Medical Biology - Theses
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ItemAdvancing a functional cure for HIV by identifying therapeutics that promote the death of latently infected cells( 2020)The persistence of a replication-competent HIV reservoir necessitates life-long antiretroviral adherence and precludes the possibility of a HIV cure via conventional therapy alone. Furthermore, recent clinical studies have made it increasingly clear that the predominant strategy for reservoir elimination, enforced transcriptional reactivation, does not diminish the size of the latent reservoir or reduce the time to viral rebound following treatment interruption. A novel approach seeks to purge the HIV reservoir by activating apoptotic pathways in latently infected cells and shifting the balance away from survival and towards cell death. Several lines of evidence implicate Bcl-2 family proteins in the long-term survival of memory CD4+ T cells – the major reservoir for HIV. Bcl-2 antagonism thus represents a viable strategy for sensitizing latent cells to death and delaying viral rebound. The development and clinical progression of BH3-mimetics, which induce apoptosis by binding pro-survival Bcl-2 homologs, has resulted in a well- characterised class of inhibitors with relatively few unknowns regarding toxicity, side effects and dosage. In this thesis, I hypothesise that there are apoptotic blocks in place, specifically a greater dependence on pro-survival Bcl-2 proteins, which prevent a minority of infected CD4+ T cells from dying during active infection. I hypothesise that latently infected cells are distinct from other infected or healthy cells, and that this pro-survival phenotype allows them to persist in such a way that renders them susceptible to pro- apoptotic therapeutics which target the intrinsic pathway, such as BH3-mimetics. In Chapter 3, I infect primary human CD4+ T cells with HIV in vitro to assess the ability of BH3-mimetics to kill actively infected cells. I demonstrate that ABT-737 and Venetoclax, but not the Mcl-1 inhibitor S63845, preferentially kill activated, HIV infected CD4+ T cells in the setting of productive viral replication. These results shed light on the pro-survival role of Bcl-2 proteins during active HIV infection, and inform our progression into a preclinical model of HIV latency. Chapter 4 uses a humanized mouse model of HIV latency to further interrogate the importance of Bcl-2 pro-survival proteins in reservoir survival. I investigate the ability of Venetoclax, a clinically-approved Bcl-2 antagonist, as well as S63845, a preclinical Mcl-1 inhibitor, to delay viral rebound following analytical treatment interruption. This work provides the first compelling evidence that BH3-mimetics, either as monotherapy or in combination, can eliminate latently infected cells in vivo. In Chapter 5 I perform a tat/rev Induced Limiting Dilution Assay (TILDA) on CD4+ T cells from latently infected mice in order to quantify the impact of Venetoclax on the magnitude of the latent HIV reservoir. I confirm the existence of an inducible reservoir in our mouse latency model, although I do not observe a significant effect of Venetoclax treatment as measured by TILDA. I also use single-cell RNA sequencing to characterize peripheral CD4+ T cells from ART-suppressed human donors following Venetoclax treatment ex vivo, arriving at the suggestion that Venetoclax may target CD4+ T cells that are enriched for a gene signature associated with activation and cell metabolism. This work lays the foundation for furthering our understanding of which cells may contribute to HIV persistence and which may be susceptible to death- inducing compounds. Overall, this thesis represents a comprehensive assessment of the ability of BH3-mimetics to kill HIV active and latently infected cells, offering a strong justification for the translation of pro-apoptotic therapeutics such as Venetoclax into a clinical setting where reservoir eradication is the goal.