Microbiology & Immunology - Theses
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ItemFc-effector functions and plasma IgA in viral pandemics (HIV-1 and SARS-CoV-2)( 2023)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - the causative agent of the COVID-19 pandemic- has caused over 6 million deaths globally since late 2019. Mutations in the receptor binding domain (RBD) of SARS-CoV-2 have resulted in the emergence of variants of concern (VOC). Following infection and/or vaccination SARS-CoV-2-specific antibodies are generated with neutralising and/or Fc functional properties. Neutralising antibodies can protect from subsequent infections. While Fc-effector functions, including antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent cellular cytotoxicity (ADCC) are important for control and resolution of many infectious diseases including SARS-CoV-2. Notably, the neutralising antibody response wanes rapidly following infection with SARS-CoV-2, however, the durability of antibody mediated Fc-effector functions including ADCP remains largely unknown. To investigate the SARS-CoV-2 Fc functional antibody response, we developed two in vitro cell based assays to assess SARS-CoV-2 specific phagocytosis and cell association (trogocytosis) over time in mild-moderate convalescent COVID-19 individuals (Chapter 2). Interestingly, we observed evidence of SARS-CoV-2 specific trogocytosis occurring during the cell association assay using confocal microscopy. We demonstrate that the SARS-CoV-2 Fc functional antibody response, specifically ADCP and cell association, were more durable than the neutralizing antibody response. All COVID-19 individuals retained detectable antibody mediated phagocytosis and cell association at ~4 months post symptom onset, while 30% of the cohort lost detectable neutralization. Therefore, highlighting the potential importance of Fc-effector functions in long-term immunity from SARS-CoV-2 reinfection. The importance of IgG antibodies for protection and control of SARS-CoV-2 has been extensively reported. However, other antibody isotypes including IgA have been poorly characterized. We aimed to examine the functional contributions of plasma IgA to neutralisation and Fc-effector functions following SARS-CoV-2 infection (Chapter 3). Using a multiplex surrogate neutralisation assay, we assessed the neutralising capacity of IgA and IgG depleted plasma and purified antibody fractions against ancestral SARS-CoV-2 Spike receptor binding domain (RBD) and RBDs with common single amino acid mutations. Notably, more than 60% of the cohort showed significantly reduced neutralising capacity following IgA depletion (p = 0.0001). Furthermore, 30% of the cohort induced stronger IgA-mediated neutralization than IgG when purified antibody fractions were tested at equivalent concentrations. Moreover, convalescent purified IgA and IgG recognized similar RBD mutations and showed comparable neutralisation of RBD mutants. Depletion of IgG significantly reduced Fc-effector functions (ADCP and cell association) of convalescent plasma, in contrast no change was observed with depletion of IgA. We demonstrate that plasma IgA has the capacity to neutralize ancestral SARS-CoV-2 RBD, however, IgA contributes minimally to SARS-CoV-2 plasma Fc-effector function. Overall, neutralizing IgA and duel functional IgG contributes to the COVID-19 antibody response after infection. A constellation of RBD mutations have resulted in enhanced transmission and/or immune escape of SARS-CoV-2 circulating strains, giving rise to new variants of concern (VOCs). Mutations within the RBD can reduce antibody recognition, leading to reduced neutralising potency and potentially altering vaccine efficacy. However, the impact of RBD mutations on Fc-effector functions following vaccination remains unknown. We examined the capacity for SARS-CoV-2 Pfizer (BNT162b2) vaccine (2 weeks post second dose) and infection induced antibodies to mediate Fc-effector functions against SARS-CoV-2 VOCs (Chapter 4). We measured IgG binding to RBDs and engagement of RBD specific antibodies with Fc gamma receptors (FcyRs) via multiplex for 6 historical VOCs (Alpha, Beta, Gamma, Delta, Kappa and Omicron BA.2). Notably, FcyRIIa and FcyRIIIa engagement was significantly reduced for the VOCs Beta, Gamma, and Omicron BA.2. Furthermore, we confirmed that reduced FcyR engagement to RBD mutants resulted in reduced cellular Fc-effector functions, via a novel competitive SARS-CoV-2 