Medicine & Health

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Now showing 1 - 9 of 9
  • (2021) Abayasingam, Arunasingam
    Neutralising antibodies (nAbs) are capable of blocking viral entry to host cells. Understanding the nAb response in early infection informs preventative and therapeutic interventions. The acute phase of hepatitis C virus (HCV) infection is characterised by two bottlenecks of viral diversity, one at the initiation of infection (selection of transmitted/founder (T/F) variants), and another within 100 days of infection (emergence of fit variants against host immunity). Three out of four patients with HCV develop chronic infection which is associated with a delayed nAb response against variants emerging from the second bottleneck. However, the ability for HCV T/F variants to induce nAbs is poorly understood. This first part of this thesis aimed to characterise T/Fs using next-generation sequencing, and determine if the immunogenicity of T/Fs was associated with infection outcome. Approximately one in four primary HCV infections were found to originate from a single T/F virus and in those with spontaneous clearance of infection, T/Fs have a stronger binding affinity towards cluster of differentiation (CD) -81, a key receptor for HCV cell entry. T/Fs of spontaneous clearers were also more susceptible to neutralisation compared to chronic progressors. The second part of this thesis describes how the techniques utilised to study nAb responses in HCV were adapted to study Severe acute respiratory syndrome (SARS) coronavirus (CoV) -2, hoping to unravel the comparable nAb protection in coronavirus disease 2019 (COVID-19) infection. A new assay to study SARS-CoV-2 neutralisation was optimised and used to characterise the longitudinal nAb responses in individuals recovering from COVID-19 infection, and up to six months post-infection. In the majority, nAb responses (titers and neutralisation capacity) were evident, but declined modestly, with most being above background at six months post-infection. This decline was associated with older age, but not with gender or illness severity. In summary, this thesis examined the role of viral and nAb traits associated with two different viruses and their varied infection outcomes. The findings contribute to better understanding of interactions between virus and host antibody responses, which is critical for vaccine design.

  • (2018) Rasoli Pirozyan, Mehdi
    The CD8+ T cell responses play a pivotal role in controlling viral replication during HCV infection. HCV evades the immune system by rapid viral evolution affording escape from immune selection pressure including at MHC-I restricted epitopes. However, some CTL epitopes remain conserved well past the time of establishment of chronic infection, implying additional mechanisms immune failure exists. CD8+ T cells exhibiting an exhausted phenotype have been extensively reported during the chronic stage of illness for chronic viral infections, such as HCV and HIV. Additionally, impaired differentiation and trafficking of CD8+ T cells is known to be associated with immune escape and exhaustion of CTLs, but the timing and mechanisms and expression patterns of inhibitory receptors as wells as impairments in differentiation during primary HCV infection remains unclear. HCV-specific CD8+ T cell responses against the transmitted founder virus identified via ELISpot. Immune escape was observed in the NGS data set in ~33% of all ELISpot identified epitopes. The majority of HCV-specific CD8+ responses identified via IFN- ELSPOT in chronic progressors were also characterised by a dominant population of terminally differentiated effector memory cells (CCR7lowCD45ROhighKLRG1highCD127low), and elevated expression of co-inhibitory markers (PD-1 and 2B4) targeting both conserved as well as escaped HCV variants at the peak of immune response (as early as 70-90 days post infection). However, evidence of long-term central memory subpopulations with moderate IFN-γ production was identified in a subset of responses. There was an association of viral escape with the magnitude (IFN- production) of the response, suggesting ongoing evolution of CTLs in response to prolonged viral exposure. Analysis of T-bet expression revealed that T-bet expression on HCV-specific CD8+ T cell was not associated with clearance. Immuno-phenotyping of liver showed that, liver was enriched with T cells expressing the chemokine receptors CCR2, CCR5, CXCR3, and CXCR6. Additionally, the studies revealed preferential expression of CXCR3 on HCV-specific CD8+ T cells in both chronic and acute HCV infection suggesting a key role for CXCR3 in regulation of HCV-specific CD8+ T cell trafficking to the site of infection in the liver. Taken together the studies in this thesis provide both consistent findings with more limited studies in HCV and comparable contexts in HIV, and clear contrasts with previous reports in murine LCMV models. The findings offer novel insights into our understanding of the immunopathgenesis of primary HCV and into HCV vaccine design.

