Fritz von Weizsäcker

T Cells with a CD4+CD25+ Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8+ T Cells during Chronic Hepatitis C Virus Infection

ABSTRACT Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8+ T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4+CD25+ regulatory phenotype in suppressing virus-specific CD8+ T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8+ T cells were inhibited by CD4+CD25+ T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8+ T cells but also to influenza virus-specific CD8+ T cells. Importantly, CD4+CD25+ T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8+ T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4+CD25+ cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4+CD25+ T cells that are able to suppress CD8+ T-cell responses to different viral antigens. Our results further suggest that CD4+CD25+ T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.

p53 mutation in hepatocellular carcinoma after aflatoxin exposure

Mutations of the p53 gene are found in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Specific mutations might reflect exposure to specific carcinogens and we have screened HCC samples from patients in 14 different countries to determine the frequency of a hotspot mutation at codon 249 of the tumour suppressor p53 gene. We detected mutations in 17% of tumours (12/72) from four countries in south Africa and the southeast coast of Asia. There was no codon 249 mutation in 95 specimens of HCC from other geographical locations including North America, Europe, Middle East, and Japan. Worldwide, the presence of the codon 249 mutation in HCCs correlated with high risk of exposure to aflatoxins and the hepatitis B virus (HBV). Further studies were completed in two groups of HBV-infected patients at different risks of exposure to aflatoxins. 53% of patients (8/15) from Mozambique at high risk of aflatoxin exposure had a tumour with a codon 249 mutation, in contrast with 8% of patients from Transkei (1/12) who were at low risk. HCC is an endemic disease in Mozambique and accounts for up to two thirds of all tumours in men. A codon 249 mutation of the p53 gene identifies an endemic form of HCC strongly associated with dietary aflatoxin intake.

Dominant influence of an HLA‐B27 restricted CD8+ T cell response in mediating HCV clearance and evolution

Virus‐specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well‐defined cohort of women inoculated with HCV from a single source showed that HLA‐B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA‐B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA‐B27 restricted hepatitis C virus (HCV)‐specific CD8+ T cell epitope that is recognized in the majority of recovered HLA‐B27 positive women. In chronically HCV‐infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA‐B27, indicating allele‐specific selection pressure at the population level. Functional analysis in 3 chronically HCV‐infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA‐B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus‐specific CD8+ T cells and the genetically determined restriction of the virus‐specific T cell repertoire in HCV infection. (HEPATOLOGY 2006;43:563–572.)

Intrahepatic CD8+ T‐cell failure during chronic hepatitis C virus infection

The precise mechanisms responsible for the failure of intrahepatic hepatitis C virus (HCV)‐specific CD8+ T cells to control the virus during persistent infection have not been fully defined. We therefore studied the CD8+ T‐cell response in 27 HLA‐A2–positive patients using four previously well‐defined HLA‐A2–restricted HCV epitopes. The corresponding HCV sequences were determined in several patients and compared with the intrahepatic HCV‐specific CD8+ T‐cell response. The results of the study indicate: (1) intrahepatic HCV‐specific CD8+ T cells are present in the majority of patients with chronic HCV infection and overlap significantly with the response present in the peripheral blood. (2) A large fraction of intrahepatic HCV‐specific CD8+ T cells are impaired in their ability to secrete interferon γ (IFN‐γ). This dysfunction is specific for HCV‐specific CD8+ T cells, since intrahepatic Flu‐specific CD8+ T cells readily secrete this cytokine. (3) T‐cell selection of epitope variants may have occurred in some patients. However, it is not an inevitable consequence of a functional virus‐specific CD8+ T‐cell response, since several patients with IFN‐γ–producing CD8+ T‐cell responses harbored HCV sequences identical or cross‐reactive with the prototype sequence. (4) The failure of intrahepatic virus–specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus‐specific CD4+ T cells at the site of disease. In conclusion, different mechanisms contribute to the failure of intrahepatic CD8+ T cells to eliminate HCV infection, despite their persistence and accumulation in the liver. (HEPATOLOGY 2005;42:828–837.)

