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Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Current gene delivery systems suffer from poor genetic delivery into resting T-cells and therefore, compensate for this by using multiple viral challenges, by activating and expanding the cells ex vivo. However, cells activated and expanded in this manner have lower engraftment, proliferative potential and potency. In contrast, the use of resting T-cells overcomes many of these short-comings, but comes with restrictions: Firstly, at the plasma membrane, due to the lack of receptor for the commonly used lentiviral envelope pseudotype, VSVg. Secondly, at reverse transcription (RTN), by intracellular restriction factor, SAMHD1.At the membrane restriction, we focused on the abundant expression of CXCR4 on resting T-cells, by testing a panel of lentiviral systems expressing various CXCR4-dependent, CD4-independent HIV envelopes. Results revealed that the above panel fused poorly or not at all within resting T-cells. The lead envelope was initially derived from HIV-2 VCP isolate, which was dependent on HIV-2 Gag to maintain high fusogenicity. Whilst HIV-2 based lentiviral systems are advantageous; they are not as developed as HIV-1 systems. Thus, we re-investigated a HIV-1 system based on a derivate of iR3A isolate that enters resting T-cells primarily through CXCR4, but to do so, it required modifications to its matrix.Fortunately, SIV and HIV-2 have evolved with the accessory gene, vpx, that directly targets SAMHD1 for degradation and can be readily incorporated into lentiviral particles. Yet it is presently unknown if all vpx genes are equal in their ability to antagonise SAMHD1 and/or enhance RTN. Through bioprospecting a genetically diverse panel of vpx, we tested SAMHD1 antagonism across several cell types. Majority of the Vpx proteins could overcome SAMHD1 restriction in dendritic cells and macrophages, but only one lead Vpx could efficiently achieve this in resting T-cells, and this was due to an additional Lysine within its nuclear localisation signal. Results fu

The HIV-1 capsid is a protein shell utilised by the virus as a binding platform to hijack host machineries to promote infection. Characterisation of novel cofactors binding to capsid demands sensitive and quantitative assays to better understand their underlying binding mechanisms. Here, I have developed three complementary fluorescence microscopy-based platforms for quantitative characterisation of capsid binding analytes using tubular, spherical or conical lattices self-assembled from recombinant capsid protein (CA) with engineered cysteine residues for cross-linking as surrogates for the authentic capsid. The first approach focused on the development of a TIRFM biosensor using CA tubes immobilised on a glass surface as the biorecognition substrate for sensitive single-molecule binding studies. The biosensor was assessed in its ability to measure binding affinity, stoichiometry and kinetics using known capsid-binding cofactors as part of its validation process. Biosensor measurements confirmed that binding of the host cell protein cyclophilin A (CypA) to the tubular lattice is substoichiometric due to steric hindrance between CypA molecules. Binding analysis of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) expressed as a full-length protein in a cell-free expression system suggested a role of oligomerisation to enhance binding via avidity. In the second approach, the biosensor surface was modified with cross-linked CA spheres as an alternative substrate containing pentameric defects and highly curved regions that are lacking in CA tubes. Binding of CypA to spheres revealed a significantly higher CypA to CA binding ratio at saturation suggesting a role of curvature in accommodating specific host cofactors. The third approach utilises confocal microscopy-based single molecule spectroscopy and was developed as a medium-throughput capsid interactor screening platform that uses conical CA self-assemblies as the substrate. Fluorescence-tagged analyte

HIV has evolved elaborate mechanisms to manipulate the host cell’s actin cytoskeleton. This supports specific steps of the viral life cycle but also leads to cell-type specific changes in leukocyte shape and behaviour that promote overall spread of the infection. In particular, actin manipulation is important for direct cell-cell transfer of HIV (CCTH), which is now recognized as a highly efficient mode of viral spread.This study aimed to further resolve the molecular players and mechanisms involved in this process.Through the use of CRISPR–Cas9-mediated gene editing, herein we systematically interrogated the loss-of-function phenotype of over 50 cytoskeletal regulators, in both HIV-donor and target cells during CCTH. This was then combined with a portfolio of complementary assays ranging from live cell microscopy through to the ultrastructural resolution of focused ion beam scanning electron microscopy. This approach identified several core actin regulators to be required on both sides of viral transfer, including the Rho-GTPases Cdc42/Rac1 (signalling), upstream of the Arp2/3 complex (actin nucleator), and Profilin (which indirectly supports F-actin elongation, e.g. via Formins). In infected myeloid cells, Cdc42-Arp2/3 and Formins collaborated to form distinct “HIV-Filopodia”, which were shown to be important for CCTH. The overall findings of this work support that, while HIV uses diverse strategies to influence numerous cytoskeletal targets, there is a high tolerance for the individual loss of many manipulated regulators in the context of CCTH. However, core regulators that secure the cellular ability to mediate F-actin polymerization remain essential to this process, in particular via their contribution to cortical F-actin membrane protrusions that facilitate intercellular contacts and later maturation of the virological synapse. To conclude, we have mapped a landscape of complex interactions between HIV and the actin cytoskeleton. This not only builds on our un

