The purpose of this project has been to investigate the mechanism of hepatitis C virus/lipoprotein complexes of lipo-viro-particles (LVP) derived from transplanted liver macerates of an HCV infected immunodeficient patient (S6b) and density-fractionated from an iodixanol gradient. Previous studies have indicated that serum derived HCV may bind to hepatocytes via the LDL-receptor (LDLr) or the scavenger receptor SR-B1. The role of these receptors in the uptake of LLVP in HepG2 cells was investigated by comparing LLVP uptake with that of dioctadecylindocarbocyanine-labeled-low density lipoprotein (DiI-LDL) and similarly labeled oxidized LDL (DiI-oxLDL), know to be taken up predominantly via the LDLr and SR-B1 respectively. DiI-LDL and DiI-oxLDL binding resembled that of LLVP in being significantly increased by insulin and LPDS treatment. DiI-LDL but not DiI-oxLDL binding was also decreased by hydroxycholesterol. These results suggest that all three lipoprotein particles may be taken up via the LDLr which binds predominantly via apolipoprotein B100. To confirm this, the inhibition of binding studies were conducted. Whilst binding of DiI-LDL was reduced by 98% by pre-incubation with anti-apoB100 antibodies, binding of both LLVP and DiI-oxLDL was enhanced. Using confocal microscopy and FACS analysis, we compared the role of glycosaminoglycans (GAGs) in the binding of the three particles types by washing cells with suramine, previously shown to remove GAG bound-LDL. Like LLVP, DiI-LDL bound at 0 degrees Celsius was washed off with suramine but became resistant at 37 degrees Celsius. In contrast, washing off DiI-oxLDL with suramine at both 0 degrees Celsius and 37 degrees Celsius had no significant effect. The binding patterns of LLVP therefore differ from those of DiI-LDL and DiI-oxLDL. To investigate whether such differences might be due to viral glycoprotein E2, the effect of polyclonal antibody to E2 and a monoclonal antibody to the E2-hypervariable region (HVR1) on binding of LLVP to HepG2 cells was assessed. Neither anti-E2 nor anti-HVR1 blocked binding.

Hepatitis C virus (HCV) infection is characterized by a broad spectrum of clinical outcomes. An estimated 14%-46% of individuals exposed to HCV are able to clear the virus, while the other portion develops chronic (persistent) infections. Among the individuals with chronic HCV who are treated with interferon-based therapies, only a portion are able to experience sustained virological suppression. Similarly, a number of chronically infected individuals have autoimmune extrahepatic manifestations such as the presence of autoantibodies. The pathological mechanisms behind these phenomena are not known, but it is believed that host genetic factors may play a role. This thesis examines the hypothesis that host genetic factors may contribute to the diverse spectrum of HCV clinical outcomes. In addition, it examines the pathogenesis of antinuclear antibodies (ANA) in chronic HCV, and the effect of ANA positivity on the natural history of HCV. Correlations were observed between female gender and geographic location and ANA positivity. No relationships were observed for an effect of ANA positivity on response rates to interferon therapy. We observed a trend of ANA positivity with faster progression of HCV-related fibrosis, although this failed to achieve statistical significance. ANA-positive individuals tended to have more plasma cells in their liver than ANA-negative individuals. This study also observed a number of correlations between genotypes of the interferon induced genes encoding the myxovirus resistance 1 protein (MxA), 2'-5'oligo-adenylate synthase 1 (OAS-1), and protein kinase (PKR), as well as genes encoding cytotoxic T-lymphocyte antigen-4 (CTLA4), and inducible nitric oxide synthase (iNOS) (encoded by the NOS2A gene) with several outcomes including self-limiting versus chronic HCV infection, along with the response to interferon therapy. This study identified several factors to be correlated with ANA positivity in HCV. These factors may serve as future points for investigation by basic scientists understanding the mechanisms of HCV-mediated autoimmunity. Importantly, this study suggests that low titre ANA positivity should not be a contraindication to therapy. This study also highlighted the importance of several genetic pathways in HCV infection. These may serve as targets for future pharmacologic interventions or genetic tests designed to screen for those who will not benefit from interferon therapies.