duplex ADCP assay. This novel SARS-CoV-2 ADCP duplex assay enables assessment of the functional capacity of the same pool of antibodies to two different SARS-CoV-2 variants in a competitive high throughput assay. Taken together, we successfully optimised a novel SARS-CoV-2 ADCP assay and show that mutations within the SARS-CoV-2 RBD may have consequences on the Fc functional capacity of vaccine induced antibodies. The human immunodeficiency virus (HIV-1) is the causative agent of the acquired immune deficiency syndrome (AIDS) epidemic which has resulted in an estimated 40.1 million deaths globally since the early 1980s. Antibodies including IgG and IgA can recognise HIV-1 to elicit antiviral functions such as neutralisation and Fc effector functions. Plasma IgA can engage with the Fc alpha receptor (FcaR) to activate Fc-effector functions including phagocytosis. However, IgA can also mediate inhibition of Fc effector functions via FcaR and potentially interfere with protective antibody functions during viral infections. Notably, elevated IgA levels were associated with reduced vaccine efficacy and inhibited ADCC in the RV144 HIV-1 human vaccine trial. We investigated the Fc functional contributions of plasma IgA to HIV-1 phagocytosis during early and chronic HIV-1 infection (Chapter 5). We depleted IgA from plasma at two early and one chronic HIV-1 timepoint and assessed the IgA functional contribution. Notably, depletion of IgA at early timepoints resulted in significantly reduced ADNP (p < 0.05), suggesting IgA contributes to HIV-1 phagocytosis during early infection. However, depletion of IgA during chronic HIV-1 plasma enhanced ADNP, suggesting the IgA at this timepoint interferes with Fc-effector functions. Furthermore, we also observed IgA mediated inhibition of ADCP for various HIV-1 mAbs with purified IgA from people living with or without HIV-1, although the magnitude of inhibition is heterogenous amongst mAbs. We suggest this inhibitory effect is at least partially mediated via the FcaRI. However, this is a preliminary study and future studies are essential to investigate this phenomenon in greater depth with a larger cohort. Overall, our studies highlight the importance of Fc-effector functions and the complexity of the functional IgA response during SARS-CoV-2 and HIV-1 infections. Plasma IgA can induce potent neutralisation and contribute to ADNP during acute/ early infections. However, plasma IgA may interfere with Fc-effector functions during chronic HIV-1. Future studies should investigate the effect of IgA on other Fc-effector functions such as trogocytosis, complement activation and ADCC in different acute and chronic viral infections.
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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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ItemNucleic acid sensing in CD4 T cells during HIV-1 and other viral infections( 2021)Viruses are small intracellular parasites and use the host cell's biosynthesis machinery to replicate and spread. Therefore, viral particles incorporate structures that are similar to the ones that naturally occur within host cells. A major mechanism to identify viral entry and replication within an infected cell is the recognition of viral nucleic acids. Sensors of the innate immune system can detect foreign nucleic acids by their unusual subcellular localisation or modification or both. Once innate immune sensors are activated, they induce distinct signalling cascades which modulate cellular responses to invading pathogens like viruses. In this thesis, we studied the role of RNA sensors RIG-I (retinoic acid-inducible gene 1) and MDA5 (melanoma differentiation-associated protein 5) in CD4 T cells during infections with SeV (Sendai virus) or HIV-1 (Human Immunodeficiency virus 1). HIV-1 is the causative agent for the acquired immunodeficiency syndrome (AIDS). Globally, more than 30 million people are living with HIV 1 and hundreds of thousands of people are newly infected every year. Today, HIV-1 infection is a chronic and manageable disease. The progression to AIDS is prevented by ART (antiretroviral therapy) which inhibits viral replication but is unable to clear the latent viral reservoir - inactive HIV-1 proviruses within long-lived subsets of immune cells. These latent viruses are not detected by innate and adaptive immunity. Furthermore, HIV-1 manipulates cellular restriction factors and sensors of viral infection to