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Human cytomegalovirus (HCMV) is the most common viral infection complicating stem cell transplantation, and if untreated frequently results in a fatal outcome. In order to identify the main risk factors for HCMV infection, a retrospective study of all allogeneic stem cell transplants performed over a five year period at a single centre was undertaken. The main risk factors for HCMV infection following transplantation were identified as a HCMV seropositive donor and/or recipient and in-vivo use of the monoclonal antibody, anti-CD52 (alemtuzumab). A prospective study of HCMV viral loads determined by real time PCR using a Taqman probe was undertaken, and the viral load dynamics of 57 patients were analysed. Despite the use of aciclovir prophylaxis, PCR monitoring and pre-emptive therapy, the peak viral load, viral replication rate and the total duration of viraemia remain significant risk factors for symptomatic HCMV infection. Peak viral load was the most significant predictor of time to viral clearance. A prospective longtitudinal study of the reconstitution of the HCMV specific immune response following allogeneic stem cell transplantation was performed in 20 patients using intracellular interferon gamma staining and flow cytometry. HCMV infection was associated with a significantly reduced HCMV specific CD4+ T cell response. Furthermore, in patients receiving in-vivo anti-CD52, HCMV specific CD4+ T cell immune recovery was significantly delayed. An HLA class II epitope mapping study was undertaken in stem cell transplant recipients and healthy controls using peptide pools consisting of 15 mer overlapping peptides spanning the entire amino acid sequence of the HCMV proteins, pp65 and IE1. Using intracellular cytokine detection in CD4+ T cells, a number of novel HCMV specific class II epitopes were identified. Transplant recipients responded to a broader range of epitopes than HCMV seropositive controls. These results have important implications for HCMV specific immunotherapy in allogeneic stem cell transplantation.

Hepatitis B virus (HBV)-specific CD8+ T cells are critical for a successful immune response to HBV infection. These cells are markedly diminished in patients that are unable to contain the virus and the mechanisms contributing to their depletion are not well-understood. The aim of this work was to further dissect this response in individuals with chronic infection. Studies of the HBV-specific CD8+ T cell immunodominance hierarchy demonstrated that persistent infection was associated with a hyporesponsiveness that was exacerbated with increasing antigenic load. The immunodominant core 18-27-specific response appeared to be particularly prone to deletion whereas a population of envelope 183-191-specific CD8+ T cells managed to persist despite excessively high viral loads but with an inability to exert effective antiviral function. cDNA microarrays were used to compare these responses in resolved and chronic infection and helped identify a group of functionally-related genes that were selectively up-regulated in HBV-peptide stimulated CD8+ T cells from individuals with chronic infection. The most striking of these was the pro-apoptotic mediator of cross-tolerance induction, Bim, whose expression was confirmed to be upregulated at the protein level. Inhibition of Bim-mediated apoptosis resulted in enhanced detection of HBV-specific CD8+ T cells both in culture and directly ex vivo. In accordance to Bim-mediated deletion of CD127low CD8+ T cells, the HBV-specific CD8+ T cells surviving in chronic HBV infection were found to be CD127hlgh. These populations had elevated levels of the anti-apoptotic protein Mcl-1, suggesting they had been subject to IL-7 mediated rescue from apoptosis. This study indicates that Bim-mediated attrition of HBV-specific CD8+ T cells contributes to the inability of these populations to persist and control viral replication.

Transplantation-associated Kaposi's sarcoma (KS), causatively associated with human herpes virus 8 (HHV-8), is particularly prevalent in Saudi Arabia, although the prevalence rate of HHV-8 infection in the general population there is comparable to that in other geographical regions. The serologic and genomic prevalences of HHV-8 in samples representing the general population (n=238), patients with end-stage renal disease (n=78), and renal allograft recipients (n=66) were investigated. To evaluate if oral shedding of HHV-8 might play a role in transplantation KS, the extent of HHV-8 shedding in the mouth compared to other anatomical compartments, and the presence of multiple HHV-8 infection were also studied. PCR protocols were applied to amplify 3 fragments of the viral genome (from open reading frames 26 and K1) from whole-mouth saliva, parotid saliva, buccal and palatal exfoliates, plasma, sub sets of peripheral blood cells, and KS lesional tissue, and to quantify the salivary viral load. Demographic and clinical data were analysed to identify risk factors for HHV-8 infection. A higher HHV-8 seroprevalence was observed in patients with renal disease compared to the general population, but no significant difference in HHV-8 DNA detection rates in CD45+ cells was found. The oral cavity was identified as a major site of HHV-8 shedding in renal disease patients regardless of a previous history of KS. In patients with end-stage renal disease, HHV-8 DNA was more frequently detectable in oral samples than in blood. They and renal allograft recipients showed evidence of being multiply infected by HHV-8. These findings suggest that iatrogenic, salivary HHV-8 transmission between patients with renal disease prior to transplantation accounts for the relatively high prevalence of HHV-8 infection. Implementation of measures to minimise contamination of oral fluid between renal disease patients may play a role in controlling HHV-8 transmission and reduce the incidence of transplantation-associated KS.

