Doherty Institute - Theses
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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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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.