Plant-produced, trans-encapsidated, replicative viral nanoparticles as a vaccine platform.

Replicative virus-based vaccines provide potential solutions to avoid the natural deficits of traditional single-protein subunit vaccines. Unlike subunit vaccines, the infectious nature of viral particles can stimulate the innate immune response, which allows enhanced and elongated stimulation of antibody and T cell adaptive immunity. When these replicative vaccines are engineered to express antigen genes naturally in the patient's cells, cell mediated immunity is stimulated via the class I antigen presentation pathway, followed by a robust CD8+ T cell activation and interferon production. In this study, I aim to develop a safe, effective and cost-efficient transencapsidated replicative vaccine platform by encapsidating within strongly immunogenic tobacco mosaic virus (TMV) nanoparticles the self-replicative RNA of Flock House virus (FHV), an insect virus that also replicates in human and plant cells. By inserting the unique packaging signal of TMV into FHV RNA, the capsid of TMV assembles around the heterologous RNA, when TMV coat protein and FHV RNA are expressed in the same cell. First, the replication of different viral delivery vectors in both mammalian cells and plant cells was investigated and their cellular co-infection was demonstrated. The resulting successfully packaged chimeric nanoparticle improved antibody production over the standards after vaccination in mice. Next I detailed the specifics and techniques required to produce and purify these nanoparticle vaccines in planta via agroinoculation. To further explore the possibility of increasing the yield of plant-produced nanoparticle vaccines, an optimized spray-on inoculation method was developed using a low-toxicity surfactant to aid agro-inoculation. Unexpectedly, this also decreased contaminating plant pathogenesis-related proteins. A continuing study is also discussed on trans-locating FHV replication to endoplasmic reticulum, which suggests potential to further increase plant yield of nanoparticle vaccines as well.