  • (2022) Van Der Byl, Willem
    This thesis aims to investigate T cell differentiation through the bioinformatic analysis of single-cell multi-omic data. T cells are an important part of the adaptive immune system, involved in the immune response to infections and cancer. Single-cell technologies have advanced to the point where multiple modes of data, such as gene and protein expression, can be assayed on the same cells. Greater understanding of T cell differentiation pathways at the single-cell level can help in the design of immunotherapies to treat cancer and autoimmune disease. The thesis begins by presenting a multi-omic workflow that combines scRNA-seq and T cell receptor (TCR) sequence extraction. The principles developed for this workflow were applied to investigate T cell differentiation in two scenarios. The first scenario was an application of single-cell multi-omics to the study of CD8+ T cell peripheral tolerance mechanisms in a mouse model. This work demonstrated that tolerance is a distinct differentiation program to functional effector responses, and T cells progressively commit to the tolerised state over the first 60hrs post exposure to triggering antigen. A gene signature for the tolerised state was identified, containing genes uniquely upregulated in tolerised cells. Quiescent and Proliferating clusters were found in tolerised cells, indicating that a proportion of cells exit cell cycle within each division. The second scenario was an investigation of the differentiation of CD4+ CAR T cells in vivo, and the evolution of a lymphoma derived from these cells. Three cell types, proliferating, cytotoxic and resting, were observed within the malignant CAR T-cells, and these types were also observed within non-malignant CAR T and endogenous CD4+ T cells. The lymphoma was characterised by expression of the NF-κB transcription factor in all three cell types, while each cell type had differing expression levels for several other known oncogenes. This thesis has contributed to the understanding of T cell differentiation in tolerance and CAR T therapy, and has helped meet the challenge of increasingly large and complex single- cell datasets through the development of bioinformatic workflows to integrate samples from multiple patients and sequencing technologies, and integrate gene, protein, TCR sequence, cell division count and somatic mutation data at the single-cell level.

  • (2023) Fichter, Christina
    Gene and cell therapies hold enormous promise for many presently incurable diseases. Although the efficiency and safety of site-specific genome editing systems have improved tremendously over the past decade, the delivery of these novel technologies to specific target cells remains a key technical bottleneck for the clinical translation. Since long-term expression of DNA-modifying enzymes can be associated with cytotoxicity, transient delivery is desirable to reduce the risk of mutagenesis from off-target activity. A main target for gene and cell therapies are T cells. In order to achieve gene transfer into human T cells, current gene delivery techniques involve significant levels of T cell activation and ex vivo expansion prior to lentiviral or retroviral transduction. However, ex vivo stimulation and expansion have been shown to significantly alter the natural physiology of T cells. To address these challenges, we have developed a non-integrative lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. To overcome barriers, present in this notoriously hard to transduce cell population, we have re-engineered lentiviral particles from the inside-out. Firstly, we have identified key viral glycoproteins that can mediate cytosolic delivery of transgenes across resting CD4+ T cell plasma membranes. Secondly, by replacing HIV nucleocapsid with the coat protein of the MS2 bacteriophage and substituting lentiviral RNA packaging motifs (LTR and packaging signal Ψ) with a series of MS2 stem loops that facilitate RNA binding to the MS2 coat protein, we have enabled the packaging of CRISPR/Cas9 components into these particles. In this setting, we observed expression of biologically active mRNA in up to 65% of resting CD4+ T cells. Furthermore, with our CRISPR/Cas9 delivery platform we have achieved genome editing efficiencies of >85% in human cell lines, and >20% in primary CD4+ T cells. With the emergence of SARS-CoV-2, we have shifted our focus on the development of a novel viral vector for gene delivery. Using a SARS-CoV-2 VLP scaffold, we have enabled the packaging and transient delivery of mRNA into target cells by tethering a reporter gene to the recently identified SARS-CoV-2 packaging signal. Equipping these VLPs with Spike glycoproteins from emerging SARS-CoV-2 variants, we observed an increase in transient mRNA delivery to >50% of target cells. This platform may give us a novel set of viral vector solutions to use for many respiratory diseases, including coronavirus disease (COVID-19) itself. Taken together, this thesis describes the development of robust delivery platforms for the transient delivery of biologically active transgenes to multiple cell types.