evade immune recognition. This highlights the demand for new approaches to restore innate immune sensing during latency reversal to allow the specific killing of infected cells to the clearance of the latent reservoir. We first studied the RIG-I signalling pathway in human CD4 T cells, the main reservoir for HIV-1 infection in vivo. Using SeV, a specific activator of RIG-I, and a cell-based type-I interferon reporter assay we showed that the RIG-I signalling pathway was functional in activated CD4 T cells. In resting CD4 T cells, we did not detect the release of type-I IFNs and used next generation sequencing (NGS) to verify the expression of members of the RIG-I signalling pathway. A typical type I IFN signature was observed in resting CD4 T cells following the stimulation of RIG I with SeV. These data also showed the downregulation of pathways relevant for T cell activation. We next evaluated how the activation of the RIG-I signalling pathway affects the biology of CD4 T cells. RIG-I activation diminished proliferation, metabolic activity and release of effector cytokine IFN in CD4 T cells. RIG-I and MDA5 are potential sensors for HIV-1 and their role during HIV-1 infection is not fully understood to date. We discovered that HIV-1 protease (PR) directly degrades RIG-I and MDA5 independently of other cellular factors. We showed this by co-expression of HIV-1 PR and RIG-I or MDA5 in HEK293T cells and in an in vitro assay using purified recombinant HIV-1 PR and RIG-I or MDA5 proteins. The degradation of RIG-I and MDA5 by HIV-1 PR sequestrated the sensing of stimulatory RNAs in an in vitro reporter assay. These data indicate that the degradation of RIG I and MDA5 is a potential immune evasion mechanism for HIV-1 which could be exploited in novel HIV-1 cure approaches. Furthermore, we generated RIG-I and MDA5 knockouts in primary human CD4 T cells and Jurkat cells and performed initial characterisations of those cell lines. Knockout cell lines will be useful in future studies on the role of RIG-I and MDA5 during HIV-1 infection.
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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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ItemThe role of Major Histocompatibility Complex class I in Human Immunodeficiency Virus-1 and Simian Immunodeficiency Virus infections( 2016)The first official report of AIDS was in 1981. Since then, the HIV-1 infection has reached a pandemic state and high rates of infection are especially seen in poor developing countries. Intensive research has greatly prolonged the life expectancy of an HIV-1+ individual, but combined antiretroviral therapy (cART) is costly and the evolution of drug resistant HIV-1 strains are major obstacles to therapy. Thus, a prophylactic anti-HIV-1 vaccine will be ideal to address these challenges. The last thirty years have seen a few vaccine trials, with the most promising of these being the Thai RV144 trial with a vaccine efficacy of 31.2%. Subsequent research has demonstrated that the protective effect was likely conferred by anti-HIV-1 antibody-dependent cell-mediated cytotoxicity (ADCC) mediating IgG antibodies against Env, in the presence of low levels of IgA against Env. Cell types, such as, natural killer cells (NK cells), monocytes and neutrophils mediate ADCC. NK cell mediated ADCC is affected by NK cell education involving major histocompatibility complex class I (MHC I) alleles, as they are ligands for most NK cell receptors. A characteristic feature of HIV-1 infection is the peak viremia observed approximately two weeks after infection, which declines with the emergence of the cellular immune response such as CD8+ T cells. CD8+ T cells recognise virus infected cells through HIV-1 peptides bound to MHC I molecules on antigen presenting cells. Population studies have also demonstrated the role played by MHC I alleles in HIV-1 infection progression. Therefore, this thesis examined the role of MHC I in HIV-1 and SIV infections. SIV infected non-human primates are a valuable research tool to study immunity to HIV-1, trial candidate vaccines and drugs. The most well studied animal model is the SIV infected Indian rhesus macaque model. However, these macaques are difficult to obtain for research purposes due to export bans in India and the limited distribution of breeding colonies in the world. The macaque model we employ is the pig-tailed macaque sourced from breeding colonies in Australia and Indonesia. SIV