Transforming growth factor-beta (TGF-β) drives the development of immature LC from hematopoietic progenitor cells and shapes the cells functions. Here I showed that two LC model cells, MuLC and MDLC, used exchangeably in the research, differ significantly in their phenotype and immune responses. Discrepancies between these models were specifically visible during stimulation with type-I IFN, where MuLC failed to up-regulate ISG levels. Yet both MuLC and MDLC demonstrated low susceptibility to HIV-1 infection, even in the absence of SAMHD-1. This post-entry restriction was conferred by the action of TGF-β on differentiation cells as indicated by our study. Indeed, in the absence of TGF-β supplementation, derived cells showed MDDC phenotype related to high susceptibility of the cells to HIV-1 infection during co-infection with SIV-Vpx. Additionally blocking of the TGF-β signalling, reversed the restrictive phenotype of LC. Importantly this pattern was also confirmed in skin extracted real epidermal LC versus dermal DC, suggesting that SAMHD-1-independent restriction activity operates in TGF-β derived cells. Accordingly to PCR analysis virus replication in LC is interrupted prior to integration, suggesting the role of additional restriction factors at early stages of virus infection or lack of essential viral dependency factors such as dNTPs. Interestingly maturation of MDLC with a synthetic bacterial triacylated lipopeptide or TNF-alpha significantly increased their susceptibility to HIV-1 infection, which may explain why HIV-1 acquisition is increased during co-infection with other STIs. In summary, our study strongly supports the action of SAMHD-1-independent HIV-1 restriction mechanisms in LC. A better understanding of the balance between HIV-1 restriction and propagation from LC to CD4+ T cells may help in the development of new microbicides or vaccines to curb HIV-1 infection at its earliest stages.

Cardiovascular disease is one of the leading causes of death in the Western world. One of the most common causes is the rupture of unstable atherosclerotic plaques, which can lead to thrombus formation, occlusion of the artery and myocardial infarction. Therefore there is a need for treatments that stabilise vulnerable plaques. Gene therapy has the potential to provide a novel treatment for this. To maximise therapeutic gene expression and minimize any potential adverse effects due to unwanted transgene expression in non-target tissues, a gene delivery vector specifically targeted to areas of atherosclerotic vasculature is required. The vector is also required to efficiently infect cells to produce relatively long term transgene expression, be stable in blood, non-toxic, non-immunogenic and producible at high titres. Viral vectors, particularly those based on adenovirus (Ad) and adeno-associated virus (AAV) have many of the desired features, but transduce vascular cells relatively inefficiently and in a non-selective manner. Methods of altering their tropism have been established and could be utilised to develop vectors with a high degree of selectivity for atherosclerotic plaques. Detargeting of vectors can be achieved by mutating regions of the virus capsid that are thought to bind the native cellular receptors and retargeting to novel cell types is achieved by inserting peptide ligands into the virus capsid. The aim of this study was to develop atherosclerotic plaque targeted vectors and to characterise Ad and AAV vector platforms in this regard. Two approaches were taken. In the first approach three previously identified plaque targeting peptides were tested for their ability to target viral vectors to atherosclerotic plaques. The second approach involved performing phage display in a mouse model of plaque rupture to identify novel peptides that specifically target unstable plaques. Further work was carried out to characterise and develop methods for using an AAV2 based peptide library as a novel tool for biopanning. This work has provided further characterisation of Ad and AAV platform vectors that may be utilised in the development of vectors with a highly selective tropism.

Hepatitis C virus (HCV) is emerging as one of the leading causes of morbidity and mortality in individuals infected with HIV and has overtaken AIDS-defining illnesses as a cause of death in HIV patient populations who have access to highly active antiretroviral therapy. For many years, the clonal analysis was the reference method for investigating viral diversity. In this thesis, a next generation sequencing (NGS) approach was developed using 454 pyrosequencing and Illumina-based technology. A sequencing pipeline was developed using two different NGS approaches, nested PCR, and metagenomics. The pipeline was used to study the viral populations in the sera of HCV-infected patients from a unique cohort of 160 HIV-positive patients with early HCV infection. These pipelines resulted in an improved understanding of HCV quasispecies dynamics, especially regarding studying response to treatment. Low viral diversity at baseline correlated with sustained virological response (SVR) while high viral diversity at baseline was associated with treatment failure. The emergence of new viral strains following treatment failure was most commonly associated with emerging dominance of pre-existing minority variants rather than re-infection. In the new era of direct-acting antivirals, next generation sequencing technologies are the most promising tool for identifying minority variants present in the HCV quasispecies populations at baseline. In this cohort, several mutations conferring resistance were detected in genotype 1a treatment-naïve patients. Further research into the impact of baseline HCV variants on SVR rates should be carried out in this population. A clearer understanding of the properties of viral quasispecies would enable clinicians to make improved treatment choices for their patients.

Immune responses towards Cytomegalovirus (CMV) often increase in magnitude with age; a phenomenon termed ‘memory inflation’. Elevated CMV-specific immunity is correlated with an increased mortality rate in elderly individuals and there is considerable interest in therapeutic approaches that may reverse this. Latent CMV infection is characterised by intermittent episodes of subclinical viral reactivation which may play a role in boosting CMV- specific immunity however, the relative importance of reactivation in the development of "memory inflation" is currently uncertain. In order to investigate these questions valaciclovir was administered as to aged mice with established murine CMV (MCMV) infection to block stochastic lytic reactivation from latency. Following 12 months of treatment there were highly significant reductions in the frequency of the MCMV-specific CD8\(^+\) T-lymphocytes and the residual MCMV-tetramer specific response exhibited a less differentiated phenotype. The accumulation of memory cells associated with untreated MCMV infection suppressed the proportion of naïve CD8\(^+\) T-cells by 60%, whereas antiviral treatment was able to completely restore this effect. Furthermore, valaciclovir treatment of MCMV reduced the elevated viral load that followed influenza virus challenge demonstrating that anti-MCMV treatment can lead to improved immunity to other pathogens in old age.

No description available.

EBV lytic replication involves the sequential expression of a large array of antigens that potentially provides a complex antigenic challenge. Despite this number, the primary CD8+ T cell response in infectious mononucleosis (IM) patients appears focused against epitopes found in immediate-early and some early expressed antigens with responses to late antigens typically subdominant. However previous approaches have only focused on a limited number of lytic antigens. To resolve these issues, this study examines the CD8+ T cell repertoire to all EBY lytic antigens in different phases of infection. Polyelonal CD8+ T cell lines were mitogenically expanded from IM patients or expanded in an antigenically unbiased way from post-1M patients and healthy carriers using autologous dendritic cells loaded with lysates of lytically infected cells. Target cells expressing individual lytic antigens along with donor HLA class I molecules were used to challenge polyclonal lines with responses measured by the release of IFNγ. These studies show that the pattern of target antigen choice varies with the phase of infection and is consistent with the idea that CD8+ T cell responses in primary infection are driven by lytically infected B cells. However over time the repertoire of responses may be influenced through antigen cross-presentation.