  • (2023) Balachandran, Harikrishnan
    The protection offered by B cell responses after viral infection or vaccination features neutralising antibodies produced by plasmablasts and memory B cells (MBCs) which can provide long lasting immunity against reinfection. Hence understanding the factors associated with MBC persistence is crucial for vaccine design. Murine and human studies have implicated initial antigen affinity, bystander activation, activation-induced cytidine deaminase (AID) expression, and chemokine levels as being associated with persistence of antigen-specific MBCs, but detailed understanding of the genesis of long-lived MBCs in humans is lacking. During the initial phase of COVID-19 pandemic there were multiple studies characterising natural immune responses against SARS-CoV-2 infection, but then the interest shifted towards understanding vaccine-induced immunity. With the spread of variants of concern and breakthrough infections in vaccinees, understanding the longevity of natural immunity is essential to design improved vaccines. The primary objective of this thesis was to develop a highly specific tetramer for flow cytometry-based isolation of SARS-CoV-2 specific MBCs, evaluate their longevity, and identify biomarkers and transcriptomic signatures present early after infection that associate with durable MBCs. The samples used in this thesis were selected from the COVID-19 Outbreak Samples in NSW (COSIN) cohort, an ongoing prospective cohort evaluating the natural history of the SARS-CoV-2 infection in NSW. This thesis observed that at one year post infection, despite declining antibody titers, maintenance of neutralisation breadth and a variant specific protection (45% - 76%) against symptomatic disease. Encouragingly 80% of participants who had SARS-CoV-2 specific MBCs during early convalescence had detectable MBCs at one year. Based on MBC persistence, the cohort was divided into two groups – “Maintained” and “Dropped”. Early antigen specific CD4+ T cell responses was associated with maintenance of antibody neutralisation breadth and MBC durability. Smart-seq2 analysis of the MBCs identified ongoing maturation of the B cell receptor in both grand transcriptomic differences between the two groups. The transcriptomic analysis revealed that an enhanced activation profile of mature, proliferating MBCs was associated with persistence at one year. These findings provide initial identification of biomarkers that can be used to predict the durability of vaccine induced immunity.

  • (2023) Sazzad, Hossain
    Prisons are a hotspot for blood-borne virus transmission. To control outbreaks of these infections, it is essential to have an in-depth understanding of risk behaviours of inmates and the impact of existing control measures, and also to apply sensitive methods for detection of new infections occurring within prisons. This thesis describes studies in these three domains to understand the spread of hepatitis C infection in the prisons in New South Wales (NSW). The clinical data and blood samples for this work originated from two prospective studies conducted in the NSW prisons: the Hepatitis C Incidence and Transmission Study in prisons (HITS-p) (2005- 2014; n=590) and the Surveillance and Treatment of Prisoners with Hepatitis C (SToP-C) (2015-2019; n=3691). The first study identified a high rate of incident infections in an at-risk cohort and documented outcomes (spontaneous clearance, chronic infection, and re-infection or superinfection), while the second study demonstrated the impact of scale up of direct acting antiviral (DAA) treatment in reducing incidence – that is treatment as prevention. The first project utilised qualitative data obtained from audiotaped interviews with inmates in the HITS-p study to understand contexts and concerns regarding violence and HCV transmission in prison. Concerns regarding violence were identified at the individual level during blood contact; triggering factors such as drug debt were identified at the network level; racial influence at the community level; and legislation such as delayed parole for violence impacted at the policy level. For the subsequent projects near-full length HCV genomes for genotypes 1a and 3a cases from both cohorts were sequenced with Oxford Nanopore Technology (ONT) using previously published protocols. A total of 211 genotype 1a sequences and 282 genotype 3a sequences were generated. Of these, 28 1a and 63 3a sequences were from samples collected within 6 months of the estimated date of infection and are hence referred to as acute infection sequences. Acute infection sequences from samples collected during 2005 – 2015 (the pre-DAA era) and 2016-2019 (the post-DAA era) were used to model the temporal evolution in the size of infected population using previously published Bayesian evolutionary analysis methods. The effective population size modelled with the genotype 3a infection samples, demonstrated a 21% reduction in the size of infected population in 2019 compared to 2014. By contrast, the trend in the genotype 1a samples was static. The SToP-C sequences (from both acute and chronically infected subjects) were used to identify molecularly related infections (clusters), before within-host viral variants were further characterised within these clusters to identify likely direct transmission events (defined as phylogenetic intermingling of within-host variants between two or more subjects). For genotype 1a, there were 131 sequences which formed 51 clusters, and for genotype 3a, 140 sequences formed 61 clusters. Each cluster had 2-4 sequences. Among these, 41 genotype 1a and 39 genotype 3a clusters were analysed for minor variants. Evidence of a direct transmission of a within-host variant was observed in only one cluster. Subjects in 45 (40%) clusters were from the same prison providing preliminary epidemiological support for the transmissions. These studies highlight the high-risk context for HCV transmissions in the prison setting, and the utility of molecular epidemiological tools for surveillance in this closed setting.

