The Size of the Viral Inoculum Contributes to the Outcome of Hepatitis B Virus Infection
病毒剂量对感染结果的影响知之甚少。在这项研究中，我们表明，对于乙型肝炎病毒（HBV），接种物的大小影响病毒传播和免疫启动的动力学，然后确定感染的结果。用连续稀释的单克隆HBV接种物感染成年黑猩猩。出乎意料的是，尽管病毒动力学差异很大，但高剂量接种物（每只动物1010个基因组当量[GE]）和低剂量接种物（每只动物10°GE）在检测到对数扩散后引发CD4+T细胞反应，允许清除病毒前100％的肝细感染和需要延长的免疫病理学。相反，中间（107和104 GE）接种物在可检测的对数扩散之前引发T细胞应答，并且在0.1％的肝细胞被感染之前以最小的免疫病理学突然终止。令人惊讶的是，在所有肝细胞被感染后，101 GE的剂量引发T细胞应答并且引起具有严重免疫病理学的延长或持续感染。最后，在接种正常快速控制的接种物之前，CD4+ T细胞耗尽排除了T细胞引发并且以最小的免疫病理学引起持续感染。这些结果表明病毒扩散动力学和CD4 T细胞引发之间的关系决定了HBV感染的结果。
乙型肝炎病毒（HBV）是一种非致细胞病变的DNA病毒，可引起急性和慢性肝炎和肝细胞癌（5）。急性HBV感染期间的病毒清除和疾病发病机制需要诱导强烈的CD8 + T细胞应答和诱导显着的肝脏免疫病理学（12,28）。相反，慢性HBV感染与HBV的特异性CD8 + T细胞反应明显减少有关（23,24），原因尚不明确。
我们之前研究过黑猩猩中HBV感染的免疫生物学和发病机制，我们接种了单一（108基因组当量[GE]）剂量的HBV单克隆接种物（12,28,33）。在所有这些动物中，无论动物的年龄，大小，性别和遗传如何，感染都追求可重复的，几乎是刻板的过程，并且在CD8 T细胞应答终止之前它会扩散到100％的肝细胞。 这些结果的可重复性表明，感染的过程和结果主要受到病毒对感染的动力学和程度的影响以及它引起的免疫应答的动力学和程度的影响。
与之前实验中108 GE剂量的高度可重复性结果相反，我们观察到这里使用的各种剂量的广泛结果，包括慢性HBV感染的发展，每只动物的细胞反应，可能与CD4+T细胞的动力学有关。 此外，在用一定剂量的病毒感染之前耗尽使病毒快速清除是CD4 +细胞，会导致持续感染。这些结果表明，通过改变动力学和感染程度与免疫应答的动力学和量级之间的平衡，病毒接种物的大小可能影响感染的结果。最近发表的类似结果是基于原位分析猿猴免疫缺陷病毒感染的猕猴和淋巴细胞脉络丛脑膜炎病毒感染的小鼠组织中病毒感染细胞与免疫效应细胞的比例（20）。
总之，这些结果表明T细胞引发的动力学相对于病毒扩散的动力学决定了HBV感染的结果。具体而言，他们提出在病毒传播之前或期间早期引发CD4 + T细胞启动，引发强烈的，同步的和功能上有效的CD8 + T细胞应答以及最终终止HBV感染的伴随免疫病理学。相反，当CD4 + T细胞延迟到所有肝细胞被感染后才启动，病毒持续存在。
值得注意的是，长期感染期间肝脏的CD8，MIG，颗粒酶B和穿孔素mRNA含量与快速控制的感染相似或更高，并且在整个感染过程中持续存在。存在适度升高的sALT活性和肝病的组织学证据，即使肝内HBV特异性CD8 + T细胞反应相对较弱且同步性差。事实上，CD8 +反应未能控制101-GE HBV感染的病毒，这表明它在功能上受到了损害。这种观察有几种可能的解释。首先，在没有足够的CD4 +帮助的情况下，HBV特异性CD8 + T细胞可能在病毒在肝脏中扩散到大量比例之前没有充分启动，延迟它们的扩增，直到所有肝细胞被感染，使其变得困难或他们不可能保持领先于感染。其次，如其他系统所述，一旦病毒扩散到所有肝细胞，连续的抗原刺激可能会使逐渐积累的T细胞（13,22）失活或耗竭，因此无法进化为功能正常的效应T细胞（ 3,21）。第三，连续抗原刺激可以诱导并维持T细胞中负调节分子的高水平表达，从而抑制它们的抗病毒功能。最近，已经描述了许多被认为有助于持续感染和免疫病理学的T细胞活性的负调节剂（7,18）。已显示上调的PD-1表达导致慢性感染人免疫缺陷病毒（8,29），HBV（1,2）和丙型肝炎病毒（31）的患者中的病毒特异性T细胞功能障碍。与此概念一致，我们观察到长期和持续感染中非常高水平的肝内PD-1 mRNA的持续升高（图3d和e）。虽然PD-1上调可能继发于长期和持续感染中的重复抗原刺激，但上调PD-1的负性信号传导可能进一步损害T细胞反应并阻止随后的病毒清除（18）。需要进一步的研究来检验这一假设，并检查其他负调控分子在持续性HBV感染发病机制中的作用。
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.
