THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTÉ UNIVERSITÉ GRENOBLE ALPES Spécialité : Virologie/Microbiologie/Immunologie Arrêté ministériel : 7 août 2006 Présentée par Deepanwita BOSE Thèse dirigée par Dr. Yahia Chebloune préparée au sein du Laboratoire Pathogénèse et Vaccination Lentivirales (PAVAL Lab) dans l'École Doctorale Chimie & Sciences du Vivant (ED218) Tat-independent lentivirus genomes for vaccination and host/pathogen interaction studies. Thèse soutenue publiquement le 26 Janvier 2017, devant le jury composé de : Dr. Giuseppe Bertoni Maitre de conférences, IVI, Bern, Suisse Rapporteur Pr. Francois Villinger Professeur, Director of New Iberia Research Center, USA, Rapporteur Pr. Philippe Sabatier Professeur à l’Université Grenoble Alpes, Président Dr.Yahia Chebloune Research Director, à l’Université Grenoble Alpes, Examinateur Dr. Corinne Ronfort Chargé de recherche, Université Lyon I, Examinateur i “Dedicated to my Mom, Dad and Brother” i Acknowledgements Acknowledgements This thesis in its current form is a result of support, assistance, collaboration, critical inputs and guidance of several people and thus, I would sincerely thank each one of them. Firstly, I would like to express my sincere gratitude to my mentor Dr. Yahia Chebloune for having his faith and confidence in me and accepting me in his research lab for doing a PhD. thesis. I am thankful to him for his support, encouragement and extraordinary guidance all through my graduate work. His insightful discussions and critical comments were an invaluable part in preparation of this thesis. I am grateful for his patience throughout the tribulations, errors, repeats and also the successes in the lab, the corrections in the redaction of the thesis. His mentorship greatly improved and enhanced my scientific grasp and outlook. I would like to thank the members of my Committee: Our collaborator Pr. François Villinger, Director of the New Iberia Research Center, for sharing his expertise in the field and providing invaluable time and suggestions with a lot of encouragement. I am also thankful to him for providing a platform for adapting the newly developed viruses that I developed in Grenoble. For performing these experiments and adapting the viruses, I would like to thank members of his team, specially Dr. Chunxia Zhao at the Yerkes National Primate Center, Margot Lavy at the New Iberia Research Center and other members of the lab working to adapt the viruses in vitro and in vivo. Dr. Giuseppe Bertoni, for his invaluable time and expertise in the field and for being part of jury and reporter of the thesis. Dr. Corinne Ronfort, for all her scientific wisdom providing valuable inputs during the thesis and for her patience, critical review and guidance throughout the correction of the thesis Pr. Phillipe Sabatier, BioHealth Computing Director, for his professional excellence in providing constructive criticism and valuable inputs into the thesis. Dr. Catherine Lemaire, for being part of my thesis committee and providing significant constructive criticisms, I would also like to thank her for allowing me free use of laboratory equipment in emergency situations. I also thank Salima, for her technical help during these visits to the LAPM, Jean Roget Institute. I am thankful to Dr. Jean Gagnon, for his expertise and unwavering kindness, with valuable suggestions throughout my graduate studies and for his precious input for the redaction of my paper and thesis. I am deeply grateful to my lab mates and colleagues Maha, Simaa and Dimitri for their help, motivation, support and encouragement during good and less good times. They were always around to discuss, and gave invaluable suggestions and technical help for which, I am more than grateful. They were a part of my day-to-day life in lab and I cannot imagine my three i Acknowledgements years of lab life without them. I want also to acknowledge the help of Takfarinas, who joined the lab recently for his help in English-French translation and his savviness in MS Word software. I am also thankful and appreciate all other current and past members of the PAVAL team including previous students Honorine, Mikael, Sara, Avika, Ivan, Amna, Nadia, Amandine, Margot, Vanessa, Kathia, and technician Abderrahim. Special thanks to Camille for repeating some experiments of this thesis during her internship. I also want to warmly thank Gour, Monalisa, Ila, Sudha, Vikas, Vinodh, Lakshman, Bharat from the Indian community in Grenoble for their homely, joyful gatherings with loads of food, fun and all their support and encouragements along with valuable inputs for living in the French system. I am thankful for the financial support that I benefited from the Presidential Scholarship, University Grenoble Alpes for the years 2013-2016. And I am thankful to my Parents and Brother in India for always being encouraging and supporting me in all my endeavors in life, and this thesis is dedicated to them. ii Abstract in English Tat-independent lentivirus