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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ItemUnderstanding the drivers of natural variation of transcription in HIV-1 latency( 2022)An estimated 37.7 million people are currently living with HIV-1, with 27.5 million of those taking antiretroviral therapy (ART) that inhibits viral replication to prevent disease progression and onward transmission. Upon infection, HIV-1 establishes a latent state of infection characterised by (near) silent viral transcription and translation to avoid immune recognition and clearance, further ensuring the virus’s survival within its host. This, together with integration of the viral genome into CD4+ T-cells (and less frequently into myeloid-derived leukocytes), creates a persistent HIV-1 reservoir. These latently infected cells can quickly and spontaneously reactivate to cause viral rebound within weeks of ART cessation. Therefore, ART is required life-long which poses significant economic needs on countries as well as financial, physical and mental health burdens on people living with HIV (PLHIV). The main goal of cure strategies is to reduce the size of the reservoir, allowing for a greater chance of immune mediated control in the absence of ART. One such strategy, the “shock and kill” approach, aims to reactivate latent proviruses to induce viral gene and protein expression and allow for elimination of infected cells by immune-mediated clearance or even direct killing by viral cytopathic effects. Clinical trials of latency reversing agents (LRAs) in PLHIV have so far demonstrated viral reactivation, though this has not resulted in meaningful reductions of the reservoir or delays to viral rebound after cessation of ART – highlighting a need for new, more potent LRAs to achieve a cure. In a prospective clinical trial of PLHIV on ART, we observed that cell-associated HIV-1 RNA - a measure of HIV-1 transcription - varied significantly on three separate occasions prior to any intervention. The variation in HIV-1 RNA was independently associated with time and visit, with higher HIV-1 RNA being measured earlier in the day. Circadian rhythms dictate numerous physiologic and behavioural changes over the 24-hour day and are entrained at a systemic level, as well as by cell-autonomous circadian molecular clocks. Such cell-autonomous circadian cycles consist of classical feedback loops driven by the major transcription factors, CLOCK and BMAL1, together with their repressors, Period and Cryptochrome. Indeed, the immune system’s activity displays circadian rhythmicity and various pathogens are known to modulate – or be modulated by – the host’s circadian cycles. In this research project, we investigated the temporal variation in HIV-1 in vivo, its source and whether this variation can be exploited to reverse latency. In a prospective observational study of virally suppressed male PLHIV on ART, we identified a circadian rhythmicity in cell-associated unspliced HIV-1 RNA and the HIV RNA-to-DNA ratio in vivo. HIV-1 DNA itself, however, remained stable over time – signifying that the circadian rhythms of HIV-1 RNA were due to variation in viral transcription or RNA clearance, rather than cell trafficking. Expression of core circadian genes, Clock, Bmal1, Period1-3, and Cryptochrome1-2 cycled in a circadian manner, indicating that PLHIV maintain intact cell-autonomous circadian cycles within peripheral CD4+ T-cells, despite chronic infection. Furthermore, there was a relationship between oestradiol’s circadian cycle and that of the HIV RNA-to-DNA ratio. We have therefore shown that cell-associated unspliced HIV-1 RNA has a circadian rhythmicity in vivo, contributing to the new paradigm that HIV-1 is not always completely latent. These observations could be leveraged for new interventions. Using the HIV-1-reporter cell line, J-Lat Tat-IRES-GFP clone A2, we next established a high-throughput assay to screen the latency-reversing potential of circadian-modulating compounds. We identified several compounds with acceptable toxicities that activated the HIV-1 long terminal repeat (LTR) promoter, including; the organic selenium compounds, methaneseleninic acid (MSA) and methylselenocysteine; the SIRT1 activator, resveratrol; and the nuclear import