Jan Grabowski

Hepatitis E virus (HEV)‐specific T‐cell responses are associated with control of HEV infection

Hepatitis E virus (HEV) infection is usually self‐limited but may lead to acute hepatitis and rarely to fulminant hepatic failure. Persistent HEV infections have recently been described in organ transplant recipients receiving immunosuppressive medications, suggesting that HEV is controlled by adaptive immune responses. However, only few studies have investigated HEV‐specific T‐cell responses and immune correlates for the failure to clear HEV infection have not been established so far. We investigated T‐cell responses against HEV in 38 subjects including anti‐HEV‐positive (exposed, n = 9) and anti‐HEV‐negative (n = 10) healthy controls, 12 anti‐HEV‐positive but HEV RNA‐negative organ transplant recipients, and seven transplant recipients with chronic hepatitis E. Proliferation as well as cytokine production of CD4+ and CD8+ T cells was studied after stimulation with overlapping peptides spanning all proteins encoded by HEV‐open reading frame (ORF)2 and HEV‐ORF3. We show that (1) strong and multispecific HEV‐specific T‐cell responses are present in exposed healthy controls, and to a lesser extent also in recovered patients after transplantation; (2) that these responses are absent in patients with chronic hepatitis E but become detectable after viral clearance; and (3) that HEV‐specific T‐cell responses can be restored in vitro by blocking the PD‐1 or CTLA‐4 pathways. However, a combination of PD‐1 and CTLA‐4 blockade had no synergistic effects. We conclude that chronic hepatitis E is associated with impaired HEV‐specific T‐cell responses and suggest that enhancing adaptive cellular immunity against HEV might prevent persistent HEV infections. (HEPATOLOGY 2012)

Dual function of the NK cell receptor 2B4 (CD244) in the regulation of HCV-specific CD8+ T cells

The outcome of viral infections is dependent on the function of CD8+ T cells which are tightly regulated by costimulatory molecules. The NK cell receptor 2B4 (CD244) is a transmembrane protein belonging to the Ig superfamily which can also be expressed by CD8+ T cells. The aim of this study was to analyze the role of 2B4 as an additional costimulatory receptor regulating CD8+ T cell function and in particular to investigate its implication for exhaustion of hepatitis C virus (HCV)-specific CD8+ T cells during persistent infection. We demonstrate that (i) 2B4 is expressed on virus-specific CD8+ T cells during acute and chronic hepatitis C, (ii) that 2B4 cross-linking can lead to both inhibition and activation of HCV-specific CD8+ T cell function, depending on expression levels of 2B4 and the intracellular adaptor molecule SAP and (iii) that 2B4 stimulation may counteract enhanced proliferation of HCV-specific CD8+ T cells induced by PD1 blockade. We suggest that 2B4 is another important molecule within the network of costimulatory/inhibitory receptors regulating CD8+ T cell function in acute and chronic hepatitis C and that 2B4 expression levels could also be a marker of CD8+ T cell dysfunction. Understanding in more detail how 2B4 exerts its differential effects could have implications for the development of novel immunotherapies of HCV infection aiming to achieve immune control.

Compromised Function of Natural Killer Cells in Acute and Chronic Viral Hepatitis

BACKGROUND  Natural killer (NK) cells are an integral part of the innate immune system. They have been suggested to play an important role in both defense against viral hepatitis and the pathogenesis of other liver diseases. METHODS  NK cells from 134 individuals including patients with acute hepatitis B and C as well as chronic hepatitis B, C, and delta (D) patients were studied. RESULTS  Infection with viral hepatitis was associated with increased frequencies of NK cells in the peripheral blood; that NK cells showed a less activated phenotype and were compromised in cytolotytic function and cytokine production in all viral hepatitis infections: Hepatitis virus infections did not alter NK cell differentiation, and the activity and severity of liver disease were reflected by alterations of NK cell surface receptors as demonstrated by principal component analysis. CONCLUSION  NK cell phenotypic and functional alterations can equally be observed in HBV, HCV, and HDV infections. Instead, patterns of NK cell alterations differ in acute and chronic infections. Thus, our data suggest a common mechanism in the alteration of NK cell phenotype and function with unique variations that depend on disease activity rather than virus-specific factors.