APOBEC‐mediated interference with hepadnavirus production

APOBEC3G is a cellular cytidine deaminase displaying broad antiretroviral activity. Recently, it was shown that APOBEC3G can also suppress hepatitis B virus (HBV) production in human hepatoma cells. In the present study, we characterized the mechanisms of APOBEC‐mediated antiviral activity against HBV and related hepadnaviruses. We show that human APOBEC3G blocks HBV production in mammalian and nonmammalian cells and is active against duck HBV as well. Early steps of viral morphogenesis, including RNA and protein synthesis, binding of pregenomic RNA to core protein, and self‐assembly of viral core protein, were unaffected. However, APOBEC3G rendered HBV core protein–associated full‐length pregenomic RNA nuclease‐sensitive. Ongoing reverse‐transcription in capsids that had escaped the block in morphogenesis was not significantly inhibited. The antiviral effect was not modulated by abrogating or enhancing expression of the accessory HBV X protein, suggesting that HBV X protein does not represent a functional homologue to the HIV vif protein. Furthermore, human APOBEC3F but not rat APOBEC1 inhibited HBV DNA production. Viral RNA and low‐level DNA produced in the presence of APOBEC3F or rat APOBEC1 occasionally displayed mutations, but the majority of clones were wild‐type. In conclusion, APOBEC3G and APOBEC3F but not rat APOBEC1 can downregulate the production of replication‐competent hepadnaviral nucleocapsids. In contrast to HIV and other retroviruses, however, APOBEC3G/3F‐mediated editing of nucleic acids does not seem to represent an effective innate defense mechanism for hepadnaviruses. (HEPATOLOGY 2005;42:301–309.)

Efficient Infection of Primary Tupaia Hepatocytes with Purified Human and Woolly Monkey Hepatitis B Virus

ABSTRACT The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.

Analysis of CD127 and KLRG1 Expression on Hepatitis C Virus-Specific CD8+ T Cells Reveals the Existence of Different Memory T-Cell Subsets in the Peripheral Blood and Liver

ABSTRACT The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1− and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.

Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.

Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide, but the study of HCV infection has been hampered by the lack of an in vitro or in vivo small animal model. The tree shrew Tupaia belangeri is susceptible to infection with a variety of human viruses in vivo, including hepatitis viruses. We show that primary Tupaia hepatocytes can be infected with serum- or plasma-derived HCV from infected humans, as measured by de novo synthesis of HCV RNA, analysis of viral quasispecies evolution, and detection of viral proteins. Production of infectious virus could be demonstrated by passage to naive hepatocytes. To assess whether viral entry in Tupaia hepatocytes was dependent on the recently isolated HCV E2 binding protein CD81, we identified and characterized Tupaia CD81. Sequence analysis of cloned Tupaia cDNA revealed a high degree of homology between Tupaia and human CD81 large extracellular loops (LEL). Cellular binding of E2 and HCV infection could not be inhibited by anti-CD81 antibodies or soluble CD81-LEL, suggesting that viral entry can occur through receptors other than CD81. Thus, primary Tupaia hepatocytes provide a potential model for the study of HCV infection of hepatocytes.

Hepatitis B Virus Nucleocapsids Formed by Carboxy-Terminally Mutated Core Proteins Contain Spliced Viral Genomes but Lack Full-Size DNA

ABSTRACT The carboxy-terminal sequence of the hepatitis B virus (HBV) core protein constitutes a nucleic acid binding domain that is rich in arginine residues and contains three serine phosphorylation sites. While dispensable for capsid assembly, this domain is involved in viral replication, as demonstrated by the effects of mutations on RNA packaging and/or reverse transcription; however, the underlying mechanisms are poorly understood. Here we tested a series of core protein mutants in which the three serine phosphorylation sites were replaced by glutamic acid, in parallel with a previously described deletion variant lacking the 19 C-terminal amino acid residues, for their ability to support viral replication in transfected hepatoma cells. Replacement of all serines and the deletion gave rise to nucleocapsids containing a smaller than wild-type DNA genome. Rather than a single-stranded DNA intermediate, as previously thought, this was a 2.0-kbp double-stranded DNA molecule derived from spliced pregenomic RNA (pgRNA). Interestingly, full-length pgRNA was associated with nucleocapsids but was found to be sensitive to nuclease digestion, while encapsidated spliced RNA and 3′ truncated RNA species were nuclease resistant. These findings suggest that HBV pgRNA encapsidation is directional and that a packaging limit is determined by the C-terminal portion of the core protein.