T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice.

BACKGROUND & AIMS: Antiviral agents suppress hepatitis B virus (HBV) replication but do not clear the infection. A strong effector T-cell response is required to eradicate HBV, but this does not occur in patients with chronic infection. T cells might be directed toward virus-infected cells by expressing HBV-specific receptors and thereby clear HBV and help to prevent development of liver cancer. In mice, we studied whether redirected T cells can engraft after adoptive transfer, without prior T-cell depletion, and whether the large amounts of circulating viral antigens inactivate the transferred T cells or lead to uncontrolled immune-mediated damage. METHODS: CD8(+) T cells were isolated from mice and stimulated using an optimized protocol. Chimeric antigen receptors (CARs) that bind HBV envelope proteins (S-CAR) and activate T cells were expressed on the surface of cells using retroviral vectors. S-CAR-expressing CD8(+) T cells, which carried the marker CD45.1, were injected into CD45.2(+) HBV transgenic mice. We compared these mice with mice that received CD8(+) T cells induced by vaccination, cells that express a CAR without a proper signaling domain, or cells that express a CAR that does not bind HBV proteins (controls). RESULTS: CD8(+) T cells that expressed HBV-specific CARs recognized different HBV subtypes and were able to engraft and expand in immune-competent HBV transgenic mice. After adoptive transfer, the S-CAR-expressing T cells localized to and functioned in the liver and rapidly and efficiently controlled HBV replication compared with controls, causing only transient liver damage. The large amount of circulating viral antigen did not impair or overactivate the S-CAR-grafted T cells. CONCLUSIONS: T cells with a CAR specific for HBV envelope proteins localize to the liver in mice to reduce HBV replication, causing only transient liver damage. This immune cell therapy might be developed for patients with chronic hepatitis B, regardless of their HLA type.

Transfer of HBV genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice.

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors.

UNLABELLED: Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. CONCLUSION: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.

Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection.

BACKGROUND & AIMS: The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown. METHODS: Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed. RESULTS: We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA. CONCLUSIONS: Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.

Apoptosis of hepatitis B virus-infected hepatocytes prevents release of infectious virus.

Apoptosis of infected cells is critically involved in antiviral defense. Apoptosis, however, may also support the release and spread of viruses. Although the elimination of infected hepatocytes is required to combat hepatitis B virus (HBV) infection, it is still unknown which consequences hepatocyte apoptosis has for the virus and whether or not it is advantageous to the virus. To study this, we designed a cell culture model consisting of both HBV-producing cell lines and primary human hepatocytes serving as an infection model. We showed that the release of mature, enveloped virions was 80% to 90% reduced 24 h after the induction of apoptosis in HBV-replicating hepatoma cells or HBV-infected hepatocytes. Importantly, HBV particles released from apoptotic hepatocytes were immature and nonenveloped and proved not to be infectious. We found an inverse correlation between the strength of an apoptotic stimulus and the infectivity of the virus particles released: the more potent the apoptotic stimulus, the higher the ratio of nonenveloped capsids to virions and the lower their infectivity. Furthermore, we demonstrated that HBV replication and, particularly, the expression of the HBx protein transcribed from the viral genome during replication do not sensitize cells to apoptosis. Our data clearly reject the hypothesis that the apoptosis of infected hepatocytes facilitates the propagation of HBV. Rather, these data indicate that HBV needs to prevent the apoptosis of its host hepatocyte to ensure the release of infectious progeny and, thus, virus spread in the liver.

Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.

UNLABELLED: With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication. CONCLUSION: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.

Hepatitis B virus promotes ß-catenin-signalling and disassembly of adherens junctions in a Src kinase dependent fashion.

Hepatitis B virus (HBV) infection is a prominent cause of hepatocellular carcinoma (HCC) but the underlying molecular mechanisms are complex and multiple pathways have been proposed such as the activation of the Wnt-/ß-catenin-signalling and dysregulation of E-cadherin/ß-catenin adherens junctions. This study aimed to identify mechanisms of how HBV infection and replication as well as HBV X protein (HBx) gene expression in the context of an HBV genome influence Wnt-/ß-catenin-signalling and formation of adherens junctions and to which extent HBx contributes to this. Regulation of E-cadherin/ß-catenin junctions and ß-catenin-signalling as well as the role of HBx were investigated using constructs transiently or stably inducing replication of HBV+/-HBx in hepatoma cell lines. In addition, HCC and adjacent non-tumorous tissue samples from HBV-infected HCC patients and drug interference in HBV-infected cells were studied. Although HBV did not alter overall expression levels of E-cadherin or ß-catenin, it diminished their cell surface localization resulting in nuclear translocation of ß-catenin and activation of its target genes. In addition, HBV gene expression increased the amount of phosphorylated c-Src kinase. Treatment with Src kinase inhibitor Dasatinib reduced HBV replication, prevented adherens junction disassembly and reduced ß-catenin-signalling, while Sorafenib only did so in cells with mutated ß-catenin. Interestingly, none of the HBV induced alterations required HBx. Thus, HBV stimulated ß-catenin-signalling and induced disassembly of adherens junctions independently of HBx through Src kinase activation. These pathways may contribute to hepatocellular carcinogenesis and seem to be more efficiently inhibited by Dasatinib than by Sorafenib.

Hepatitis B Virus Activates Signal Transducer and Activator of Transcription 3 Supporting Hepatocyte Survival and Virus Replication.

BACKGROUND & AIMS: The human hepatitis B virus (HBV) is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection. METHODS: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3) was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA-mediated knockdown of STAT3. RESULTS: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6-dependent and -independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA-mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression. CONCLUSIONS: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis.