The Size of the Viral Inoculum Contributes to the Outcome of Hepatitis B Virus Infection

The impact of virus dose on the outcome of infection is poorly understood. In this study we show that, for hepatitis B virus (HBV), the size of the inoculum contributes to the kinetics of viral spread and immunological priming, which then determine the outcome of infection. Adult chimpanzees were infected with a serially diluted monoclonal HBV inoculum. Unexpectedly, despite vastly different viral kinetics, both high-dose inocula (10¹⁰ genome equivalents [GE] per animal) and low-dose inocula (10° GE per animal) primed the CD4 T-cell response after logarithmic spread was detectable, allowing infection of 100% of hepatocytes and requiring prolonged immunopathology before clearance occurred. In contrast, intermediate (10⁷ and 10⁴ GE) inocula primed the T-cell response before detectable logarithmic spread and were abruptly terminated with minimal immunopathology before 0.1% of hepatocytes were infected. Surprisingly, a dosage of 10¹ GE primed the T-cell response after all hepatocytes were infected and caused either prolonged or persistent infection with severe immunopathology. Finally, CD4 T-cell depletion before inoculation of a normally rapidly controlled inoculum precluded T-cell priming and caused persistent infection with minimal immunopathology. These results suggest that the relationship between the kinetics of viral spread and CD4 T-cell priming determines the outcome of HBV infection.The hepatitis B virus (HBV) is a noncytopathic DNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma (5). Viral clearance and disease pathogenesis during acute HBV infection require the induction of a vigorous CD8⁺ T-cell response and the induction of significant hepatic immunopathology (12, 28). In contrast, chronic HBV infection is associated with a markedly diminished CD8⁺ T-cell response to HBV (23, 24) for reasons that are not well defined.We have previously studied the immunobiology and pathogenesis of HBV infection in chimpanzees that we inoculated with a single (10⁸ genome equivalents [GE]) dose of a monoclonal inoculum of HBV (12, 28, 33). In all of these animals, the infection pursued a reproducible, almost stereotypical course irrespective of the age, size, sex, and genetics of the animals, and it spread to 100% of the hepatocytes before it was terminated by the CD8 T-cell response. The reproducibility of these results suggested that the course and outcome of infection were dominated by the impact of the virus on the kinetics and magnitude of the infection and on the kinetics and magnitude of the immune response that it elicited.Because a high viral load has a negative impact on the outcome of other virus infections (reviewed in references 19 and 32), we examined in the present study the impact of the size of the viral inoculum on the outcome of HBV infection in HBV-naive, immunocompetent adult chimpanzees using a wide dose range of the same monoclonal inoculum that we used in our earlier studies.In contrast to the highly reproducible outcome to the 10⁸ GE dose in our previous experiments, we observed a wide range of outcomes to the various dosages used here, including the development of chronic HBV infection, that we could relate to the kinetics of the CD4 T-cell response in each animal. Furthermore, depletion of CD4⁺ cells before infection with a dose of virus that is otherwise rapidly cleared led to persistent infection. These results suggested that the size of the viral inoculum may contribute to the outcome of infection by altering the balance between the kinetics and magnitude of infection versus the kinetics and magnitude of the immune response. Similar results have been recently published based on in situ analysis of the ratio of virus-infected cells to immune effector cells in the tissues of simian immunodeficiency virus-infected macaques and lymphocytic choriomeningitis virus-infected mice (20).Collectively, these results suggest that the kinetics of T-cell priming relative to the kinetics of viral spread determines the outcome of HBV infection. Specifically, they suggest that early priming of the CD4⁺ T-cell response before or during viral spread initiates a vigorous, synchronized, and functionally efficient CD8⁺ T-cell response and the accompanying immunopathology that ultimately terminates HBV infection. In contrast, the virus persists when CD4⁺ T-cell priming is delayed until after all of the hepatocytes are infected.