PBMC.

Prior to infection and each week thereafter for up to 32 weeks, 40 ml of citric acid-sodium citrate-dextrose anticoagulated blood were obtained for isolation of peripheral blood mononuclear cells (PBMC) and shipped to La Jolla, California, on wet ice for processing the next day. PBMC were isolated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in Hanks balanced salt solution (Gibco Laboratories, Grand Island, N.Y.), and stained for surface markers CD3, CD4, and CD8 immediately. Briefly, 2 × 10⁵ PBMC per well were plated in a 96-well V-bottom plate, stained with the respective antibodies for 30 min on ice, washed three times with phosphate-buffered saline containing 1% fetal calf serum (FCS), and analyzed directly thereafter. Fluorescence-activated cell sorter (FACS) analysis was performed at the Scripps Research Institute Flow Cytometry Core Facility by using FACSort and FACScan flow cytometers and analyzed with Cell Quest software (Becton Dickinson, San Jose, Calif.).

Liver biopsy.

Prior to infection and each week thereafter, liver tissue was obtained by hepatic needle biopsy. In most instances, two to three tissue fragments 5 to 10 mm in length were obtained. One fragment was immediately placed into RPMI medium (Gibco Laboratories) containing 10% AB serum and cooled on wet ice for shipment to Scripps; another fragment was fixed in 10% zinc formalin solution for subsequent histological examination; and the final fragment was snap frozen for subsequent RNA isolation.

Isolation and expansion of intrahepatic T cells.

At weekly intervals, liver-infiltrating lymphocytes were isolated from approximately 0.5 to 1 cm of hepatic needle biopsy sample. The tissue was homogenized in 2 to 3 ml of Dulbecco's phosphate-buffered saline (Gibco Laboratories) with a Dounce tissue grinder. The average number of intrahepatic lymphocytes was between 5 × 10⁴ and 5 × 10⁵ cells. Cell suspensions were then incubated with magnetic beads coupled to either anti-CD8 or anti-CD4 antibodies (Dynabeads; Dynal, Oslo, Norway) for 20 min at 4°C, and bound CD8⁺ or CD4⁺ T cells were isolated by using a particle magnetic concentrator. The isolated intrahepatic CD4⁺ and CD8⁺ T cells were then plated in separate wells of 24-well plates (Corning, Corning, N.Y.) in 1 ml of 10% human AB⁺ serum, 100 U of interleukin-2 (IL-2; Hoffmann-La Roche, Inc., Nutley, N.J.)/ml, and 0.04 μg of anti-human CD3 monoclonal antibody (Immunotech, Marseilles, France)/ml as a stimulus to T-cell growth and 2 × 10⁶ irradiated autologous PBMC per well as feeder cells. Twice a week, 1 ml of media was exchanged and 100 U of IL-2/ml was added. After 2 weeks of expansion, 4 to 6 million cells were recovered and the expanded T cells were tested for HBV-specific T-cell responses (see below). The purity of each T-cell subset was always >95%.

Recombinant HBV proteins.

A recombinant preparation of hepatitis B core antigen (HBcAg) prepared from bacterial extracts of Escherichia coli K-12 strain HB 101 harboring the recombinant plasmid carrying the HBcAg-encoding gene was obtained from Biogen (Cambridge, Mass.) as previously described (29). Purity was over 98% as determined by scanning densitometry of Coomassie blue-stained sodium dodecyl sulfide-polyacrylamide gels.

EBV B-cell lines and recombinant expression vectors.

Autologous Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines (B-LCL) were established as described previously (16). All target cells were maintained in RPMI with 10% (vol/vol) heat-inactivated FCS (Gibco Laboratories). Recombinant vaccinia viruses expressing the HBV core (c-vac), polymerase, and large envelope region (ayw subtype) have been previously described (16). Wild-type vaccinia viruses were used as a negative control. Briefly, B-LCL were infected at a multiplicity of infection of 25 for 1 h at room temperature with the respective vaccinia viruses. After 1 h, 1 ml of 10% FCS medium was added and the infected B-LCL were cultured (at 37°C) overnight and then washed once before they were added as stimulators to the intracellular IFN-γ staining assays (see below).

