宿主对HBV感染反应的染色体组分析

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Microbiology
Genomic analysis of the host response to hepatitis B virus infection
Stefan Wieland *, Robert Thimme * , Robert H. Purcell  and Francis V. Chisari *  

*Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; and Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8009

Contributed by Francis V. Chisari, March 12, 2004

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[B]Abstract  [/B]

Previous studies in hepatitis B virus (HBV)-infected humans and chimpanzees suggest that control of HBV infection involves the cells, effector functions, and molecular mediators of the immune response. The objective of the current study was to identify, in the liver of acutely HBV-infected chimpanzees, the spectrum of virus-induced and immune response-related genes that regulate the infection. The results demonstrate that HBV does not induce any genes during entry and expansion, suggesting it is a stealth virus early in the infection. In contrast, a large number of T cell-derived IFN--regulated genes are induced in the liver during viral clearance, reflecting the impact of an adaptive T cell response that inhibits viral replication and kills infected cells, thereby terminating the infection.



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The hepatitis B virus (HBV) is a noncytopathic hepatotropic DNA virus that causes acute chronic hepatitis and hepatocellular carcinoma (1). Viral clearance and disease pathogenesis during HBV infection are tightly associated with the appearance of a vigorous T cell response to all viral proteins (2, 3). In contrast, viral persistence and chronic hepatitis are associated with a markedly diminished HBV-specific T cell response (4, 5). CD8+ T cells are the main immune effector cells during HBV infection, because viral clearance and liver disease are blocked by depletion of CD8+ T cells in acutely infected chimpanzees (6). Furthermore, the onset of viral clearance in these animals is tightly associated with the appearance of virus-specific T cells (6) as well as CD3, CD8, and IFN- mRNA (6, 7) in the liver, indicating again that the adaptive T cell response plays a key role in this process.
Although cytolytic T cell functions certainly contribute to viral clearance, noncytolytic T cell functions also play a role, because adoptively transferred HBV-specific CD8+ T cells inhibit viral replication in HBV transgenic mice by a noncytopathic IFN--mediated mechanism (8), and because HBV DNA largely disappears from the liver and blood long before the peak of liver disease in acutely HBV-infected chimpanzees (6, 7). Furthermore, IFN-/-mediated mechanisms also inhibit HBV replication noncytopathically in transgenic mice (9), and they do so by inhibiting viral capsid assembly (ref. 10 and S.W. and F.V.C., unpublished observations). Interestingly, genomic analysis of the livers and hepatocyte cell lines from those mice demonstrated a close association between the antiviral effects of both IFN- and IFN-/ and the induction of hepatocellular genes that might mediate the antiviral effects (11). These genes include GTP-binding proteins (e.g., GBP-1 and TGTP) known to inhibit other viral infections (12, 13), as well as components of the immunoproteasome (LMP2, LMP7, and MECL-1), IFN-stimulated protein 15 (ISG15), ubiquitin-specific protease 18 (Usp18), the chemokine IP-10, and the signal transducer and activator of transcription (STAT)-1 (11).

The current study was prompted by the desire to validate these findings in the setting of an acute HBV infection where the genomic changes occurring during viral entry, spread, and clearance can be uniquely identified. We did so by serially profiling the liver transcriptome in three acutely HBV-infected chimpanzees, searching especially for two distinct groups of cellular genes: those whose expression might correlate with the entry and expansion of the virus that might reflect the innate immune response, and those whose expression correlates with viral clearance reflecting the adaptive immune response that terminates infection.


[B]Materials and Methods[/B]   