inhibitor, ivermectin. Of these, MSA exhibited the greatest increase in LTR activation with tolerable toxicities and was therefore characterised further. In the latently infected cell lines, J-Lat 10.6 and ACH2, MSA potently induced HIV-1 RNA and protein expression, as well as cell-associated unspliced HIV-1 RNA in primary CD4+ T-cells from PLHIV on ART ex vivo. Expression of the major circadian activator, Bmal1, was also increased by MSA, demonstrating that latency reversal was associated with perturbations to cell-autonomous circadian cycles. Additionally, MSA did not induce sustained cellular activation or proliferation. Together, this research identified a novel LRA that induced both viral and circadian gene transcription, in the absence of cellular activation or proliferation. Finally, using molecular techniques, we addressed the source of circadian variation in HIV-1 RNA and the association between circadian disruption and latency reversal. Co-expression of both Clock and Bmal1 transcription factors activated the HIV-1 LTR in vitro. By generating various LTR mutants, we identified that this activation was entirely dependent on a single E-box motif within the LTR recognised by CLOCK:BMAL1 heterodimers, indicating that cell-autonomous circadian cycles may directly interact with the integrated HIV-1 provirus to initiate transcription and contribute to the circadian rhythmicity of HIV-1 transcription observed in vivo. Collectively, this research has demonstrated circadian rhythmicity in HIV-1 transcription in vivo despite suppressive ART and the cell-autonomous circadian cycles of latently infected cells may directly activate their harboured provirus. These data highlight the dynamic nature of viral activity throughout the 24-hour day, and necessitate consideration when designing future clinical trials. Moreover, we demonstrated that cell-autonomous circadian clocks offer a novel, druggable pathway to target as part of the shock and kill approach to a cure for HIV-1.
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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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ItemCharacterisation of Neutralising and Functional Antibody Responses to Different HIV-1 Env Vaccines in Bovines( 2020)Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic hypermutation in germline genes compared to AD8 Unc gp140, with each animal presenting a unique antibody profile, and with germline antibodies already presenting high affinity towards HIV-1 Env trimers, as high levels of affinity maturation were not required to obtain antibodies with high neutralising activity. Overall, the results in this work show that open structured trimers elicit antibodies which highly activate antibody-effector functions, while SOSIP trimers focus antibody responses to concealed neutralising epitopes. The high neutralising responses observed in bovines against HIV-1 Env are due to antibodies which do not need high levels of somatic hypermutations and, in particular for KNH1/BG505 SOSIP, this antigen induced high levels of affinity maturation, probably favouring the improvement of both binding and neutralisation. Our study suggests that an effective vaccine regimen may include both uncleaved gp140 and SOSIP gp140, in order to target epitopes required for antibody-dependent effector functions as well as neutralisation, or a new trimeric structure with flexibility in the gp120-gp41 interface, exposing both epitopes involved in Fc-effector functions as well as neutralising ones.
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ItemFc-functional antibody immunity to HIV: the role of neutrophils and IgA( 2020)A safe and effective prophylactic vaccine against HIV-1 is an essential component to limit the HIV-1 epidemic. The RV144 HIV vaccine efficacy trial has highlighted the importance of generating Fc functional antibodies to prevent the further spread of HIV infection. Fc functional antibody responses have also been shown to correlate with delayed HIV disease progression. Despite the intensification of interest in Fc-mediated responses to HIV infection, there has been limited research focused on the Fc functional capacity of neutrophils, which are a key innate immune cell at mucosal surfaces and in the blood. The majority of Fc-effector studies in HIV focus upon examining NK cells and/or monocytes responses, while other effector cells such as neutrophils remain understudied. