Microbiology & Immunology - Theses
Permanent URI for this collection
Search Results
1 - 5 of 5
-
ItemTargeting the untargetable: Eliminating HIV latency using nanoparticle delivery systems( 2023-04)T cells form an important therapeutic target for the development of immunotherapies or the treatment of T cell-implicated pathologies. Specifically, CD4 T cells are subject to human immunodeficiency virus (HIV) infection. Whilst treatment with antiretroviral therapy (ART) successfully represses the viral load to undetectable levels, HIV treatment is lifelong, posing a medical, social and financial burden to those 38.5 million people currently living with HIV globally. The ability of HIV to establish a reservoir of latently infected cells is the foremost barrier to finding a cure for HIV. One approach towards eliminating HIV latency is the reactivation of viral transcription and subsequent elimination of infected cells through immune-mediated clearance or viral-mediated cytotoxicity. However, this approach so far has suffered from a lack of potency and dose-limiting toxicities due to the use of compounds that affect both host and viral transcription and the inability to specifically target the infected cells. One solution involves the use of nanoparticles for the targeted delivery of existing therapeutics or to advance the development of HIV-specific mRNA-based therapeutics including CRISPR-Cas. However, the generally low rate of endocytosis in CD4+ T cells forms a challenge to efficient nanoparticle-based drug delivery to CD4+ T cells in vitro and in vivo. This thesis describes our efforts towards rationally designing a nanoparticle platform capable of delivering HIV latency-reversing therapeutics to CD4+ T cells with high efficiency. We first established a methodology to improve the assessment of nanoparticle performance in vitro through the generation of absolutely quantitative, comparable data on nanoparticle-cell interactions. We then used this methodology to screen for nanoparticle designs with enhanced uptake kinetics in CD4+ T cells in vitro, using a novel, high-throughput assay to quantify nanoparticle internalization over time. We found that targeting sub-100 nm nanoparticles to T cell surface receptors undergoing rapid receptor cycling can be exploited to actively trigger nanoparticle uptake through receptor-mediated endocytosis and identified CD2 and CD7 as potent candidate receptors for future in vivo T cell targeting. We next aimed to use translate these findings to lipid nanoparticles, a well-established platform for the delivery of nucleic acid-based therapeutics. We investigated whether lipid nanoparticles could be used to deliver a next-generation latency-reversing agent based on CRISPR activation, which specifically targets the HIV proviral genome without affecting host-cell transcription. We identified a novel lipid nanoparticle formulation that can efficiently transfect T cell lines as well as resting CD4+ T cells. We showed that this lipid nanoparticle can co-encapsulate the three RNA components of the CRISPR activation system and induce strong latency reversal in a cell line model for HIV latency. We finally presented preliminary evidence that targeting lipid nanoparticles to rapidly cycling surface receptors increases mRNA delivery, further supporting our findings that targeting receptor-mediated endocytosis could be a viable strategy to increase nanoparticle-mediated drug delivery to traditionally hard-to-transfect T cells. These findings provide a compelling justification for the further assessment of CRISPR activation lipid nanoparticles for the elimination of the latent HIV reservoir, and more broadly contribute to the development of T cell-targeted nanomedicine for HIV and beyond.
-
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.
-
ItemHIV persistence, inflammation and the gut( 2021)Human Immunodeficiency Virus (HIV) remains incurable despite antiretroviral therapy (ART) due to its persistence as integrated provirus within long-lived and proliferating CD4+ T cells. Following initial infection, it replicates efficiently within and depletes Th17 cells which are enriched within the gut and play an important role in maintaining gut barrier integrity. This depletion leads to gut barrier permeability, bacterial translocation across the gut wall and local and systemic inflammation. These findings do not completely resolve on ART and may contribute to HIV persistence by promoting proliferation of infected CD4+ T cells and/or exhaustion of an effective immune response against HIV. Integrated provirus is enriched within Th17 cells in people living with HIV on ART. Th17 cells express CCR6 which promotes their migration towards CCL20 secreted by gut epithelium. Our laboratory has previously shown that CCL20 can promote HIV latency establishment in vitro likely through depolymerisation of the cortical actin barrier of resting CD4+ T cells allowing HIV to traverse this barrier and migrate to the nucleus. It is unknown to what extent CCL20-mediated gut migration or actin depolymerisation might contribute to HIV persistence in vivo. We compared the half-maximal effective concentration (EC50) of CCL20 for HIV latency establishment within resting memory CD4+ T cells with that of AKT phosphorylation, polarisation (a surrogate for actin depolymerisation) and migration of these cells. We reasoned that if the EC50 for HIV latency establishment were considerably lower than the EC50 for any of these cellular processes then that cellular process may not be required for CCL20-induced HIV latency establishment. We found progressively increasing EC50s for AKT phosphorylation, polarisation and migration; however, we were unable to demonstrate an effect of CCL20 on latency establishment. This was independent of viral tropism. Discrepancy with our laboratory’s previous findings may have