GENOME-WIDE HOST GENE EXPRESSION ANALYSIS BEFORE AND AFTER THE INITIATION OF HIGHLY ACTIVE ANTIRETROVIRAL THERAPY AND NATURAL CONTROL OF HIV IN THERAPY NAÏVE HIV+ NON-PROGRESSORS The University of Sydney Viviane Nascimento da Conceição A thesis submitted for fulfilment of the requirements for the degree of Doctor of Philosophy, 2013. Faculty of Medicine, The University of Sydney, Sydney, NSW, Australia. Preface The work described in this thesis was performed by the author under the supervision of Dr Nitin K Saksena and Dr Bin Wang at the Centre for Virus Research, Westmead Millennium Institute, Westmead, NSW, Australia. These studies were completed between March 2009 and March 2013 to fulfil the requirements for the degree of Doctor of Philosophy in the Faculty of Medicine, The University of Sydney, NSW, Australia. This study was approved by the Sydney West Area Health Services Research Ethics Committee. None of this work has been submitted previously for the purpose of obtaining another degree, and the author is entirely responsible for the work in this thesis unless otherwise stated. Signed: _________________________ Viviane Nascimento da Conceição, 30th August, 2013 i The University of Sydney Acknowledgements I wish to thank first and foremost my supervisor, Dr Nitin Saksena, who without even knowing who I was, believed in me and brought me to Sydney, Australia. He made this dissertation possible with his guidance, support and help throughout my long PhD journey. Your strength is an inspiration to me and I really admire you for the successful professional that you are. I also would like to acknowledge my co-supervisor Dr Bin Wang, for his assistance with all my technical work; he was a great advisor during the course of my work, helping me with some laboratory techniques and making them look much easier. I cannot find words to express my gratitude to Dr Kaushal Gandhi with his incredible patience in teaching me statistical analysis; without him I would not have come so far. I also owe an enormous gratitude to Dr Monica Miranda-Saksena for all the time and effort she has spent with me. I became the “trouble” student she never had, and she devoted so much of her precious time to me, in order to get the perfect pictures in confocal and DeltaVision microscopy. Thanks so much Mon!! Without your help, a lot of this work would not make it to these final stages. I cannot forget to thank all my special “team” of co-workers and fellow students, especially the ones who stayed with me until the end and those who eventually moved on. I could never forget Dr Katherine Lau, Dr Jingqin Wu, Maly Soedjono and Tara Sasse. Thank you guys for giving me a great welcome in the laboratory and in the city, and also for helping me in the beginning with the training I much needed!! You guys made my life so much easier!! Thank you, Dr Julie Zhou, for the help anytime I needed. And I have to say it was not only one time I asked for help, but you were always ready to advise me. Thanks for the talks as well, I really appreciate that!! My very special thanks go to the following people, for their advice, timely help and suggestions reading my work and providing technical help throughout my PhD studies: Dr Maggie Wang for flow cytometer, Dr Hong Yu in using the confocal laser scanning microscope and also the Deltavision microscope, Dr Laurence Cantrill in using the confocal microscope in the Children‟s Hospital, Dr Andrew Harman for the use of his Interferon primers in some of my PCR experiments, Dr Russel Diefenbach and Dr Barbara Kelly in using their equipment for Western Blot. A special thanks to my fellow PC3 friends: Kabo Matlho, thank you for the long hours late at night, you always helped me whenever you could, especially processing patient samples; Priyanka Gupta, thanks for being so friendly and helpful and for the time it took you to help me with PCR when I was away, and also for reading my work; Amber Campbell, Manu Pokharna and Josh Givorshner for the help with Western Blot even though it didn‟t work - you guys were the best in trying with me, even being very busy with your work. Thank you so much!! Raany Rahme, thank you for your friendship, always having a nice thing to say to me and listening to my problems; Suneth ii The University of Sydney “Sunnybear” Perera, you stayed with me until the end and it was so much easier to “suffer” the writing process with you. Thanks for the snacks during the long nights in the end!! With you guys, I had the best time I could ever have asked for. Thank you all for the long PC3 hours with the jokes and talks and the after hour help whenever I needed. I would also like to acknowledge