  • (2020) Riaz, Nasir
    Rapidly mutating RNA viruses such as hepatitis C (HCV) and dengue virus exist as a population of variants within a single infected host. Yet this within-host diversity is not captured in most genomic analyses due to technical difficulties in sequencing full length within-host viral variants. The existing bioinformatics algorithms for generating within host haplotypes from “short read” next generation sequencing data have poor agreement with each other, and the gold standard of single genome amplification is too laborious to process large sample numbers. The work presented in this thesis overcomes this barrier by improvising nanopore sequencing (a third generation long read sequencing technology) as a cost-effective strategy to sequence full-length hepatitis C virus, alongside a novel in-house developed bioinformatics pipeline to differentiate within-host variants from long-read sequencing data. The generalizability of this workflow to other RNA viruses is demonstrated by adapting the pipeline to generate and study within host variants of the dengue virus. To demonstrate the value of this advancement, this thesis also presents two advanced virological analyses using the output of HCV within-host sequence data generated from the improvised workflow. The first is a high resolution, within-host evolutionary analysis of chronic HCV infection demonstrating ongoing bottlenecks in viral diversity in the chronic phase of infection and the capacity to observe extinction and survival of specific within-host variants over time. The second analysis demonstrates how mutations at specific sites of the viral genome results in distinct viral populations with potential alternate open reading frames and the survival of these populations across serial sampling timepoints, extending to months or years. In conclusion, the methods developed in this project open novel and exciting opportunities to observe changes in HCV within-host viral variants which will help to understand the virological determinants of immune escape (in vaccine trials) and drug resistance. These methods are also adaptable to study the within-host evolution of similar RNA viruses such as dengue.