infection in pig-tailed macaques closely mimics that in Indian rhesus macaques. Unlike in the SIV infected Indian rhesus macaque model, the MHC I alleles that control SIV infection progression in the pig-tailed macaque model are poorly defined. To date, only one allele, Mane-A1*084, has been linked with CTL escape in Gag and Tat proteins of SIV. In Chapter 2, we examined the role played by MHC I alleles in CTL escape in 44 SIVmac251 infected pig-tailed macaques. We extracted cellular RNA from 44 pig-tailed macaques and used 454 Roche pyrosequencing to determine the Mane haplotypes. Viral RNA was extracted from the plasma of these SIVmac251 infected pig-tailed macaques and we sequenced the whole genome of the virus using Illumina Nextera XT MiSeq sequencing. We also sequenced the challenge stock virus for the purposes of a reference genome during the bioinformatics analysis. Using a novel approach we first identified the well known CTL escape epitopes linked with Mane-A1*084, KP9 in Gag and KSA10 and KVA10 in Tat to validate our approach. We found over 70 potential CTL escape mutations. We validated one such result, a Nef CTL escape mutation associated with Mane-B028 using an intracellular cytokine staining (ICS) assay. Analysis of viral load data demonstrated that CTL escape driven by Mane-A1*084, as previously published by our group does not affect SIV infection control. Tetramer staining of expanded epitope specific CD8+ T cells, with tetramers synthesised for use in Indian rhesus macaques demonstrated that reagents used in rhesus macaques can also be used in pig-tailed macaques. Many studies have shown that cell-associated virus is more infectious than cell-free virus. We developed an assay to measure anti-HIV-1 NK cell activity against allogeneic T cells (Chapter 3) using a 29 cohort of HIV-1- individuals. We used gp120 coated T cells from donor 1, and combined whole blood from donor 2 in the presence of HIV-1+ plasma, brefeldin A, monensin and CD107a. NK cell activation was measured using intracellular IFNγ staining. NK cells were activated only in the presence of gp120 coated T cells and HIV-1+ plasma. We were able to modify this assay to investigate the contribution of different HLA/KIR interactions on anti-HIV-1 NK cell activation using an HIV-1- cohort. Epidemiological studies have demonstrated that the presence of KIR3DL1 and its ligand HLA-Bw4 confers protection and/or controls HIV-1 infection. We observed that educated KIR3DL1+ NK cells were able to overcome the inhibitory interaction between KIR3DL1 and HLA-Bw4, to show NK cell activation against HLA-Bw4+ gp120 coated allogeneic T cells. We also used a LDH cytotoxicity assay and showed cytolysis of gp120 coated CEM cells, in the presence of HIV-1+ antibodies. Another KIR/HLA combination of interest is the inhibitory KIR2DL1 and HLA-C2, which may protect from or control HIV-1. Studies on HIV-1+ African cohorts have shown that this KIR population expands following HIV-1 infection. In Chapter 4, we examined the role of this HLA/KIR combination using the above-mentioned modified assay. KIR2DL1+ NK cells from HLA-C2 donors showed higher levels of activation than KIR2DL1- NK cells, in the presence of gp120 coated CEM cells and HIV-1+ plasma. NK cell activation was not limited to antibody dependent stimulation, as a HLA devoid cell line called 721.221 also elicited NK cell activation in educated KIR2DL1+ NK cells. CD57 is a NK cell differentiation marker, and CD57+ NK cells showed higher NK cell activation than CD57- NK cells. CD57+ NK cells also had a higher frequency of KIR2DL1 expression as expected. Despite this, the enhanced activation observed in the educated KIR2DL1+ NK cell subset was not a result of differentiation, as CD57- KIR2DL1+ also showed high NK cell activation. An autologous ADCC assay showed that the functional advantage observed in educated KIR2DL1+ NK cells is a function of education. In conclusion, it is clear that host MHC I alleles play an important role in HIV-1 and SIV infections. We identified a number of MHC I alleles with the potential to drive CTL escape in SIV infection. Future studies to validate these findings will be useful for vaccine studies. This will enable the selection of macaques for vaccine trials and help determine the effect of the vaccine independent of host MHC I alleles. The Thai RV144 trial indicated that anti-HIV-1 ADCC, may be an important immune mechanism to target in anti-HIV-1 vaccine development. As detailed in Chapters 3 and 4, NK cell education plays an important role in the anti-HIV-1 NK cell activation in the presence of allogeneic T cells. Anti-HIV-1 ADCC was observed in the presence of low levels of surface Env on target T cells as discussed in Chapter 3, which is promising as HIV-1+ CD4 T cells are known to down-regulate the expression of CD4 and hence display low levels of surface HIV-1 Env. The studies in this thesis show that it is important to consider the influence of host genes in HIV-1 infection progression, in future anti-HIV-1 vaccine development studies.