This thesis explores the firs t-ever administration of a prime/ boost combination of aden ovirus and MVA vector based vaccin es that target hepatitis C virus (HeV) infection to humans. Therapeutic application of these vaccines in patients with chronic HeV infection is first detailed. together with an interrogation of the immune responses genera ted. Vaccination safely ind uces novel T cells that target HeV immunogen in 5/11 vacci nated patients. However, assessment of circulating virus reveals significant sequence differences between autologous virus and the corresponding peptide in the vaccine immunogen. Further analysis shows that T cells induced by vaccination are poorly cross-reactive aga inst autologous virus epitopes. Compared with vaccination in healthy individuals, T cell responses are red uced in their frequency, magnitude and breadth in Hev infected patients who receive combination with standard HCV treatment IF N) / ribaviri n). vaccination, (pegylated either alone or in interferon-a (PEGTo help explain the reason for attenuated vaccin e responses in Hev infected patients, I next explored baseline and PEG-IFN/ribavirin induced differences in circulating lymphocyte subpopulations. At baseline, high proportions of regulato ry T cells and NK~ rl&ht cells suggest both adaptive and innate immunity is altered by chronic HCV infection. During the first 28 days of therapy, complex changes in the activation, differentiation status and cytokine profiles of circulating lymphocytes are observed over tim e. Intriguingly, PEG-IFN causes a transient but significant decrease in circulating lymphocytes that express CXCR3, a liver homing ch emokine receptor. Finally, the hypothesis that HCV th erapy ind uces CXCR3 mediated hepatic sequestration of lymphocytes was explored. In-vivo, PEG-IFN caused a transient but profound elevation of plasma IP-10 (a CXCR3-b inding chemokin e) and this co rrelated with declines in circulating CXCR3· CDS· T cells in HCV infected patients. Although in-vitro experiments show IFN-a drives hepatocytes to produce IP-10, it also causes lymphocytes to down-regulate CXCR3. Unfortunately, exogenous IFN-a does not objectively demonstrate hepatic enrichment of CXCR3·lymphocytes in a murine model.

Hepatitis C virus (HCV) infection is common with more than 180 million people infected worldwide. The majority of individuals who acquire the virus develop chronic infection leading to chronic hepatitis, and are at risk of developing cirrhosis and hepatocellular carcinoma. In the minority who clear infection acutely, an effective adaptive immune response against the virus is paramount. There is presently no available preventative vaccine against HCV infection. In the developed world, injection drug use (IOU) of illicit substances is the dominant source of new infections with high rates of HCV transmission. Rates of HCV infection are particularly high in long term I DU who frequently share drug injecting equipment such as needles and syringes. Despite this there is a cohort of long term I DU with high risk injecting practises who remain negative for any markers of HCV infection when tested for by commercial assays. It is possible they are being exposed to HCV yet remain uninfected, and have been termed exposed uninfected (or EU). It is possible that they possess immune mechanisms of protection. Understanding these mechanisms could be important in developing preventative strategies against HCV infection. This thesis has set out work to identify the potential immune mechanisms that may underlie this resistance to HCV infection in this unique cohort of IOU. Earlier studies had demonstrated HCV specific T cell reactivity in up to 50% of this cohort. These responses were of low amplitude and thus did not entirely explain this apparent resistance. To further study these T cell responses, HCV specific T cell reactivity against recombinant HCV proteins and overlapping peptides spanning the entire HCV polyprotein were studied in EU subjects using the ELlSPOT assay. Multispecific T cell responses were confirmed in up to 50% EU subjects, however were still of comparatively lower amplitude when compared to those seen in spontaneous resolvers of HCV. A large panel of serum cytokines were analysed in 22 EU subjects and levels compared to those found in cases with spontaneous clearance of HCV and chronically infected patients. This demonstrated a distinct cytokine profile in EU with raised levels of innate immune cytokines and proinflammatory chemokines, particularly Interleukin-6 and interleukin-8. These findings suggested an upregulation of the innate immune system in these individuals. To investigate host innate immunogenetic factors possibly related to protection from HCV in this cohort, a functional single nucleotide polymorphism (SNP) in the promoter region of the IL-6 gene as well as the recently discovered IL-28B related SNPs, rs12979860 and rs8099917, were studied. No over-expression or association was demonstrated betvveen these variants and this cohort. As studies had demonstrated an association with EU and a NK cell receptor:HLA compound genotype that favours viral clearance, immunophenotyping of NK cells and dendritic cells was performed in EU cases. The predominantly cytotoxic NK cell subset, CD56dim NK cells, was found to be expanded in EU, suggesting a possible phenotypic alteration in NK cell subsets in this cohort. Further studies on NK cell cytotoxicity against NK-sensitive K562 cell line demonstrated enhanced I L-2 induced cytotoxicity in EU compared to chronic viraemics. Overall, the studies performed in this thesis demonstrated a distinct immune phenotype in EU SUbjects with low amplitude HCV specific T cell reactivity, upregulated innate immune cytokines and cytotoxic NK cell subsets with enhanced cytotoxicity, thus pointing to the initial innate immune responses as the predominant factor in preventing HCV infection in this cohort.

