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Developing a Novel Hepatitis B Core – Based Antigen Presentation System

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Developing a Novel Hepatitis B Core – Based Antigen Presentation System Developing a novel hepatitis B core – based antigen presentation system by Hadrien Peyret 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 2014 © 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 therefrom must be in accordance with current UK Copyright Law. In addition, any quotation of extract must include full attribution. 1 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. Hadrien Peyret ii Abstract Abstract Plant-produced proteins of pharmaceutical interest are beginning to reach the market, and the advantages of transient plant expression systems are gaining increasing recognition. In parallel, the use of virus-like particles (VLPs) has become standard in vaccine design. The pEAQ-HT vector derived from the cowpea mosaic virus – based CPMV-HT expression system has been shown to allow the production of large amounts of recombinant proteins, including VLPs, in Nicotiana benthamiana. Moreover, previous work demonstrated that a tandem fusion of the core antigen (HBcAg) of hepatitis B virus (HBV) could direct the formation of core-like particles (CLPs) in plants. The work presented here demonstrated that the tandem core system is better suited for the plant-based production of CLPs presenting foreign antigens than SplitCore technology. It was shown that tandem core technology allows the plant-based production of CLPs which are suitable for the presentation of antigens either via chemical coupling or through antibody- antigen interactions. Of particular significance was the successful display of single domain antibody fragments of camelid origin (nanobodies, or VHH). The resulting “tandibody” particles, as they are named here, can bind to their cognate antigen to yield CLPs covered in the antigen of interest. Furthermore, it was shown that target antigens can be attached to CLPs via a fusion partner, raising the possibility of the development of a universal generic antigen-display platform. In addition to the transient expression work on HBcAg, lines of stably transformed N. benthamiana were created which constitutively expressed human gastric lipase (hGL), an enzyme of medical importance. In the future, this approach could be used to create transgenic plants constitutively expressing a universal tandibody, thus obviating the need for infiltration. iii Acknowledgements Acknowledgements First and foremost, I would like to thank my supervisor, George P. Lomonossoff, for being much more than a supervisor. I could not have done any of this without his encouragement, insight, enthusiasm, and advice. George has given me the freedom to explore new paths, try new things, gain new skills, and put myself forward in the scientific community. Of equal importance, he has given me the freedom to fail, which has given me invaluable experience as a scientist. I hope I never forget the lessons that I learned from George; from the importance of planning experiments properly, to the importance of drinking at conferences. I would also like to thank my secondary supervisor, Alison Smith, for her helpful insight and reassurance. Pooja Saxena also deserves a big thank you for being the first person to tell me about all the cool things that happen in the Lomonossoff lab, and for supervising me during my rotation. And speaking of rotations, I would like to thank Nick Brewin, Jonathan Jones, Alexandre Robert-Seilaniantz, Michael McArthur, and all of the rotation students for giving me the opportunity to make an informed decision about what to do with my life. I have been hugely fortunate to work in such a fun lab, and I want to thank all Lomonossoff group members past and present, as well as the members of the Biological Chemistry department, for creating such a lovely environment, both during coffee breaks and at the bench. Of particular importance in this regard is Keith Saunders, who was, on a daily basis, a friendly, supportive and helpful guide as I walked in the shadow of the valley of data. I am also hugely grateful to Eva Thuenemann, for all of her help and advice, as well as for writing the bible. I also wish to thank all of my collaborators, Dave Rowlands, Nicola Stonehouse, Clare Stevenson, and Robert Gilbert. Of course, I also acknowledge all those professionals without whom my job would have been much harder: Andrew Davis, Gerhard Saalbach, Sarah Tolland, Kim Findlay, Elaine Barclay, and the staff in the media kitchen and the glasshouse. Last but not least, special thanks go to my family and Lucie for all of their love and support. iv Table of Contents Table of Contents Declaration ................................................................................................................................. ii Abstract ..................................................................................................................................... iii Acknowledgements ................................................................................................................... iv Table of Contents ....................................................................................................................... v List of Figures .......................................................................................................................... viii Abbreviations ............................................................................................................................. x Chapter 1 : General Introduction .................................................................................................. 1 I - Plant molecular farming........................................................................................................ 1 II - The CPMV-HT system and the pEAQ vectors ....................................................................... 8 III - Virus-like particles (VLPs) .................................................................................................. 15 IV - Antigen display ................................................................................................................. 20 V - The HBcAg CLP as an antigen carrier ................................................................................. 23 A) N-terminal fusions .......................................................................................................... 25 B) C-terminal fusions ........................................................................................................... 26 C) Insertions in the e1 loop ................................................................................................. 28 D) Other insertion strategies .............................................................................................. 30 E) Other antigen carriers and the advantages of HBcAg .................................................... 33 VI - Aims of this thesis ............................................................................................................. 35 Chapter 2 : Materials and Methods ............................................................................................ 36 I - DNA constructs ................................................................................................................... 36 II - DNA isolation/purification ................................................................................................. 36 III - Restriction digests ............................................................................................................. 36 IV - Ligation ............................................................................................................................. 36 V - Media ................................................................................................................................. 37 VI - Polymerase Chain Reaction (PCR) .................................................................................... 39 VII - Agarose gel electrophoresis ............................................................................................ 40 VIII - DNA sequencing.............................................................................................................. 41 IX - Bacterial strains, growth and storage conditions ............................................................. 41 X - Transformation of competent bacterial cells .................................................................... 42 XI - Plasmids used.................................................................................................................... 42 XII - Agroinfiltration................................................................................................................. 43 v Table of Contents XIII - Plant growth conditions .................................................................................................. 43 XIV - Small-scale protein extraction ........................................................................................ 43 XV - Large-scale protein extraction ........................................................................................
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