In the present study we examined the impact of the size of the viral inoculum on the kinetics and magnitude of viral spread and the immune response to HBV and the impact of that relationship on the course and outcome of HBV infection in nine young chimpanzees. To rule out the possible impact of viral genetic diversity on the outcome of infection, we inoculated the animals with a monoclonal viral inoculum. Importantly, this is the same inoculum that we previously used to infect five other chimpanzees with a single 108
-GE dose (12
). Irrespective of their age, size, sex, and genetic background, all five of these animals developed highly reproducible infections in which the virus spread with a doubling time of ~2.0 days and reached 75 to 100% of the hepatocytes within 9 weeks after inoculation (see Table ). All of these animals mounted a strong T-cell response to the virus (12
) that terminated the infection, like the response in the animal inoculated with 1010
GE of HBV in the present study that had a similar course of infection. The salient features of the infections in these animals are included in Table and Tables S1 and S4 in the supplemental material. The reproducibility of these results suggested that any interanimal differences that might have influenced the course and outcome of infection at this dose were overshadowed by the impact of the virus on the kinetics and magnitude of viral spread and on the kinetics and magnitude of the immune response that it elicited. Thus, if the dose of the inoculum is sufficiently high (e.g., ≥108
GE), it appears to exert a dominant influence on the course and outcome of infection.
To test the hypothesis that the size of the viral inoculum influences the outcome of infection, in the present study we examined the kinetics, magnitude, host response, and outcome of infection over a wide dose range of the same inoculum that we used in our earlier studies. All 12 of the animals in the present and previous studies that we inoculated with 1010, 108, 107, 104, or 10° GE of HBV cleared the virus within 8 to 30 weeks after its first detection in a virus dose-related fashion (Table ). In contrast, both of the animals that were inoculated with 101 GE became chronically infected, one of which (like many chronically infected humans) ultimately cleared the virus in the context of an acute disease flare 42 weeks after first detection, whereas the other remained heavily infected for at least 55 weeks at which point the study was terminated. This suggests that a virus dose window exists between 104 and 10° GE within which the host-virus dynamics favor persistent infections and on either side of which viral clearance occurs.
Interestingly, in two of the three animals inoculated with the 107- and 104-GE doses, the infection was apparently contained before it spread beyond 0.1% of the hepatocytes, and their peak virus titers never exceeded 108 GE/ml. The huge differences in the magnitude and course of infection between these two animals and the twelve animals that received either higher or lower doses of the inoculum could imply that 107 and 104 GE represent the upper and lower boundaries of a dosage window within which the virus can be so rapidly controlled by the immune response that its spread is interrupted before it reaches 0.1% of the hepatocytes. The fact that the virus spread to 100% of hepatocytes in one of the two animals that received the 104-GE dose, similar to all of the lower dose animals, suggests that 104 GE may be a transitional dose at the lower end of the rapidly controllable dose range of HBV in chimpanzees. This could imply that interanimal variation in host genetics, age, weight, etc., may be dominant over virological influences at this dose and explain the differential course of infection in these two animals (see Tables S1 and S4 in the supplemental material). For example, the animal that rapidly cleared the infection before it spread to all of the hepatocytes (Fig. ) was older and larger (see Table S1 in the supplemental material) than the animal in which the virus spread to all of the hepatocytes before terminating the infection, implying greater immunological maturity and possibly a lower multiplicity of infection because of its larger liver. Additional studies in multiple animals infected with this dose are needed to clarify this issue.
Unexpectedly, we found that the 101-GE HBV inoculum caused persistent infection, whereas infections with both higher- and lower-dose inocula were cleared with kinetics that corresponded to the number of infected hepatocytes and the maximal virus titers attained at the peak of infection. Infection in an animal inoculated with 107 and one of two animals inoculated with 104 GE of HBV DNA was rapidly terminated before it reached 0.1% of the hepatocytes. Clearance was heralded by early CD4+ T-cell priming either before or at the onset of detectable viral spread, and it coincided with a sharply synchronized influx of HBV-specific CD8+ T cells into the liver and a corresponding increase in intrahepatic CD8 mRNA, sALT activity, and histological evidence of acute viral hepatitis. Indeed, the interval between the first measurable levels of HBV DNA and the first detectable CD4 T-cell response in the animals that cleared the infection in the present study was ≤3 weeks (Table ), i.e., during or before the phase of detectable viral expansion. In contrast, the interval was 7 to 8 weeks in the two animals that developed persistent infection (Table ), and these responses were detected at or after the peak of infection at which point the virus had infected 100% of the hepatocytes.