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ItemElucidating unique HIV-1 subtype C genotypic and phenotypic characteristics to develop novel HIV-1 prognostics( 2015)Human immunodeficiency virus type 1 (HIV-1) causes acquired immunodeficiency syndrome (AIDS) by infecting cells of the immune system. HIV-1 infects target cells via an interaction between its envelope glycoproteins (Env), a cell-surface CD4 receptor and one of two coreceptors, CCR5 or CXCR4. The global spread of HIV-1 has enabled its evolution into a number of distinct subtypes. HIV-1 subtype C (C-HIV) accounts for >50% of HIV-1 infections worldwide and >95% of infections in Southern Africa and Central Asia. While relatively little is known about C-HIV, current evidence suggests that C-HIV pathogenesis is distinct from other HIV-1 subtypes. For example, although the pathogenesis of C-HIV is thought to predominantly involve CCR5-using (R5) viruses, we do not have a firm understanding of how frequently CXCR4-using viruses emerge during progressive C-HIV infection, nor do we completely understand the molecular mechanisms associated with coreceptor switching or cellular tropism. Within this thesis, these major gaps in the literature were addressed. Firstly, the coreceptor usage of a large panel of HIV-1 Envs (n=300) cloned from longitudinally collected plasma samples of 21 antiretroviral (ARV) therapy naïve subjects who progressed from chronic to advanced C-HIV infection was characterised. CXCR4-using C-HIV viruses emerged in only one individual. Subsequent Env mutagenesis studies showed that sequence alterations within the third variable loop (V3) region of Env were critical for determining C-HIV CCR5- or CXCR4-usage. Moreover, by demonstrating a statistically significant linkage between the ability of C-HIV Envs to use CCR5 and the alternative coreceptors CCR3, CCR8 and FRPL1 in vitro, these data suggest that in the absence of coreceptor switching, an altered more flexible use of CCR5 by R5 C-HIV Envs may be selected for during progressive C-HIV infection, which may contribute to C-HIV pathogenicity. Naïve and memory CD4+ T-cell subsets are the major cellular targets for infection by HIV-1. This thesis describes the first analysis of R5 and CXCR4-using C-HIV tropism for naïve and memory CD4+ T-cell subsets, including the recently described stem cell memory CD4+ T-cell (TSCM) subset. The studies described herein show that R5 and CXCR4-using C-HIV viruses preferentially infect memory and naïve CD4+ T-cell subsets, respectively, and that TSCM are susceptible to infection by R5 and CXCR4-using viruses. Mutagenesis and sequence analysis studies established sequence alterations within the V3 region of Env as the major determinants of C-HIV tropism for naïve and memory CD4+ T-cell subsets. Finally, the findings of these studies were used to inform the development of PhenoSeq; a suite of in silico prognostic tests that predict whether HIV-1 uses CCR5 or CXCR4 for entry into cells, based on distinctive sequence mutations within the V3 region of Env. Notably, because the ARV drug maraviroc (MVC) is contraindicated in patients harbouring CXCR4-using HIV-1 strains, a pre-MVC treatment prognostic test is required to determine whether patient’s circulating HIV-1 use CCR5 or CXCR4 for entry into cells. Since the current gold standard prognostic tests are expensive, time consuming and highly specialised, the comparatively rapid, user-friendly and free-to-use PhenoSeq platform (www.burnet.edu.au/phenoseq) has the potential to greatly improve patient access to MVC, in particular for HIV-1 infected individuals residing in resource-constrained regions that are burdened the most by the HIV-1 pandemic. In conclusion, the published outcomes of this thesis have the potential to expedite future research into C-HIV pathogenicity, HIV-1 vaccines and ARV treatments, and may improve patient access to MVC.
