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

doi:10.1128/JVI.00867-09

J Virol. 2009 October; 83(19): 9652–9662.

Published online 2009 July 22. doi: 10.1128/JVI.00867-09

PMCID: PMC2748002

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

Shinichi Asabe,¹ Stefan F. Wieland,¹ Pratip K. Chattopadhyay,² Mario Roederer,² Ronald E. Engle,³ Robert H. Purcell,³ and Francis V. Chisari¹^*

Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037,¹ Immuno Technology Section, Laboratory of Immunology, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,² Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892³

^*Corresponding author. Mailing address: The Scripps Reserach Institute, SBR-10, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-8228. Fax: (858) 784-2160. E-mail: fchisari/at/scripps.edu

Received April 30, 2009; Accepted July 13, 2009.

This article has been cited by other articles in PMC.

Abstract

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.

MATERIALS AND METHODS

Chimpanzees.

Nine healthy, young adult, HBV-seronegative chimpanzees (A0A006, A0A007, 1622, 1603, 1616, 1618, A2A014, A3A005, and A2A007) were studied. The sex, age, and body weight before inoculation are given in Table S1 in the supplemental material. The animals were handled according to humane use and care guidelines specified by Animal Research Committees at the National Institutes of Health, The Scripps Research Institute, and Bioqual Laboratories. They were individually housed at Bioqual Laboratories (Rockville, MD), an American Association for Accreditation of Laboratory Animal Care International-accredited institution under contract to the National Institute of Allergy and Infectious Diseases. The animals were inoculated with a serial dilution of an HBV-positive serum from chimpanzee 5835 that was previously inoculated with a monoclonal HBV isolate (genotype D, ayw subtype; GenBank accession no. V01460) (9) contained in HBV transgenic mouse serum (11). The dilutions were prepared in preinoculation serum obtained from animal 5835. Before inoculation and weekly thereafter, blood was obtained by venipuncture and analyzed for serum alanine aminotransferase (sALT), HBV antigens, and anti-HBV antibodies as described previously (10).

PBMC and liver tissue.

Prior to infection and every other week thereafter, 20 to 40 ml of acid-citrate-dextrose anticoagulated blood was obtained and shipped to The Scripps Research Institute for isolation of peripheral blood mononuclear cells (PBMC) the next day, as described previously (28). Tissue fragments 5 to 10 mm in length were obtained by needle biopsy and shipped to The Scripps Research Institute after processing. One fragment was immediately placed into RPMI containing 10% AB serum and cooled on wet ice, another fragment was fixed in 10% zinc formalin for histological examination exactly as previously described (10, 12, 34), and the final fragment was snap-frozen for 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 as described in Thimme et al. (28) and in the supplemental material.

Serum HBV DNA detection.

HBV DNA was extracted from serum as described in the supplemental material and quantified by HBV-specific quantitative real-time PCR as described previously (28).

RNA isolation and quantitation.

Total liver RNA was isolated as described previously (6), and 0.5 μg was analyzed for intrahepatic gene expression using gene-specific primers by reverse transcription quantitative real-time PCR exactly as described previously (16, 17, 33). The fold changes of intrahepatic gene expression were normalized to the average baseline expression in all chimpanzees for each gene in at least two preinoculation time points. Baseline expressions for all genes varied (5.3 ± 1.6)-fold among all chimpanzees.

IFN-γ ELISPOT assay.

Cryopreserved PBMC were thawed and placed into round-bottom 96-well plates at 2 × 10⁵ per well and cultured in RPMI 1640, 10% AB serum, and 2 mM l-glutamine with or without recombinant HBV core antigen (as described in the supplemental material) at 1 μg/ml for 6 days. Enzyme-linked immunospot (ELISPOT) plates (BD Bioscience) were coated overnight at 4°C with primary antibody against human gamma interferon (IFN-γ; BD Bioscience) and blocked for 2 h at 25°C with RPMI and 5% AB serum. Cultured cells were transferred into coated plates and cultured at 37°C for 18 h. The plates were processed according to the manufacturer's protocol, and spots were counted by using an immunospot analyzer (Cellular Technology, Ltd., Shaker Heights, OH). The specificity of CD4⁺ T-cell response was confirmed by depletion of CD4⁺ cells using magnetic beads (BD Biosciences). Samples in which the ratio of spot-forming cells (SFC) with versus without antigen was higher than 2.5 were considered positive, and the number of specific SFC was calculated as follows: (SFC with antigen) - (SFC without antigen).