Hepatitis Delta: Immunopathogenesis and Clinical Challenges

Hepatitis delta is caused by infection with the hepatitis D virus (HDV) and is considered to be the most severe form of viral hepatitis in humans. Hepatitis delta occurs only in hepatitis B virus (HBV) surface antigen (HBsAg)-positive individuals as HDV is a defective RNA virus which requires the HBsAg for complete replication and transmission. Eight different HDV genotypes have been described with specific geographic distributions and distinct clinical courses. HDV/HBV co-infection can be associated with complex and dynamic viral dominance patterns. While HDV is frequently the dominating virus not only in HBV/HDV co-infection, but also in HBV/HCV/HDV triple-infected patients, the fluctuating courses of HDV and HBV viremia can be observed in other patients. Chronic HDV infection leads to more severe liver disease than HBV monoinfection with accelerated fibrosis progression, earlier hepatic decompensation and an increased risk for the development of hepatocellular carcinoma. However and in contrast to HCV infection, hepatic decompensation, rather than development of liver cancer, is the first clinical endpoint that develops during the course of infection. So far, only interferon-α treatment has proven antiviral activity against HDV in humans and has been linked to improved long-term outcome. Recent studies on the use of pegylated interferon showed a sustained virological response concerning HDV-RNA in about one quarter of the patients. HDV-specific immune responses might be associated with the response to treatment. Novel alternative treatment options, including prenylation inhibitors, are still awaiting clinical development for delta hepatitis. So far, only few studies have investigated immune responses against HDV and HBV in humans.

Hepatitis D virus-specific cytokine responses in patients with chronic hepatitis delta before and during interferon alfa-treatment.

Background: Hepatitis delta is caused by infection with the hepatitis D virus (HDV) and is considered the most severe form of viral hepatitis. Treatment options for hepatitis delta are limited, with only 25% of patients responding to interferon (IFN)‐alfa‐based therapies. The role of the adaptive immune system in controlling HDV infection during spontaneous or treatment‐induced viral clearance is not well understood.

Effects of HDV infection and pegylated interferon α treatment on the natural killer cell compartment in chronically infected individuals

Objective Although hepatitis delta is considered an immune-mediated disease, adaptive immune responses to hepatitis delta virus (HDV) are hardly detectable. Thus, the role of other immune responses, including those mediated by natural killer (NK) cells, must be considered in HDV pathogenesis and in treatments with immune-stimulating agents such as interferon (IFN)α. However, the phenotype and function of NK cells in chronic HDV infection, or in HDV-infected individuals undergoing IFNα treatment, have not been extensively studied. Design We performed an extensive analysis of NK cells in chronically HDV-infected patients before and during treatment with IFNα, and compared the results with those for patients with HBV mono-infection as well as healthy controls. Results In untreated HDV-infected patients, a higher than normal frequency of NK cells was observed in peripheral blood with unaltered phenotypic NK cell differentiation status. In contrast, long-term IFNα treatment of HDV-infected patients caused a significant change in NK cell differentiation status, with selective loss of terminally differentiated NK cells and, in parallel, a relative enrichment in immature NK cell subsets. Treatment was associated with marked functional impairment of the NK cells, which was independent of the changes in NK cell differentiation status. Furthermore, treatment polarised NK cell IFN signalling from STAT4 towards STAT1 dependency. Strikingly, a high frequency of CD56dim NK cells at baseline was positively associated with IFNα treatment outcome in the patients. Conclusions We describe in detail how HDV infection, and IFNα treatment of this infection, affects the NK cell compartment and what consequences this has for the functional capacity of NK cells.

Anti-HDV IgM as a Marker of Disease Activity in Hepatitis Delta

Background Hepatitis delta frequently leads to liver cirrhosis and hepatic decompensation. As treatment options are limited, there is a need for biomarkers to determine disease activity and to predict the risk of disease progression. We hypothesized that anti-HDV IgM could represent such a marker. Methods Samples of 120 HDV-infected patients recruited in an international multicenter treatment trial (HIDIT-2) were studied. Anti-HDV IgM testing was performed using ETI-DELTA-IGMK-2-assay (DiaSorin). In addition, fifty cytokines, chemokines and angiogenetic factors were measured using multiplex technology (Bio-Plex System). A second independent cohort of 78 patients was studied for the development of liver-related clinical endpoints (decompensation, HCC, liver transplantation or death; median follow up of 3.0 years, range 0.6–12). Results Anti-HDV IgM serum levels were negative in 18 (15%), low (OD<0.5) in 76 (63%), and high in 26 (22%) patients of the HIDIT-2 cohort. Anti-HDV IgM were significantly associated with histological inflammatory (p<0.01) and biochemical disease activity (ALT, AST p<0.01). HDV replication was independent from anti-HDV IgM, however, low HBV-DNA levels were observed in groups with higher anti-HDV IgM levels (p<0.01). While high IP-10 (CXCL10) levels were seen in greater groups of anti-HDV IgM levels, various other antiviral cytokines were negatively associated with anti-HDV IgM. Associations between anti-HDV IgM and ALT, AST, HBV-DNA were confirmed in the independent cohort. Clinical endpoints occurred in 26 anti-HDV IgM positive patients (39%) but in only one anti-HDV IgM negative individual (9%; p = 0.05). Conclusions Serum anti-HDV IgM is a robust, easy-to-apply and relatively cheap marker to determine disease activity in hepatitis delta which has prognostic implications. High anti-HDV IgM levels may indicate an activated interferon system but exhausted antiviral immunity.