Antibodies.

Fluorescein isothiocyanate (FITC)-conjugated anti-CD8, phycoerythrin (PE)-conjugated anti-CD8, PE-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-CD3, PE-conjugated anti-IFN-γ, and FITC-, APC-, or PE-conjugated isotype antibodies were obtained from Pharmingen (San Diego, Calif.) and used for immunostaining and FACS analysis according to the manufacturer's instructions.

CD4⁺- and CD8⁺-T-cell depletion.

The ability of CD4⁺ and CD8⁺ T cells to control HBV infection was monitored in chimpanzees 1615 and 1620, respectively. CD4 depletion was achieved with humanized chimeric monoclonal anti-human CD4 antibody cM-T412, which contains the antigen binding region of a murine antibody, M-T412, and the constant region of a human immunoglobulin G1 κ chain (20). This antibody has been previously used to deplete CD4⁺ cells in chimpanzees (20) according to a schedule of three intravenous injections of 5 mg/kg of body weight administered once every other day for 1 week. CD8 depletion was performed with a humanized chimeric monoclonal anti-human CD8 antibody cM-T807, which contains the variable region of a murine antibody, M-T807, and the constant region of a human immunoglobulin G1-κ (34, 35). This antibody has been previously used to deplete CD8⁺ T cells in macaque monkeys (34, 35) according to a schedule of 3 injections of 5 mg/kg over 7 days. Chimpanzee 1627 was treated with an isotype-matched mouse and human chimeric monoclonal antibody directed against respiratory syncytial virus (MedImmune, Inc., Gaithersburg, Md.) as a control.

HBV-specific proliferative T-cell response.

Intrahepatic CD4⁺ T cells were resuspended together with 10⁵ autologous irradiated (3,000 rads) PBMCs as APC (10⁶ cells/ml) in complete medium with 10% human AB serum, plated at a concentration of 2 × 10⁵ cells/200 μl/well and stimulated with 10 μg of HBcAg/ml or no antigen in five replicate wells of 96-well U-bottom plates. [³H]thymidine (1 μCi; Dupont NEN, Boston, Mass.) was added to each well on day 5. Cultures were harvested on day 6 after 16 h of [³H]thymidine incorporation. The degree of cellular proliferation was expressed as a stimulation index (SI) in which the average [³H]thymidine uptake (in counts per minute) in replicate wells stimulated with HBcAg was divided by the average [³H]thymidine uptake in replicate wells stimulated without antigen. Based on prior analysis of uninfected and infected chimpanzees, an SI of >3.0 was used as a positive cutoff value.

HBV-specific CD8⁺-T-cell responses.

The intrahepatic HBV-specific CD8⁺-T-cell response was analyzed by intracellular IFN-γ staining of polyclonally expanded CD8⁺ T lymphocytes (see above) after 5 h of in vitro stimulation with autologous EBV-transformed B-LCL that were infected with recombinant HBV vaccinia viruses that express the HBV envelope, core, and polymerase proteins or with wild-type vaccinia virus as a control. Briefly, previously expanded CD8⁺ T cells were plated in 96-well plates at 2 × 10⁵ cells per well and stimulated with 2 × 10⁵ cells of autologous EBV-transformed B cells infected with vaccinia viruses that express the HBV envelope, core, or polymerase proteins for 5 h (37°C, 5% CO₂) in the presence of 50 U of recombinant human IL-2 (Hoffmann-La Roche)/ml and 1 μg of brefeldin A (Pharmingen)/ml, and then the cells were stained with antibodies to human CD8 and IFN-γ (Pharmingen). Duplicate wells incubated with B-LCLs infected with wild-type vaccinia virus were also included to determine the background level of IFN-γ production. In addition, one well containing only autologous EBV B cells plus CD8⁺ T cells was included for isotype antibody staining, and another well was stimulated with 10 ng of phorbol 12-myristate 13-acetate (Sigma Chemical Co.)/ml and 200 ng of ionomycin/ml to serve as a positive control for IFN-γ staining. FACS analysis was performed at the Scripps Research Institute Flow Cytometry Core Facility by using FACSort and FACScan flow cytometers and analyzed with Cell Quest software (Becton Dickinson). The frequency of HBV-specific CD8⁺ T cells was defined as the percentage of CD8⁺ T cells that produce IFN-γ in response to stimulation by B-LCL coinfected by each of the viral HBV constructs after subtraction of the IFN-γ⁺ CD8⁺ T cells detected after stimulation by wild-type vaccinia virus.

