- 现金
- 3700 元
- 精华
- 16
- 帖子
- 1790
- 注册时间
- 2002-12-9
- 最后登录
- 2021-4-14
|
2楼
发表于 2004-8-26 10:27
[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.
--------------------------------------------------------------------------------
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. |
|