been due to a shorter duration of exposure to chemokine in our experiments and/or a shorter resting time between cell sorting and infection which may have influenced cellular activation state and hence permissiveness to infection independent of chemokine. Vitamin D3 is a steroid hormone with pleiotropic effects on the immune system including reductions in CD4+ and CD8+ T cell activation, proliferation and exhaustion and reductions in frequency of Th17 and Th1 cells, both of which are important HIV reservoirs. It has also been shown in animal models to promote gut barrier integrity and in humans to reduce gut dysbiosis. We hypothesised that vitamin D3 may be able to deplete the HIV reservoir through these systemic and gut anti-inflammatory effects. We performed a randomised placebo-controlled trial evaluating the effect of 10,000 international units vitamin D3 per day for 24 weeks on markers of HIV persistence and immunology. Participants were followed for an additional 12 weeks post treatment. The primary endpoint was the difference between treatment arms in the change in frequency of total HIV DNA within CD4+ T cells from baseline to week 24. We found no effect of vitamin D3 on the primary endpoint. However, we found an increase at week 12 and a decrease at week 36 in frequency of total HIV DNA in the vitamin D3 arm relative to placebo. Importantly, 25-hydroxyvitamin D levels were still elevated at week 36 in the vitamin D3 arm relative to the placebo arm likely due to its long half-life. We also found a shift away from more differentiated subsets towards central memory CD4+ and CD8+ T cells at all time points including a reduction in frequency of effector memory CD4+ T cells at week 36. Other findings included a reduction in frequency of Th1 cells and levels of monocyte activation as expected but a paradoxical increase in frequency of Th17 cells and activated CD8+ T cells and NK cells in the vitamin D3 arm relative to the placebo arm. We hypothesise that the decrease in total HIV DNA frequency at week 36 may be due to the reduction in frequency of effector memory CD4+ T cells, known to be enriched in HIV DNA, which in turn may reflect the known anti-proliferative activity of vitamin D3. Increases in CD8+ T cell and NK cell activation may also have contributed to depletion of the HIV reservoir at week 36. Our findings support larger clinical trials which could incorporate an analytical treatment interruption to determine whether vitamin D3 is able to exert a clinically meaningful impact on the HIV reservoir.
-
ItemCharacterisation of Neutralising and Functional Antibody Responses to Different HIV-1 Env Vaccines in Bovines( 2020)Two main challenges have impeded the development of an effective HIV-1 envelope (Env) vaccine, with antibodies eliciting neutralisation of virions as well as Fc-effector functions, such as antibody-dependent cytotoxicity (ADCC), phagocytosis (ADP) or complement deposition (ADCD). On one hand, designing the right Env vaccine to elicit humoral or cellular protection has been challenging and, to date, SOSIP-Env trimers which are covalently constrained in the closed, pre-fusion conformation are the best vaccine candidate over uncleaved (Unc), open-structured trimers. On the other hand, eliciting heterologous neutralising antibodies in several animal models (including humans) has been difficult. Cows nevertheless produce unique antibodies with long CDRH3 regions, capable of accessing neutralising epitopes beneath the glycan shield, inaccessible for other animals. We tested how differences in clade and/or structure of HIV-1 Env vaccines affect the neutralising activity and Fc-effector functions of antibodies elicited, using recombinant trimers of clades A (KNH1,BG505), B (AD8, PSC89) and C (MW), which exposed either an open structure (Unc gp140) or a closed structure (SOSIP gp140). KNH1/BG505 SOSIP gp140 vaccine elicited the best neutralising IgGs against heterologous tier-2 pseudoviruses with high potency and breadth. While AD8 Unc gp140 also induced neutralisation, it was against only tier-1 pseudoviruses. Nevertheless, it was the only vaccine able to elicit IgGs that engaged CD32 (FcgRIIa), induced phagocytosis and complement-activation. The different antibody profile observed with both vaccines was explained by the Env immunogen structure, as KNH1/BG505 SOSIP gp140 induced mostly IgGs targeting the V1/V2 loop, whereas AD8 Unc gp140 induced antibodies targeting CD4-binding site and CD4-induced epitopes. In addition, analysis of IgG repertoires from animals of KNH1/BG505 SOSIP 100 and AD8 Unc 500 groups showed that KNH1/BG505 SOSIP gp140 induced higher rates of somatic hypermutation in germline genes compared to AD8 Unc gp140, with each animal presenting a unique antibody profile, and with germline antibodies already presenting high affinity towards HIV-1 Env trimers, as high levels of affinity maturation were not required to obtain antibodies with high neutralising activity. Overall, the results in this work show that open structured trimers elicit antibodies which highly activate antibody-effector functions, while SOSIP trimers focus antibody responses to concealed neutralising epitopes. The high neutralising responses observed in bovines against HIV-1 Env are due to antibodies which do not need high levels of somatic hypermutations and, in particular for KNH1/BG505 SOSIP, this antigen induced high levels of affinity maturation, probably favouring the improvement of both binding and neutralisation. Our study suggests that an effective vaccine regimen may include both uncleaved gp140 and SOSIP gp140, in order to target epitopes required for antibody-dependent effector functions as well as neutralisation, or a new trimeric structure with flexibility in the gp120-gp41 interface, exposing both epitopes involved in Fc-effector functions as well as neutralising ones.
