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Transient Expression of Poliovirus-Like Particles in Plants

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Transient Expression of Poliovirus-Like Particles in Plants Transient expression of poliovirus-like particles in plants. Developing a synthetic polio vaccine by Johanna Dörte Marsian This thesis is submitted in partial fulfilment of the requirements of the degree of Doctor of Philosophy at the University of East Anglia. John Innes Centre, Norwich, UK September 2016 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Declaration DECLARATION I hereby certify that the work contained within this thesis is my own original work, except where due reference is made to other contributing authors. This thesis is submitted for the degree of Doctor of Philosophy at the University of East Anglia and has not been submitted to this or any other university for any other qualification. Johanna D. Marsian 2 Abstract ABSTRACT Plants, or cell suspension cultures derived from them, are a promising platform for the production of biologics and pharmaceuticals. In this work transient expression utilising the pEAQ vector system was deployed for the expression of virus-like particles (VLPs) in Nicotiana benthamiana or N. tabacum BY-2 cell suspension cultures. The results presented in this thesis demonstrate the potential of plant systems for the production of VLP-based vaccines. VLPs of the fish virus, Nervous necrosis virus (NNV), were successfully produced in plants by transient expression of the coat protein. The protein self-assembled into T = 3 particles, which appeared to be morphologically identical to the wild-type NNV when analysed by high resolution microscopy but were devoid of nucleic acid. In addition, transgenic BY-2 cell suspension lines were generated expressing correctly assembled NNV VLPs. Poliovirus (PV)-like particles from all three PV serotypes, containing either the wt coat protein or coat proteins with stabilising mutations, were successfully expressed in plants. These were generated by co-expression of the structural polyprotein P1 and the proteinase 3CD. Sufficient quantities of purified particles could be obtained for structural and immunological analysis. Mice carrying the gene for the human PV receptor were protected from wild-type PV when immunised with the plant-made stabilised PV VLPs. Structural analysis of the stabilised mutant of PV3 at 3.6 Å resolution revealed a structure almost indistinguishable from wild-type PV3, with the stabilising mutations having no effective on the antigenic surface of the particle. To make the product more attractive to the vaccine industry, tobacco BY-2 cells have been successfully tested for the transient expression of the above-mentioned PV mutant VLPs using the cell-pack method. 3 Acknowledgements ACKNOWLEDGEMENTS First and foremost, I would like to thank Prof. George Lomonossoff for being a great supervisor and mentor. His positivity and enthusiasm is truly inspirational. He gave me free rein to follow my own ideas and to learn from my own mistakes, yet his door was always open when I needed help. I am very thankful for the numerous conferences and meetings I was able to attend, where George taught me how to network; a skill that will always be useful throughout my life. I would like to thank the other members of my supervisory committee, Prof. Nick Brewin and Dr. Keith Saunders for providing advice and expertise throughout my degree. Special thanks go to my colleagues, in particular Keith, Hadrien, Eva, Yulia and Milena for all the laughter and banter in the lab. I have been very fortunate to work in such a supportive, diverse and extremely friendly department and I would like to thank all the members of Biological Chemistry for the great company. I also want to acknowledge the staff in the media kitchen and the horticultural services, I am very grateful for their support. Through my project I got the opportunity to collaborate and meet inspirational scientists which has certainly enriched my work. For great collaboration and support, I would especially like to thank Prof. Nicola Stonehouse and Prof. Dave Rowlands (University of Leeds), Helen Fox and Dr. Andrew Macadam (NIBSC) and Dr. Mohammad Bahar (University of Oxford). I want to include in my gratitude all the other members of the WHO polio consortium and the members of our advisory committee. I am also very grateful to Dr. Anneli Ritalla and Jaana Rikkinen (VTT Finland), as well as Markus Sack (IME Fraunhofer, Aachen) for introducing me to BY-2 cell suspension cultures. I want to thank my Mum and Dad, my brother Julius and my sister Franziska for their love and support and to Charlie for always believing in me. 4 Table of Contents TABLE OF CONTENTS DECLARATION ..................................................................................................................... 2 ABSTRACT……. ..................................................................................................................... 3 ACKNOWLEDGEMENTS ........................................................................................................ 4 TABLE OF CONTENTS ........................................................................................................... 5 TABLE OF FIGURES .............................................................................................................. 9 TABLE OF TABLES .............................................................................................................. 11 LIST OF ABBREVIATIONS .................................................................................................... 12 CHAPTER 1: GENERAL INTRODUCTION ............................................................................ 14 Molecular pharming ................................................................................................................14 Plants as expression system ..................................................................................................... 14 Plant suspension cultures ......................................................................................................... 16 Stable- vs. transient-expression ................................................................................................ 17 Viral vectors used for VLP production ......................................................................................18 Virus-like particles as vaccines .................................................................................................22 Nervous necrosis virus .............................................................................................................24 Poliovirus.................................................................................................................................26 200 years of polio history ......................................................................................................... 28 Need for new vaccines .............................................................................................................. 31 Aims of this thesis ...................................................................................................................34 CHAPTER 2: MATERIALS AND METHODS ......................................................................... 35 Media, buffers and solutions ...................................................................................................35 Molecular cloning techniques ..................................................................................................36 Plasmid construction ................................................................................................................ 36 DNA/plasmid isolation and purification ................................................................................... 36 Restriction digest ...................................................................................................................... 37 DNA ligation .............................................................................................................................. 37 Polymerase chain reaction (PCR) .............................................................................................. 37 Agarose gel electrophoresis ..................................................................................................... 38 DNA sequencing ........................................................................................................................ 38 Site-directed mutagenesis (SDM) ............................................................................................. 39 5 Table of Contents Growth, storage and transformation of competent cells ......................................................... 39 Plasmids used ........................................................................................................................... 39 Transient expression of proteins in Nicotiana bethamiana .....................................................40 Plant growth conditions ............................................................................................................ 40 Agroinfiltration ......................................................................................................................... 40 Protein extraction ..................................................................................................................... 40 Transient
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