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses therefore, other immune cells like neutrophil are necessary for IgA to be studied. Neutrophils are highly functional innate effector cells with the potential to induce both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis. In chapter 2, methods were optimized to evaluate antibody-dependent neutrophil phagocytosis (ADNP) and neutrophil-mediated rapid fluorometric antibody-dependent cellular cytotoxicity (RFADCC) effector responses, using freshly isolated primary human neutrophils from blood. In vitro, neutrophil-mediated RFADCC responses peaked at 4 hours, which was faster than primary NK cells or monocyte mediated responses. There was a large spectrum of responses of both ADNP and neutrophil-mediated RFADCC responses across a cohort of 41 viremic antiretroviral-therapy naive HIV positive subjects. ADNP and RFADCC responses correlated well with each other, suggesting that they measure overlapping functions. The viral load of the patients inversely correlated with the ADNP responses, suggesting that these antibody-mediated neutrophil-based assays could prove useful in dissecting HIV-specific immunity. The role that IgA plays in active HIV infection remains controversial, with some reports of HIV-specific IgA being able to inhibit HIV infection and potentially being protective. Chapter 3 investigated if HIV progression was influenced by HIV-specific ADNP and neutrophil-mediated RFADCC responses and the effects of IgA on these responses. It was shown that, although neutrophil-mediated RFADCC responses were higher in the plasma of subjects who controlled their viremia levels (viremic controllers), IgA from both viremic controllers and viremic non-controllers inhibited both ADNP and neutrophil-mediated RFADCC responses similarly. The IgG mediated ADNP responses from both viremic controller and viremic non-controllers were broadly inhibited by both autologous HIV positive IgA and HIV negative pooled purified IgA. The IgA inhibition was able to be blocked by pretreating neutrophils with an Fc alpha receptor (FcalphaR) blocking antibody. This suggests that IgA inhibition of ADNP responses can be mediated by 2 mechanisms; 1) antigen dependent, FcalphaR independent and 2) antigen independent, FcalphaR dependent. The RV144 vaccine trial has generated interest in Fc functional antibodies and in the role that HIV-specific IgA can play during HIV vaccination strategies and in HIV infection. The RV144 vaccine induced IgG antibodies that were able to mediate ADCC responses. However, the vaccine efficacy was reduced in the presence of high concentrations of HIV-specific IgA. Monoclonal IgA that was isolated from the plasma of the RV144 vaccinees was able to block the potentially protective IgG antibodies from binding similar epitopes, thus preventing ADCC responses with NK cells. This indicates there was epitope competition between IgA and IgG antibodies in the RV144 vaccine trial. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses. Chapter 4 assessed plasma samples from the RV144 vaccine trial for their ability to induce neutrophil-mediated responses and if IgA was able to inhibit these responses. IgG from the RV144 vaccinees was able to induce modest HIV-specific ADNP and neutrophil-mediated RFADCC responses. Plasma IgA from the vaccinees was able to inhibit ADNP responses but not neutrophil-mediated RFADCC responses. Using pooled IgG from the vaccinees, it was shown that pooled purified IgA from vaccinees, pooled purified IgA from HIV positive donors and pooled purified HIV negative IgA were able to inhibit the IgG mediated ADNP responses. Overall, this thesis shows that neutrophils can mediate HIV-specific antibody-dependent phagocytosis and neutrophil-mediated RFADCC responses. HIV-specific IgG mediated neutrophil responses, induced by either infection or vaccination, can be inhibited by plasma IgA in an antigen dependent mechanism and an antigen independent mechanism that is a FcalphaR dependent mechanism. The inhibitory effects of IgA may assist in understanding HIV pathogenesis and improving future HIV vaccine designs.
