Microbiology & Immunology - Theses
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ItemIdentifying novel compounds to eliminate latent HIV infection( 2019)There are currently an estimated 36.9 million people living with human immunodeficiency virus (HIV) (PLWH) worldwide. In the past few decades, the advent of antiretroviral therapy (ART) has significantly reduced the number of deaths associated with this virus. However, ART is not curative. The persistence of HIV latently infected CD4+ T-cells presents the major barrier towards a cure for HIV. Latently infected T-cells are formed when the virus integrates into the host genome of infected cells without ensuing productive infection. Due to these latently infected cells, viral gene expression and production infection rebounds from the integrated viral DNA if ART is ceased. Thus, ART must currently be taken life-long, posing a tremendous economic burden. The “shock and kill” approach is an extensively studied cure strategy that involves the use of pharmacological agents termed latency reversing agents (LRAs) to reactivate or “shock” the latent virus to express viral RNA and proteins. Following the reactivation of latently infected cells, the production of HIV proteins and viral particles was proposed to result in the elimination of these cells through immune-mediated clearance or cytopathic events. Results from clinical trials that involve a single LRA to reactivate latently infected cells in PLWH have not yielded any significant impact on the HIV DNA reservoir. This can be attributed to a number of different reasons that include the potency of the LRAs to reactivate latency, the failure to elicit an effective immune response and the inhibition of T-cell clearance by cytopathic viral proteins. There is clearly a need for more potent LRAs as well as novel strategies that will result in the clearance of these latently infected cells once reactivated. In this thesis, we investigate several novel pro-apoptotic compounds in isolation as well as in combination with LRAs to clear latently infected cells. We have also developed two new methods in which to study the effects of LRAs and pro-apoptotic drugs on latently infected cells. Dual-fluorescent reporter viruses have proven to be useful tools in studying latent HIV infection in vitro. Here we have modified a dual-fluorescent reporter HIV aiming to improve its functional characteristics in a pre-activation model of HIV latency. The new virus termed, DuoAdvance, contains two fluorescent viral reporters: a latent GFP reporter driven by elongation factor 1-alpha (EF1-alpha) and a productive E2 Crimson reporter driven by the HIV long terminal repeat (LTR) (Chapter 2). Using DuoAdvance, we demonstrate that DuoAdvance can successfully infected Jurkat T-cell lines. In a pre-activation model of HIV latency in primary resting CD4+ T-cells, DuoAdvance infection resulted in little to no latent GFP expression. Subsequent analysis of the GFP negative population of cells revealed DuoAdvance infection can result in the production of latently infected cells carrying latent provirus but the expression of the GFP latency reporter was perturbed. Due to the partial expression of this GFP latent reporter in primary resting T-cells, DuoAdvance is limited to use in dividing T-cell lines and potentially a post-activation model of HIV latency using activated CD4+ T-cells, where better expression of the GFP latency and E2 Crimson reporters were seen. Latency reversing agents can reactivate latent HIV but the effects on decreasing HIV DNA in PLWH has been less encouraging. In this thesis, we examine the effects of different pro-apoptotic drugs combined with different LRAs on decreasing HIV DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. Here we tested a number of LRAs together with several phosphoinositide-3 kinase (PI3K) inhibitors: IPI-443, IPI-3063 and wortmannin, as well as a B-cell lymphoma-2 (Bcl-2) inhibitor venetoclax as our pro-apoptotic drugs. The LRA romidepsin combined with all pro-apoptotic drugs resulted in synergistic decreases in the levels of integrated HIV DNA in the PLWH CD4+ T-cells ex vivo (Chapter 3). Additionally, several other LRA and pro-apoptotic combinations also decreased integrated HIV DNA in CD4+ T-cells ex vivo. All drugs were able to induce HIV viral transcription. Interestingly, we show that the pro-apoptotic drugs alone also led to an increase in HIV transcription and a decrease in HIV DNA. These data demonstrated the select combinations of pro-apoptotic drugs and LRAs together or pro-apoptotic drugs alone can result in a decrease in HIV integrated DNA in CD4+ T-cells from PLWH on ART ex vivo. However, we were unable to distinguish if there was selective death of the reactivated latently infected cells with minimal impacts on uninfected T-cells also in the cell cultures. In order to explore this, we developed a new approach to detect selective cell death (Chapter 4). This method involves the use of PrimeFlow, a HIV RNA in situ hybridisation method combined with branched-DNA technology, together with a cell death stain and analysis of stained cells using flow cytometry. Using this approach, we were able to demonstrate selective cell death in ACH2 T-cell lines treated with a combination of the PMA LRA, and venetoclax or IPI-443 PI3K inhibitor pro-apoptotic drug in a latently infected T-cell line. However, due to the elaborate staining procedure and large cell loss from the multi-step staining procedure, further investigation is required to move this staining approach into testing these drugs upon inducing the selective death of latently infected CD4+ T-cells from PLWH ex vivo. In summary, we have developed two new methods to investigate the effects of LRAs and/or pro-apoptotic drugs on HIV latency. Although further work is required to optimise these methods for use of the novel DuoAdvance fluorescent reporter virus with primary resting CD4+ T-cells for drug testing, or for use of the novel PrimeFlow assay to study the selective impact of these drugs upon latently infected CD4+ T-cell samples from PLWH ex vivo. Most importantly, our work demonstrates novel combinations of pro-apoptotic drug and LRA combinations that can decrease HIV integrated DNA in cultures of CD4+ T-cells from PLWH on ART ex vivo. This has important therapeutic implications for using these drug combinations to deplete latently infected cells in PLWH on ART and additional studies that investigate these combinations in a clinical setting is warranted. In conclusion, our work demonstrates that latency reversal combined with a drug-based strategy to promote apoptosis can eliminate HIV latently infected CD4+ T-cells from PLWH on ART ex vivo and thus this approach holds important potential to lead to HIV remission off ART in PLWH.