Hepatitis C virus (HCV) is a growing problem affecting thousands of people in the UK. The majority of individuals infected develop chronic liver disease, which can progress to cirrhosis and cancer. Treatment is available for some, but as it is not always successful many people continue to live with the virus in the long term. Experiences of living with hepatitis C are poorly understood yet necessary in order to best meet the need of an increasing number of people who will be living with the disease. Descriptive phenomenology underpinned the design ofthis study which aimed to explore experiences of living with hepatitis C, both for those treated and not treated for the disease over time. A purposeful sample of 23 adults with medically confirmed hepatitis C participated in unstructured interviews, conducted twice over a 9 month period. An analytical framework based on Colaizzi (1978) revealed six themes: HCV and Self; HCV, Self and Others; Self and Handling HCV; Self and Handling HCV Treatment Issues; Living with the Consequences ofHCV and Self, HCV and Thoughts of the Future. For many, diagnosis with HCV can disrupt personal and social identity which triggers a life transition, following which uncertainty exists until a valued self identity is reconstructed. The ability to handle life ~ith HCV, treatment issues, the consequences of living with the disease and its impact on thoughts about the future serve to sustain uncertainty, leaving many failing to make a healthy transition to life with the condition. The understanding reached in this study is important to improve the care of those with hepatitis C, it can enable healthcare provision to focus on supporting individuals with the disease in a chronic care framework which could better facilitate their transition to life with HCV and improve experiences of living with the virus.

The majority of Hepatitis C Virus (HCV) infections result in chronic infection, which can ultimately, cause hepatocellular carcinoma and liver failure. Within the infected host, the virus exists as a population of heterogeneous viral variants called a quasispecies. The envelope region of HCV genome is the most heterogeneous among the whole genome. The envelope proteins mediate viral entry and are also the target of the host immune response. Studying envelope gene evolution can provide important molecular insights into. entry and antibody escape, which ultimately can inform vaccine and therapy developments. Despite this, the influence of host antibody response in driving envelope gene evolution and its role in immune evasion is unclear. To address this shortfall we studied the evolution of HCV envelope genes, over a period of 7-15 years, in treatment naive chronically infected patients. The pattern of HCV evolution was patient specific and clustering of quasispecies was not dependant on the sampling time, which is in stark contrast with other related viruses. The hyper variable region I (HVR-l) was under strong selective pressure. The residues from HVR-l and downstream region, in and around receptor and antibodies binding sites were positively selected during viral evolution. The isolated envelope genes from only two patients (40%) were functional in the retroviral pseudo-particle assay (HCVpp). The functional envelope genes isolated from sequential samples showed variations in infectivity, which could be attributed to substitutions at multiple residues acquired during the course of evolution. Construction of El E2 chimeras derived from infectious and non-infectious clones showed that the region downstream of HVR-l was an important determinant of in vitro infectivity. The patients' isolated HCVpp functional envelope genes were efficiently neutralised by autologous IgGs, and there was no obvious evidence of antibody escape. One of the EIE2 chimeras showed differential sensitivity to well-defined CD81 binding-site-specific neutralising mono clonal antibody, demonstrating the existence of complex mechanisms underpinning antibody resistance. Finally, studies of quasi species transmission in a xenomouse model showed the occurrence of a transmission bottleneck. In multiple transmission experiments a minor variant within the inoculum consistently became established within the recipient mouse host. The established variant harboured amino acid substitutions at amino acid residues 198, 448 and 474 of the virus polyprotein, including loss of a predicted glycosylation site (N448D). It was not possible to define the phenotypic effects of the substitutions as the EIE2 clones were non-infectious in the HCVpp assays. Defining viral determinants involved in successful transmission will help the design of vaccines.

