Medicine (St Vincent's) - Theses
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ItemAssociations between Hepatitis B virus mutations and infection kinetics, host responses and outcomes( 2020)The hepatitis B virus (HBV) has infected more than 2 billion people worldwide and is associated with a significant global burden of disease due to its main associated complications of cirrhosis and hepatocellular carcinoma (HCC). The risk of developing cirrhosis or HCC is highly variable, with some people who have quiescent disease throughout their lifetime, whilst others develop aggressive disease associated with high mortality and morbidity rates. HBV is prone to mutations during viral replication and, as a result, it acquires multiple mutations due to pressures either from exogenous vaccines or therapeutic agents or from the host’s immune system. This propensity to mutate has resulted in the formation of genotypes and viral variants. Genotypes of HBV have a sequence variation of 8% or more and they have a specific geographical distribution. The most common viral variants of HBV are the basal core promoter (BCP) and precore (PC) mutations. BCP diminishes the production of the e antigen (HBeAg) whilst PC abolishes it completely. The role of the HBeAg is not completely understood. It is not required for infectivity or any major steps of the viral life cycle. It is thought to be an immune-modulator although the mechanisms of immune modulation are yet to be elucidated. There is a robust body of epidemiological evidence that links viral variants and genotypes to distinct clinical phenotypes. However, the actual host-virus mechanistic interactions are unknown. The HBV, in itself, is not generally cytopathic. It is the immune response to the virus that is thought to be responsible for the inflammatory response and subsequent scarring and liver fibrosis. Currently, there is no definitive cure for chronic hepatitis B (CHB) infection. Antiviral therapy suppresses viral replication but does not eliminate the virus. Due to its close interaction with the immune response, an understanding of the host-virus immuno-pathogenesis is needed in order to explore novel immune-based therapies. The first aim of this thesis was to understand the characteristics of CHB in Australia. Using a population sample from a large tertiary Victorian hospital, a detailed study of the virological characteristics including genotypes, and BCP and PC mutations was performed. We focused on the association between virological factors including genotypes and mutations and clinical phenotypes. Genotypes A, B, C and D were the most prevalent, with a clear preservation of the relationship between genotype and country of origin of the patient. Genotypes B and C were the most common, a reflection of the large migrant population from South East Asia. HBeAg-negative CHB was common and was strongly associated with the presence of the PC variant. A large proportion of patients with HBeAg-negative CHB had an active hepatitis and they were in the immune escape phase. BCP and PC mutations were highly prevalent and were independently associated with the presence of advanced fibrosis, regardless of age, sex or genotype. These findings suggest that there may be a role for sequencing for these mutations in clinical practice and use them as biomarkers. Based on this clinical observation, we also hypothesized that HBeAg was modulating the immune response to enable chronicity in early stages of disease but then becomes a liability in later phases. This formed the basis of studies of the immune response with a specific focus on HBeAg. The second aim of this thesis was to explore some of the key immune pathways that modulate HBV infection. The hypothesis was that HBeAg modifies innate host immunity to establish chronic infection in the early phases of disease. Much of the literature in HBV has focused on the adaptive immune response but we were interested in the early phases of the natural history, where HBeAg is almost universally present and its absence is associated with reduced infectivity. The role of HBeAg in the early stages of HBV infection was studied using an immunocompetent mouse model transfected with HBeAg- negative strains of HBV. Pertinent findings included a sudden, early drop in viraemia in HBeAg- negative mutants compared to wild type (WT) HBV. A corresponding transaminitis strongly suggested that in absence of HBeAg, there was a more robust innate immune and inflammatory response to the virus. Complementation studies confirmed that the effect was due to HBeAg and no other viral components. Using gene-modified animals and selective antibody depletion, a series of studies were performed to examine individual arms of the innate immune response. We showed that the Type 1 interferon (IFN-I) response was much stronger in absence of HBeAg. Correspondingly, transcriptomic analysis revealed the downregulation of multiple IFN-I pathways and proteome/ phosphor-proteome studies showed changes in the expression of interferon regulating genes associated with HBeAg. Collectively our data showed that HBeAg allows the virus to evade the innate immune response by antagonising IFN-I in the initial stages of infection. The emergence of HBeAg- negative mutants in the later stages of the natural history of CHB suggests that HBeAg presents a liability to the virus. In our mouse model, following a significant drop in viraemia in the initial phases of infection, there was a recrudescence in viral load in the later stages of disease in HBeAg deficient mutants. We hypothesized that in the later, adaptive immune response, HBeAg is an immune target and there is a strong selective pressure that favours HBeAg- deficient viral variants. A series of experiments using gene-targeted animals was performed to examine the roles of various mediators of the adaptive immune system. These studies showed that the absence of HBeAg was associated with reduced function of CD4+ T cells and a reduction in TNF and IFN- gamma activity. HBeAg deficiency was also associated with an escape from the antibody response. In conclusion, HBeAg is an important antigen in the modulation of the immune response in HBV. Sequencing for HBeAg negative variants may have a role in clinical practice and further longitudinal, prospective studies are warranted. Further dissection of the proteomic and transcriptomic signatures associated with HBeAg negative mutants and their correlation with clinical samples would be the next steps to elucidate the intricate role of HBeAg in modulating disease behaviour and outcomes.