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ItemThe potential role of antibody dependent cellular cytotoxicity in protection from HIV-1( 2013)Background: Antibodies (Ab) mediating cellular cytotoxicity (ADCC) could potentially provide protection from HIV-1 acquisition in a vaccine setting. ADCC Abs mediate the cytolysis of HIV-1 infected target cells through the cross-linking of the Fc portion of the Abs by effector cell populations bearing Fc receptors (FcRs) such as NK cells. ADCC activated effector cells also secrete pro-inflammatory cytokines, which helps to recruit other immune cells to aid in the clearance of infected cells. ADCC responses occur naturally in HIV-1 positive people and have often, but not always, been associated with slower progression of HIV-1 to AIDS. In addition, ADCC responses were elicited by the recent RV144 HIV-1 vaccine trial carried out in Thailand. This trial provided a modest 31.2% reduction in HIV-1 acquisition in the vaccinated cohort. By studying ADCC responses elicited naturally and by vaccination, the potential of ADCC responses to protect from HIV-1 acquisition can be explored. Furthermore a better understanding of ADCC targets and methods for manipulating or enhancing this potentially beneficial immune response will enable the development of more effective HIV-1 vaccines. Methods: ADCC responses against whole Env glycoprotein (gp) 140 and multiple linear HIV-1 overlapping peptide pools were analysed in 65 Long term slow progressors (LTSP) and 74 subjects with progressive HIV-1 disease. Studies were carried out using an assay that assessed ADCC activity based on the activation of NK cells. We evaluated the effect of Ab purification from the plasma of HIV+ subjects and the effect of a number of HIV-relevant soluble factors on the activation profile of healthy donor NK cells using the NK cell activation ADCC assay. This assay was used in conjunction with a rapid fluorescent ADCC (RFADCC) assay, which measures ADCC based on target cell cytolysis, to assess ADCC responses elicited by the phase II RV135 and phase III RV144 HIV-1 vaccine trials. Results: We found that the breadth but not the magnitude of ADCC responses was correlated with slow progression of HIV-1. In addition regulatory/accessory HIV-1 proteins were targeted more frequently in LTSPs. ADCC responses targeting HIV-1 Vpu were over represented in LTSPs and Vpu-specific ADCC Abs were mapped to 3 distinct epitopes within the protein. The cytokines interleukin (IL)-15 and IL-10 were found to consistently enhance both the cytokine production and degranulation of ADCC activated NK cells. Envspecific ADCC responses were detected in plasma samples from the RV135 and RV144 trials, consistent with recent reports in the literature, although improved methods will be needed to map epitope-specific responses. Conclusions: Broader ADCC responses may play a role in long-term control of HIV-1 progression in a subset of LTSPs. Conserved ADCC epitopes within HIV-1 Vpu may represent potentially beneficial vaccine targets. The cytokine milieu influences the ADCC effector cell response and those involved with enhancing the magnitude of ADCC responses may be useful vaccine adjuvants. Improvements to the breadth, specificity and durability of the HIV-1-specific ADCC responses elicited by the RV144 trial could improve vaccine efficacy to the point where it would be a viable vaccine to administer to large human populations.