genomes for vaccination and host/pathogen interaction studies. Abstract in English Our lab has previously described the generation of a unique vaccine prototype against HIV- 1/AIDS. It is a non-integrative lentivector DNA vaccine, tested in pilot studies in animal models of HIV vaccine. The non-human primate study showed protection of all 6/6 macaques and immune response correlates were composed of a variety of effector (EM) and central memory (CM) T cells. More importantly, they also contained high proliferating antigen specific cells containing a type of stem cell-like memory T cells (TSCM). Here in my thesis work, the specificity of the vaccine was enhanced further by switching the CXCR4 envelope of the vaccine to CCR5 tropic envelopes such as the clade B WARO obtained from a chronically infected patient and three transmitted/founder (T/F) HIV Clade C strains from Zambia. To improve the vaccine further, we developed new strategies to incorporate cytokine genes as molecular adjuvants able to enhance and sustain the newly elicited immune responses. Since HIV-1 has developed multiple complex strategies to persist, the focus of the next part of my thesis was to develop tools to ease and better understand the underlying mechanisms of latency in infected memory CD4+ T cells. Latently infected cells have integrated in their genomes the complete viral DNA sequences but viral genes are not expressed. One of the main mechanisms of this latency is the absence of Tat transactivation of the LTR promoter. A recent focus in the era of efficient highly active antiviral therapy (HAART) is the cure of infection by targeting the reservoir of latently infected cells. One of the obstacles faced, is a lack of primate lentivirus prototypes incapable of undergoing latency as an extreme infection model to allow for teasing apart the various mechanisms contributing to latency. We hypothesized that a replication-competent SHIV genome driven by Tat-independent constitutive-expression LTRs such as the one of the caprine arthritis encephalitis virus, CAEV, the natural lentivirus of goats, will be a valuable tool for such studies. We designed chimeric CAEV LTRs bearing terminal sequences of SIV at their extremities, and used them to drive the transcription of the complete genome of SHIV-KU2. The reverse transcription of such a genome generates double stranded viral DNA bearing SIV attachment sequences (att) used efficiently by SIV IN for provirus integration. The resulting construct termed SHIV- YCC is expected to generate a viral infection that will not undergo latency due to uncoupling from Tat activation. We found that cells transfected with the SHIV-YCC DNA produce SHIV proteins that assemble into infectious particles released from the producer cells. These virions are able to infect new target CD4+ T cells both in primary PBMCs and in cell lines. SHIV- YCC passaged in macaques PBMCs increased virus replication and infectivity. SHIV-YCC is the first chimeric primate replication-competent lentivirus that constitutively expresses all viral proteins. This new model offers the possibility of studying the early events by which provirus undergoes latency and will serve as backbone for the construction of chimeric SHIVs with T/F CCR5 tropic HIV-1 envelopes more representative of early infecting isolates. Another major focus of this thesis was the development of replication competent (Tat driven) SHIVs that also more closely represent new HIV infections in humans. There is a need for CCR5 tropic SHIVs that not only infect monkeys but also induce sustained viral load set- points and pathogenesis, as many of the current R5-tropic SHIVs tend to undergo spontaneous control in vivo. In an attempt to palliate this issue in this thesis, SHIV-AD-T/F-31 and SHIV- AD-T/F-18 were developed using the SHIV-AD8EO backbone, which is a recent clade B SHIV with promising replication kinetics in vivo. The tat-env region of SHIV-AD8EO was iii Abstract in English exchanged with those of two HIV-1 clade C isolates: Z331T/F and Z3618T/F. These CCR5- tropic SHIVs will also provide tools to determine the mechanisms of latency/persistence when used in parallel with CAEV LTR chimeric constructs that are independent of the Tat/TAR transactivation. In addition, the replicative SHIV may provide invaluable reagents for testing HIV vaccines and microbicide efficacy in the macaque models. Keywords : HIV/SIV ; Tat ; CAEV ; CCR5 SHIV iv Abstract in French Génomes de lentivirus Tat indépendants pour des études de vaccination et les interactions hôte / pathogène Abstract in French Notre laboratoire a développé un prototype vaccinal unique contre le VIH-1 responsable du SIDA chez l’homme. C'est un lentivecteur ADN non-intégratif qui a été testé dans des études pilotes utilisant des modèles animaux. Une desq études a montré la protection de tous les macaques (6/6) vaccinés et la réponse immunitaire qui corrèle avec cette protection était composée de cellules effectrices (EM) et des cellules T centrales mémoires (CM).