HCV has highly structured RNA elements at the 3' and 5' end of the genome. These conserved regions are not translated and are termed 3' and 5' untranslated regions (UTRs). The terminal and penultimate nucleotides of other flaviviruses are conserved; mutation of these nucleotides compromises RNA replication. The HCV replicon was used to study the effects of mutating the 5' terminal nucleotide on HCV RNA replication. This is a bicistronic RNA molecule containing HCV non structural proteins NS3-58 in conjunction with a selectable neomycin phosphotransferase gene. Replicon transfected cells were selected by adding the antibiotic G418 to culture media. Replicons with a full range of 5' terminal nucleotide mutants were made. A replicon with a purine nucleotide (A or G) could replicate efficiently. A terminal U could support replication at low levels whereas a terminal C could not. Replicons containing a luciferase reporter gene were generated to assess levels of RNA replication at early time points after transfection. These suggested that a terminal A and a terminal G allowed similar levels of RNA replication even at early time points, although a terminal G appeared more efficient at colony formation. In contradiction of previous work, this study found that a terminal A or G was maintained stably in culture. The ability of a replicon with a terminal U to be maintained in culture was dependent on mutation of this nucleotide to A. Studies using the JFH-1 strain of HCV revealed that conventional transcription methods placed non templated nucleotides immediately upstream of the 5'UTR. The native 5' end is restored rapidly in culture and defining this 5'end with a hammerhead ribozyme did not improve rescue efficiency. Analysis of the nucleotides at the +2, +3 and +4 positions of the HCV 5'UTR also suggests that there is some conservation at these sites.

The hepatitis C virus protein p7 is a 63 amino acid membrane protein that is able to oligomerise and functions as an ion channel in artificial bilayer systems, displaying sensitivity to amantadine as well as a range of other small molecule inhibitors. Consequently, p7 has been categorised as a member of the viroporin family; small virally encoded proteins that increase membrane permeability via their ability to oligomerise within host cell membranes.

The hepatitis C virus is a worldwide cause of liver disease by infecting approximately 2 % of the population. One of its non-structural protein, NS5A, Is known to interact with many cellular proteins involved in cell signaling pathways. Doing so modifies the outcome of the signalling for the benefit of virus replication and survival. We have previously demonstrated that NSSA contains two Class II poly-proline motifs in the second low complexity sequence, with the consensus sequence Pro-X-X-Pro-X-Lys/ Arg, which mediate the interaction with SH3 domain containing proteins. Substitution of the prolines with alanines did not have an effect on RNA replication but disrupted interactions with SH3 domain containing proteins. In order to identify novel NSSA binding partners, genotype 2a (JFH1) NSSA was expressed with a biotin tag individually or in the context of the replicon as an experimental requirement to screen an all human SH3 domain displaying library. In total, sixteen SH3 domain containing binding partners were identified, from which Mlk3, RelA, Vinexin, PACSIN1 RBP2 and STAC1 were novel targets. The interaction of NS5A with Amph1, CMS, Nck1 and Ponsin were investigated in more details. Interactions of NS5A with Amph1 and CMS were confirmed using immunoprecipitation, immunofluorescence and biochemical assays. Interestingly these proteins have been implicated in clathrin-mediated endocytosis and epidermal growth factor receptor trafficking. Upon overexpression of wt but not the proline mutant NSSA the cellular localization of Amph1 was altered and interaction with dynamin2, a well characterized partner of Amph1, was disrupted. Furthermore, as it was reported before, NS5A diverts the epidermal growth factor receptor away from the late endosomes thereby modifying its degradation rate and signalling capability towards amongst other targets the Ras-Erk mitogen-activated protein kinase pathway, a signalling cascade known to be inhibited by NS5A in a PxxPxR dependent manner. NS5A-CMS and EGFR was found to co-localise to vesicles upon EGF stimulation and overexpression of CMS was found to reverse the effect of NS5A on EGFR.

No description available.