Comment on "Immunomodulation by bepirovirsen may induce killing of infected hepatocytes (B-Together study)".

The hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and hepatocellular carcinoma. Most infected individuals become lifelong carriers of HBV as the drugs currently used to treat the patients can only control the disease, thereby achieving functional cure (loss of the hepatitis B surface antigen) but not complete cure (elimination of infected hepatocytes). Therefore, we aimed to identify the target genes for the selective killing of HBV-positive hepatocytes to develop a novel therapy for the treatment of HBV infection. Our strategy was to recognize the conditionally essential genes that are essential for the survival of HBV-positive hepatocytes, but non-essential for the HBV-negative hepatocytes. Using microarray gene expression data curated from the Gene Expression Omnibus database and the known essential genes from the Online GEne Essentiality database, we used two approaches, comprising the random walk with restart algorithm and the support vector machine approach, to determine the potential targets for the selective killing of HBV-positive hepatocytes. The final candidate genes list obtained using these two approaches consisted of 36 target genes, which may be conditionally essential for the cell survival of HBV-positive hepatocytes; however, this requires further experimental validation. Therefore, the genes identified in this study can be used as potential drug targets to develop novel therapeutic strategies for the treatment of HBV, and may ultimately help in achieving the elusive goal of a complete cure for hepatitis B.

74. The Mechanism of Hepatotoxicity from IV Administration of High Dose VSV

Targeting Innate Immunity: A New Step in the Development of Combination Therapy for Chronic Hepatitis B

Hepatitis B virus (HBV) causes a productive, non-cytopathic infection of the hepatocyte population that can lead to chronic liver disease and, after many years, cirrhosis and hepatocellular carcinoma (HCC). The primary cause of chronic liver disease and cirrhosis is the immune killing of infected hepatocytes. The cause of HCC is unclear. As reviewed here, possible contributors include oxidative damage/mutation of hepatocyte DNA, accelerated turnover of this largely self-renewing population, expression of wild type and mutant viral proteins that alter host gene expression, hepatocyte repopulation due to selective immune killing of virus-susceptible hepatocytes and, in some patients, cirrhosis itself. Important clues to hepatocyte transformation and HCC have appeared over the past 20 years, mostly from studies in model systems. It now appears very likely, though not a surprise, that the antiviral immune response and associated increases in liver regeneration are major factors in HCC. Other factors remain elusive, primarily because it is not a simple matter to show that results in model systems are applicable to a natural infection, where progression from HBV infection to HCC may take 40 years or more. This creates an obvious difficulty in classifying, never mind proving, the major steps in neoplastic transformation of just a few hepatocytes to HCCs in a liver population of nearly 5 × 1011. Current results highlight the need not only for more translational research, but also for improved antiviral therapy of HBV carriers, and earlier detection (and treatment) of HCCs before they have caused major liver impairment and have reached a size that is untreatable.

A View to Natural Killer Cells in Hepatitis C

Eliminating cccDNA to cure hepatitis B virus infection

Background & AimsAntigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy.MethodsPrimary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity.ResultsUsing unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides.ConclusionsWe present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general.Lay summaryImmunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.

SARS-CoV-2 infection is accompanied by elevated liver enzymes, and patients with pre-existing liver conditions experience more severe disease. While it was known that SARS-CoV-2 infects human hepatocytes, our study determines the mechanism of infection, demonstrates viral replication and spread, and highlights direct hepatocyte damage. Viral replication was readily detectable upon infection of primary human hepatocytes and hepatoma cells with the ancestral SARS-CoV-2, Delta, and Omicron variants. Hepatocytes express the SARS-CoV-2 receptor ACE2 and the host cell protease TMPRSS2, and knocking down ACE2 and TMPRSS2 impaired SARS-CoV-2 infection. Progeny viruses released from infected hepatocytes showed the typical coronavirus morphology by electron microscopy and proved infectious when transferred to fresh cells, indicating that hepatocytes can contribute to virus spread. Importantly, SARS-CoV-2 infection rapidly induced hepatocyte death in a replication-dependent fashion, with the Omicron variant showing faster onset but less extensive cell death. C57BL/6 wild-type mice infected with a mouse-adapted SARS-CoV-2 strain showed high levels of viral RNA in liver and lung tissues. ALT peaked when viral RNA was cleared from the liver. Liver histology revealed profound tissue damage and immune cell infiltration, indicating that direct cytopathic effects of SARS-CoV-2 and immune-mediated killing of infected hepatocytes contribute to liver pathology.