These fascinating results led us to hypothesize that an early CD4+
T-cell response to HBV infection may be necessary to induce the CD8+
T-cell response required to clear the infection. To test this hypothesis, we inoculated an animal that was immunodepleted of CD4+
cells with a virus dose (104
GE of HBV) that should have been terminated in the context of a T-cell response. Interestingly, CD4 depletion resulted in persistent HBV infection, whereas the same 104
-GE HBV inoculum, as expected, caused an acute resolving HBV infection in an animal that had received an isotype control antibody. Importantly, CD4 T-cell immunodepletion using the same antibody that we used in the present study had no impact on the outcome of infection when it was performed 6 weeks after inoculation with the same inoculum in another chimpanzee (animal 1615), i.e., at the peak of HBV infection (28
) (Table ). Collectively, these results suggest that the timing of CD4+
T-cell priming relative to the kinetics of viral spread was the key element that determined the magnitude and quality of the subsequent CD8+
T-cell response to HBV and, therefore, the outcome of infection.
The significance of CD4+
T cells in acute viral infections is somewhat controversial (4
). Several studies suggest that CD4+
T cells are dispensable for the early expansion of CD8+
effector T cells but required for the generation of a functional memory CD8+
T-cell pool (14
). Other studies suggest that an early CD4+
T-cell response is required for clearance of acute hepatitis C virus infection (26
). We present here cellular and molecular evidence that an early CD4+
T-cell response to HBV is required for the development of optimal CD8+
T-cell responses which then determine the outcome of HBV infection. Since early CD4+
T-cell priming was observed in some animals prior to detectable viremia and antigenemia, we suggest it may have been triggered by noninfectious subviral antigens that are in large molar excess (4 logs) relative to the number of infectious virions in the inoculum (see Table S1 in the supplemental material). Studies to test this hypothesis are currently under way. We also point out that host genetic influences could easily contribute to the relative kinetics of the T-cell response, since chimpanzee 1616 that developed persistent infection is homozygous for MHC class 2 alleles at three loci (see Table S4 in the supplemental material).
It is important to note that the CD8, MIG, granzyme B, and perforin mRNA content of the liver during the prolonged infections was comparable to or greater than that seen in the rapidly controlled infections, and it persisted throughout the course of the infections in the presence of modestly elevated sALT activity and histological evidence of liver disease, even though the intrahepatic HBV-specific CD8+
T-cell response was relatively weak and poorly synchronized. The fact that the CD8+
response failed to control the virus in the 101
-GE HBV infections suggested that it was functionally compromised. There are several possible explanations for this observation. First, in the absence of adequate CD4+
help, HBV-specific CD8+
T cells may not have been adequately primed before the virus had spread to massive proportions in the liver, delaying their expansion until after all of the hepatocytes were infected, making it difficult or impossible for them to stay ahead of the infection. Second, as described in other systems, once the virus had spread to all of the hepatocytes, continuous antigen stimulation might have anergized or exhausted the progressively accumulating T cells (13
), which therefore failed to evolve into functionally competent effector T cells (3
). Third, continuous antigen stimulation could have induced and maintained high levels of expression of negative regulatory molecules in the T cells, thereby suppressing their antiviral function. Recently, a number of negative regulators of T-cell activity have been described that are thought to contribute to persistent infection and immunopathology (7
). Upregulated PD-1 expression had been shown to contribute to virus-specific T-cell dysfunction in patients chronically infected by human immunodeficiency virus (8
), HBV (1
), and hepatitis C virus (31
). Consistent with this concept, we observed prolonged elevation of very high levels of intrahepatic PD-1 mRNA in the prolonged and persistent infections (Fig. ). Although PD-1 upregulation was possibly secondary to repetitive antigen stimulation in the prolonged and persistent infections, negative signaling by the upregulated PD-1 could further impair the T-cell response and prevent subsequent viral clearance (18
). Additional studies are required to test this hypothesis and to examine the role of other negative regulatory molecules in the pathogenesis of persistent HBV infection.