Chimpanzees. Three healthy young adult HBV-seronegative chimpanzees (Ch1615, -1627, and -5835) were used in this study. The animals were handled according to humane use and care guidelines specified by the Animal Research Committees at the National Institutes of Health and The Scripps Research Institute. They were 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 108 genome equivalents of a monoclonal HBV isolate (genotype ayw) contained in pooled serum from HBV transgenic mice (14). Before inoculation and weekly thereafter, blood was obtained by venipuncture and analyzed for serum alanine aminotransferase activity (sALT) as described (8). Six weeks after inoculation, Ch1615 and -1627 received three daily i.v. injections of either a humanized chimeric monoclonal anti-human CD4+ antibody (cM-T412) or an irrelevant control antibody, respectively, as described (6). The course of infection, inflammatory infiltrate, and kinetics of viral clearance were not affected in these animals (6). Liver biopsy and DNA and RNA preparation protocols are available in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Gene Expression Analysis. At selected time points, 100 ng of total liver RNA isolated from frozen liver biopsies was used to prepare cRNA by the reduced-volume RNA amplification protocol described by Baugh et al. (15). The cRNA was hybridized to high-density oligonucleotide arrays (HG-U133A Human GeneChips, Affymetrix, Santa Clara, CA), which interrogate the expression of 22,000 human genes. Primary image analysis was performed with GENECHIP VERSION 5.0 (Affymetrix). Relative gene expression levels were normalized throughout the entire data set by using algorithms in the DCHIP software package (16, 17). Genes not called "present" by GENECHIP at least at one time point were excluded from further analysis, and the remaining values were logarithm-transformed to base 10. Genes were identified whose expression correlated (by Pearson's correlation) either with the pattern of log-transformed intrahepatic HBV DNA or with the expression profile of specific prototype cellular genes described in the text. A Pearson correlation coefficient of 0.7 over all time points in all three chimpanzees was required for a gene to be further analyzed. Finally, selected genes had to be called "present" at the peak of intrahepatic HBV DNA levels or at the expression maxima of specific prototype genes, and they had to be called "induced" by GENECHIP at the same time point relative to the preinfection sample in all three animals. Genes were selected as "induced during viral clearance" if they were called "present" and "induced" relative to preinfection levels in weeks 12, 14, and 16 in Ch1627, 14 and 16 in Ch1615, and 14 and 18 in Ch5835. In addition, the expression level of these genes had to be higher during viral clearance than during the weeks before peak viremia in the same animal (i.e., weeks 4 and 6 in Ch1627 and 2 and 4 in Ch1615 and -5835). For Ch1627, HBV clearance-associated genes were grouped according to their induction kinetics into early, middle, and late genes based on the week (i.e., 12, 14, or 16) when peak induction was observed. Gene expression profiles were visualized by using GENESPRING VERSION 5.0 (Silicon Genetics, Redwood City, CA) by using gene expression values normalized to the 10th and 90th percentiles for each gene.

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[B]Results  [/B]

Course of HBV Infection in Acutely Infected Chimpanzees. Ch1615, -1627, and -5835 were inoculated i.v. with 0.5 ml of HBV-transgenic mouse serum containing 1 x 108 genome equivalents of HBV DNA (6). As shown in Fig. 1, all three animals, including the CD4-depleted animal, became infected, developed acute hepatitis, and cleared the infection with remarkably similar kinetics (liver HBV DNA levels are shown in Table 2, which is published as supporting information on the PNAS web site). Viral spread in Ch1627 (Fig. 1A) and -5835 (Fig. 1C) was not accompanied by induction of CD3, IFN-, or 2'5' oligoadenylate synthetase (2'5'OAS) mRNA, which was monitored by RNase protection analysis (Fig. 1), suggesting that the virus was not recognized by the innate or adaptive immune response in those animals at that time. In contrast, CD3, IFN-, and 2'5'OAS mRNA were all transiently induced in Ch1615 during week 5, coinciding with a transient spike of elevated sALT activity (Fig. 1B). All of these events were abruptly terminated by antibody-mediated CD4 depletion during week 6 (Fig. 1B, vertical arrow), suggesting that they reflected the atypically early onset of an adaptive immune response in this animal. This notion is further supported by the observation that, several weeks later, viral clearance in all animals coincided with the appearance of CD3, IFN-, and 2'5'OAS mRNA (Fig. 1), elevated sALT (Fig. 1), and HBV-specific T cells in the liver (ref. 6 and data not shown). Collectively, these results suggest that these T cells and their products contributed to viral clearance in these animals. Importantly, in all three animals, the viral DNA decreased 10- to 50-fold in the liver before the number of hepatitis B core antigen-positive hepatocytes began to decrease (Fig. 1), indicating that the decrease in viral DNA did not reflect the destruction of infected cells.

