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Structural Presentation of Influenza Epitopes on Modular Virus-Like Particles Doan Thanh Tam Master of Biotechnology

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Structural Presentation of Influenza Epitopes on Modular Virus-Like Particles Doan Thanh Tam Master of Biotechnology Structural presentation of influenza epitopes on modular virus-like particles Doan Thanh Tam Master of Biotechnology A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Australian Institute for Bioengineering and Nanotechnology i Abstract Vaccine development has increasingly developed toward subunit vaccine approach which uses highly-purified and well-characterised components, such as antigenic peptides, to elicit an immune response. An antigenic peptide requires a protein carrier or platform e.g. virus-like particle (VLP), to improve its poor immunogenicity. Based on this approach, murine polyomavirus (MuPyV) VLP and its subunit (capsomere) have been employed as vaccine platforms to present heterogeneous epitopes or antigenic elements from targeted pathogens. The design rule for epitope presentation on VLPs still remains unclear. A tandem repeat display strategy, in which antigenic element was repetitively arrayed, led to the induction of a significant antibody titer against its native structure, compared with the use of flanking sequence. Tandem repeat display strategy might retain the epitope conformation that is close to its native structure. This work further investigates the structural presentation of Helix 190 (H190) and Helix A (HA2A), from influenza hemagglutinin, using tandem repeat strategy. Different tools are exploited to characterise the structures of presented epitopes. The major outcomes of this research are: (i) demonstrating the limitation of phage antibody panning as a tool to inform the structural presentation of epitope on modular VLPs; (ii) developing a simple tool for rapid screening of presented epitope conformation using circular dichroism spectroscopy coupled with singular value decomposition algorithms (CD-SVD); (iii) demonstrating the capability of tandem repeat strategy in promoting the helicity of the helical structure; (iv) establishing initial steps in determining atomic structure of presented epitope using X-ray crystallography. This work introduces, to the best of our knowledge, a CD-SVD method, a simple and rapid tool to characterise epitope conformation, particularly helical structure, on modular capsomeres. Due to the simplicity and speed, it enables the CD-SVD method to be employed as a screening tool for the structural presentation of antigenic modules. It allows the rapid identification of potential vaccine constructs in the early stage of vaccine development and therefore, reduces the further cost of pre-clinical and clinical trials. This work also demonstrates the structural presentation of HA2A using tandem repeat display strategy led to induce antibody titer against full-length hemagglutinin in the chicken trial. The finding reveals that a tandem repeat strategy promotes the helicity of inserts derived from native helical regions; this approach deserves further research into its potential as a generic design for the presentation of the helical antigenic elements on modular VLPs. ii .Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Publications during candidature Conference abstract Tam T. Doan, Nani Wibowo, Linda H.L. Lua, Anton P.J. Middelberg* (2014). Structural presentation of an influenza antigenic element on the virus-like particle. 5th International NanoBio Conference. 6-10 July 2014. Brisbane, Australia. *Corresponding author Publications included in this thesis None. iv Contributions by others to the thesis This PhD project was performed by the candidate under the guidance of supervisory team including Prof. Linda Lua as a primary supervisor and Prof. Anton Middelberg as co-supervisors. This thesis was written by the candidate under the supervision of the supervisory team. Modular capsomeres simulations in chapter 4 were performed by Dr. Natalie Connors. Animal handling during in vivo immunogenicity study in chapter 4 was performed by the candidate with the assistance of Dr. Nani Wibowo, Jarurin Waneesom, Andrea Schaller and Dr. Natalie Connor. Technical supports in animal handling were provided by Dr. Arun Kumar in Poultry Research Unit, Gatton campus, The University of Queensland. The daily animals care the was performed by Vivek Guruswamy in Poultry Research Unit, Gatton campus, The University of Queensland. The screening of crystallisation conditions in chapter 5 was performed in collaboration with Dr. Santosh Panjikar from the Beamline team of Macromolecular Crystallography (Protein crystallography), the Australian Synchrotron (Melbourne, Australia). All of the data and results presented in the figures and tables of this thesis are solely the work of the candidate with the exception of the following figures and table in which the data collected with collaborators: • Figure 5.18: Protein crystallisation screening was performed by Dr. Santosh Panjikar. • Figure 5.19: X-ray diffraction was performed by Dr. Santosh Panjikar. • Table 5.5: Collection of X-ray diffraction was performed by Dr. Santosh Panjikar. Statement of parts of the thesis submitted to qualify for the award of another degree None. v Acknowledgements I would like to thank and express my gratitude to my principal supervisor Prof. Linda Lua and my co-supervisor Prof. Anton Middelberg for their valuable guidance and encouragement during my PhD A special thanks to my former principal supervisor, Dr. Nani Wibowo, for her mentorship and helpful comments and suggestions. Thank you very much Prof. Linda Lua, Prof. Anton Middelberg and Dr. Nani Wibowo for your support during my pregnancy. I would like to acknowledge The University of Queensland for providing me The University of Queensland International (UQI) Scholarship including tuition fee and living allowance during my study. Thanks to all the CBE members for their support and friendship. Thanks to all the members of Protein Expression Facility (PEF) who provided me helpful technical support. A special thanks to Prof. Steve Mahler and Dr. Martina Jones for your technical support and useful suggestions during my work with phage antibody panning in Ritchie Lab. Thanks to all my friends in Australia for your friendship and encouragement. Finally, a very big thanks to my family. Cám ơn ba mẹ đã vất vả cực nhọc dạy dỗ và nuôi con ăn học cho đến ngày học nay. Những thành tựu mà con đạt được đều nhờ vào công sức quý báu của ba mẹ. Em xin cám ơn anh, người bạn đời của em là chồng em, Lê Lâm Đức Kế, người đã cùng em chia sẽ biết bao nhiêu khó khăn vất vả trong cuộc sống. Xin cám ơn anh trong những đêm khuya hay những sáng sớm giá lạnh mùa đông đã không ngần ngại đưa đón em đến lab. Mọi lúc em cần anh đều bên cạnh em hỗ trợ động viên em. Without your support and encouragement, I would not be able to fulfill my PhD degree. vi Keywords Virus-like particle, influenza, peptide antigen, structural presentation, vaccine design Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 860801, Human Biological Preventives (e.g. Vaccines), 10% ANZSRC code: 100403, Medical Molecular Engineering of Nucleic Acids and Proteins, 50% ANZSRC code: 090403, Chemical Engineering Design, 40% Fields of Research (FoR) Classification FoR code: 0904, Chemical Engineering, 60% FoR code: 1004, Medical Biotechnology, 40% vii Table of Contents ABSTRACT ...................................................................................................................................... ii DECLARATION BY AUTHOR ..................................................................................................... iii PUBLICATION DURING CANDIDATURE .................................................................................. iv PUBLICATION INCLUDED IN THIS THESIS ............................................................................. iv CONTRIBUTIONS BY OTHERS TO THE THESIS ....................................................................... v STATEMENT OF PARTS OF THE THESIS SUBMITTED TO QUALIFY FOR THE AWARD OF ANOTHER DEGREE .................................................................................................................
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