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ItemAntigenicity and immunogenicity of envelope glycoproteins from HIV-1 preseroconversion strains: potential novel vaccine candidates( 2012)An efficacious vaccine is needed to combat the spread of HIV-1 and novel immunogens are required to facilitate this. Immune selection pressure modifies HIV-1 envelope glycoproteins (Envs) during the course of infection to drive escape from neutralizing antibodies (NAbs). However HIV-1 preseroconversion (PSC) strains proliferating in the absence of selection pressure promote Envs featuring reduced glycosylation and shorter variable loops (VLs). These features may potentially favour exposure of neutralization sensitive epitopes and hence PSC Envs as immunogens could elicit NAbs. Blood samples were collected from 38 HIV-1 MSM (men who have sex with men) subjects of the PSC cohort. Based on preliminary env sequence analysis, five samples with fewer potential N-linked glycosylation sites (PNGS) compared to pNL4.3 reference strain were chosen to generate env gp160 clones. These were genotyped for changes in PNGS and length of VLs in comparison to HXB2 (X4) and AD8 (R5) strains and also coreceptor (CoR) usage and changes in binding sites of CD4 and known broadly NAbs (bNAbs) were analyzed. PSC Envs were phenotyped for Env mediated entry, CoR usage, receptor affinity and neutralization sensitivity to CD4 and bNAbs. PSC gp140 clones were constructed and engineered to secrete soluble gp140 trimers and these were assessed for exposure of CD4 binding site (CD4bs) and bNAb epitopes by ELISA and surface plasmon resonance. PSC gp140 DNA and protein immunogens were tested in mice and sera were assessed for binding antibody titres and avidity by ELISA and NAb responses against HIV-1 pseudoviruses by TZM-bl assay. PSC Envs were further tested in rabbits as systemic and mucosal immunogens. A novel lipopeptide R4Pam2Cys adjuvant was primarily used for both systemic and mucosal routes. Montanide and R4Pam2Cys adjuvant combinations for systemic and mucosal routes respectively were also tested. Sera, nasal lavages, cervicovaginal lavages and faecal pellets were evaluated for binding antibody titres and NAb responses against HIV-1 pseudoviruses. Genotypic analysis showed that PSC env clones at intrapatient level were highly identical with fewer PNGS in V1 and V4, shorter VLs in V1, V3 and V4 and fewer changes in binding sites of CD4 and bNAbs. Phenotypic analysis showed that PSC Envs mediated functional entry, were R5 tropic with high efficiency for CCR5 usage and were neutralization sensitive to CD4 and bNAbs b12, 2F5 and 4E10. Antigenicity studies by ELISA and SPR showed exposure of CD4bs and epitopes of bNAbs b12, 2F5 and 447-52D. Immunogenicity studies testing PSC Envs in mice raised high titres of IgG with high avidity, moderate IgA and IgM and bNAbs against clade B and C pseudoviruses. Systemic and mucosal immunization of PSC Envs in rabbits raised effective humoral responses and the group receiving combination of Montanide and R4Pam2Cys for systemic and mucosal routes respectively showed sustained responses. Systemic responses included very high IgG titres with high avidity, low IgA and moderate IgM responses and NAb responses were highly potent but not broad. Mucosal responses included moderate to low IgG, low IgA and low/no IgM responses and NAb responses were broad but the potency varied across the different mucosal compartments. Thus the PSC Envs studied here displayed less glycosylation and shorter VLs compared to HXB2 and AD8 reference strains and exposed bNAb epitopes. When these PSC Envs were tested in mice and rabbit vaccination experiments these antigenic features promoted effective systemic and mucosal humoral responses. Challenge studies in non-human primates will allow further evaluation of the protective potential of PSC Env immunogens.