RNA isolation and RNase protection assay.

Frozen liver tissues were mechanically pulverized, and RNA was extracted by the acid guanidinium-phenol-chloroform method (6). Total RNA (20 μg) was analyzed by the RNase protection assay for quantitation of mRNA exactly as previously described (15).

Course of HBV infection in the control animal.

As shown in Fig. ​Fig.1A1A on a linear scale and in Fig. ​Fig.2A2A on a logarithmic scale, hepatic HBV DNA levels became detectable in chimpanzee 1627 by quantitative PCR during week 5 after inoculation when the intrahepatic HBV DNA content was 249 GE/ng of liver DNA and approximately 0.2% of the hepatocytes were HBcAg positive. The intrahepatic content of HBV DNA peaked at 3.9 × 10⁴ copies/ng of liver DNA in week 8, at which time 82% of the hepatocytes were HBcAg positive. Based on these figures, there were approximately 285 HBV DNA molecules per infected hepatocyte, or 1.3 × 10¹³ GE of HBV in the liver at that time point, representing a >133,000-fold amplification of the inoculum at the peak of the infection. Between weeks 8 and 11, the HBV DNA content of the liver abruptly decreased by more than 95% while the fraction of HBcAg-positive hepatocytes fell by less than 40% and there was only a slight increase in serum alanine amino transferase (sALT) activity. Subsequently, however, sALT activity increased, reached a peak of 381 U/liter by week 14, and returned rapidly to the baseline by week 17. This delayed episode of liver disease coincided with the final elimination of HBV DNA and HBcAg-positive hepatocytes from the liver at week 17. It is noteworthy that all of these events occurred in the absence of an anti-HBs antibody response, which was not detected until week 26 (not shown), long after viral clearance had occurred.

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FIG. 1.

Course of acute HBV infection in chimpanzees after experimental inoculation with HBV in the presence or absence of CD4⁺ and CD8⁺ cells. All animals were inoculated with 10⁸ GE of HBV intravenously in week 0 and with a monoclonal antibody (see below) in week 6. (A) Chimpanzee (Ch.) 1627 was injected with irrelevant control antibody (Ab). (B) Chimpanzee 1615 was injected with a CD4-specific monoclonal antibody. (C) Chimpanzee 1620 was injected with a CD8-specific monoclonal antibody. Intrahepatic HBV DNA (black triangles) is expressed as a percentage (%max) of the corresponding peak HBV DNA levels in the liver of each animal. sALT activity (black squares) is expressed in units per liter. HBcAg-positive hepatocytes are expressed as a percentage of the total number of hepatocytes. The number of CD3⁺ CD4⁺ or CD3⁺ CD8⁺ T cells is expressed as a percentage of the total number of CD3⁺ T cells in the peripheral blood. Vertical arrows indicate the time of antibody treatment.

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FIG. 2.

