Microbiology & Immunology - Theses
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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.
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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.