Doherty Institute - Theses
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ItemImmunity and protection against Influenza B viruses( 2018)Broad immunity to influenza viruses can be conferred by both humoral and cellular components of the immune system. Thus, universal vaccines, need to elicit both antibody and T cell responses with broad cross-reactivity across both influenza A and influenza B viruses (IAV and IBV, respectively). To achieve this, a thorough understanding of the immune response to IAV and IBV is required at the cellular and molecular level. In contrast to IAV, immune responses to IBV are understudied, despite the clinical relevance of IBV. Therefore, the overall aim of this PhD thesis was to characterize B cell and T cell immunity to IBV, in comparison to IAV, following influenza vaccination and virus infection in order to inform the rational design of future universal influenza vaccines. Using longitudinal blood samples from healthy adults vaccinated with the inactivated influenza vaccine, the cellular events that precede the generation of protective immunity were dissected in Chapter 3. This was achieved using recombinant haemagglutinin probes to quantitively and qualitatively assess influenza- specific B cells directly ex vivo. While vaccination induced humoral immunity, comprising of a three-pronged B cell response (CXCR5-CXCR3+ antibody-secreting B cells, CD21hiCD27+ B cells and CD21loCD27+ B cells) and a specialized subset of ICOS+PD-1+CXCR3+ circulating T follicular helper cells, components of cellular immunity like CD8+ T cells were not elicited. Furthermore, vaccine-induced B cells were not maintained in peripheral blood at one year after vaccination. Furthermore, analysis of influenza-specific memory B cells across human tissue compartments demonstrated the enrichment of memory B cells in human secondary lymphoid organs (SLOs) and the lung. In Chapter 4, conservation analysis of known CD8+ T cell epitopes derived from IAV identified a set of conserved peptides across IAV, IBV and is some cases influenza C (the A2/PB1413-421 epitope), providing the first evidence of universal cross-reactivity across IAV, IBV and ICV. Such CD8+ T cell cross-reactivity across influenza A, B and C viruses is broader than any known antibody or T cell specificity known for influenza viruses. Cutting-edge immunopeptidomics approach was used to further identify immunodominant CD8+ T cell epitopes from IBV in the context of the highly prevalent HLA-A*02:01 allele, with high conservation in IBV and the ability to accelerate viral clearance in HLA-A2-transgenic mice. Prominent memory CD8+ T cells towards both universal and influenza type-specific epitopes were detected in blood, lungs and SLOs of healthy humans, with lung-derived CD8+ T cells displaying a tissue-resident memory phenotype. Importantly, in IAV- and IBV-infected pediatric and adult patients, CD8+ T cells against these novel epitopes were readily detected with an effector CD38+Ki67+ phenotype. In Chapter 5, the quality of broadly cross-reactive CD8+ T cell responses was assessed by analyzing their TCR/pMHC avidity, functional avidity, polyfunctionality and TCRab repertoire. The TCRab repertoires of CD8+ T cells specific for broadly cross-reactive influenza-specific CD8+ T cells varied in terms of the intra-individual clonality and inter-individual sharing, in a manner dependent on underlying TCRab genetics and/or cognate pMHC-I structure, without however, significantly compromising functionality. Having characterized CD8+ T cell responses to IAV and IBV, the ability of influenza viruses to downregulate MHC-I was assessed in Chapter 6, as this is a common immune evasion mechanism. While IAV causes a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the loss of MHC-I molecules only from the cell surface, whereby MHC-I trafficking to the cell surface is delayed by retaining MHC-I intracellularly during IBV infection. Overall, this PhD thesis investigated immunity to IAV and IBV viruses by dissecting humoral and cellular immunity in healthy individuals, vaccinated adults, influenza-infected patients as well as human lymphoid and mucosal tissues. This work provided novel insights for the rational design of universal influenza vaccines which do not require annual reformulation.
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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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ItemPermanent silencing of HIV transcription using triplex forming oligonucleotides: a novel strategy for an HIV cure( 2018)Combination antiretroviral therapy (cART) for HIV infection has significantly reduced morbidity and mortality, however, treatment is lifelong. The main barrier to a cure for HIV is the persistence of long lived latently infected T-cells. Virus can integrate in the host genome and be transcriptionally silenced however, upon reactivation of transcription virus can re-emerge from these latently infected cells. In individuals on ART, reactivation of virus goes undetected but once ART is stopped, reactivation of virus leads to virus replication and rebound. One strategy to eliminate virus rebound after cessation of ART is to permanently silence HIV transcription. Here we explore an alternative approach to silence HIV transcription in CD4+ T cells using triplex formation oligonucleotides (TFO). We hypothesize that TFOs can bind irreversibly to the integrated provirus in a sequence specific manner with limited off-target effects. We assessed TFO activity against the green fluorescent protein (GFP) and HIV in vitro by using uninfected and latently infected cell lines and determined the effects of gold nanoparticles to enhance nuclear localization.