Intrahepatic cytokine profile and HBV-specific T-cell responses during acute HBV infection in control chimpanzee (Ch.) 1627. (A) The courses of intrahepatic HBV DNA and sALT activity during acute infection are displayed as described in the legend to Fig. ​Fig.1,1, except for the intrahepatic HBV DNA content, which is displayed on a logarithmic scale in this figure and in Fig. ​Fig.33 and ​and44 in order to facilitate comparison of the duration of the infection in the 3 animals. (B) Total RNA isolated from liver biopsy samples was analyzed for the expression of CD3, IFN-γ, and L32 by an RNase protection assay. The L32 signals reflect the amount of RNA used in the assay. (C) The intrahepatic CD4⁺-T-cell response to HBcAg is expressed as the SI. (D) The intrahepatic CD8⁺-T-cell response is shown as the percentage of intrahepatic CD8⁺ cells that produce IFN-γ after stimulation with autologous EBV B cells that were infected with recombinant vaccinia viruses expressing the HBV core, polymerase, and large envelope proteins after subtraction of their responsiveness to the same B cells infected by wild-type vaccinia virus. The vertical arrow indicates control antibody (Ab) treatment.

Depletion of CD4⁺ cells does not alter the duration or outcome of acute HBV infection.

As shown in Fig. ​Fig.1B1B and ​and3A,3A, the course of infection in chimpanzee 1615 was almost identical to that in chimpanzee 1627, despite the fact that chimpanzee 1615 was depleted of CD4⁺ cells in week 6 after infection, at which time hepatic HBV DNA levels were rapidly rising. Unexpectedly, a small peak of sALT activity was detectable in this animal during the period of logarithmic viral expansion prior to CD4 depletion. Interestingly, circulating CD4⁺ cells became undetectable, and the early sALT peak subsided in parallel with CD4 depletion (Fig. ​(Fig.1B,1B, week 7), suggesting that CD4⁺ cells may have contributed to the atypically early liver disease in this animal. The absence of CD4⁺ cells, however, didn't affect the rate of viral spread or the magnitude of the infection. Specifically, HBV DNA levels peaked at 2.7 × 10⁴ copies/ng of liver DNA in week 8, at which time 92.5% of hepatocytes were HBcAg positive, reflecting the presence of about 175 copies of HBV DNA per infected cell or 9.3 × 10¹² GE of HBV in the entire liver, i.e., slightly lower than that in the control animal at the same time point. Like in the control animal, the hepatic HBV DNA levels fell by >90% between weeks 8 and 11, coincident with the return of CD4⁺ cells to the circulation, while the level of HBcAg-positive hepatocytes decreased less than 7% and there was a slight increase in sALT activity. Subsequently, the sALT activity rose, reached a peak of 534 U/liter in week 13, and returned to baseline by week 19, 2 weeks after the disappearance of HBV DNA from the liver, coinciding with the disappearance of HBcAg-positive hepatocytes and the absence of an anti-HBs antibody response which was not detectable until week 32 after inoculation (data not shown). It is important to emphasize that the profound decrease in HBV DNA between weeks 8 and 11 occurred as CD4⁺ T cells reappeared, but the dynamic changes in sALT activity during weeks 10 to 19 occurred even though the number of circulating CD4⁺ cells was less than 23% of the baseline level and relatively stable at that time. It is also important to note that the number of peripheral CD8⁺ cells did not change during the period of profound CD4 depletion (data not shown). Thus, the course of HBV infection was almost identical in the control and CD4-depleted animals, both of whose CD8⁺-T-cell populations were intact.

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FIG. 3.

Intrahepatic cytokine profile and HBV-specific T-cell responses during acute HBV infection and CD4 depletion in chimpanzee (Ch.) 1615. (A) The courses of intrahepatic HBV DNA and sALT activity during acute infection and CD4 depletion are displayed as described in the legend to Fig. ​Fig.2A.2A. (B) Analysis of intrahepatic CD3, IFN-γ, and L32 expression. (C) The intrahepatic CD4⁺-T-cell response to HBcAg during acute infection and CD4 depletion. (D) Intrahepatic CD8⁺-T-cell responses during acute infection and CD4 depletion. See the legend to Fig. ​Fig.22 for all other details. Ab, antibody.

Depletion of CD8⁺ cells dramatically alters the duration and outcome of acute HBV infection.