Patr multimer staining and flow cytometry analysis.

Cryopreserved cells were thawed and suspended in RPMI plus 4% fetal calf serum, followed by the addition of a mixture of Patr/peptide multimers corresponding to previously identified Patr-restricted epitopes (see the supplemental material) and stained at 37°C for 10 min. Cells were washed and stained with a cocktail of antibodies for the surface staining at 4°C for 30 min. Dead cells were excluded by either propidium iodide or a Live/Dead Fixable Aqua dead cell stain kit (Invitrogen). The flow cytometric acquisition was done at the Vaccine Research Center or The Scripps Research Institute by using Digital LSR II (BD Biosciences), and the analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR).

CD4⁺ T-cell depletion.

CD4 depletion was achieved by administration of a humanized chimeric monoclonal anti-human CD4 antibody cM-T412 that we have previously described (15, 28). The antibody was administered three times during the week prior to HBV inoculation and weekly thereafter at 5 mg/kg. A control chimpanzee was treated with an isotype-matched chimeric monoclonal antibody to respiratory syncytial virus (MedImmune, Inc., Gaithersburg, MD) as described previously (15, 28). CD4 depletion was monitored in PBMC using a cocktail of anti-CD4 (clone: OKT4), anti-CD3, CD8, and CD14 antibodies. The absolute number of CD4⁺ T cells was calculated based on the total number of lymphocytes in the blood, and the percentage of CD4⁺ T cells was determined by fluorescence-activated cell sorting analysis.

RESULTS

Massive spread and delayed clearance after inoculation with 10¹⁰ GE of HBV.

Chimpanzee A0A006 (see Table S1 in the supplemental material) was inoculated with a monoclonal HBV inoculum of 10¹⁰ GE of HBV DNA as described in Materials and Methods. As shown in Fig. Fig.1a,1a, viral DNA (black line) and viral antigens (horizontal black bars) increased immediately in the serum of chimpanzee A0A006, indicating rapid viral spread. The virus spread to virtually 100% of the hepatocytes (HBV core antigen immunostaining [data not shown]), and serum HBV DNA levels reached 3.3 × 10¹⁰ GE/ml by 6 weeks after inoculation. This was followed by a sharp, synchronous rise in sALT activity on week 9 heralding the elimination of HBV by week 16. A brief resurgence of virus was eliminated by week 26 after the appearance of anti-HBs antibodies.

FIG. 1.

Course of acute HBV infection in chimpanzees after experimental intravenous inoculation of various doses of HBV indicated as GE of HBV in parentheses next to the chimpanzee identification number. Serum HBV DNA levels were determined by quantitative real-time (more ...)

Limited spread and rapid clearance after inoculation with 10⁷ and 10⁴ GE of HBV.

Chimpanzees A0A007 and 1622 (see Table S1 in the supplemental material) received doses of 10⁷ and 10⁴ GE, respectively (Fig. 1b and c). Virus spread was delayed, the peak serum HBV DNA titers in these chimpanzees reached only 3.9 × 10⁷ and 2.5 × 10⁷ GE of HBV/ml, respectively, and, correspondingly, fewer than 0.1% of the hepatocytes were HBcAg positive. At this point, viral spread was abruptly interrupted 6 to 8 weeks after inoculation, and the infection was rapidly terminated, coincident with a sharp, synchronous rise in sALT activity, long before the appearance of anti-HBs antibodies.

Massive spread and persistent infection after inoculation with 10¹ GE of HBV.

