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.