As shown in Fig. ​Fig.1C1C and ​and4A,4A, chimpanzee 1620 was treated with a monoclonal anti-CD8 antibody during week 6, when HBV DNA levels were rapidly rising in the liver. During the infusion, the CD8⁺ cells immediately became undetectable, and they didn't begin to reappear until week 12 when they started a slow, prolonged, and somewhat halting recovery. Despite the CD8⁺ cell depletion, the early kinetics of the infection, until week 8, were almost indistinguishable from those in the control and CD4-depleted animals. Indeed, at week 8, 96% of the hepatocytes were HBcAg positive and HBV DNA replicative intermediates were present at a level of 3.3 × 10⁴ GE/ng of liver DNA, reflecting 207 copies per hepatocyte or 1.3 × 10¹³ copies in the entire liver, almost identical to the number of copies in control chimpanzee 1627 (Fig. ​(Fig.1A1A).

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FIG. 4.

Intrahepatic cytokine profile and HBV-specific T-cell responses during acute HBV infection and CD8 depletion in chimpanzee (Ch.) 1620. (A) The courses of intrahepatic HBV DNA and sALT activity during acute infection and CD8 depletion are displayed as described in the legend to Fig. ​Fig.2A.2A. (B) Analysis of intrahepatic CD3, IFN-γ, and L32 expression. (C) The intrahepatic CD4⁺-T-cell response to HBcAg during acute infection and CD8 depletion. (D) Intrahepatic CD8⁺-T-cell responses during acute infection and CD8 depletion. See the legend to Fig. ​Fig.22 for all other details. Ab, antibody.

Subsequently, however, the virological and pathogenic events in the CD8-depleted chimpanzee were vastly different from those in the control and CD4-depleted animals. As illustrated in Fig. ​Fig.1C1C and especially in Fig. ​Fig.4A,4A, which replots the course of infection on a logarithmic scale in order to display the virological changes after week 15 when HBV DNA levels were very low, there was a striking relationship between the number of circulating CD8⁺ cells and both virus titer and sALT activity at the later time points in the infection. For example, in the absence of CD8⁺ cells, the time span of peak infection (weeks 8 to 11) was prolonged, the time of onset of the initial decrease in HBV DNA levels and increase in sALT activity (week 13) was delayed, and the time required for the first phase of viral clearance (week 15) and the eventual termination of infection (week 31) were markedly delayed and prolonged. Importantly, the total intrahepatic HBV DNA content and the number of HBV DNA GE per hepatocyte remained at their initial peak levels until week 11, i.e., for as long as CD8⁺ cells were undetectable in the circulation. The reappearance of CD8⁺ cells between weeks 11 and 17 was associated with the onset of a mild liver disease and with a 50-fold reduction in total liver HBV DNA to 2.5 × 10¹¹ copies in the entire liver, even though the number of HBcAg-positive hepatocytes decreased only 18% during the same time period, suggesting that, as in the previous two animals, HBV replication was inhibited noncytopathically at this stage of the infection.

Surprisingly, after their initial recovery, the number of CD8⁺ cells stabilized at approximately 40% of baseline levels between weeks 16 to 27, and this was associated with a comparably stable plateau in sALT activity and intrahepatic HBV DNA content (Fig. ​(Fig.1C1C and ​and4A).4A). In contrast, the number of HBcAg-positive hepatocytes decreased progressively in the same time period despite a stable total HBV DNA content of 2.8 × 10¹¹ copies in the entire liver on week 27. These results, together with the absence of serum anti-HBs antibodies in this animal until weeks 42 to 45 (data not shown), suggest that HBcAg-positive hepatocytes apparently were being destroyed and replaced during this prolonged period of time, yet the infection wasn't eliminated, presumably because of unopposed viral spread to new hepatocytes. Unexpectedly, on week 29, the number of CD8⁺ cells suddenly rebounded to baseline levels, and this coincided with a surge in sALT activity and the disappearance of HBV DNA from the liver. As in the previous animals, all of these events occurred in the absence of anti-HBs neutralizing antibodies (data not shown), suggesting that viral clearance could be attributed to the effects of the CD8⁺ cells in this animal. It is also important to note that the number of peripheral CD4⁺ cells remained normal during the period of profound CD8 depletion when the infection was greatly prolonged, further supporting the notion that the virological events in this animal were CD8 dependent.