Chimpanzees 1603 and 1616 (see Table S1 in the supplemental material) were inoculated with 10¹ GE of HBV. As expected, the appearance of HBV DNA was greatly delayed (Fig. 1d and e) compared to the high dose animals (Fig. 1a to c), although the doubling time was the same (~2.0 days). Unexpectedly, however, instead of being rapidly controlled as seen with the 10⁷- and 10⁴-GE inocula, the virus spread to 100% of the hepatocytes and produced extremely high peak serum HBV DNA titers of 1.3 × 10¹⁰ to 1.9 × 10¹⁰ GE/ml, similar to chimpanzee A0A006 that received 9 logs more virus. Unlike that animal, however, HBV DNA persisted in both animals for 42 and more than 50 weeks, respectively, and it was accompanied by a slow, asynchronous increase in sALT activity. Chimpanzee 1603 was unavailable for analysis between weeks 29 and 34; however, on week 34, sALT activity was sharply elevated, and the viral DNA titer was several logs lower, suggesting that an immunological flare had occurred during the observational hiatus and that virus clearance was in progress. Indeed, by week 42 viral DNA was no longer detectable in the serum of this animal, sALT activity returned to baseline, and anti-HBs antibodies became detectable at week 47. In contrast, chimpanzee 1616 became persistently infected with histological evidence of chronic active hepatitis (see Fig. S1 in the supplemental material) for more than 55 weeks, at which point the study was discontinued.

Massive spread and delayed clearance after inoculation with 10° GE of HBV.

Chimpanzees 1618 and A2A014 (see Table S1 in the supplemental material) were inoculated with 10° GE of HBV (i.e., a single infectious virion). As shown in Fig. 1f and g, after a prolonged delay due to the low-titer inoculum, the virus spread to virtually 100% of the hepatocytes (HBV core antigen immunostaining [data not shown]), and serum HBV DNA levels reached 1.4 × 10¹⁰ to 2 × 10¹⁰ GE/ml by 10 to 13 weeks after inoculation. This was followed by an initially slow and then a sharp, synchronous rise in sALT activity heralding the elimination of HBV. Except for the delayed viral kinetics and more prolonged infection plateau, these results are remarkably similar to those for chimpanzee A0A006, which received 10 logs more virus.

Early peripheral CD4⁺ T-cell responses are predictive of viral clearance.

We tested the ability of the peripheral CD4⁺ T cells of the infected animals to produce IFN-γ after in vitro stimulation by recombinant HBcAg. As shown in Fig. Fig.2,2, strong HBcAg-specific CD4⁺ T-cell responses were first detectable in chimpanzees A0A007 and 1622 as early as 1 to 3 weeks after inoculation (Fig. 2b and c, black bars in the upper panels), coincident or before the period of detectable viral spread (Table (Table1)1) and before the corresponding HBe antigens or HBV DNA could be detected in the serum (Table (Table11 and Fig. 1b and c) or HBcAg in the liver (data not shown). Interestingly, both animals terminated the infection before the virus reached 0.1% of the hepatocytes. In contrast, HBcAg-specific CD4⁺ T-cell responses were first observed after the onset of detectable viral spread in chimpanzees A0A006, 1618, and A2A014 (see Fig. 2a, f, and g, upper panels, and Table Table1)1) in which the virus spread to all of the hepatocytes before it was slowly cleared. Importantly, HBcAg-specific CD4⁺ T-cell responses were not detectable in chimpanzees 1603 and 1616, both of which became persistently infected, until 13 weeks after inoculation (Fig. 2d and e, upper panels, and Table Table1),1), when peak serum HBV DNA levels had been reached, and 100% of the hepatocytes were infected. Interestingly, onset of the CD4 response occurred slightly before the peak of infection in chimpanzee 1603 (Fig. (Fig.2d)2d) that spontaneously cleared the virus after 42 weeks in the context of a sALT flare (Fig. (Fig.2d),2d), a finding reminiscent of an acute disease flare as is observed in many chronically infected patients, whereas it began after the peak of infection in chimpanzee 1616 (Fig. (Fig.2e),2e), who remained persistently infected. These results suggest that timing of the priming of HBV-specific CD4⁺ T-cell responses relative to the timing of viral spread (summarized in Table Table1)1) is closely related to the outcome of infection.

FIG. 2.

Peripheral CD4⁺ T-cell responses against HBV core protein and intrahepatic CD8⁺ T-cell responses. The upper panel in each figure represents the serum HBV DNA as a black line and sALT as a yellow shaded area, and the results of peripheral (more ...)