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Fig. 1. Course of acute HBV infection in chimpanzees after experimental i.v. inoculation of 108 genome equivalents of HBV in week 0. (A) Ch1627 was also injected with an irrelevant control antibody in week 6. (B) Ch1615 was injected with a CD4-specific monoclonal antibody as described in the text. (C) Ch5835 did not receive any additional treatment. Intrahepatic HBV DNA (black squares) is expressed as a percentage (% max) of the peak HBV DNA levels in the liver of each animal (actual liver HBV DNA levels are presented in Table 2). Hepatitis B core antigen-positive (HbcAg) hepatocytes (gray bars) are expressed as percentage of the total number of hepatocytes. sALT (open circles) is expressed in units per liter. Intrahepatic expression profiles for IFN-, CD3, and 2'5'OAS were determined by RNase protection assay. Vertical arrows indicate the time of antibody injection. Stars indicate the time points for which gene expression profiling was performed.

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Liver Gene Expression Profiles in Acutely Infected Chimpanzees. The stars in Fig. 1 indicate the time points when liver gene expression profiling was performed in the animals. In this study, we attempted to identify two classes of genes: one group whose expression correlated with hepatic viral DNA content and therefore could reflect the initial (innate) host response to HBV in the liver, and a second group whose expression correlated with viral clearance and who are likely to be induced by the adaptive immune response to the virus.
Liver Genes Induced by HBV. The first group of genes displayed expression patterns that correlated (directly or inversely) with the amount of HBV DNA in the liver over the entire time course profiled. To reduce the chance of misinterpreting random fluctuations in gene expression in individual animals, we calculated the Pearson correlation between the expression of each gene and the amount of HBV DNA at each time point in all three animals, and we restricted our focus to genes whose changing expression levels correlated with the changing viral DNA content in the liver with a correlation coefficient of at least 0.7. Importantly, as shown in Fig. 2A, no genes fulfilled these criteria. Furthermore, no transcripts were uniformly induced or repressed if we limited our focus solely to the lag phase of infection (weeks 0-2 in Ch1615 and -5835) or to the phase of logarithmic expansion (weeks 4-6 in Ch1615 and -5835 and weeks 4-7.5 in Ch1627) of the virus (data not shown). Because virtually 100% of the hepatocytes were infected in all three animals (see hepatitis B core antigen-positive hepatocytes in Fig. 1), the failure of the virus to induce cellular gene expression as it spread throughout the liver suggests that HBV does not induce an innate immune response at the site of infection.

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Fig. 2. Liver gene expression profile during acute HBV infection. (A) Genes correlated with viremia in all chimpanzees. No genes correlated positively or negatively with the level of intrahepatic HBV DNA during the time course of acute HBV infection in all three animals. (B) Liver gene expression profile associated with viral clearance in all three acutely HBV-infected chimpanzees. The gene identities are shown in Table 1 and the primary data in Tables 3 and 4. Green lines show the intrahepatic HBV DNA as a percentage (% max) of the corresponding peak levels. For optimal visualization, gene expression levels (red lines) are shown after normalization to the 10th and 90th percentiles for each gene. Values on the x axis represent weeks after inoculation with HBV.

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Liver Genes Associated with Viral Clearance. To identify genes associated with viral clearance, we searched for transcripts that were either induced or suppressed when viral DNA disappeared from the liver. We identified 59 unique genes ("Selected as induced during viral clearance," Table 1, and indicated with a Y in column 5 of Table 3, which is published as supporting information on the PNAS web site) that were induced during viral clearance in all three animals (average peak fold change, 10.7), and none that were suppressed. The expression profile of these genes reflects a type 1 T cell response, because it was heavily weighted toward genes expressed by / T cells (e.g., T cell receptor ), / T cells (e.g., T cell receptor ), the cytolytic effector molecule granzyme A, and many genes known to be induced by IFN-, which itself correlated with viral clearance (Fig. 1). The IFN--induced genes include STAT1, MHC class I, MHC class II, immunoproteasome subunits LMP2, PA28 and -, ubiquitin D, E2L6, tapasin, the chemokines monokine induced by IFN- (MIG), IP-10, and RANTES, the GTPase GBP2, and several other IFN--induced genes [e.g., IFI27 (18) and IFI30 (19), and tryptophanyl-tRNA synthetase (20)], whose relevance to the T cell response and viral clearance is not immediately apparent. IFN- itself, however, is not in this list because it was scored present in only a few samples (data not shown and Table 4, which is published as supporting information on the PNAS web site), suggesting that the absolute level of intrahepatic IFN- expression (Fig. 1) was too low to be consistently detected by the microarrays used. 2'5'OAS, which is induced during viral clearance (Fig. 1), also is not part of this list, because its induction during viral clearance in Ch1615 was very brief (Table 4) and therefore did not satisfy our selection criteria. In addition, several genes not known to be inducible by IFN- (e.g., apolipoprotein L3, butyrophilin A3, cathepsin C, cofilin 1, complement component C1q, galectin 3, lysozyme, Plac8, superoxide dismutase, and Ig heavy and light chain alleles) were also induced during viral clearance.