An HIV-1 cure using gene therapy is presently proposed. Past and present inroads to genetically manipulate immune cells have specified the use of generalised gene therapy vectors, alongside extensive and prolonged cellular activation to impart vector compatibility with immune cells. Whilst these methods increase gene delivery outcomes, the harsh cellular conditioning regimen lowers cellular engraftment and proliferative potential.Given these consequences, perhaps optimising the viral vector, and not the target cell, could improve gene therapy outcomes. Two major challenges were firstly identified: overcoming a block at the viral membrane that inhibited particle delivery; and circumvention of a restriction factor, SAMHD1, that inhibited gene delivery post-entry. It was hypothesised the first barrier could be overcome by selecting an optimised pseudotype. Indeed, from many prospective pseudotypes, one lead candidate was identified. This pseudotype, consistently enabled cellular entry in greater than 80% of cellular targets.Whilst this solved the limitation of inefficient particle delivery, gene delivery was still inhibited by SAMHD1. By incorporating the lentiviral accessory protein Vpx into vectors, SAMHD1 could be depleted and this enabled achievement of gene delivery efficiencies approaching 40%. Importantly, these observed from the window of untouched resting CD4+ T cells.Further inroads into the characterisation of diverse Vpx variants yielded additional insights into how Vpx increases gene delivery. Firstly, several variants exist that enhance gene delivery greater than the canonical variant (SIVmac239). Secondly, T cell-tropic Vpx variants exist, that do not enhance gene delivery in macrophages. And finally, Vpx-mediated enhancements are mechanistically biphasic.Overall, this thesis described the development of a highly modular, custom gene therapy particle platform with broad- reaching applications encompassing efficient protein or gene delivery into multiple

Current anti-retroviral treatment (ART) for HIV-1 is highly effectively at controlling the infection. However, during early infection the virus establishes a latent reservoir, which is not impacted by ART. Any treatment interruption rapidly results in virus rebound from the latent reservoir to pre-therapy levels and thus ART does not constitute an HIV-1 cure. Alternate treatments are currently being explored in the form of gene therapy, following the success of the Berlin patient who has had undetectable virus since 2007. This review will describe the contrasting cure approaches that are currently the focus of multiple studies to control HIV, then focus in on functional cure gene therapy strategies; specifically, epigenetic silencing of HIV-1 by various methods, including RNA-directed transcriptional gene silencing. The various delivery strategies for targeting cells of the latent reservoir will be reviewed and finally, the clinical status and current challenges for ex vivo versus in vivo gene therapy delivery approaches will be discussed.

The entry of HIV-1 into target cells and viral spread among cells of the immune system are two criticalevents in the viral life cycle. This study aimed to further characterise HIV-1 pathogenesis three-fold.Firstly, development of fluorescent markers for HIV-1 content and lipid resulted in a novel system togenerate fluorescent HIV-1 particles with near wild-type infectivity for use in single particle imaging.Through stringent analysis of the viral progeny, two key caveats of HIV-1 particle tracking wereidentified. Firstly, the viral content was compartmentalised both within and outside of the HIV-1capsid core. Also, although improvements were made to the lipid label, significant numbers offluorescent microvesicles that co-associate with HIV-1 were recognised. These key findings influencethe interpretation of these particles when used for single particle tracking.Secondly, this fluorescent virus was used to characterise one of the most potent forms of viral spread,transfer across the dendritic cell and CD4+ T cell synapse. In this context, a new pathogenic structurewas identified, which we now term HIV-1 filopodia. These finger-like structures protrude from themembrane and are used by the newly formed virions as a means of cell-cell transfer. The spread ofHIV-1 via viral filopodia was found to be reliant on the viral protein Nef, specifically in the domainthat interacts with the cellular protein Dynamin-II.Finally, using a panel of novel small molecule inhibitors, along with well characterised virologicalmethods, it was investigated whether endocytosis was a pre-requisite for entry into primary cell targetsof HIV-1. Significant post-entry blocks of viral infection were detected when using Dynamin-IIinhibitors. In studies of primary cells, limited inhibition of HIV-1 entry was observed in macrophagesand resting CD4+ T cells, while viral entry was blocked in HeLa-based and activated CD4+ T cells.This thesis provides new, stringent techniques for visualising HIV-1 entry