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.
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. 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.
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 PCR and are shown as GE/ml. sALT activity (yellow shaded area) is shown as units/liter. Horizontal bars represent serum HBe and HBs antigen levels and the open horizontal bars represent the presence of anti-HBc, anti-HBe, and anti-HBs antibodies. The amount of each protein is reflected by the thickness of each bar as indicated in the legend. The sex, age, and body weight at the inoculation time of each animal is shown in Table S1 in the supplemental material. The superscript “a” indicates the percentage of HBc antigen-positive hepatocytes determined by immunohistochemical analysis of paraffin-embedded liver biopsy tissue.
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. 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 1) and before the corresponding HBe antigens or HBV DNA could be detected in the serum (Table 1 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 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 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. 2d) that spontaneously cleared the virus after 42 weeks in the context of a sALT flare (Fig. 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. 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 1) is closely related to the outcome of infection.
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 CD4+ T-cell ELSIPOT assays are overlaid as black bars. Cryopreserved PBMC were thawed and stimulated in vitro with HBV core protein, and the numbers of IFN-γ producing cells were determined by an ELISPOT assay. The data are shown as number of spots at each time point minus the number of spots before inoculation per million PBMC. The lower panel shows the total number of intrahepatic HBV-specific CD8+ T cells per 102 total CD8+ T cells as filled blue bars (right axis) and the fold induction of intrahepatic CD8 mRNA compared to two preinoculation time points as a shaded red area (left axis). Intrahepatic lymphocytes were expanded antigen nonspecifically in vitro and tested with all of the corresponding Patr/peptide multimer complexes shown in Table S5 in the supplemental material. Frequencies of CD8+ T cells for each Patr/peptide multimer are shown in Table S3 in the supplemental material (a to f). nt, Not tested. *, Tested and negative.
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. 2, and the results for individual multimer stainings are shown in Table S3 in the supplemental material.
As shown in Fig. 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).
Intrahepatic mRNA induction profiles in infected animals. The first panel in each figure represents the virological and sALT data as in Fig. 1. The fold induction of intrahepatic mRNA compared to two preinoculation time points was calculated and shown as a shaded gray area for CD8 (second panel), MIG (third panel), granzyme B (fourth panel), perforin (fifth panel), FAS (sixth panel), FAS-ligand (seventh panel), and PD-1 (eighth panel).
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. 1c and 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. 4a, top panel) developed an acute self-limited (<28 weeks) infection (Fig. 4a, top panel) that was heralded by an early CD4+ T-cell response that coincided with detectable viral expansion (Fig. 4a, middle panel) and was terminated by a highly synchronized intrahepatic CD8+ T-cell response (Fig. 4a, bottom panel). In contrast, the peripheral CD4+ T-cell population (Fig. 4b, top panel) and the HBV-specific CD4+ T-cell response (Fig. 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.
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 the presence of anti-HBc, anti-HBe, and anti-HBs antibodies are shown as in Fig. 1 (top panel). The numbers of CD4+ T cells per μl of whole blood are indicated as closed squares (top panel, right axis). Arrows on the top panels represent injections of control antibody (a) or anti-CD4 antibody (b). Peripheral CD4+ T-cell IFN-γ ELISPOT assays against HBV core protein (second panel) and detection of intrahepatic HBV-specific CD8+ T cells (bottom panel, left axis) were performed as described in Fig. 2 except that freshly prepared cells were used instead of cryopreserved cells. The fold induction of intrahepatic CD8 mRNA compared to two preinoculation time points is shown as a shaded red area (bottom panel, right axis). *, Tested and negative.
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 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 1 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 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. 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 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 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 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.
Pathogenesis and Immunity
Shinichi Asabe, Stefan F. Wieland, Pratip K. Chattopadhyay, Mario Roederer, Ronald E. Engle, Robert H. Purcell, Francis V. Chisari
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The Size of the Viral Inoculum Contributes to the Outcome of Hepatitis B Virus Infection | Journal of Virology https://jvi.asm.org/content/83/19/9652