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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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ItemTranscriptional regulation and co-stimulatory signaling in antiviral T cell immunity( 2018)Special AT-rich binding protein-1 (SATB1) is a global chromatin organizer, promoting or repressing gene transcription in mice and human. In this PhD thesis, SATB1 expression was examined in humans across ages and tissues (Chapter 3). The molecular (Chapter 4) and functional (Chapter 5) role of SATB1 was investigated during anti-viral immunity in mice using an influenza (IAV) infection model. Additionally, the effect of CD27-mediated co-stimulation was studied in the context of HIV-1 infection (Chapter 6). SATB1 has pivotal roles during T cell development and maturation, with lineage fate decision in hematopoietic stem cells and gradual changes in SATB1 expression contributing to T cell development in the thymus in mice. In Chapter 3, SATB1 expression was analyzed across lymphocyte compartments from different human tissues and correlated with PD-1 expression in virus-specific CD8+ T cells. SATB1 expression in pediatric and adult donors showed that SATB1 expression was highest in the human thymus with differential expression levels from DN to DP thymocytes and down-regulation of SATB1 in peripheral T cells. Chapter 3 shows that SATB1 expression in the periphery is not static but follows fine-tuned expression dynamics with downregulation from naïve to antigen-specific CD8+ T cells, likely to be antigen- and tissue-dependent. These data led to the hypothesis that fine-tuned SATB1 expression is necessary for maintaining fate-potential in developing and mature, peripheral T cells. Several molecular mechanisms have been identified for gene regulation by SATB1 with wide-range impacts on the overall chromatin landscape. Previous studies in our laboratory showed that SATB1 mRNA levels are high in naïve, but low in effector CD8+ T cells. The impact of SATB1 in repressing transcriptional programs in naïve CD8+ T cells, prior to its downregulation in effector T cells, was addressed in Chapter 4 of this study. ChIP-Sequencing analysis was performed to decipher genomic binding sites of SATB1 in naïve and effector CD8+ T cells. SATB1 ChIP-Seq data demonstrated that SATB1 binding sites were predominately distal to transcriptional start sites, likely to harbor transcriptional enhancer sites, with reduced SATB1 binding sites in effector over naïve CD8+ T cells. To understand the effects of SATB1 on the transcriptional regulation in naïve and IAV-specific CD8+ T cells, SATB1 imposter mice (SATB1imp/imp) were used in this PhD study. In these mice, Satb1 contains a point mutation in the DNA-binding domain encoding position. SATB1 protein expression in SATB1imp/imp mice persists but is dysfunctional with reduced DNA-binding capability. CD8+ T cells from SATB1imp/imp mice showed up-regulation of certain gene profiles, especially at the naïve stage, such as Pdcd1, Ctla4 and Ccl5, characteristic of activated or exhausted T cells. In Chapter 5, an IAV infection model was used, to examine the effects of dysfunctional SATB1 in IAV-specific CD8+ T cell response generation. CD8+ T cell numbers were consistently reduced in SATB1imp/imp mice with significantly reduced IAV-specific CD8+ T cell numbers in lungs on d10 post-infection. SATB1imp/imp CD8+ T cells exhibited an early overexpression of PD-1 from the naïve stage and reduced polyfunctionality within IAV-specific SATB1imp/imp CD8+ T cells. Using a bone marrow chimera approach, in which mice were reconstituted with a mixture of wildtype and SATB1imp/imp-derived lymphocytes, data showed that reduced T cell numbers and PD-1 overexpression are T cell intrinsic in SATB1imp/imp mice. Immunotherapies, including anti-PD-1, anti-CD27 and histone deacetylase inhibitors, are often used in clinical trials to manipulate activation of T cells. In Chapter 6, we used CD27-mediated stimulation to understand the effect on CD4+ T cells with and without HIV-1 infection. CD27 is a co-stimulatory receptor of the TNF-family, expressed on naïve and central memory T cells. Non-permanent stimulation via CD27 leads to increased primary and memory antiviral CD8+ T cell responses in mice. Here, in humans, CD27-mediated stimulation of CD4+ T cells via its ligand CD70 exhibited profound activation potential in vitro, with high CD4+ T cell proliferation and GzmB production. To examine whether this high activation potential could trigger re-activation of viral transcription in latently infected CD4+ T cells, we re-stimulated CD4+ T cells with conventional α-CD28 or CD27-mediated co-stimulation in an in vitro latency model. Unexpectedly, re-stimulation via CD27 of CD4+ T cells led to reduced viral reactivation compared to α-CD28 stimulation of CD4+ T cells. However, similar transcriptional reactivation levels were obtained when CD4+ T cells isolated from HIV+ individuals on ART were re-stimulated with the two protocols. Strikingly, pre-stimulation of CD4+ T cells prior to in vitro HIV-1 infection showed a trend towards reduced HIV-DNA integration and overall infection. This suggests that CD27-mediated stimulation could lead to activation of antiviral mechanisms that reduces CD4+ T cells HIV-1 infection. Overall, this PhD study provides an in-depth understanding of the transcriptional and co-stimulatory regulations of T cell differentiation in response to viral infections. SATB1’s ability to regulate immune checkpoint molecules, such as PD-1 by its DNA-binding capability in antiviral immunity highlights its significance in future PD-1-related cancer and HIV-1 immunotherapy trials used to reverse T cell exhaustion.
