Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees

Abstract

We have previously shown that hepatitis B virus (HBV) replication is controlled by noncytolytic mechanisms that depend primarily on the effector functions of the CD8(+) T cell response, especially the production of IFN-gamma in the liver. The mechanisms that control the nuclear pool of viral covalently closed circular DNA (cccDNA) transcriptional template of HBV, which must be eliminated to eradicate infection, have been difficult to resolve. To examine those mechanisms, we quantitated intrahepatic HBV cccDNA levels in acutely infected chimpanzees whose virological, immunological, and pathological features were previously described. Our results demonstrate that the elimination kinetics of the cccDNA are more rapid than the elimination of HBV antigen-positive hepatocytes during the early phase of viral clearance, and they coincide with the influx of small numbers of IFN-gamma producing CD8(+) T cells into the liver. In contrast, terminal clearance of the cccDNA is associated with the peak of liver disease and hepatocellular turnover and with a surge of IFN-gamma producing CD8(+) T cells in the liver. Collectively, these results suggest that cccDNA clearance is a two-step process mediated by the cellular immune response. The first step reduces the pool of cccDNA molecules noncytolytically, probably by eliminating their relaxed circular DNA precursors and perhaps by destabilizing them. The second step enhances this process by destroying infected hepatocytes and triggering their turnover. Surprisingly, despite this multipronged response, traces of cccDNA persist indefinitely in the liver, likely providing a continuous antigenic stimulus that confers lifelong immunity.

Fig. 1.

Southern blot analysis of cccDNA isolated from chimpanzee liver biopsies. Low-molecular-weight DNA was isolated from a liver biopsy obtained from Ch1620 at the peak of infection (week 12), as described in Materials and Methods. Ten microliters of extracted nucleic acids was subjected to HBV-specific Southern blot analysis (lane 1). Another 10 μl of the week 8 sample was linearized by XhoI digestion in a 100-μl reaction before Southern blot analysis (lane 2). Lane 3 shows HBV replicative intermediates in 10 μg of total HBV DNA (T) in the liver of Ch1620 in week 12. A linear EcoRI HBV fragment (F) was included as a size marker (lane 4). rcDNA, relaxed circular HBV DNA; linear, one genome-copy-length HBV fragment; ssDNA, single-stranded HBV DNA.

Fig. 2.

Kinetics of HBV cccDNA during acute HBV infection in the control Ch1627. (A) Intrahepatic HBV cccDNA (shaded area with black triangles) was quantitated by real-time PCR and is expressed as a percentage (%max) of the corresponding peak of cccDNA in the liver of this animal. HBcAg-positive hepatocytes (black squares) are expressed as a percentage (%) of the total number of hepatocytes. (B) Total RNA isolated from liver biopsy samples was analyzed for the expression of IFN-γ, CD3, and L32 by a RNase protection assay, as described in Materials and Methods. The L32 signals reflect the amount of RNA used in the assay. (C) Total HBV DNA replicative intermediates (black diamonds) were quantitated by real-time PCR and are expressed as a percentage (%max) of the corresponding peak value. (D) PCNA-positive (open squares) hepatocytes are displayed as a percentage (%) of the total number of hepatocytes. sALT activity (black circles) is expressed in units per liter (U/l).

Fig. 3.

Kinetics of HBV cccDNA during acute HBV infection and CD8 depletion in Ch1620. (A) Intrahepatic HBV cccDNA and HBcAg-positive hepatocytes are displayed as described in the legend to Fig. 2 A. The number of CD3⁺ CD8⁺ T cells is expressed as a percentage of the total number of CD3⁺ T cells in the peripheral blood. (B) Analysis of IFN-γ, CD3, and L32 expression as described in Fig. 2B.(C) Intrahepatic levels of pgRNA were determined by RNase protection assay. Total HBV DNA (black diamonds) and pgRNA (open circles) are expressed as a percentage (%max) of their corresponding peak value. (D) PCNA-positive hepatocytes are expressed as a percentage of the total number of hepatocytes and sALT activity (black circles) are displayed as units per liter (U/l). See the legend to Fig. 2 for all other details. The arrow indicates time of CD8 depletion.