Monocytes transduced with lentiviral vectors expressing hepatitis C virus non-structural proteins and differentiated into dendritic cells stimulate multi-antigenic CD8+ T cell responses

Halting the spread of hepatitis C virus (HCV) and also eradicating HCV in subjects with chronic infection are major goals for global health. To this end, several years of research on HCV vaccine development have led to the conclusion that multi-antigenic and multi-functional vaccine types are necessary for effectiveness against HCV infection. In this study, we evaluated lentiviral vectors (LV) expressing clusters of HCV structural (LV-HCV-S) and non-structural (LV-HCV-NS) genes for future vaccine development. Batches of high titer LV were used to transduce differentiated dendritic cells (DC) and monocytes. We report successful delivery of HCV gene clusters, particularly into monocytes, leading to >80% LV-HCV-NS and >70% LV-HCV-S and transduced cells, respectively. Intracellular expression of HCV proteins in monocyte-derived DC resulted in immunophenotypic changes, such as downregulation of CD83 and CD86. Monocytes expressing NS proteins and differentiated into DC stimulated allogeneic and autologous CD8(+) and CD4(+) T cells in vitro and resulted in antigen-specific CD8(+) T cell responses against NS3, NS4a and NS5b. Hence, lentiviral-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality.

The third signal cytokine IL-12 rather than immune checkpoint inhibitors contribute to the functional restoration of hepatitis D virus-specific T-cells.

Background. Hepatitis D virus (HDV) infection affects 15–20 million individuals worldwide and causes severely progressive hepatitis. It is unknown to what extent cellular immune responses contribute to liver disease and control of viral replication in HDV infection. Methods. Immune cell frequencies and phenotypes were determined in 49 HDV-infected patients, 25 individuals with hepatitis B virus monoinfection and 18 healthy controls. T-cell proliferative and cytokine-producing capacities were analyzed by stimulation with overlapping peptides spanning the large HDV antigen. To restore T-cell responses, blocking antibodies (anti-cytotoxic T-lymphocyte antigen 4, anti-programmed death ligand 1) or proinflammatory cytokines (interleukin [IL] 12) were used. Results. Immune cell frequencies and phenotypes did not vary between the groups. Exclusively, the senescence marker CD57 was significantly up-regulated in CD8+ T cells from patients with hepatitis delta. HDV-specific T-cell proliferation and cytokine production were weak and could only partly be rescued by blockade of the programmed death 1 pathway. However, a more robust and consistent increase in HDV-specific CD4+ and CD8+ T-cell responses was evident when the third signal cytokine IL-12 was added, which also affected cytomegalovirus- and Epstein-Barr virus–specific T cells. Conclusions. This investigation of virus-specific T-cell immunity in patients with HDV infection, the largest to date, revealed premature aging of immune cells and impaired T-cell functionality. This could be restored by blocking inhibitory pathways and, in particular, by supplementing with IL-12.

Immunopathogenesis of Hepatitis D

Infection with the hepatitis D virus (HDV) causes hepatitis delta, which is considered to be the most severe form of viral hepatitis. HDV is a defective virus, using the hepatitis B virus (HBV) surface antigen (HBsAg) as its envelope protein; thus, hepatitis delta affects only carriers of the HBsAg. Of the 350 million people infected with HBV worldwide, 15–20 million are estimated to be coinfected with HDV; thus, hepatitis delta infection represents a global health burden. Immune responses to HDV are less well studied than for HCV and HBV, but in recent years, advances have been made to gain more knowledge on the interaction of HDV and the immune system. Since HDV is noncytopathic, immune responses play a key role not only in the control of the infection but also in the pathogenesis of liver disease.