Microbiology & Immunology - Theses
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ItemTargeting the untargetable: Eliminating HIV latency using nanoparticle delivery systems( 2023-04)T cells form an important therapeutic target for the development of immunotherapies or the treatment of T cell-implicated pathologies. Specifically, CD4 T cells are subject to human immunodeficiency virus (HIV) infection. Whilst treatment with antiretroviral therapy (ART) successfully represses the viral load to undetectable levels, HIV treatment is lifelong, posing a medical, social and financial burden to those 38.5 million people currently living with HIV globally. The ability of HIV to establish a reservoir of latently infected cells is the foremost barrier to finding a cure for HIV. One approach towards eliminating HIV latency is the reactivation of viral transcription and subsequent elimination of infected cells through immune-mediated clearance or viral-mediated cytotoxicity. However, this approach so far has suffered from a lack of potency and dose-limiting toxicities due to the use of compounds that affect both host and viral transcription and the inability to specifically target the infected cells. One solution involves the use of nanoparticles for the targeted delivery of existing therapeutics or to advance the development of HIV-specific mRNA-based therapeutics including CRISPR-Cas. However, the generally low rate of endocytosis in CD4+ T cells forms a challenge to efficient nanoparticle-based drug delivery to CD4+ T cells in vitro and in vivo. This thesis describes our efforts towards rationally designing a nanoparticle platform capable of delivering HIV latency-reversing therapeutics to CD4+ T cells with high efficiency. We first established a methodology to improve the assessment of nanoparticle performance in vitro through the generation of absolutely quantitative, comparable data on nanoparticle-cell interactions. We then used this methodology to screen for nanoparticle designs with enhanced uptake kinetics in CD4+ T cells in vitro, using a novel, high-throughput assay to quantify nanoparticle internalization over time. We found that targeting sub-100 nm nanoparticles to T cell surface receptors undergoing rapid receptor cycling can be exploited to actively trigger nanoparticle uptake through receptor-mediated endocytosis and identified CD2 and CD7 as potent candidate receptors for future in vivo T cell targeting. We next aimed to use translate these findings to lipid nanoparticles, a well-established platform for the delivery of nucleic acid-based therapeutics. We investigated whether lipid nanoparticles could be used to deliver a next-generation latency-reversing agent based on CRISPR activation, which specifically targets the HIV proviral genome without affecting host-cell transcription. We identified a novel lipid nanoparticle formulation that can efficiently transfect T cell lines as well as resting CD4+ T cells. We showed that this lipid nanoparticle can co-encapsulate the three RNA components of the CRISPR activation system and induce strong latency reversal in a cell line model for HIV latency. We finally presented preliminary evidence that targeting lipid nanoparticles to rapidly cycling surface receptors increases mRNA delivery, further supporting our findings that targeting receptor-mediated endocytosis could be a viable strategy to increase nanoparticle-mediated drug delivery to traditionally hard-to-transfect T cells. These findings provide a compelling justification for the further assessment of CRISPR activation lipid nanoparticles for the elimination of the latent HIV reservoir, and more broadly contribute to the development of T cell-targeted nanomedicine for HIV and beyond.
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ItemHIV persistence, inflammation and the gut( 2021)Human Immunodeficiency Virus (HIV) remains incurable despite antiretroviral therapy (ART) due to its persistence as integrated provirus within long-lived and proliferating CD4+ T cells. Following initial infection, it replicates efficiently within and depletes Th17 cells which are enriched within the gut and play an important role in maintaining gut barrier integrity. This depletion leads to gut barrier permeability, bacterial translocation across the gut wall and local and systemic inflammation. These findings do not completely resolve on ART and may contribute to HIV persistence by promoting proliferation of infected CD4+ T cells and/or exhaustion of an effective immune response against HIV. Integrated provirus is enriched within Th17 cells in people living with HIV on ART. Th17 cells express CCR6 which promotes their migration towards CCL20 secreted by gut epithelium. Our laboratory has previously shown that CCL20 can promote HIV latency establishment in vitro likely through depolymerisation of the cortical actin barrier of resting CD4+ T cells allowing HIV to traverse this barrier and migrate to the nucleus. It is unknown to what extent CCL20-mediated gut migration or actin depolymerisation might contribute to HIV persistence in vivo. We compared the half-maximal effective concentration (EC50) of CCL20 for HIV latency establishment within resting memory CD4+ T cells with that of AKT phosphorylation, polarisation (a surrogate for actin depolymerisation) and migration of these cells. We reasoned that if the EC50 for HIV latency establishment were considerably lower than the EC50 for any of these cellular processes then that cellular process may not be required for CCL20-induced HIV latency establishment. We found progressively increasing EC50s for AKT phosphorylation, polarisation and migration; however, we were unable to demonstrate an effect of CCL20 on latency establishment. This was independent of viral tropism. Discrepancy with our laboratory’s previous findings may have been due to a shorter duration of exposure to chemokine in our experiments and/or a shorter resting time between cell sorting and infection which may have influenced cellular activation state and hence permissiveness to infection independent of chemokine. Vitamin D3 is a steroid hormone with pleiotropic effects on the immune system including reductions in CD4+ and CD8+ T cell activation, proliferation and exhaustion and reductions in frequency of Th17 and Th1 cells, both of which are important HIV reservoirs. It has also been shown in animal models to promote gut barrier integrity and in humans to reduce gut dysbiosis. We hypothesised that vitamin D3 may be able to deplete the HIV reservoir through these systemic and gut anti-inflammatory effects. We performed a randomised placebo-controlled trial evaluating the effect of 10,000 international units vitamin D3 per day for 24 weeks on markers of HIV persistence and immunology. Participants were followed for an additional 12 weeks post treatment. The primary endpoint was the difference between treatment arms in the change in frequency of total HIV DNA within CD4+ T cells from baseline to week 24. We found no effect of vitamin D3 on the primary endpoint. However, we found an increase at week 12 and a decrease at week 36 in frequency of total HIV DNA in the vitamin D3 arm relative to placebo. Importantly, 25-hydroxyvitamin D levels were still elevated at week 36 in the vitamin D3 arm relative to the placebo arm likely due to its long half-life. We also found a shift away from more differentiated subsets towards central memory CD4+ and CD8+ T cells at all time points including a reduction in frequency of effector memory CD4+ T cells at week 36. Other findings included a reduction in frequency of Th1 cells and levels of monocyte activation as expected but a paradoxical increase in frequency of Th17 cells and activated CD8+ T cells and NK cells in the vitamin D3 arm relative to the placebo arm. We hypothesise that the decrease in total HIV DNA frequency at week 36 may be due to the reduction in frequency of effector memory CD4+ T cells, known to be enriched in HIV DNA, which in turn may reflect the known anti-proliferative activity of vitamin D3. Increases in CD8+ T cell and NK cell activation may also have contributed to depletion of the HIV reservoir at week 36. Our findings support larger clinical trials which could incorporate an analytical treatment interruption to determine whether vitamin D3 is able to exert a clinically meaningful impact on the HIV reservoir.
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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.