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ItemVariation in killer cell immunoglobulin-like receptors and their human leukocyte antigen class I-encoded ligands impacts natural killer cell education and the control of viral infection( 2018)The interaction of HLA class I (HLA-I) proteins with inhibitory receptors such as those of the Killer cell Immunoglobulin-like Receptor (KIR) family play a key role in effector function acquisition and target cell identification by Natural Killer (NK) cells. However, the extent to which polymorphism in the genes encoding both KIR and their HLA-I encoded ligands impacts the capacity of NK cells to respond to virus-infected or transformed cells with reduced expression of HLA-I proteins is unclear. Analyses of KIR expression on NK cells from a large panel of HLA-I typed healthy donors, found little evidence to suggest that the presence of a HLA-I ligand significantly impacted the frequency of KIR expression. In contrast, the NK cell’s capacity to respond to HLA-I-deficient targets was elevated by the presence of cognate KIR/HLA-I-ligand pairs. Focussed analyses of the interaction between KIR3DL1 and HLA-Bw4 allotypes demonstrated that polymorphisms in both the receptor and ligand impacted the strength of the interaction. Moreover, functional analyses showed that the proportion of KIR3DL1+ve NK cells responding to HLA-I-deficient targets strongly correlated with the strength of the interaction between KIR3DL1 and HLA-Bw4 alloypes, a phenomenon often described as education. Similarly, analyses of KIR3DL1/HLA-Bw4 pairs in a cohort of HIV+ve individuals showed a strong correlation with viral loads, implicating NK cell education in the control of HIV. Analysis of viral loads in HIV-infected HLA-B*57:01+ve individuals revealed that those with KIR3DL1 alleles encoding a valine at position 47 had reduced viral loads relative to individuals that possessed alleles with isoleucine at this position. Binding and reporter cell analyses showed that KIR3DL1*005, a common Ile47 encoding allotype, interacted more strongly with HLA-B*57:01 than other allotypes. Strikingly, functional analyses showed that NK cells expressing Ile47 allotypes were more strongly inhibited by HLA-B*57:01 when expressed at low levels than Val47 variants, suggesting that allotypic variation in KIR impacts the capacity of NK cells to mediate immunosurveillance of HLA-I levels. Finally, although HLA-Bw4 allotypes show extensive conservation across residues that made direct contacts with KIR3DL1, the capacity of allotypes such as HLA-B*57:01 and HLA-A*24:02 to inhibit NK cell activation varied markedly. Since immune synapse formation relies on discerning between structurally similar ectodomains, as may be the case for HLA-Bw4 allotypes, the membrane behaviour of these two HLA-I was compared by fluorescence recovery after photobleaching (FRAP). The data revealed that the mobility of HLA-I was influenced by the cell in which they were expressed, however allotypic differences between the mobility of HLA-B*57:01 and -A*24:02 were observed and, via the analyses of chimeric and mutant HLA-I proteins, was found in part to be dependent on their ectodomains. Taken together, the data suggest that polymorphisms in both KIR and HLA-I impact the strength of their interaction, which in turn influences NK cell education and target cell recognition, particularly with regard to the sensitivity to altered HLA-I expression. In the case of KIR3DL1/HLA-Bw4 interaction, this is manifested in changes in viral replication in HIV-infected individuals and likely has significance in other settings including infection and cancer.