-
ItemFc-functional antibody immunity to HIV: the role of neutrophils and IgA( 2020)A safe and effective prophylactic vaccine against HIV-1 is an essential component to limit the HIV-1 epidemic. The RV144 HIV vaccine efficacy trial has highlighted the importance of generating Fc functional antibodies to prevent the further spread of HIV infection. Fc functional antibody responses have also been shown to correlate with delayed HIV disease progression. Despite the intensification of interest in Fc-mediated responses to HIV infection, there has been limited research focused on the Fc functional capacity of neutrophils, which are a key innate immune cell at mucosal surfaces and in the blood. The majority of Fc-effector studies in HIV focus upon examining NK cells and/or monocytes responses, while other effector cells such as neutrophils remain understudied. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses therefore, other immune cells like neutrophil are necessary for IgA to be studied. Neutrophils are highly functional innate effector cells with the potential to induce both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis. In chapter 2, methods were optimized to evaluate antibody-dependent neutrophil phagocytosis (ADNP) and neutrophil-mediated rapid fluorometric antibody-dependent cellular cytotoxicity (RFADCC) effector responses, using freshly isolated primary human neutrophils from blood. In vitro, neutrophil-mediated RFADCC responses peaked at 4 hours, which was faster than primary NK cells or monocyte mediated responses. There was a large spectrum of responses of both ADNP and neutrophil-mediated RFADCC responses across a cohort of 41 viremic antiretroviral-therapy naive HIV positive subjects. ADNP and RFADCC responses correlated well with each other, suggesting that they measure overlapping functions. The viral load of the patients inversely correlated with the ADNP responses, suggesting that these antibody-mediated neutrophil-based assays could prove useful in dissecting HIV-specific immunity. The role that IgA plays in active HIV infection remains controversial, with some reports of HIV-specific IgA being able to inhibit HIV infection and potentially being protective. Chapter 3 investigated if HIV progression was influenced by HIV-specific ADNP and neutrophil-mediated RFADCC responses and the effects of IgA on these responses. It was shown that, although neutrophil-mediated RFADCC responses were higher in the plasma of subjects who controlled their viremia levels (viremic controllers), IgA from both viremic controllers and viremic non-controllers inhibited both ADNP and neutrophil-mediated RFADCC responses similarly. The IgG mediated ADNP responses from both viremic controller and viremic non-controllers were broadly inhibited by both autologous HIV positive IgA and HIV negative pooled purified IgA. The IgA inhibition was able to be blocked by pretreating neutrophils with an Fc alpha receptor (FcalphaR) blocking antibody. This suggests that IgA inhibition of ADNP responses can be mediated by 2 mechanisms; 1) antigen dependent, FcalphaR independent and 2) antigen independent, FcalphaR dependent. The RV144 vaccine trial has generated interest in Fc functional antibodies and in the role that HIV-specific IgA can play during HIV vaccination strategies and in HIV infection. The RV144 vaccine induced IgG antibodies that were able to mediate ADCC responses. However, the vaccine efficacy was reduced in the presence of high concentrations of HIV-specific IgA. Monoclonal IgA that was isolated from the plasma of the RV144 vaccinees was able to block the potentially protective IgG antibodies from binding similar epitopes, thus preventing ADCC responses with NK cells. This indicates there was epitope competition between IgA and IgG antibodies in the RV144 vaccine trial. NK cells lack the FcalphaR and cannot mediate any IgA-dependent Fc-mediated effector responses. Chapter 4 assessed plasma samples from the RV144 vaccine trial for their ability to induce neutrophil-mediated responses and if IgA was able to inhibit these responses. IgG from the RV144 vaccinees was able to induce modest HIV-specific ADNP and neutrophil-mediated RFADCC responses. Plasma IgA from the vaccinees was able to inhibit ADNP responses but not neutrophil-mediated RFADCC responses. Using pooled IgG from the vaccinees, it was shown that pooled purified IgA from vaccinees, pooled purified IgA from HIV positive donors and pooled purified HIV negative IgA were able to inhibit the IgG mediated ADNP responses. Overall, this thesis shows that neutrophils can mediate HIV-specific antibody-dependent phagocytosis and neutrophil-mediated RFADCC responses. HIV-specific IgG mediated neutrophil responses, induced by either infection or vaccination, can be inhibited by plasma IgA in an antigen dependent mechanism and an antigen independent mechanism that is a FcalphaR dependent mechanism. The inhibitory effects of IgA may assist in understanding HIV pathogenesis and improving future HIV vaccine designs.