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ItemDevelopment of humoral immunity to severe and uncomplicated malaria in children from different geographical locations( 2018)Malaria is one of the leading causes of childhood morbidity and mortality, and Plasmodium falciparum infection is responsible for nearly all malaria deaths. Pregnant women and children under 5 years of age are mainly affected by its severe pathological complications. Although a considerable number of previous studies have focused on pregnancy related malaria, there is scarcity on the number of studies in infants and the mechanism of development of naturally acquired immunity to malaria in young children. Naturally acquired immunity to malaria is developed by the continuous acquisition of antimalarial antibodies in response to exposure which may protect the children against severe complications. Moreover, immunity to severe malaria is attained quite early in life compared to uncomplicated malaria infections which require longer time to get immune to. The mechanisms of developing naturally acquired immunity to malaria in young children remain elusive and there are many factors (e.g. undernutrition, clinical infections etc.) to consider which may influence this acquisition. This thesis aims to understand the influence of nutritional supplements and symptomatic malaria infections on the development of naturally acquired immunity to malaria in infancy, and to identify antibody responses that may protect against severe malaria. To address these research questions, antibodies to several blood stage merozoite antigens, schizont extract and variant surface antigens were measured by enzyme-linked immunosorbent assays and flow cytometry-based assays on plasma samples from young children residing in Malawi and Papua New Guinea. The 1st part of the thesis evaluated antibodies in a subset of children enrolled to mothers in iLiNS-DYAD study. In this longitudinal study, children from rural Malawi and their mothers were enrolled in a randomized clinical trial, Antibody levels and seroprevalence were tested at 6 and 18 months of age and compared for nutrient supplementation groups lipid nutrient supplement (LNS), multiple micronutrient supplement (MMN) and iron and folic acid (IFA). The study found no association between intake of additional nutrient supplementation and improved malarial immunity in 6 and 18-month-old children. Secondly, to understand whether early life exposure to malaria has an impact on the antibody acquisition in infants, weekly follow up was done for children from the same parent study from birth to 18 months. Febrile episodes were reported and confirmed cases of malarial episodes were related to the antibody measurements at 18 months. The results suggested that both antibody levels and seroprevalence to tested antimalarial antigens increased in early childhood following infection. But the levels and seroprevalence of antibodies at 18 months of age did not differ depending on the age of the child at the time of malaria episodes. Finally, to understand whether the severity of previous malarial infections influence the antibody acquisition in young children, a subset of Papua New Guinean children (0.5 to 10 years) from a case control study were tested for antibody responses to circulating parasites by flow cytometry. Var gene transcription levels by the parasites were also determined by qRTPCR. Homologous boosting of antibody in convalescence was common in children regardless of the severity of infection or blood groups of children. Among children with non-O blood groups, in comparison to children on presentation (acute) with severe malaria, there were broadly higher levels of antibody to IE surface antigens in convalescence from severe malaria and in acute or convalescent samples from uncomplicated malaria. By contrast, in children with blood group O, only convalescent plasma from severe malaria showed higher recognition of tested isolates. These differences may relate to the differential susceptibility of children with blood group O to severe malaria. Several var genes were upregulated in severe malaria including mostly var type A, but also some var types B/A, B and B/C, while none were upregulated in uncomplicated relative to severe malaria. Increased transcription, predominantly of var genes associated with severe malaria in Africa, was common in severe malaria in PNG. Overall, this thesis provides considerable understanding on the mechanism of acquisition of antimalarial antibodies in young children from birth to infancy and at an older age covering two separate geographical locations from Africa and Oceania. The results suggested that additional nutrient supplementation for both mothers and children didn’t improve the malaria antibody immunity at 6 and 18 months of age in Malawian children. But the subsequent malaria infections lead to the acquisition of antimalarial antibodies in 18 months of age indicating that some of this blood stage antigens might be used as a sero-surveillance tools for biomarkers of malaria exposure. Antibody responses to circulating parasite isolates in some Papua New Guinean children suggested that broad response to severe isolates is acquired in some children with severe malaria. Upregulation of mostly group A var genes were also observed in Papua New Guinean children with severe malaria. Findings from this study can be used as a basis for further clinical studies using larger sample size to provide useful information on the dynamics of naturally acquired immunity to malaria in children.