Mycobacterium tuberculosis (M. tuberculosis) is responsible for 2 million deaths annually. In recent years co-infection with HIV and drug-resistant strains has led to the increase in clinical cases of tuberculosis. BCG is the only vaccine currently available but is ineffective against adult pulmonary forms of the disease. Understanding the principle components of the host immune response against the pathogen will aid in the design of a better vaccine. Dendritic cells are potent antigen presenting cells that play a key role in priming naïve T cells. Effective T cell priming is necessary for a successful protective immune response in the host against the pathogen. I have been investigating the interaction of dendritic cells with M. tuberculosis, with a view to better understanding the signalling pathways affecting priming of anti-mycobacterial T cells by dendritic cells. Using DNA microarray and bio-informatics, I have been able to study early transcriptional signatures of bone marrow-derived dendritic cells (BMDCs) in response to M. tuberculois infection. Interferon Regulatory Factors (IRFs) and NFkB (Nuclear Factor kappa B) appear to be the principal transcription factors involved in regulating the cellular responses in M. tuberculosis infected BMDCs. It has been found that IRFs can function independent of the adaptor MyD88 (Myeloid Differentiation Factor 88) in M. tuberculosis infected BMDCs, which is surprising considering the important role played by MyD88 in the Toll mediated signalling pathway. Exvivo experiments also show that MyD88 may not be absolutely essential for mounting anti-mycobacterial T cell responses. Hence immune responses generated by M. tuberculosis infected BMDCs appear to be mediated via the MyD88 dependent and independent pathways.

KSHV infection is associated with both endothelial and B cell tumours. In KSHV the genes expressed in latency have been implicated in cell transformation. vFLIP is one of a small number of viral proteins expressed in latently infected tumour cells. In KSHV-infected primary effusion lymphoma (PEL) cells, vFLIP binds to, and persistently activates the IkB kinase complex, leading to constitutive activation of the canonical NF-kB pathway. We have previously shown in our lab that vFLIP directly interacts with the IKKy subunit of the IKK complex (Field, et al., 2003) to activate IKK. In this report, we demonstrate that vFLIP also activates the alternative NF-kB pathway, which involves processing of the p100 protein precursor and generation of the p52 subunit. Stable vFLIP expression in Jurkat cells stimulates expression of endogenous p100 and nuclear accumulation of p52 and RelB. Metabolic radiolabelling of transiently transferred 293T cells indicates that vFLIP promotes proteolysis of p100 and active generation of p52. Moreover, we show that vFLIP associates with p100 when over-expressed in Jurkat cells, or when endogenously expressed in PEL cells, and a region in the C-terminus of p100, which includes the p100 DD, is identified as the vFLIP binding region. Finally, inhibition of p100 and p52 production mediated by siRNA knockdown leads to the induction of apoptosis in PEL cells, inferring that vFLIP activation of the alternative NF-kB pathway contributes to PEL survival. These data demonstrate that vFLIP activates both canonical and alternative NF-kB pathways, a property shared with the Tax oncoprotein of HTLV-1 and LMP1 of EBV. In addition, we have examined the effect of vFLIP on primary human dermal microvascular endothelial cell (MVEC) survival, as vFLIP is expressed in the KSHV-infected cells within KS lesions. Stable vFLIP expression in MVECs induces the activation of the classical NF-kB pathway and the nuclear translocation of RelA/p65. vFLIP-mediated NF-kB activation prevents detachment-induced apoptosis (anoikis) of MVECs, but does not inhibit growth factor removal-induced apoptosis, by inducing the secretion of an additional paracrine survival factor(s). These data strongly support an important role for vFLIP in NF-kB activation, which may be crucial for cell transformation by KSHV, for the survival of infected cells, and for metastasis.

Herpes Simplex Virus (HSV) – 1 also known as Human Herpes Virus (HHV) – 1 is a common infectious agent of humans which can cause a wide variety of clinical outcomes, ranging from mild mucocutaneous lesions to long term morbidity and possible mortality. Viral entry into cells requires the co-ordinated action of at least four HSV-1 envelope glycoproteins: gB, gD and the heterodimer gHgL. Dendritic cells (DC) are the most potent antigen presenting cells and are likely to encounter the virus early after entry into the host. HSV-1 readily infects DC leading to a series of both morphological and functional changes, and yet the pathway induced by HSV-1 that leads to DC maturation has not been elucidated. This thesis aims to understand the role of the viral entry glycoproteins in the activation of DC and the consequent initiation of an immune response. Defining this role has important implications not only in understanding immunopathogenesis, but also in the study of HSV-1 as an immunotherapeutic vector, and in the design of an efficient HSV-1 vaccine. Monocyte-derived DCs (MDDC) were found to recognise and respond to the complex of four essential viral glycoproteins, independent of other viral proteins or nucleic acids. MDDC recognition of these four glycoproteins leads to the upregulation of a maturation phenotype and the production of type I interferon (IFN) as well as the induction of a strongly polarised TH1, IFN-γ dominated allogeneic T cell response. In contrast, monocyte-derived Langerhans cells (MDLC), display only partial maturation phenotype and do not produce type I IFN. Plasmacytoid DC (pDC) induce a strong type I IFN response to a viral infection, but not to the viral surface glycoproteins. In the context of natural HSV-1 infection, these results suggest a model in which at the site of HSV-1 infection, different DC sub-populations respond differently to a viral infection and to glycoproteins expressed on the surface of infected cells in order to provide an effective immune response.