Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Drs. Aiming Wang

2nd Supervisor

Norman P.A. Huner

Joint Supervisor

Abstract

Virus-based expression systems have been widely exploited for the production of recombinant proteins in plants during the last three decades. Advances in technology have boosted scale-up manufacturing of plant-made pharmaceuticals to high levels, via the complementation of transient expression and viral vectors. This combination allows proteins of interest to be produced in plants within a matter of days and thus, is well suited for the development of plant-made vaccines or therapeutics against emerging infectious diseases and potential bioterrorism agents. Several plant-based products are currently in varying stages of clinical development. To investigate the viability of virus-based expression systems for plant-made subunit vaccines against Porcine reproductive and respiratory syndrome virus (PRRSV), I have developed several viral vectors which express partial PRRSV glycoprotein 5 (GP5).

In this thesis, I demonstrated that viral vectors can increase the expression of proteins for vaccine development in a variety of host plants. Green fluorescent protein (GFP) and the C-terminus of GP5 fused with GFP (GP5C-GFP) were expressed at up to 37.29 mg/kg and 4.36 mg/kg fresh weight of leaf tissue, respectively, in soybean plants via biolistic bombardment, using viral vectors derived from Bean pod mottle virus (BPMV). A Cucumber green mottle mosaic virus (CGMMV)-based vector was employed to display chimeric virus particles, presenting the neutralizing epitope (NE) of GP5, at levels as high as 35.84 mg/kg of cucumber leaf fresh weight, via agroinfiltration. In addition, GFP and the ectodomain of GP5 fused with GFP (GP5e-GFP) were successfully produced at up to 2.03 g/kg and 36.53 mg/kg fresh weight of leaf tissue, respectively, in Nicotiana benthamiana using transient expression of Pepino mosaic virus (PepMV)-based vectors. Co-infiltration of viral vectors with p19, a silencing suppressor, can help to mitigate the toxicity of GP5e to plant tissue and enhance the accumulation of this protein. These plant-made products may be tested as oral subunit vaccines against PRRSV in pigs during future experimental trials. Overall, the results demonstrate that viral systems can produce low-cost, versatile and robust vaccines that have a great impact in the fight against viral diseases, especially in developing countries.

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