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Table 1. Liver genes associated with HBV clearance in all three chimpanzees

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Next, to identify viral clearance-associated genes that might have been excluded by our stringent selection criteria, we searched for genes whose expression correlated with the profiles of prototype genes, specifically T cell antigen receptor (TCR), TCR, CD3D, IFN-, MIG, RANTES, IP-10, and sALT, as indicated in columns 6-13 of Table 3. This increased the number of genes induced during viral clearance to 110, all of which are shown in Fig. 2B and identified in Tables 1 and 3.
This list contains additional T cell (CD38, CD53, and granzyme A), natural killer (NK) cell (CD53 and granzyme K), B cell (CD48 and CD83), macrophage (CD68), chemokine (CXCL11 and CXCR4), GTPase (GBP1), complement (C3a), IFN-stimulated (ISG20 and IFI16), and immunoproteasome (MECL-1) genes that were not included in the first-pass analysis. Importantly, 84 of these 110 genes were closely correlated with the TCR and - mRNA expression profiles (Table 3), 88 correlated with the induction profiles of MIG, IP-10, or RANTES, which are known to be highly induced by IFN- (21, 22), and 79 correlated with sALT levels in all three chimpanzees (Table 3 and Fig. 1). It is noteworthy that all of these genes returned to baseline when the viral DNA was eliminated (Fig. 2B). Because that correlation was not required for a gene to be designated as "clearance-associated," it strongly supports the notion that these genes were functionally related to viral clearance.

Sequential Induction of Clearance-Associated Liver Genes in Ch1627. Examination of the clearance-associated gene expression profile of Ch1627 (Figs. 2B and 3) reveals three distinct gene clusters whose expression peaked in week 12, 14, or 16 and were designated early, middle, and late clearance genes, accordingly (Fig. 3). The spacing of the sampled time points in the other chimpanzees did not permit sequential gene clusters to be identified in those animals. As shown in Table 1 and Fig. 3 (in blue), the early gene cluster consisted of 35 genes that included the TCR locus, several IFN--induced genes including MHC class I and II genes, the TAP-binding protein (tapasin), the immunoproteasome components PA28 and -, the chemokine RANTES, and IFN--inducible protein 30, suggesting that they are mainly T cell-related and IFN--induced genes that mediate antigen processing and presentation and inflammatory cell recruitment.

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Fig. 3. Kinetics of liver gene expression during viral clearance in Ch1627. Early (blue), Middle (red), and Late (green) genes displayed maximal expression in weeks 12, 14, and 16, respectively.

The middle gene cluster [Fig. 3 (shown in red) and Table 1] consisted of 73 genes, including CD3D, the TCR locus, a T cell activation marker (CD38), and the cytolytic effector granzymes A and K, as well as a T-LAK cell-originated protein kinase, a macrophage antigen (CD68), and a B cell membrane protein (CD48). Several additional IFN--induced genes were also detected, including STAT1, the chemokines IP-9, IP-10, and MIG, the immunoproteasome component MECL-1, several ubiquitin-related genes, the GTPase GBP1, and its closely related family member GTPase guanylate-binding protein 2. Thus, the second wave of genes expressed during viral clearance in this animal reflects the amplification and diversification of the intrahepatic inflammatory response and the induction of antiviral activity within the infected cells.
Only two of the clearance-associated genes (i.e., Ig heavy and light chains) reached peak induction late in infection in this animal [Fig. 3 (shown in green) and Table 1], reflecting the expansion of a B cell infiltrate when the viral DNA was disappearing from the liver.

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