our collaborators: Dr. Wayne Dyer (Australian Red Cross) for the frozen PBMCs for this work, to Professor Dominic Dwyer and Dr Choo Beng Chew, for all the samples from HIV patients. A very special acknowledgement goes to all the study participants who agreed to give blood for these studies. If I have forgotten anybody, I just wish to say that you are not forgotten. A big thank you to all who participated in helping me and enabled me to pursue my PhD studies. Without the mention of Endeavour International Postgraduate Research Scholarship (EIPRS) from the University of Sydney, Top-up Grant from the Millennium Foundation, this work would not have finished. I owe my gratitude to them. I also want to thank my dear friends in Brazil, who always supported me even though is hard to be so far away - you guys never made me guilty for coming to Australia. Thank you for all your love and faith in me! My heartfelt thanks go to my „aussie‟ family: Erika, Shirlley, Indinna, Ranni - you guys made my life so much better. Without your support I would not have made it this far. To my flatmate and great friend Jeana, who put up with my mood changes and the fact that I was never around, thank you for always being there for me. You guys were rock solid in supporting me throughout!! Finally, my utmost acknowledgement goes to my own beloved family, without whom all this would not have taken place. Your support and love were the pillars of my strength and inspiration I needed to finish this long journey. My very loving Mum and Dad, who always supported me in everything I did and taught me to believe that I could do anything. Thank you for all the talks and encouragement over the phone. To my loving brother and sister, I owe you a special “thank you” for not stopping to believe in me, even when I doubted myself. A special consideration to my big sis, Fabs, for her assistance in reviewing my thesis, you did a great job, thank you so much for your time. The end to this great journey could not have been achieved without your undoubting love, faith and encouragement. iii The University of Sydney Abstract Since its discovery 25 years ago, the HIV virus has infected more than 34 million people in the world; the infection caused by this virus has led the world to its most terrible pandemic, and the greatest global health crises of our times. The host-virus interaction and natural history of the disease are influenced by the distinctive interface the virus has with each infected individual. The infection with HIV-1 is characterized by the destruction of CD4+ T-cells during the typical course of the infection, but HIV has the arsenal to infect practically all the major blood leukocytes. Taken from these observations, it is apparent that HIV has the innate ability to subvert and manipulate the host gene machinery at the transcriptomic level. The highly active anti-retroviral therapy (HAART) consists of a combination of powerful drugs, which serve as potent defence mechanism against the ways in which the HIV virus attacks the human body. Although these drugs are not able to rid the body of HIV virus, they can significantly delay the onset of AIDS and reduce the incidence of opportunistic infections, morbidity and mortality related to HIV infection. After the introduction of HAART treatment, it is observed that most patients with good adherence respond to HAART, which is defined by a decrease of plasma viral load to undetectable levels and an immune reconstitution with a significant increase of CD4+ T cell levels. Around 30% of the patients fail to achieve this response and continue to express high plasma viral load and low CD4+ T cell numbers. In contrast, some rare HIV+ patients maintain below detectable levels of plasma viremia without the treatment. These are termed long-term non- progressors (LTNPs), less commonly called elite controllers. These rare individuals are infected with HIV, but have the natural ability to control the infection with the strength of their immune system. Many of these patients have been HIV positive for 30 years or more and off therapy for the entire duration of their infection, showing high CD4+T cells counts and no progression to the disease. In this context, it is important to mention that the genomic basis of this natural effective immunological control of viremia in LTNPs, as opposed to drug-mediated control of HIV, remains unknown. The development of high throughput microarray platforms and bioinformatic platforms to visualize and analyse the complex dataset has enabled considerable progress in the field of viral genomics, and also the visualization of host-virus interactions at the molecular level.