TABLE 1.

Timing of the priming of CD4 T-cell responses versus HBV kinetics

Viral clearance is associated with a synchronized CD8⁺ T-cell response.

We have previously shown that viral clearance in acute HBV infection is strictly dependent on the CD8⁺ T-cell response to HBV antigens (28). To examine the influence of the dose of the viral inoculum and the kinetics of CD4⁺ T-cell priming on the influx of HBV-specific CD8⁺ T cells into the liver of the infected animals, we monitored intrahepatic CD8⁺ T-cell responses with the HBV-specific major histocompatibility complex (MHC) class I multimers shown in Table S5 in the supplemental material and the intrahepatic content of CD8 mRNA and the mRNA of an array of functional T-cell markers (see Table S2 in the supplemental material). The sum of the frequencies of all HBV-specific MHC class I multimer-positive CD8⁺ liver-infiltrating lymphocytes is shown as blue bars in the bottom panels of Fig. Fig.2,2, and the results for individual multimer stainings are shown in Table S3 in the supplemental material.

As shown in Fig. Fig.2,2, the chimpanzees whose CD4 responses developed before or soon after the onset of detectable virus spread and that cleared the infection coincident with a sharp increase of sALT activity also showed a simultaneous influx of HBV-specific CD8⁺ T cells and CD8 mRNA into the liver (Fig. 2a, b, and c, lower panel). The influx of HBV-specific CD8⁺ T cells and CD8 mRNA into the liver of the chimpanzees that showed massive viral spread and delayed clearance after inoculation with 10° GE of HBV (chimpanzees 1618 and A2A014) also correlated with surges in sALT activity, which ultimately exceeded 500 U/liter during the phase of viral clearance (Fig. 2f and g). Not surprisingly, the magnitude and duration of the infection reflected the number of infected cells and serum HBV DNA titer at the peak of infection. Unfortunately, for technical reasons, CD8⁺ T-cell multimer analysis could not be tested in the animal receiving the 10⁷-GE inoculum.

Interestingly, the intrahepatic mRNA content for monokine induced by IFN-γ (MIG), granzyme B, FAS-L, and PD-1 corresponded to the CD8 mRNA content and sALT activity in the animals that ultimately cleared the infection (Fig. 3a to c, f, and g), indicating the influx of functionally activated CD8⁺ T cells into the liver. Together, these results suggest that viral clearance was associated with an apparently synchronized influx of HBV-specific CD8⁺ T cells into the liver in all animals whose CD4⁺ T cells had been primed to HBV before or soon after the onset of logarithmic viral spread. Thus, although the extent and duration of infections were strikingly different in these animals, viral clearance always occurred in the context of CD4⁺ T-cell priming before or shortly after the first appearance of serum HBV DNA (Fig. 2a to c, f, and g, upper panels) and by well-coordinated and highly synchronized CD8⁺ T-cell response (Fig. 2a to c, f, and g, lower panels, and Fig. 3a to c, f, and g).

FIG. 3.

Intrahepatic mRNA induction profiles in infected animals. The first panel in each figure represents the virological and sALT data as in Fig. Fig.1.1. The fold induction of intrahepatic mRNA compared to two preinoculation time points was calculated (more ...)

Viral persistence is associated with a poorly synchronized CD8 T-cell response.

In contrast to the surge in sALT activity in the chimpanzees that cleared the infection, the increase in sALT activity was gradual and strongly delayed until long after the peak of infection in chimpanzees 1603 and 1616 that received 10¹ GE of HBV and failed to control the infection for more than 6 months (Fig. 2d and e, upper panels). Similarly, the appearance of intrahepatic HBV-specific CD8⁺ T cells, CD8 mRNA (Fig. 2d and e, lower panels), MIG, granzyme B, perforin, FAS-L, and PD-1 mRNA (Fig. 3d and e) also increased gradually over several months, implying that the entry of HBV-specific CD8⁺ T cells into the liver of these animals was both slow and poorly synchronized. Importantly, the elevated sALT levels, histological evidence of chronic hepatitis (see Fig. S1 in the supplemental material), and the upregulation of MIG, granzyme B, and perforin (Fig. 3d and e) suggests that the CD8⁺ T cells were activated in these animals. Nonetheless, there was little or no decrease in the magnitude of infection for several months, implying that the antiviral activity of the CD8⁺ T-cell response was functionally ineffective in these animals, perhaps because it was not synchronized because their CD4⁺ T cells were not primed before the virus spread to 100% of the hepatocytes or because prolonged antigen stimulation induced prolonged expression of high levels of PD-1 (Fig. 3d and e).

