.forename: Brooke
authors.initials: B
authors.lastname: Hjelm
authors.forename: Charles
authors.initials: C
authors.lastname: Arntzen
authors.forename: Hugh
authors.initials: H
authors.lastname: Mason
mesh_terms: D001709:Biotechnology / Q000379:methods*; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D004261:DNA Replication; D017791:Geminiviridae / Q000235:genetics*; D005822:Genetic Vectors; D006512:Hepatitis B Core Antigens / Q000096:biosynthesis / Q000235:genetics; D009663:Norwalk virus / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*; D022701:Virosomes / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D006512:Hepatitis B Core Antigens; D022701:Virosomes