Although the liver's function as unique immune organ regulating immunity has received a lot of attention over the last years, the mechanisms determining hepatic immune surveillance against infected hepatocytes remain less well defined. Liver sinusoidal cells, in particular, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), serve as physical platform for recruitment and anchoring of blood-borne immune cells in the liver. Liver sinusoidal cells also function as portal of entry for infectious microorganisms targeting the liver such as hepatotropic viruses, bacteria or parasites. At the same time, liver sinusoidal cells actively contribute to achieve immune surveillance against bacterial and viral infections. KCs function as adhesion hubs for CD8 T cells from the circulation, which initiates the interaction of virus-specific CD8 T cells with infected hepatocytes. Through their phagocytic function, KCs contribute to removal of bacteria from the circulation and engage in cross talk with sinusoidal lymphocyte populations to achieve elimination of phagocytosed bacteria. LSECs contribute to local immune surveillance through cross-presentation of viral antigens that causes antigen-specific retention of CD8 T cells from the circulation. Such cross-presentation of viral antigens activates CD8 T cells to release TNF that in turn triggers selective killing of virus-infected hepatocytes. Beyond major histocompatibility complex (MHC)-restricted T-cell immunity, CD1- and MR1-restricted innate-like lymphocytes are found in liver sinusoids whose roles in local immune surveillance against infection need to be defined. Thus, liver sinusoidal cell populations bear key functions for hepatic recruitment and for local activation of immune cells, which are both required for efficient immune surveillance against infection in the liver.

Bepirovirsen is an investigational drug; its multimodal mechanism of action (MoA) is under evaluation. Observations in treated participants show transient alanine aminotransferase (ALT) increases, alongside hepatitis B surface antigen (HBsAg) declines. We investigated bepirovirsen's MoA in relation to virological response, hepatocyte death, and ALT increases. In B-Together (NCT04676724), 108 participants on stable nucleos(t)ide analogs received bepirovirsen for 24 (Arm 1) or 12 (Arm 2) Weeks, then up to 24Weeks of pegylated interferon-α-2a. This post hoc peripheral longitudinal biomarker exploratory analysis examined serum proteomics and whole blood transcriptomics from peripheral blood mononuclear cell samples from 82 participants. Relative expressions of immune- and disease-related biomarkers were measured, and differential expression determined across arms and response subgroups. Increases from baseline in mean expression of serum proteins with immune effector and apoptotic functions (Week 3) and transcripts associated with immune cell proliferation and activation (Week 5) were observed regardless of arm or response subgroup. By Week 8, serum liver and apoptosis-specific proteins were increased; this was more pronounced in responders than non-responders, with the difference more marked in Arm 1 versus Arm 2. Increased abundance of these proteins was highly correlated with ALT levels, which were often associated with transient hepatitis B virus (HBV) DNA elevations and HBsAg decreases. These findings provide evidence that bepirovirsen may modulate the immune system to facilitate infected hepatocyte killing in chronic HBV infection in addition to its direct antiviral effects; therefore, ALT increases could reflect a therapeutic response to bepirovirsen. NCT04676724 and NCT04449029.

The most reliable method of making a specific aetiological diagnosis of chronic viral hepatitis would be to identify virus specific cytotoxic T lymphocytes responsible for the killing of virus infected hepatocytes in each patient's liver. Unfortunately, this can not be proposed for routine diagnosis and surrogate tests are required. The detection of virus markers, and even of the virus itself, does not imply that liver damage is caused by virus infection. Indirect markers of the host's antiviral immunoresponse have to be used to confirm more specifically the diagnosis of viral hepatitis. IgM antibodies against viral antigens implicated in the elimination of the virus seem to be suitable alternative candidates. Significant changes in the serum values of viraemia and aminotransferases occur within a few days, while a significant variation in liver histology takes much longer. Only the kinetics of the highly variable parameters can be used for an appropriate study of the relationship between viraemia, antiviral immunoresponse, and liver cell necrosis. Quantitative and dynamic analyses of hepatitis virus markers seem the most suitable and reliable methods of monitoring the patients eligible for antiviral treatment and identifying the most appropriate time to start this.

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell-mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8(+) T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8(+) T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8(+) T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8(+) T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8(+) T cells produce an array of cytokines, among which IFN-gamma and TNF-alpha are responsible for HBV inactivation in the target cells. Blockade of IFN-gamma and TNF-alpha abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-gamma and rTNF-alpha resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8(+) T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-gamma and TNF-alpha.

Current treatments efficiently control chronic HBV infection but they do not lead to its elimination. Now, Ebert and colleagues have shown that cellular inhibitor of apoptosis proteins (cIAPs) prevent TNF-mediated killing of infected hepatocytes and that cIAP antagonists might lead to HBV cure by promoting death of infected cells.

The Pf72/Hsp70-1 antigen is a major target in the naturally acquired immunity against Plasmodium falciparum malaria. We carried out an extensive analysis of the responses to several epitopes on the least conserved C-terminal domain, according to the mode of sensitization: malaria infection or immunization with different immunogens. We found significant differences in the panel of B-cell epitopes recognized by animal models including primates, and by humans sensitized by natural infection. We focused the analysis on one epitope that is unique to Plasmodium species. It is specifically recognized by a monoclonal antibody that mediates the killing of infected hepatocytes in vitro. We produced a polymeric multiple antigenic peptide (MAP) form of this sequence, which enabled us to identify a new B-cell epitope not detected by ELISA with linear peptides. The polymer was strongly recognized by sera from monkeys or humans sensitized by natural infection, whereas the monomer was not. We modelled the three-dimensional structure of the Pf72/Hsp70-1 sequence, using known Escherischia coli DnaK structures as a template. This predicted that the corresponding region would form a loop in the native antigen. The results presented here suggest that the MAP strategy is also particularly useful as a means of obtaining suitable synthetic models for conformation-dependent epitopes.

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