Intrahepatic T-cell and cytokine profiles during acute HBV infection.

In order to determine the extent to which the foregoing virological and pathogenic events were related to the adaptive intrahepatic T-cell response to HBV, we studied the magnitude and kinetics of the HBV-specific CD4⁺- and CD8⁺-T-cell response at multiple time points in these animals. In addition, total liver RNA was subjected to RNase protection analysis in order to monitor changes in global T-cell and cytokine gene expression in the liver.

Control animal chimpanzee 1627.

As shown in Fig. ​Fig.2C,2C, an intrahepatic HBcAg-specific CD4⁺-T-cell response was first detectable in chimpanzee 1627 in week 6 and it surged by week 10 to levels that were, for the most part, sustained for the duration of the study. Importantly, the onset of the CD4⁺-T-cell response was not associated with a decrease in the HBV DNA titer or an increase in sALT activity (Fig. ​(Fig.2A).2A). In contrast, HBV DNA levels started to decrease, and sALT activity started to rise, when HBV envelope-specific CD8⁺ T cells became detectable in the liver on week 10 (Fig. ​(Fig.2D),2D), and there was a corresponding surge in the intrahepatic CD4⁺-T-cell response (Fig. ​(Fig.2C)2C) and the appearance of CD3 and IFN-γ mRNA (Fig. ​(Fig.2B)2B) in the liver. The intrahepatic CD8⁺-T-cell response persisted, strengthened, and diversified to include HBV core and polymerase specificities over the next several weeks (Fig. ​(Fig.2D),2D), as did the intrahepatic content of CD3 and IFN-γ mRNA (Fig. ​(Fig.2B),2B), until viral DNA was no longer detectable and sALT activity returned to normal in week 17 (Fig. ​(Fig.2A).2A). Interestingly, HBV-specific CD4 and CD8⁺-T-cell responses remained easily detectable in the liver for at least several weeks after viral clearance, implying that traces of virus may persist after apparent eradication of the infection.

CD4-depleted animal chimpanzee 1615.

As shown in Fig. ​Fig.3C,3C, the intrahepatic HBcAg-specific CD4⁺-T-cell response wasn't detectable in the CD4-depleted chimpanzee 1615 until week 11, at which time the total HBV DNA content of the liver had already decreased by more than 92% and sALT activity had already started to rise (Fig. ​(Fig.3A),3A), suggesting that CD4⁺ T cells did not play an effector role in these events. Unfortunately, we were unable to expand the intrahepatic CD8⁺-T-cell population between week 8 (when HBV-specific CD8⁺ T cells were undetectable) and week 15 (when they were strongly positive) (Fig. ​(Fig.3D),3D), so we can't determine whether the changing virological and pathological status of the animal during that period was temporally related to the CD8⁺-T-cell response. Between weeks 11 and 18, however, HBV-specific CD4⁺ (Fig. ​(Fig.3C)3C) and CD8⁺ T cells (Fig. ​(Fig.3D)3D) were detectable in the liver, corresponding with the appearance of CD3 and IFN-γ mRNA (Fig. ​(Fig.3B),3B), the peak of liver disease, and the final elimination of detectable viral DNA (Fig. ​(Fig.3A3A).

We thank Max Shapiro (Bioqual, Inc.) for animal care and Rick Koch, Janell Pemberton, and Ronald Engle for excellent technical assistance. We thank Luca G. Guidotti and H. C. Spangenberg for helpful discussions.

This study was supported by grant R01-AI20001 from the NIH. R.T. was also supported by grants TH 719/1-1 and TH 719/2-1 EN from the Deutsche Forschungsgemeinschaft, Bonn, Germany, and by a postdoctoral training fellowship from the Cancer Research Institute, New York, N.Y.