  • (2023) Adhikari, Anurag
    Fc-mediated effector antibody functions including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent complement deposition (ADCD), and antibody-dependent respiratory burst (ADRB) have been recognised to play a key role in antiviral immunity. Among these effector functions, ADCP alone or together with neutralisation function was shown to correlate with immune protection against several RNA viruses. However, methods that comprehensively study ADCP in HCV and SARS-CoV-2 using whole patient plasma are unavailable thereby precluding understanding to its role in the immunopathogenesis of these diseases. To address these, a robust high throughput flow cytometry-based ADCP assay was developed using THP-1 monocyte cell line as effector cells. This easy-to-use assay was then used to characterise ADCP in patients with HCV and SARS-CoV-2 infections. In patients with chronic HCV infection there was higher envelope 2 (E2) antibody titres while patients that cleared the virus produced antibodies with higher affinities. ADCP function was significantly higher in patients with chronic diseases than clearers and strongly correlated with antibody titre and neutralisation function. Multiple regression analysis showed that anti-E2 IgG is the most important variable for predicting ADCP regardless of disease severity, sex or age. Longitudinal study over 14 months showed anti-E2 antibody titres and ADCP function was maintained in both chronically infected patients and those who cleared the virus while there were variable changes in antibody affinities. In COVID-19, patients with acute disease mounted ADCP early as 10 days after symptom onset, primarily driven by heat labile components in the plasma. In convalescent patients, increased ADCP response significantly correlated with high anti-Spike IgG titres, older age, neutralisation and severe disease. Multiple regression analysis showed that anti-Spike antibody titre was the most important variable for predicting high ADCP. Longitudinal study over 12 months showed an increase in plasma antibody affinity and preservation of ADCP and neutralisation functions despite a decline in the anti-Spike IgG titres by >90%. Collectively, anti-E2 and anti-Spike antibody titres in plasma were the most important predictors of ADCP and neutralisation functions in HCV and SARS-CoV-2 infections respectively. Patients with chronic HCV infection showed higher ADCP when compared to clearers and there was a positive correlation between disease severity and ADCP in SARS-CoV-2 infection. Antibody titres in patients with HCV were maintained longitudinally together with ADCP while changes in affinities of the antibodies were variable. By contrast, in COVID-19 affinities increased overtime while. It is therefore likely preservation of ADCP function in HCV is due maintenance of high antibody titres while in COVID-19 it is due to the improvement in the quality of the antibodies.

  • (2022) Gupta, Money
    The emergence of single cell RNA sequencing technologies has opened a vast number of strategies to look at various aspects of immunity to infectious diseases, especially into the adaptive immune system. Here, we applied scRNA-seq to study rare antigen-specific memory B cells in Hepatitis C and COVID-19 disease. Firstly, a tool for the reconstruction of single T cell and B cell receptor (TCR and BCR) sequencing method was developed that can obtain full-length repertoire from short read sequencing of T and B cell at single cell resolution. This tool, VDJPuzzle was able to report the detection of V(D)J genes, isotypes, somatic hypermutations, membrane vs secreted exon isoforms. VDJPuzzle showed highly accurate detection of V(D)J when benchmarked against available tools together. The utility of this tool to explore infection dynamics associated between BCR and disease outcome was then explored for two diseases: Hepatitis C (HCV) and COVID-19. In the HCV study, properties associated with rare HCV-specific memory B cells and varied HCV infection outcome in primary infection and reinfection were examined. In the primary infection cohort, a higher maturation of memory B cells and a reduced level of aromatic residues was observed in individuals that developed chronic infection. In the reinfection cohort, no association of BCR recall was observed, however transcriptomic differences were observed prior to re-infection across clearers and chronics where, clearers had a pre-existing enrichment of genes associated with the TNF-α signaling via NF-kB pathway. There was also a population of memory B cells with increased CD95 surface expression that showed migration of these cells towards effector long-lived plasma cells showing higher expression of ZBTB32, TBX21, FAS, ITGAX, etc. In the COVID-19 study, we successfully extracted rare SARS-CoV-2 specific memory B cells from mild/moderate and severely infected subjects, at 1 month and 4 months convalescence. Interestingly, the VDJPuzzle tool observed a higher percentage of dual kappa-lambda chains in SARS-CoV-2-specific B cells. The presence of unique phenotypes was also identified in SARS-CoV-2-specific memory B cells from subjects with severe disease. In particular, there was an increase in two populations defined as ‘actBC1’ (CD80hiTNFAIP3hi) and ‘actBC2’ (CD11chiCD95hi) which we hypothesized might be associated with increased longevity of the memory B cell response. The severe patients also maintained a higher level of activation genes associated with TNF-α signaling via NF-kB pathway, which may relate to the ‘cytokine storm’ that is often observed in severe SARS-CoV-2 infected individuals. Whereas, in mild/moderate infected individuals a decline in activated genes was observed over time. In summary, this thesis took advantage of unique sets of scRNA-seq techniques to identify the causes associated with HCV-specific memory B cells towards the clearance and chronic infection outcome in primary and reinfection cohort. Furthermore, we looked at the potential mechanistic insights associated with SARS-CoV-2-specific memory B cell response towards how transcriptome contributes towards the long-term immunity in a mild/moderate or severe infected individuals up to 4 months at convalescence.