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ItemThe role of CD8+ tissue-resident memory T cells in melanoma immune surveillance.( 2018)In addition to its role in protecting the body from infection, the immune system can prevent the development of cancer in a process termed tumour immune surveillance. During this process, immune cells can either recognise and completely eliminate cancerous cells, or can suppress the outgrowth of malignant cells without completely eradicating them. This latter mode of control, designated ‘cancer-immune equilibrium’, can be sustained for extended periods of time in a manner dependent upon adaptive immune cells such as T cells. The vast majority of human cancers are spawned from epithelial tissues. However, long-lived CD8+ circulating memory T (TCIRC) cells such as effector memory T (TEM) cells and central memory T (TCM) cells are typically excluded from epithelial tissue compartments in the absence of robust inflammation. In contrast, CD8+ tissue-resident memory T (TRM) cells are a population of non-migratory immune cells that permanently occupy epithelial tissue sites without recirculating. CD8+ TRM cells provide efficacious protection against peripheral viral and bacterial infections and have recently been identified in a variety of human solid tumours, where they associate with improved disease outcome. However, a direct role for TRM cells in promoting natural immunity to cancer has yet to be demonstrated. In this thesis, we examined the contribution of CD8+ TRM cells to peripheral cancer immune surveillance and the mechanisms through which these cells protect against tumour progression. In order to study the peripherally localised anti-tumour immune response, we developed and characterised an orthotopic epicutaneous (e.c.) model of melanoma in mice that targets tumour growth to the outermost layers of skin. We found that a portion of mice receiving tumour cells e.c. remained free of macroscopic cancer long after inoculation, in a manner that depended upon immune cell mediated control. Spontaneous protection from progressive tumour development was associated with the formation of melanoma-specific CD69+CD103+ CD8+ skin TRM cells, whereas mice genetically deficient in TRM cell formation were highly susceptible to tumour growth. Importantly, tumour-specific skin TRM cells could protect against tumour development independently of TCIRC cells. Closer inspection of macroscopically tumour-free mice revealed that many harboured occult melanoma cells in their skin long after e.c. inoculation. These dormant melanoma cells were retained in the epidermis, where they were dynamically surveyed by tumour-primed CD8+ skin TRM cells. Ablation of skin TRM cells from macroscopically tumour-free mice that were initially protected from tumour development triggered late-stage tumour outgrowth, demonstrating that CD8+ TRM cells can suppress cancer progression by promoting a state of subclinical cancer-immune equilibrium. Further, our findings suggest that the cytokine tumour necrosis factor (TNF) may play a role in the induction and maintenance of this equilibrium state. Overall, we show that CD8+ TRM cells contribute to immune surveillance of peripherally localised cancers by upholding tumour-immune equilibrium. As such, our findings elucidate how cancers arising in epithelial compartments are subject to long-term and ongoing immune suppression. Collectively, our work provides critical insight and the impetus necessary to exploit CD8+ TRM cells as targets of cancer immunotherapies in order to improve solid cancer treatments in patients.
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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.