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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.
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ItemThe role of antibody-dependent cellular cytotoxicity in curing latent HIV-1 infection( 2018)As of 2017, 36.9 million people worldwide were living with HIV-1, with approximately 21.7 million people treated with antiretroviral therapy (ART) and only a single individual cured of HIV-1. HIV-1 infection has proven difficult to cure as HIV-1 integrates into the genome of host cells and establishes a latent viral reservoir despite ART, necessitating lifelong therapy for the millions of people living with HIV-1. This continues to place a massive economic burden on the health sector, substantiating the need for an HIV-1 cure to end the HIV-1/AIDS pandemic. One of the most extensively studied cure strategies is the ‘shock and kill’ approach, which aims to reactivate HIV-1 expression from latently infected cells and subsequently eliminate the reactivated cells through immune-mediated mechanisms. We hypothesised that antibody Fc effector functions including antibody-dependent cellular cytotoxicity (ADCC) could be harnessed to eliminate the reactivated viral reservoir. This thesis first examined whether antibodies within HIV-1-infected individuals could recognise and eliminate cells reactivated from latency (Chapter 2). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-dependent natural killer (NK) cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on ACH-2 cells likely resulted in the concealment of CD4-induced (CD4i) epitopes on HIV-1 envelope (Env) that are highly targeted by ADCC antibodies within HIV-1-infected individuals. We found that ex vivo-expanded primary CD4+ T cells from HIV-1-infected individuals were modestly susceptible to ADCC mediated by autologous serum antibodies and effector cells. Importantly, ADCC against these ex vivo-expanded CD4+ T cells could be enhanced following incubation with a small-molecule CD4 mimetic compound that exposes CD4i ADCC epitopes on Env. Since HIV-1-specific ADCC antibodies decline in individuals on long-term ART, this thesis next examined whether latency reversal with the histone deacetylase inhibitor panobinostat could provide sufficient antigenic stimulus to boost ADCC antibodies (Chapter 3). We found that in vivo treatment with panobinostat or a short analytical treatment interruption (ATI) of median 21 days was not sufficient to stimulate an increase in HIV-1-specific ADCC antibodies, despite viral rebound in all individuals who underwent the short ATI. In contrast, a longer ATI of 2 to 12 months robustly boosted HIV-1-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a delay between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies aiming to utilise autologous ADCC responses against latently infected cells. Given that the CD4-bound conformation of Env is predominantly recognised by ADCC antibodies within HIV-1-infected individuals, this thesis next examined whether early-stage infected cells in the process of downregulating CD4 were more susceptible to ADCC compared to late-stage infected cells that have fully downregulated CD4 (Chapter 4). We found that both early- and late-stage infected cells were eliminated by antibodies within plasma from HIV-1-infected individuals, even though there was higher binding of plasma antibodies to the CD4-intermediate early-stage infected cells. Our results show that soluble Env and virions derived from the viral inoculum could sensitise early-stage infected cells to ADCC prior to de novo Env expression, resulting in artefactual in vitro ADCC measurements. Future studies will need to develop improved models to address the potential in vivo role for ADCC against cells with nascent HIV-1-infection. Collectively, our studies highlight a potential role for HIV-1-specific ADCC in curative HIV-1 strategies and in controlling HIV-1 infection. Our results suggest that sufficient expression of Env and the expression of appropriate epitopes will be needed for cells reactivated from latency to be eliminated by autologous ADCC antibodies. Future studies will need to optimise latency reversal in vivo and examine strategies to either boost or administer ADCC antibodies to ensure efficient elimination of the reactivated viral reservoir.