Characterisation of Hepatitis B virus from chronically infected African children living in Australia

Abstract

One third of the worlds’ population have evidence of hepatitis B infection making it one of the most important global health problems of this century. The risk of developing chronic hepatitis B is greatest if acquired as a newborn or in early childhood. Untreated, 1 in 4 people with chronic hepatitis B (CHB) will die as a result of the infection. This is despite the fact that an effective vaccine has been available for 30 years. Children in the poorest countries are often not vaccinated, including much of Africa, where 60 million people are chronically infected. With immigration of people from highly endemic regions including sub-Saharan Africa, Australia’s prevalence is continuing to rise.

Different Hepatitis B virus (HBV) strains, or genotypes, are present throughout the world and vary in terms of natural history of CHB, disease complications, and treatment response. Most of what is known about this small, yet stealthy virus has been studied in genotypes from Asia and Europe, and little is known of the natural history of this virus in African children. Moreover, genotypes of this virus in African children with CHB living in Australia are unknown.

The studies in this thesis involved patients from Royal Children’s Hospital Melbourne, examining the natural history of chronic hepatitis B infection in African children living in Australia from clinical, serological, and virological perspectives. We performed comprehensive molecular analysis to identify the HBV genotype from these patients and specifically looked for mutations that have been associated with more severe disease phenotypes. We then created African HBV clones based on the genotypes identified in our patients to study their replication and protein expression in cell culture transfection models. We also investigated potential mechanisms for virally-mediated host immune evasion.

67 treatment-naive children were studied, with more than half already transitioned into the second phase of CHB infection at a median age of 12.5 years. Genotype E was the dominant HBV genotype identified which has been associated with vaccine escape, and another subgenotype, the A1, was also detected which has been associated with early development of hepatocellular carcinoma. There was a high frequency of clinically-significant mutations in the precore and basal core promotor regions detected in these patients despite their young age. Replication-competent HBV clones of the African genotypes identified were created and successfully replicated in a transient transfection cell culture model showing marked differences in replication phenotype and protein expression between the genotypes relative to a European HBV reference clone. Functional assays examining the role of the precore and surface proteins of HBV genotype E have identified possible mechanisms for immune modulation that could enhance viral persistence and account for differences in the clinical phenotype of this important African genotype.

Our findings have important public health implications for patient monitoring and treatment guidelines, and potential vaccine efficacy in the Australian setting, as well as translation to the broader global community.