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ItemImmune control of Legionella pneumophila lung infection( 2018)Legionnaire’s Disease is a life-threatening lung infection caused by the bacterium Legionella pneumophila. Recently, studies have revealed that cytokines such as IL-1α, TNF and IFNγ secreted by innate immune cells facilitate the restriction of intracellular replication of L. pneumophila. However, the exact mechanisms by which innate immune cells and cytokines contribute to the control of L. pneumophila lung infection are still unclear. In this study, we demonstrated that the bactericidal activity of monocyte-derived cells (MCs) was IFNγ-dependent while alveolar macrophages (AMs) did not respond to IFNγ during L. pneumophila infection. To understand IFNγ-mediated killing machinery in MCs, we sorted MCs from the lungs of wild type C57BL/6 (WT) mice and Ifng-/- mice 3 days after L. pneumophila infection and performed RNA-sequencing. We found that a set of genes belonging to the Interferon-stimulated GTPase (ISGase) superfamily that functions in cell-autonomous immunity was highly expressed in WT MCs compared to Ifng-/- MCs. Using Real-time quantitative reverse transcription-PCR (qRT-PCR), we confirmed that expression of these genes in MCs was induced by IFNγ during L. pneumophila infection. Further, we observed that Guanylate-binding protein 1 (Gbp1) was predominantly expressed in MCs compared to AMs during L. pneumophila lung infection. These findings suggested that Gbp1 might have an important role in the clearance of L. pneumophila by MCs in vivo. Mechanistically, we observed that IFNγ could disrupt the translocation of bacterial Dot/Icm effector proteins by L. pneumophila in macrophages. Dot/Icm effectors are required for biogenesis of the intracellular replicative Legnionella-containing vacuole (LCV). Interestingly, MCs were refractory to the translocation of Dot/Icm effectors even in the absence of IFNγ in vivo. This suggested that L. pneumophila could not establish the LCV and replicate in MCs. One possible explanation for increased bacterial numbers in Ifng-/- MCs might be lack of induction of the killing and degradation machinery mediated by ISGases. We observed that Gbp1, Gbp2 and Irga6 promoted LCV lysis and lysosomal clearance of intracellular L. pneumophila in the presence of IFNγ. In addition, we observed that the IFNγ receptor 1 (IFNGR1) was dramatically downregulated in AMs but not MCs during L. pneumophila infection in vivo, which might explain why AMs showed little response to IFNγ in the control of L. pneumophila intracellular replication in vivo. Further studies in vivo and in vitro, suggested that the downregulation of IFNGR1 in macrophages was dependent on both MyD88 and Trif signalling and NF-κB activation. Constitutive expression of IFNGR1 in AMs significantly improved the capacity of AMs to restrict intracellular replication of L. pneumophila in the presence of IFNγ in vivo. Overall this study demonstrated that MCs are critical innate immune cells that respond to IFNγ stimulation to control intracellular replication of L. pneumophila in vivo and that ISGases might contribute to the lysosome mediated clearance of intracellular L. pneumophila in MCs. In addition, the downregulation of IFNGR1 in AMs dampened their response to IFNγ stimulation, which may partially explain the susceptibility of AMs to L. pneumophila infection in vivo. Hence, this study has uncovered potential mechanisms underpinning IFNγ-mediated cell-autonomous immunity against L. pneumophila. This permits future study into understanding the detailed molecular and cellular mechanisms that contribute to the control of L. pneumophila intracellular replication.
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ItemFollowing the HIV-1 RNA footprint in cells with latent provirus: reversing silent infection through Tat( 2018)Globally, the HIV-1 epidemic remains robust and the size of the infected population continues to grow, particularly in sub-Saharan Africa. Although viral suppression is achieved through administration of cART, therapy is lifelong. A compartment of cells that carry HIV-1 in a transcriptionally inactive state, but which retains replicative potential, persists in infected individuals and re-emerges to seed infection when treatment is interrupted. Use of latency reversing agents for perturbation of this reservoir has been shown to be ineffective in the clinical context. This stresses the need for the development of more refined approaches to reactivate latent infection. Multiple layers of repression are present in the cell and at the latent HIV-1 promoter. A central aspect of HIV replication that is blocked during latency is the process of transcriptional elongation. In productive infection, this stage of transcription is enhanced by the action of the viral protein, Tat. Hence, reactivation of latency may be possible through inducing the expression of Tat in a latently infected cell. From the pre-existing DNA template in cells with silenced proviral genomes, tat sequences can be transcribed by a process independent of the 5’ LTR. Generation of readthrough transcripts containing sequences of both human and viral origin is a consequence of HIV-1’s propensity to integrate into introns of transcriptionally active genes. These transcripts are an HIV-1 RNA footprint that may provide the means for expression of Tat in latently infected cells. Here, the HIV-1 RNA footprint in the CCL19-induced primary cell model of HIV-1 latency and in ex vivo samples from individuals on suppressive therapy was studied using target enrichment and next-generation sequencing technologies. Chimeric cellular:tat mRNAs were detected in the primary cell model that had some stretches of tat sequence incorporated in a variety of different configurations. A subset of these have the potential to translate Tat in their original unfragmented forms. In addition, as expected for the generation of readthrough transcripts, HIV-1 integration was predominantly parallel to the human gene, although a small proportion was attributed to the convergent orientation. No cellular:tat mRNAs were detected in the ex vivo samples, however, 3’ LTR activation and the use of the splice donor 1 (SD1) site were the major mechanisms leading to the generation of chimeras. The restrictive context of incorporation of tat sequences into a chimeric cellular:HIV transcript would impede translation through canonical 5’ cap-dependent ribosome scanning modalities. An internal ribosome entry site (IRES) located within Tat encoding sequences has been described and its properties in the context of chimeric cellular:tat mRNA was investigated. Robust, but low-level expression of Tat from an IRES-dependent mechanism was observed using luciferase-based assay systems, and a correspondingly weak reactivation of viral production from the J-Lat10.6 T-cell line model of HIV-1 latency was detected. In addition, SRP14 and HMGB3, two cellular RNA-binding proteins, are putative co-factors of Tat IRES translation detected by affinity purification-mass spectrometry, were shown to be positive and negative regulators of Tat expression respectively and may have roles in the regulation of HIV replication. In cells with quiescent proviruses this novel pathway of Tat expression could be targeted as part of a more biologically relevant combinatorial strategy for reversing latency.