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ItemPathways to HIV latency and reactivation in vitro( 2018)Human immunodeficiency virus (HIV) infection remains a major global health issue. Antiretroviral drugs improve life expectancy and significantly reduce the rate of viral transmission; however, we are far from finding a cure for HIV. The major barrier to finding a cure is the persistence of the replication-competent yet transcriptionally silent latent reservoir. Current latency reversal agents (LRA) lack efficacy to eliminate all the latent proviruses from the reservoir. The response to the same LRAs is varied in latently infected cells ex vivo or in vitro. We hypothesised that HIV could generate different populations of latently infected cells that differ in HIV integration sites and response to reactivation by LRAs. We used a Nef-competent EGFP reporter virus to generate infection and to determine the latently infected cells in chemokine-treated CD4+ T cells in vitro. We first demonstrated that EGFP expression is dependent on viral integration and can be used to determine productively expressed and latently induced infected cells in our culture system. Infection and latency were established in both resting untreated and CCL19-treated CD4+ T cells in vitro. Addition of integrase inhibitor, raltegravir, at time of infection reduced the levels of EGFP expression in both T cell conditions, providing evidence that in our culture system EGFP expression is dependent on viral integration. There was a 4-fold reduction in EGFP expression in the CCL19-treated compared to the matched resting untreated cells. The reduction in the EGFP expression following addition of integrase inhibitor strongly suggested that incubating CD4+ T cells with CCL19 favors viral integration in vitro. We subsequently showed that the addition of IL-7 significantly increases the levels of latency in the chemokine-treated CD4+ T cells. Thus, we clearly showed that both resting and chemokine-treated CD4+ T cells are permissive to direct infection with HIV in vitro. However, the effect of CCL19 in the induction of latency is more pronounced with the addition of IL-7. We further asked whether the establishment of latency affects the response to reactivation by LRAs or T cell receptor (TCR) signalling. We used resting CD4+ T cells to establish infection in the pre-activation pathway and used activated T cells as a model for the establishment of infection in the post-activation pathway. Co-culturing EGFP- cells with allogeneic monocytes alone or in combination with an antibody against CD3 (aCD3); we showed a significant increase in EGFP expression from latently infected cells in the pre-activation latency model. Response to allogeneic monocytes in combination with signals derived from aCD3 significantly correlated with T cell proliferation and there was a minimal spontaneous EGFP expression from latently infected cells in this culture. In contrast, allogeneic monocytes alone or in combination with aCD3 reduced the EGFP expression from latently infected cells in the post-activation latency model. There was no correlation between T cell proliferation and viral expression. The level of spontaneous EGFP expression from latently infected cells was high, and the inhibition of EGFP expression by monocytes was dependent on the direct contact between monocytes and T cells. We further showed that the interaction between T cells and monocytes at time of infection induced spontaneous expression, providing evidence that monocyte-T cell interaction at an early time post infection maintains latency in activated T cells. By direct comparison of pre- and post-activation latency in vitro we, therefore, demonstrated that effective strategies to reverse latency would depend on how latency is established. We further profiled the HIV integration sites in pre- and post-activation latency models and showed a significant enrichment of the sites in genic, exon and intron; in sense direction in the introns of pre-activation latency compared to the post-activation models, suggesting preferential integration of proviral DNA in these locations. By indexing genes with integration sites with gene expression available for these genes in GEO dataset using RNA-Seq analysis, we found a set of genes that are not expressed during activation of T cells in response to TCR stimulation. This observation was found across all T cell subsets in the GEO datasets and suggests there is a common mechanism in T cells that allows for viral entry and integration in non-expressing genes. Our study has clearly shown that how latency is established is a critical factor affecting how latency is maintained or reversed in response to LRAs. Understanding the relationship between chromatin status of the genes that are never expressed during activation of T cells and establishment of infection or latency is of interest for designing strategies to induce the expression from latency or to permanently silence the virus.