Hans Christian Spangenberg

Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease.

To define the early events that determine the outcome of acute hepatitis C virus (HCV) infection, we compared the course of viremia with the peripheral and intrahepatic T cell response and intrahepatic cytokine profile in six acutely infected chimpanzees. Three different outcomes were observed after peak viral titers were reached: sustained viral clearance, transient viral clearance followed by chronic infection, and chronic infection that persisted at initial peak titers. The results indicate that HCV spread outpaces the T cell response and that HCV rapidly induces but is not controlled by IFN-α/β; that viral clearance follows the entry and accumulation of HCV-specific IFN-γ-producing T cells in the liver; and that it may not require the destruction of infected cells.

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.

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.)

Expression of the Interleukin-7 Receptor Alpha Chain (CD127) on Virus-Specific CD8+ T Cells Identifies Functionally and Phenotypically Defined Memory T Cells during Acute Resolving Hepatitis B Virus Infection

ABSTRACT Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.

Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees

We have previously shown that hepatitis B virus (HBV) replication is controlled by noncytolytic mechanisms that depend primarily on the effector functions of the CD8+ T cell response, especially the production of IFN-γ in the liver. The mechanisms that control the nuclear pool of viral covalently closed circular DNA (cccDNA) transcriptional template of HBV, which must be eliminated to eradicate infection, have been difficult to resolve. To examine those mechanisms, we quantitated intrahepatic HBV cccDNA levels in acutely infected chimpanzees whose virological, immunological, and pathological features were previously described. Our results demonstrate that the elimination kinetics of the cccDNA are more rapid than the elimination of HBV antigen-positive hepatocytes during the early phase of viral clearance, and they coincide with the influx of small numbers of IFN-γ producing CD8+ T cells into the liver. In contrast, terminal clearance of the cccDNA is associated with the peak of liver disease and hepatocellular turnover and with a surge of IFN-γ producing CD8+ T cells in the liver. Collectively, these results suggest that cccDNA clearance is a two-step process mediated by the cellular immune response. The first step reduces the pool of cccDNA molecules noncytolytically, probably by eliminating their relaxed circular DNA precursors and perhaps by destabilizing them. The second step enhances this process by destroying infected hepatocytes and triggering their turnover. Surprisingly, despite this multipronged response, traces of cccDNA persist indefinitely in the liver, likely providing a continuous antigenic stimulus that confers lifelong immunity.

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.)

Virological and Immunological Determinants of Intrahepatic Virus-Specific CD8+ T-Cell Failure in Chronic Hepatitis C Virus Infection

Virus‐specific CD8+ T‐cells play an important role in the outcome of acute hepatitis C virus (HCV) infection. In the chronic phase, however, HCV can persist despite the presence of virus‐specific T‐cell responses. Therefore, we set out to perform a full‐breadth analysis of the intrahepatic virus‐specific CD8+ T‐cell response, its relation to the peripheral T‐cell response, and the overall influence of viral escape and the genetic restriction on intrahepatic CD8+ T‐cell failure. Intrahepatic and peripheral CD8+ T‐cells from 20 chronically HCV infected patients (genotype 1) were comprehensively analyzed using overlapping peptides spanning the entire HCV polyprotein in concert with autologous viral sequences that were obtained for all targeted regions. HCV‐specific CD8+ T‐cell responses were detectable in most (90%) chronically HCV‐infected patients, and two thirds of these responses targeted novel previously undescribed epitopes. Most of the responses were detectable only in the liver but not in the peripheral blood, indicating accumulation and enrichment at the site of disease. Of note, only approximately half of the responses were associated with viral sequence variations supported by functional analysis as viral escape mutations. Escape mutations were more often associated with HLA‐B alleles. Conclusion: Our results show an unexpected high frequency of intrahepatic virus‐specific CD8+ T‐cells, a large part of which continue to target the present viral antigens. Thus, our results suggest that factors other than mutational escape contribute to the failure of intrahepatic virus‐specific CD8+ T‐cells. (HEPATOLOGY 2008.)

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.

Targeted therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The major etiologies and risk factors for development of HCC are well defined and some steps of hepatocellular carcinogenesis have been elucidated. Despite these scientific advances and the implementation of measures for early detection of HCC in patients who are at risk of this disease, survival of patients has not improved greatly over the past three decades. This situation is partly due to the limited therapeutic options available. While surgery and percutaneous or transarterial interventions are effective for patients with limited or compensated underlying liver disease, more than 80% of patients present with multifocal HCC and/or advanced liver disease, or have comorbidities at the time of diagnosis. Treatment options for these patients have previously been limited to best supportive care. The effectiveness of targeted therapy with monoclonal antibodies or small-molecule kinase inhibitors has now been demonstrated for the treatment of different tumors. In 2007, the multitargeted kinase inhibitor, sorafenib, was found to prolong survival significantly for patients with advanced HCC. This Review discusses the mechanisms of targeted therapies and clinical studies that have investigated these therapies in patients with HCC. Perspectives for future developments are also provided.

Previously Infected Chimpanzees Are Not Consistently Protected against Reinfection or Persistent Infection after Reexposure to the Identical Hepatitis C Virus Strain

ABSTRACT Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.