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ItemFalling down the cascade: the gaps in care delivery for people living with chronic hepatitis B in Australia( 2017)In Australia, an estimated 239,000 people were living with chronic hepatitis B (CHB) in 2016. Most of the affected community were born overseas and acquired their infection early in life, in countries with high and intermediate prevalence of hepatitis B. The mortality attributable to CHB is from both liver cancer (hepatocellular carcinoma) and cirrhosis. Hepatocellular carcinoma has poor five-year survival, increasing incidence globally, and was projected to become the sixth most common cause of cancer death in Australia in 2016. The first “Global Hepatitis Health Sector Strategy” was signed in 2016 by all the member states of the World Health Assembly including Australia, with the aim of eliminating viral hepatitis, including hepatitis B, as a public health concern by 2030. The five studies presented in this thesis use the cascade of care framework to approach different aspects of the health system response to chronic hepatitis B in Australia. The studies have used different data sources and methodologies to measure the cascade and explore factors associated with the delivery of care. The first study presents an analysis of national data from 2012. It proposed, for the first time, a cascade of care for chronic hepatitis B in Australia that included a novel “enrolled in care” indicator. The second study presents findings from a multicentre retrospective study of adherence to antiviral therapy for chronic hepatitis B from 2010-2013. The study measured the proportion of people adherent to treatment in tertiary settings and analysed the demographic and health system factors associated with poor adherence. The third study analysed the association of a pharmacy-based adherence measure (the medication possession ratio) with viral outcomes using a time-to-event analysis for favourable and unfavourable viral outcomes. The fourth study presents findings from a retrospective analysis of primary care data in a community health centre that received external support from a tertiary service to improve the delivery of guideline-based care for chronic hepatitis B, including surveillance for hepatocellular carcinoma. This study evaluated four and a half years of data focusing on hepatocellular carcinoma surveillance participation and adherence. The fifth study presents findings from a qualitative study: semi-structured interviews of African-Australians living with chronic hepatitis B. This study explored participants’ understanding of health risks associated with hepatitis B, including their perceptions of their risk of developing hepatocellular carcinoma. Findings from this thesis have shown that few people living with chronic hepatitis B in Australia were enrolled in care. It provided the first multicentre estimates of the adherence of people on antiviral therapy for chronic hepatitis B in our health system (using medication possession ratio as the measure of adherence) and that factors associated with poor adherence were younger age and poor continuity of clinician. In a further study, the association between medication possession ratio and unfavourable viral outcomes was demonstrated for the first time. This analysis found that there was no true cut-off point or threshold to define adherence and the risk of poor outcomes. Rather, there was an increasing hazard ratio for unfavourable events with decreasing medication possession ratio. The findings also include results from a study that demonstrated hepatocellular carcinoma surveillance in a tertiary-supported general practice – with both participation and adherence to six-monthly scans with supported recall and reminder systems – is hard to achieve. Finally, the fifth study presented as part of this thesis found that African-Australians living with chronic hepatitis B perceived and experienced significant risks to social and emotional wellbeing from the shock of diagnosis, fear of infectiousness, and discrimination from telling others about their illness, as well as physical or liver-related problems. The results from this thesis have informed the development of the current Australian cascade of care for chronic hepatitis B and provided insights into the challenges of delivering health services to people living with chronic hepatitis B. These findings have led to recommendations for further development of the cascade at a national and regional level, and the need for further research and evaluation of the health system response to chronic hepatitis B. The work presented demonstrates that Australia has failed to meet the targets of the 2014-2017 National Strategy and needs to rapidly improve essential elements of the cascade, including increasing the proportion diagnosed and enrolled in care, to reach the targets of elimination of chronic hepatitis B as a public health concern by 2030.