The HBV-specific CD4⁺ T-cell response determines the outcome of HBV infection.

To test the hypothesis that CD4⁺ T-cell priming before or during early viral spread is necessary to induce the synchronized intrahepatic CD8⁺ T response that appears to be required to clear the infection, we immunodepleted CD4⁺ T cells in chimpanzee A2A007 (see Table S1 in the supplemental material) before and for several months after inoculation with 10⁴ GE of HBV, a dose that typically results in a self-limited infection (Fig. (Fig.1c1c and and2c),2c), and we compared the course, duration and outcome of infection with a similarly inoculated animal (chimpanzee A3A005) that received an irrelevant control antibody.

As expected, the chimpanzee that received the control antibody (Fig. (Fig.4a,4a, top panel) developed an acute self-limited (<28 weeks) infection (Fig. (Fig.4a,4a, top panel) that was heralded by an early CD4⁺ T-cell response that coincided with detectable viral expansion (Fig. (Fig.4a,4a, middle panel) and was terminated by a highly synchronized intrahepatic CD8⁺ T-cell response (Fig. (Fig.4a,4a, bottom panel). In contrast, the peripheral CD4⁺ T-cell population (Fig. (Fig.4b,4b, top panel) and the HBV-specific CD4⁺ T-cell response (Fig. (Fig.4b,4b, middle panel) were virtually abolished in the CD4-depleted chimpanzee. Importantly, this animal became persistently (>70 weeks) infected, but without the sALT elevation or CD8⁺ T-cell responses we observed in all of the other animals. 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 in another chimpanzee, i.e., at the peak of HBV infection (28). Together with the results of the dosage experiment, these results strongly suggest that the relative kinetics of viral spread and the CD4⁺ T-cell response determines the outcome of HBV infection.

FIG. 4.

Course of HBV infections, peripheral CD4⁺ T-cell responses against HBV core protein, and intrahepatic CD8⁺ T-cell responses in chimpanzees with or without CD4 immunodepletion. Serum HBV DNA level, serum HBe and HBs antigen levels, and (more ...)

DISCUSSION

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, 28, 33). 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 Table1).1). All of these animals mounted a strong T-cell response to the virus (12, 28, 33) 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 Table11 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 (Table1).1). 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. (Fig.1c)1c) 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 (Table1),1), 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 (Table1),1), 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 (Table1).1). 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, 25, 27). Other studies suggest that an early CD4⁺ T-cell response is required for clearance of acute hepatitis C virus infection (26, 30). 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, 22), which therefore failed to evolve into functionally competent effector T cells (3, 21). 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, 18). Upregulated PD-1 expression had been shown to contribute to virus-specific T-cell dysfunction in patients chronically infected by human immunodeficiency virus (8, 29), HBV (1, 2), 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. 3d and e). 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.

Supplementary Material

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Acknowledgments

This study was supported by grants AI20001 and CA76403 and by contracts N01-AI-52705, N01-AI-45180 and N01-CO-56000 from the National Institutes of Health (NIH); by the Intramural Research Program of the National Institute of Allergy and Infectious Disease, NIH; and by the Sam and Rose Stein Charitable Trust.

This is manuscript number 19937-IMS from the Scripps Research Institute.

We thank C. Shaver (Bioqual, Inc.) for animal care; K. A. Masterman, L. Friske, and B. Boyd for technical assistance; and the NIH Tetramer Facility for providing Patr/peptide monomers. We also thank J. Ghrayeb (Centocor) for the anti-CD4 antibody.

Footnotes

Published ahead of print on 22 July 2009.

^†Supplemental material for this article may be found at http://jvi.asm.org/.

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